Annex B 

Spirotetramat 

B.9 Ecotoxicology 

Reviewed by: 

AGES, Austria 

Peer – reviewed by: 

PMRA, Canada 

US EPA, USA 
– 2 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9 Ecotoxicology .........................................................................................................................1

B.9.1 Effects on birds (OECD IIA 8.1, IIIA 10.1)..............................................................................4

B.9.1.1 Acute oral toxicity (OECD IIA 8.1.1) .........................................................................................4

B.9.1.2 Short-term dietary toxicity (OECD IIA 8.1.2; OECD IIA 8.1.3)..................................................5

B.9.1.3 Sub-chronic toxicity and reproduction (OECD IIA 8.1.4) ..........................................................9

B.9.1.4 Supervised cage or field trials (OECD IIA 10.1.7) ..................................................................19

B.9.1.5 Acceptance of bait, granules or treated seeds by birds (OECD IIA 10.1.8) ...........................30

B.9.1.6 Effects of secondary poisoning (OECD IIA 10.1.9) ................................................................30

B.9.1.7 Summary of effects on birds ...................................................................................................30

B.9.2 Effects on aquatic organisms (OECD IIA 8.2, IIIA 10.2) ....................................................31

B.9.2.1 Acute toxicity of active substance, metabolites and formulations to aquatic organisms........31

B.9.2.2 Chronic toxicity of active substance, metabolites and formulations to aquatic 

organisms ...............................................................................................................................70

B.9.2.3 Bioaccumulation (OECD IIA 8.2.6, IIA 8.2.7)..........................................................................76

B.9.3 Effects on other terrestrial vertebrates (OECD IIIA 10.3)..................................................77

B.9.4 Effects on bees (OECD IIA 8.7; OECD IIIA 10.4.) ...............................................................78

B.9.4.1 Acute Toxicity (OECD IIA 8.7.1; OECD IIA 8.7.2; OECD III 10.4.2 )......................................78

B.9.4.2 Residue test (OECD IIA 8.7.3)................................................................................................83

B.9.4.3 Bee brood feeding test (OECD IIA 8.7.4) ...............................................................................83

B.9.4.4 Cage test (OECD IIIA 10.4.4) .................................................................................................83

B.9.4.5 Field tests (OECD IIIA 10.4.5) Formulation: Spirotetramat OD 100.....................................83

B.9.4.6 Tunnel tests (OECD IIIA 10.4.7) .............................................................................................93

B.9.4.7 Summary of effects on honey-bees......................................................................................106

B.9.5 Effects on other arthropod species (OECD IIA 8.8, IIIA 10.5).........................................109

B.9.5.1 Laboratory, extended laboratory and semi-field tests (OECD IIA 8.8.1; OECD IIIA 

10.5.1)...................................................................................................................................109

B.9.5.2 Semi-field or field tests (OECD IIIA 10.5.3) ..........................................................................120

B.9.5.3 Summary...............................................................................................................................122

B.9.6 Effects on earthworms (OECD IIA 8.9, IIIA 10.6.3)...........................................................124

B.9.6.1 Acute toxicity (OECD IIA 8.9.1, IIIA 10.6.1.1).......................................................................124

B.9.6.2 Sublethal effects (OECD IIA 8.9.2, IIIA 10.6.4) ....................................................................127

B.9.6.3 Field studies..........................................................................................................................129

B.9.7 Effects on soil non-target macro-organisms (OECD IIA 8.14, IIIA 10.6.6).....................130

B.9.7.1 Effects on a species level (gamasid mites)...........................................................................130

B.9.7.2 Effects on a functional level (degradation in litter bags) (OECD IIIA 10.6.7)........................131

B.9.7.3 Summary of effects on soil non-target macro-organisms.....................................................131

B.9.8 Effects on soil non-target micro-organisms (OECD IIA 8.10, IIIA 10.7).........................132

B.9.8.1 Nitrogen mineralization .........................................................................................................132

B.9.8.2 Carbon mineralization...........................................................................................................133

B.9.8.3 Summary of effects on non-target micro-organisms.............................................................134

B.9.9 Effects on other non-target organisms (flora and fauna) believed to be a risk 

(OECD IIA 8.12, IIIA 10.8)....................................................................................................135

B.9.9.1 Effects on terrestrial non-target plants (OECD IIIA 10.8.1) ..................................................135
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.10 Effects on biological methods of sewage treatment (OECD IIA 8.15) ...........................146

B.9.10.1 Activated sludge, respiration inhibition .................................................................................146

B.9.11 References relied on.............................................................................................................148

Appendix 

B.9.1.8 Risk assessment for birds.....................................................................................................163

B.9.2.4 Risk assessment for aquatic organisms ...............................................................................176

B.9.3.1 Risk assessment for mammals.............................................................................................182

B.9.4.8 Risk assessment for honey bees..........................................................................................186

B.9.5.4 Risk assessment for non-target arthropods..........................................................................188

B.9.6.4 Risk assessment for earthworms..........................................................................................190

B.9.7.4 Risk assessment for other soil non-target macro-organisms ...............................................193

B.9.8.4 Risk assessment for non-target micro-organisms ................................................................194

B.9.9.2 Risk assessment for terrestrial non-target plants .................................................................195

B.9.10.2 Risk assessment for biological methods of sewage treatment.............................................196
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.1 Effects on birds (OECD IIA 8.1, IIIA 10.1) 

B.9.1.1 Acute oral toxicity (OECD IIA 8.1.1) 

Active substance – Spirotetramat (BYI 08330) 

Reference: Acute Oral Toxicity for Bobwhite Quail (Colinus virginianus) for the Test 

Item BYI 08330 (techn.) 

Author(s), year: Barfknecht, R. (2003) 

Report/Doc. number: BAR / LD 047 

Guideline(s): US EPA, subdiv. E, § 71-1; EPA Series 850- OPPTS No. 850.2100 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat (techn.), batch No.: MIX-Batch 08045/0014, TOX-no. 6344-00; 

purity 97.5 % 

Material and methods: 

Test organism: Bobwhite quail (Colinus virginianus), sexually mature 

Dosage levels: 0, 500, 1000 and 2000 mg/kg body weight (nominal) 

Number of birds and 

replicates/group: 

five males and five females / treatment group and control 

Administration: single oral administration in gelatine capsules 

Duration: 14 d post-treatment observation period 

Test conditions: acclimation 6 weeks; Feeding: 15 h starvation prior to dosing, standard diet ad 

libitum; housing singly in pens with air-condition; 10 h photoperiod, daylight visual 

spectrum, 18.6 - 21.1 oC, 46.6 - 80.8 % relative humidity 

Test parameter: 

 Observations: Observations on mortality and signs of intoxication were made hourly on the day 

of dosing and at least once work-daily throughout the observation period. 

 Body weight: recorded prior to test initiation and on days 7 and 14 

 Feed consumption: determined on days 3 and 7 

 Necropsy: gross necropsy performed at termination 

Statistics: none 

Findings: 

Regurgitation: None. 

Mortalities: A female of the highest dose died on day 6 and a male on day 10. 

Clinical signs: The birds that died suffered from diarrhoea from day 1 after administration on. 

Before death they showed reduced activity, fluffed feathers, ptosis and apathy. 

Diarrhoea and soft excrements were also observed at 500 and 1000 mg a.s./kg 

bw and ceased on day 3 and 10, respectively. 

Body weight: Birds at 2000 mg a.s./kg bw lost body weight from day 0 to 7, but recovered until 

test termination. 

Feed consumption: Birds of all test doses reduced their food consumption from day 0 to 3, most 

evidently in the highest dose group. 

Gross post-mortem 

examination: 

no treatment-related findings at necropsy 

Conclusion: LD50 > 2000 mg a.s./kg bw, NOEL(mortality): 1000 mg a.s./kg bw, NOEL(sublethal 

effects) < 500 mg a.s./kg bw (lowest dose tested, effects: diarrhoea, soft 
– 5 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

excrement, reduced feed consumption) 

B.9.1.2 Short-term dietary toxicity (OECD IIA 8.1.2; OECD IIA 8.1.3) 

Active substance - Spirotetramat (BYI 08330) 

Reference: BYI 08330 techn. a.s.: 5-Day-Dietary LC50 Bobwhite Quail (Colinus 

virginianus) 

Author(s), year: Barfknecht, R. (2004) 

Report/Doc. number: BAR / LC017, M-084796-01-2 

Guideline(s): EPA, OPPTS No.: 850.2200; OECD No. 205 

GLP: Yes (certified laboratory) 

Deviations: None relevant; relative humidity not reported, temperatures were warmer (32.6 – 

38.3 °C) than EPA-recommended (22 - 27 °C), acclimation period was only 3 d 

and not 7 days as recommended by EPA guidelines. 

Validity: Acceptable 

Test substance: Spirotetramat (techn.), batch No.: MIX-Batch 08045/0014, TOX-no. 6344-00; 

purity 97.5 % 

Material and methods: 

Test organism: Bobwhite quail (Colinus virginianus), 10 days old and weighing 15 - 20.9 g 

Dosage levels: control (untreated diet); 313, 625, 1250, 2500 and 5000 ppm a.s. (nominal) 

corresponding to 301, 624, 1218, 2552 and 4998 mg measured a.s./kg feed 

Number of birds and 

replicates/group: 

10 birds per diet group, 2 control groups, sex undetermined 

Administration: test substance mixed in diet 

Duration: 5 days administration period and 3 days post-exposure period 

Test conditions: acclimation: 3 d; feeding: standard rearing diet for quails ad libitum; housing in 

stainless steel wire cages; temperature: 32.6 - 38.3 °C, relative humidity: not 

reported, photoperiod 14 h 

Analytics: Concentration, homogeneity and stability of the test substance in the diet were 

sufficiently verified by analytical methods 

Test parameter: 

 Observations: Observations for signs of intoxication were made daily during acclimation, twice 

on the first exposure day and continued at least once daily until termination. 

 Body weight: Individual body weights were measured at test initiation, on day 5 and at 

termination of the test on day 8 

 Feed consumption: daily feed consumption per group for the periods day 0-4 and day 5-7 determined 

Necropsy: Gross necropsy performed at termination 

Statistics: Statistics was performed in 2008 upon request of US EPA (not included in study 

report): The data were subjected to an analysis of equal variances (Bartlett‘s test). 

In case of equal variances (which was the case for all tests) subsequent analyses 

were conducted using parametric techniques (Dunnett’s test). 

The program was ToxRat Professional Version 2.09 Software. 

Findings: 

Mortalities: None treatment related. One chick of the 1250 ppm group was sacrificed because 

of an injury. 

Clinical signs: During the exposure period the chicks of the highest dose group showed signs of 
– 6 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

intoxication like soft excrements (reversible). 

Body weight: Body weights were statistically analysed for day 0, +5 and +8. At the beginning 

the birds at 625 ppm and at 5000 ppm had a slightly higher body weight, which 

was statistically significant. After the exposure (day +5) only the birds at 2500 ppm 

showed significantly lower body weights while at day +8 (end of recovery) no 

differences between control and treatment groups were found. From these results 

it was concluded that there was no impact on body weight. 

A significantly reduced body weight change during the exposure period for the 

birds at 2500 ppm and at 5000 ppm was detected. This finding is is in good 

compliance with the reduced food consumption at these concentrations. 

In the recovery period, the birds of the two highest concentrations grew more than 

the control or the lower concentration groups. At 5000 ppm the increased growth 

rate was even statistically significant. 

These findings indicated a severe starvation effect at the high test concentration. 

These effects were compensated as soon as the birds were switched to normal 

diet. 

Feed consumption: Food avoidance in the 2500 ppm a.s. group and above. Chicks consumed 30 - 

50 % less of the amount of the control groups. No differences in the postexposure period. 

Necropsy: No findings except the injured bird (not treatment related). 

Table B.9.1.2-1a: Summary of effects of spirotetramat on Bobwhite Quail following short-term 

dietary exposure 

Treatment

(measured)

[ppm a.s.] 

Cumulative 

mortality 

Mean bw 

per bird

d 0 – 4 [g]

Mean bw 

gain d 0 - 5 

[%] 

Mean bw 

gain d 6 - 8 

[%] 

Mean feed 

consumption 

d 0 - 4 [g/bird/d]

Mean feed 

consumption 

d 5 - 7 [g/bird/d]

Estimated 

intake d 0 - 4 

[mg as/kg bw/d]

Control 0/20 21.4 46 23.5 3.1 3.8 0 

301 0/10 22.6 43 23.7 3.1 3.9 39.8 

624 0/10 23.0 43.4 26.2 3.1 4.1 82.6 

1218 1/10 20.8 41.3 25.5 3.1 3.6 182.7 

2552 0/10 19.6 28.7 28.2 1.8 3.4 234.7 

4998 0/10 21.1 24.5 37.2 2.1 4.3 497.4 

Table B.9.1.2-2b: Body weight data

Nominal dietary Body Weights (g) 

Concentration Day 0 ⇐ ∆ ⇒ Day 5 ⇐ ∆ ⇒ Day 8 

0 mg ai./kg diet mean 17.4 25.4 31.4 

 SD 1.5 

(n=20) +46.0 % 

2.5 

(n=20) +23.5 % 

4.3 

(n=20) 

313 mg ai./kg diet mean 18.6 26.6 32.9 

 SD 1.3 

(n=10) +43.0 % 

2.7 

(n=10) +23.5 % 

3.7 

(n=10) 

625 mg ai./kg diet mean 18.9* 27.1 34.2 

 SD 1.4 

(n=10) +43.3 % 

2.3 

(n=10) +26.1 % 

2.9 

(n=10) 

1250 mg ai./kg diet mean 17.2 24.3 30.5 

 SD 0.9 

(n=10) +41.6 % 

1.5 

(n=9) +25.5 % 

1.4 

(n=9) 

2500 mg ai./kg diet mean 17.1 22.0* 28.2 

 SD 1.2 

(n=10) +28.6 %* 

3.4 

(n=10) +28.1 % 

4.5 

(n=10) 

5000 mg ai./kg diet mean 18.8* 23.4 32.1 

 SD 1.3 

(n=10) +24.5 %* 

1.8 

(n=10) +37.1 %* 

2.6 

(n=10) 

* statistically significant 
– 7 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.1.2-3c: Estimated daily food consumption per bird

Conclusion: LC50 > 4998 mg a.s./kg food, equivalent to > 497 mg a.s./kg bw/d, based on a 

mean feed consumption of 2.1 g/bird/d, a mean body weight of 21.1 g and on 

measured test substance concentrations; no mortality level = 4998 mg a.s./kg 

food (measured); NOEC: 1218 mg a.s./kg food (measuerd) based on food 

avoidance and reduced body weight gain. 

Reference: Technical BYI 08330: A Subacute Dietary LC50 with Mallards 

Author(s), year: Stoughton, T.L.; 2005 

Report/Doc. number: EBFNX012; M-274385-01-1 

Guideline(s): EPA 71-2, OPPTS No.: 850.2200; OECD No. 205 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat (techn.), batch No.: 08045/0014; purity 97.1 % 

Material and methods: 

Test organism: Mallard Duck (Anas platyrhynchos), 10 day-old hatchlings 

Dosage levels: control (untreated diet); 313, 625, 1250, 2500 and 5000 ppm a.s. (nominal) 

corresponding to 344, 708, 1436, 2843 and 6050 mg measured a.s./kg feed 

Number of birds and 

replicates/group: 

10 birds per diet group, one control group, sex undetermined 

Administration: test substance mixed in diet 

Duration: 5 days administration period and 3 days post-exposure period 

Test conditions: acclimation: 5 d; feeding: starter ration ad libitum; housing in stainless steel wire 

cages; average temperature: 22 °C, relative humidity: 56 %, photoperiod 14 h 

Analytics: Concentration, homogeneity and stability of the test substance in the diet were 

sufficiently verified by analytical methods 

Test parameter: 

 Observations: Observations for signs of intoxication were made daily during acclimation, twice 

on the first exposure day and continued at least once daily until termination. 

 Body weight: individual body weights were measured at test initiation, on day 5 and at 

termination of the test on day 8 

 Feed consumption: daily feed consumption per group recorded for the pre-exposure, exposure and 
– 8 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

recovery periods 

Necropsy: Gross necropsy performed at termination 

Statistics: Body weight and growth data were analysed using TOXSTAT (if normally 

distributed, data were subjected to parametric analysis (one-way ANOVA); if not 

normally distributed after transformation, data were subjected to nonparametric 

analysis (Kruskal-Wallis ANOVA)) 

Findings: 

Mortalities: None 

Clinical signs: None 

Body weight: Body weights for Day 5 and body weight change from Day 0 to Day 5 exhibited a 

statistical difference at all treatment levels relative to the control. 

Body weight change from Day 5 to Day 8 exhibited a statistical difference at all 

treatment levels relative to the control. However, these were not an adverse effect 

since the increase in bodyweight at the treatment levels was greater than in the 

control. 

Feed consumption: Due to the fact that there were no replicates for feed consumption (based on 

guideline study design) no statistical analysis could be performed on this 

parameter. A reduction in feed consumption was observed in all treatment groups 

during the exposure period as compared to the control. During the recovery period 

the groups treated with up to 2843 ppm showed a significant increase in food 

consumption and returned to normal or higher levels as compared to the controls. 

Necropsy: No findings. 

Table B.9.1.2-2a: Summary of effects of spirotetramat on Mallard Duck following short-term dietary 

exposure 

Treatment

(measured)

[ppm a.s.] 

Cumulative 

mortality 

Mean bw

d 0 – 5

[g] 

Mean bw 

gain d 0 - 5 

[g] 

Mean bw 

gain d 6 - 8 

[g] 

Mean feed 

consumption 

d 0 - 4 [g/bird/d] 

Mean feed 

consumption 

d 5 -7 [g/bird/d]

Estimated intake 

d 0 - 4 

[mg as/kg bw/d]

Control 0/10 199.2 116.4 25.4 45.2 41.5 / 

344 0/10 172.4 70.1* 53.9* 32.6a 44.1 65 

708 0/10 162.8 51.2* 64.1* 27.7a 41.9 120 

1436 0/10 149.4 25.6* 82.4* 24.2a 56.4 233 

2843 0/10 147.2 7.6* 71.1* 19.9a 46.4 384 

6050 0/10 126.2 -15.1* 50.6* 9.9a 30.2 475 

* statistically significantly different 

a

 Apparent significant reduction in feed consumption based on empirical analysis as compared to the control group. 

Since there were no replicates for feed consumption foreseen in the used test guideline no statistical analysis could be 

performed on this parameter. 

Table B.9.1.2-2b: Mean body weight data (g)

Treatment (measured) 

[ppm a.s.] Mean bw on day 0 Mean bw on day 5 Mean bw on day 8 

Control 141.0 257.4 282.8 

344 137.3 207.5* 261.3 

708 137.2 188.4* 252.5* 

1436 136.6 162.2* 244.6* 

2843 143.4 151.0* 222.1* 

6050 133.7 118.6* 169.2* 

* statistically significantly different compared to control 
– 9 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Conclusion: LC50 > 6050 mg a.s./kg food, equivalent to > 475 mg a.s./kg bw/d, based on a 

mean feed consumption of 9.9 g/bird/d, a mean body weight of 126.2 g and on 

measured test substance concentrations; no mortality level = 6050 mg a.s./kg 

feed; NOEC < 344 mg a.s./kg feed (food avoidance and resulting reduced body 

weight gain already at the lowest test concentration observed). 

B.9.1.3 Sub-chronic toxicity and reproduction (OECD IIA 8.1.4) 

Active substance – Spirotetramat 

Reference: Effect of Technical BYI 08330 on Northern Bobwhite Reproduction 

Author(s), year: Sabbert, T.J. and Nuessle C.; 2006a 

Report/Doc. number: EBFNO220, M-277757-01-1 

Guideline(s): OPPTS Nr. 850.2300, FIFRA Guideline 71-4, OECD 206 

GLP: Yes 

Deviations: Minor deviation: age of birds lower than recommended in guidelines (16 weeks 

instead 20 w according to OECD 206 and 7 months according to EPA). 

Eggs were not candled on day 0 (deviation to all guidelines). 

Validity: Acceptable 

Test substance: Spirotetramat (techn.), batch No.: 08045/0014, purity 97.2 % 

Material and methods: 

Test organism: Bobwhite quail (Colinus virginianus),16 weeks of age 

Dosage levels: Control (untreated diet); 80, 240 and 720 mg ai/kg feed (nominal), corresponding 

to 0, 85, 264 and 802 mg ai/kg feed (measured). The amount of a.s. technical was 

adjusted to reflect 100 % a.s.. The daily dietary doses were 0, 8, 23 and 74 mg 

a.s./kg bw/day. 

Number of birds and 

replicates/group: 

18 pairs (1 pair in one pen) / treatment group 

Administration: Test substance mixed in the diet, prepared weekly 

Analytics: Concentration, homogeneity and stability of the test substance in the diet were 

sufficiently verified by analytical methods. 

Study phases: Acclimation: 23 days; Pre-photostimulation: 7 weeks; 11 weeks prior to egg-laying 

and for 11 weeks during the reproductive phase of the study Pre-egg laying (with 

photostimulation): 3 weeks; Egg-laying: approximately 11 weeks; Post-adult 

termination: 6 weeks 

Test conditions: Feeding: standard commercial diet ad libitum; adult housings: average 22°C, 

relative humidity: 56 %, 13 air changes per hour, photoperiod 7 h increasing to 

17 h, wide spectrum illumination 

Duration: Approximately 22 weeks (pre-egg laying until termination) 

Test parameter: 

 Observations: Clinical appearance and mortality of the parental generation were monitored daily 

throughout the study. Reproductive parameters included egg production, egg 

fertility, embryo survival, hatchability, hatchling body weight, 14-day survival, 14-

day survivor body weight, and eggshell strength/thickness. 

 Adult Body weight: Adult body weights were measured at study initiation, on weeks 3, 5, 7, 9, and at 

adult termination. 

 Adult feed 

 consumption: 

Feed consumption for each pen was measured weekly throughout the test. 
– 10 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Egg parameter: Eggs were collected daily and candled weekly to detect egg shell cracks or 

abnormal eggs. Eggs were candled later to determine embryo viability and to 

determine embryo survival. Egg shell thickness was measured. 

 Hatchling parameter The group body weight of the surviving hatchlings per pen was determined. At 14 

days of age, the average body weight by parental pen of all surviving chicks was 

determined. 

 Post-mortem 

 examination: 

All adult birds were subjected to a gross necropsy.

Statistics: A SAS based statistical program was used for data analysis (USEPA, 2002). Data 

from treatment groups were compared to controls using the Shapiro-Wilk`s and 

Levene`s test for equal variance to determine if dose groups had unequal 

variances. If normality and variances were equal, then parametric analyses were 

conducted using ANOVA followed by Dunnett`s or William`s test. If variances 

were unequal, then the nonparametric analyses were conducted using the 

Jonckheere or Mann-Whitney procedures. 

Findings: 

Mortalities: No treatment-related mortalities. Seven incidental mortalities occurred (2 controls, 

three at 85 ppm, two at 802 ppm). 

Clinical signs: No treatment-related signs of toxicity were observed in any test group. 

Body weight: Parents: There was an approximate 14 % reduction in the mean female weight 

gain in the 802 ppm group as compared to the control (statistically significant, 

Jonckheere`s test). 

Hatchlings: An approximate 5 % reduction in the median body weights was noted 

in the highest treatment level as compared to the control (statistically significant, 

Dunnett`s test). This small reduction is considered to be not biologically relevant. 

Feed consumption: No treatment-related effects upon feed consumption. 

Gross necropsy: No treatment-related findings. 

Reproductive results: See Table B.9.1.3-1. 

For the percent eggs set of eggs laid, the Jonckheere and Mann-Whitney`s test 

indicated a statistical difference from the control in the 85, 264 and 802 ppm 

levels. However, the difference in medians between the treatment levels and the 

control was 1.85 % at maximum. This difference is considered to be not 

biologically significant. 

The Jonckheere`s test indicated a statistical difference from the control in the 802 

ppm level for the percent viable embryos of eggs set endpoint. The approximate 

3 % reduction in means between the high treatment level as compared to the 

control, however, is considered not biologically significant. 93 % of this parameter 

is acceptable even for control levels. 

Table B.9.1.3-1: Summary of reproductive results of spirotetramat in the bobwhite quail 

Test concentration (measured, ppm a.s.) 

Reproductive parameter 

Control 85 264 802 

Overall mean food consumption (g/bird/day) 20.6 20.2 19.3 19.5 

Mean adult body weight total change (g) 

 male 

28 27 23 25 

 female 62 54 57 49* 

Total eggs laid 867 785 805 972 

Eggs set / Pen 44.61 39.17 40.83 48.94 

Eggs set / Eggs laid [%] 93.04 89.75* 86.4* 90.38* 
– 11 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test concentration (measured, ppm a.s.) 

Reproductive parameter 

Control 85 264 802 

Viable embryos / Eggs set [%] 95.59 94.99 94.44 93.13* 

Viable embryos / Pen 42.33 37.44 38.72 45.72 

Live 3-week embryos / Pen 42.11 37.11 38.50 45.17 

Hatchlings / Pen 40.11 34.61 35.06 42.28 

14-day old survivors 718 614 625 759 

Eggs laid / Hen 48.17 43.61 44.72 54 

Eggs cracked / Pen 0.22 0.67 0.06 0.5 

14-day survivors / Hen 42.24 40.93 36.76 42.17 

Mean egg shell thickness [mm] 0.21 0.20 0.20 0.20 

Mean hatchling body weight [g] 7.2 6.9 7.0 6.8* 

inhibition of above compared to control [%] / 4.2 2.8 5.6 

Mean 14-day survivor body weight [g] 35.5 35.6 35.5 34.5 

Live 3-week embryos / Viable embryos [%] 99.43 98.93 99.57 98.94 

Hatchlings / Live 3-week embryos [%] 92.73 93.03 89.57 93.64 

14-day old survivors / Hatchlings [%] 99.43 98.80 98.07 99.80 

Hatchlings / Eggs laid [%] 81.72 79.26 73.11 78.26 

Hatchlings / Eggs set [%] 88.01 87.97 84.77 86.54 

14-day old survivors / Eggs set [%] 87.46 86.87 83.72 86.35 

* Significantly different from the control, p< 0.05

Conclusion: Based on all parameters, and the consideration of biological relevance, the 

NOAEC for adult bobwhite quails was 264 mg a.s./kg feed, based on statistically 

significant reductions in adult female body weight gain (equivalent to 23 mg 

a.s./kg bw/d, based on measured concentrations). 

The NOAEC for reproductive parameters was the highest dosage tested, 802 mg 

a.s./kg feed. This is equivalent to 74 mg a.s./kg bw/d, based on a mean body 

weight of 218 g and mean feed consumption of 20 g/bird/d. 

Comments: US EPA recommends reporting the reproductive NOAEC as 264 ppm (23 mg/kg 

bw/d), based on a statistically significant effect on mean hatchling body weight 

and a reduction of the embryos/eggs set (%) endpoint. 

Reference: Effect of Technical BYI 08330 on Mallard Reproduction 

Author(s), year: Sabbert, T.J. and Nuessle C.; 2006b 

Report/Doc. number: EBFNX013, M-277794-01-2 

Guideline(s): USEPA FIFRA 71-4, OPPTS 850.2300 (Draft), OECD 206, ASTM standard E 

1062-86 

GLP: Yes 

Deviations: None relevant, minor deviation to EPA guideline: age of birds lower than 

recommended 

Validity: Acceptable 

Test substance: Spirotetramat (techn.), batch No.: 08045/0014, purity 97.2 % 

Material and methods: 
– 12 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test organism: Mallard duck (Anas platyrhynchos), 19 weeks of age 

Dosage levels: Control (untreated diet); 80, 240 and 720 mg ai/kg feed (nominal), corresponding 

to 0, 89, 269 and 869 mg ai/kg feed (measured). The amount of a.s. technical was 

adjusted to reflect 100 % a.s.. The daily dietary doses were 0, 9, 22 and 50 mg 

a.s./kg bw/day (calculated). 

Number of birds and 

replicates/group: 

15 pairs (1 pair in one pen) / treatment group 

Administration: Test substance mechanically mixed in the diet, prepared weekly. 

Analytics: Concentration, homogeneity and stability of the test substance in the diet were 

sufficiently verified by analytical methods. 

Study phases: Acclimation: 3 weeks; Pre-photostimulation: 7 weeks; Pre-egg laying (with 

photostimulation): 3 weeks; Egg-laying: approximately 11 weeks; Post-adult 

termination: 6 weeks 

Test conditions: Feeding: standard commercial diet ad libitum; adult housings: average 22°C, 

relative humidity: 55 %, 13 air changes per hour, photoperiod 7 h increasing to 

17 h, wide spectrum illumination 

Duration: Approximately 21 weeks (pre-egg laying until termination) 

Test parameter: 

 Observations: Adult birds were observed at least once daily for signs of toxicity, injuries, illness 

or mortality. Reproductive parameters included egg production, egg fertility, 

embryo survival, hatchability, hatchling body weight, 14-day survival, 14-day 

survivor body weight, and eggshell strength/thickness. 

 Adult Body weight: Adult body weights were measured at study initiation, on weeks 3, 5, 7, 9, and at 

adult termination. 

 Adult feed 

 consumption: 

Feed consumption for each pen was measured weekly throughout the test. 

 Egg parameter: Eggs were collected twice daily and candled weekly to detect egg shell cracks or 

abnormal eggs. Eggs were candled later to determine embryo viability and to 

determine embryo survival. Egg shell thickness and strength were measured. 

 Hatchling parameter Body weights were measured on the day they were removed from the hatcher and 

on day 14. Hatchlings were observed daily for signs of toxicity, injuries, illness or 

mortality. 

 Post-mortem 

 examination: 

All adult birds were subjected to a gross necropsy.

Statistics: A SAS based statistical program was used for data analysis (USEPA, 2002). Data 

from treatment groups were compared to controls using the Shapiro-Wilk`s and 

Levene`s test for equal variance to determine if dose groups had unequal 

variances. If normality and variances were equal, then parametric analyses were 

conducted using ANOVA followed by Dunnett`s or William`s test. If variances 

were unequal, then the nonparametric analyses were conducted using the 

Jonckheere or Mann-Whitney procedures. 

Findings: 

Mortalities: A total of five adult mallards died during the exposure period. One in the control 

group, one in the 269 mg a.s./kg treatment level and three in the 869 mg a.s./kg 

treatment level (prior to week 12 adult sacrifice). The mortalities up to 269 ppm 

were considered not treatment-related. The birds at the highest dose level died as 

a result of emaciation. 

Clinical signs: Approximately week 7 post study initiation, nine of the thirty adult mallard in the 

869 mg a.s./kg treatment level began to express signs of ataxia (loss of balance). 

The birds in this treatment level were also noted to have severe peeling and 

cracking of the foot webbing. During study week 7, one hen was found dead. By 
– 13 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

study week 8, nineteen of the remaining twenty eight birds in the 869 mg a.s./kg 

treatment level had severe peeling and cracking on the bottoms of their feet. The 

birds could not feed or drink and by week 9, body weights of these adult birds 

showed a marked decrease from week 1 body weights. The foot symptoms were 

originally diagnosed as “bumblefoot” by the attending veterinarian although they 

were noted to be atypical of this foot condition which is occasionally noted in 

laboratory-housed mallards. A foot treatment of 3 % hydrogen peroxide was 

provided to the 869 mg a.s./kg treatment level birds to reduce the foot symptoms 

prior to reproductive phase. At week 12, two more adult mortalities occurred and 

approximately 79 % of the remaining 24 birds were observed with moderate to 

severe foot symptoms. All of the adult mallard in the 869 mg a.s./kg treatment 

level were sacrificed at the end of week 12 post study initiation for humane 

reasons due to the condition of the feet resulting in emaciation. 

The severe peeling and cracking of feet were observed in one bird at the 269 ppm 

treatment level beginning week 9 post study initiation. By adult sacrifice (week 

21), approximately 54 % of the 269 mg a.s./kg study birds had the foot condition. 

One mortality occurred during this time period. Adults were able to feed and drink, 

did not show the same signs of ataxia as seen in the 869 ppm treatment level and 

were able to complete the reproductive phase of the study. 

A light to moderately severe form of the peeling and cracking foot symptoms were 

observed in one bird at week 14 post study initiation at the 89 ppm treatment 

level. At the time of adult sacrifice (week 21) in the 89 ppm treatment level, 

approximately 27 % of the adult birds showed foot symptoms. No bird mortalities 

occurred at this treatment level and the adult body weights at termination were not 

statistically different from the control birds. 

At week 16 post study initiation in the control group, one adult mallard was 

observed to have a bumblefoot-like condition. At adult sacrifice (week 21), six of 

twenty eight (one mortality occurred) control birds were indicated as having 

bumblefoot-like foot symptoms. These symptoms, however, were not the peeling 

and cracking foot symptoms that was seen in the 269 and 869 mg a.s./kg 

treatment levels. These symptoms were indistinguishable from foot cage wear that 

is typically seen due to housing conditions. 

Excluding the foot symptoms that were indistinguishable from cage wear or 

“typical” bumblefoot, the foot symptoms appeared related to dose. However, it 

was unclear whether the foot symptoms had an effect on the outcome of the other 

endpoints analysed for this study. The NOEC based on foot symptoms was 

< 89 mg a.s./kg and the LOEC was 89 mg a.s./kg. 

No clinical signs were observed in hatchlings. 

Body weight: Parents: A significant statistical difference was determined at the 269 ppm 

treatment level for both female and male body weight gain. 

Hatchlings: In the 89 ppm treatment level a statistical difference was indicated for 

the body weight (approximate 6.5 % reduction). For 14-d-old hatchlings the 

difference was not significant at 89 ppm, but at 269 ppm it was significant. 

Feed consumption: A significant statistical difference was determined at the 269 ppm treatment level 

for adult feed consumption as compared to the control. 

Gross necropsy: No apparent treatment-related findings except for the foot lesions. Birds of the 

highest treatment level were emaciated. 

Reproductive results: See Table B.9.1.3-2. 

For the eggs laid and eggs set per pen endpoints, the respective tests indicated a 

statistical difference from the control in the 269 ppm level. 

The Jonckheere`s test indicated a statistical difference from the control in the 269 
– 14 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

ppm level for the number of live and viable embryos and percent live embryos of 

viable embryos endpoints. 

Table B.9.1.3-2: Summary of reproductive results of spirotetramat in the mallard duck 

Test concentration (measured, ppm a.s.) 

Reproductive parameter 

Control 89 269 869 

Overall mean food consumption (g/bird/day) 113 118 86* 57* 

Mean adult body weight total change (g) 

 male 

17 5 -113* -315* 

 female 197 140 -8* -124* 

Total eggs laid 758 767 427* n.d. 

Eggs laid / Hen 50.5 51.1 28.5* n.d. 

Eggs cracked 2 1 2 n.d. 

Eggs cracked / Pen 0.14 0.07 0.13 n.d. 

Eggs set / Pen 48.1 50.5 25.4* n.d. 

Eggs set / Eggs laid [%] 88.4 92.2 87.1 n.d. 

Viable embryos / Eggs set [%] 79.4 67.3 54.1 n.d. 

Viable embryos / Pen 40.0 25.2 16.1* n.d. 

Live 3-week embryos / Pen 39.6 40.8 23.3* n.d. 

Hatchlings / Pen 27.8 32.2 8.1* n.d. 

14-day old survivors 386 386 80* n.d. 

14-day survivors / Hen 29.7 32.2 8.9* n.d. 

Mean egg shell thickness [mm] 0.364 0.364 0.344 n.d. 

Mean hatchling body weight [g] 37.2 34.7* 29.0* n.d. 

Mean 14-day survivor body weight [g] 260.6 250.8 217.4* n.d. 

Live 3-week embryos / Viable embryos [%] 98.9 99.2 94.9* n.d. 

Hatchlings / Live 3-week embryos [%] 69.5 80.4 32.2* n.d. 

14-day old survivors / Hatchlings [%] 99.2 100 99.5 n.d. 

Hatchlings / Eggs laid [%] 48.5 56.6 20.7* n.d. 

Hatchlings / Eggs set [%] 54.5 61.1 23* n.d. 

14-day old survivors / Eggs set [%] 58.2 61.1 25.3* n.d. 

* Significantly different from the control, p< 0.05

n.d. not determined for the 869 ppm treatment level due to adult sacrifice 12 weeks post study initiation 

Conclusion: The study authors consider neither the foot effects nor the slightly reduced 

hatchlings body weight as biologically adverse and propose a NOAEC of 89 mg 

a.s./kg feed, based on several statistically significant reproductive parameters and 

reduced adult body weight gain. This is equivalent to 9.4 mg ai/kg bw/d, based on 

a mean body weight of 1122 g and mean feed consumption of 118 g/bird/d. 

The RMS considers the slight reduction in the hatchlings body weight in the lowest 

dose group as biologically not adverse, therefore the reproductive NOAEC is 89 

ppm. However, no NOEC based on foot symptoms could be determined and the 

LOEC was 89 mg a.s./kg. 
– 15 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: BYI 08330 Technical: A Reproduction Study with the Mallard 

Author(s), year: Temple, D.L.; Martin, K.H.; Beavers, J.B.; Jaber, M.; 2006 

Report/Doc. number: EBFNP024, M-278058-01-1 

Guideline(s): US EPA FIFRA 71-4, OPPTS 850.2300 (Draft), OECD 206 

GLP: Yes 

Deviations: None relevant. Minor deviation: relative humidity lower than required according to 

guidelines. Minor deviation to EPA guideline only: age of birds lower than 

recommended. 

Validity: Acceptable 

Test substance: BYI 08330 (techn.), batch No.: 08045/0014, purity 97.99 % 

Material and methods: 

Test organism: Mallard duck (Anas platyrhynchos), 19 weeks of age 

Dosage levels: Control (untreated diet); 30, 80, 200 and 720 mg a.s./kg feed (nominal), equivalent 

to measured concentrations of 28.8, 78.8, 201 and 727 mg a.s./kg feed. Due to 

the debilitated condition of the birds, the 720 ppm a.s. treatment group was 

terminated in week 5. The amount of a.s. technical was adjusted to reflect 100 % 

a.s.. The daily dietary doses were 0, 4.2, 10 and 23.6 mg a.s./kg bw/day. 

Number of birds and 

replicates/group: 

15 pairs (1 pair in one pen) / treatment group 

Administration: Test substance mechanically mixed in the diet, prepared weekly. 

Analytics: Concentration, homogeneity and stability of the test substance in the diet were 

sufficiently verified by analytical methods. 

Study phases: Acclimation: 4 weeks; Pre-photostimulation: 9 weeks; Pre-egg laying (with 

photostimulation): 3 weeks; Egg-laying: approximately 10 weeks; Post-adult 

termination: 6 weeks 

Test conditions: Feeding: standard commercial diet ad libitum; adult housings: average 21.5 - 

23.3°C, relative humidity: 27.3 - 64.5 %, 15 air changes per hour, photoperiod 7 h 

increasing to 17 h, wide spectrum illumination 

Duration: Approximately 22 weeks (pre-egg laying until termination) 

Test parameter: 

 Observations: Adult birds were observed daily for signs of toxicity and abnormal behaviour. 

Photographs of the duck`s feet were taken at every body weight interval for 

potential comparison of foot lesions. Reproductive parameters included egg 

production, egg fertility, hatchability, hatchling body weight, 14-day survival, 14-

day survivor body weight. 

 Adult Body weight: Adult body weights were measured at study initiation, on weeks 2, 4, 6, 8, 9, and 

at adult termination. 

 Adult feed 

 consumption: 

Feed consumption for each pen was measured weekly throughout the test. 

 Egg parameter: Eggs were collected twice daily and candled weekly to detect egg shell cracks or 

abnormal eggs. Eggs were candled later to determine embryo viability and to 

determine embryo survival. Egg shell thickness and strength were measured. 

 Hatchling parameter Body weights were measured on the day they were removed from the hatcher and 

on day 14. Hatchlings were observed daily for signs of toxicity. 

 Post-mortem 

 examination: 

All adult birds were subjected to a gross necropsy.

Statistics: An analysis of variance (ANOVA) was performed to determine statistically 

significant differences between groups. Dunnett`s multiple comparison procedure 

was used to compare the treatment means with the control group mean. 
– 16 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Percentage data were examined using Dunnett`s method following arcsine square 

root transformation. See also “Appendix”: US EPA re-analyzed the data applying 

different test procedures. 

Findings: 

Mortalities: A single mortality occurred in the 200 ppm a.s. treatment group. 

Clinical signs: Birds in the 720 ppm a.s. treatment group were first noted exhibiting clinical signs 

of toxicity, which ultimately included loss of coordination, lower limb weakness and 

cracked and bleeding lesions on the bottoms of their feet, on Day 3 of Week 3. 

Marked, continued loss of body weight was also apparent at the Week 4 body 

weight interval. On Day 3 of Week 5, as a result of clinical observations and loss 

of condition noted, all birds in that group were euthanized. 

Incidental clinical observations noted during the test included those that normally 

are associated with injuries and penwear. Such signs included bumblefoot (plantar 

pododermatitis), lameness, feather loss, molting and neck, back and shoulder 

lesions. 

Treatment-related findings in the 80 ppm treatment and above groups included 

lesions on the bottoms of the feet characterised by thickened areas that appeared 

rough, dry and hard, with cracking, fissuring and/or bleeding evident. 

Body weight: Parents: Slight and transient statistically significant body weight changes in the 30 

and 80 ppm treatment groups were considered not treatment related. A significant 

statistical difference in body weight and bw changes was determined in hens of 

the 200 ppm treatment group. Birds in the 720 ppm treatment group exhibited 

marked weight loss. 

Hatchlings: In the 80 ppm treatment level a slight but statistically significant 

reduction was seen in the hatchling`s body weight. 

Feed consumption: At the 80 ppm test concentration, there was a slight and transient, statistically 

significant reduction in mean feed consumption during Week 15. At the 200 ppm 

test concentration, there were treatment-related reductions in feed consumption 

during Week 4 and from Week 11 through 18 that were statistically significant. At 

the 720 ppm test concentration there were statistically significant treatmentrelated reductions in feed consumption from Week 1 to Week 5 (termination of the 

adult birds for the high dose group). According to the US EPA`s re-analysis (see 

Appendix), the mean overall feed consumption was statistically significantly 

reduced at 80 and 200 ppm. 

Gross necropsy: Treatment related findings included lesions on the bottoms of the feet 

characterised by thickened areas that appeared rough, dry and hard, with 

cracking, fissuring and/or bleeding evident. These lesions were noted in four 

males and six females from the 80 ppm a.s. treatment group, 13 males and 14 

females from the 200 ppm a.s. treatment group and 14 males and 12 females 

from the 720 ppm a.s. treatment group. 

The numbers of hens with regressed ovaries and the numbers of drakes with 

reduced testes were increased in both the 80 and 200 ppm a.s. treatment groups. 

Reproductive results: See Table B.9.1.3-3. 
– 17 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.1.3-3: Summary of reproductive results of spirotetramat in the mallard duck 

Test concentration (measured, ppm a.s.) 

Reproductive parameter 

Control 28.8 78.8 201 

Number of replicates 15 15 15 14 

Overall mean food consumption (g/bird/day) 162 159 142 + 131 +

Mean adult body weight total change (g) 

 male 

-32 9 4 -34 

 female 58 84 -2 -73* 

Total eggs laid1686 629 456 276 

Eggs laid / Hen146 42 30 20 

Eggs cracked14 6 3 3 

Eggs cracked / Eggs laid [%] 2 1 1 2 

Viable embryos / Eggs set [%] 87 83 91 48* 

14-day old survivors 1 426 333 266 76 

14-day survivors / Hen 1 28 22 18 5 

Mean egg shell thickness [mm] 0.389 0.382 0.389 0.373 

Mean hatchling body weight [g] (sample size) 34 (12) 32 + (13) 31* + (14) 28* + (6) 

Mean 14-day survivor body weight [g] 274 266 257 200* 

Live 3-week embryos / Viable embryos [%] 98 99 98 96

Hatchlings / Live 3-week embryos [%] 79 77 63 + 41*+

14-day old survivors / Hatchlings [%] 100 99 94 91 +

Hatchlings / Eggs set [%] 69 63 60 25* 

14-day old survivors / Eggs set [%] 69 62 57 + 23*+

* Significantly different from the control, p< 0.05, Dunnett`s test, study author`s analysis 

+ Significantly different from the control, p< 0.05, Williams`s test or Jonckheere`s test, according to EPA analysis (see Appendix) 

1

 no statistical analysis on this parameter 

Conclusion: The NOAEC was 28.8 mg a.s./kg feed (measured), based on treatment related 

foot lesions, effects upon offspring body weights and on egg production and 

hatchability at 78.8 mg a.s./kg feed. This is equivalent to a NOAEL of 4.0 mg 

a.s./kg bw/d, based on a measured feed concentration of 28.8 ppm, a mean body 

weight of 1147 g and mean feed consumption of 159 g/bird/d. The statistically 

significant reduction of the hatchlings body weight at the 28.8 ppm dose level is 

considered biologically not relevant. 

Appendix: statistical analysis of the reproductive data by the US EPA:

The reproductive data of this study were re-analyzed according to US EPA Environmental Fate and Effects 

Division’s (EFED) standard protocol, since not all endpoints typically considered by the EPA were initially 

statistically analyzed and included in the study report. The study authors have explained that parameters that 

were raw count data endpoints were not statistically analyzed because they were inherently non-normally 

distributed. Instead, these raw count data were converted to proportions (e.g., by dividing the counts by the 

number of eggs set from a pen). All data that were inherently proportions or were converted to a proportion 

were arcsine-transformed to meet assumptions of normality. However, no tests of normality or homogeneity 

of variance were performed on any endpoint parameter even after these transformations. Once the data 

were transformed, an analysis of variance (ANOVA) was performed to determine statistically significant

differences between groups. Then Dunnett's multiple comparison procedure was used to compare the 
– 18 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

treatment means with the control group mean and assess the statistical significance of the observed 

differences. 

In general, the EPA prefers not to transform data, but rather tests the assumptions of normality and 

homogeneity prior to analysis to determine whether parametric or non-parametric tests should be performed. 

Therefore the US EPA developed a SAS program for analyzing data from avian reproduction studies, Chicks 

version 3.1, which was employed for the re-analysis. 22 variables were analyzed on a ‘per pen’ basis. 

The reproductive parameters for which the EPA’s and study authors’ conclusions diverged are the following: 

Overall mean food consumption, Mean hatchling body weight [g], Hatchlings / Live 3-week embryos [%],14-

day old survivors / Hatchlings [%] and 14-day old survivors / Eggs set [%]. 

The EPA notes the following: First, according to the results of Levene’s test (equality of variance) and 

Shapiro-Wilks’ test (normality), most of the count data (# of eggs set, # of eggs laid, # of viable embryos, # of 

live embryos, # of eggs hatched, and hatchling survival) that were converted to proportions and arcsinetransformed by the study authors, did not violate the basic assumptions of normality and homogeneity and 

could be analyzed using parametric tests without transformations. 

Second, all of the study authors’ findings could be reproduced by the EPA when strictly employing Dunnett’s 

test or its non-parametric analog, the Mann-Whitney test with a Bonferroni adjustment. However, the EPA’s 

statistical analyses did result in lower NOAEC values in a number of cases, primarily resulting from the 

EPA’s application of the more-sensitive Williams’ (parametric) and Jonckheere’s (non-parametric) tests when 

the assumption of a negative dose-response relationship could reasonably be supported, as compared to the 

study authors’ reliance on Dunnett's multiple comparison procedure. 

Most importantly it should be noted that for the endpoint “mean hatchling bodyweight” there was a significant 

reduction at all test levels and a NOAEC could not be determined (based on a ~6 % reduction at the lowest 

test level). Since this study results in the most sensitive NOAEC of all the available avian reproduction 

studies for spirotetramat, and a definitive NOAEC could not be identified for this endpoint, the EPA 

recommends that a request for another avian reproduction study with mallards that tests at lower levels 

should be considered. 

In a response to the EPA re-analysis Bayer Crop Science express their concern on the use of the Williams` 

test in the evaluation of avian reproduction data. They argue that the Williams test is susceptible to

increasing type 1 error (false positive difference) when used to make multiple comparisons and when sample 

sizes are unequal. For the hatchling weight parameter, sample sizes were unequal (see table B.9.1.3-3). 

According to a published article, the Williams test maintains a predetermined α-level less and less as the 

imbalance increases. Another publication recommends applying the Williams test at 2.5 % to quantitative 

parameters of repeated toxicity studies. If this recommendation is followed the p-value calculated for the 30 

ppm dose level (p=0.031) of the hatchling weight parameter is higher than the recommended statistical level 

(p=0.025). 

Comment (RMS): A statistician of AGES (RMS authority) looked at the issue and, overall, agreed with the 

EPA`s position. Hence the parameter “hatchling body weight” is considered to show a statistically significant 

difference at 30 ppm compared to the control. 

Hovever, in the RMS opinion the question of statistical significance of this difference in hatchling weight is a 

borderline case anyway, if the p-values of both tests applied are looked at. While the outcome of the 

statistical analysis is important for the interpretation of the study, for the derivation of a “No observed adverse 

effect level” the magnitude and biological relevance of an observed effect should also be considered. The 

bodyweight reduction compared to the control is 6 %. BCS compared the hatchling bodyweight data 

obtained at the 30 ppm level with historical control bodyweight data from 20 duck reproduction studies

provided by Wildlife International. This suggests that the hatchling bodyweights were not adverse, since they 

were in the range of historical control hatchling bodyweights (mean of 20 studies = 33 ± 3 g). Further, it is 

important to note that the NOEC for the 14-day old duckling weight is 80 ppm. This supports that any 

reduction in the hatchling bodyweight was temporary and recovery took place in the ducklings of the lowest 
– 19 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

dose level. Hence, the dose level of 30 ppm, corresponding to 4 mg/kg bw/d, is considered as NOAEL with 

regard to reproductive effects in the mallard duck. 

During joint review discussions between the authorities, PMRA Canada agreed with the RMS position to 

consider the 30 ppm level as NOAEL for the purpose of risk assessment. 

B.9.1.4 Supervised cage or field trials (OECD IIA 10.1.7) 

- Further studies on the Mallard Duck 

Introduction:

During avian reproduction studies with the mallard duck dietarily exposed to spirotetramat the ducks 

experienced severe foot problems, expressed as dried and deeply cracked skin on the underside of the feet. 

These dermal effects to the feet may have been exacerbated by an interaction between the wire mesh of the 

caging (possible physical damage) and food contact with excreta (possible chemical exposure) on the cage 

floors. However, the causal mechanism is not fully understood, and the notifier performed two further studies 

to gain more information. 

Reference: Long-Term Mallard Duck Dermal Exposure to BYI 08330

Author(s), year: Stafford, J. M.; 2007a 

Report/Doc. number: EBFNP033; M-290165-01-1 

Guideline(s): Not applicable 

GLP: Yes 

Deviations: Not applicable 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 98.10 %, lot no. 08045-0014 

Material and methods: 

Test organism: Mallard Duck (Anas platyrhynchos), age: 35 weeks 

Test design: The objective of the study was to determine if dermal exposure to treatment 

excrement alone is sufficient to elicit a dermal response in the feet of ducks not 

exposed via diet. Four groups were incorporated into this study: 

1. Dietary control: received untreated diet, excrements collected every other day 

for transfer to dermal control cages; 

2. Dermal control: received untreated diet, application of excrements from group 1 

to the floor of their cages 

3. Dietary exposure: received nominal dietary concentration: 720 mg a.s./kg feed, 

corresponding to 760 mg a.s./kg feed (measured), housed in isolated room, 

excrements collected every other day for transfer to dermal exposure cages. Two 

sets of ducks were sequentially exposed due to severe reactions necessitating 

euthanisation of the first set of ducks at week 4 of the study. 

4. Dermal exposure: housed in isolated room, received untreated diet, application 

of excrements from group 3 to the floor of their cages 

Number of birds 

/group: 

Twelve males and twelve females/group. One pair/cage. 

Administration: Test substance mixed in diet / dermal exposure to excrements from dietary 

exposure group 

Duration: Eight weeks of treatment. Dietary exposure group: 4 weeks exposure, then 

replacement with another group. 
– 20 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test conditions: Acclimation: 2 weeks; feeding: basal diet and water ad libitum; housing in cages 

with the dimensions 76 x 83 x 44 cm. The floor, back wall and top of the cages 

were made of polycarbonate-coated welded-wire mesh, the sides of galvanised 

steel. Isolated environmental conditions were provided by establishing one room 

for the two control groups and one room each for dermal and dietary exposure 

group. Temperature: 14 - 33 °C, relative humidity: 45 - 94 %, photoperiod 7 h. 

During the experimental period, excrements were catched in plastic trays. Every 

other day, all cages were scrubbed with brushes dipped in water. Separate 

equipment were used in each room. The excrement collected from the dietary 

control and exposure cage was spread with spatulas evenly and throroughly 

across the entire floor of the dermal group cages. 

Analytics: Dietary analysis confirmed recoveries of 95 to 116 % of nominal. 

Excrement sample analysis: Composite excrement samples were collected from 

the dietary groups every two weeks following treatment initiation. These samples 

were analysed to provide a general idea of the degree of exposure, not for precise 

quantification. Excreta were analysed to determine concentrations of 

Spirotetramat and its enol metabolite using an LC/MS/MS system. 

Test parameters: 

 Observations: All birds were monitored every two weeks for change in body weight and changes 

in the condition of the feet. Daily monitoring for behavioural abnormalities. On the 

day of treatment and at the end of each two-week interval all birds were captured 

and their feed closely examined. Notations were made in greater detail regarding 

unique conditions of the feet and digital photos were taken. 

 Feed consumption: Not reported. 

Necropsy: None. 

Statistics: Body weights were compared using one-way analysis of variance. 

Findings: 

Analytics: As result of the excreta sample analysis in the dietary treatment group residues of 

0.11 (week 2) and 0.6 (week 8) mg a.s./kg were detected. The residue level of 

BYI 8330-enol was between 61.6 (week 2) and 97.6 (week 8) mg/kg. Residues in 

the dietary control were < 0.01 mg/kg. 

Mortalities: Severe foot symptoms progressed to warrant in extremis euthanisation of both 

sets of ducks of the dietary exposure group after 4 weeks of exposure. 

Clinical signs: Behaviour: One female in the dermal exposure group appeared emaciated and 

weak and showed diminished appetite from week 5 on, however, the condition of 

her feet was normal and this observation was considered not treatment-related. 

Ducks in both dietary exposure sets displayed abnormal behaviour and adverse 

reactions in their feet: From week 3 on, limping was observed. From week 4 on 

additional symptoms appeared: using wings to aid locomotion, decreased 

ambulation, piloerection, lacrimation, cracking on feet (cracks wrap around edges 

of feet). After 4 weeks of exposure, ducks were euthanised. 

Detailed foot observations: Dietary control ducks did not experience any cracking 

or drying. In the dermal control group two notations were made of minor cuts 

along the foot digits. The study author states it is not unusual for ducks to 

experience temporary cracking and healing of the skin of their feet from time to 

time. In the dermal exposure group four occurrences of minor cracking along the 

digits were noted at week 4, and three occurrences of moderate cracking during 

the middle two weeks. Effects seemed to have been transient. During week four 

10 cases of inflamed webbing (transient) were noted. Dietary exposure group: 

Moderate to extreme dermal cracking along the digits, drying of the skin and 

impaired mobility and perceived discomfort due to the sores of the feet were 
– 21 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

observed. This severe cracking was distinctly different from transient minor 

cracking observed in other groups. A transient effect of inflamed webbing during 

week 4 was also seen. Some birds became reluctant to feed and became 

emaciated and weak. 

Body weight: Body weights of the dietary exposure group were significantly reduced compared 

to the control. 

Conclusion: Two separate sets of ducks exposed to Spirotetramat at 720 mg/kg feed exhibited 

symptoms of progressively worsening of cracking and drying of the feet, and 

impaired mobility within 2 weeks. The symptoms progressed to warrant in 

extremis euthanisation of both sets of ducks after 4 weeks of exposure. Ducks 

dermally exposed to the excrement of dietarily exposed ducks experienced fewer 

cases of minor to moderate cracking and drying of the feet during 8 weeks of 

repeated exposure to the excrement, and symptoms were transient, less severe 

and did not lead to behavioural symptoms. 

Reference: Semi-Field Study of the Effects of Dermal and Dietary Exposure to BYI 08330 

in Mallard Ducks (Anas platyrhynchos) 

Author(s), year: Stafford, J. M.; 2007b 

Report/Doc. number: EBFNP034; M-290000-01-1 

Guideline(s): Not applicable 

GLP: Yes 

Deviations: Not applicable 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) techn., purity: 98.10 %, lot no. 08045-0014 

Movento formulation: BYI 08330 150 OD, lot no. 08030/0189(0125), purity: 14.7 % 

a.s. 

Material and methods: 

Test organism: Mallard Duck (Anas platyrhynchos), age: 27 weeks 

Test design: The objective of the study was to determine wether the dermal effects observed in 

laboratory cages would occur in mallards in a more natural outdoor, field-pen 

environment. Ducks in this study received dermal exposure by walking on turf 

(mowed grass) treated with the test substance as well as by dietary exposure from 

feed mixed with the test substance. 

Birds in the treatment group received a dietary concentration of 200 mg a.s./kg 

feed. Formulated spirotetramat was applied in spray solution to the turf in the 

pens. The birds were placed in holding cages before the application and the sides 

of the pens were covered with plastic cover. Turf applications took place on three 

occasions spaced 14 d apart and at a rate of 158 g a.s./ha (event 1), 86.8 g 

a.s./ha (event 2) and 140 g a.s./ha (event 3). Additionally, treated feed was placed 

in the treatment pens immediately after turf application. 

At the end of 10 weeks, all treated feed was replaced with clean feed and 

remaining birds were left for observation through a 4-week recovery period. 

Number of birds 

/group: 

Three treatment and control replicate pens. Six males and six females/pen (36 

birds / treatment or control group). 

Administration: Test substance mixed in diet and exposure to oversprayed turf. 

Duration: Ten weeks of treatment followed by a 4-week recovery period. 

Test conditions: Acclimation: 2 weeks and 4 days; feeding: basal diet and water ad libitum; 

housing in predator-proof aviaries enclosing a total area of approximately 1.03 ac 
– 22 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

(91 x 46 m). This aviary was evenly divided into 6 replicate pens measuring 46 x 

15 m providing a total area of approx. 0.17 ac. each. At the opposite end of the 

pens where the feed was placed a 9.14 m untreated zone was maintained to 

simulate edge or adjacent habitat. At the center of the untreated zone a small 

pond was constructed. The ducks had to cross 36.6 m (120 ft) of treated turf from 

the pond to obtain feed. Natural temperature and humidity conditions, natural 

photoperiod. Weather data as well as rain events were recorded. 

Analytics: Dietary analysis confirmed recoveries of 82.7 to 98.9 % of nominal. Homogeneity 

was demonstrated. 

Test parameters: 

 Observations: Daily observations for mortality and behavioural abnormalities. All birds were 

monitored every two weeks for changes in the condition of the feet and body 

weight measurement. Notations were made in great detail regarding unique 

conditions of the feet and digital photos were taken. Notations were also made to 

document bird moving across the treated field and two sessions on birds 

behaviour and movements were captured by digital video. 

 Feed consumption: Feed consumption was measured daily and average values were calculated per 

bird and day for the exposure and post-exposure periods. 

Feet preservation:

 

At the end of 10 weeks, the birds in Control pen 2 and Treatment pen 4 were 

euthanised and their feet were removed and preserved. After the recovery period 

the same was done with birds of other treatment and control groups. 

Statistics: Body weights were compared using one-way analysis of variance followed by a 

multiple comparison test. 

Findings: 

Mortalities: None. 

Clinical signs: Behavioural observations: Minor abnormal behaviours were first observed in week 

6 in 2 birds and in most affected treatment birds by week 8. These included 

limping, reluctance to walk, birds sitting down immediately upon landing and 

stumbling while walking. The tendency to fly rather than walk increased as 

treatment progressed. Limping had entirely ceased one week post-treatment. 

Foot condition: Abnormalities were noted by week 2 of treatment including 

cracking of the skin along the underside of the digits. Though mild cracks are 

apparently normal, as supported by the occurrence among control birds, the 

treated birds experienced more minor cracking than the controls, and also showed 

moderate and severe cracking. 

The incidence of abnormal foot conditions among treated birds ranged from 29.2 

(week 14) to 63.9 % (week 8). The number of severe cases increased until week 

10. During the recovery period, significant improvements were made in the 

condition of the feet. By week 14, only 7 minor cases of feet cracking were 

recorded. During the entire study, no infection was noted in cracked feet. 

Feed consumption: Food consumption followed the same general pattern in all pens during the course 

of the study, though average weekly food consumption in the control pens was 

slightly higher than in treatment pens. 

Body weight: Variations of group body weights between different pens occurred, but being 

inconsistent and transient these effects were not considered treatment related. 

Conclusion: Ducks that received spirotetramat-treated feed and were exposed to oversprayed 

turf, showed minor, moderate and severe cracking of the skin of the feet from the 

second week on. Behavioural effects as limping and the reluctance to walk were 

seen in affected birds. During a 4 week recovery period, significant improvements 

were made in the condition of the feet. 
– 23 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

- Further studies for the refinement of the risk assessment 

Reference: BYI08330 150 OD - Magnitude of the Residue in/on Soybeans/Wheat, 

Potential Wildlife Feed Items 

Author(s), year: Fischer, D. R., Ramanarayanan, T. & Ortego, L.; 2006 

Report/Doc. number: RAFNP002; M-277315-01-1 

Guideline(s): Not applicable 

GLP: Yes (certified laboratory) 

Deviations: Not applicable 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) OD 150, 150 g a.s./L (nominal), batch no. 

08030/0189(0152) 

Material and methods: 

Four soybean field trials and four wheat field trials were conducted in NAFTA Growing Regions 2 (one 

soybean trial), 3 (one wheat trial), 4 (one soybean trial and one wheat trial), and 5 (two soybean trials and 

two wheat trials) in multiple regions througout the US. The locations of the soybean trials were in Georgia, 

Mississippi, Kansas and Nebraska, the locations of the wheat trials in Florida, Mississippi, Kansas and 

Nebraska. At each test site, two foliar applications of Spirotetramat 150 OD were made to established 

soybeans or wheat at a target application rate of 0.176 kg/ha/application (0.157 Ib a.s./A) and with an 

application interval of 19 to 21 days. All applications were made using spray volumes of 110 to 167 L/ha and 

used Dyne-Amic (0.25 % v/v) as an additive. For the soybean trials, the first application was made either at 

BBCH growth stage 15 to 16 or at stage 69 to 74. For the wheat trials, the first application was made at 

BBCH growth stage 25 to 32. The wheat trials included two treated plots; one plot was mowed one to eight 

days prior to the applications and represented short grass; the other plot was not mowed and represented 

tall grass. Duplicate forage samples were collected from each of the treated plots at each of the sampling 

intervals from the soybean and wheat trials. The forage samples were collected prior to the first application 

(control samples) and at various time intervals after the first and second applications, up to 35 days following 

the second application. The samples were homogenized, and the residues of BYI 08330 and its metabolites 

BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy, and BYI 08330-enol-glucoside were 

quantified by high pressure liquid chromatography/triple stage quadrupole mass spectrometry (Ic-ms/ms)

using the stable isotopically labelled analytes as internal standards and the method of external standard 

quantitation. The individual analyte residues were converted to Spirotetramat molar equivalents and summed 

to give a total Spirotetramat residue. 

For each trial, the total residue was measured for each sample collected at each sampling interval. The 

mean total Spirotetramat residue values at each sample interval were calculated from the measured total 

residues from the duplicate samples collected at that interval. 

The foliar dissipation half-life of the total Spirotetramat residue in soybean forage (broad leaf plants) and 

wheat forage (short and tall grass) following the second application of Spirotetramat 150 OD was calculated 

using an MS Excel 2002 electronic speadsheet. 
– 24 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Findings:

Table B.9.1.4-1: Residues of spirotetramat in soybean forage (broad leaf plants) and wheat forage 

(short and tall grass) following the second application of Spirotetramat 150 OD and calculated halflives 

Crop Mean Total Spirotetramat 

Residue Immediately 

Following the Second 

Application [ppm]a

Maximum Total 

Spirotetramat Residue 

Following the Second 

Application [ppm]b

Mean Half-Life 

[days]c

Soybean Forage 8.32 10.28 3.42 / 4.45 d

Wheat Forage 

(Short Grass) 

14.35 20.35 2.13 

Wheat Forage 

(Tall Grass) 

13.04 21.65 1.96 

a

 Mean total spirotetramat residue immediately following the 2nd application is the average from all trials of the total 

spirotetramat residue from the sample interval immediately following the 2nd application of Spirotetramat 150 OD. 

b

 Maximum total Spirotetramat residue following the second application is the maximum total a.s. residue from all of the 

trials for the sample intervals following the second application of Spirotetramat 150 OD. 

c

 Mean half-life is the average of the half-lives calculated for each trial from the total Spirotetramat residue at each of the 

sample intervals following the second application of Spirotetramat 150OD. 

d

 The mean half-life for total a.s. residue in soybean forage is 4.45 days without inclusion of the half-life from trial FN333-

05W which received 1.3 inches of rain 1 day following the second application of Spirotetramat 150 OD. 

The highest peak residue level detected was in unmown wheat (21.65 ppm) which was slightly higher as 

compared to mown wheat (20.35 ppm) and soybean foliage (10.28 ppm). 

Observations:

Precipitation totals between the time of the first application and the final harvest date ranged from 2.58 to 

6.07 inches. Rainfall did not occur on the day of application for any of the trials. For any of the trials, the 

earliest time that rainfall occurred following application of Spirotetramat 150 OD was in trials FN331-05W and 

FN333-05W which received rain (0.02 inches and 1.3 inches, respectively) one day following the second 

application. 

Conclusions:

Total Spirotetramat residues dissipated with half-lives of between 2 and 4.5 days in soybean forage (broad 

leaf plants) and wheat forage (short and tall grass) after two foliar applications of Spirotetramat 150 OD at 

the highest proposed seasonal use rate. Mean and maximum residue values determined immediately after 

the second application as well as the calculated half-lives are shown in the table above. 

Reference: Residues of Spirotetramat in arthropods after spray application in citrus – 

magnitude and time course of decline 

Author(s), year: Wilkens, S. and Schneider, K.; 2008 

Report/Doc. number: RA07031 (Rifcon) 

Guideline(s): Not applicable 

GLP: Yes (certified test facility) 

Deviations: Not applicable 

Validity: Acceptable 

Test substance: Spirotetramat OD150B G formulation, 150 g a.s./L (nominal), 152.8 g a.s./L 

(analytical), batch no. 2007-0002474 

Material and methods: 

The aim of this field study was to determine residue levels of Spirotetramat (BYI08330) and its metabolites 

BYI08330-enol and BYI08330-ketohydroxy on foliage and ground dwelling arthropods in citrus orchards after 

spray application of Spirotetramat OD150B G. 
– 25 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Study area: citrus orchards in the region Valencia/Spain. Two orchards (1.2 and 1.3 ha) with two plots each 

(a total of 4 replicates, approx. 0.6 ha each). 

Application rate: One application on each plot at a rate of 99.6 and 121.3 g a.s. per ha and canopy height 

(equivalent to nominal 270 g a.s./ha) with a spray volume of 3000 L/ha. The BBCH principle growth stage of 

the trees was 7. 

Arthropod sampling: Ground dwelling arthropods were sampled using pitfall traps with a diameter of approx. 

12.5 cm, which were dug at surface level and protected with a cover. 86 to 90 traps were installed per plot 

within the orchard rows. The traps were activated approx. 24 h before sampling. 

Foliage dwelling arthropods were collected by inventory spray sampling (full cover application of a 

“knockdown” insecticide containing natural pyrethrum). Trees were sprayed until shortly before run-off. 

Sampling was carried out also before application of the test substance, sampling took place for ground

dwellers: DAT -1, DAT 0 (before application), DAT +1, +2, +3, +4, +5, +7, +9, +11, +14 and +21; and for 

foliage dwellers: DAT -1, DAT 0 (after application), DAT +1, +2, +3, +4, +5, +7, +9, +11, +14 and +21. After 

swift identification and quantification of the main taxonomic groups, the samples were stored deep frozen 

until residue analysis. 

Residue analysis: All samples were analysed for their content of Spirotetramat (BYI08330) and its 

metabolites BYI08330-enol and BYI08330-ketohydroxy via HPLC/MS-MS. Residues are reported in terms of 

mg a.s./kg f.w. of matrix and mg metabolite/kg f.w., respectively. The LOQ was 0.01 mg/kg for both a.s. and 

metabolites. 

Calculation and statistics: Initial and maximum measured values of the mean residues (n=4) were expressed 

for parent, parent+enol and parent+enol+ketohydroxy (total residue). The mean residue values were used to 

calculate the TWAs of daily intervals between DAT 0/1-21, expressed as percentage of the maximum 

measured residue value. TWAs of residue values were calculated by interpolating values for days for which 

no experimental data were available (linear interpolation) and by calculating the mean over a given period. 

The residue decline (DT50) of parent, parent+enol and total residue in foliage dwelling arthropods was 

determined to assess the potential exposure of foliage dwellers to the a.s.. The data used are based on the 

arithmetic mean of the residue values of 4 replicates. First order kinetic was assumed. 

In ground dwelling arthropods residues were low and the kinetic profile was more complex and did not allow 

the calculation of a SFO-DT50. 

Climate data (precipitation, temperature) were recorded during the study. 

Findings:

Abundance of arthropod groups: Arachnida, Isopoda, Myriapoda, Coleoptera, Dermaptera and Lepidoptera 

larvae were the most abundant groups in the pitfall traps, whereas Ephemeroptera, Diptera, Coleoptera,

Neuroptera (adults and larvae) and Hymenoptera (without Formicidae) were most abundant in the samples 

from inventory spraying. 

Precipitation: Between DAT 8 and 20, three slight rainfalls (0.5-1 mm) occurred, and on day 15 one medium 

rainfall (6 mm) took place. 

Validation of the analytical method: The individual recovery values for Spirotetramat for arthropods ranged 

from 78 to 97 %, with an overall recovery of 92 ± 3.2 %. For Spirotetramat-enol recovery was 99 ± 3.5 %, 

and for Spirotetramat-ketohydroxy 79 ± 8.6 %. 

Residue concentrations and TWAs in arthropods are shown in the table below: 
– 26 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.1.4-2: Residue concentrations in ground and foliage dwelling arthropods after application 

of spirotetramat in the field 

Mean residue concentrations [mg/kg f.w.] in ground dwelling arthropods 

Parent Parent+enol Total measured residue 

Initial measured value 

(DAT+1) 

0.21 0.52 0.54 

Maximum measured 

value (MMV) 

0.32 (DAT +2) 0.60 (DAT +2) 0.62 (DAT +2) 

TWA (DAT1-21) 0.15 (46 % of MMV) 0.35 (59 % of MMV) 0.39 (63 % of MMV) 

Mean residue concentrations [mg/kg f.w.] in foliage dwelling arthropods 

Parent Parent+enol Total measured residue 

Initial measured value 

(DAT+1) 

4.0 5.2 5.3 

Maximum measured 

value (MMV) 

4.0 (DAT +0) 6.0 (DAT +1) 6.2 (DAT +1) 

TWA (DAT1-21) 1.07 (27 % of MMV) 1.90 (32 % of MMV) 2.03 (33 % of MMV) 

DT50 2.8 d 4.7 d 5.1 d 

Conclusion:

After application of 99.6 or 121.3 g spirotetramat per ha and meter canopy height (equivalent to nominal 270 

g a.s./ha) to citrus orchards, maximum measured residues in ground dwellers were 0.32, 0.60 and 0.62 

mg/kg f.w. for parent, parent+enol and total measured residues, respectively; and for foliage dwellers 4.0, 6.0 

and 6.2 mg/kg f.w. for parent, parent+enol and total measured residues, respectively. The decline of residue 

levels in foliage dwellers followed a first-order kinetic, the DT50 for residue dissipation was 2.8, 4.7 and 5.1 d 

for parent, parent+enol and total measured residues, respectively, under the climatic conditions prevalent in 

Spain. 

Comment (RMS): In several instances residues exceeding the LOQ were found in arthropods collected 

before application of spirotetramat (both ground and foliage dwelling). These residues amounted up to 0.11 

mg/kg f.w. in the case of ground dwellers. Upon request the notfier tried to find an explanation and admitted 

that some kind of contamination of samples must have taken place, however they were unable to identify the 

situation or location of contamination. Because this shortcoming of the study does not lead to an 

understimation of the residues, the study is considered acceptable. 

Reference: Insectivorous birds in southern European citrus plantations - focal species 

and their diet composition (Position Paper) 

Author(s), year: Wolf, Ch. & Gerlach, J.; 2006 

Report/Doc. number: RC06-067; M-277503-01-1 

Guideline(s): Not applicable 

GLP: Yes (certified laboratory) 

Deviations: Not applicable 

Validity: Acceptable. 

The position paper gives a summary and evaluation of literature sources on the Great Tit (Parus major) as 

focal species in citrus orchards. 

As a typical insectivorous bird in Europe inhabiting a wide range of habitats with trees, like woodlands, 

gardens and orchards, the great tit (Parus major) can be regarded as a representative focal species also in 

citrus plantations. Great tits usually breed in holes (Perrins 1979). Tree holes, as well as nestboxes are 

accepted, and both constellations can be observed in orange groves in Spain (Gil-Delgado & Barba 1987). 

Two investigations on breeding biology and success have been made regarding both the occurrence in tree 

holes and in nestboxes. Moreover observations of open nesting in this species have also been reported 

(Monrós et al. 1999). 
– 27 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Some publications deliver information on the occurrence of great tits in citrus plantations: The population 

density of great tits in the orange grove was ranging between 13.0 and 13.6 pairs per 10 ha, respectively 

(Monrós et al. 1999). A comparative study on breeding biology of great tits revealed an earlier onset of 

breeding and a larger clutch size (mean 7.7 eggs) in orange groves, compared to four natural habitats (two 

holm oak forests (Quercus ilex) at low and high altitudes, a pine forest (Pinus sylvestris) at high altitude and 

a zeen oak forest (Quercus faginea) at high altitude) (Belda et al. 1998). 

The population of great tits breeding in tree holes in the same study area (Sagunto, Spain) reached a density 

of 5 - 8 pairs per 10 ha and average clutch size is 6.4 eggs (Gil-Delgado & Barba 1987). 

Comparable to the results from Spain, a study of La Manita (1982) on the breeding bird community in a 

suburban area around Palermo, Italy, revealed a similar population density of great tits (5 - 6 pairs per 10 ha) 

nesting in similar sites (holes in citrus trees, wall crevices). 

Therefore, based on the results of the above cited papers, great tits obviously inhabit citrus orchards and can 

serve as a representative for a small insectivorous bird for pesticide risk assessment in citrus. 

Data for refinement of the portion of diet (PD) are evaluated in the position paper. 

In Spanish citrus orchards, the diet of Great Tits largely consists of arthropods. The mean size of the prey 

brought to the nestlings was 15.5 mm on the average (Monrós et al. 1997). Another study on the diet 

composition of great tits foraging in modern citrus orchards in Spain revealed that great tit nestlings in a 

modern orange grove near Sagunto, East Spain, from April to August 1988 were predominantly fed with 

Lepidoptera to about 87.8 % in numbers of 526 prey items (Barba and Gil-Delgado 1990). Among the 

lepidopterans 49.8 % were imagines, almost exclusively Noctuidae, 23.6 % were lepidopteran caterpillars, 

14.3 % lepidopteran pupae (Barba & Gil-Delgado 1990). Furthermore the nestlings obtained 5.7 % spiders

and 6.5 % other prey items including Hymenoptera, Coleoptera (Curculionidae), Orthoptera (grass-hoppers), 

egg cocoons and orange pieces (Barba & Gil-Delgado 1990). 

Caterpillars were the most abundant prey delivered to the young only during 6-15 May, while Lepidoptera 

imagines were more abundant during the rest of the season and thus were considered as an important 

resource for the great tits breeding in orange groves (Barba & Gil-Delgado 1990). 

Thus, the diet of Great tits in Spanish citrus orchards consisted at ca. 88 % of large insects (lepidopteran 

caterpillars 23.6 %, pupae 14.3 %, and imagines 49.8 %). For the size class of the remainder it is also 

reasonable to expect preferential feeding on larger prey (Monrós et al. 1997) 

Conclusion:

Based on the results of the above cited papers, great tits obviously inhabit citrus orchards and can serve as 

a representative for a small insectivorous bird for pesticide risk assessment in citrus. 

Remark (RMS): The diet composition of great tits, as discussed in this position paper, was not used in the 

final risk assessment. This is discussed under B. 9.1.8.2. 

References:

Barba & Gil-Delgado (1990): Seasonal variation in nestling diet of the great tit Parus major in orange groves in eastern 

 Spain. Ornis Scandinavica 21: 296-298. 

Belda et al. (1998): Laying date and cluth size of great tits Parus major in the Mediterranean region: a comparison of four 

 habitat types. J. Ornithologie 139: 269-276. 

Gil-Delgado & Barba (1987): Aves nidificantes en huecos de los naranjos. Mediterranea Ser. Biol. 9: 29-40.

La Manita (1982): Dati quantitative sull` avifauna nidificante in una zona suburbana di Palermo. Avocetta 6: 41-46. 

Monrós et al. (1999): Open nestings in Great Tits Parus major. Ardeola 46 (1): 89-91. 

Perrins (1979): British Tits. Collins, London. 
– 28 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: Generic field monitoring of birds in potato, cultivation in northern Germany 

Author(s), year: Wolf, Ch. 2004 

Report/Doc. number: WFC/FS 019; M-090336-02-1 

Guideline(s): Not applicable. The test was especially designed for the purpose of this study. 

GLP: Yes (certified laboratory) 

Deviations: Not applicable 

Validity: Acceptable. 

Material and methods: 

The study has been conducted in and around six different potato fields in the “Wildeshauser Geest” region 

near the village Dötlingen between the towns of Wildeshausen and Oldenburg, in Lower Saxony, Germany. 

This region is a typical area of potato cultivation in Europe and known to hold a population of Yellow

Wagtails (Motacilla flava). 

To identify bird species of concern for potato fields (besides the Yellow Wagtail) and to appraise the

relevancy of potato fields and other habitats as feeding habitats for birds, census counts were carried out 

along different transects, representing all main agrarian habitats within the study area. Birds were counted 

and observed during a slow-going walk on 4 defined transects within and in the vicinities of all 6 monitored 

fields at least once a week during the whole study period. In summary the transects had a length of 6300 m 

and covered a crop area of 48.58 ha. The species, the number of individuals, the location (e.g. flying over, 

vegetation, ground) and the behaviour (e.g. foraging, singing/breeding/feeding young, locomotion, resting, 

others) per passed field were recorded. 

20 Yellow Wagtails were trapped on and in the margin of potato fields, tagged with radio transmitters and 

tracked for one to five daylight periods each. The transmitters, weighting 0.7 g, are known not to influence 

bird`s behaviour significantly. The location, habitat and behaviour was recorded continuously to get 

information of the home range, habitat selection and time budget of birds using potato fields living in a 

cultivable area for potatoes. Birds were followed continuously by car. Emphasis was placed on spatial and 

temporal feeding activities and habitat used. 

To analyse the habitat selection of the Yellow Wagtails while “potentially foraging” (categories “active”, 

“foraging” and “unknown” combined) the Jacob`s preference index (D) (Jacobs 1974) for each habitat and

tracking session was calculated based on the minimum convex polygon (MCP) home range. The Jacob`s 

index indicates the relation between the portion of time a given habitat was used and the spatial portion of 

this habitat within the birds` homerange: 

D = (r - p) / (r + p - 2rp), where r is the portion the habitat was used by the bird and p is the portion of the 

same habitat within the homerange of the bird. 

To get information about the food items, actually selected by the Yellow Wagtails, samples of faeces were 

analysed quantitatively for composition (taxonomic orders of arthropods, other items). Reference samples of 

arthropods living in potato fields and sampled unselectively were obtained from another GLP study, 

conducted at the same time in the same fields. 

At two fields, rainfall was recorded daily. A logger for temperature and humidity measurement was installed. 

Findings:

The results of the monitoring are shown in the table below: 
– 29 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

PORTION OF TIME (PT) in habitat of Yellow Wagtails after radio tracking 

potato fields 38.4 % (72.3) 

cereal fields 39.8 % (44.1) 

oil seed rape fields 6.8 % (22.2) 

street and field path 3.7 % (10.7) 

potential foraging time 

(sum of behaviour categories 

“foraging”+“active”+“unknown”) 

spent per habitat; based on 20 individuals, 1 to 

5 24-hour sessions each [mean of individuals], 

(90%ile) other habitats 11.3 % (16.0) 

FEEDING HABITAT of Yellow Wagtails after transect counts (based on population) 

potato fields 0.60 

cereal fields 0.41 

sugar beet fields 0.32 

abundance of potentially foraging individuals of 

the population after 9 transect counts covering 

48.58 ha field crops each [individuals/ha] 

oil seed rape fields 0.22 

PREFERENCE OF POTATO FIELDS in Yellow Wagtails after radio tracking 

preference of potato fields as a feeding habitat 

[Jacobs’ index (D), MCP (100%)] 

0.16 

DIET of Yellow Wagtails 

taxonomic order steadiness1 mean number2

Diptera 100% 3.96 

Coleoptera 84% 3.60 

Aphidoidea 72% 5.28 

Hymenoptera 52% 0.80 

main arthropod orders actually eaten by 

Wagtails foraging in and around potato fields 

(based on 25 samples of faeces) 

Aranea 48% 0.64 

SIZE OF ARTHROPODS actually chosen by Yellow Wagtails 

taxonomic order food 

[mm] (n) 

population 

[mm] (n, stratum3) 

Diptera 7.49 (99) 2.78 (523, f) 

Coleoptera 7.22 (90) 5.85 (128, g) 

Aphidoidea 3.00 (132) 1.98 (350, f) 

Hymenoptera 6.33 (20) 3.13 (25, f) 

Mean sizes of actually eaten individuals (after 

samples of faeces) compared to mean sizes of 

unselective samples of the population in potato 

fields 

Aranea 4.38 (16) 3.12 (58, g) 

MAIN BIRD SPECIES in potato fields potentially foraging

Yellow Wagtail 0.60 individuals per ha 

Pied Wagtail 0.29 individuals per ha 

mean abundance of main species after 9 

transect counts covering 9.98 ha potato fields 

each 

Whitethroat 0.12 individuals per ha 

1

 portion of samples containing this type 

2

 mean number of individuals per sample 

3

 f = foliage dwelling arthropods, obtained by inventory spraying; g = ground dwelling arthropods, obtained by pitfall 

trapping 

Conclusion 

Radio-tracking of 20 individual Yellow Wagtails (each for a minimum of 24 and a maximum of 120 hours) in 

an agrarian landscape with a high number of potato fields in the north-western part of Lower Saxony showed 

that this field type was used as a main feeding habitat by Yellow Wagtails. However, cereal fields (barley, 

wheat and rye) have been used as well to a (in summary) similar high proportion by Yellow Wagtails, which 

foraged for chick provisioning or the bird’s own use. Only one individual fed almost exclusively on potato 

fields while tracking, while one individual did not use potato fields as feeding habitat at all. 

Potato fields were on average selected to a slightly higher proportion for foraging as to be derived from the 

available proportion in their home range [Jacobs’ index (D)]. Thus Yellow Wagtails positively selected

potatoes as a foraging habitat, but only to a little extent. 
– 30 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Tracking data of individual Yellow Wagtails were confirmed by census data of the population of this species 

within the study area. Moreover census data confirmed the Yellow Wagtail to be the most relevant bird 

species in potato, followed by the Pied Wagtail and the Whitethroat. 

To sum up, it can be ascertained, that potato fields offered a significant but not exclusive feeding habitat for 

the tracked Yellow Wagtails. 

For risk assessment purposes a value for portion of time spent foraging in potato fields (PT) can be derived 

for Yellow Wagtails from the study results: Yellow Wagtails settling in or in close vicinity to potato fields have 

been on average 38.4 % of their potentially foraging time in potato fields (90th percentile 72.3 %). 

Food of Yellow Wagtails in and around potato fields was dominated by Diptera, Coleoptera and Aphidoidea. 

In all taxonomic arthropod orders large individuals were selected distinctly. 

B.9.1.5 Acceptance of bait, granules or treated seeds by birds (OECD IIA 10.1.8) 

Not required. 

B.9.1.6 Effects of secondary poisoning (OECD IIA 10.1.9) 

Not required. See B.9.1.8.4. 

B.9.1.7 Summary of effects on birds 

Table B.9.1.7-1: Summary of avian toxicity endpoints for spirotetramat 

Study type Test species NOAEC 

(mg/kg feed)

NOAEL 

(mg/kg bw) 

LC50 

(mg/kg feed) 

LD50 

(mg/kg bw) Reference 

Acute oral bobwhite quail / < 500 / > 2000 Barfknecht, 

2003 

bobwhite quail 1250 183 > 5000 > 497 Barfknecht, 

2004 Short-term 

dietary 

toxicity mallard duck < 344 < 65 > 6050 > 475 Stoughton, 

2005 

bobwhite quail Adults: 264 

repro: 802 

Adults: 23 

repro: 74 / / Sabbert & Nuessle, 2006a 

mallard duck Adults: < 89*

repro: 89 

Adults: < 9.4*

repro: 9.4 / / Sabbert & Nuessle, 2006b 

Subchronic / 

reproductive 

toxicity 

mallard duck Adults+repro: 

28.8 

Adults+repro: 

4.0 / / Temple et al., 2006 

All values except for acute oral study based on measured figures; 

* foot lesions in all treatment groups 

B.9.1.8 Risk assessment for birds 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 
– 31 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 

B.9.2 Effects on aquatic organisms (OECD IIA 8.2, IIIA 10.2) 

B.9.2.1 Acute toxicity of active substance, metabolites and formulations to aquatic organisms 

B.9.2.1.1 Acute toxicity to fish (OECD IIA 8.2.1, IIA 8.11.1, IIIA 10.2.2) 

Active substance 

Reference: Acute toxicity of BYI 08330 (tech.) to fish (Oncorhynchus mykiss) 

Author(s), year: Dorgerloh, M., 2004 a 

Report/Doc. number: DOM 24025, Edition Number: M-182649-01-2, 16.12.2004 

Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92/69/EEC C.1 

GLP: Yes 

Deviations: Measured test concentration dropped below 80 % of nominal concentration. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 

Material and methods: 

Test species: Rainbow trout (Oncorhynchus mykiss) 

 Number of organisms: 10 fish per concentration and control 

 Weight, length: 2.3 ± 1.1 g (mean ± SD) and 5.8 ± 0.9 cm (mean ± SD) 

 Loading: 0.58 g fish/L test medium 

Type of test, duration: Semi static test (renewal of test media every 24 hours), 96 hours 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L 

 Measured (mean): -- (control and solvent control), 0.409,0.825, 1.5, 3.14 and 6.44 mg/L 

 Solvent Aceton 0.2 mL/L 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaCO3

 Temperature: 11.3 – 12.3 °C 

 pH: 7.0 – 7.1 (0 h, new medium), 7.0 (96 h, aged medium) 

 O2 content: 89 – 99 % saturation 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330 in test 

solutions samples were taken daily from new and aged test media. Test samples 

were directly injected into the HPLC-UV without centrifugation. 

Statistics: EC50: Probit analysis, NOEC: directly from raw data 

Findings: 

Analytical data: Over the whole test period the mean measured concentrations were in the range 

from 77 to 85 % of nominal. 

Behavioural effects: Controls and concentration levels up to 0.825 mg/L: No sublethal effects were 

reported over the whole test period. 

At test concentration ≥ 1.5 mg/L: After 96 hours following symptoms were noted: 

Remained on the bottom of aquarium, loss of equilibrium, labored respiration, 

remained at water surface, turned in a vertical position. 

Thus the NOEC was 0.825 mg/L based on sublethal effects. 
– 32 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Mortality: See table B.9.2.1.1-1 

Table B.9.2.1.1-1: Effects on rainbow trout (O. mykiss) exposed to spirotetramat 

Spirotetramat Cumulative mortality (%) 

[mg a.s./L] 

(mean measured) 24 hours 48 hours 72 hours 96 hours 

Control 0 0 0 0 

Solvent control 0 0 0 0 

0.409 0 0 0 0 

0.825 0 0 0 0 

1.5 0 0 0 0 

3.14 0 30 30 80 

6.44 60 100 100 100 

NOLC: 1.5 mg/L 

LC50 (96 h): 2.54 mg/L (95 % C.I. 2.11 – 3.05 mg/L) 

Details of the Probit: Slope b: 8.4 

95 %CI of b: not stated 

Variance of b: 3.85 

Intercept a: 1.6 

Goodness of fit, Chi2: 1.25 

Degrees of freedom: 3 

p (Chi2): 0.74 

Conclusion: LC50 (96 h): 2.54 mg/L 

NOEC (96 h): 0.825 mg/L 

based on mean measured concentration 

Comments: The US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 

Reference: Acute toxicity of BYI 08330 (tech.) to fish (Cyprinus carpio) 

Author(s), year: Dorgerloh, M., 2004 b 

Report/Doc. number: DOM24022, Edition Number: M-128667-01-2, 25.11.2004 

Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92/69/EEC C.1 

GLP: Yes 

Deviations: Measured test concentration dropped below 80 % of nominal concentration. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 

Material and methods: 

Test species: Common carp (Cyprinus carpio) 

 Number of organisms: 10 fish per concentration and control 

 Weight, length: 0.9 ± 0.3 g (mean ± SD) and 4.0 ± 0.5 cm (mean ± SD) 

 Loading: 0.23 g fish/L test medium 

Type of test, duration: Semi static test (renewal of test media every 24 hours), 96 hours 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 1.25, 2.5, 5.0, 10.0 and 20.0 mg/L 

 Measured (mean): -- (control and solvent control), 1.02, 1.71, 3.65, 8.13 and 13.4 mg/L 

 Solvent Aceton 0.2 mL/L 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaCO3
– 33 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Temperature: 20.5 – 23.2 °C 

 pH: 7.1 – 7.2 (0 h, new medium), 7.1 (96 h, aged medium) 

 O2 content: 97 – 103 % saturation 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330 in test 

solutions samples were taken daily from new and aged test media. Test samples 

were directly injected into the HPLC-UV without centrifugation. 

Statistics: EC50: Probit analysis, NOEC: directly from raw data 

Findings: 

Analytical data: Over the whole test period the mean measured concentrations were in the range 

69 – 83 % of nominal. 

Behavioural effects: Controls and at 1.02 mg/L: No sublethal effects were observed over the whole test

period. 

At concentration levels ≥ 1.71 mg/L: After 96 hours following symptoms were 

noted: Remained on the bottom of aquarium, loss of equilibrium, labored 

respiration, dark coloration, laying on the sides or backs, turned in a vertical 

position. 

Thus the NOEC was 1.02 mg/L based on sublethal effects. 

Mortality: See table B.9.2.1.1-2 

Table B.9.2.1.1-2: Effects on common carp (C. carpio) exposed to spirotetramat 

Spirotetramat Cumulative mortality (%) 

[mg a.s./L] 

(mean measured) 24 hours 48 hours 72 hours 96 hours 

Control 0 0 0 0 

Solvent control 0 0 0 0 

1.02 0 0 0 0 

1.71 0 0 0 0 

3.65 0 10 50 100 

8.13 100 100 100 100 

13.4 100 100 100 100 

NOLC: 1.71 mg/L 

LC50 (96 hours): 2.59 mg/L (95 % C.I.: not determined due to mathematical reasons) 

Details of the Probit: Slope b: 16.09 

95 %CI of b: not stated 

Variance of b: 29.9 

Intercept a: -1.66 

Goodness of fit, Chi2: 17.69 

Degrees of freedom: 3 

p (Chi2): < 0.001 

Conclusion: LC50 (96 h): 2.59 mg/L 

NOEC (96 h): 1.02 mg/L 

based on mean measured concentration 

Comments: The US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. (95 %CI: not stated) 
– 34 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: Acute toxicity of BYI 08330 (tech.) to fish (Lepomis macrochirus) 

Author(s), year: Dorgerloh, M., 2005 a 

Report/Doc. number: DOM24056, Edition Number: M-242689-01-2, 12.01.2005 

Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92/69/EEC C.1 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods: 

Test species: Bluegill sunfish (Lepomis macrochirus) 

 Number of organisms: 10 fish per concentration and control 

 Weight, length: 1.7 ± 0.7 g (mean ± SD) and 4.9 ± 0.6 cm (mean ± SD) 

 Loading: 0.43 g fish/L test medium 

Type of test, duration: Semi static test (renewal of test media every 24 hours), 96 hours 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L 

 Measured (mean): -- (control and solvent control), 0.48, 0.95, 1.90, 3.56 and 7.61 mg/L 

 Solvent Aceton 0.2 mL/L 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 40 – 60 mg/L as CaCO3

 Temperature: 17.9 – 20.5 °C 

 pH: 7.4 – 7.5 (0 h, new medium), 7.3 (96 h, aged medium) 

 O2 content: 96 – 105 % saturation 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330 in test 

solutions samples were taken daily from new and aged test media. Test samples 

were directly injected into the HPLC-UV without centrifugation. 

Statistics: EC50: Probit analysis, NOEC: directly from raw data 

Findings: 

Analytical data: Over the whole test period the mean measured concentrations were in the range 

91 – 99 % of nominal. 

Behavioural effects: Controls and at 0.5 mg/L: No sublethal effects were observed over the whole test 

period. 

At concentration levels ≥ 1.0 mg/L: Transient effects like remaining on the bottom 

of aquarium, laying on the sides or backs, labored respiration, hyperactivity, 

inactivity, convulsions and remaining on the water surface were observed. 

Thus the NOEC was 0.5 mg/L based on sublethal effects. 

Mortality: See table B.9.2.1.1-3 
– 35 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.2.1.1-3: Effects on bluegill sunfish (L. macrochirus) exposed to spirotetramat 

Spirotetramat Cumulative mortality (%) 

[mg a.s./L] 

(nominal) 24 hours 48 hours 72 hours 96 hours 

Control 0 0 0 0 

Solvent control 0 0 0 0 

0.5 0 0 0 0 

1.0 0 20 20 20 

2.0 0 10 10 10 

4.0 20 50 100 100 

8.0 80 100 100 100 

NOLC: 0.5 mg/L 

LC50 (96 hours): 2.2 mg/L (95 % C.I.: not determined due to mathematical reasons) 

Details of the Probit: Slope b: 4.44 

95 %CI of b: not stated 

Variance of b: 1.21 

Intercept a: 3.48 

Goodness of fit, Chi2: 8.93 

Degrees of freedom: 3 

p (Chi2): 0.0302 

Conclusion: LC50 (96 h): 2.2 mg/L 

NOEC (96 h): 0.5 mg/L 

based on nominal concentration 

Comments: The US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 

Metabolites 

Reference: Acute toxicity of BYI 08330-enol to fish (Oncorhynchus mykiss) 

Author(s), year: Nieden, D., 2005 a 

Report/Doc. number: Report No.: EBFNM013, Edition Number: M-251850-01-2, 18.05.2005 

Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92/69/EEC C.1 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005 

Material and methods: 

Test species: Rainbow trout (Oncorhynchus mykiss) 

 Number of organisms: 2 x 15 fish per concentration and control 

 Weight, length: 1.3 ± 0.6 g (mean ± SD) and 4.9 ± 0.7 cm (mean ± SD) 

 Loading: 0.49 g fish/L test medium 

Type of test, duration: Static limit test, 96 hours

Applied concentrations: 

 Nominal: 0 (control), 100 mg/L 

 Measured (mean): -- (control), 98 mg/L 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaCO3. 
– 36 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

After the addition of test substance the pH was adjusted to about 7 with NaOH. 

 Temperature: 11.1 – 13 °C 

 pH: 6.7 – 7.2 (0 h), 6.8 – 6.9 (96 h) 

 O2 content: 86 – 102 % saturation 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

At 0, 48 and 96 hours samples were taken for chemical analysis (HPLC-MS/MS 

and HPLC-UV) of BYI 08330-enol in test solutions. 

Statistics: None, because a limit test was performed 

Findings: 

Analytical data: Over the whole test period the mean measured concentration was 98.5 % 

(replicate A) and 97.7 % (replicate B) of nominal. 

Behavioural effects: No effects were observed in control and 100 mg/L treatment replicates. 

Mortality: No mortality was reported in control and 100 mg/L treatment replicates. 

Conclusion: LC50 (96 h): > 100 mg/L 

NOEC (96 h): 100 mg/L 

based on nominal concentration 

Reference: 4-Methoxycyclohexanone - Acute fish toxicity 

Author(s), year: Neuhahn, A., 2006 a 

Report/Doc. number: Report No.: 2006/0032/02, Edition Number: M-274305-01-1, 10.07.2006 

Guideline(s): OECD 203, EU Directive 92/69/EEC C.1 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: 4-Methoxycyclohexanone (metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV5506-001 

 Solubility: Within the test, the item was soluble at the limit concentration of 100 mg/L in test 

medium after ultra turrax treatment. There was no indication for any unsolved 

material after mixing. Substance specific GC analysis showed recoveries of >80% 

for all samples taken. 

Material and methods: 

Test species: Zebra fish (Danio rerio) 

 Number of organisms: 10 fish per concentration and control 

 Weight, length: Not reported, 3.4 ± 0.26 cm (mean ± SD) 

 Loading: 0.64 g fish/L test medium 

Type of test, duration: Static limit test, 96 hours

Applied concentrations: 

 Nominal: 0 (control), 100 mg/L 

 Measured (mean): -- (control), 98.5 mg/L 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 246 mg/L as CaCO3. 

 Temperature: 20.5 – 21.0 °C 

 pH: 7.6 – 7.9 

 O2 content: 88 – 101 % saturation 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 2, 24, 48, 72 and 96 hours. 

At 0, 24 and 96 hours samples were taken for chemical analysis (GS analysis and 
– 37 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

MS detection) of 4-Methoxycyclohexanone in test solutions. Test samples were 

analysed directly without centrifugation. 

Statistics: None, because a limit test was performed 

Findings: 

Analytical data: The mean measured concentration was 105 % (0 hours) and 89.8 % (96 hours) of 

nominal. 

Behavioural effects: After 2 hours abnormalities in swimming were noted for 50 % of fish. However this

effect was transient and no effects were observed at later time points. 

Mortality: No mortality was reported in control and 100 mg/L treatment replicates. 

Conclusion: LC50 (96 h): > 100 mg/L 

NOEC (96 h): 100 mg/L 

based on nominal concentration 

Comments While the study not deviated from recommendations given in OECD 202 and EU 

guideline, deviation from USEPA guidelines are notable: 

-Minor: The temperature was slightly lower than the recommended levels for 

zebra fish (23 ±2oC), and the weight of fish wasn’t reported. 

-Major: The guidelines require at least 30 fish to be tested, and only ten were 

tested at the 100 mg/L treatment level..

Comments: US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 

Formulation 

Reference: Acute toxicity of BYI 08330 OD 150 to fish (Oncorhynchus mykiss) under 

static conditions 

Author(s), year: Nieden, D., 2005 d 

Report/Doc. number: EBFNM008, Edition Number: M-253916-01-2, 29.06.2005 

Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92/69/EEC C.1 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330 OD 150 (formulation), spirotetramat content: 148.89 g/L (15.1 %), 

batch: 08030/0189(0152) 

Material and methods: 

Test species: Rainbow trout (Oncorhynchus mykiss) 

 Number of organisms: 10 fish per concentration and control 

 Weight, length: 0.8 ± 0.2 g (mean ± SD) and 4.6 ± 0.3 cm (mean ± SD) 

 Loading: 0.20 g fish/L test medium 

Type of test, duration: Static test, 96 hours 

Applied concentrations: 

 Nominal: Formulation: 0 (control), 2.50, 5.00, 10.0, 20.0, 40.0 mg product/L 

 Spirotetramat: 0 (control), 0.378, 0.755, 1.51, 3.02, 6.04 mg a.s./L 

 Measured (mean): Spirotetramat: -- (control), 0.394, 0.783, 1.57, 3.10, 6.45 mg a.s./L 

Control: Untreated test water, no blank formulation control was used 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaCO3

 Temperature: 11.6 – 12.2 °C 

 pH: 6.9 – 7.2 

 O2 content: 95 – 102 % saturation 
– 38 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC-MS/MS and HPLC-UV) of spirotetramat in test 

solutions samples were taken at 0, 48 and 96 hours from each concentration. 

Statistics: EC50: Logit and Probit analysis, NOEC: directly from raw data 

Findings: 

Analytical data: Over the whole test period the mean measured concentrations of active 

substance were in the range from 103 to 107 % of nominal. 

Behavioural effects: Controls and 2.5 mg product/L concentration level: No sublethal effects were 

observed over the whole test period. 

At test concentrations of 5.0 and 10 mg product/L: After 96 hours following 

symptoms were noted: Remained on the bottom of aquarium, dark coloration, loss 

of equilibrium, labored respiration, hyperactivity, laying on sides or backs. 

Thus the NOEC was 2.5 mg product/L based on sublethal effects. 

Mortality: See table B.9.2.1.1-4: 

Table B.9.2.1.1-5: Effects on rainbow trout (O. mykiss) exposed to BYI 08330 OD 150 (formulation) 

BYI 08330 OD 150 Cumulative mortality (%) 

[mg product/L] 

(nominal) 

Spirotetramat 

mg a.s./L 

(mean meas.) 24 hours 48 hours 72 hours 96 hours 

Control 0 0 0 0 

2.5 0.394 0 0 0 0 

5.0 0.783 0 0 0 10 

10.0 1.57 0 0 10 10 

20.0 3.1 70 80 90 100 

40.0 6.45 100 100 100 100 

NOLC: 2.5 mg product/L corresponding to 0.397 mg a.s./L 

LC50 (96 h): 9.48 mg/L (95 % C.I. 5.84 – 15.7 mg/L) corresponding to 1.41 mg a.s./L calculated by Logit 

LC50 (96 h): 11.8 mg/L (95 % C.I. n.d.) corresponding to 1.76 mg a.s./L calculated by Probit 

Details of the Probit: Slope b: 5.607 

95 % C.I. of b: 0.994 – 10.221 

Variance of b: 2.27 

Intercept a: -0.898 

Goodness of fit, Chi2: 7.498 

Degrees of freedom: 3 

p (Chi2): 0.0576 

Conclusion: LC50 (96 h): 9.48 mg product/L, 1.41 mg a.s./L 

NOEC (96 h): 2.5 mg product/L, 0.378 mg a.s./L 

based on nominal concentration 

B.9.2.1.2 Acute toxicity to aquatic invertebrates (OECD IIA 8.3.1, IIA 8.5.1, IIIA 10.2.2.2) 

Active substance 
– 39 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: Acute toxicity of BYI 08330 (tech.) to the waterflea Daphnia magna under 

static conditions 

Author(s), year: Dorgerloh, M., 2005 c 

Report/Doc. number: DOM 24004, Edition Number: M-242683-01-2, 14.01.2005 

Guideline(s): OECD 202, EPA 72-2, OPPTS 850.1010, EU Directive 92/69/EEC C.2 

GLP: Yes 

Deviations: Minor deviation from EPA guideline: The pH is lower and the hardness is higher 

than EPA-recommended range. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods:

Test species: Waterflea (Daphnia magna) 

 Number of organisms: 6 replicates each with 5 daphnids per treatment and control 

 Age: First instar < 24 hours old 

Type of test, duration: Static test, 48 h 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 6.25, 12.5, 25.0, 50, 100 mg/L 

 Measured (mean): -- (control and solvent control), 4.7, 8.77, 20.3, 38.8, 42.7 mg/L 

 Solvent 0.4 mL/L Aceton 

Test conditions: 

 Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring 

test), hardness: 196 mg/L as CaCO3

 Temperature 20.5 – 20.6 °C 

 pH 6.3 – 6.4 (0 h), 6.6 – 6.7 (48 h) 

 O2 content: 96 – 102 %, 8.5 – 9.0 mg O2/L 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Immobility and sublethal effects were assessed after 24 and 48 hours. For 

chemical analysis (HPLC-UV) of BYI 08330 in the test media samples were taken 

at test initiation (0 h) and termination (48 h). The water samples were analysed by 

direct injection into the HPLC-UV instrument without centrifugation. 

Statistics: EC50: None, because at highest test concentration (100 mg/L nominal) the 

immobilisation were < 50 %, NOEC: directly from the raw data 

Findings: 

Analytical data: The mean measured concentrations at the start and end of the test were in the 

range of 22 – 90 %, overall mean measured concentration ranged from 42.7 – 

81.0 % of nominal. 

Effects: After 48 hours no immobility was observed in the control, solvent control and in 

test concentrations up to 20.3 mg/L. At 38.8 and 42.7 mg/L the immobilisation was 

7 and 23 %. No sublethal effects were noted during the whole study. 

Thus the NOEC was determined to be 20.3 mg/L and the EC50 was > 42.7 mg/L. 

Conclusion: EC50 (48 h): > 42.7 mg/L 

NOEC: 20.3 mg/L 

based on mean measured concentration 

Comments: US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 
– 40 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: Acute toxicity of BYI 08330 (tech.) to larvae of Chironomus riparius in a 48 h 

water-only study 

Author(s), year: Dorgerloh, M., 2005 g 

Report/Doc. number: EBFNX072, Edition Number: M-262632-01-2, 08.12.2005 

Guideline(s): No specified guideline, the study is following the procedure of OECD 202 

GLP: Yes 

Deviations: None of relevance considering OECD 202 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 

Material and methods: 

Test species: Midge (Chironomus riparius) 

 Number of organisms: 4 replicates each with 10 larvae per treatment and control 

 Age: L1 (first instar) larvae 

Type of test, duration: Static (water-only) test, 48 h 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.56, 1.00, 1.80, 3.20, 5.60, 10.0 mg/L 

 Measured (mean): -- (control and solvent control), 0.53, 0.96, 1.72, 3.04, 5.37, 9.47 mg/L 

 Solvent 0.1 mL/L Aceton 

Test conditions: 

 Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring 

test), hardness: not stated 

 Temperature 20.4 – 21 °C 

 pH 8.2 – 8.6 (0 h), 8.2 – 8.9 (48 h) 

 O2 content: > 80 %, (8.6 – 8.9 mg O2/L) 

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Test parameters: Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330 in the test media samples 

were taken at test initiation (0 h) and termination (48 h). 

Statistics: Correction of mortality: Abbott’s formula, LC10 and LC50: Probit analysis, NOEC: 

not determined, comparison of controls: Student-t test for homogeneous 

variances. 

All statistical analyses are based on data of pooled control. 

Findings: 

Analytical data: The mean measured concentrations at the start and end of the test were in the 

range of 106 – 109 % and 82 – 83 % of nominal, respectively. Overall mean 

measured concentrations ranged from 95 to 96 % of nominal. 

Effects: After 48 hours the mortality in pooled controls was 8.7 %, thus the validity criteria 

of maximum control mortality of 10 % is met, 

further information see table B.9.2.1.2-1 

Table B.9.2.1.2-1: Effects on midge (C. riparius) exposed to spirotetramat 

Spirotetramat [mg a.s./L] Mortality after 48 hours 

mean measured 

Exposed chironomids 

(= 100 %) [n] [%] 

Control* 40 3 7.5 

Solvent control* 40 4 10.0 

0.528 40 4 10.0 

0.958 40 13 32.5 
– 41 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Spirotetramat [mg a.s./L] Mortality after 48 hours 

mean measured 

Exposed chironomids 

(= 100 %) [n] [%] 

1.715 40 29 72.5 

3.035 40 39 97.5 

5.365 40 40 100 

9.465 40 40 100 

LC10 (48 h): 0.699 mg/L (95% C.I. 0.536 – 0.913 mg/L) 

LC50 (48 h): 1.299 mg/L (95% C.I. 1.120 – 1.506 mg/L) 

* Result of t-test is indicated no significant differences between control and solvent control, thus pooled control are used 

for statistical analysis. 

Details of the Probit: Slope b: 4.76 

95 % CI of b: not stated 

Variance of b: 0.54 

Intercept a: 4.46 

Goodness of fit, Chi2: 6.62 

Degrees of freedom: 4 

p (Chi2): 0.158 

Conclusion: LC10 (48 h): 0.699 mg/L 

LC50 (48 h): 1.299 mg/L 

NOEC (48 h): < 0.528 mg/L 

based on mean measured concentration 

Comments: While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

-Age of the test organisms is 2nd or 3rd instar, 1st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Mortality and sublethal effects should be measured every 24 hours.

Metabolites 

Reference: Acute toxicity of BYI 08330-enol to the waterflea Daphnia magna in a static 

laboratory test system - limit test 

Author(s), year: Dorgerloh, M., 2005 d 

Report/Doc. number: EBFNM012, Edition Number: M-254487-01-2, 14.07.2005 

Guideline(s): OECD 202, EPA 72-2, OPPTS 850.1010, EU Directive 92/69/EEC C.2 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005, water solubility (at pH 7): 2.7g/L 

Material and methods: 

Test species: Waterflea (Daphnia magna) 

 Number of organisms: 6 replicates each with 5 daphnids per treatment and control 

 Age: First instar < 24 hours old 

Type of test, duration: Static limit test, 48 h 

Applied concentrations: 

 Nominal: 0 (control), 100 mg/L 

 Measured (mean): -- (control), 111.5 mg/L 

Test conditions: 
– 42 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring 

test), hardness: 196 mg/L as CaCO3, adjusted to pH 7 before use for dilution 

procedure 

 Temperature 20.6 – 21.2 °C 

 pH 7.0 – 6.9 (0 h), 7.5 – 7.9 (48 h) 

 O2 content: 89 – 96 %, 8.1 – 8.7 mg O2/L 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Immobility and sublethal effects were assessed after 24 and 48 hours. For 

chemical analysis (HPLC-UV) of BYI 08330-enol in the test media samples were 

taken at test initiation (0 h) and termination (48 h). Test samples were analysed by 

direct injection into a HPLC-UV instrument without a centrifugation. 

Statistics: EC50: None, because at the limit concentration (100 mg/L nominal) no 

immobilisations were observed, NOEC: directly from the raw data 

Findings: 

Analytical data: The measured concentrations at the start and end of the test were 113 and 110 % 

of nominal, overall mean measured concentration was 111.5 % of nominal. 

Effects: After 48 hours no immobility and no sublethal effects were observed in the control, 

and in the limit test concentrations of 100 mg/L. 

Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. 

Conclusion: EC50 (48 h): > 100 mg/L 

NOEC: 100 mg/L 

based on nominal concentration 

Reference: Acute toxicity of BYI 08330-enol to larvae of Chironomus riparius in a 48 h 

water-only study 

Author(s), year: Dorgerloh, M., 2005 h 

Report/Doc. number: EBFNX073, Edition Number: M-262638-01-2, 08.12.2005 

Guideline(s): No specified guideline, the study is following the procedure of OECD 202 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005, water solubility (at pH 7): 2.7g/L 

Material and methods: 

Test species: Midge (Chironomus riparius) 

 Number of organisms: 4 replicates each with 10 larvae per treatment and control 

 Age: L1 (first instar) larvae 

Type of test, duration: Static (water-only) test, 48 h 

Applied concentrations: 

 Nominal: 0 (control), 1.0, 2.0, 4.0, 8.0, 16, 32, 64 mg/L 

 Measured (mean): -- (control), 0.95, 1.93, 4.06, 8.3, 15.65, 32.25, 66.45 mg/L 

Test conditions: 

 Water quality: M7 medium (according to OECD 202), hardness: not stated 

 Temperature 20.8 – 21 °C 

 pH 7.8 – 8.2 (0 h), 8.1 – 8.4 (48 h) 

 O2 content: > 90 %, (8.5 – 8.7 mg O2/L) 

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Only on time 0.01 mL aqueous fish food solution directly after insertion of the 
– 43 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

larvae 

Test parameters: Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330-enol in the test media 

samples were taken at test initiation (0 h) and termination (48 h). Test samples 

were analysed by direct injection into a HPLC-UV instrument without a 

centrifugation. 

Statistics: LC10 and LC50: Probit analysis, NOEC: not determined 

Findings: 

Analytical data: The mean measured concentrations at the start and end of the test were in the 

range of 98 – 105 % and 88 – 103 % of nominal, respectively. Overall mean 

measured concentrations ranged from 95 to 104 % of nominal. 

Effects: See table B.9.2.1.2-2 

Table B.9.2.1.2-2: Effects on midge (C. riparius) exposed to BYI 08330-enol 

BYI 08330-enol 

[mg/L] Mortality after 48 hours 

nominal mean measured 

Exposed chironomids 

(= 100 %) 

[n] [%] 

Control 40 0 0 

1.0 0.95 40 0 0 

2.0 1.93 40 0 0 

4.0 4.06 40 0 0 

8.0 8.28 40 0 0 

16.0 15.68 40 5 12.5 

32.0 32.16 40 11 27.5 

64.0 67.59 40 16 40.0 

LC10 (48 h): 17.07 mg/L (95% C.I. 12.15 – 23.98 mg/L) based on nominal 

11.62 mg/L (95% C.I. 4.74 – 28.48 mg/L) based on mean measured 

LC50 (48 h): 74.85 mg/L (95% C.I. 48.86 – 114.64 mg/L) based on nominal 

97.78 mg/L (95% C.I. 43.15 – 221.60 mg/L) based on mean measured 

Details of the Probit: Slope b: 1.38 

95 % CI of b: not stated 

Variance of b: 0.248 

Intercept a: 2.246 

Goodness of fit, Chi2: 0.175 

Degrees of freedom: 1 

p (Chi2): 0.676 

Conclusion: LC50 (48 h): 74.9 mg/L 

LC10 (48 h): 17.1 mg/L 

NOEC (48 h): not determined 

based on nominal concentration 

Comments: While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

-Age of the test organisms is 2nd or 3rd instar, 1st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Mortality and sublethal effects should be measured every 24 hours.
– 44 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: Acute toxicity of BYI 08330-ketohydroxy to larvae of Chironomus riparius in 

a 48 h static laboratory test system 

Author(s), year: Dorgerloh, M., 2006 a 

Report/Doc. number: EBFNO347, Edition Number: M-263956-01-2, 16.01.2006 

Guideline(s): No specified guideline, the study is following the procedure of OECD 202 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330-ketohydroxy (metabolite of spirotetramat), purity: 98.6 %, batch: 

NLL7549-7, water solubility (in distilled water): 0.228g/L 

Material and methods: 

Test species: Midge (Chironomus riparius) 

 Number of organisms: 4 replicates each with 10 larvae per treatment and control 

 Age: L1 (first instar) larvae 

Type of test, duration: Static (water-only) test, 48 h 

Applied concentrations: 

 Nominal: 0 (control), 1.0, 3.6, 10.0, 36, 100 mg/L 

 Measured (mean): -- (control), 0.94, 3.47, 10.01, 33.6, 93.2 mg/L 

Test conditions: 

 Water quality: M7 medium (according to OECD 202), hardness: not stated 

 Temperature 20.5 – 21.6 °C 

 pH 7.8 – 8.4 (0 h), 8.0 – 8.1 (48 h) 

 O2 content: > 80 %, (6.9 – 7.7 mg O2/L) 

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Test parameters: Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330-ketohydroxy in the test 

media samples were taken at test initiation (0 h) and termination (48 h). Test 

samples were analysed by direct injection into a HPLC-UV instrument without a 

centrifugation. 

Statistics: LC10 and LC50: Probit analysis, NOEC: directly from raw data 

Findings: 

Analytical data: The mean measured concentrations at the start and end of the test were in the 

range of 93 – 105 % and 92 – 96 % of nominal, respectively. Overall mean 

measured concentrations ranged from 93 to 100 % of nominal. 

Effects: See table B.9.2.1.2-3 

Table B.9.2.1.2-3: Effects on midge (C. riparius) exposed to BYI 08330- ketohydroxy 

Mortality after 48 hours BYI 08330-enol 

[mg/L] (nominal) 

Exposed chironomids 

(= 100 %) [n] [%] 

Control 40 1 2.5 

1.0 40 2 5.0 

3.6 40 1 2.5 

10 40 1 2.5 

36 40 2 5.0 

100 40 11 27.5 

LC10: 23.9 mg/L (95% C.I.: not determined due to mathematical reasons) 

LC50: > 100 mg/L (95% C.I.: not determined due to mathematical reasons) 
– 45 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Conclusion: LC50 (48 h): > 100 mg/L 

LC10 (48 h): 23.9 mg/L 

NOEC (48 h): < 1 mg/L 

based on nominal concentration 

Comments: While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

-Age of the test organisms is 2nd or 3rd instar, 1st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Mortality and sublethal effects should be measured every 24 hours.

Reference: 4-Methoxycyclohexanone - Acute daphnia toxicity 

Author(s), year: Neuhahn, A. 2006 b 

Report/Doc. number: 2006/0032/01, Edition Number: M-274275-01-1, 10.07.2006 

Guideline(s): OECD 202, EU Directive 92/69/EEC C.2 

GLP: Yes 

Deviations: OECD: None of relevance 

US EPA: 

Validity: Acceptable 

Test substance: 4-Methoxycyclohexanone (metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV5506-001 

 Solubility: Within the test, the item was soluble at the limit concentration of 100 mg/L in test 

medium after ultra turrax treatment. There was no indication for any unsolved 

material after mixing. Substance specific GC analysis showed recoveries of >80% 

for all samples taken. 

Material and methods: 

Test species: Waterflea (Daphnia magna) 

 Number of organisms: 2 replicates each with 10 daphnids per treatment and control 

 Age: First instar < 24 hours old 

Type of test, duration: Static limit test, 48 h 

Applied concentrations: 

 Nominal: 0 (control), 100 mg/L 

 Measured (mean): -- (control), 111.5 mg/L 

Test conditions: 

 Water quality: M4 medium, hardness: 273 mg/L as CaCO3

 Temperature 21.4 – 21.5 °C 

 pH 7.8 (control) and 9.2 (100 mg/L) 

 O2 content: 98 %,8.7 mg O2/L 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Immobility and sublethal effects were assessed after 24 and 48 hours. For 

chemical analysis (GC analysis and MS detection) of 4-methoxycyclohexanone in 

the test media samples were taken at test initiation (0 h) and termination (48 h). 

Test samples were analysed directly without centrifugation. 

Statistics: None, because at the limit concentration (100 mg/L nominal) no immobilisation 

were observed 

Findings: 

Analytical data: The measured concentrations at the start and end of the test were 103 and 90 % 
– 46 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

of nominal, overall mean measured concentration was 97 % of nominal. 

Effects: After 48 hours no immobility and no sublethal effects were observed in the control 

and in the limit test concentrations of 100 mg/L. 

Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. 

Conclusion: EC50 (48 h): > 100 mg/L 

NOEC: 100 mg/L 

based on nominal concentration 

Comments: While the study not deviated from recommendations given in OECD 202 and EU 

guideline, significant deviation from USEPA guidelines are notable: 

The EPA recommends that at least 30 test organisms are used at each test level 

in a limit test, and only 20 were used here. 

Furthermore US EPA has general concerns regarding uncertainties surrounding 

exposure concentrations in test media. 

Reference: Acute toxicity of 4-Methoxycyclohexanon to Larvae of Chironomus riparius

in a 48 h Static Laboratory Test System (Limit-Test) 

Author(s), year: Dorgerloh, M., 2007 a 

Report/Doc. number: EBFNX104, Edition Number: M-287218-01-1, 27.04.2007 

Guideline(s): No specified guideline, the study is following the procedure of OECD 202 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: 4-Methoxycyclohexanon (photo-metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV55506-001 

 Solubility: > 100 mg (pH 8, 20 °C) 

Material and methods: 

Test species: Midge (Chironomus riparius) 

 Number of organisms: 6 replicates each with 10 larvae per treatment and control 

 Age: L1 (first instar) larvae, < 2 – 3 days old 

Type of test, duration: Static (water-only) limit-test, 48 h 

Applied concentrations: 

 Nominal: 0 (control), 100 mg/L 

 Measured (mean): -- (control), 111 mg/L 

Test conditions: 

 Water quality: M7 medium (according to OECD 202), hardness: 231 mg/L CaCO3

 Temperature 20.3 – 20.9 °C 

 pH 8.0 (after 0 and 48 hours) 

 O2 content: > 90 %, (8.6 – 8.9 mg O2/L) 

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Test parameters: Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MS/MS) of 4-Methoxycyclohexanon in the test media samples 

were taken at test initiation (0 h) and termination (48 h). Test samples were 

analysed by direct injection into a HPLC-UV instrument without a centrifugation. 

Statistics: A limit-test was performed, thus no statistic is applicable. 

Findings: 

Analytical data: The mean measured concentrations at the start and end of the test were 109 % 
– 47 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

and 112 % of nominal, respectively. Overall mean measured concentrations is 

111 % of nominal. 

Effects: After 48 hours no mortality and no sublethal effects were observed in the control 

and in the limit test concentrations of 100 mg/L. 

Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. 

Conclusion: LC50 (48 h): > 100 mg/L 

NOEC (48 h): 100 mg/L 

based on nominal concentration 

Comments: While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

-Age of the test organisms is 2nd or 3rd instar, 1st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Mortality and sublethal effects should be measured every 24 hours.

Furthermore US EPA has general concerns regarding uncertainties surrounding 

exposure concentrations in test media. 

Reference: Acute toxicity of BYI 08330-cis-methoxy-cyclohexylamino carboxylic acid to 

Larvae of Chironomus riparius in a 48 h Static Laboratory Test System 

(Limit-Test) 

Author(s), year: Dorgerloh, M., 2007 b 

Report/Doc. number: EBFNX105, Edition Number: M-287220-01-1, 27.04.2007 

Guideline(s): No specified guideline, the study is following the procedure of OECD 202 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330-cis-methoxy-cyclohexylamino acid (photo-metabolite of spirotetramat), 

purity: 93.7 %, batch: KTS10330-1-4 

 Solubility: > 100 mg (pH 8, 20 °C) 

Material and methods: 

Test species: Midge (Chironomus riparius) 

 Number of organisms: 6 replicates each with 10 larvae per treatment and control 

 Age: L1 (first instar) larvae, < 2 – 3 days old 

Type of test, duration: Static (water-only) limit-test, 48 h 

Applied concentrations: 

 Nominal: 0 (control), 100 mg/L 

 Measured (mean): -- (control), 88.6 mg/L 

Test conditions: 

 Water quality: M7 medium (according to OECD 202), hardness: 231 mg/L CaCO3

 Temperature 20.3 – 20.9 °C 

 pH 6.9 – 8.0 (0 h), 7.7 – 8. 0 (48 h) 

 O2 content: > 90%, (8.7 – 8.9 mg O2/L) 

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Test parameters: Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MS/MS) of BYI 08330-cis-methoxy-cyclohexylamino acid in the 

test media samples were taken at test initiation (0 h) and termination (48 h). Test 
– 48 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

samples were analysed by direct injection into a HPLC-UV instrument without a 

centrifugation. 

Statistics: A limit-test was performed, thus no statistic is applicable. 

Findings: 

Analytical data: The mean measured concentrations at the start and end of the test were 87.8 % 

and 89.4 % of nominal, respectively. Overall mean measured concentrations is 

88.6 % of nominal. 

Effects: After 48 hours no mortality and no sublethal effects were observed in the control 

and in the limit test concentrations of 100 mg/L. 

Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. 

Conclusion: LC50 (48 h): > 100 mg/L 

NOEC (48 h): 100 mg/L 

based on nominal concentration 

Comments: While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

-Age of the test organisms is 2nd or 3rd instar, 1st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Mortality and sublethal effects should be measured every 24 hours.

Furthermore US EPA has general concerns regarding uncertainties surrounding 

exposure concentrations in test media. 

Formulation 

Reference: Acute toxicity of BYI 08330 OD 150 to larvae of Chironomus riparius in a 

48 h static laboratory test system 

Author(s), year: Dorgerloh, M., 2006 b 

Report/Doc. number: EBFNM007, Edition Number: M-264018-01-2, 19.01.2006 

Guideline(s): No specified guideline, the study is following the procedure of OECD 202 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08330 OD 150 (formulation), spirotetramat content: 148.89 g/L (14.89 % 

active ingredient), batch: 08030/0189(0152), density: 0.986 g/mL 

Material and methods: 

Test species: Midge (Chironomus riparius) 

 Number of organisms: 4 replicates each with 10 larvae per treatment and control 

 Age: L1 (first instar) larvae 

Type of test, duration: Static (water-only) test, 48 h 

Applied concentrations: 

 Nominal: Formulation: 0 (control), 2.12, 3.71, 6.62, 11.9, 21.2, 37.1, 66.2 mg product/L 

 Spirotetramat: 0 (control), 0.32, 0.56, 1.0, 1.80, 3.20, 5.60, 10.0 mg a.s./L 

 Measured (mean): Spirotetramat: -- (control), 0.30, 0.51, 0.87, 1.38, 2.67, 4.83, 8.98 mg a.s./L 

Control: Untreated test water, no blank formulation control was used 

Test conditions: 

 Water quality: M7 medium (according to OECD 202), hardness: not stated 

 Temperature 20.6 – 21.4 °C 

 pH 8.1 – 8.4 (0 h), 7.8 – 8.4 (48 h) 

 O2 content: > 90 %, (8.4 – 8.6 mg O2/L) 
– 49 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Test parameters: Mortality and sublethal effects were assessed after 48 hours. 

For chemical analysis (HPLC-MS/MS and HPLC-UV) of the actual concentration 

of spirotetramat in the test media samples were taken at test initiation (0 h) and 

termination (48 h). 

Statistics: LC10 and LC50: Probit analysis, NOEC: directly from raw data, correction of control 

mortality: Abbott’s formular 

Findings: 

Analytical data: The mean measured concentrations of spirotetramat at the start and end of the 

test were in the range of 86 – 109 % and 68 – 80 % of nominal, respectively. 

Overall mean measured concentrations ranged from 77 to 95 % of nominal. 

Effects: See table B.9.2.1.2-6 

Table B.9.2.1.2-4: Effects on midge (C. riparius) exposed to BYI 08330 OD 150 (formulation) 

BYI 08330-OD 150 

[mg product/L] Mortality after 48 hours 

(nominal) (mean 

measured) 

Spirotetramat 

[mg a.s./L] 

(nominal) 

Exposed 

chironomids 

(= 100 %) [n] [%] 

Control Control 40 4 10 

2.12 2.00 0.32 40 4 10 

3.71 3.40 0.56 40 13 32.5 

6.62 5.75 1.00 40 33 82.5 

11.9 9.14 1.80 40 39 97.5 

21.2 17.6 3.20 40 40 100 

37.1 32.0 5.60 40 40 100 

66.2 59.3 10.0 40 40 100 

LC10 (48 h)*: 2.39 mg product /L (95% C.I. 1.83 – 3.1 mg/L), corresponding to 0.36 mg a.s./L 

LC50 (48 h)*: 4.41 mg product/L (95% C.I. 3.45 – 7.68 mg/L), corresponding to 0.66 mg a.s./L 

Details of the Probit: Slope b: 6.735 

95 % CI of b: 4.925 – 8.544 

Variance of b: 0.37 

Intercept a: 1.91 

Goodness of fit, Chi2: 6.10 

Degrees of freedom: 5 

p (Chi2): 0.971 

Conclusion: LC50 (48 h): 4.41 mg product/L, 0.66 mg a.s./L 

LC10 (48 h): 2.39 mg product/L, 0.36 mg a.s./L 

NOEC (48 h): < 2.0 mg/product, < 0.30 mg a.s./L 

based on mean measured concentration 

Comments: While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

-Age of the test organisms is 2nd or 3rd instar, 1st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Mortality and sublethal effects should be measured every 24 hours.
– 50 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.1.3 Effects on algal growth and growth rate (OECD IIA 8.4, IIIA 10.2.2.3) 

Active substance 

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330 

(tech.) 

Author(s), year: Dorgerloh, M., 2004 c 

Report/Doc. number: DOM 23092, Edition Number: M-128874-01-2, 29.11.2004 

Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400, JMAFF guideline (12 

Nousan No 8147) 

GLP: Yes 

Deviations: EPA guideline: 

• The temperature is less than the EPA-specified temperatures (24-25oC) and the 

lighting intensity is higher than EPA specified lighting. 

• The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-120 hour tests. 

• Test concentrations dropped as low as 22% of the nominal, and test solutions 

were not centrifuged prior to analysis 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods: 

Test species: Green alga (Pseudokirchneriella subcapitata)

 Number of organisms: 1 x 104 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Type of test, duration: Static test, 72 h 

Applied concentrations: 

 Nominal: 0 (medium control and solvent control), 0.31, 1.0, 3.1, 10, 31, 100 mg/L 

 Measured (mean): -- (medium control and solvent control), 0.141, 0.471, 1.46, 5.38, 18.5, 63.2 mg/L 

 Solvent: 0.5 mL/L Aceton 

Test conditions: 

 Water quality: Nutrient medium (according to OECD guideline) 

 Temperature 22.5 – 23.1 °C 

 pH 7.8 – 8.0 (0 h), 7.5 – 8.3 (96 h) 

 Incubation: Continuous “cool white” illumination with fluorescent light (8000 lux ± 15 %) 

Test parameters: Cell numbers per volume were estimated photometrically (measuring the 

extinction in a single-beam-photometer at 578 nm) at 24, 48 and 72 hours. 

Morphological examinations were made under a microscope on each study day. 

For chemical analysis (HPLC method) of test the substance, samples of test 

solution were taken on day 0 and day 3. Test samples were analysed by direct 

injection into a HPLC-UV instrument without a centrifugation. 

Statistics: LOEC: ANOVA Procedure, t-test; ECx: Probit analysis, comparison of control 

groups: Kolmogorov-Smirnov-test (normal distribution) and Cochran’s test 

procedure on variance homogeneity 

All statistical analyses are based on data of pooled control. 

Findings: 

Analytical data: Mean measured concentrations were in the range of 84 – 94 % and 22 – 48 % of 

nominal concentrations on day 0 and day 3, respectively. 

Effects: 

Morphological effects: None 

Biomass & growth rate: See table B.9.2.1.3-1 
– 51 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.2.1.3-1: Effects of spirotetramat (BYI 08330) on the green alga P. subcapitata

BYI 08330 [mg/L] Inhibition after 72 h 

(mean measured) Biomass (AUC) Yield1) Growth rate 

0 (pooled control) 2) -- -- -- 

0.141 -51.1 -54.7 -11.3 

0.471 -26.0 -22.2 -5.3 

1.46 -15.1 -8.6 -2.2 

5.38 40.9* 46.7* 18.1* 

18.5 93.9* 101.2* 122.7* 

63.2 98.5* 101.5* 130.7* 

NOEC 1.46 mg/L 1.46 mg/L 1.46 mg/L 

LOEC 5.38 mg/L 5.38 mg/L 5.38 mg/L 

EC50

(95% C.I.) 

6.58 mg/L 

(6.29 – 6.91 mg/L) 

5.6 mg/L 

(5.46 – 5.83 mg/L) 

8.15 mg/L 

(7.56 – 8.81 mg/L) 

1) Yield = biomass at the end of exposure minus the biomass at the start of exposure 

2) Results statistical analyses of control groups indicated no significant differences, thus pooled control are used for 

statistical analysis. 

* Significant difference (alpha = 0.05) from the control 

. 

Conclusion: EbC50 (0-72 h): 6.58 mg/L 

ErC50 (0-72 h): 8.15 mg/L, 

NOEC (0-72 h): 1.46 mg/L (biomass and growth rate) 

based on mean measured concentrations 

Reference: Toxicity of BYI 08330 to the freshwater diatom Navicula pelliculosa

Author(s), year: Kern, M.E.; Lam, C.V., 2005 

Report/Doc. number: EBFNX008, Edition Number: M-252794-01-1, 15.06.2005 

Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FIFRA 123-2 

GLP: Yes 

Deviations: OECD: None of relevance 

US EPA: The number of replicates (3) were fewer than are accepted by EPA 

protocol (4 for Navicula sp.). 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 

Material and methods: 

Test species: Freshwater diatom (Navicular pelliculosa)

 Number of organisms: 1 x 104

 cells/mL; 3 replicates for each concentration, medium control and solvent 

control 

Type of test, duration: Static test, 96 h 

Applied concentrations: 

 Nominal: 0 (medium control and solvent control), 0.20, 0.51, 1.3, 3.2, 8.0, 20 mg/L 

 Initial measured: -- (medium control and solvent control), 0.22, 0.47, 1.25, 3.2, 8.23, 20.4 mg/L 

 Solvent: 0.5 mL/L Aceton 

Test conditions: 

 Water quality: AAP Nutrient medium (according to OECD guideline) 

 Temperature 23.5 – 24.1 °C 

 pH 7.7 – 7.9 (0 h), 8.0 – 9.5 (96 h) 

 Incubation: Continuous illumination (4026 – 4693 lux), orbital shaking at 100 rpm 

Test parameters: Cell densities were determined using a electronic particle counter after 24, 48, 72 
– 52 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

and 96 hours. In addition the cell counts using a haemocytometer and a 

microscope were performed in order to confirmed measured cell densities. 

Morphological examinations were made under a microscope on each study day. 

For chemical analysis (HPLC method) of test the substance, samples of test 

solution were taken on day 0 and day 4. The test samples were directly analysed 

without a centrifugation. 

Statistics: NOEC: ANOVA followed by Dunnett’s test; EC50: Regression analysis, 

comparison of control groups: T-test 

All statistical analyses are based on data of pooled control. 

Findings: 

Analytical data: Mean measured concentrations were in the range of 92 – 110 % and 0 (< LOQ) – 

59 % of nominal concentrations on day 0 and day 4, respectively. 

Effects: 

Morphological effects: None 

Biomass & growth rate: See table B.9.2.1.3-2 

Table B.9.2.1.3-2: Effects of spirotetramat (BYI 08330) on the freshwater diatom N. pelliculosa

BYI 08330 [mg/L] 

(initial measured) 

BYI 08330 [mg/L] 

(geomean day 0/day 4) 

Cumulative biomass 

Inhibition after 96 h [%] 

Growth rate 

Inhibition after 96 h [%] 

0 (pooled control) a) 0 (pooled control) a) -- -- 

0.22 0.03 11 3.2 

0.47 0.193 10 2.4 

1.25 0.55 15* 4.4** 

3.2 1.00 16* 3.5 

8.23 4.73 62* 13* 

20.4 15.4 99* 74* 

NOEC 0.19 mg/L 1.0 mg/L 

LOEC 0.55 mg/L 4.73 mg/L 

EC50

(95% C.I.) 

4.05 mg/L 

(3.73 – 4.38 mg/L) 

15.0 mg/L 

(0 – 903 mg/L) 

a) Result of t-test is indicated no significant differences between control and solvent control, thus pooled control are used 

for statistical analysis. 

* Significant difference (alpha = 0.05) from the control 

** Statistically significant from control but not considered biologically significant 

Conclusion: EbC50 (0-96 h): 4.05 mg/L 

ErC50 (0-96 h): 15.0 mg/L, 

NOEC (0-96 h): 0.19 mg/L (biomass); 1.0 mg/L (growth rate) 

based on geometric mean measured concentrations (day 0 and day 4) 

Reference: Toxicity of BYI 08330 to the blue-green alga Anabaena flos-aqua

Author(s), year: Kern, M.E.; Lam, C.V., 2006 

Report/Doc. number: EBFNX007, Edition Number: M-264055-01-1, 12.01.2006 

Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FIFRA 123-2 

GLP: Yes 

Deviations: All guidelines: The study results did not exhibit 50% inhibition at the highest test 

concentration for growth rate. 

US EPA: The temperature is less than the EPA-specified temperatures (24-25oC). 

Validity: Acceptable, even if the inhibiton for growth rate is lower than recommended the 

enpoints are acceptable for EU risk assessment. 
– 53 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test substance: Spirotetramat (BYI 08330), purity: 97.99 %, batch: 08045/0014 

Material and methods: 

Test species: Blue-green alga (Anabaena flos-aqua)

 Number of organisms: 1 x 104 cells/mL; 3 replicates for each concentration, medium control and solvent 

control 

Type of test, duration: Static test, 96 h 

Applied concentrations: 

 Nominal: 0 (medium control and solvent control), 0.51, 1.3, 3.2, 8.0, 20 mg/L 

 Initial measured: -- (medium control and solvent control), 0.45, 1.29, 3.32, 9.05, 21.7 mg/L 

 Solvent: 0.5 mL/L Aceton 

Test conditions: 

 Water quality: AAP Nutrient medium (according to OECD guideline) 

 Temperature 23.3 – 23.8 °C 

 pH 7.7 – 7.9 (0 h), 8.1 – 8.3 (96 h) 

 Incubation: Continuous illumination with cool white fluorescents (2200 lux ± 10 %), orbital 

shaking at 100 rpm 

Test parameters: Cell densities were determined using a electronic particle counter after 24, 48, 72 

and 96 hours. In addition the cell counts using a hemocytometer and a 

microscope were performed in order to confirmed measured cell densities. 

Morphological examinations were made under a microscope on each study day. 

For chemical analysis (HPLC method) of test the substance, samples of test 

solution were taken on day 0 and day 4. The test samples were directly analysed 

without a centrifugation. 

Statistics: NOEC: ANOVA followed by Dunnett’s test; EC50: Regression analysis, 

comparison of control groups: T-test 

All statistical analyses are based on data of pooled control. 

Findings:

Analytical data: Mean measured concentrations were in the range of 87 – 113 % and 31 – 52 % of 

nominal concentrations on day 0 and day 4, respectively. 

Effects: 

Morphological effects: None 

Biomass & growth rate: See table B.9.2.1.3-3 

Table B.9.2.1.3-3: Effects of spirotetramat (BYI 08330) on the blue-green algae Anabaena flos-aquae

BYI 08330 [mg/L] 

(initial measured) 

BYI 08330 [mg/L] 

(geomean day 0/day 4) 

Cumulative biomass 

Inhibition after 96 h [%] 

Growth rate 

Inhibition after 96 h [%] 

0 (pooled control) a) 0 (pooled control) a) -- -- 

0.45 0.3 -43 3 

1.29 0.718 -15 -13 

3.32 2.08 -51 -13 

9.05 5.68 -6 -3 

21.7 15.1 48** 14 

NOEC 5.68 mg/L 15.1 mg/L 

LOEC 15.1 mg/L > 15.1 mg/L 

EC50

(95% C.I.) 

15.2 mg/L 

(14.9 – 15.5 mg/L) > 15.1 mg/L 

a) Result of t-test is indicated no significant differences between control and solvent control, thus pooled control are used 

for statistical analysis. 

** Biological significant based on % inhibition compared to the pooled controls 
– 54 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Conclusion: EbC50 (0-96 h): 15.2 mg/L 

ErC50 (0-96 h): > 15.1 mg/L, 

NOEC (0-96 h): 5.68 mg/L (biomass); 15.1 mg/L (growth rate) 

based on geometric mean measured concentrations (day 0 and day 4) 

Metabolites 

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330-enol 

Author(s), year: Dorgerloh, M. 2005 f 

Report/Doc. number: EBFNM010, Edition Number: M-252531-01-2, 06.06.2005 

Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400 

GLP: Yes 

Deviations: US EPA: 

• The temperature is less than the EPA-specified temperatures (24-25oC) and the 

lighting intensity is higher than EPA specified lighting. 

• The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-120 hour tests. 

Validity: Acceptable 

Test substance: BYI 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005 

Material and methods: 

Test species: Green alga (Pseudokirchneriella subcapitata)

 Number of organisms: 1 x 104 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Type of test, duration: Static test, 72 h 

Applied concentrations: 

 Nominal: 0 (medium control and solvent control), 1.0, 3.1, 10, 31, 100 mg/L 

 Measured (mean): -- (medium control and solvent control), 1.02, 2.9, 10.1, 29.4, 102 mg/L 

 Solvent: 0.5 mL/L Dimethylformamide (DMF) 

Test conditions: 

 Water quality: Nutrient medium (according to OECD guideline) 

 Temperature 22.3 – 23.5 °C 

 pH 7.1 – 7.9 (0 h), 7.6 – 8.5 (96 h) 

 Incubation: Continuous “cool white” illumination with fluorescent light (5560 - 7350 lux), orbital 

shaking at 100 rpm 

Test parameters: Cell numbers per volume were estimated photometrically (measuring the 

extinction in a single-beam-photometer at 578 nm) at 24, 48 and 72 hours. 

Morphological examinations were made under a microscope on each study day. 

For chemical analysis (HPLC method) of test the substance, samples of test 

solution were taken on day 0 and day 3. The test samples were analysed by direct 

injection into an HPLC-MA/MS instrument without a centrifugation. 

Statistics: LOEC: ANOVA Procedure, t-test; EC50: Not applicable because the inhibition at 

highest test concentration was clearly below 50 %, comparison of control groups: 

Student- t-test for homogenous variances 

All statistical analyses are based on data of pooled control. 

Findings:

Analytical data: Mean measured concentrations were in the range of 94 – 104 % and 94 – 102 % 

of nominal concentrations on day 0 and day 3, respectively. 

Effects: 
– 55 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Morphological effects: None 

Biomass & growth rate: See table B.9.2.1.3-4 

Table B.9.2.1.3-4: Effects of BYI 08330-enol (metabolite of spirotetramat) on the green alga P. 

subcapitata

Inhibition after 72 hours 

BYI 08330 [mg/L] 

(nominal) Biomass (AUC) Growth rate 

0 (pooled control) 1) -- -- 

1.0 -9.2 -1.0 

3.1 -15.1 -3.4 

10 0.2 0.5 

31 1.9 1.5 

100 32.9* 13.0* 

NOEC 31 mg/L 31 mg/L 

LOEC 100 mg/L 100 mg/L 

EC50 > 100 mg/L) > 100 mg/L) 

1) Result of t-test is indicated no significant differences between control and solvent control, thus pooled control are used 

for statistical analysis. 

* Significant difference (alpha = 0.05) from the control 

. 

Conclusion: EbC50 (0-72 h): > 100 mg/L 

ErC50 (0-72 h): > 100 mg/L, 

NOEC (0-72 h): 31 mg/L (biomass and growth rate) 

based on nominal concentrations 

Reference: 4-Methoxycyclohexanone - Alga growth inhibition test 

Author(s), year: Weyers, A., 2006 

Report/Doc. number: 2006/0032/03, Edition Number: M-275954-01-1, 28.07.2006 

Guideline(s): EU Directive 92/69/EEC C.3 

GLP: Yes 

Deviations: EPA guideline: 

• The temperature is less than the EPA-specified temperatures (24-25oC) and the 

lighting intensity is higher than EPA specified lighting. 

• The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-120 hour tests. 

Validity: Acceptable 

Test substance: 4-Methoxycyclohexanone (metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV5506-001 

 Solubility: Within the test, the item was added to the limit concentration of 100 mg/L in test 

medium by weighing-in. There was no indication for any unsolved material after 

mixing. Substance specific GC analysis showed recoveries of >80% for all 

samples taken. 

Material and methods:

Test species: Green alga (Desmodesmus subspicatus)

 Number of organisms: 5 x 103

 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Type of test, duration: Static limit test, 72 h 

Applied concentrations: 

 Nominal: 0 (medium control), 100 mg/L 
– 56 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Measured (mean): -- (medium control), 92.7 mg/L 

Test conditions: 

 Water quality: Nutrient medium (according to OECD guideline) 

 Temperature 22.3 – 23.5 °C 

 pH 8.1 (0 h), 8.6 (72 h) 

 Incubation: Continuous illumination with a spectral range of 400 – 700 nm and an intensity of 

4000 - 8000 lux 

Test parameters: Cell densities were measured in a microcell counter or alternatively, were 

determined by means of a microscopic counting chamber at 24, 48 and 72 hours. 

For chemical analysis (GC analysis and MS detection) of test the substance, 

samples of test solution were taken on day 0 and day 3. The test samples were 

directly analysed without a centrifugation. 

Statistics: None, limit test 

Findings:

Analytical data: Mean measured concentration was 92.1 % on day 0 and 93.9 % day 3 of nominal 

concentration 

Effects: 

Morphological effects: None 

Biomass & growth rate: No significant effects were observed at the limit concentration of 100 mg/L 

Conclusion: EbC50 (0-72 h): > 100 mg/L 

ErC50 (0-72 h): > 100 mg/L, 

NOEC (0-72 h): 100 mg/L (biomass and growth rate) 

based on nominal concentrations 

Comments: US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 

Formulation 

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330 OD 

150 

Author(s), year: Dorgerloh, M., 2006 c 

Report/Doc. number: EBFNM006, Edition Number: M-264263-01-2, 16.01.2006 

Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400, JMAFF guideline (12 

Nousan No 8147) 

GLP: Yes 

Deviations: EPA guideline: 

• The temperature is less than the EPA-specified temperatures (24-25oC) and the 

lighting intensity is higher than EPA specified lighting. 

• The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-120 hour tests. 

Validity: Acceptable 

Test substance: BYI 08330 OD 150 (formulation), spirotetramat content: 148.89 g/L (15.1 %), 

batch: 08030/0189(0152) 

Material and methods:

Test species: Green alga (Pseudokirchneriella subcapitata)

 Number of organisms: 1 x 104 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Type of test, duration: Static test, 72 h 

Applied concentrations: 
– 57 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Nominal: Formulation: 0 (control), 9.53, 17.1, 30.9, 55.6, 100 mg product/L 

 Spirotetramat: 0 (control), 1,44, 2,58, 4.67, 8.4, 15.1 mg a.s./L 

 Measured (mean): Spirotetramat: -- (control), 0.61, 1.14, 2.02, 4.05, 8.2 mg a.s./L 

Control: Untreated test medium, no blank formulation control was used 

Test conditions: 

 Water quality: Nutrient medium (according to OECD guideline) 

 Temperature 22.3 – 23.3 °C 

 pH 8.0 – 8.9 (0 h) 

 Incubation: Continuous “cool white” illumination with fluorescent light (8000 lux ± 15 %) 

Test parameters: Cell numbers per volume were estimated by direct cell counting under a 

microscope at 24, 48 and 72 hours. In addition morphological examinations were 

performed. 

For chemical analysis (HPLC method) of test the substance, samples of test 

solution were taken on day 0 and day 3. The test samples were analysed by direct 

injection into an HPLC-UV instrument without a centrifugation. 

Statistics: LOEC: ANOVA procedure; ECx: Probit analysis (for growth rate an EC50 could 

not be determined because the inhibition at highest test concentration was < 50%) 

Findings: 

Analytical data: Mean measured concentrations of the active substance were in the range of 91 – 

96 % and 19 – 31 % of nominal concentrations on day 0 and day 3, respectively. 

The geometric mean concentrations of the active substance over the whole test 

period were 42 – 54 % of nominal. 

Effects: 

Morphological effects: At 100 mg/L (nominal) some cells were enlarged. 

Biomass & growth rate: See table B.9.2.1.3-5 

Table B.9.2.1.3-6: Effects of BYI 08330 OD 150 (formulation) on the green alga P. subcapitata

BYI 08330 OD 150 Inhibition after 72 hours 

[mg product/L] 

(nominal) 

BYI 08330 

[mg a.s./L] 

(mean measured) Biomass (AUC) Growth rate 

9.53 0.61 3.6 -0.2 

17.1 1.14 8.0 2.4 

30.9 2.02 14.1 3.7 

55.6 4.05 29.8* 10.8* 

100 8.2 60.2* 27.1* 

NOEC 2.02 mg a.s./L 2.02 mg a.s./L 

LOEC 4.05 mg a.s./L 4.05 mg a.s./L 

EC50

(95% C.I.) 

6.56 mg a.s./L 

(5.19 – 9.37 mg/L) > 8.2 mg a.s./L 

* Significant difference (alpha = 0.05) from the control 

1) Yield = biomass at the end of exposure minus the biomass at the start of exposure 

. 

Conclusion: EbC50 (0-72 h): 6.56 mg a.s./L corresponding to 43.4 mg product/L 

ErC50 (0-72 h): > 8.2 mg a.s./L corresponding to 54.3 mg product/L 

NOEC (0-72 h): 2.02.mg a.s./L (biomass and growth rate) corresponding to 

13.4 mg product/L 

based on geometric mean measured concentrations of active substance 
– 58 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.1.4 Effects on aquatic plants (OECD IIA 8.6, IIIA 10.8.2) 

Active substance 

Reference: Lemna gibba G3 - Growth inhibition test with BYI 08330 (tech.) under staticrenewal test conditions 

Author(s), year: Dorgerloh, M., 2005 j 

Report/Doc. number: DOM 24019, Edition Number: M-255296-01-2, 29.07.2005 

Guideline(s): OECD 221, EPA OPPTS 850.4400 

GLP: Yes 

Deviations: EPA guideline: 

-The light intensity was higher than the EPA recommended light intensity. 

-All test concentrations have not been centrifuged prior to analysis. 

-Precipitate was observed in the highest test level. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods: 

Test species: Duckweed (Lemna gibba) 

 Number of organisms: 12 fronds per vessel; 3 replicates for each concentration, control and solvent 

control 

Type of test, duration: Static renewal test (renewal of test medium on day 0, 3 and 5), 7 d 

Applied concentrations: 

 Nominal: 0 (medium and solvent control), 0.30, 0.95, 3.05, 9.77, 31.3, 100 mg/L 

 Measured (mean): -- (medium and solvent control), 0.14, 0.47, 1.54, 6.06, 20.5, 71.4 mg/L 

 Solvent 0.5 mL/L Aceton 

Test conditions: 

 Water quality: 20X-AAP growth medium (according to OECD guideline), the pH was adjusted to 

7.5 ± 0.1 

 Temperature 23.5 – 24.8 °C 

 pH 7.2 – 7.6 (new test media), 8.0 – 8.7 (aged test media); control: 7.4 (0 d), 8.7 (7 d) 

 Incubation: Continuous illumination with white fluorescent light (mean intensity: 8.04 kLux) in 

a growth incubator. 

Test parameters: Biomass: Frond numbers and total frond area were determined using LemnaTec 

Scanalyzer machine on day 0, 3, 5 and 7, respectively. 

Dry weights of plants (needed for yield calculations) were measured at the end of 

the study (final biomass). 

For chemical analysis (HPLC-MS/MS, HPLC-UV) of test the substance (BYI 

08330) samples of test solution were taken from freshly prepared test media (0 d, 

3 d, 5 d) and from aged test media (3 d, 5 d, 7 d). Test samples were analysed by 

direct injection into an HPLC instrument without centrifugation. 

Statistics: LOEC: ANOVA (α = 0.05, one sided) and Dunnett’s multiple t-tests 

Findings: 

Analytical data: Overall mean measured concentrations ranged from 47 % to 71 % of nominal. An 

overview of all measured concentrations is given in table B.9.2.1.4-1 
– 59 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.2.1.4-1: Analytical results of spirotetramat 

Effects: 

Morphological effects: None visual effects were observed in concentration levels up to 0.47 mg/L. At 1.54 

mg/l slight chlorosis was observed at day 7. At 6.06 mg/L and higher 

concentration levels strong chlorosis and necrosis were noted. 

Biomass & growth rate: Doubling time Td of the control = 2.2 d (fulfilled the validity criterion), 

see also table B.9.2.1.4-2 

Table B.9.2.1.4-2: Effects of spirotetramat on duckweed Lemna gibba

Growth rate% inhibition in 7 d Spirotetramat [mg/L]

(nominal) 

Spirotetramat [mg/L] 

time weighted mean 

measured 

Yield 

% inhibition in 7 d based on frond 

number 

based on total 

frond area 

0.30 0.14 -3.5 2.4 -3.6 

0.95 0.47 -5.5 5.8 -4.0 

3.05 1.54 -2.3 5.7 -2.7 

9.77 6.06 45.7* 34* 50.1* 

31.3 20.5 87.4* 70.2* 91.8* 
– 60 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Growth rate% inhibition in 7 d Spirotetramat [mg/L]

(nominal) 

Spirotetramat [mg/L] 

time weighted mean 

measured 

Yield 

% inhibition in 7 d based on frond 

number 

based on total 

frond area 

100a) 71.4 97.6* 113.5* 116.1* 

* Significant difference (alpha = 0.05) from the control 

Table B.9.2.1.4-3: Toxicity of spirotetramat on duckweed Lemna gibba based on time weighted mean 

measured concentrations 

Endpoint Time 

scale 

NOEC 

[mg/L] 

LOEC 

[mg/L] 

EC50 (95% C.I.) 

[mg/L]a)

EC50 (95% C.I.) 

[mg/L]b) 

Growth rate (based on frond 

number) 

7 d 1.54 6.06 10.4 (7.96 – 13.8) 9.96 (7.85 – 12.49) 

Growth rate (based on total area 

of plants 

7 d 1.54 6.06 6.21 (5.33 – 7.18) 6.21 (5.55 – 6.91)

Yield (based on frond number) 7 d 1.54 6.06 4.62 (2.04 – 10.3) 4.49 (2.70 – 7.20) 

Yield (based on biomass – dry 

weight) 

7 d 1.54 6.06 6.96 (5.76 – 8.38) 7.04 (6.07 – 8.12)

Visual effects on plants 7 d 0.47 1.54 -- -- 

a) The 100 mg/L concentration level was excluded from EC50 calculations because of exceeded water solubility 

b) The 100 mg/L concentration level was included into the EC50 calculations. 

Conclusion: EyC50 (7 d): 4.62 mg/L 

ErC50 (7 d): 6.21 mg/L 

NOEC (7d): 1.54 mg/L (for biomass/yield and growth rate) 

based on time weighted mean measured concentrations

Comments: Spirotetramat is an insecticide and therefore a study on higher aquatic plants is 

not required according to directive 91/414/EEC. However, in general an exposition 

of Lemna gibba is possible and therefore the study is considered in the aquatic 

risk assessment. 

Metabolites 

Reference: Lemna gibba G3 - Growth inhibition test with BYI 08330-enol under static 

test conditions 

Author(s), year: Dorgerloh, M., 2005 k 

Report/Doc. number: EBFNX065, Edition Number: M-252412-01-2, 07.06.2005 

Guideline(s): OECD 221, OPPTS 850.4400 

GLP: Yes 

Deviations: OECD: None of relevance 

US EPA: The light intensity was higher than the EPA recommended light intensity. 

Validity: Acceptable 

Test substance: BYI 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005 

Material and methods:

Test species: Duckweed (Lemna gibba) 

 Number of organisms: 12 fronds per vessel; 3 replicates for each concentration, control and solvent 

control 

Type of test, duration: Static test, 7 d 

Applied concentrations: 

 Nominal: 0 (medium control), 0.09, 0.30, 0.95, 3.05, 9.77, 31.3, 100 mg/L 

 Measured (mean): -- (medium control), n.a., n.a., 1.02, 3.41, 11.7, 37.6, 119 mg/L 
– 61 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test conditions: 

 Water quality: 20X-AAP growth medium (according to OECD guideline), the pH was adjusted to 

7.5 ± 0.1 

 Temperature 22.3 – 24.0 °C 

 pH 7.2 – 7.8 (day 0), 8.2 – 8.8 (day 7) 

 Incubation: Continuous illumination with white fluorescent light (mean intensity: 8.96 kLux) in 

a growth incubator. 

Test parameters: Biomass: Frond numbers and total frond area were determined using LemnaTec 

Scanalyzer machine on day 0, 3, 5 and 7, respectively. 

Dry weight of plants (needed for yield calculations) was measured at the end of 

the study (final biomass). 

For chemical analysis (HPLC-MS/MS, HPLC-UV) of test the substance samples of 

test solution were taken on day 0 and on day 7 (test termination). Test samples 

were analysed by direct injection into an HPLC instrument without centrifugation. 

Statistics: LOEC: ANOVA (α = 0.05, one sided) and Dunnett’s multiple tests 

Findings: 

Analytical data: Overall mean measured concentrations ranged from 114 % to 120 % on day 0 

and from 97 % to 120 % on day 7 of nominal. 

Effects: 

Morphological effects: None visual effects were observed in concentration levels up to 0.95 mg.L. At 

≥ 3.5 mg/l chlorosis was noted. 

Biomass & growth rate: Doubling time Td of the control = 1.9 d (fulfilled the validity criterion), 

see also table B.9.2.1.4-3 

Table B.9.2.1.4-4: Effects of BYI 08330-enol on duckweed Lemna gibba

BYI 08330-enol [mg/L] Growth rate inhibition [%] 

(nominal) Yield inhibition [%] 

based on frond number based on total frond area 

0.09 0.1 -0,9 -2,9 

0.30 0.1 -2,7 -2,4 

0.95 3.1 0,1 0,1 

3.05 27.8* 8,3 9,5 

9.77 80.8* 38,5* 55,2* 

31.3 84.4* 50,3* 60,8* 

100 88.6* 45,8* 67,0* 

NOEC 0.95 mg/L 3.05 mg/L 3.05 mg/L 

LOEC 3.05 mg/L 9:77 mg/L 9:77 mg/L 

EC50 (95% C.I.) 5.4 (3.3 – 9.1) mg/L 60.8 (21.6 - 1643) mg/L 19.3 (6.9 – 74.2) mg/L 

* Significant difference (α=0.05) from the control 

Conclusion: EyC50 (7 d): 5.4 mg/L 

ErC50 (7 d): 19.3 mg/L 

NOEC (7d): 0.95 mg/L (yield); 3.05 mg/L (growth rate) 

based on nominal concentrations 

Comments: BYI 08330-enol is a metabolite of spirotetramat which is an insecticide and 

therefore a study on higher aquatic plants is not required according to directive 

91/414/EEC. However, in general an exposition of Lemna gibba is possible and 

therefore the study is considered in the aquatic risk assessment. 
– 62 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.1.5 Effects on marine or estuarine organisms (OECD IIA 8.11, IIIA 10.2.2.4, IIIA 10.2.2.5) 

General comment: 

Studies on four marine species (fish, shrimp, oyster, algae) with the active substance are available. 

Usually these studies are no required according to directive 91/414/EEC and the EU Aquatic Ecotoxicology 

Guidance Document. However in the aquatic Guidance document SANCO/3268/2001 rev.4 final it is stated 

“...in some cases, data are available on estuarine/marine species. At present, there is no requirement under 

directive 91/414/EEC to perform these studies, but if data are available, they must be submitted and should 

be considered in the risk assessment.” In general marine organisms are regarded as representative for 

freshwater ecosystem. Thus the RMS has evaluated the studies and the results have been considered in the 

risk assessment. 

B.9.2.1.5.1 Effects on saltwater fish 

Active Substance 

Reference: Acute toxicity of BYI 08330 technical to the sheepshead minnow 

(Cyprinodon variegatus) under flow-trough conditions 

Author(s), year: Banman, C. S.; Lam, C. V., 2005 

Report/Doc. number: EBFNX006, Edition Number: M-255363-01-1, 27.07.2005 

Guideline(s): OECD 203, US EPA OPPTS 850.1075, FIFRA 72-2 

GLP: Yes 

Deviations: OECD: None of relevance 

USEPA: Based on EFED’s guidance document, “Estuarine Fish 96-Hour Acute 

Toxicity Standard Evaluation Procedure’s the recommendation of fish weight is 

between 0.5 and 5 g. 

Validity: Acceptable. 

Test substance: Spirotetramat (BYI 08330), purity: 97.99 %, batch: 08045/0014 

Material and methods: 

Test species: Sheepshead minnow (Cyprinodon variegatus) 

 Number of organisms: 20 fish per concentration and control 

 Weight, length: 0.13 ± 0.03 g (mean ± SD), 1.72 ± 0.14 cm (mean ± SD) 

 Loading: 0.03 g fish/L test material passing through tank each day 

Type of test, duration: Flow-through test (renewals approx. 5.3 x per day), 96 hours 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.63, 1.25, 2.50, 5.0, 10 mg/L 

 Measured (mean): -- (control and solvent control), 0.52, 1.17, 2.52, 4.54, 9.77 mg/L 

 Solvent 0.1 mL/L Aceton 

Test conditions: 

 Water quality: Artificial sea salts mixed with reverse osmosis water or blended soft water to 

produce a salinity of 17 ‰ 

 Temperature: 21.0 – 22.5 °C 

 pH: 8.1 – 8.4 (0 h), 7.7 - 8.1 (96 h) 

 O2 content: 5.3 – 6.9 mg O2/L (67 – 87 %) 

 Light regime: 16 hours light / 8 hours darkness, 30 min light/dark transition period 

Test parameters: Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC method) of BYI 08330 in test solutions samples 

were taken at 0, 24 and 96 hours. Test samples were analysed by direct injection 

into an HPLC instrument without centrifugation. 
– 63 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Statistics: LC50: Moving average method, NOEC: directly from raw data 

Findings: 

Analytical data: Mean measured concentrations were in the range of 92 – 115 % of nominal 

concentrations. 

Behavioural effects None in controls and at 0.52 mg/L concentration level. 

After 96 hours at ≥ 1.17 mg/L effects like loss of equilibrium and lying on the 

bottom of the aquarium were observed. Thus the NOEC is 0.52 mg/L. 

Mortality: See table B.9.2.1.5-1 

Table B.9.2.1.5-1: Cumulative mortality of sheepshead minnow exposed to spirotetramat 

Spirotetramat Cumulative mortality (%) 

(mean measured) 

[mg/L] 4 hours 24 hours 48 hours 72 hours 96 hours 

Blank control 0 0 0 0 0 

Solvent control 0 0 0 0 0 

0.52 0 0 0 0 0 

1.17 0 0 0 0 10 

2.52 0 0 0 35 60 

4.54 0 10 100 100 100 

9.77 0 100 100 100 100 

NOEC = 0.52 mg/L 

LC50 = 1.96 mg/L (95%C.I. 1.64 – 2.37) based on moving average method 

LC50 = 2.10 mg/L (95%C.I. 1.73 – 2.52) based on Probit analysis 

Details of the Probit: Slope b: 5.696 

95 % C.I. of b: 3.495 – 7.897 

Variance of b: 1.12 

Intercept a: -1.838 

Goodness of fit, Chi2: 1.282 

Degrees of freedom: 3 

p (Chi2): 0.733 

Conclusion: LC50 (96 h): 1.96 mg/L 

NOEC: 0.52 mg/L 

based on mean measured concentration 

B.9.2.1.5.2 Effects on saltwater invertebrates 

Active substance 

Reference: BYI 08330 - Acute toxicity to mysids (Americamysis bahia) under flowthrough conditions 

Author(s), year: Cafarelle, M. A., 2005 a 

Report/Doc. number: EBFNX010, Edition Number: M-270200-01-1, 06.01.2005 

Guideline(s): EPA OPPTS 850.1035, FIFRA 72-3 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable. 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods: 

Test species: Saltwater mysid (Americamysis bahia) 
– 64 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 Number of organisms: 2 replicates each with 10 mysids per treatment and control 

 Age: 5- to 6-days old 

Type of test, duration: Flow-through test, 96 h 

Applied concentrations: 

 Nominal: 0 (control and solvent control) 0.63, 1.3, 2.5, 5.0, 10.0 mg/L 

 Measured (mean): -- (control and solvent control), 0.73, 1.2, 2.6, 4.6, 9.0 mg/L 

 Solvent 0.1 mL/L Dimethylformamide (DMF) 

Test conditions: 

 Water quality: Filtered natural seawater diluted with well water, salinity: 21 – 20 ‰ 

 Temperature 24 – 25 °C 

 pH 8.1 (0 h), 7.8 – 8.1 (96 h) 

 O2 content: > 60 % saturation, 4.9 – 7.4 mg O2/L 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Mortality and sublethal effects were assessed after 0, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC method) of spirotetramat in the test solution samples 

were taken at 0 and 96 hours. Test samples were analysed by direct injection into 

an HPLC instrument without centrifugation. 

Statistics: EC50: Binominal probability and probit analysis, NOEC: directly from the raw data 

Findings: 

Analytical data: Overall mean measured concentrations were in the range of 90 – 120 % of 

nominal concentrations. 

Behavioural effects At 2.6 mg/L and higher concentrations sublethal effects like erratic swimming, 

lethargy and laying on the bottom of test vessel were observed. Thus the NOEC 

was determined to be 1.2 mg/L. 

Mortality: See table B.9.2.1.5-2 

Table B.9.2.1.5-2: Cumulative mortality of saltwater mysids exposed to spirotetramat 

Spirotetramat (BYI 08330 Cumulative mortality (%) 

Nominal 

[mg/L] 

Mean measured 

[mg/L] 24 hours 48 hours 72 hours 96 hours 

Control 0 0 0 0 

Solvent control 0 0 0 0 

0.63 0.73 5 5 5 5 

1.3 1.2 0 0 5 5 

2.5 2.6 10 20 20 30 

5.0 4.6 15 20 25 25 

10 9.0 60 100 100 100 

NOLC (96 hours) < 0.73 mg/L 

LC50 (96 h) = 5.5 mg/L (95%C.I. 1.2 – 9.0 mg/L) based on binominal probability 

LC50 (96 h) = 4.27 mg/L (95%C.I. not calculated due to mathematical reasons) based on probit analysis 

Details of the Probit: Slope b: 2.926 

95%C.I. of b: 0.793 and 5.059 

Intercept a: -1.845 

Goodness of fit, Chi2: 13.25 

Degrees of freedom: 3 

p (Chi2): 0.0041 

Conclusion: LC50 (96 h): 5.5 mg/L 

NOLC: < 0.73 mg/L 

based on mean measured concentration 
– 65 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: BYI 08330 - Acute toxicity to eastern oysters (Crassostrea virginica) under 

flow-through conditions 

Author(s), year: Cafarella, M. A., 2005 b 

Report/Doc. number: EBFNX011, Edition Number: M-257677-01-1, 15.06.2005 

Guideline(s): EPA OPPTS 850.1025, FIFRA 72-3 

GLP: Yes 

Deviations: • During the final 24 hours of exposure the oxygen saturation drop below 60 % 

(ranged from 52 – 96 %). 

• The measured concentrations drop below 59% of nominal concentration. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 

Material and methods: 

Test species: Eastern Oyster (Crassostrea virginica) 

 Number of organisms: 20 oysters per treatment and control 

 Valve height: 37 ± 5 mm (mean ± SD) 

Type of test, duration: Flow-through test, 96 h 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.19, 0.38, 0.75, 1.5, 3.0 mg/L 

 Measured (mean): -- (control and solvent control), 0.20, 0.33, 0.55, 0.89, 2.1 mg/L 

 Solvent 0.1 mL/L Dimethylformamide (DMF) 

Test conditions: 

 Water quality: Filtered natural seawater, salinity: 31 – 32 ‰ 

 Temperature 20 – 22 °C 

 pH 7.6 – 8.0 

 O2 content: 3.8 – 7.3 mg O2/L (52 – 96 % saturation) 

 Light regime: 16 hours light / 8 hours darkness 

Test parameters: Observations of mortalities and sublethal effects were made at 0, 24, 48, 72 and 

96 hours and the shell growth was determined at test termination. For chemical 

analysis (HPLC method) of spirotetramat in the test solution samples were taken 

at the beginning and at the end of the test. Test samples were analysed by direct 

injection into an HPLC instrument without centrifugation. 

Statistics: EC50: Linear regression analysis, NOEC: Williams test, comparison of control 

groups: T-test. 

All statistical analyses are based on data of pooled control. 

Findings: 

Analytical data: Overall mean measured concentrations were in the range of 59 – 100 % of 

nominal concentrations. 

Mortality: None 

Shell Deposition: See table B.9.2.1.5-3 

Table B.9.2.1.5-3: Mean shell deposition of eastern oysters (C. virginica) exposed to spirotetramat 

Spirotetramat [mg/L] 

(mean measured) 

Mean shell deposition 

(mm ± SD) Inhibition of shell growth [%] 

Pooled control a) 3.5 ± 1.2 -- 

0.20 3.8 ± 1.2 +7.9 

0.33 3.4 ± 1.1 3 

0.55 2.4 ± 1.0 31* 

0.89 1.6 ± 0.9 54* 

2.1 0.4 ± 0.7 89* 
– 66 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Spirotetramat [mg/L] 

(mean measured) 

Mean shell deposition 

(mm ± SD) Inhibition of shell growth [%] 

NOEC = 0.33 mg/L 

EC50 (96 h)= 0.85 mg/L (95%C.I. 0.59 – 1.3 mg/L) 

a) no statistically significant difference between dilution control and solvent control 

* statistically significant difference from the pooled control (p ≤ 0.05) 

Conclusion: EC50 (48 h): 0.85 mg/L 

NOEC: 0.33 mg/L 

based on mean measured concentration 

Comments: US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. (95 %CI: not stated) 

B.9.2.1.5.3 Effects on saltwater algae 

Reference: Toxicity of BYI 08330 technical to the saltwater diatom Skeletonema 

costatum

Author(s), year: Banman, C. S. & Lam, C. V., 2006 

Report/Doc. number: EBFNX009-1, Edition Number: M-271037-02-1, 16.06.2006 

Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FIFRA 123-2 

GLP: Yes 

Deviations: OECD: None of relevance 

USEPA: 

-The photoperiod for studies involving Skeletonema costatum is 14 hour light/10 

hour dark cycle; however, continuous illumination was used in this study. 

-Test samples were not centrifugated prior to chemical analyses. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.99 %, batch: 08045/0014 

Material and methods: 

Test species: Marine diatom (Skeletonema costatum)

 Number of organisms: 1 x 104

 cells/mL; 3 replicates for each concentration, medium control and solvent 

control 

Type of test, duration: Static test, 96 h 

Applied concentrations: 

 Nominal: 0 (medium control and solvent control), 0.256, 0.640, 1.60, 4.0, 10 mg/L 

 Initial measured: -- (medium control and solvent control), 0.240, 0.622, 1.63, 4.15, 10.6 mg/L 

 Solvent: 0.5 mL/L Aceton 

Test conditions: 

 Water quality: ASTM saltwater media (slightly modified: 40 mg/L silicates instead of 20 mg/L 

silicates, salinity 27 ‰ 

 Temperature 19.4 – 21.0 °C 

 pH 8.0 – 8.4 

 Incubation: Photoperiod: 16:8 (light/dark), intensity (3897 - 4682 lux ), orbital shaking at 

100 rpm 

Test parameters: Cell densities were determined using an electronic particle counter after 24, 48, 72 

and 96 hours. In addition the cell counts using a haemocytometer and a 

microscope were performed in order to confirmed measured cell densities. 

Morphological examinations were made under a microscope on each study day. 

For chemical analysis (HPLC method) of test the substance, samples of test 
– 67 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

solution were taken on day 0 and day 4. . Test samples were analysed by direct 

injection into an HPLC instrument without centrifugation. 

Statistics: NOEC: ANOVA followed by Dunnett’s test; EC50: Linear regression analysis, 

comparison of control groups: T-test 

All statistical analyses are based on data of pooled controls. 

Findings: 

Analytical data: Mean measured concentrations were in the range of 94 – 106 % and 25 – 46 % of 

nominal concentrations on day 0 and day 4, respectively. 

Effects: 

Morphological effects: None 

Biomass & growth rate: See table B.9.2.1.5-4 

Table B.9.2.1.5-4: Effects of spirotetramat (BYI 08330) on the marine diatom S. costatum.

BYI 08330 [mg/L] 

(initial measured) 

BYI 08330 [mg/L] 

(geomean) 

Cumulative biomass 

Inhibition after 96 h [%] 

Growth rate 

Inhibition after 96 h [%] 

0 (pooled control) a) 0 (pooled control) a) -- -- 

0.24 0.124 -22 -10 

0.622 0.394 57* 27* 

1.63 1.03 87* 61* 

4.15 2.7 105* 115* 

10.6 6.95 104* 111* 

NOEC 0.124 mg/L 

LOEC 0.394 mg/L 

EC50

(95% C.I.) 

0.36 mg/L 

(0.27 – 0.46 mg/L) 

0.98 mg/L 

(0.92 – 1.05 mg/L) 

a) no statistically significant difference between dilution control and solvent control 

* Significant difference (alpha = 0.05) from the control 

Conclusion: EbC50 (0-96 h): 0.36 mg/L 

ErC50 (0-96 h): 0.98 mg/L 

NOEC (0-72 h): 0.12 mg/L (biomass and growth rate) 

based on geometric mean measured concentrations 
– 68 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.1.6 Summary of acute toxicity 

Table B.9.2.1.6-1: Summary of acute toxicity data for aquatic organisms; values which are relevant 

for risk assessment are in bold 

Test organism Test 

condition Time Endpoint Testconc. 

NOEC 

[mg/L] 

EC50/LC50 

[mg/L] Reference study acceptable 

Spirotetramat

Oncorhynchus mykiss 

Rainbow trout semi static 96 hr mortality mm 0.825 2.54 Dorgerloh, 2004a yes 

Cyprinus carpio 

Common carp semi static 96 hr mortality mm 1.02 2.59 Dorgerloh, 2004b yes 

Lepomis macrochirus 

Bluegill sunfish semi static 96 hr mortality nom 0.5 2.2 Dorgerloh, 2005a yes 

Cyprinodon variegatus 

Sheephead minnow

flow 

through 96 hr mortality mm 0.52 1.96 Banman& Lam, 2005 yes 

Daphnia magna 

Waterflea static 48 hr immobility mm 20.3 > 42.7 Dorgerloh, 2005c yes 

Chironomus riparius 

Midge

static 

(wateronly) 

48 hr mortality mm <0.53 1.30 Dorgerloh, 

2005g yes 

Americamysis bahia 

Saltwater mysid

flow 

through 96 hr mortality mm < 0.73 4.27 Cafarelle, 2005a yes 

Crassostrea virginica 

Eastern oyster

flow 

through 96 hr shell deposition mm 0.33 0.85 Cafarelle, 2005b yes 

P. subcapitata 

Green alga static 72 hr biomass 

growth rate mm 1.46 6.58 8.15 

Dorgerloh, 

2004c yes 

Navicular pellicullosa 

Diatom (freshwater) static 96 hr biomass 

growth rate mm 

0.19 

1.00 

4.05 

15.0 

Kern&Lam, 

2005 yes 

Anabaena flos-aqua 

Blue-green alga static 96 hr biomass 

growth rate mm 

5.68 

15.1 

15.2 

> 15.1 

Kern&Lam, 

2006 yes 

Skeletonema costatum 

Diatom (marine) static 96 hr biomass 

growth rate mm 0.124 0.36 0.96 

Banman& 

Lam, 2006 yes 

Lemna gibba 

Duckweed semi static 7 d yield 

growth rate mm 1.54 4.49 6.21 

Dorgerloh, 

2005j yes 

BYI 08330-enol

Oncorhynchus mykiss 

Rainbow trout staticb) 96 hr mortality nom 100 > 100 Nieden, 

2005a yes 

Daphnia magna 

Waterflea staticb) 48 hr immobility nom 100 > 100 Dorgerloh, 2005d yes 

Chironomus riparius 

Midge

static 

(wateronly) 

48 hr mortality nom 17.1a) 74.9 Dorgerloh, 

2005h yes 

P. subcapitata 

Green alga static 72 hr biomass 

growth rate nom 31 > 100 Dorgerloh, 

2005f yes 

Lemna gibba 

Duckweed static 7 d yield 

growth rate mm 

0.95 

3.05 

5.4 

19.3 

Dorgerloh, 

2005k yes 

BYI 08330-ketohydroxy

Chironomus riparius 

Midge

static 

(wateronly) 

48 hr mortality nom 23.9 a) > 100 Dorgerloh, 

2006a yes 

4-Methoxycyclohexanone

Danio rerio 

Zebra fish staticb) 96 hr mortality nom 100 > 100 Neuhahn, 

2006a yes 

Daphnia magna 

Waterflea staticb) 48 hr immobility nom 100 > 100 Neuhahn, 2006b yes 

Chironomus riparius 

Midge

static b)

(wateronly) 

48 hr mortality nom 100 > 100 Dorgerloh, 

2007a yes 

D. subspicatus 

Green alga staticb) 72 hr biomass 

growth rate nom 100 > 100 Weyers, 

2006 yes 
– 69 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test organism Test 

condition Time Endpoint Testconc. 

NOEC 

[mg/L] 

EC50/LC50 

[mg/L] Reference study acceptable 

4-Methoxycyclohexylamino caboxylic acid

Chironomus riparius 

Midge

static b)

(wateronly) 

48 hr mortality nom 100 > 100 Dorgerloh, 

2007b yes 

Formulation - BYI 08830 OD 150

Oncorhynchus mykiss 

Rainbow trout static 96 hr mortality nom 2.5 

(0.38 a.s.) 

9.48 

(1.41 a.s.) 

Nieden, 

2005b yes 

Chironomus riparius 

Midge

static 

(wateronly) 

48 hr mortality mm 2.39a) 

(0.36 a.s.) 

4.41 

(0.66 a.s.) 

Dorgerloh, 

2006b yes 

P. subcapitata 

Green alga static 72 hr 

biomass 

growth rate 

nom 

13.4 

(2.02 a.s.) 

43.4 

(6.56 a.s.) 

> 54.3 

(> 8.2 a.s.) 

Dorgerloh, 

2006c yes 

Test conc.: test concentration based on mean measured (mm) or nominal (nom) concentration 

a) LC10, NOEC was not determined 

b) Limit test 
– 70 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.2 Chronic toxicity of active substance, metabolites and formulations to aquatic 

organisms 

B.9.2.2.1 Chronic toxicity to fish to fish (OECD IIA 8.2.2., IIA 8.2.3, IIIA 10.2.5.1) 

No study submitted. Not required, because a fish early life stage toxicity test has been carried out. 

B.9.2.2.2 Fish early life stage toxicity test (OECD IIA 8.2.4, IIIA 10.2.5.2) 

Active substance 

Reference: Early-life stage toxicity of BYI 08330 tech. to fish (Pimephales promelas)

Author(s), year: Dorgerloh, M., 2005 b 

Report/Doc. number: EBFN0305, Edition Number: M-260676-01-2, 16.11.2005 

Guideline(s): EPA 72-4a, EPA OPPTS 850.1400, OECD 210 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 

Material and methods: 

Test species: Fathead minnow (Pimephales promelas) 

 Number of organisms: 4 replicates x 25 eggs per test concentration and control, 

after completion of hatch larvae were thinned to 15 individuals per egg cup 

 Age: Freshly fertilized eggs < 24 hours old 

Type of test, duration: Flow-through test, 33 days (28 days post hatch) 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.0625, 0.125, 0.250, 0.500, 1.00 mg/L 

 Measured (mean): -- (control and solvent control), 0.0607, 0.121, 2.243, 0.486, 0.971 mg/L 

 Solvent Dimethylformamide (DMF) 0.1 mL/L 

Test conditions: 

 Water quality: Reconstituted water (according to ISO), hardness: 45 – 52 mg/L as CaCO3

 Temperature: 24.5 (23 – 26) °C 

 pH: 7.0 – 7.3 during the total test period 

 O2 content: 98 – 100 % saturation 

 Light regime: 16 hours light / 8 hours darkness, 30 min transient period 

 Feeding Larvae were fed 5 x daily (except on weekends/holidays: 2 x daily) with live newly 

hatched shrimp nauplii ad libidum. Feeding was started shortly after hatching. 

Uneaten food an faeces were siphoned in order to minimise microbiological 

growth and biodegradation of test substance. 

Test parameters: Embryo mortality, hatched larvae, fry mortality and abnormal appearance or 

behaviour were assessed weekdays. At test termination the length and the weight 

were determined. 

Determined endpoints were: Time to hatch, hatching success, overall fry survival 

(fry survival before and after thinning), standard length and dry weight. 

For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330 in test 

solutions samples were taken at -1, 0, 7, 14, 21, 28 and 33 days 

Statistics: If control and solvent control can be pooled: T-test 

Testing for normal distribution: R/s procedure 

Time to hatch, hatching success, fry survival: Williams test with previous acrisine 
– 71 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

transformation 

Growth data: Williams test without previous data transformation 

Findings: 

Analytical data: Overall mean measured concentrations in test media were 93 – 119 % of nominal. 

Biological observation: Time to hatch: In control and all treatment levels hatching began on day 4 and

continued until day 5. 

Over the total test period no morphological and behavioural effects were 

observed. 

Effects: See table B.9.2.2.2-1 and table B.9.2.2.2-2 

Table B.9.2.2.2-1: Hatching success and fry survival 

Spirotetramat [mg/L] 

(mean measured) 

Egg hatch [%] 

phd 0 

Mean (SD) 

Fry survival [%] 

phd 0 

Mean (SD) 

Fry survival [%] 

phd 28 

Mean (SD) 

Overall fry survival 

[%]a)

Mean (SD) 

pooled control b) 85 (8.8) 98 (2.6) 88 (11.1) 87 (11.1) 

0.0566 85 (11.5) 94 (9.0) 87 (16.3) 82 (18.4) 

0.115 84 (8.0) 95 (5.9) 85 (6.4) 81 (6.0) 

0.247 88 (5.7) 100 (0) 85 (17.5) 85 (17.5) 

0.534 91 (5.0) 92 (4.7) 78 (19.9) 73 (21.7) 

1.16 84 (8.0) 83 (7.1) 78 (14.8) 65 (11.5)* 

NOEC 1.16 mg/l 0.534 mg/L 

LOEC > 1.16 mg/L 1.16 mg/l 

a) calculated as follows: (fry survival before thinning/number of fry hatched)*(fry survival/number of fry after thinning)*100 

phd – post hatch day 

b) no statistically significant difference between dilution control and solvent control 

* significant difference when compared to pooled control (alpha = 0.05) 

Table B.9.2.2.2-2: Length and Weight 

Spirotetramat [mg/L] 

(mean measured) 

Length [mm] 

Test termination (phd 28) 

Mean (SD) 

Dry weight [mg] 

Test termination (phd 28) 

Mean (SD) 

pooled control a) 18.0 (3.5) 20.4 (7.4) 

0.0566 18.4(3.4) 21.8 (11.4) 

0.115 18.3 (3.4) 21.4 (11.3) 

0.247 18.3 (3.9) 21.5 (11.4) 

0.534 19.1 (2.2) 22.1 (7.5) 

1.16 18.6 (3.4) 21.9 (10.4) 

NOEC 1.16 mg/l 

LOEC > 1.16 mg/L 

a) no statistically significant difference between dilution control and solvent control 

* significant difference when compared to pooled control (alpha = 0.05) 

Conclusion: Fry survival: NOEC: 0.534 mg/L, LOEC: 1.16 mg/L 

Hatchability and growth: NOEC : 1.16 mg/L, LOEC > 1.16 mg/L 

based on mean measured concentrations 

B.9.2.2.3 Fish life cycle test (OECD IIA 8.2.5, IIIA 10.2.5.3) 

No study submitted. The logPOW of spirotetramat is 2.51 and the potential of bioaccumulation is expected to 

be low. Furthermore spirotetramat is not persistent in water or sediment of aquatic systems and therefore a 

fish life-cycle test is not required. 
– 72 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.2.4 Chronic toxicity to aquatic invertebrates (OECD IIA 8.3.2, IIIA 10.2.6) 

Active substance 

Reference: Influence of BYI 08330 (tech.) on development and reproductive output of 

the waterflea Daphnia magna in a static renewal laboratory test system 

Author(s), year: Dorgerloh, M., 2005 e 

Report/Doc. number: EBFN0245, Edition Number: M-251843-01-2, 13.05.2005 

Guideline(s): OECD 211, EEC Directive C.20, US EPA 72-4, OPPTS 850.1300 

GLP: Yes 

Deviations: The concentraton of the solvent (acetone) is slightly higher than recommended in 

OPPTS 850.1300 (0.2 ml/L instated of 0.1 ml/L). However an additional solvent 

control was added to make sure that the solvent was not initiating any toxic 

response, thus no negative impact on the results can be concluded. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 

Material and methods: 

Test species: Waterflea (Daphnia magna) 

 Number of organisms: 12 replicates each with one daphnid per treatment and control 

 Age: First instar < 24 hours old 

Type of test, duration: Semi static test, 21 d, renewals of test solutions: 0, 2, 5, 7, 9, 12, 14, 16, 19 days 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.05, 0.13, 0.32, 0.8, 2.0, 5.0, 12.5 mg/L 

 Measured (mean): -- (control and solvent control), 0.055, 0.129, 0.322, 0.796, 1.84, 4.41, 10.9 mg/L 

 Solvent 0.2 mL/L Aceton 

Test conditions: 

 Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring 

test), hardness: 196 mg/L as CaCO3

 Temperature 20 – 20.9 °C 

 pH 7.0 – 7.3 (new test solution) 7.4 – 8.1(aged test solution) 

 O2 content: 6.3 – 10.8 mg O2/L (> 60 % saturation) 

 Light regime: 16 hours light / 8 hours darkness 

 Feeding Daily with Desmodesmus subspicatus suspension (1 x 108

 cells/L), one exception 

was noted: On day 2 the three fold amount was fed for the first weekend

Test parameters: Parent mobility, reproduction (mean time to first brood, age at first brood, offspring 

per surviving parental) and sublethal effects were observed daily (with exception 

of the first weekend after test initiation), at study termination body length and 

parental body mass (dry weight) were reported. 

For chemical analysis (HPLC/UV) of spirotetramat in test media duplicate samples 

were taken from fresh (on days 0, 9, 19) and old solutions (on days 2, 12, 21) from 

each test concentration. 

Statistics: In general data for parent mobility were acrisine transformed before further 

evaluation. Variance homogeneity and normal distribution were analysed by 

Bartletts test and Kolmogorroff-Smirnov test. 

If control groups can be pooled a t- test was performed. 

All NOEC were derived by comparing each treatment group with pooled controls: 

Parent mortality: Fishers Exact Binominal Test with Bonferroni Correction 

Reproduction: T-test 

Body length: Dunnett’s Test 

Parental body mass (dry weight): U-test after Bonferroni-Holm 
– 73 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Statistical analyses concidering nominal concentrations are based on data of 

pooled controls. 

Statistical analyses concidering mean measured concentrations are using only 

negative control. 

Findings: 

Analytical data: The mean measured concentrations ranged from 87.2 – 110.7 % 

Effects: See table B.9.2.2.4-1 

Table B.9.2.2.4-1: Summary of effects of long-term exposure of spirotetramat on Daphnia magna

Spirotetramat 

[mg/L] 

(nominal) 

Spirotetramat 

[mg/L] 

(mean 

measured) 

Parent 

mortality 

at day 21 [%] 

Mean time to 

first brood 

[days] 

Offspring 

per 

surviving 

parental 

Mean dry 

weight of 

parent after 

21 d 

[mg] 

Mean length 

of parent 

after 21d 

[mm] 

Blank control Blank control 0 8.7 ± 1.85 117.3 ± 27.6 1.07 ± 0.14 4.50 ± 0.13 

Solvent control Solvent control 0 8.7 ± 1.19 115.2 ± 16.4 0.98 ± 0.15 4.42 ± 0.17 

Pooled control Pooled control 0 8.7 ± 1.52 116.3 ± 22.1 1.02 ± 0.15 4.46 ± 0.15 

0.05 0.055 0 9.9 ± 0.92 127.3 ± 16.6 1.09 ± 0.17 4.55 ± 0.07 

0.13 0.012 0 9.1 ± 1.10 127.4 ± 17.6 0.89 ± 0.27 4.56 ± 0.15 

0.32 0.322 0 8.7 ± 1.78 113.0 ± 25.5 0.90 ± 0.34 4.55 ± 0.06 

0.8 0.796 0 9.1 ± 1.11 118.7 ± 14.0 1.00 ± 0.12 4.58 ± 0.09 

2.0 1.84 0 9.4 ± 1.09 122.4 ± 16.8 1.05 ± 0.12 4.61 ± 0.11 

5.0 4.41 10 7.9 ± 3.40 115. ± 25.0 1.14 ± 0.32 4.61 ± 0.11 

12.5 10.9 20 7.2 ± 2.00 93.5 ± 30.5* 0.81 ± 0.12* 4.21 ± 0.13* 

NOEC (based on nominal) 2.0 mg/L 12.5 mg/L 5.0 mg/L 5.0 mg/L 5.0 mg/L 

LOEC (based on nominal) 5.0 mg/L > 12.5 mg/L 12.5 mg/L 12.5 mg/L 12.5 mg/L 

NOEC (based on mean measured) a) 10.9 mg/L 10.9 mg/L 4.41 mg/L 4.41 mg/L 4.41 mg/L 

LOEC (based on mean measured) a) > 10.9 mg/L > 10.9 mg/L 10.9 mg/L 10.9 mg/L 10.9 mg/L 

* Statistically significantly different from pooled control (alpha = 0.05) 

a) Statisics were performed using only negative control 

Conclusion: Mortality adult: NOEC 2.0 mg/L, LOEC 5.0 mg/L 

Reproduction (offspring per surviving parental): NOEC: 5.0 mg/L, LOEC: 12.5 mg/L

Growth (weight and length): NOEC: 5.0 mg/L, LOEC: 12.5 mg/L 

All endpoints are based on nominal concentrations. 

B.9.2.2.5 Chronic toxicity to sediment dwelling organisms (OECD IIA 8.5.2) 

Reference: Chironomus riparius 28-day chronic toxicity test with BYI 08330 (tech.) in a 

water-sediment system using spiked water 

Author(s), year: Dorgerloh, M., 2005 i 

Report/Doc. number: M-248099-02-2, 16.03.2005, Amended: 29.06.2006 

Guideline(s): OECD 219 

GLP: Yes 

Deviations: The concentration of the used solvent control was higher than recommended (0.2 

mL/L instead of 0.1 mL/L), however no significant differences between solvent 

control and the water only control were noted. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods: 
– 74 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test species: Midge (Chironomus riparius) 

 Number of organisms: 4 replicates each with 20 larvae per treatment and control 

 Age: First instar (L1) larvae 

Type of test, duration: Static test (spiked water exposure), 28 d 

Applied concentrations: 

 Nominal: 0 (control and solvent control), 0.05, 0.10, 0.20, 0.40, 0.80, 1.60, 3.20 mg/L 

 Initial measured: -- (control and solvent control), 0.058, n.a., n.a., 0.401, n.a., n.a., 2.95 mg/L 

 Solvent: 0.2 mL/L Aceton 

Test system: 

 Water quality: M7 medium (according to OECD), the height of water column was 6.0 cm 

 Sediment: 74 % quartz sand, 4 – 5 % sphagnum peat, 20 % Kaolin, 1 % CaCO3 to adjust the 

pH to 7 ± 0.5; the wet sediment layer was 1.5 cm 

 Size of test vessels: 0.6 L glass beakers (9.5 cm diameter) 

 No. of replicates: 4 for biological evaluation and 1 for chemical analysis per each concentration and

control 

Test condition: 

 Temperature: 19.8 – 20.1 °C 

 pH: 8.3 – 8.7 

 Hardness: 320 – 338 mg CaCO3/L 

 O2 content: 6.9 – 8.5 mg O2/L (> 60 % saturation) 

 Light regime: 16 hours light / 8 hours darkness, intensity: 500 – 1000 lux 

 Feeding: 3 x weekly with a commercial ornamental fish food extract (0.5 – 1 mg/larvae/day)

Test parameters: The sex, the time point of emergence and the number of emerged midges were 

recorded daily. Behavioural differences compared to control were observed 3 

times per week. 

For chemical analysis (HPLC/UV) of spirotetramat in the overlaying water column 

and the pore water of sediment samples were taken from fresh after 1 hours, 7 

and 28 days from 0.05, 0.4 and 3.2 mg/L treatment. 

Statistics: Sex ratio: Chi2-test 

EC50: Probit analysis 

LOEC: ANOVA and multiple t-tests 

Comparison of control groups: Student-t test for homogeneous variances 

All statistical analyses are based on data of pooled controls. 

Findings:

Analytical data: See table B.9.2.2.5-1 

Table B.9.2.2.5-1: Measured concentration of spirotetramat in the overlaying water and the pore water 

1 hour 7 days 28 days Spirotetramat 

[mg a.s./L] Analysed 

conc. [mg/L]a) % of nominal Analysed 

conc. [mg/L]a) % of nominal Analysed conc. [mg/L]a) % of nominal

Overlaying water 

Control < LOQ -- n.a. -- n.a. -- 

Solvent control < LOQ -- n.a. -- n.a. -- 

0.05 0.0579 115.8 < LOQ 0 < LOQ 0 

0.40 0.401 100.3 < LOQ 0 < LOQ 0 

3.20 2.95 92.2 0.0529 1.7 < LOQ 0 

Pore water b)

Control < LOQ -- n.a. -- n.a. -- 

Solvent control < LOQ -- n.a. -- n.a. -- 

0.05 < LOQ 0 < LOQ 0 < LOQ 0 

0.40 < LOQ 0 < LOQ 0 < LOQ 0 

3.20 0.155 0.3 0.0639 0.1 < LOQ 0 

LOQ <0.00636 
– 75 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

a) means of two samples 

b) calculated to the real volume of pore water and the applied amount of a.s. 

Effects: Sex ratio: No relationship between treatment and sex ratio was found, therefore 

number of males and females midges was pooled for further endpoint 

calculations. 

Start of emergence: In controls the emergence started on day 14; at concentration 

levels up to 0.2 mg/L first midges emerged on day 15; at 0.4 and 0.8 mg/l 

emergence started on day 17 and 18, respectively 

For further details see table B.9.2.2.5-2. 

Table B.9.2.2.5-2: Effects of spirotetramat on midge (C. riparius) in a water-spiked test 

Spiroteramat Emergence of inserted larvae 

[mg a.s./L] 

initial nominal 

concentration 

(overlying water) 

Number of 

emerged midges 

(out of 80) total [%] male [%] female 

[%] 

Emergence 

rate (ER) 

Development 

rate (1/d) 

pooled sex 

controls (pooled) a) 144 90.0 43.1 46.9 0.90 0.061 

0.05 65 72.3 42.50 38.75 0.81 0.063 

0.10 72 90.0 40.00 50.00 0.90 0.062 

0.20 59 74.8 42.50 31.25 0.74* 0.062 

0.40 54 67.5 36.25 31.25 0.68* 0.062 

0.80 6 7.5 3.75 3.75 0.08* 0.061 

1.60 0 0 - - - - 

3.20 0 0 - - - - 

NOEC 0.1 mg/L 0.8 mg/L 

LOEC 0.2 mg/L 1.6 mg/L 

EC50

(95% C.I.) 

0.46 mg/L 

(n.d)** > 0.8 mg/L 

ER = midge emerged per vessel/number of introduced larvae 

n.d. = not determined due to mathematical reasons 

a) Result of t-test indicated no significant differences between control and solvent control, thus pooled control are used for 

statistical analysis. 

* significant different from pooled control (alpha = 0.05) 

** slope b (probit analysis): 4.429 

Conclusions: Emergence of pooled sexes: 

NOEC: 0.1 mg/L, LOEC: 0.2 mg/L, EC50: 0.46 mg/L 

Development rate of pooled sexes, males and females: 

NOEC: 0.8 mg/L, LOEC: 1.6 mg/L, EC50: > 0.8 mg/L 

Endpoints based on initial nominal concentrations. 

Comments: While no major deviations are noted from guidline OECD 219 significant deviation 

from USEPA recommendations are notable: 

The EPA requires that the sediment be spiked with the test compound and 

allowed to equilibrate with overlying water prior to test initiation; the overlying 

water was spiked in this test and equilibrium was not achieved. 

EPA also requires that mean-measured, rather than initial measured values, be 

used to perform statistical analyses and report endpoints. 

EPA also recommends having endpoints calculated based on measured 

concentrations in overlying water, bulk sediment, and interstitial water. 

EPA recommends a pH in the range of 6 to 7, the pH values measured in the test 

were outside this range. 
– 76 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.2.2.6 Microcosmos or mesocosmos study (OECD IIIA 10.2.3) 

No study submitted. Not required, because the risk assessment for intended uses (see B.9.2.4) indicated a 

low risk for aquatic organisms and no further refinement is necessary. 

B.9.2.2.7 Summary of chronic toxicity 

Table B.9.2.2.7-1: Summary of chronic toxicity data for aquatic organisms; values which are relevant 

for risk assessment are in bold 

Test organism Test 

condition Time Endpoint Test

conc. 

NOEC 

[mg/L] 

LOEC

[mg//L] 

Reference/ 

study 

acceptable 

Spirotetramat (BYI 08330) 

Pimephales promelas 

Fathead minnow flowthrough 33 d 

hatchability 

fry survival 

growth 

mm 

1.16 

0.534 

1.16 

> 1.16 

1.16 

> 1.16 

Dorgerloh 2005b/ 

yes 

Daphnia magna 

Waterflea 

semi 

static 21 d 

mortality adults 

reproduction 

growth 

nom 

2.0 

5.0 

5.0

5.0 

12.5 

12.5 

Dorgerloh 2005e/ 

yes 

Chironomus riparius 

Midge (larvae)

Static 

(spiked 

water) 

28 d emergence 

development nom 

0.1 

0.8 

0.2 

1.6 

Dorgerloh 2005i/ 

yes 

Test conc.: test concentration based on mean measured (mm) or nominal (nom) concentration 

B.9.2.3 Bioaccumulation (OECD IIA 8.2.6, IIA 8.2.7)

B.9.2.3.1 Bioconcentration potential of active substance in fish (OECD IIA 8.2.6.1) 

No study submitted. Not required, because spirotetramat has a logPOW 2.51 and a bioaccumulation study is 

not triggered. 

B.9.2.3.2 Bioconcentration potential of metabolites in fish (OECD IIA 8.2.6.2) 

Environmental relevant metabolites are BYI 08330-enol, BYI 08330-ketohydroxy (both metabolites are major 

metabolites in water and sediment of water/sediment study), 4-methoxycyclohexanone and 4-

methoxycyclohexylamino carboxyl acid (both are major metabolites in photolysis study).

The logPOW of metabolites BYI 08330-enol and BYI-ketohydroxy is 0.3 (at pH 7) and 1.3 (at pH 7), 

respectively. For the two major photolysis metabolites information about the logPOW was not provided. 

However for these metabolites the logPOW was estimated by RMS using the program ALOGPS 2.1. The 

logPOW for 4-methoxycyclohexanone and 4-methoxycyclohexylamino carboxyl acid was calculated to be in 

the range from -0.04 to 0.74 and -2.49 to 1.0, respectively. 

B.9.2.4 Risk assessment for aquatic organisms (OECD IIIA 10.2, IIIA 10.2.1) 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 
– 77 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 

B.9.3 Effects on other terrestrial vertebrates (OECD IIIA 10.3) 

Toxicity to mammals 

Table B.9.3-1: Ecotoxicologically relevant mammalian toxicity endpoints for spirotetramat 

 Species mg/kg bw/day Reference 

Acute oral LD50 Rat, female > 2000 Eigenberg (2004) 

Acute oral LD50 formulation Rat, female > 2000* Schüngel (2005) 

Reproductive NOAEL Rat 70.7 / 82.5 (m / f) Young (2006) 

* equivalent to 306 g a.s. 

B.9.3.1 Risk assessment for mammals 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 
– 78 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.4 Effects on bees (OECD IIA 8.7; OECD IIIA 10.4.) 

B.9.4.1 Acute Toxicity (OECD IIA 8.7.1; OECD IIA 8.7.2; OECD III 10.4.2 ) 

Active Substance 

Reference: Effects of BYI 08330 (Acute Contact and Oral) on Honey Bees (Apis mellifera 

L.) in the Laboratory 

Author(s), year: Schmitzer, S.; 2004 

Report/Doc. number: 20091035; M-081227-01-2 

Guideline(s): OECD 213 & 214 (1998) 

GLP: Yes 

Deviations: ventral application in the contact test instead of dorsal as recommended in 

guideline 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) tech., Batch no.: 08045/0014 (mixed batch), 

Tox no.: 06695-00, purity: 97.2 % w/w 

Material and Methods: 

Test species: Apis mellifera L. (Hymenoptera: Apidae), 4 - 6 week old adult worker honey bees 

Type of test: Acute contact and oral limit toxicity test 

Number of organisms: 5 replicates with 10 bees for controls, toxic reference and the test item treatment 

groups 

Dosage: Control: oral test: sugar solution with tap water and sugar solution with solvent 

acetone, contact test: tap water with 1 % Adhaesit and solvent acetone; test item: 

oral test: 107.3 μg a.s./bee, based on the quantity of food actually consumed; 

contact test: 100 μg a.s./bee (nominal); toxic reference: 0.04 - 0.3 μg 

dimethoate/bee (Dimethoate 400 g/L) 

Exposure route: Application in the contact test: a 5 μL droplet of a.s. in solvent and Adhaesit was 

placed on the ventral bee thorax. Application in the oral test: Syrup was mixed 

with the test substance, tap water and acetone and offered to the bees in syringes 

which were weighted before and after introduction in the cages. 

Feeding: Commercial ready-to-use syrup (30 % saccarose, 31 % glucose, 39 % fructose) 

Test conditions: Test units were stainless steel cages of 10 cm x 8.5 cm x 5.5 cm, the front side 

was a removable glass sheet, the bottom was perforated with 98 ventilation holes 

(Ø 1 mm), the inner walls were lined with filter paper. 

Bees were anaesthetised with CO2 in the contact test. 

Exposure time: 48 h. Temperature: 25°C, relative humidity: 58 - 72 %, darkness 

(except during observation), ventilation to avoid possible accumulation of pesticide 

vapour 

Test parameter: Mortality and behavioural abnormalities were assessed 4, 24 and 48 h after 

treatment application. 

Statistics: The contact and oral LD50 of the toxic standard was estimated with Moving 

Average Computations (after Thompson 1947). The LD50 calculation was carried 

out taking into account the mortality data corrected by control mortality using 

Abbott's formula (1925). The software used to perform the statistical analysis was 

ToxRat Professional, Version 2.07. 

Findings: 
– 79 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 The results of the mortality and behavioural assessment are presented in Table 

B.9.4.1-1 and -2. The oral and contact LD50 of the toxic reference were 0.15 μg 

a.s./bee and 0.17 μg a.s./bee, respectively. 

Table B.9.4.1-1: Mortality and behavioural abnormalities of the bees in the contact toxicity test 

(results are average from 5 replicates per dosage/control) 

after 4 hours after 24 hours after 48 hours 

mortality behav. 

abnorm. 

mortality behav. 

abnorm. 

mortality behav. 

abnorm. 

Dosage 

[μg a.s./bee] 

mean [%] mean [%] mean [%] mean [%] mean [%] mean [%] 

Test item 

100 

0.0 0.0 0.0 0.0 2.0 0.0 

Water 0.0 0.0 0.0 0.0 2.0 0.0 

Solvent 0.0 0.0 0.0 0.0 2.0 0.0 

Toxic standard 

0.30 

20.0 34.0 94.0 4.0 96.0 0.0 

0.20 28.0 12.0 70.0 2.0 82.0 0.0 

0.15 18.0 6.0 46.0 8.0 62.0 0.0 

0.10 0.0 0.0 0.0 0.0 2.0 0.0 

Table B.9.4.1-2: Mortality and behavioural abnormalities of the bees in the oral toxicity test (results 

are average from 5 replicates per dosage/control) 

after 4 hours after 24 hours after 48 hours 

mortality behav. 

abnorm. 

mortality behav. 

abnorm. 

mortality behav. 

abnorm. 

Dosage 

[μg a.s./bee] 

mean [%] mean [%] mean [%] mean [%] mean [%] mean [%] 

Test item 

107.3 

0.0 0.0 2.0 0.0 4.0 0.0 

Water 0.0 0.0 0.0 2.0 2.0 0.0 

Solvent 0.0 0.0 0.0 0.0 0.0 0.0 

Toxic standard 

0.24 

28.0 24.0 94.0 0.0 94.0 0.0 

0.12 0.0 0.0 22.0 0.0 26.0 0.0 

0.07 0.0 0.0 14.0 0.0 22.0 0.0 

0.04 0.0 0.0 2.0 0.0 2.0 0.0 

Conclusion: 48 h oral LD50 > 107.3 μg a.s./bee 

48 h contact LD50 > 100 μg a.s./bee 

Comments As deviations to EPA OPPTS guidelines the test substance (oral) was 

offered in syrup instead of a 50 % sugar solution and Adhäsit®is not an 

EPA-recommended solvent. 
– 80 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Formulation: Movento 150 OD 

Reference: Acute toxicity of BYI 08330 150 OD to the honeybee Apis mellifera L. under 

laboratory conditions. 

Author(s), year: Barth, M.; 2005a+b 

Report/Doc. number: 05 10 48 032; M-259122-01-2 

Guideline(s): OECD 213 & 214 (1998) 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) OD 150, Batch no.: 08030/0189(0152), TOX no.: 

07034-00; Analytical content of a.s.: 148.89 g/L 

Material and Methods: 

Test species: Apis mellifera carnica P. (Hymenoptera: Apidae), 4 - 6 week old adult worker 

honey bees 

Type of test: Acute contact and oral toxicity test 

Number of organisms: 3 replicates with 10 bees for controls, toxic reference and the test item treatment 

groups 

Dosage: Control: oral test: 50 % sucrose solution, contact test: water and 0.1 % v/v Tween 

solution; test item: oral test: applied volume - 20 μL sucrose solution/bee; contact 

test: applied volume - 4 μL/bee (0.1 % v/v Tween solution); toxic reference: 

0.0625 - 0.5 μg dimethoate/bee (Perfection EC 400) 

Application rates for contact and oral toxicity test (based on analysed content of 

a.s.): 

Contact toxicity Oral toxicity* 

Test item 

[μg product/bee] 

BYI 08330 

[μg a.s./bee] 

Test item 

[μg product/bee] 

BYI 08330 

[μg a.s./bee] 

1324.5 200.0 1317.0 198.9 

662.2 100.0 661.3 99.9 

331.1 50.0 330.7 49.9 

165.6 25.0 165.6 25.0 

82.8 12.5 82.7 12.5 

*based on actual intake 

Exposure route: Application in the contact test: Anaesthetised bees were treated individually by 

topical application with a micropipette to the dorsal thorax. Application in the oral 

test: Bees were fed with a defined quantity of sucrose solution containing the test 

item. Groups of 10 bees were provided with 200 μL of the test solution in a glass 

ampoule. 

Feeding: 50 % aqueous sucrose solution; continuous feeding. 

Test conditions: Test units were disposable cages of cardboard with holes in the bottom for 

ventilation and a glass plate in front for observation of the bees (dimensions 

inside: 80 mm x 45 mm x 65 mm). 

Exposure time: 48 h. Temperature: 25 - 26°C, relative humidity: 59 - 61 %, 

darkness (except during observation), ventilation 

Test parameter: The number of dead and affected bees was counted at 4, 24 and 48 h. During 

assessments times any behavioural abnormalities of the bees were recorded, 

considering the following parameters compared to control: healthy or affected 

(paralysis, lateral position, lying on the back), any differences in activity, in 

position within the cage (all on the bottom), abnormal amount and colour of 
– 81 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

excretion. 

Statistics: Mortality was corrected according to Abbott (1925). For statistical calculation of 

the mortality results the Fisher’s Exact Binomial test was used. The accepted 

significance level was p ≤ 0.05 (one-sided greater). The median lethal dose (LD50) 

along with the 95 % confidence limits was calculated by Probit analysis according 

to the maximum likelihood method (FINNEY 1971) or according to the linear 

weighed regression. The goodness-of-fit of the model was evaluated by Pearson’s 

X

2

 test. The calculation of statistical significance and the LD50 was performed with 

the computer program ToxRat Professional 2.09. 

Findings: 

 The results of the mortality and behavioural assessment are presented in Tables 

B.9.4.1-3 and -4. The validity criteria were met as mortality in the control was ≤

10 % after 48 h and the contact and oral LD50 of the toxic reference were 0.263 μg 

a.s./bee and 0.194 μg a.s./bee, respectively. 

In the oral toxicity test statistically significant effects on the survival were found at 

consumed doses of 330.7, 661.3 and 1317 μg product/bee (23.3, 50.0 and 90.0 % 

mortality, respectively) during 48 h. No effects on behaviour were observed in 

bees consuming doses up to 330.7 μg product/bee. After consuming doses of 

661.3 and 1317 μg product/bee most bees were affected at the 4 and 24 h 

assessment. Bees had recovered at the 48 h assessment. 

In the contact toxicity test no effects on behaviour were observed after exposure 

to doses up to 331.3 μg product per bee. Bees exposed to doses equal or greater 

than 662.2 μg product/bee were affected at the 4 and 24 h assessment. Bees had 

recovered at the 48 h assessment. 

Acute Oral Test: Results Based on EPA’s Probit Analyses

LD50 = 607.18 ug/bee

95% Confidence Limits of LD50= 506.17, 737.88 

Intercept = -10.21 

Slope = 3.67; Standard error of slope=0.55

95 % Confidence Limits of Slope = 2.59, 4.74 

Goodness-of-Fit Test: 

Pearson Chi-Square value= 1.95; Degrees of freedom= 3; p(Chi-Square)=0.58 

Acute Contact Test: Results Based on EPA’s Probit Analyses

LD50 = 1073 ug/bee

95 % Confidence Limits of LD50 = 903.59, 1351 

Intercept=-13.83 

Slope = 4.56; Standard error of slope =0.89

95 % Confidence Limits of Slope = 2.82, 6.30 

Goodness-of-Fit Test: 

Pearson Chi-Square value= 2.90; Degrees of freedom= 3; p(Chi-Square)=0.41 
– 82 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.1-3: Oral and contact toxicity LD50 values of bees treated with BYI 08330 150 OD after 24 

and 48 h 

treatment contact toxicity test oral toxicity test 

time μg product/bee μg a.s./bee μg product/bee μg a.s./bee 

24 h 

95%-cl lower 

Upper 

Slope b 

1151.0 

933.8 

1418.8 

4.1 

- 607.1 

506.7 

727.4 

Slope b 

- 

3.7 

Test item 

BYI 08330 

150 OD 

48 h 

95%-cl lower 

Upper 

Slope b 

1072.9 

894.3 

1287.3 

4.6 

- 607.1 

506.7 

727.4 

Slope b 

- 

3.7 

24 h 

95%-cl lower 

Upper 

Slope b 

- 0.263 

0.225 

0.308 

4.7 

- 0.194 

0.171 

0.220 

7.2 

Reference item 

Dimethoate 

EC 400 

48 h 

95%-cl lower 

Upper 

Slope b 

- 0.243 

0.209 

0.283 

5.0 

- 0.189 

0.166 

0.214 

6.7 

cl: confidence limits 

Table B.9.4.1-4: Mortality of bees in the contact toxicity test (results are average from 3 replicates per 

dosage/control) 

dosage 

Test item [μg test 

item/bee]

Mortality [%] after 4 h 

(= corrected mortality) 

Mortality [%] after 24 h 

(= corrected mortality) 

Mortality [%] after 48 h 

(= corrected mortality) 

82.78 0.0 0.0 0.0 

165.56 0.0 0.0 0.0 

331.12 3.3 3.3 3.3 

662.24 10.0 10.0 10.0 

1324.48 0.0 63.3 70.0 

Control (Tween) 0.0 0.0 0.0 

Toxic standard [μg a.s. 

/bee] 

0.0625 0.0 0.0 0.0 

0.125 0.0 0.0 3.3 

0.250 33.3 63.3 63.3 

0.500 73.3 83.3 90.0 

Control (Tween) 0.0 0.0 0.0 
– 83 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.1-5: Mortality of bees in the oral toxicity test (results are average from 3 replicates per 

dosage/control) 

dosage 

Test item [μg consumed 

test item/bee]

Mortality [%] after 4 h 

(= corrected mortality) 

Mortality [%] after 24 h 

(= corrected mortality) 

Mortality [%] after 48 h 

(= corrected mortality) 

82.66 0.0 0.0 0.0 

165.56 0.0 0.0 0.0 

330.65 0.0 23.3 23.3 

661.30 50.0 50.0 50.0 

1316.99 90.0 90.0 90.0 

Control (sucrose solution) 0.0 0.0 0.0 

Toxic standard [μg 

consumed a.s. /bee] 

0.0625 0.0 0.0 0.0 

0.125 0.0 3.3 6.7 

0.249 53.3 90.0 90.0 

0.495 96.7 96.7 96.7 

Control (sucrose solution) 0.0 0.0 0.0 

Conclusion: 48 h oral LD50 = 607.1 product/bee equivalent to 91.7 μg a.s./bee

48 h contact LD50 = 1072.9 μg product/bee equivalent to 162.0 μg a.s./bee 

Comments: As deviation to EPA guidelines, Tween®is not an EPA-recommended 

solvent. 

B.9.4.2 Residue test (OECD IIA 8.7.3) 

Not submitted. 

B.9.4.3 Bee brood feeding test (OECD IIA 8.7.4) 

Studies with similar design are reported under B. 1.4.5. 

B.9.4.4 Cage test (OECD IIIA 10.4.4) 

Not required. 

B.9.4.5 Field tests (OECD IIIA 10.4.5) 

Formulation: Spirotetramat OD 100 

 Reference: Assessment of the Side-Effects of BYI 08330 OD 100 on the Honey Bee (Apis 

mellifera L.) in a Feeding Test 

Author(s), year: Schur, A.; 2004a 

Report/Doc. number: 20031168/01-BFEU; M-000345-01-2

Guideline(s): OEPP/EPPO Bulletin No. 22 (Oomen et al, 1992) 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 
– 84 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test substance: Spirotetramat OD 100 (a.s. BYI 08330), Batch no.: 08030/0038(0035), Tox no.: 

06293-00, content: 101.54 g/L 

Material and Methods: 

Test species: Apis mellifera L. (Hymenoptera: Apidae) 

Type of test: Feeding test, field study 

Method aimed at products with insect growth-regulating properties 

Number of organisms: 3 replicates (= bee colonies, approx. 12000 bees per colony) for control, toxic 

reference and the test item treatment group 

Dosage: Control: Water/sucrose solution, test item: dissolved in water and mixed in pure 

sucrose solution at a concentration of 0.0144 % a.s. (analysed; 1371 mg product 

per colony); toxic reference: 300 mg Insegar 25 WG per colony (a.s.: 25 % 

fenoxycarb) (dissolved in water and mixed in sucrose solution). 

Experimental 

procedure: 

The study was located in Southern Germany (location: Niefern). Bees from the 

test colonies were free-flying, with access to natural nectar sources. No main 

sources of nectar were found in the near surroundings. All colonies were in good 

condition at start of the test. Colonies of honey bees were fed with the test 

substance. 

Feeding: A quantity of 1 kg sugar solution was offered per colony. 

Test conditions: Meteorological data (temperature, relative humidity, rainfall) were recorded. 

Test parameters, 

observations: 

Mortality of adult bees, pupae and larvae, behaviour of the bees around the hives, 

condition of the colonies and the development of the bee brood were assessed 

before and after the feeding of the test substance. 

Wooden bee traps (Münster design) with gauze on bottom and on 100 % of the 

top were attached to the entrance of the colonies in order to register those dead 

bees which were carried out of the hives. Evaluation of mortality took place over 

three days before start of feeding (once a day at the same time in the morning), at 

the day of start of feeding (twice before start of feeding) and for 21 days after start 

of feeding (once a day at the same time in the morning). The influence of the test 

substance was evaluated by comparing the results in the test substance treatment 

to the control data and those from the toxic standard treatment and furthermore by 

comparing the pre- and post-feeding results. Dead larvae and pupae were 

examined for specific effects of IGR`s. 

Statistics: None in original study report. In 2007 statistics was performed using t-test and 

Dunnett`s t-test in an one-sided approach. 

Findings: 

 The test was considered to be valid because the following parameters were met: 

A detectable effect of the toxic standard was found (brood termination before a 

successful hatch, mortality of pupae). After 7 days at the latest all colonies had 

taken up the whole test solution. 

A clear difference was found in the brood termination rate in cells between the 

colonies in the control and the colonies in the toxic standard treatment. 
– 85 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.5-1: Mortality and flight activity of honey bees in a feeding study with 

Spirotetramat OD 100 

Treatment group Test substance 

(BYI 08330 OD 100) Control Toxic standard 

Feeding rate per colony 

(in pure sugar solution) 

0.0144% a.s. - 300 mg product 

pre 

(DAF –3 to 

0bf): 

rep. 1 

6.8±4.1

rep. 2 

6.3±4.6

rep. 3 

7.3±5.2

rep. 1 

7.5±2.5

rep. 2 

4.3±4.0

rep. 3 

9.5± 

10.3 

rep. 1 

5.0±6.1

rep. 2 

8.5± 

14.4 

rep. 3 

8.0±7.0 

 average 

 of 3 rep. 

6.8 7.1 7.2 

post ± SD 

(DAF 1 to 

21) 

rep. 1 

8.6±5.7

rep. 2 

15±16.8

rep. 3 

7.4±8.3

rep. 1 

5.7±4.2

rep. 2 

6.3±4.5

rep. 3 

2.8±1.9

rep. 1 

54.1±53

rep. 2 

115.2± 

129.9 

rep. 3 

104.2± 

115.3 

 average 

 of 3 rep. 

10.4 ± 11.6 4.9 ± 4.0 91.2 ± 106.5 

Average Mortality 

[dead bees/ 

colony/day]

QM(average): 1.5 0.7 12.7 

∑ dead adult bees and dead 

pupae (post-feeding, DAF 1 to 

21) 

597 + 55P 282 + 29P 321 + 5425P 

0bf 46.67 28.67 38.00 

DAF 1 57.00 35.00 57.00 

Average 

Flight activity* 

DAF 3 17.67 23.33 28.00 

DAF days after start of feeding 

0bf mortality on the day of start of feeding before feeding 

QM (average) ∅ post-feeding mortality ÷ ∅ pre-feeding mortality 

Pre average value for the pre-feeding period DAF –3 to 0bf 

post (DAF 1 to 21) average value for the post-feeding period DAF1 to 21 

P = dead pupae 

SD = Standard deviation 

rep. = replicate hive 

* mean number of bees leaving/entering the hive per minute 

 Regarding the total daily mortality in the test substance treatment, a slightly 

increased mean mortality was noticed in the test substance group on DAF 6, 13 

and 15. Throughout the entire post-feeding period a sum of 55 dead pupae were 

counted in the test substance treatment compared to 29 dead pupae in the control 

group. A sum of 5425 dead pupae were counted during the entire post-feeding 

period in the toxic standard which is evidence that Insegar 25 WG massively 

disturbed the brood development of these test colonies. 

The sum of dead pupae and adults were analysed seperately over the postfeeding period (control vs treatment and control vs toxic standard). As result, no 

statistically significant effect on pupae mortality was detected, but there was a 

statistically significant difference in adult bee mortality between control and 

treatment (t-test, pooled, one-sided, p=0.0447). 

Honey bee flight intensity: Regarding the flight intensity observed in front of the 

hives no remarkable differences were observed at both post-feeding assessment 

dates DAF 1 and 3 between the control, the test substance and the toxic standard 

treatment. 

Condition of the colonies: During the assessments of the condition of the colonies 

throughout the study no remarkable observations were made regarding the 

strength of the colonies and the brood nest size in the test substance treatment 
– 86 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

compared to the control treatment. The colonies of the test substance treatment, 

control and toxic standard treatment showed all brood stages at the assessment 

dates during the experimental phase of the study. 

Honey bee brood development: By comparing the individual brood assessments 

of single cells, the indices (the values of the different brood stages of all cells in 

each treatment group, assessed at the same date, summed up and divided by the 

number of observed cells) of the control group showed the course to be expected 

in a natural bee development cycle. In hive 2 and 3 of the test substance 

treatment a termination of the bee brood in more than three quarters of the cells 

observed was noticed from BFD (brood fixing day) to BFD 5. The cells which 

showed a terminated brood development were empty at the first assessment after 

start of feeding of the test substance. Even at the following assessments a 

massively disturbed brood development was observed with empty cells, cells 

refilled or refilled again with new brood. Hive 2 and 3 of the test substance 

treatment were not able to compensate the effect on the brood development 

caused by the test substance until the end of the observation period. No clear 

effect which could be attributed to the test substance was observed at hive 1. 

Honey bee behaviour in front of the colonies: No differences regarding the 

behaviour of the bees were noticed between the test substance treatment, the 

toxic standard treatment and the control. 

Conclusion: It was concluded that the feeding of Spirotetramat OD 100 had a slight but in the 

context not biologically significant effect on adult and pupae mortality of the bees 

at the dosage tested and did not result in an adverse effect on the flight activity 

and the behaviour of the bees in front of the hives. Regarding the brood 

development a high termination rate was noticed 4 days after start of feeding in 

two of the three test substance colonies. In these colonies the further brood 

development was massively disturbed at the following assessments and they 

were not able to compensate the effect on the brood development until the end of 

the observation period. No effect was detected by the regular brood assessments. 

Formulation: Spirotetramat SC 240 

Reference: Assessment of the Side-Effects of BYI 08330 SC 240 on the Honey Bee (Apis 

mellifera L.) in a Feeding Test. 

Author(s), year: Schur, A.; 2004b 

Report/Doc. number: 20031166/01-BFEU; M-121877-01-2

Guideline(s): OEPP/EPPO Bulletin No. 22 (Oomen et al, 1992) 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat SC 240 (a.s. BYI 08330), Batch no.: 07218/0013, Tox no.: 06294-

00, content: 243.85 g/L (analysed); Adjuvant: RME EW 500 (rape methyl ester) 

Material and Methods: 

Test species: Apis mellifera L. (Hymenoptera: Apidae) 

Type of test: Feeding test, field study 

Method aimed at products with insect growth-regulating properties 

Number of organisms: 3 replicates (= bee colonies, approx. 12000 bees per colony) for control, toxic 

reference and the test item treatment group 
– 87 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Dosage: Control: Water/sucrose solution, test item: dissolved in water and mixed in pure 

sucrose solution at a concentration of 0.0144 % a.s. plus 0.200 % of the adjuvant 

RME EW 500 (analysed; 637 mg product per colony); toxic reference: 300 mg 

Insegar 25 WG per colony (a.s.: 25 % fenoxycarb) (dissolved in water and mixed 

in sucrose solution). 

Experimental 

procedure: 

The study was located in Southern Germany (location: Niefern). Bees from the 

test colonies were free-flying, with access to natural nectar sources. No main 

sources of nectar were found in the near surroundings. All colonies were in good 

condition at start of the test. Colonies of honey bees were fed with the test 

substance. 

Feeding: A quantity of 1 kg sugar solution was offered per colony. 

Test conditions: Meteorological data (temperature, relative humidity, rainfall) were recorded. 

Test parameters, 

observations: 

Mortality of adult bees, pupae and larvae, behaviour of the bees around the hives, 

condition of the colonies and the development of the bee brood were assessed 

before and after the feeding of the test substance. 

Wooden bee traps (Münster design) with gauze on bottom and on 100 % of the 

top were attached to the entrance of the colonies in order to register those dead 

bees which were carried out of the hives. Evaluation of mortality took place over 

three days before start of feeding (once a day at the same time in the morning), at 

the day of start of feeding (twice before start of feeding) and for 21 days after start 

of feeding (once a day at the same time in the morning). The influence of the test 

substance was evaluated by comparing the results in the test substance treatment 

to the control data and those from the toxic standard treatment and furthermore by 

comparing the pre- and post-feeding results. Dead larvae and pupae were 

examined for specific effects of IGR`s. 

Statistics: None. 

Findings: 

 The test was considered to be valid because the following parameters were met: 

A detectable effect of the toxic standard was found (brood termination before a 

successful hatch, mortality of pupae). After 7 days at the latest all colonies had 

taken up the whole test solution. 

A clear difference was found in the brood termination rate in cells between the 

colonies in the control and the colonies in the toxic standard treatment. 
– 88 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.5-2: Mortality and flight activity of honey bees in the feeding study with 

Spirotetramat SC 240 

Treatment group Test substance 

(BYI 08330 OD 100) Control Toxic standard 

Feeding rate per colony 

(in pure sugar solution) 

0.0144 % a.s. 

+ 0.200 % adjuvant - 300 mg product 

pre 

(DAF –3 to 0bf): 6.9 7.1 7.2 

post ± SD 

(DAF 1 to 21) 15.8 ± 24.1 4.9 ± 4.0 91.2 ± 106.5 

Average Mortality 

[dead bees/ 

colony/day]

QM(average): 

2.3 0.7 12.7 

∑ dead adult bees and dead pupae (postfeeding, DAF 1 to 21) 988 + 10P 282 + 29P 321 + 5425P 

0bf 36.33 28.67 38.00 

DAF 1 45.00 35.00 57.00 

Average 

Flight activity* 

DAF 3 19.00 23.33 28.00 

DAF days after start of feeding 

0bf mortality on the day of start of feeding before feeding 

QM (average) ∅ post-feeding mortality ÷ ∅ pre-feeding mortality 

Pre average value for the pre-feeding period DAF –3 to 0bf 

post (DAF 1 to 21) average value for the post-feeding period DAF1 to 2 

P = dead pupae 

SD = Standard deviation 

* mean number of bees leaving/entering the hive per minute 

 Regarding the total daily mortality in the test substance treatment an increased 

daily mean mortality was noticed on DAF 6 as well as on DAF 15 to 17 with 23.3 

to 71.0 dead bees/colony. Only 10 dead pupae were counted in the test 

substance treatment throughout the entire post-feeding period compared to 29 

dead pupae in the control group. A sum of 5425 dead pupae were counted during 

the entire post-feeding period, mainly between DAF 10 and 21, in the toxic 

standard which is evidence that Insegar 25WG massively disturbed the brood 

development. 

Honey bee flight intensity: Regarding the flight intensity observed in front of the 

hives no remarkable differences were observed at both post-feeding assessment 

dates DAF 1 and 3. 

Condition of the colonies: During the assessments of the condition of the colonies 

throughout the study no remarkable observations were made regarding the 

strength of the colonies and the brood nest size in the test substance treatment 

compared to the control treatment. The colonies of the test substance treatment, 

control and toxic standard treatment showed all brood stages at the assessment 

dates during the experimental phase of the study. 

Honey bee brood development: By comparing the individual brood assessments 

of single cells, the indices (the values of the different brood stages of all cells in 

each treatment group, assessed at the same date, summed up and divided by the 

number of observed cells) of the control group showed the course to be expected 

in a natural bee development cycle. In all colonies of the test substance treatment 

a termination of the brood in almost all cells was noticed from BFD (Brood area 

fixing day) to BFD 5. Hive 1 and 2 were able to compensate this lack of brood and 

the main part of the empty cells was filled again with eggs and larvae at the 
– 89 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

following assessment on BFD 11. In hive 3 of the BYI 08330 SC 240 treatment the 

situation of the marked brood area was similar on BFD 11 compared to the 

previous assessment because the cells remained empty until this assessment day 

and even at BFD 15 the brood index was still below the level of 1 (egg stage). 

These observations indicate an effect of BYI 08330 SC 240 to the brood 

development in the first 5 to 15 days after start of feeding. Nevertheless the brood 

indices continued to rise from BFD 11 to 22 in all test substance colonies and at 

the final brood assessment the big part of the cells contained brood. 

Honey bee behaviour in front of the colonies: No differences regarding the 

behaviour of the bees were noticed between test substance treatment, toxic 

standard treatment and control. Regarding the flight intensity observed in front of 

the hives no remarkable differences were observed at both post-feeding 

assessment dates DAF 1 and 3. 

Conclusion: It was concluded that the feeding of BYI 08330 SC 240 caused an increase of 

adult honey bee mortality 6 and 15 to 17 days after start of feeding but did not 

result in an adverse effect on the flight activity and the behaviour of the bees in 

front of the hives. An almost total termination of the brood development was 

noticed 4 days after start of feeding in all colonies but the colonies showed the 

tendency to recover until the end of the observation period since most of the cells 

observed contained developing brood at the final brood assessment and no effect 

was detected by the regular brood assessments. 

Formulation: Movento 150 OD 

Reference: Assessment of Side Effects of Spirotetramat OD 150 on the Honey Bee (Apis 

mellifera L.) in the Field.

Author(s), year: Schur, A.; 2006 

Report/Doc. number: 20061133/S1-BFEU; M-277194-02-1

Guideline(s): OEPP/EPPO No. 170 (3) (2001) 

GLP: Yes (certified laboratory) 

Deviations: No 

Validity: Acceptable 

Test substance: Spirotetramat OD 150 (a.s. BYI 08330), Batch no.: 08030/0189(0152), content of 

a.s.: 15.1 % (w/w) (analysed) 

Material and Methods: 

Test species: Apis mellifera mellifera (Hymenoptera: Apidae) 

Type of test: field study with special attention to bee brood development 

Number of organisms: Four normally developed, healthy and queen-right colonies were used per test 

field. Each colony contained one body with 12 frames and approx. 20000 bees / 

colony. 

Treatment: Three test fields: 

- test item treated field T1 (4 × 72 g a.s./ha, two times before flowering (no bees in 

the field) and 2 x during flowering of the crop and foraging activity of the bees) 

- test item field T2 (2 × 96 g a.s./ha during flowering of the crop and foraging 

activity of the bees) 

- untreated control field 

The applications were carried out in approximately 7-day intervals in 200 L 

water/ha. Calibrated commercial spraying equipment was used. 

Experimental The field test was located in Spain (region: Valencia). The crop Phacelia 
– 90 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

procedure: tanacetifolia was used in flowering stage. The field sizes were: test item field T1: 

2025 m2, test item field T2: 2016 m2 and the control field C: 3306 m2. The test 

fields were not close to other flowering crops or extensive blooming weeds, which 

may be attractive to bees and were separated by a distance of at least 2 km. 

Commercial bee colonies were placed near the test fields 3 days before the 3rd

application in the test item field T1 and before the 1st application in the test item 

field T2, respectively. To ensure that the bees are exposed to the test field 

detailed assessments of foraging activity were done before as well as after the 

application. 

Test conditions: Temperature and rainfall data were recorded at the governmental weather station 

in Jalance, approximately 15 km (T1 field), 17 km (T2 field) and 18 km (control 

field) away from the field site. The humidity data were recorded at the 

governmental weather station in Almansa, approximately 20 km (T1 and T2 field) 

and 25 km (control field) away from the field site. The degree of cloud coverage 

and wind speed during applications was assessed at the test fields.

Test parameters, 

observations: 

Exposure of the bees was assessed by visual inspection of the P. tanacetifolia

pollen on combs entered in the hives during the study. 

The influence of the test item on the honey bees was evaluated by comparing the 

results of the test item treatments to those of the control treatment. The following 

points were assessed: 

• Condition of the colonies (strength) and development of the bee brood 

• Mortality in the field and in the bee traps in front of the hives 

• Foraging activity (number of forager bees/m2/minute flowering crop) 

• Behaviour of the bees on the crop and around the hive 

Flight intensity and foraging activity were assessed in 5 marked squares (1 m2) 

that were regularly distributed. Mortality and foraging activity of the bees were 

checked prior to first applications in the test fields (over 2 days and shortly before 

first application) and followed up after application (foraging activity: over 10 days 

after first application during exposure, mortality: over 28 days after first application 

during exposure). In order to record the number of dead bees water-permeable 

linen sheets were spread out in front of the hives. Dead bee traps were attached 

to the entrance of the hives in order to register those dead bees which were 

carried out of the hives. Furthermore, mortality was recorded at three impartially 

selected places in each test field using linen sheets. Dead bees were 

differentiated in adult worker bees, males, freshly emerged bees, pupae and 

larvae during each assessment. 

The condition of the colonies and the bee brood development were checked once 

before first application during exposure and five times afterwards (up to 26 days 

after first application during exposure). 

Collection of flowers: For the possible analysis/determination of residues of the 

test item in flower clusters of P. tanacetifolia samples were taken during the trial. 

The residue analysis was not part of the present study. 

Statistics: None. 

Findings: 

 Data on mortality and flight activity are presented in the table below. 
– 91 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.5-3: Mortality and flight activity of honey bees in the feeding study with 

Movento 150 OD 

Treatment group T1 T2 Control 

1

st application date (01May06, before flowering): application 

rate g a.s./ha 

72.0 - - 

2

nd application date (08May06, before flowering): application 

rate g a.s./ha 

72.0 - - 

3

rd application date (17May06, during flowering/bee flight): 

application rate g a.s./ha 

72.0 96.0 - 

4

th application date (24May06, during flowering/bee flight): 

application rate g a.s./ha 

72.0 96.0 - 

Pre-appl. [DAA -2 to 0ba]: 43.4 135.4 87.7 

Pre-appl. [DAA 0ba]: 42.0 94.8 65.0 

Post-appl. [DAA 0aa]: 15.0 23.0 29.0 

Pre-appl. [DAA 7ba]: 22.0 17.8 2.8 

Post-appl. [DAA 7aa]: 3.8 6.8 2.3 

Post-appl. [DAA 0aa to 28]: 5.7 6.5 5.6 

Mean mortality 

[dead 

bees/colony/day]

QM: 0.1 <0.1 0.1 

Mean number dead pupae/larvae per colony/day 

(post-application, DAA 0aa to 28) 

0.3 0.1 <0.1 

Pre-appl. [DAA -2 to 0ba]: 11.3 11.5 13.7 

Pre-appl. [DAA 0ba]: 15.0 13.0 12.4 

Post-appl. [DAA 0aa]: 11.9 14.4 14.4 

Pre-appl. [DAA 7ba]: 12.0 15.0 1.0 

Post-appl. [DAA 7aa]: 14.6 23.0 11.1 

Mean flight intensity 

[foraging 

bees/minute/m2]

Post-appl. [DAA 0aa to 10]: 11.9 16.3 12.2 

ba = before application in the test item fields during the exposure period 

aa = after application in the test item fields during the exposure period 

DAA = days after first applications in the test item fields during the exposure period 

QM = post-application [DAA 0aa to 28] / pre-application [DAA –2 to 0ba] 

To insure that the bees were exposed to the test field and the test item detailed assessments of foraging 

activity were done before as well as after the application during the exposure period. The daily mean 

foraging activity after the applications (DAA 0aa to DAA 10) was 11.9 foraging bees/minute/m2

 in the test 

item group T1, 16.3 foraging bees/minute/m2

 in the test item group T2 compared to 12.2 foraging 

bees/minute/m2 in the control group. The degree of P. tanacetifolia pollen from the total amount of pollen per 

colony ranged from 10 % up to 33 % during time of exposure (brood assessments on 19May06 and 

25May06) at the test field in the test item group T1, from 8 % up to 40 % at the test field T2 and from 6 % up 

to 85 % in the control field. The results of the pollen assessments in the colonies confirm that the bees were 

actively foraging on the test fields. A quantitative comparison between the results of the treatments is not 

possible, because the foraging and storage of pollen in a bee colony depends on outside conditions as well 

as on the individual need of pollen in the bee colony. 

Honey bee mortality 

The number of dead bees on the day before as well as shortly before the first application during exposure in 

the test fields was increased in single colonies of the test item treatment group T2 as well as in the control 

group. Since in both of the test fields in which this slightly increased mortality has been observed, no preexposure application was carried out, the temporarily increased mortality in those colonies was clearly not 

caused by the test item. However, on the assessments after first application during exposure (day 0 after 

application) the mean mortality in all treatment groups was on a low level and no test item related increase in 

the number of dead bees was observed (T1: 15.0 dead bees/colony, T2: 23.0, control: 29.0). On the second 

application date during exposure (7 days after the first one) the mortality in all treatment groups was on a 

similar low level at the assessments after applications. No test item related difference in the number of dead 

bees of the treatment groups T1 and T2 compared to the mortality in the control group was observed at any 
– 92 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

time after the applications. This resulted in a daily mean post-application mortality (day 0 after application to 

day 28) in the test item treatment group T1 of 5.7 dead honey bees/colony, 6.5 dead honey bees/colony in 

the test group T2 and 5.6 dead honey bees/colony in the control group. 

The value for QM (mean mortality post-application during exposure divided by the mean mortality preapplication during exposure) was calculated as 0.1 in the test item treatment group T1, <0.1 in the test item 

group T2 and 0.1 in the control group. 

The mean number of dead pupae and larvae from day 0 to 28 after first application during exposure was on 

a low level in all treatments with 0.3 dead pupae and larvae/colony/day in the treatment group T1, 0.1 dead 

pupae and larvae/colony/day in the treatment group T2 and <0.1 dead pupae and larvae/colony/day in the

control group. Since larval and pupae mortality was at a comparable level in all treatment groups, there were 

no treatment-related effects. 

Honey bee flight intensity: 

The daily mean flight intensity (foraging bees/minute/m2) before the first application during exposure was 

15.0 in the test item treatment group T1, 13.0 in the test item treatment group T2 and 12.4 in the control 

group. The mean flight intensity after the applications on DAA 0 was 11.9 foraging bees/minute/m2 in the 

treatment group T1, 14.4 foraging bees/minute/m2 in the treatment group T2 as well as in the control group. 

On the second application date (DAA 7) no reduction in the mean flight intensity was noticed after 

applications in the test item group T1 and T2. The daily mean post-application (DAA 0aa to DAA 10) flight 

intensity was 11.9 foraging bees/minute/m2 in the test item group T1, 16.3 foraging bees/minute/m2 in the 

test item group T2 compared to 12.2 foraging bees/minute/m2 in the control group. 

The intense flight and foraging activity of the bees on the test fields was also supported by the fact that the 

bees stored actively P. tanacetifolia pollen in the combs of the colonies of all treatment groups. 

Condition of the colonies and honey bee brood development

Assessments of the colony strength as judged by number of bee ways between combs filled with bees and 

the brood nest size (number of brood combs per colony) did not indicate significant differences between 

treatment groups T1, T2 and the control colonies. On the frequent assessments during exposure in the test 

fields and afterwards (up to 29 days after the first brood assessment) 3 colonies of the test item treatment T1 

and all colonies in the test item group T2 and in the control group showed all brood stages and a similar 

development. One colony in the test item treatment T1 showed a lack of eggs or/and larvae on the last 2 

assessment dates. In that colony a tendency to swarm or to remove the old queen was recorded (queen 

cells on combs) starting from the first brood assessment after set-up of the colonies at the test fields. It is 

very likely that this was caused by a lack of space for the colony to grow, or that the colony tried to breed a 

new queen because they were not satisfied with the old queen. Since the other 3 colonies of the treatment 

group T1 were in good condition over the entire test period it is very unlikely that the lack of brood was 

caused by the application of the test item. 

Honey bee behaviour in front of the colonies and within the crop area

No differences regarding the behaviour of the bees were observed between the test item treatment groups 

T1 and T2 and the control group. 

Conclusion: The test item treatments (T1 and T2) did not result in an adverse effect on honey 

bees as determined by mortality and flight intensity. Differences of bee behaviour 

between control and treatment groups were not observed. The condition of the 

colonies as assessed by colony strength and size of the brood nest was not 

affected by any of the treatments. From the detailed assessment of the honey bee 

brood status one colony in the test item treatment group T1 showed a lack of 

brood on the last assessments. In that colony a tendency to swarm or to remove 

the old queen was recorded (queen cells on combs) starting from the first brood 

assessment after set-up of the colonies at the test fields. It is very likely that this 

was caused by a lack of space for the colony to grow, or that the colony tried to 

breed a new queen because they were not satisfied with the old queen. No 

evidence of an irritation or termination of the development based upon exposure 

to treated crops was obtained in the other colonies of the test group T1 as well as 
– 93 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

in the test item group T2 and the control colonies.

B.9.4.6 Tunnel tests (OECD IIIA 10.4.7) 

Formulation: Spirotetramat OD 100 

Reference: Assessment of side effects of BYI 08330 OD 100 on the honey bee (Apis 

mellifera L.) in the semi-field. 

Author(s), year: Schur, A.; 2005 

Report/Doc. number: 20041144/01-BZEU; M-244490-01-2

Guideline(s): EPPO No. 170 (3) and Bulletin of Insectology 56 (1), 91 – 96; 2003: Honey bee 

brood ring-test in 2002: method for the assessment of side effects of plant 

protection products on the honey bee brood under semi-field conditions. 

GLP: Yes (certified laboratory) 

Deviations: No 

Validity: Acceptable 

Test substance: Spirotetramat OD 100 (a.s. BYI 08330), Batch no.: 08030/0110(0073) 

Tox no.: 06593-00, content: 102.02 g/L (analysed) 

Material and Methods: 

Test species: Apis mellifera L. (Hymenoptera: Apidae) 

Type of test: Semi-field test (tunnel) with special attention to the development of the bee brood 

Number of organisms: Small healthy colonies ["Mini-Plus-Beuten"; 1 queen and approximately 1 kg bees 

(6000 - 8000 bees per colony)] with 12 combs were used. 3 replicates (= bee 

colonies) for control, toxic reference and the test item treatment group. 

Treatments: Control: water, test item: treatment group T1: Test item applied 3 x on the crop 

before flowering at 72 g a.s. (692.33 g product)/ha and 2 x after set-up of the 

colonies at 96 g a.s. (923.11 g product)/ha. Treatment group T2: test item applied 

once, 3 d after set-up of the colonies at a rate of 96 g a.s./ha; toxic reference: 

Insegar WG 25 (a.s.: fenoxycarb, 25 %) 150 g a.s. per ha. All treatments were 

applied in 400 L water/ha using a plot sprayer. 

Experimental 

procedure: 

The semi-field test was located in the south of Germany at Niefern near 

Pforzheim. The crop used was oil-seed spring rape (Brassica napus). Before full 

flowering and start of bee-exposure tunnel tents were set-up in the test field. The 

area covered with rape was 47.52 m2 per tunnel. The tunnels had a size of 5.0 x 

12 m and a height of about 3.5 m. The tunnel frames were covered with light 

plastic gauze (mesh size: 1.5 mm). Before the start of bee-exposure in the tunnels 

a path was created along the tunnel walls at both front sides and through the 

middle of each tunnel by removing the plants and levelling the ground. 

Subsequently, the path was covered with linen sheets. For the test, small healthy 

colonies with 12 combs were used. All nuclei were produced at the same time. 

The corresponding queens originated from one breeding line in order to guarantee 

uniform bee material in all treatment groups. Each bee hive consisted of two 

bodies with one bottom and one lid (height: 40 mm). The outer dimensions of this 

hive were 300 mm x 300 mm, the inner dimensions are 230 mm x 230 mm and 

the height is 170 mm. Each body contained six frames (130 mm x 200 mm). 

The control and toxic standard applications were performed on the same day as 

the forth test item application in treatment group T1 (main application day). There 
– 94 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

were three tunnels (one hive per tunnel) per treatment. 

Feeding: No supplementary feeding. 

Test conditions: Meteorological data (temperature, relative humidity, rainfall) were recorded. 

Test parameters, 

observations: 

Mortality, flight intensity, condition of the colonies and the development of the bee 

brood were assessed before and after the main application day. Particular 

attention was directed to the brood development of the colonies. 

The influence of the test item was evaluated by comparing the results in the tunnel 

tents of the test item treatments to the control data and those of the toxic standard 

treatment. The following points were assessed: 

• Condition of the colonies (strength) and development of the bee brood 

• Mortality at the edge of the treated area and in the bee traps 

• Foraging activity (number of forager bees/m2 flowering rape) 

• Behaviour of the bees on the crop and around the hive 

Wooden bee traps with gauze on bottom and on the top were attached to the 

entrance of the hives in order to register those dead bees which were carried out 

of the hives. Furthermore, the mortality was recorded in the crop. Therefore the 

oilseed spring rape (Brassica napus) was removed prior to the set-up of the hives 

and water-permeable linen sheets (covered area approximately 15 m2) were 

spread out in the array. The mortality of adult honey bees found on the linen 

sheets and in the bee trap at the entrance of the colonies was assessed. After an 

exposure period of 10 days the bee colonies were taken out of the tunnels and 

transferred to an area without flowering main crops and where no pesticides were 

applied during the time of assessments. Mortality of adult honey bees in the bee 

trap at the entrance of the colonies was assessed for another 13 days [until day 

+23 after BFD (brood area fixing day)]. The condition of all colonies was checked 

two days before the main application day and five times afterwards (4, 9, 16, 23 

and 29 days after BFD). Assessment of the development of the bee brood took

place at the BFD (egg), 4 days (young to old larvae), 9 days (capped cells), 16 

days (capped cells shortly before hatch) and 23 days after BFD (empty cells or 

cells containing eggs) using the acetate sheet method. After an exposure period 

of 10 d, the bee colonies were taken out of the tunnels and transferred to an area 

without flowering main crops and pesticide application. Mortality at the entrance of 

the colonies was assessed for another 13 d (BFD +23). 

Statistics: None in original study report. In 2007 statistics was performed using t-test and 

Dunnett`s t-test in an one-sided approach.. 

Findings: 

 The test was considered to be valid because a detectable effect of the toxic 

standard was found (brood termination before a successful hatch, increased 

mortality of pupae). Results are presented in the table below. 
– 95 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.6-1: Mortality and flight activity of honey bees in the feeding study with 

Spirotetramat OD 100

Exposure T1: five spray treatments of BYI 08330 OD 100 three before 

 and two during foraging activity in flowering oilseed 

 spring rape 

T2: one spray treatment of BYI 08330 OD 100 during 

 foraging activity in flowering oil-seed spring rape 

Treatment group Test substance 

(BYI 08330 OD 100) 

Control 

(water) 

Toxic standard 

(Insegar 25 WG) 

T1 T2 C R 

Single application rate 

[in 400 L water/ha] 

3 × 72 g a.s./ha

2 × 96 g a.s./ha 

96 g a.s./ha - 150 g a.s. 

pre 13.8 12.9 7.9 11.2 

post (BFD +2aa) 5.0 2.7 2.7 1.7 

post (BFD +2aa to 

+23) 

5.5 4.5 4.1 8.1 

Mean Mortality 

[dead bees/ 

replicate/day]

QM(average): 0.4 0.3 0.5 0.7 

∑ dead pupae (post-application, 

BFD +2aa to +23)

12 29 8 300 

pre 10.3 8.3 8.4 10.7 

post (BFD +2aa) 19.0 12.2 15.0 15.3 

Daily mean flight 

intensity [foraging 

bees/m2/replicate]

post (BFD +2aa to 

+9) 

14.5 14.8 14.1 13.8 

aa after application 

pre average values for BFD to BFD +2ba (before application) 

post (BFD +2aa) main application day after application 

post (BFD +2aa to +9) mean value for BFD +2aa to BFD +9 

post (BFD +2aa to +23) mean value for BFD +2aa to BFD +23 

QM(average) mean mortality per day after application divided by mean mortality per day before appl. 

BFD brood area fixing day 

Regarding the total daily adult bee mortality observed during the exposure of the bees to the test item 

treated plants (T1 and T2) in the tunnels and in the time afterwards no remarkable observations were made 

compared to the control or toxic standard treatment. The mean post-application adult bee mortality was

comparable in all treatment groups (T1: 5.5 dead bees/replicate/day, T2: 4.5, control: 4.1, toxic standard: 

8.1). In the toxic standard, all three colonies showed an increased number of dead pupae in approximately 

the same period of time from BFD +12 to BFD +19 which is a typical effect of Insegar WG 25. The total 

number of dead pupae per colony from BFD +2aa to BFD +23 was 12 dead pupae in the test item treatment 

T1, 29 dead pupae in treatment T2, 8 dead pupae in the control treatment compared to 300 dead pupae in

the toxic standard treatment. 

The sum of dead pupae and adults were analysed separately over the post-treatment period (control vs 

treatment and control vs toxic standard). As result, no statistically significant effect on pupae mortality nor 

adult bee mortality between control and treatment (Dunnett`s test, test) was found, whereas pupae mortality 

was significantly higher in the toxic standard group. 

Honey bee flight intensity:

The application(s) of the test item and the toxic standard had no effects on flight intensity compared to the 

control treatment. The daily mean post-application flight intensity was on a similar level in all treatment 

groups (see table above). 

Condition of the colonies:

During the assessments of the condition of the colonies throughout the study no remarkable observations 

were made regarding the strength of the colonies and the brood nest size in the test item treatment groups 
– 96 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

T1 and T2 compared to the control treatment. The colonies of the test item treatment groups and control 

showed all brood stages at the assessment dates during the experimental phase of the study. In one colony 

of the toxic standard no eggs were observed and queen cells were noticed at the last brood assessment 

which is the evidence that the queen of this colony died. 

Honey bee brood development:

By comparing the individual brood assessments of single cells, the indices (the values of the different brood 

stages of all cells in each treatment group, assessed at the same date, summed up and divided by the 

number of observed cells) showed the course to be expected in a natural bee development cycle in two 

colonies of the test item treatment group T1, in the colonies of treatment group T2, in two colonies of the 

control group and in one colony of the toxic standard. In one control colony and in one colony of T1 a

termination of brood in single cells was observed during the observation period which resulted in a retarded 

increase of the brood index from BFD to BFD +9 compared to the other test hives of the same treatment (for 

details see table B.1.4.1.1.-4). In the colonies of T2 about 25 % of the marked cells showed a termination of 

the bee brood during the test. However, since one control colony showed a high termination rate between 

BFD and BFD +4 and the other two colonies of the control showed a termination of about 20 % during the

test, the effects noticed in the test item treatments can not be clearly ascribed to the application of the test 

item. One colony of the toxic standard treatment was obviously affected by the treatment with Insegar WG 25 

as can be seen in a decreasing index between the first assessment and BFD +4, as well as in a very low

index on the following assessment on BFD +9. 

Table B.9.4.6-2: Summary of the brood indices calculated based on individual brood assessments of 

single cells in the test treatment groups T1, T2, the control and toxic standard treatment

Mean brood indices at x d after BFD 

Treatment Replicate 

0 +4 +9 +16 +23 

1T1 1.00 1.85 3.57 3.70 2.23 

2T1 1.00 2.03 3.87 4.00 2.07 

3T1 1.00 1.37 2.56 3.21 3.42 

T1 

Mean 1.00 1.75 3.33 3.64 2.57 

1T2 1.00 1.67 3.16 3.78 2.40 

2T2 1.00 1.85 3.17 3.41 1.96 

3T2 1.00 1.73 3.24 3.52 2.01 

T2 

Mean 1.00 1.75 3.19 3.57 2.12 

1C 1.00 1.81 3.60 3.98 2.96 

2C 1.00 1.72 3.29 3.90 3.26 

3C 1.00 1.21 2.20 3.79 1.80 

Control 

Mean 1.00 1.58 3.03 3.89 2.67 

1R 1.00 1.96 3.36 3.71 1.45 

2R 1.00 1.76 2.86 3.37 1.96 

3R 1.00 0.29 0.41 2.13 3.81 

Toxic 

standard 

Mean 1.00 1.34 2.21 3.07 2.41 

(categories of brood indices: 0 - termination of development, 1 - egg stage, 2 - young larvae, 3 - old larvae, 4 - pupae 

stage (capped cell), 5 - empty after hatch) 

Honey bee behaviour in front of the colonies and within the crop area:

No differences regarding the behaviour of the bees were noticed between the test item treatment groups T1 

and T2, the toxic standard treatment and the control. 
– 97 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Conclusion: It is concluded that the application of BYI 08330 OD 100 applied five times 

(treatment group T1) and once (treatment group T2) on a bee-attractive flowering 

crop did not result in an adverse effect on the mortality of adult honey bees and 

brood, flight intensity of the bees on the crop and the behaviour of the bees in the 

crop area or in front of the hive. An irritation of the brood development at the 

earlier assessments was noticed in one colony of the test item treatment group 

T1, and about one quarter of the marked cells showed a termination of bee brood 

in the colonies of treatment group T2. However, since one control colony showed 

a high termination rate between BFD and BFD +4 and the other two colonies of 

the control showed a termination of about 20 % during the test, the effects noticed 

in the test item treatments can not be ascribed to the application of the test item. 

The colonies were in good condition during the observation period of 29 days. 

Additional non-GLP studies on the effects of spirotetramat on the bee brood:

Introduction:

For research and product positioning purposes Bayer CropScience AG performed orientating honey bee 

semi-field (tunnel) studies with Spirotetramat. The objectives of these studies were to investigate potential 

side effects of different formulations and rates of spirotetramat, and different pre-application intervals, to 

honey bees in flowering crops under tunnel conditions. 

Nine honey bee tunnel studies were performed between 2002 and 2007, seven in Germany, two in Spain. 

For all studies Phacelia tanacetifolia was used as flowering crop with the exception of one study performed in 

2007 on strawberry. All studies were carried out following the guidance of the EPPO 170 guideline under 

non-GLP conditions but according to GEP (good experimental practice). 

The general test performance was as follows: Honeybee colonies of an appropriate size (approx. 3000 - 3500 

bees) were confined in tunnels (44 m2 and 60 m2 in Spain and 50 m2 (100 m2 for the strawberry trial, due to 

lower density of the flowering crop) in Germany) with a flowering crop on a field. For the reported studies one or 

two replicates were set up for each treatment group. Each study comprised one untreated control, one toxic 

reference (fenoxycarb) and one to six different test item treatment groups. Honey bees were introduced into the 

tunnels some days prior to application to get familiar with the new environmental conditions (unless preflowering applications were tested). The bee colonies were examined for potential test item related effects for at 

least 7 days after the application inside the tunnels and for at least additional 14 days after removal from the 

tunnels. In particular, the endpoints adult, larval and pupal mortality, flight intensity and brood development 

were assessed, furthermore honey and pollen storage, egg laying and breeding activity, colony strength, hive 

weight and behavioural anomalies of the honey bees were recorded. The obtained results for each endpoint 

and treatment group from the post-application period were compared to those of the pre-application phase. The 

post-application results of the test item treatment rate(s) were also compared one with each other as well as to 

the control and the toxic reference. Only the results of the endpoints related to bee brood development with 

regard to the larval development and mortality of pupae (where observed) are reported here. All other brood 

(e.g. egg and capped cells, storage area) as well as all non-brood related endpoints (e.g. adult mortality, 

flight density, behavioural abnormalities, hive weight) were not impacted and therefore were not reported in 

the notifier`s summary, which is focussing on the potential of brood effects. 

Due to the fact that only one or two replicates per treatment group were used in the reported studies, a 

statistical evaluation was not feasible. Hence, the determination of effects was performed on the basis of 

expert judgment. 
– 98 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Trials in Germany or Spain with different formulations of Spirotetramat and partly with addition of 

RME 500 EW (rape oil methyl ester):

Trial 1: Spirotetramat 240 SC as spray application, 300 L/ha, additive RME 500 EW (rape oil methyl ester), 

2 replicates, Germany 

Results: 

Table B.9.4.6-3: Evaluation of Larval Cell Comb Area Proportion for Trial 1 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 0 DAA* 3 DAA 7 DAA 15 DAA 21 DAA 

untreated (control) 18.3 9.2 6.7 30.8 14.2 

36 g a.i./ha 

+ RME 500 EW, 0.2 % a.i./v 17.5 2.5 5.0 17.5 12.5 

 72 g a.i./ha 

+ RME 500 EW, 0.2 % a.i./v 21.7 1.7 5.0 20.8 20.0 

144 g a.i./ha 

+ RME 500 EW, 0.2 % a.i./v 17.5 1.7 6.7 7.5 16.7 

144 g a.i./ha 23.3 5.8 3.3 20.8 17.5 

Insegar 25 WP, 100 g a.i./ha 

(toxic reference) 18.3 0.8 0.0 18.3 10.0 

DAA: days after application 

* assessment on 0 DAA was done shortly before application 

Effect 

Unclear effect 

Conclusion:

The application of three different rates of Spirotetramat SC 240 in combination with the additive RME 

500 EW (0.2% a.i./v) to flowering Phacelia caused transient effects on honey bee larval abundance. The 

application of Spirotetramat at a rate of 144 g a.i./ha without the additive RME 500 EW caused a less 

pronounced reduction of larval comb area proportion. The reduction of larval comb area proportion was 

visible 3 days after application. Seven days after application no more differences compared with the control 

were observed. The toxic standard showed a clear effect on brood development also 7 DAA. All other brood 

and non-brood endpoints in Spirotetramat treated groups were not impacted and the survival of the colonies 

was not endangered. 

Trial 2: Spirotetramat 240 SC as spray application, 300 L/ha, additive RME 500 EW (rape oil methyl ester), 

2 replicates, Germany 

Results: 
– 99 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.6-4: Evaluation of Larval Cell Comb Area Proportion for Trial 2

Treatment Proportion of comb area covered by larval cells ± SD [%] 

(at X days after application) 

 0 DAA* 3 DAA 7 DAA 15 DAA 21 DAA 

untreated (control) 6.7 7.5 3.3 2.5 1.7 

36 g a.i./ha + 

RME 500 EW, 0.2% a.i./v 10.0 2.7 0.0 0.2 0.0 

72 g a.i./ha + 

RME 500 EW, 0.2% a.i./v 3.3 0.8 2.5 3.7 3.3 

144 g a.i./ha + 

RME 500 EW 0.2% a.i./v 6.0 3.3 4.2 3.3 1.7 

144 g a.i./ha 9.2 7.5 7.5 5.8 5.8 

Insegar 25 WP, 100 g a.i./ha 

(toxic reference) 5.8 1.0 0.0 0.3 0.0 

* assessment on 0 DAA was done shortly before application 

Conclusion:

Spirotetramat SC 240 in combination with 0.2 % a.i./v RME 500 EW caused transient effects on honey bee

larvae when applied to flowering Phacelia at 36 and 72 g a.i./ha. The reduction of larval comb area 

proportion was visible 3 days after application. At the application rate of the tank mixture of 72 g a.i./ha 

differences compared to the control were no longer observed 7 days after application. At an application rate 

of 36 g a.i./ha the effect seemed to last until 7 to 21 days after application. After the application of 

Spirotetramat SC 240 at a rate of 144 g a.i./ha with the additive RME 500 EW an unclear effect was 

observed 7 days after application, and the application at the same rate without the additive RME 500 EW 

caused no reduction of larval cell comb area proportion. The survival of the colonies was not endangered by 

the effects observed. 

Due to unfavourable weather conditions and therefore disadvantageous conditions for appropriate foraging, 

the breeding activity was low and thus the total brood area of the bee hives during the monitoring period until 

the end of the study was quite low, as well, with an overall decreasing tendency in all colonies. 

For spirotetramat SC 240 in combination with 0.2 % a.i./v RME 500 EW at 36 g a.i./ha and 144 g a.i./ha, a 

slight increase in dead pupae was found in front of the bee hives from 4 days after application to 7 days after 

application. The effects were less pronounced compared to the toxic standard. All other (brood and nonbrood related) endpoints were not impacted. 

Trial 3: Spirotetramat 240 SC or 100 OD as spray application, 300 L/ha, additive RME 500 EW (rape oil 

methyl ester), 1 replicate, Germany 
– 100 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Results: 

Table B.9.4.6-5: Evaluation of Larval Cell Comb Area Proportion for Trial 3a 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 0 DAA* 4 DAA 7 DAA 14 DAA 22 DAA 28 DAA 

untreated 

(control) 10.0 6.7 5.0 9.2 8.3 4.2 

240 SC, 144 g a.i./ha + 

RME 500 EW 0.4% a.i./v 8.3 1.0 0.0 0.8 4.2 3.3 

240 SC, 144 g a.i./ha + 

RME 500 EW 0.2% a.i./v 11.7 2.5 2.5 4.2 6.7 4.2 

RME 500 EW, 0.4% a.i./v 9.2 11.7 4.2 4.2 10.0 4.2 

100 OD, 140 g a.i./ha 10.8 2.5 0.5 2.5 5.8 4.2 

Insegar 25 WP, 100 g a.i./ha 

(toxic reference) 10.8 0.8 0.0 2.5 6.7 2.5 

Table B.9.4.6-5 (continued): Evaluation of Larval Cell Comb Area Proportion for Trial 3b 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 0 DAA* 4 DAA 7 DAA 15 DAA 22 DAA 30 DAA 

untreated 

(control) 5.0 7.5 6.7 6.7 4.2 2.5 

240 SC, 144 g a.i./ha + 

RME 500 EW 0.4% a.i./v 7.5 0.0 2.5 2.5 2.5 1.7 

240 SC, 144 g a.i./ha + 

RME 500 EW 0.2% a.i./v 5.8 0.0 0.0 1.7 0.8 2.5 

RME 500 EW, 0.4% a.i./v 6.7 3.3 5.0 5.8 3.3 2.5 

100 OD, 140 g a.i./ha 7.5 0.0 5.0 5.0 4.2 1.7 

Insegar 25 WP, 100 g a.i./ha 

(toxic reference) 9.2 0.0 0.0 2.8 2.5 0.8 

* assessment on 0 DAA was done shortly before application 

Conclusion:

The application of 144 g a.i./ha Spirotetramat SC 240 as a tank mixture with 0.4 % a.i./v RME 500 EW to 

flowering Phacelia caused transient effects on honey bee larvae. The reduction of larval comb area 

proportion was visible from 4 days until 14 days after application in the first trial, whereas in the second trial 

these transient effects were pronounced 4 days after application, but unclear at the following assessment 

days. For the second tank mixture of 144 g a.i./ha Spirotetramat SC 240 plus 0.2 % a.i./v RME 500 EW, 

transient effects on honey bee larvae were observed in one trial up to 22 DAA, more pronounced from 4 

days until 7 days after application; in the first trial effects were unclear. Spirotetramat OD 100 applied at 140 

g a.i./ha to flowering Phacelia led to a short and transient depression in larval abundance in both trials. No 

effect on the larval comb area was observed when 0.4 % a.i./v RME 500 EW was applied alone. In all 

Spirotetramat treatment groups the survival of all colonies was not endangered. 

In the second trial a slight increase in dead pupae found in front of the bee hive 15 days after application was 

found for 144 g a.i./ha Spirotetramat SC 240 as a tank mixture with 0.4 % a.i./v RME 500 EW. The effects 

were less pronounced compared to the toxic reference Insegar. A full recovery was recorded at 22 days after 

application at the latest. All other brood and non-brood related endpoints were not impacted. 
– 101 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Trial 4: Spirotetramat 240 SC or OD 100 as spray application, 300 L/ha, additive RME 500 EW (rape oil 

methyl ester), 1 replicate, Spain 

Results: 

Table B.9.4.6-6: Evaluation of Larval Cell Comb Area Proportion for Trial 4 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 -1 DAA 2 DAA 7 DAA 14 DAA 21 DAA 

untreated 

(control) 3.1 1.0 1.8 5.0 5.6 

240 SC, 144 g a.i./ha + 

RME 500 EW, 0.1% a.i./v 5.0 5.0 1.9 3.4 6.3 

100 OD, 

 140 g a.i./ha 5.0 2.1 0.5 1.9 9.4 

100 OD, 

100 g a.i./ha 8.1 2.8 1.4 6.1 7.5 

Insegar 25 WP, 100 g a.i./ha 

(toxic reference) 3.8 6.3 0.9 2.1 7.5 

Conclusion:

The evaluation of potential effects of the test items on the larval cell comb area proportions is problematic in 

this case and results cannot be unambiguously interpreted, since the larval abundance in the control was 

very low during the exposure phase in the tunnel and the toxic reference did not cause a distinct effect on 

the larvae. In the two Spirotetramat OD 100 treatment groups, the figures for 7 days after application may 

indicate a slight reduction of larval cells, however, this cannot be diagnosed with certainty. 

Trial 5: Spirotetramat OD 150 as spray application, 300 - 400 L/ha, no additive, 1 replicate, Germany 

Results: 

Table B.9.4.6-7: Evaluation of Larval Cell Comb Area Proportion for Trial 5a 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 0 DAA* 3 DAA 8 DAA 15 DAA 21 DAA 29 DAA 

Untreated (control) 16.7 6.7 9.2 23.3 12.5 6.7 

 50 g a.i./ha 19.2 0.0 0.0 11.7 15.0 5.0 

 75 g a.i./ha 18.3 0.0 0.0 11.7 15.0 5.8 

 100 g a.i./ha 19.2 0.0 0.0 7.5 16.7 5.0 

 150 g a.i./ha 20.8 0.0 0.0 13.3 20.8 5.0 

Insegar, 150 g a.i./ha 

(toxic reference) 18.3 0.0 0.0 10.0 13.3 5.0 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 -1 DAA 3 DAA 7 DAA 14 DAA 20 DAA 28 DAA 

untreated (control) 8.3 19.2 10.8 10.0 15.8 6.7 

 50 g a.i./ha 10.0 2.5 0.0 1.7 -** -** 

 75 g a.i./ha 12.5 5.8 0.0 5.8 16.7 10.0 

100 g a.i./ha 13.3 7.5 0.0 11.7 23.3 7.5 
– 102 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 0 DAA* 3 DAA 8 DAA 15 DAA 21 DAA 29 DAA 

150 g a.i./ha 10.0 7.5 0.0 5.8 20.0 13.3 

Insegar 25 WP, 150 g a.i./ha 

(toxic reference) 14.2 0.0 0.0 5.0 20.0 5.8 

* : assessment on 0 DAA was done shortly before application 

-**: no egg-laying activity after loss of the queen (not treatment related) 

Assessments of pupal mortality were performed on -3, -1, 0, 1, 2, 3, 6, 7, 8, 9, 13, 14, 17, 20, 23 and 28 

DAA. Only assessment dates during which increased pupal mortality was recorded, are listed in the following 

table. 

Table B.9.4.6-8: Pupal Mortality Evaluation for Trial 5b 

Treatment Number of dead pupae in bee trap (at X days after application) 

 4 DAA* 7 DAA 10 DAA 14 DAA 15 DAA 

Untreated (control) 0 0 0 0 0 

 50 g a.i./ha 1 12 34 0 1 

 75 g a.i./ha 0 15 35 2 0 

 100 g a.i./ha 6 48 47 2 0 

 150 g a.i./ha 5 43 33 1 1 

Insegar 25 WP, 150 g 

a.i./ha (toxic reference) 2 16 12 27 46 

Treatment Number of dead pupae in bee trap (at X days after application) 

 3 

DAA* 

6 

DAA 

7 

DAA 

8 

DAA 

9 

DAA 

13 

DAA 

14 

DAA 

17 

DAA 

20 

DAA 

Untreated (control) 1 0 0 0 1 0 0 0 0 

 50 g a.i./ha 0 12 13 18 14 2 1 16 0 

 75 g a.i./ha 0 19 15 15 18 4 2 0 0 

 100 g a.i./ha 2 25 20 19 20 3 2 0 0 

 150 g a.i./ha 1 21 25 21 21 3 1 0 0 

Insegar 25 WP,150 g 

a.i./ha (toxic reference) 1 27 21 22 14 0 8 8 2 

*: first observed dead pupae in bee trap 

DAA: days after application 

Conclusion:

In both trials the application of Spirotetramat OD 150 to flowering Phacelia at four different rates (50 g, 75 g, 

100 g and 150 g a.i./ha) caused transient effects to honey bee larvae. The reduction of larval comb area 

proportion was visible from 3 days after application to 8 to 15 days after application. The depression was 

transient and full recovery of the bee colony and brood status was recorded at 20 days after application at 

the latest. 

There was an increase in numbers of dead pupae found in front of the bee hive from 7 days and 6 days after 

application (trial 1, 2) which lasted until 10 and 9 days after application, respectively. A normal pupal 

mortality level was recorded from 14 days after application on. All other brood and non-brood related 

endpoints were not impacted and the survival of all colonies was not endangered. 
– 103 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Trial 6: Spirotetramat OD 150 as spray application, 300 L/ha, no additive, 1 replicate, Germany 

Results: 

Table B.9.4.6-9: Evaluation of Larval Cell Comb Area Proportion for Trial 6 

Treatment 

Proportion of comb area covered by larval cells [%] 

(at X days after beginning of exposure) 

 0 4 DAE 7 DAE 15 DAE 22 DAE 28 DAE 

untreated (control) 13.3 21.7 16.7 8.3 19.2 13.3 

150 g a.i./ha 

20 days prior to flowering 10.8 15.0 10.0 5.0 19.2 23.3 

150 g a.i./ha 

15 days prior to flowering 10.0 11.7 10.0 10.0 18.3 17.5 

150 g a.i./ha 

10 days prior to flowering 16.7 14.2 7.5 8.3 15.8 19.2 

150 g a.i./ha 

5 days prior to flowering 12.5 0.8 1.7 3.5 12.5 12.5 

150 g a.i./ha 

on flowering crop at bee flight 16.7 2.5 5.0 4.2 7.5 11.7 

150 g a.i./ha on flowering crop 

after daily bee flight 11.7 7.5 3.3 5.8 16.7 12.5 

Insegar 25 WP, 150 g a.i./ha 

on flowering crop at bee flight 

(toxic reference) 

12.5 1.7 0.0 0.0 16.7 7.5 

DAE: days after beginning of exposure 

Conclusion:

The application of 150 g a.i./ha of Spirotetramat OD 150 five days prior to flowering (start of exposure) and 

on the flowering crop during and after bee flight caused transient reductions of larval cell comb area

proportion. The effects were different regarding the time of application. 

When applied 5 days before flowering the effects were obvious on the fourth and seventh days of exposure 

and less clear on the 15th day. The application of 150 g Spirotetramat/ha to flowering crop after bee flight 

caused effects on the fourth and the seventh day after beginning of exposure. When Spirotetramat OD 150 

was applied at the same rate during bee flight to the flowering crop, a clear reduction of the larval comb area 

was observed on the fourth and seventh day and less clear effects on the 15th and 22nd day. All other brood 

and non-brood related endpoints were not impacted. 

Spray intervals of 10, 15 and 20 days before flowering of Phacelia did not cause effects to the honey bee 

brood or any other endpoint. 

Trial 7: Spirotetramat OD 150 as spray application, 300 L/ha, no additive, 1 replicate, Germany 

Results: 

Table B.9.4.6-10: Evaluation of Larval Cell Comb Area Proportion for Trial 7 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after beginning of exposure) 

 0 DAE 3 DAE 7 DAE 15 DAE 21 DAE 28 DAE 

untreated 

(control) 20.0 10.8 16.7 21.7 11.7 6.7 

100 g a.i./ha 

20 days prior to flowering 20.8 12.5 4.2 9.2 8.3 7.5 

100 g a.i./ha 

15 days prior to flowering 25.0 10.0 5.0 20.0 15.0 11.7 

100 g a.i./ha 32.5 10.0 10.0 10.8 14.2 15.0 
– 104 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after beginning of exposure) 

 0 DAE 3 DAE 7 DAE 15 DAE 21 DAE 28 DAE 

10 days prior to flowering 

100 g a.i./ha 

5 days prior to flowering 16.7 0.8 4.2 16.7 13.3 8.3 

100 g a.i./ha 

on flowering crop at bee flight 27.5 2.5 0.0 3.3 5.0 4.2 

100 g a.i./ha on flowering crop 

after daily bee flight 22.5 14.2 3.3 10.0 15.8 8.3 

Insegar 25 WP, 150 g a.i./ha 

on flowering crop at bee flight 

(toxic reference) 

17.5 0.8 0.0 10.8 9.2 8.3 

OD: oil dispersion 

DAE: days after beginning of exposure 

Conclusion:

Application of Spirotetramat OD 150 at 100 g a.s./ha 5 days prior to flowering, and application to the

flowering crop after daily bee flight caused transient effects to honey bee brood in terms of reduction of larval 

abundance at 3 and 7 days, or at 7 days after start of exposure, respectively. Spirotetramat OD 150 applied 

at the same rate to the flowering crop during bee flight showed likewise transient effects to honey bee brood 

starting at three days and up to 15 days after start of exposure. A full recovery was observed 21 days after 

start of exposure in this treatment group. Spray intervals of 10 days before flowering of Phacelia did not 

cause effects to the honey bee brood. After application 15 and 20 days before flowering, abundance of 

larvae 7 days after start of exposure was distinctly lower than compared with the larval abundance on the 

day of exposure and in the control group. However, due to the lack of a clear application date - response 

relationship (no effects after application 10 days before flowering) and under consideration of the intrinsic 

variation of this endpoint, it is difficult to unambiguously assess whether these observation have to be 

considered to be treatment effects, in particular since the study was not replicated. Therefore, the figures 

here are classified as “unclear effects”. All other brood or non-brood related endpoints were not impacted. 

Trial 8: Spirotetramat OD 150 as spray application, 300 L/ha, 1 replicate, Spain 

Results: 

Table B.9.4.6-11: Evaluation of Larval Cell Comb Area Proportion for Trial 8 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 -3 DAA 7 DAA 14 DAA 21 DAA 31 DAA 

untreated (control) 9.3 5.6 11.4 10.6 6.0 

75 g a.i./ha 6.3 0.0 0.0 2.4 1.3 

100 g a.i./ha 0.0 0.0 15.0 10.5 6.3 

150 g a.i./ha 0.2 0.0* -* -* -* 

Insegar 25 WP, 150 g a.i./ha (toxic reference) 5.0 0.0 0.0 -* -* 

DAA: days after application 

*: dead bee hive 

Conclusion:

The application of Spirotetramat OD 150 at a rate of 75 g a.i./ha to flowering Phacelia caused transient 

effects on honey bee larvae. No larval comb area was recorded on 7 and 14 days after application. 21 and 
– 105 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

31 days after application the area with larval cells was still rather low, but due to the lack of replication it is 

unclear whether this has to be attributed to natural variability. 

No evaluation of potential effects of the test item on the larval comb area proportions can be given for the 

application rates of 100 and 150 g a.i./ha. At the colony of the lower rate no larvae were found shortly before 

and 7 days after application. This might be explained by a re-queening of the colony since at 14 days after 

application larvae in sufficient proportions were recorded. The colony of the highest application rate had 

nearly no larval cells shortly before the application and died thereafter, which is uncommon for a bee trial and 

might be explained by the presence of a disease (A treatment effect is not to be assumed here, since the 

affected colony did lack any larval stages from the beginning of the study). Presumably, the same happened 

for the colony of the toxic reference, which died 21 days after application. 

Due to the above mentioned observations, the results of this trial have to be interpreted with care. 

Additionally, the overall brood development was weak in all hives (see e.g. bee brood evaluation at -3 days 

after application). Therefore and due to lack of replication, it is difficult to attribute changes in larval 

abundances as seen in this study, to potential treatment effects or rather to natural variation, respectively. 

Trial 9: strawberry; Spirotetramat OD 150 as spray application, 400 L/ha, 1 replicate, Germany 

Results: 

Table B.9.4.6-12: Evaluation of Larval Cell Comb Area Proportion for Trial 9 

Treatment Proportion of comb area covered by larval cells [%] 

(at X days after application) 

 -1 DAA 4 DAA 9 DAA 16 DAA 21 DAA 30 DAA 

untreated 16.7 20.0 9.2 11.2 21.7 11.7 

100 g a.i./ha 9.6 14.6 4.2 7.1 22.9 13.3 

Insegar 25 WP, 150 g a.i./ha 11.7 4.2 1.4 5.2 12.0 8.8 

Conclusion:

After application of Spirotetramat OD 150 at a rate of 100 g a.i./ha to flowering strawberries under tunnel 

conditions, a transient decline of honey bee larval abundance was observed. However, since there was also 

a significant decrease of larval abundance seen in the control group at the same assessment date, it is 

doubtful whether this finding is indicating a treatment-related effect. The survivals of the colonies were not 

endangered at any time. There was a slight increase in dead pupae found in front of the test item bee hives 

at 9 days after application. But the mortalities were less pronounced compared to the toxic reference Insegar 

(1 dead pupa in the control, 11 dead pupae in the test item and 14 in the toxic reference). All other brood and 

non-brood related endpoints were not impacted. 
– 106 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.4.7 Summary of effects on honey-bees 

Table B.9.4.7-1: Summary of acute effects of Spirotetramat (technical and formulated) on honey-bees 

Test substance Exposure route Endpoint Value 

(μg a.s./bee) Reference 

Spirotetramat techn. Oral 

Contact 

48 h LD50 

48 h LD50

> 107.3 

> 100 Schmitzer, 2004a 

Spirotetramat OD150 Oral 

Contact 

48 h LD50 

48 h LD50

91.7 

162 Barth, 2005a 

Table B.9.4.7-2: Summary of semi-field and field data of Spirotetramat effects on honey-bees 

Test substance Exposure route Observed effects Reference 

Spirotetramat OD100 Feeding test, 

mixed in sucrose 

solution at 0.0144 % 

a.s. 

Slightly increased mortality of adults and pupae 

during 21 d after start of feeding 

No effect on flight acivity, bee behaviour. 

Brood development: High termination rate in two 

of three colonies. 

Schur, 2004a 

Spirotetramat SC240 Feeding test, 

mixed in sucrose 

solution at 0.0144 % 

a.s. 

Increased adult mortality. 

Brood development: termination of brood 

development with tendency to recover. 

No effects on flight activity and behaviour of 

bees. 

Schur, 2004b 

Spirotetramat OD100 Semi-field study, 

2 appl. on crop 

before flowering at 

72 g a.s./ha + 2 x 72 

g a.s./ha after flow. 

or 1 x 96g a.s./ha 

no adverse effects on adult and brood mortality, 

condition of the colonies, flight activity and 

behaviour of bees. 

Schur, 2006 

Spirotetramat OD100 Semi-field study 

(tunnel design), 3 

appl. on crop before 

flowering at 72 g 

a.s./ha + 2 x 96 g 

a.s./ha after flow. or 

1 x 96g a.s./ha 

no adverse effects on adult and brood mortality, 

condition of the colonies, flight activity and 

behaviour of bees. 

Irritation of brood development in the earlier 

assessments in one of 3 colonies of the 5x 

treatment, no clear treatment relation, recovery. 

Schur, 2005 

Table B.9.4.7-3: Summary of effects to honey bee brood (larvae) from additional non-GLP studies of 

Spirotetramat 

Trial Country Treatment 

Formulation 

Type and 

content 

Crop Effect Unclear 

effect 

Recovery 

until 

Spirotetramat 36 g a.i./ha 

RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 

Spirotetramat 72 g a.i./ha 

RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 

1 Germany/2

002 

Spirotetramat 144 g a.i./ha 

SC 

240 g a.i./L Phacelia 

- 3 DAA 7 DAA 

Spirotetramat 36 g a.i./ha 

RME 500 EW 0.2% a.i./v 

3 DAA - 

7 DAA 

15 DAA - 

21 DAA - 

Spirotetramat 72 g a.i./ha 

RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 

2 Germany/ 

2002 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.i./v 

SC 

240 g a.i./L Phacelia 

- 3 DAA 7 DAA 
– 107 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Trial Country Treatment 

Formulation 

Type and 

content 

Crop Effect Unclear 

effect 

Recovery 

until 

Spirotetramat 144 g a.i./ha - - - 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.4% a.i./v 

4 DAA - 

14 DAA - 22 DAA 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.i./v - 

4 DAA - 

7 DAA 14 DAA 

RME 500 EW 0.4% a.i./v 

SC 

240 g a.i./L 

- - - 

3a Germany/ 

2002 

Spirotetramat 140 g a.i./ha OD 

100 g a.i./L 

Phacelia 

7 DAA 4 DAA/ 

14 DAA 22 DAA 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.4% a.i./v 4 DAA 7 DAA - 30 DAA - 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.i./v 

4 DAA - 

7 DAA 

15 DAA - 

22 DAA 30 DAA 

RME 500 EW 0.4% a.i./v 

SC 

240 g a.i./L 

- - - 

3b Germany/ 

2003 

Spirotetramat 140 g a.i./ha OD 

100 g a.i./L 

Phacelia 

4 DAA - 7 DAA 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.1% a.i./v 

SC 

240 g a.i./L - 7 DAA 14 DAA 

Spirotetramat 140 g a.i./ha - 7 DAA 14 DAA 4 Spain/ 2003 

Spirotetramat 100 g a.i./ha 

OD 

100 g a.i./L 

Phacelia 

- - - 

Spirotetramat 50 g a.i./ha 3 DAA - 

8 DAA 

15 DAA 21 DAA 

Spirotetramat 75 g a.i./ha 3 DAA - 

8 DAA 

15 DAA 21 DAA 

Spirotetramat 100 g a.i./ha 3 DAA - 

15 DAA 

- 21 DAA 

5a Germany/ 

2004 

Spirotetramat 150 g a.i./ha 

OD 

150 g a.i./L Phacelia 

3 DAA - 

8 DAA 15 DAA 21 DAA 

Spirotetramat 50 g a.i./ha 3 DAA - 

14 DAA 

-

1 

-

1 

Spirotetramat 75 g a.i./ha 3 DAA - 

 7 DAA 

14 DAA 20 DAA 

Spirotetramat 100 g a.i./ha 3 DAA - 

7 DAA 

- 14 DAA 

5b Germany/ 

2004 

Spirotetramat 150 g a.i./ha 

OD 

150 g a.i./L Phacelia 

3 DAA - 

7 DAA 

14 DAA 20 DAA 

Spirotetramat 150 g a.i./ha 

20 days prior to flowering - - - 

Spirotetramat 150 g a.i./ha 

15 days prior to flowering - - - 

Spirotetramat 150 g a.i./ha 

10 days prior to flowering - - - 

Spirotetramat 150 g a.i./ha 

5 days prior to flowering 

4 DAE - 

7 DAE 

15 DAE 22 DAE 

Spirotetramat 150 g a.i./ha 

on flowering crop at bee flight 

4 DAE - 

7 DAE 

15 DAE - 

22 DAE 28 DAE 

6 Germany/ 

2004 

Spirotetramat 150 g a.i./ha 

on flowering crop after bee 

flight

OD 

150 g a.i./L Phacelia 

4 DAE - 

7 DAE - 15 DAE 

7 Germany/ 

2004 

Spirotetramat 150 g a.i./ha 

20 days prior to flowering 

OD 

150 g a.i./L Phacelia - 7 DAE 15 DAE 
– 108 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Trial Country Treatment 

Formulation 

Type and 

content 

Crop Effect Unclear 

effect 

Recovery 

until 

Spirotetramat 150 g a.i./ha 

15 days prior to flowering - 7 DAE 15 DAE 

Spirotetramat 150 g a.i./ha 

10 days prior to flowering - - - 

Spirotetramat 150 g a.i./ha 

5 days prior to flowering 

3 DAE - 

7 DAE - 15 DAE 

Spirotetramat 150 g a.i./ha 

on flowering crop at bee flight 

3 DAE - 

15 DAE 

- 21 DAE 

Spirotetramat 150 g a.i./ha 

on flowering crop after bee 

flight 

7 DAE - 15 DAE 

Spirotetramat 75 g a.i./ha 7 DAA - 

14 DAA 

21 DAA - 

31 DAA - 

Spirotetramat 100 g a.i./ha - - -

8 Spain/ 2004 

Spirotetramat 150 g a.i./ha 

OD 

150 g a.i./L Phacelia 

-

2 

-

2 

-

2 

9 Germany/ 

2007 Spirotetramat 100 g a.i./ha OD 150 g a.i./L 

Strawberry - 9 DAA 16 DAA 

RME: rape oil methyl ester 

DAA: days after application 

DAE: days after beginning of exposure 

-: no influence on bee brood observed

1

 No egg laying activity after loss of the queen (not treatment related) 

2

 bee in hive died presumably not due to treatment related effects 

B.9.4.8 Risk assessment for honey bees 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 
– 109 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.5 Effects on other arthropod species (OECD IIA 8.8, IIIA 10.5) 

B.9.5.1 Laboratory, extended laboratory and semi-field tests (OECD IIA 8.8.1; OECD IIIA 

10.5.1) 

Reference: Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) 

(Hymenoptera: Braconidae) in the laboratory; BYI 08330150 OD

Author(s), year: Waltersdorfer, A.; 2005a 

Report/Doc. number: CW05/023; M-254442-01-1 

Guideline(s): Mead-Briggs et al. 2000 (IOBC) 

GLP: Yes (certified laboratory) 

Deviations: No reproduction assessment. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030/0189(0152), Tox no.: 

TOX07034-00, analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Adult wasps Aphidius rhopalosiphi (Hymenoptera: Braconidae), < 48 h old 

Type of test: Acute contact laboratory test 

Number of organisms: 4 replicates with 15 wasps for each treatment group 

Treatments: Control: deionised water; toxic standard: 0.1 g a.s./ha (37 % Dimethoate, EC); test 

item: 2, 9, 24, 70 and 200 g a.s./ha; deionised water as diluent; treatments applied 

with a calibrated sprayer in 200 L water/ha 

Exposure route, 

duration: 

Adult wasps were exposed to dried residues on treated glass plates for 48 h. The 

test units consisted of two treated glass plates (100 x 100 x 3 mm) and an 

untreated acryl frame (inner size 92 x 92 x 14 mm) with 3 ventilation holes on 

each side. These holes were covered with gauze (mesh size 80 μm) which was 

jammed in the orifices by pieces of Teflon tubing (8 mm outer diameter). 

Feeding: Honey/water solution 

Test conditions: Temperature: 19 - 22°C; relative humidity: 62 - 83 %, 16 h light, 570 – 1001 lux 

Test parameter: Observations of mortality were performed approximately 24 and 48 h after start of 

exposure. The number of alive, affected, moribund and dead wasps was 

recorded. No reproduction assessment. 

Statistics: Mortality corrected according to Abbott. Fisher`s Exact test, two-sided. 

Findings: 

 Results are summarised in table 9.5.1-1. 

Table B.9.5.1-1: Toxicity of dried residues of Spirotetramat 150 OD on glass plates to Aphidius 

rhopalosiphi

Treatment Mean mortality at 48 h [%] Mean corrected mortality at 48 h [%] 

Control 8.3 - 

2 g ai/ha 5 -3.6 

9 g ai/ha 1.7 -7.3 

24 g ai/ha 8.3 0 

70 g ai/ha 21.7 14.5 

200 g ai/ha 86.7 85.5* 

Toxic standard 100 100 

* statistically significant 

Conclusion: 48 h LR50 = 114.676 g a.s./ha; 95 % Confidence Interval: (86 – 145), based on the 
– 110 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

nominal content of the active ingredient 

Reference: Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, 

Phytoseiidae) in the laboratory; BYI 08330150 OD

Author(s), year: Waltersdorfer A., 2005b 

Report/Doc. number: CW05/024; M-254269-01-1 

Guideline(s): Blümel et al. 2000 

GLP: Yes (certified laboratory) 

Deviations: Short deviation in relative humidity. No reproductive assessment. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030/0189(0152), Tox no.: 

TOX07034-00, analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Typhlodromus pyri protonymphs 

Type of test: Acute contact laboratory test 

Number of organisms: 4 replicates with 20 individuals for each treatment group 

Treatments: Control: deionised water; toxic standard: 4 g a.s./ha (37 % Dimethoate); test item: 

0.19, 0.43, 0.97, 2.21 and 5.00 g a.s./ha. The test item was sprayed in 200 L/ha 

on the glasses (after calibration). 

Exposure route, 

duration: 

One exposure unit consisted of two cover slides (glass) which were put together 

at the longer sides with a narrow gap between the slides. Cover slides were 

placed on tissue paper which again was placed on top of a wet foam rubber. To 

avoid the escape of the mites a barrier of sticky material was added. This test 

arena was transferred to a plastic tray filled with deionised water. The narrow gap 

between the two cover slides was filled with water by capillary forces to provide 

the mites with water. The test units were prepared one day before application to 

make sure that the foam rubber was saturated with water. 

Protonymphs were exposed to dried residues for 14 d. 

Feeding: Pollen (birch + pine) 

Test conditions: Temperature: 24 - 25.5°C; relative humidity: 63 - 71 % (very short deviation to 

35°C due to opening of the door), 16 h photo period, 795 – 1060 lux 

Test parameter: The number of dead and living mites was counted on days 1, 3, and 7. The 

number of escaped mites was calculated as the difference from the total number 

exposed. On day 7 the number of females, males, eggs and juveniles was 

counted. 

Statistics: Mortality corrected according to Abbott. Fisher`s Exact test, two-sided. 

Findings: 

 Results are summarised in table 9.5.1-2. The results can be considered as valid 

as the mortality/escaping rate in the control chambers up to day 7 after treatment 

was 3.0%. 
– 111 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.5.1-2: Toxicity of dried residues of Spirotetramat 150 OD on glass plates to Typhlodromus 

pyri

Mortality [%] 7 DAA 

Treatment g a.s./ ha Uncorr. Abbott P-Value(*) 

Control 0 3.0 0 - 

Test item 0.19 13.0 10.3 0.016 sign. 

Test item 0.43 72.0 71.1 <0.0001 sign. 

Test item 0.97 100 100 <0.0001 sign. 

Test item 2.21 100 100 <0.0001 sign. 

Test item (DAA 1) 5 100 100 <0.0001 sign. 

Reference item 4 88.0 87.6 - 

n.d. not determined 

Conclusion: 48 h LR50 = 0.333 g a.s./ha; 95 % Confidence Interval: (0.298 – 0.367) 
– 112 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Extended laboratory studies 

Reference: Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) 

(Hymenoptera: Braconidae) using an extended laboratory test; BYI 08330 150 

OD.

Author(s), year: Waltersdorfer A., 2006a 

Report/Doc. number: CW05/071; M-264179-01-1 

Guideline(s): Mead-Briggs et al. 2000; Mead-Briggs et al. 2002 

GLP: Yes (certified laboratory) 

Deviations: Increase in temperature due to technical reasons 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030/0189, Tox no.: TOX07034-

00; (0152), analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Adult wasps Aphidius rhopalosiphi (Hymenoptera: Braconidae) 

Type of test: Acute contact extended laboratory test 

Number of organisms: 6 replicates with 5 females for each treatment group 

Treatments: Control: deionised water; toxic standard: 2.5 g a.s./ha (37 % Dimethoate); test 

item: 22, 42, 80, 151 and 288 g a.s./ha, deionised water as diluent. The 

substances were applied in the equivalent of 400 L/ha after calibration of the 

sprayer. 

Exposure route, 

procedure: 

Adult wasps were exposed to freshly dried residues on barley plants for 48 h. 

One day after the start of the study, oat plants were infected with Rhopalosiphum

padi. 

The test units consisted of a pot with treated barley plants which were enclosed 

within a clear polyacrylic cylinder with a hole for the introduction of the parasitoids. 

After introduction the hole was closed by a stopper. The top of the cylinder was 

closed with fine mesh gauze. The test animals were introduced from prepared test 

tubes by slightly blowing them. When all wasps were in the test unit, the glass 

tube was removed and the orifice closed with a feeding stopper. During the 

exposure phase of 2 days the test animals had access to the sugar solution. 

From the water control and the dose rates 80, 151 and 288 g a.s./ha, 15 

impartially chosen females per treatment were each transferred to a cylinder 

containing untreated cereal plants infested with Rhopalosiphum padi for a period 

of 24 hours. The number of mummies was assessed 11 days later. 

Feeding: Honey/water solution 

Test conditions: Temperature: 19 – 23.5°C; relative humidity: 62 - 85 %, photo period 16 h, 688 – 

1718 lux in the parasitation phase, 5590 – 19730 lux in the reproduction phase 

Test parameter: Mortality was assessed after 2, 24 and 48 h. The number of alive, affected, 

moribund and dead wasps was recorded. Repellence of the test item was 

determined during the initial 3 h after the release of the females. Five separate 

observations were made at 30-minute intervals starting approximately 15 - 30 min 

after the introduction of all wasps. 

Reproduction assessment: number of mummies. 

Statistics: Mortality corrected according to Schneider-Orelli. One-way ANOVA, p-values are 

adjusted according to Dunnett.

Findings: 

 All validity criteria were met. No dose related repellent effect of the test item was 

observed. Results are summarised in table 9.5.1-3. 
– 113 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.5.1-3: Mortality and reproduction after exposure of Aphidius rhopalosiphi to freshly dried 

residues of Spirotetramat OD 150 applied onto barley plants 

 Mortality Reproduction Repellency 

Treatment Uncorr. Abbott Rate Rel. to 

control 

[%] 

P-value * % wasps 

on plant 

Rel. to 

control 

[%] 

P-value 

Control 0 0 23 0 - 75.3 - - 

22 g a.s./ha 3.3 3.3 n.d.** n.d.** - 75.1 0.4 1 (n.s.) 

42 g a.s./ha 3.3 3.3 n.d.** n.d.** - 69.0 8.4 0.892 (n.s.) 

80 g a.s./ha 0 0 29.5 - 28.1 0.435 (n.s.) 56.7 24.8 0.071 (n.s.) 

151 g a.s./ha 6.7 6.7 24.6 - 7 0.967 (n.s.) 60.7 19.5 0.195 (n.s.) 

288 g a.s./ha 0 0 16.6 27.8 0.265 (n.s.) 60.6 19.5 0.244 (n.s.) 

Reference 80 80 n.d.* n.d.* n.d.* 70.3 6.6 - 

n.s. not significant *:n.d. not detected, **:n.d. not detected due to capacity reasons 

Conclusion: 48 h LR50 > 288 g a.s./ha. 

There was no reduction in reproductive success relative to the control at the 

tested treatment rates. 

Reference: Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, 

Phytoseiidae) using an extended laboratory test BYI 08330150 OD.

Author(s), year: Waltersdorfer A., 2005c 

Report/Doc. number: CW05/030; M-256306-01-1 

Guideline(s): Blümel et al. 2000 

GLP: Yes (certified laboratory) 

Deviations: very short declines in rel. humidity due to opening of the door 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; 

Batch no.: 08030/0189, (0152), Tox no.: TOX07034-00; 

analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Typhlodromus pyri protonymphs 

Type of test: Acute contact extended laboratory test 

Number of organisms: 4 replicates with 20 individuals for each treatment group 

Treatments: Control: deionised water; toxic standard: 39.5 g a.s./ha (37 % Dimethoate EC); 

test item: 0.15, 0.51, 1.7, 5.9 and 20 g a.s./ha. The test concentrations and the 

suspension for the reference item were prepared stepwise on the day of 

application. Deionised water was used as diluent. The items were applied to 

Phaseolus vulgaris leaves in the equivalent of 200 L/ha using a sprayer after 

calibration. 

Exposure route, 

duration: 

Protonymphs were exposed to freshly dried residues on leaves of Phaseolus 

vulgaris for 14 d. 

Feeding: Pollen (birch, pine). The water supply for the mites was insured sticking a pin into 

each of the leaves. Additional food was added on days 3, 7 and 12. 

Test conditions: Temperature: 24 - 25°C; relative humidity: 67 - 72 % (with short declines to 40 % 

due to opening of the door), 16 h photo period, 968 – 1026 lux 

Test parameters, 

procedure: 

One exposure unit consisted of an intact leaf of Phaseolus vulgaris, which was 

laid after application on a layer of wet filter paper on top of a water soaked foam. 

A circle of insect glue (ø approx. 40 mm) was formed on the leaves. Sets of such 
– 114 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

cylinders were placed on a plastic tray such that the filter paper was constantly 

provided with deionised water. 

After the test units were set up (day 0) the protonymphs were placed onto the 

plant surface. The mites were transferred with a fine brush under a 

stereomicroscope and immediately afterwards examined to ensure they were 

undamaged and in good health. 

Mortality of 80 protonymphs was assessed 1, 3, 7, 10, 12 and 14 days after 

exposure by counting the number of living and dead mites. The number of 

escaped mites was calculated as the difference from the total number exposed. 

Additionally on days 7, 10, 12 and 14 the number of females, males, eggs and 

juveniles was counted. Eggs and juveniles were removed. The reproduction rate 

of surviving mites was evaluated over the period of 7-14 days after treatment by 

counting the total number of females and eggs. 

Statistics: Mortality corrected according to Schneider-Orelli. Fisher`s Exact test, two-sided, 

p-values are adjusted according to Bonferroni-Holm; one-way ANOVA, p-values 

adjusted according to Dunnett 

Findings: 

 Results are summarised in table 9.5.1-4. All validity criteria were met. 

Table B.9.5.1-4: Mortality and reproduction after exposure of Typhlodromus pyri to freshly dried 

residues of Spirotetramat OD 150 applied onto bean leaves 

Mortality Reproduction 

Treatment Uncorr. Corr. Schn.-

Orelli 

P-value * Rate Rel. to control 

[%] 

P-value ** 

Control 7.5 - - 7.3 - 

0.15 g a.s./ha 11.3 4.1 n.s. 4.1 43.2 0.081 (n.s.) 

0.51 g a.s./ha 8.8 1.4 n.s. 3.7 48.7 0.060 (n.s.) 

1.7 g a.s./ha 60.0 56.8 <0.0001 sign. n.d. / / 

5.9 g a.s./ha 98.8 98.6 <0.0001 sign. n.d. / / 

20 g a.s./ha 100 100 <0.0001 sign. n.d. / / 

Reference 100 100 / / 

n.s. not significant, sign. = significant, * Fisher`s Exact test, ** one-way ANOVA 

Conclusion: 7 d LR50 1.588 g a.s./ha (95 % CI: 1.311 – 1.860) 

There was a significant reduction of reproduction at the low rates of 0.15 and 0.51 

g a.s./ha. 

Reference: BYI 08330 150 OD; Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN 

(Acari, Phytoseiidae) using an extended laboratory test (under semi-field 

conditions aged residues on apple trees)

Author(s), year: Waltersdorfer A., 2006c 

Report/Doc. number: CW05/028 

Guideline(s): Blümel et al. 2000 

GLP: Yes (certified laboratory) 

Deviations: The test design was modified - potted apple trees were treated instead of glass 

plates; mites were exposed to freshly applied and under semi-field conditions 

aged residues on excised apple leaf discs 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; 

Batch no.: 08030/0189(0152), Tox no.: TOX07034-00; product code: AE 1302943 
– 115 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

00 OD15 A101; analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Typhlodromus pyri protonymphs 

Type of test: Acute + aged residue contact extended laboratory test 

Number of organisms: 100 protonymphs in each treatment group (5 replicates with 20 individuals) 

Treatments: Control: deionised water; toxic standard: 40 g a.s./ha (37 % Dimethoate EC) in 

200 L water; test item: 4 x 72 g a.s./ha applied to potted apple trees (7 days 

interval) (Malus sylvestris) under semi-field conditions in 600 L water/ha using a 

plot sprayer after calibration. 

Exposure route, 

duration: 

Protonymphs were exposed to aged residues on apple leaves under laboratory 

conditions for 7 d. Aging of the spray residues on the potted apple trees took 

place under natural semi-field conditions with rain protection during the whole 

study. 

Feeding: Pollen (birch, pine)

Test conditions: Weather data (temperature and rel. humidity) in the outdoor area were recorded. 

Lab conditions: Temperature: 24.5 - 28°C; relative humidity: 61 - 73 %, 990 – 

1567 lux 

Test parameters, 

procedure: 

Bioassays were initiated on DAA (days after last test substance application) 0; 7; 

14; 21; 28; 42; 49 and 56: leaves were taken from the apple trees, and predatory 

mites were exposed on these leaves. In each bioassay, mortality was assessed 

over a period of 7 days after exposure on excised leaf discs by counting the 

number of living and dead mites for each bioassay on days 1, 3, 7, 10, 12 and 14. 

One exposure unit consisted of an intact apple leaf, which was laid on a layer wet 

filter paper on top of a water soaked foam. A circle of insect glue was formed on 

the leaves. These sets were placed on a plastic tray such that the filter paper was 

constantly provided with deionised water. The number of escaped mites was 

calculated as the difference from the total number exposed. At day +7 of each 

test, percentage mortality (including escapers) was calculated as mean of the 4 

replicates. 

The reproduction rate of surviving mites was then evaluated over the period of 7-

14 days by counting the total number of offspring (eggs and larvae) produced. 

From these data the endpoints mortality and effects on reproduction were 

calculated. 

Statistics: Mortality corrected according to Schneider-Orelli. 

Findings: 

 The results are considered as valid since the mortality/escaping value for the 

control stated in the laboratory method on glass was reached in this study 

(< 20 %) and the average number of eggs/female (calculated as sum of 4 

assessment dates – from day 7 on) in the control group exceeded 4 eggs per 

female. 

The mean corrected mortality of the nymphs, and the mean reproduction rate of 

the surviving females exposed to the test item and the toxic reference are given in 

the tables below: 
– 116 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.5.1-5: Mortality of T. pyri after exposure to freshly applied and aged residues of 

Spirotetramat OD 150 on apple leave discs 

Days after last application 0 7 14 21 28 42 49 56 

Treatment Mortality after 7 days (%) 

Control 5 4 5 17 6 4 3 7 

Corr. Mortality [%] 

Test item 93.7 100 90.5 92.8 100 58.3 50.5 10.8 

Reference item (DAA1) 100 100 100 100 100 100 100 100 

DAA: days after last application 

Table B.9.5.1-6: Reproduction of T. pyri after exposure to freshly applied and aged residues of 

Spirotetramat OD 150 on apple leave discs 

Treatment Control Test item 

 Mean no. of eggs 

/female 

Mean no. of eggs 

/female 

Reduction rel. to control [%] 

DAA 0 to day 42 n.d. n.d. n.d. 

DAA 49 5.19 5.94 -14.48 

DAA 56 6.88 7.87 -14.35 

n.d.: not determined 

Conclusion: In this extended laboratory test the lethal and sublethal effects of Spirotetramat 

OD 150 residues (aged under semi-field conditions) on Typhlodromus pyri were 

determined after application of 4 x 72 g a.s./ha onto apple trees. 58.3, 50.5 and 

10.8 % corr. mortality were found on DAA 42, 49 and DAA56. After 7 weeks of 

aging, no effects > 11 % on survival or reproduction were observed any longer. 

Reference: Dose-response toxicity (LR50) of BYI 08330 150 OD to the green lacewing 

Chrysoperla carnea (STEPH.) under extended laboratory conditions (including 

food-application).

Author(s), year: Röhlig, U.; 2005 

Report/Doc. number: 051048082; M-262650-01-1 

Guideline(s): Vogt et al. 2000 (IOBC), modified 

GLP: Yes (certified laboratory) 

Deviations: Guideline modified for the extended laboratory test (exposure on natural 

substrate) in such a way that bean leaves were used instead of glass plates and 

that treated food was given 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; 

Batch no.: 08030/0189 (0152), Tox no.: TOX07034-00; 

analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Chrysoperla carnea, larvae, age 2-3 d (L1) 

Type of test: Acute contact extended laboratory test 

Number of organisms: 40 replicates with 1 larvae for each treatment group 

Treatments: Control: deionised water; toxic standard: 30 mL product/ha (Dimethoate EC400); 

test item: 44, 72, 112, 184 and 288 g a.s./ha; deionised water as diluent. The test 

substance was applied in 200 L/ha. 

Exposure route, Larvae were exposed to freshly dried residues on bean leaves and Sitotroga eggs 
– 117 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

duration: until pupation. 

Feeding: Larvae were fed with treated eggs of Sitotroga cerealella 3 times per week, adults 

with artificial food (diet according to the guideline) at each assessment day. 

Test conditions: Temperature: 23 - 26°C; relative humidity: 66 - 77 %, 16 h photo period, 2100 lux 

Test parameters, 

procedure: 

The test consisted of two phases. In the exposure phase of the test, mortality of 

the larvae and pupae and adult emergence was determined following exposure to 

dried spray deposits on bean leaves and feeding with treated Sitotroga eggs. In 

the reproduction phase, the sublethal effects on reproductive performance (egg 

laying and hatching success) were assessed. 

Exposure cages consisted of glass cylinders with gauze cover and a treated bean 

leaf on moistened filter paper as bottom. Cages were fixed to a glass plate and an 

acrylic plate. Oviposition cages consisted of glass beakers covered with cotton 

gauze during egg laying. Hatching cages for F1 larvae consisted of plastic cages 

with clear cover. Oviposition was started one week after the first egg laying was 

observed (pre-oviposition period). 

Test plants were kidney beans (Phaseolus vulgaris), grown in the laboratory of the 

test facility under controlled conditions. Primary leaves were cut shortly before the 

application, when the plants were 3 weeks old. 

The number of surviving larvae and hatched adults, as well as the number of eggs 

laid and larvae hatched (F1) were recorded over a period of 43 days. From these 

data the endpoints mortality and fecundity were calculated. 

Statistics: Mortality corrected according to Abbott. Statistical analysis of mortality data 

(Fisher`s Exact Binomial Test, 1-sided, p ≤ 0.05). The software ToxRat 

Professional, Version 2.09 (RATTE, 2004) was used. 

Findings: 

 All validity criteria were met. Results are summarised in table 9.5.1-7. 

Statistical analysis revealed no significant differences concerning mortality 

between control and test item treatment groups. Also the reproductive output was 

above the lower limit given as validity criterion for the glass plate method (≥ 15 

eggs/female/d) and therefore considered as not impacted by the treatment. 

Table B.9.5.1-7: Toxicity of dried residues of Spirotetramat 150 OD on Chrysoperla carnea in an 

extended laboratory test

Reproduction 

Fecundity Fertility 

Treatment Mortality after 

24 days 

[%] [mean number of eggs/ 

female] 

mean hatching rate 

[%]

Control 5.0 18.9 81 

Application rate 

[g a.s./ha]

Corrected mortality [%] 

44 5.3 19.5 80 

72 7.9 18.7 81 

112 2.6 20.2 81 

184 0 18.6 80 

288 5.3 19.0 80 

Reference item 63.2 - - 

LR50 > 288 g a.s./ha 

Conclusion: LR50 > 288 g a.s./ha. 

No negative effects on reproduction after exposure to rates up to 288 g a.s. 

Spirotetramat 150 OD were observed. 
– 118 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Reference: Toxicity to the ladybird beetle Coccinella septempunctata L. (Coleoptera, 

Coccinellidae) using an extended laboratory test including exposure to and oral 

uptake of BYI 08330 150 OD.

Author(s), year: Waltersdorfer, A.; 2006b 

Report/Doc. number: CW05/053; M-264175-01-1 

Guideline(s): Schmuck et al., 2000 (IOBC), modified

GLP: Yes (certified laboratory) 

Deviations: Guideline modified for the extended laboratory test in such a way that bean leaves 

were used for exposure instead of glass plates; in addition, the larvae got treated 

food (aphids) for 4 days of the study to include a possible oral uptake of the test 

item and afterwards untreated food was given; on day 7 aphids on new plants 

were sprayed and this food was offered to larvae which had not yet pupated. 

Validity: Acceptable. The validity criterion for control mortality was slightly not met. 

However, validity criteria have been defined for glass plate studies only and 

considering the special design the study can be considered valid. 

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; 

Batch no.: 08030/0189 (0152), Tox no.: TOX07034-00; 

analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: Coccinella septempunctata, larvae, 4 d old 

Type of test: Acute contact extended laboratory test 

Number of organisms: 40 replicates with 1 larvae for each treatment group 

Treatments: Control: tap water; toxic standard: 80 mL product/ha (Dimethoate EC37); test 

item: 33, 57, 97, 168 and 288 g a.s./ha; tap water as diluent. The test substance 

was applied in 200 L/ha. Application was done using a specially constructed 

sprayer, permitting application on a defined area. 

Exposure route, 

duration: 

Larvae were exposed to freshly dried residues on bean leaves and Sitotroga eggs 

until pupation. 

Feeding: Larvae were fed with aphids (Acyrthosiphon pisum) ad libitum which had been 

treated for four days and afterwards fed with untreated aphids. Adults were fed 

with aphids and pollen. 

Test conditions: Temperature: 23.6 – 26.6°C; relative humidity: 42 - 85 %, 16 h photo period, 1050 

- 1760 lux 

Test parameters, 

procedure: 

Exposure units consisted of a detached leaf of Phaseolus vulgaris, which was laid 

on a wet cotton wool pad in a petri dish (9 cm diameter) after application. A 

constant supply of deionised water from the bottom was assured. Per individual 

test unit one steel ring (about 3 cm diameter and 3 cm high) was placed on the 

leaf. The top inner edge of the rings was coated with a lubricant (Hostaflon) in 

order to prevent the larvae from escaping. 

Once dry, the steel rings were placed onto them into the petri dishes and one 

larva was added to each test unit. 

Additionally potted bean plants with aphids (Acyrthosiphon pisum on Vicia faba) 

were sprayed on the day of application with the corresponding rates of the test 

item. The aphids were brushed from the leaves and were used as food for 4 days. 

Afterwards untreated aphids were fed until day 7. On day 7 new food was sprayed 

and offered to those larvae which had not yet pupated. After 3 days the remaining 

larvae and the adults were fed with untreated food.
– 119 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

All test units were assessed daily and the condition of the beetles recorded. At 

every feeding session, dead aphids and exuviae from earlier feeding sessions 

were removed, in order to maintain a constant contact between the larvae and the 

treated surface. Once the larvae had pupated, the emerged beetles were 

transferred to plastic boxes at the day of their individual hatching. 

After this feeding period all beetles were transferred to glass jars. Their sex was 

determined and fresh stems of bean plants with aphids and untreated pollen were 

introduced as required. Sheets of black paper were offered to the beetles for egglaying, which was checked daily. Pieces of paper with freshly laid eggs were cut 

out of the paper and stored in petri dishes. Mortality during the egg laying period 

was recorded and the sex of the dead animals was determined. 

Statistics: Mortality corrected according to Schneider-Orelli. 

Findings: 

 The mortality value for the control was slightly above that mentioned in the 

guideline for glass plate studies (32.5 % mortality instead of max. 30 %). 

However, validity criteria have been defined for glass plate studies only and 

considering the special design the study can be considered valid. 

As recommended by the guideline (for glass plate studies) the reproductive 

performance parameters can only be evaluated qualitatively, due to the very high 

species-inherent variability in egg-laying performance. As all values for the three 

highest and the lowest dose rate were within the historical data base for control 

beetles (≥ 2 fertile eggs per female and day), this parameter is considered as not 

impacted by the test item rates. 

Table B.9.5.1-8: Mortality and reproduction after exposure of Coccinella septempunctata to freshly 

dried residues of Spirotetramat OD 150 applied onto detached bean leaves: 

Mortality [%] Reproduction 

Treatment [g a.s./ha] Uncorr. corrected Fertility (hatching 

rate) [%] 

Fertile eggs per 

female and day 

Control (tap water) - 32.5 - 99.3 6.0 

BYI 08330 150 OD 33 22.5 -14.8 88.9 3.8 

BYI 08330 150 OD 57 17.5 -22.2 91.7 1.8 

BYI 08330 150 OD 97 32.5 0 89.3 3.6 

BYI 08330 150 OD 168 47.5 22.2 99.0 2.0 

BYI 08330 150 OD 288 50.0 25.9 86.7 4.1 

Reference item 80 mL prod./ha 100 100 n.d.* n.d.* 

LR50 > 288 g a.s./ha 

* n.d.: not determined 

Conclusion: LR50 > 288 g a.s./ha. 

No negative effects on reproduction after exposure to rates up to 288 g a.s. 

Spirotetramat 150 OD were observed. 
– 120 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.5.2 Semi-field or field tests (OECD IIIA 10.5.3) 

Reference: Evaluating effects of BYI 08330 OD 150 applications on mite fauna (Acari) in 

the field (grape vines, France). 

Author(s), year: Aldershof, S.; 2006 

Report/Doc. number: B127AFG; M-269107-01-1 

Guideline(s): Blümel et al., 2000; Hassan, 1985 ; Candolfi et al., 2000 

GLP: Yes (certified laboratory) 

Deviations: No 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) OD 150, purity: 148.89 g a.s./L (analysed), density 

0.986 g/mL, batch no. 08030/0189(0152); sample no. TOX07034-00, development 

no. 30-00364846 

Material and Methods: 

Test species: Predatory mites in grapes 

Type of test: Field study 

Location: Commercial vineyard in Fources in the Armagnac region, South-West France 

Test design Treatments: water control; reference item: Dimethoate applied at 1 L product/ha 

(400 g a.s./L EC) applied during the first and the last application of the fourth test 

scenario (3 week interval); test item: randomised design with 5 replicates per 

treatment. Application volumes were 500 L/ha; spraying equipment was calibrated 

before use. Plot layout: size: 20 m length, 40 m2

 surface. Plots consisted of 20 

adjacent vine positions. Plots were separated by untreated vines or a corridor of at 

least 5 m. The first applications were performed at BBCH 65 (flowering), the last 

applications (scenario 4) at BBCH 73. Crown height was 1.5 to 2 m. 

Applied concentrations: An overview on the application rates is provided in table B.9.5.2-1. 

Table B.9.5.2-1: Spray scenarios 

Scenario Description No of applications Spray interval Nominal Rate 

1 Full (2x) 2 2 weeks 96 g a.s./ha 

2 Drift 1 

(2x high) 

2 2 weeks Application 1: 36.4 g a.s./ha 

Application 2: 35 g a.s./ha 

3 Drift 2 (2x low) 2 2 weeks 9.1 g a.s./ha 

4 Drift 3 (4x low) 4 1 week 4.8 g a.s./ha 

Test conditions: Throughout the experiment the climatic conditions (rel. humidity, temperature) 

were measured at the test site with a portable device. Rainfall data were obtained 

from a local weather station. 

Sampling: The effects of BYI 08330 OD 150 were expressed in terms of population changes 

relative to the water control. Population samples were taken shortly before each 

application, 1 week after the first application and approximately 1, 4 and 8 or 9 

weeks after the last application. For test item scenario 4 and water an additional 

sample was taken 11 weeks after the last treatment. The evaluation was based on 

time to recovery (= population density similar to control). 

Three leaves of average size were collected from each of the 8 innermost vines of 

a plot ( 

24 leaves per plot). The material collected from one plot was pooled for each 

sampling point. 

The leaf washing method according to Boller (1984) was applied. The collected 
– 121 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

arthropods were inspected under a binocular microscope. The first 16 phytoseiid 

mites observed in each plot were mounted for species identification. 

Statistics: Effect values were calculated from total phytoseiid mite numbers according to 

Abbott (1925) and Henderson-Tilton (1955). In addition, total phytoseiid mite 

numbers were analysed statistically using the non-parametric Man-Whitney U test. 

Findings:

Test conditions: Weather parameters were in an acceptable range; there was no rain on the 

application days. 

Predatory mites: Several acarine taxa were identified. The most abundant taxon prevailing in the 

vineyard was the predatory mite family Phytoseiidae. Phytoseiid populations 

almost exclusively consisted of the species Typhlodromus pyri (98 % - 100 % 

throughout the study period). Other mite taxa encountered were Eriophyoidea 

(rust mites), Tydeidae, Tarsonemidae and Oribatida (fungivorous mites), however, 

their numbers were too low for evaluation. 

BYI 08330 OD 150 had no effect on any of the taxa found, in any of the spray 

scenarios tested. No statistically significant differences with the water control were 

found for any of the BYI 08330 OD 150 treatments on all sampling dates, except 

on 16 June, 5 days after the first treatment, when the 3rd drift rate (4 applications 

at 4.8 g a.s./ha) showed an effect value of 41 % (Abbott) or 31 % (HendersonTilton). However, on later sampling dates this treatment no longer showed 

statistically significant effects. Moreover, Abbott effects remained below 50 % 

(Henderson-Tilton effects below 40 %) throughout the study. This finding was 

probably due to natural variation of mite field populations, rather than to an actual 

treatment effect. Population dynamics observed in other test item treatment plots 

closely resembled fluctuations detected in water control plots. The different spray 

scenarios of the test item neither induced effects on Eriopheiid mites. 

The reference item severely affected Phytoseiid mites in a negative way, with a 

statistically significant reduction in phytoseiid numbers of 43 % to 96 %. Thus, the 

trial is considered valid for the purpose of evaluating potential consequences of 

test item treatments to predatory mites. Populations of Eriophydoidea could 

however increase, probably due to reduced predation by phytoseiids. 

All effects in the reference item group were statistically significantly different to the 

control. 
– 122 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.5.2-2: Summary results 

08.06.05 16.06.05 02.07.05 12.07.05 25.07.05 01.08.05 29.08.05 

Abbott values 

drift 1 (high 2x) -1.5 % -11.4 % -5.8 % 2.1 % 

drift 2 (low 2x) 17.3 % 27.6 % 14.2 % -1.0 % 

drift 3 (low 4x) 13.9 % 41.1 % 43.8 % 31.9 % -17.2 % 

full (2x) 4.1 % 16.3 % 36.2 % 13.9 % 12.8 % 

reference 18.9 % 43.1 % 94.1 % 95.6 % 

Henderson-Tilton values 

drift 1 (high 2x) -10.1 % -4.4 % 3.5 % 

drift 2 (low 2x) 12.5 % -3.8 % -18.2 % 

drift 3 (low 4x) 31.7 % 34.7 % 21.0 % -28.7 % 

full (2x) 12.7 % 33.4 % 10.3 % 94.6 % 9.0 % 

reference 29.8 % 92.7 % 

P-values (Mann-Whitney U test, comparison to water) 

drift 1 (high 2x) 0.834 0.965 0.917 0.917 

drift 2 (low 2x) 0.175 0.076 0.465 0.917 

drift 3 (low 4x) 0.465 0.028 0.327 0.117 0.754 

full (2x) 0.754 0.347 0.175 0.465 0.754 

reference 0.347 0.009 0.014 0.009 

P-values in bold italics are statistically significant (P < 0.05) 

Conclusion: It is concluded that Spirotetramat OD 150, applied 2 times at a nominal rate of 96 

g a.s./ha or lower, or 4 times at a nominal rate of 4.8 g a.s./ha, has no effects on 

the acarine mite fauna detected with leaf sampling in vineyards in France. 

B.9.5.3 Summary 

Table B.9.5.3-1: Summary of effects of spirotetramat on non-target-arthropods (lab + ext lab) 

Test species Exposure Test item 

Rate 

[g a.s./ha] 

Type of effect Effect [%] Reference 

Corrected 

mortality 

-3.6 

-7.3 

0 

14.5 

85.5 

Aphidius 

rhopalosiphi 

adults

contact with 

dried residues 

on treated 

glass plates 

Spirotetramat 

150 OD 

2 

9 

24 

70 

200 

48 h LR50 = 114.7 g a.s./ha 

Waltersdorfer 

2005a 

Corrected 

mortality 

10.3 

71.1 

100 

100 

100 

Typhlodromus 

pyri 

protonymphs

contact with 

dried residues 

on treated 

glass plates 

Spirotetramat 

150 OD 

0.19 

0.43 

0.97 

2.21 

5 

48 h LR50 = 0.333 g a.s./ha 

Waltersdorfer 

2005b 

Corrected 

mortality / 

reproduction 

3.3 / n.p. 

3.3 / n.p. 

0 / -28.1 

6.7 / -7 

0 / 27.8 

Aphidius 

rhopalosiphi 

adults

contact with 

dried residues 

on treated 

barley plants 

Spirotetramat 

150 OD 

22 

42 

80 

151 

288 

48 h LR50 > 288 g a.s./ha 

Waltersdorfer 

2006a 
– 123 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test species Exposure Test item 

Rate 

[g a.s./ha] 

Type of effect Effect [%] Reference 

Typhlodromus 

pyri 

protonymphs

contact with 

dried residues 

on treated 

bean leaves 

Spirotetramat 

150 OD 

0.15 

0.51 

1.7 

5.9 

20 

Corrected 

mortality / 

reproduction 

4.1 / 43.2 

1.4 / 48.7 

56.8 / n.d. 

98.6 / n.d. 

100 / n.d. 

Waltersdorfer 

2005c 

Typhlodromus 

pyri 

protonymphs

contact with 

dried residues 

(acute and 

aged) on 

treated apple 

leaves 

Spirotetramat 

150 OD 

4 x 72 (7d) 

Corrected 

mortality 

aged 0d: 93.7 

aged 7d: 100 

aged 14d: 90.5

aged 21d: 92.8

aged 28d: 100

aged 42d: 58.3

aged 49d: 50.5

aged 56d: 10.8

no reduction of 

reproduction on 

DAA 49+56 

Waltersdorfer 

2006c 

Chrysoperla 

carnea 

larvae

contact with 

dried residues 

on treated 

bean leaves 

Spirotetramat 

150 OD 

44 

72 

112 

184 

288 

Corrected 

mortality 

5.3 

7.9 

2.6 

0 

5.3 

no effect on 

reproduction 

Roehlig 2005 

Coccinella 

septempunctata

larvae 

contact with 

dried residues 

on treated 

bean leaves, 

feeding with 

treated aphids 

Spirotetramat 

150 OD 

33 

57 

97 

168 

288 

Corrected 

mortality 

-14.8 

-22.2 

0 

22.2 

25.9 

no effect on 

reproduction 

Waltersdorfer 

2006b 

n.d. not determined, n.p. no assessment performed 

negative figures indicate a positive effect compared to the control 

B.9.5.4 Risk assessment for non-target arthropods 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 
– 124 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.6 Effects on earthworms (OECD IIA 8.9, IIIA 10.6.3) 

B.9.6.1 Acute toxicity (OECD IIA 8.9.1, IIIA 10.6.1.1) 

Active substance 

Reference: BYI 08830 (techn.): Acute toxicity to earthworms (Eisenia fetida) tested in 

artificial soil with 5 % peat 

Author(s), year: Lechelt-Kunze, C., 2004a 

Report/Doc. number: LKC/RG-A-18/03, Edition Number: M-104105-01-2, 07.01.2004 

Guideline(s): OECD 207 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.5 %, batch: 08045/0014 

Material and methods: 

Test species: Earthworm (Eisenia fetida) 

 Number of organisms: 4 replicates each with 10 individuals per treatment and control 

 Weight, age: Average weight: 360 mg/worm, clitellate adults > 2 months 

Type of test, duration: Laboratory acute test, 14 days 

Applied concentrations: Nominal: 0 (control, quartz sand), 32, 100, 178, 316, 562, 1000 mg/kg dw soil 

 Solvent/vehicle: Quartz sand 

Toxic standard: 2-Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mg/kg dw soil 

Test conditions: 

 Test substrate: 5 % sphagnum peat, 20 % kaolinite clay, 74 % quartz sand, about 0.2 – 1 % 

CaCO3 to adjust pH to 6.0 ± 0.5, max. WHC between 40 - 60 % , pH and the water 

content were measured at the beginning and the end of the study 

 Substrate/test vessel: 625 g wet weight (500 g dw) in 1.5 L test containers 

 Temperature: Measured temperatures were not stated

 Light regime: Continuous illumination (400 – 800 Lux) 

Test parameters: Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 14 

days) and mean body weight (measured at 0 and 14 days) 

Statistics: NOEC: Dunnett’s’ Test (mortality, mean body weight) 

LC50 (toxic standard): Probit Analysis 

Findings: 

 Water content: About 21 % (0 h), 19.6 – 20.1 % (14 d) 

 pH: 6.4 (0 h), 6.4 (14 d) 

 Mortality: Spirotetramat: No mortality was noticed in the control and all treatments up to 562 

mg/kg dw soil. At 1000 mg/kg dw soil the mortality was 3 % (do not differ 

statistically significantly in Dunnett test). 

Toxic standard: LC50: 23 mg/kg dw soil (95% C.I.: 21 – 24 mg/kg dw soil) 

 Behavioural effects: No effects were observed in the control and all treatments 

 Body weight: See table B.9.6.1-1 
– 125 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.6.1-1: Effects of BYI 08330 (spirotetramat) on the mean body weight of Eisenia fetida

Mean body weight (SD) Spirotetramat 

[mg/kg dw soil] Day 0 [g] Day 14 [g] 

Mean change in body 

weights at day 14, 

compared to control 

0 (control) 0.35 (0.02) 0.32 (0.02) -- 

32 0.36 (0.03) 0.33 (0.02) +2.3 % 

100 0.37 (0.03) 0.34 (0.03) +5.5 % 

178 0.37 (0.02) 0.33 (0.02) +3.9 % 

316 0.36 (0.02) 0.33 (0.02) +2.3 % 

562 0.36 (0.02) 0.33 (0.03) +3.1 % 

1000 0.35 (0.03) 0.32 (0.02) ± 0.0 % 

NOEC (body weight): 1000 mg/kg soil dw 

EC50 > 1000 mg/kg soil dw

Conclusions: LC50 > 1000 mg/kg soil dw, NOEC (body weight, mortality): 1000 mg/kg soil dw

Comments: While no deviations to the OECD guideline 207 are notable, deviations to the 

OPPTS guideline are remarkeable: 

The recommended test duration is 28 days, with observations and measured 

concentrations taken on Days 0, 7, 14, 21, and 28; this study only lasted 14 days 

and measured concentrations are not reported 

Metabolites 

Reference: BYI 08830 cis-ketohydroxy: Acute toxicity to earthworms (Eisenia fetida) 

tested in artificial soil 

Author(s), year: Lechelt-Kunze, C., 2005 

Report/Doc. number: LKC/RG-A-47/05, Edition Number: M-258925-01-2, 17.10.2005 

Guideline(s): OECD 207 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08830 cis-ketohydroxy (metabolite of spirotetramat), purity: 98.6 %, batch: 

NLL7549-7 

Material and methods: 

Test species: Earthworm (Eisenia fetida) 

 Number of organisms: 4 replicates each with 10 individuals per treatment and control 

 Weight, age: Average weight: 0.34 g/worm, clitellate adults > 2 months 

Type of test, duration: Laboratory acute test, 14 days 

Applied concentrations: Nominal: 0 (control, quartz sand), 32, 100, 316, 1000 mg/kg dw soil 

 Solvent/vehicle: Quartz sand 

Toxic standard: 2-Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mg/kg dw soil 

Test conditions: 

 Test substrate: 10 % sphagnum peat, 20 % kaolinite clay, 70 % quartz sand, about 0 % CaCO3 to 

adjust pH to 6.0 ± 0.5, max. WHC between 40 - 60 % , pH and the water content 

were measured at the beginning and the end of the study 

 Substrate/test vessel: 625 g wet weight (500 g dw) in 1.5 L test containers 

 Temperature: Measured temperatures were not stated

 Light regime: Continuous illumination (400 – 800 Lux) 
– 126 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test parameters: Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 7 

and 14 days) and mean body weight (measured at 0 and 14 days) 

Statistics: NOEC: Dunnett’s’ Test (mortality, mean body weight) 

LC50 (toxic standard): Probit Analysis 

Findings: 

 Water content: 23.3 % (0 h, mean), 24.7 % (14 d, mean) 

 pH (mean): 6.4 (0 h), 6.2 (14 d) 

 Mortality: No mortality was noticed in the control and all treatments. 

 Behavioural effects: No effects were observed in the control and all treatments 

 Body weight: See table B.9.6.1-2 

Table B.9.6.1-2: Effects of BYI 08330-ketohydroxy on the mean body weight of Eisenia fetida

Spirotetramat- Mean body weight (SD) 

ketohydroxy 

[mg/kg dw soil] Day 0 [g] Day 14 [g] 

Mean change in body 

weights at day 14, 

compared to control 

0 (control) 0.35 (0.01) 0.36 (0.01) -- 

10 0.33 (0.01) 0.34 (0.01) -5.9 % 

32 0.35 (0.01) 0.36 (0.02) -0.7 % 

100 0.35 (0.01) 0.35 (0.01) -2.8 % 

316 0.35 (0.01) 0.35 (0.01) -4.2 % 

1000 0.35 (0.01) 0.36 (0.01) ± 0.0 % 

NOEC (body weight): 1000 mg/kg soil dw 

EC50 > 1000 mg/kg soil dw

Toxic standard: LC50: 23 mg/kg dw soil (95% C.I.: 21 – 24 mg/kg dw soil) 

Conclusions: LC50 > 1000 mg/kg soil dw, NOEC (body weight, mortality): 1000 mg/kg soil dw

Comments: While no deviations to the OECD guideline 207 are notable, deviations to the 

OPPTS guideline are remarkeable: 

The recommended test duration is 28 days, with observations and measured 

concentrations taken on Days 0, 7, 14, 21, and 28; this study only lasted 14 days 

and measured concentrations are not reported 

Reference: Spirotetramat – 4-methoxycyclohexanone: Acute toxicity to earthworms 

(Eisenia fetida) tested in artificial soil with 5 % peat 

Author(s), year: Leicher, T., 2008 

Report/Doc. number: LRT-Rg-A-98/07, Edition Number: E310 3411-3, 09.01.2008 

Guideline(s): OECD 207 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: 4-methoxycyclohexanon (metabolite of spirotetramat), purity: 97.9 %, batch: 

SVA 5506-001 

Material and methods: 

Test species: Earthworm (Eisenia fetida) 

 Number of organisms: 4 replicates each with 10 individuals per treatment and control 

 Weight, age: Average weight: 0.29 g/worm, clitellate adults > 2 months 

Type of test, duration: Laboratory acute test, 14 days 

Applied concentrations: Nominal: 0 (control, quartz sand), 62.5, 125, 250, 500, 1000 mg/kg dw soil 

 Solvent/vehicle: Quartz sand 
– 127 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Toxic standard: Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mg/kg dw soil 

Test conditions: 

 Test substrate: 5 % sphagnum peat, 20 % kaolinite clay, 74.8 % quartz sand, about 0.2 % CaCO3 

to adjust pH to 6.0 ± 0.5, max. WHC between 40 - 60 % , pH and the water 

content were measured at the beginning and the end of the study 

 Substrate/test vessel: 580 g wet weight (500 g dw) in 1.5 L test containers 

 Temperature: Measured temperatures were not reported 

 Light regime: Continuous illumination (400 – 800 Lux) 

Test parameters: Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 7 

and 14 days) and mean body weight (measured at 0 and 14 days) 

Statistics: NOEC: Dunnett’s’ Test (mortality, mean body weight) 

LC50 (toxic standard): Probit Analysis 

Findings: 

 Water content: 20.7 % (0 h, mean), 20.4 % (14 d, mean) 

 pH (mean): 5.76 (0 h), 5.53 (14 d) 

 Mortality: No mortality was noticed in the control and all treatments. 

 Behavioural effects: No effects were observed in the control and all treatments 

 Body weight: See table B.9.6.1-3 

Table B.9.6.1-3: Effects of 4-methoxycyclohexanone on the mean body weight of Eisenia fetida

4-methoxycyclohexanone [mg/kg dw soil] Weight alteration of the surviviors 

Mean ± standard devision 

0 (control) + 8,93 ± 6,07 

62.5 + 3,19 ± 2,32 

125 + 2,51 ± 3,14 

250 - 4,24 ± 2,32* 

500 - 13,66 ± 5,36* 

1000 - 20,88 ± 7,45* 

NOEC (body weight): 125 mg/kg soil dw 

EC50 > 1000 mg/kg soil dw 

* significant difference when compared to control (Dunnett test, alpha = 0.05) 

Toxic standard: LC50: 11.1 mg/kg dw soil (95% C.I.: 10.1 – 12.2 mg/kg dw soil) 

Conclusions: LC50 > 1000 mg/kg soil dw, NOEC (body weight, mortality): 125 mg/kg soil dw

Comments: While no deviations to the OECD guideline 207 are notable, deviations to the 

OPPTS guideline are remarkeable: 

The recommended test duration is 28 days, with observations and measured 

concentrations taken on Days 0, 7, 14, 21, and 28; this study only lasted 14 days 

and measured concentrations are not reported 

B.9.6.2 Sublethal effects (OECD IIA 8.9.2, IIIA 10.6.4) 

Active substance 

Not submitted. Not required because the maximum application number is two times per year and 

spirotetramat is not persistent in soil. 
– 128 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Metabolites 

Reference: BYI 08330-Enol: Reproduction toxicity to the earthworm Eisenida fetida in 

artificial soil 

Author(s), year: Moser, T.; Scheffczyk, A., 2005 

Report/Doc. number: P18RR, Edition Number: M-243305-01-2, 19.01.2005 

Guideline(s): ISO Guideline 11268-2 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08830-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005 

Material and methods: 

Test species: Earthworm (Eisenia fetida) 

 Number of organisms: 4 replicates each with 10 individuals per treatment and control 

 Weight, age: Mean: 404 mg/worm, clitellate adults 

Type of test, duration: Laboratory sublethal test, 8 weeks 

Applied concentrations: Nominal: 0 (control, quartz sand), 10, 32, 100, 316, 1000 mg/kg dw soil 

 Solvent/vehicle: Quartz sand 

Toxic standard: Carbendazim (applied as SC formulation Derosal): 1, 3, 5 mg a.s./kg dw soil 

Test conditions: 

 Test substrate: 10 % sphagnum peat, 20 % kaolinite clay, 69.55 % quartz sand, 0.45 % CaCO3 to 

adjust pH to 6.0 ± 0.5, WHCmax: 50 ± 10 %, pH and the water content were 

measured at the beginning and the end of the study 

 Substrate/test vessel: 500 g dry weight/test container 

 Temperature: 17 – 22 °C 

The temperature was only for few and short time period below the recommended 

18 °C, thus this did not affected the results of the test. 

 Light regime: 16 hours light (485 – 558 Lux)/8hours dark 

 Feeding: At 24 hours and then weekly (until 4 weeks after application): 5 g manure per test 

vessel on the soil surface, sprinkled with 10 ml deionised water 

Test parameters: Mortality of adults (assessed after 28 days), mean body weight of adults 

(measured at day 0 and after 28 days), morphological and behavioural changes of 

adults (observed at day 28), number of juvenile earthworms (counted after 8 

weeks) and condition and behaviour of juveniles (observed after 8 weeks) 

Statistics: NOEC: Dunnett’s multiple test, EC50 (reproduction): Probit analysis using max. 

likelihood regression 

Findings: 

 Water content: Start: 32.1 – 35.4 % (53 – 58.6 % of WHCmax) 

End: 28.9 – 33.8 % (49 – 60.7 % of WHCmax) 

 pH: 6.1 – 6.2 (start), 6.8 – 7.0 (end) 

 Effects test item: See table B.9.6.2-1 
– 129 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.6.2-1: Effect of BYI 08330-enol (metabolite of spirotetramat) on mortality, growth and 

reproduction of Eisenia fetida 

BYI 08330-enol 

[mg/kg dw soil] 

Mean mortality 

[%] 

Mean biomass increase 

after 4 weeks [%] Mean number of juveniles (SD) 

Control 0 34.7 148 (43.3) 

10 0 33.7 133 (30.3) 

32 0 40.0 133 (9.6) 

100 0 34.5 130 (25.2)* 

316 2.5 42.6 113 (18.8)* 

1000 0 41.5 76 (20.9)* 

NOEC [mg/kg dw] 1000 32 

LOEC [mg/kg dw] > 1000 100 

LC50 [mg/kg dw] > 1000 1269 (95% C.I. 1007 – 1896) 

* significantly different to control (alpha = 0.05)

 Effects toxic standard: Reproduction: LOEC = 1 mg a.s./kg dw soil 

 Validity criteria: Adult mortality (≤ 10 %): 0 % after 4 weeks 

Weight loss of adult worms (≤ 20 %): + 37.7 % after 4 weeks 

Number of juveniles/vessel (≥ 30 %): 148 ± 43.3 % 

Coefficient of variation (≤ 30 %): 29.3 % 

Conclusions: EC50 (reproduction): 1269 mg/kg soil dw 

NOEC (reproduction) 32 mg/kg soil dw 

B.9.6.3 Field studies 

No study submitted. Not required, because the risk assessment for earthworms (see B.9.6.5) indicated an 

acceptable risk for the intended uses and no refinement is necessary. 

B.9.6.4 Risk assessment for earthworms (OECD IIIA 10.6.1) 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 
– 130 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.7 Effects on soil non-target macro-organisms (OECD IIA 8.14, IIIA 10.6.6) 

B.9.7.1 Effects on a species level (gamasid mites) 

Metabolites 

Reference: BYI 08330-Enol: Effects on survival and reproduction of the predaceous 

mite Hypoaspis aculeifer Canestrini (Acari: Laelapidae) in standard soil 

(LUFA 2.1) 

Author(s), year: Moser, T.; Scheffczyk, A., 2005 

Report/Doc. number: P11HR, Edition Number: M-243307-01-1, 19.01.2005 

Guideline(s): SECOFASE, final report (Lokke & von Gestel 1996), 

Guidance document on regulatory testing procedures for pesticides with nontarget arthropods (Barret et al. 1994) 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: BYI 08830-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09-

0005 

Material and methods: 

Test species: Gamasid mite (Hypoaspis aculeifer) 

 Number of organisms: 4 replicates with 20 mites per treatment, 5 replicates with 20 mites per control and 

3 replicates with 20 mites per toxic reference 

 Age: Protonymphes 2 day old 

Type of test, duration: Laboratory reproduction test, 34 days 

Applied concentrations: Nominal: 0 (control, deionised water), 10, 32, 100, 316, 1000 mg /kg dw soil 

 Solvent: Deionised water 

Toxic standard: Dimethoat (applied as EC formulation “Perfekthion”): 5 mg/kg soil dw 

Test conditions: 

 Test substrate: LUFA 2.1 standard soil (type: sand): WHCmax: 34.6 %, pH 6.2 ± 0.7, Corg: 1.23 ± 

0.3 % 

 Substrate/test vessel: 5.1 – 5.3 g wet weight (approx. 3 mm layer) / 30 mL glass container 

 Temperature: 25 ± 2 °C 

 Light regime: Permanent dark 

 Feeding: At day 0 mites were fed with prey mites (Tyrophagus putrescentiae). Twice a 

week the food was checked and was added on demand. After 21 days 

enchytraeids (Enchytraeus crypticus) were fed. 

 Test procedure: 0-day: Introduction of 20 individuals on the soil surface of each test unit randomly.

14-day: Surviving adult mites were transferred to untreated mating units (replicate 

groups together with sex ratio of 1:5 male to female). 

21-day: Females from the control and the three highest concentration levels which 

caused < 50 % mortality (i.e. 100, 316, 1000 mg/kg) were transferred individually 

to the first series of reproduction units (one female per unit). 

24-day: Females from the first series of reproduction units were transferred 

individually to a second reproduction unit (one female per unit). 

Day 28 – 30: Counting of juveniles from the first series. 

Day 32 – 34: Counting of juveniles from the second series.. 

Test parameters: Mortality of adults after 14 days, reproduction after a 7-day period 

Statistics: NOEC (mortality adults): ANOVA followed by Dunnetts test, NOEC (reproduction): 
– 131 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Welch-t-test with Bonferroni Adjustment 

Findings: 

 Water content: Start: 18.8 – 20.9 % (= 54.2 – 60.2 % WHC) 

End: Not reported 

 pH: Start: 5.4 – 5.5 

End: not reported 

 Effects test item: See table B.9.7.1-1 

Table B.9.7.1-1: Effects of BYI 08830-enol (metabolite of spirotetramat) on mortality and reproduction

of gamasid mites 

Mortality of parental mites Number of juveniles per female after 7 d BYI 08330-enol 

[mg a.s./kg dw] Number of dead 

mites at 14 days 

Corrected 

mortalitya) [%] Mean ± SD Reduction [%] 

Control 3 0.0 25.6 ± 7.3 0.0 

10 8 7.2 n.d. n.d. 

32 12 12.4 n.d. n.d. 

100 4 2.1 30.1 ± 3.2 -14.8 

316 8 7.2 26.4 ± 8.6 -3.4 

1000 34 40.7* 23.7 ± 5.9 7.4 

NOEC 316 mg/kg soil dw 1000 mg/kg soil dw 

LOEC 1000 mg/kg soil dw > 1000 mg/kg soil dw 

LC/EC50 > 1000 mg/kg soil dw > 1000 mg/kg soil dw 

a) corrected according to Abbott (1925) 

*Significant different by Dunnetts test (alpha = 0.05) 

 Effects toxic standard: 88 % corrected mortality at 5 mg/kg soil dw 

 Validity criteria: Adult mortality in control after 14 days (≤ 25 %): 3 % 

Mean reproduction rate in control (≥ 10 fertile eggs per female): 25.6 ± 7.3 

Adult mortality in toxic standard after 14 days (50 – 99.5 %): 88 % 

Conclusions: NOEC (mortality): 316 mg/kg soil dw 

LC/EC50 > 1000 mg/kg soil dw

B.9.7.2 Effects on a functional level (degradation in litter bags) (OECD IIIA 10.6.7) 

Not submitted. Study is not required because spirotetramat is not persistent and the effects on soil microand macro-organisms are acceptable. 

B.9.7.3 Summary of effects on soil non-target macro-organisms 

Substance Species Endpoint Time EC/LC50

[mg/kg dw] 

NOEC 

[mg/kg dw] 

Reference/study 

acceptable 

BYI 08330-enol Gamasid mite 

(Hypoaspis aculeifer)

Mortality 

Reproduction 28 d > 1000 316 1000 

Moser & Scheffczyk 

2005/yes 
– 132 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.7.4 Risk assessment for other soil non-target macro-organisms 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 

B.9.8 Effects on soil non-target micro-organisms (OECD IIA 8.10, IIIA 10.7) 

B.9.8.1 Nitrogen mineralization 

Active Substance 

Reference: BYI 08330 tech.: Determination of effects on nitrogen transformation in soil 

Author(s), year: Lechelt-Kunze, C., 2004 b 

Report/Doc. number: LKC-N-32/04, Edition Number: M-087831-01-1, 01.09.2004 

Guideline(s): OECD 216 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 

Material and methods:

Test species: Soil microflora 

Type of test, duration: Nitrogen transformation test, 28 days 

Applied concentrations: 0 (control, purified water), 0.13 and 1.32 mg/kg dry soil 

 Solvent/vehicle: Quartz sand 

Toxic standard: Sodium chloride: 16 g/kg soil dw 

Test conditions: 

 Test substrate: Natural Soil, collected on April 13

th, 2004 at a depth of 0-20 cm, from 

experimental field on Lacherhof Germany: 

Corg: 0.7, pH: 5.6, 

texture (DIN): 6.3 % clay, 15.6 % silt, 78.1 % sand, 

microbial biomass: 393 mg microbial C/kg soil dw, 

additionally 5 g/kg soil dw Lucerne-grass-green meal was added after the 

application. 

 Substrate/test vessel: 300 g soil dw/500 mL brown glass bottles 

 Incubation: 20 ± 2 °C in the dark, about 40 % MHC 

Test parameters: Samples (10 g dry soil) were extracted with 50 mL 1M KCl and soil particles were 

removed by filtration, NH4

+

 and NO3

-

 were determined using a “Bran & Lübbe 

Autoanalyzer 3”. Samples were taken at 0, 7, 14 and 28 days after incubation. 

Statistics: F-test (homogeneity of variance), T-test (significant differences) 
– 133 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Findings: 

 pH: 5.6 (start), 5.9 – 6.0 (end) 

 Effects test item: See table B.9.8.1-1 

Table B.9.8.1-1: Effects of spirotetramat on nitrogen transformation (mean values) 

Treatment Time 

(days) 

NH4-N 

(mg N/kg dw) 

NO3-N 

(mg N/kg dw) 

Nitrification rate 

(mg NO3-N/kg soil 

dw/d) 

% Deviation from 

control 

0 3.27 ± 0.00 28.34 ± 0.41 - - 

7 1.93 ± 0.08 13.35 ± 1.18 -2.14 ± 0.22 - 

14 1.56 ± 0.07 13.46 ± 1.47 0.02 ± 0.37 - 

Control 

28 1.48 ± 0.14 26.02 ± 1.46 0.90 ± 0.07 - 

0 3.42 ± 0.17 30.07 ± 0.55 - - 

7 1.95 ± 0.07 14.76 ± 0.65 -2.19 ± 0.02 2 

14 1.58 ± 0.06 13.77 ± 1.24 -0.14 ± 0.22 980 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 

28 1.57 ± 0.02 25.94 ± 0.76 0.87 ± 0.08 3 

0 3.37 ± 0.21 30.00 ± 0.22 -2.38 ± 0.12 11 

7 1.93 ± 0.13 13.32 ± 1.02 - - 

14 1.76 ± 0.33 13.60 ± 0.54 0.04 ± 0.09 143 

1.32 mg a.s./kg 

(0.99 kg a.s./ha) 

28 1.71 ± 0.18 28.19 ± 1.22 1.04 ± 0.05 16 

 Validity criteria: Coefficient of variation in the control (≤ 15 %): max. 11 % 

Conclusions: After 28 days of incubation the deviations between control soil and soil treated 

with spirotetramat (applied with dose rates of 0.13 mg/kg soil and 1.32 mg/kg soil, 

corresponding to an application rate of 0.099 kg a.s./ha and 0.99 kg a.s./ha) was 

< 25 %, thus spirotetramat had no adverse effects on N-transformation of soil 

microorganisms.

B.9.8.2 Carbon mineralization 

Active substance 

Reference: BYI 08330 tech.: Determination of effects on carbon transformation in soil 

Author(s), year: Lechelt-Kunze, C., 2004 c 

Report/Doc. number: LKC-C-30/04, Edition Number: M-087840-01-1, 01.09.2004 

Guideline(s): OECD 217 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 

Material and methods:

Test species: Soil microflora 

Type of test, duration: Carbon transformation test, 28 days 

Applied concentrations: 0 (control, purified water), 0.13 and 1.32 mg/kg dry soil 

 Solvent/vehicle: Quartz sand 

Toxic standard: Sodium chloride: 16 g/kg soil dw 

Test conditions: 

 Test substrate: Natural Soil, collected on April 13

th, 2004 at a depth of 0-20 cm, from 

experimental field on Lacherhof Germany: 
– 134 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Corg: 0.7, pH: 5.6, 

texture (DIN): 6.3 % clay, 15.6 % silt, 78.1 % sand, 

microbial biomass: 393 mg microbial C/kg soil dw, 

in addition glucose was added to the soil samples (2 g/kg soil dw) to induce 

maximum respiration rate. 

 Substrate/test vessel: 350 g soil dw/500 mL brown glass bottles, three replicates per treatment 

 Incubation: 20 ± 2 °C in the dark, about 40 % MHC 

Test parameters: Evolved CO2 was measured with a gas analyser for at least 12 hours. 

Samples were taken at 0, 7, 14 and 28 days after incubation. 

Statistics: F-test (homogeneity of variance), T-test (significant differences) 

Findings:

 pH: 5.6 (start), 5.5 – 5.6 (end) 

 Effects test item: See table B.9.8.2-1 

Table B.9.8.2-1: Effects of NOA446510 on glucose-induced short-term respiration 

Day 0 Day 7 Day 14 Day 28 

Treatment Mean 

CO2

(mg/h/kg) 

% 

variation 

from 

control 

Mean CO2

(mg/h/kg)) 

% 

deviation 

from 

control 

Mean CO2

(mg/h/kg)) 

% 

deviation 

from 

control 

Mean 

CO2

(mg/h/kg) 

% 

deviation 

from 

control 

Control 268.3±4.2 - 284.6±2.3 - 290.56.4 - 264.5±1.7 - 

0.13 mg a.s./kg 251.7±7.7 6 256.9±3.1 10 267.85.9 8 247.3±5.2 6 

1.3 mg a.s./kg 248.7±0.6 7 253.8±5.1 11 269.54.6 7 245.9±2.6 7 

 Validity criteria: Coefficient of variation in the control (≤ 15 %): max. 2.3 % 

Conclusions: After 28 days of incubation the deviations between control soil and soil treated 

with spirotetramat (applied with dose rates of 0.13 mg/kg soil and 1.33 mg/kg soil, 

corresponding to an application rate of 0.099 kg a.s./ha and 0.99 kg a.s./ha) was 

< 25 %, thus spirotetramat had no adverse effects on C-transformation of soil 

micro-organisms.

B.9.8.3 Summary of effects on non-target micro-organisms 

Test substance Test parameter Test concentration Time 

Effects (deviation from 

control) 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 3 % 

Spirotetramat Nitrogen mineralization 

1.3 mg a.s./kg 

(0.99 kg a.s./ha) 

28 days 

16 % 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 6 % 

Spirotetramat Carbon mineralization 

1.3 mg a.s./kg 

(0.99 kg a.s./ha) 

28 days 

7 % 

B.9.8.4 Risk assessment for non-target micro-organisms 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 
– 135 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 

B.9.9 Effects on other non-target organisms (flora and fauna) believed to be a risk (OECD 

IIA 8.12, IIIA 10.8) 

B.9.9.1 Effects on terrestrial non-target plants (OECD IIIA 10.8.1) 

B.9.9.1.1 Formulation 

Reference: Non-target terrestrial plants: an evaluation of the effects of BYI 08330 OD 

150 in the vegetative vigour test (Tier 1) 

Author(s), year: Gosch, H., Nguyen, D.H. (2006a) 

Report/Doc. number: VV06/03-Tier 1; M-273381-01-1 

Guideline(s): OECD 208 B (July 2000, draft) 

GLP: No 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD, Batch no.: 08030/0233(0152), Tox no.: 

TOX07087-00, content: 148.28 g a.s./L 

Material and Methods: 

Test species: Corn Zea mays, oats Avena sativa (Monocotyledonae: Poaceae), cucumber 

Cucumis sativus (Dicotyledonae: Cucurbitaceae), oilseed rape Brassica napus

(Dicotyledonae: Brassicaceae), soybean Glycine max (Dicotyledonae: 

Leguminosae), sunflower Helianthus annuus (Dicotyledonae: Asteraceae) 

Type of test: Vegetative vigour test 

Number of organisms: 4 to 5 replicates with 4 to 5 plants per pot for each species (19 - 20 plants per

treatment group) 

Treatments: Control: deionised water; test item: 288 g a.s./ha in 200 L/ha. The test item was 

sprayed on the leaves and above-ground portions of the plants. 

Duration: 21 days after treatment application 

Soil characteristic: Standard soil: 20.7 % clay, 65.1 % silt, 14.2 % sand, organic carbon: 1.19 %, 

pH: 7.4 

Test design: At the 2 - 4 leaf stage the test item was sprayed on the leaves and above-ground 

portions of the plants. 

Test conditions: Glasshouse conditions: temperature: 18°C at night, 23°C at day; natural daylight 

supplemented by artificial lighting, 16 h photoperiod; Plants were watered from the 

bottom. 

Test parameter: Visual phytotoxicity ratings were done at days 7, 14 and 21 after application. Plant 

biomass was determined per replicate (pot). 

Statistics: Pairwise Mann-Whitney-U-test, one sided smaller 

Findings: 

 There were no adverse effects on survival. 
– 136 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

There were visible signs of phytotoxicity such as chlorosis, necrosis, leaf 

deformation, bleaching, and/or stunting in all crops tested. There were differences 

in growth stages in corn and oats. 

Biomass was statistically significantly reduced in all species tested. The 

differences exceeded the 50 % trigger in three of the species tested. 

A summary of all the assessments for the day 21 vegetative vigour test (Tier 1) for 

the effects of BYI 08330 OD 150 is shown in the table below. 

Table B.9.9.1.1-1: Phytotoxicity assessments for the day 21 of the vegetative vigour test (Tier 1) for

the effects of BYI 08330 OD 150 

 Cucumber Oilseed rape Soybean Sunflower Corn Oats 

Survival in number 

(out of 20) and (%) 19 (95) 20 (100) 20 (100) 20 (100) 20 (100) 20 (100) 

Inhibition of 

survival * (%) 5 0 0 0 0 0 

Phytotoxicity 

(see below) B-C D A B-C C B 

Dry Weight ** 

(% inhibition) 41.3 83.3 40.2 43.1 56.9 56.6 

* compared to the control, where survival was 100 % (20 plants) in all cases 

** inhibition expressed on a per plant basis (figures in parentheses indicate that there was an increase in dry weight 

when compared to the control) 

phytotoxicity ratings: 

0 = no injury or effect 

A = slight symptom(s) throughout the whole plant or more moderate symptom(s) on a small area, i.e. one leaf 

B = moderate symptom(s) throughout the whole plant or severe symptom(s) on a limited area, i.e. one-two leaves 

C = severe symptom(s) throughout the whole plant with younger or newly developed leaves growing normally 

D = total plant symptom(s) with the plant showing poor vigour. 

E = moribund 

Conclusion: An application rate of 288 g a.s./ha BYI 08330 OD 150 showed several significant 

adverse effects (i.e. greater than 50 %) to representative non-target plants in the 

vegetative vigour test. Oilseed rape was the most sensitive species tested. These 

results trigger further testing. 

Comments: Following deviations from US guideline requirements are notable: 

- The EPA recommends using at least 4 monocot species including corn, and at 

least six dicot species in four families including soybean and a root crop. 

- US EPA recommends that shoot length/plant height effects be measured and 

reported. 

Reference: Tier I and Tier II Vegetative Vigor Non-Target Plant Study using BYI 08330 

150 OD 

Author(s), year: Christ, M.T., Lam, C.V. (2006a) 

Report/Doc. number: EBFNX016; M-276131-01-1 

Guideline(s): OECD 208 B (July 2000, draft), US EPA Guideline 122-1 & 123-1 

GLP: Yes 

Deviations: None. 

Validity: Acceptable 

Test substance: BYI 08330 150 OD, Batch no.: 08030/0189(0152), Tox no.: TOX07087-00, purity: 

15.1 % w/w 

Material and Methods: 
– 137 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test species: 4 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avena sativa, 

Gramineae), ryegrass (Lolium perenne, Poaceae), onion (Allium cepa, Alliaceae) 

6 dicotyledonous plants: cucumber (Cucumis sativus, Cucurbitaceae), canola 

(Brassica napus, Brassicaceae), soybean (Glycine max.), sunflower (Helianthus 

annuus L., Asteraceae), sugarbeet (Beta vulgaris, Chenopodiaceae), tomato 

(Lycopersicon esculentum, Solanaceae) 

Type of test: Vegetative vigour test 

Number of organisms: 8 replicates with 5 plants per pot for each species (40 seeds per treatment) 

Treatments: Control: deionised water; test item (nominal): monocotyledones: 11, 22, 44, 88, 

176 g a.s./ha; Dicotyledones: 176 g a.s./ha. Nominal test concentrations (mg/L): 0, 

118, 235, 471, 941, 1882; measured test concentrations (mg/L): 0. 123, 252, 527, 

1072, 2113; Calibration had been performed before application. 

Duration: 21 days after treatment application 

Soil characteristic: Silt loam: 22 % clay, 50 % silt, 28 % sand, organic matter: 2.4 % (organic carbon

1.2 %), pH: 7.8 

Test design: At the 2 - 4 leaf stage the test item was sprayed using an Allen Track Sprayer 

from above the plant canopy. The test units were then transferred to the 

greenhouse. 

Test conditions: Glasshouse conditions: temperature: 18.6 - 22.5°C; 37.1 - 61.2 % relative 

humidity; 16 h photoperiod; bottom watering. 

Test parameter: Visual phytotoxicity ratings were done at days 7, 14 and 21 after application using 

the following rating system: 0 (no injury/effect), 10 to 20 % (indicates slight effect), 

30 to 40 % (indicates moderate effect), 50 to 60 % (indicates severe effect), 70 to 

80 % (indicates total plant effect), 90 % and above (plant death). Shoot height 

measurements were performed on day 21. Plant biomass was determined per 

replicate (pot). 

Statistics: Descriptive statistics were performed using EXCEL. One-way-ANOVA and 

Dunett`s test were applied. Statistical analyses were performed using SAS 

procedure. 

In Dec 2007 the applicant was asked to calculate, if possible, EC05 values. Due to 

the nature of the non-linear regression program, the dose-response slope and 

standard error values could not be calculated. Most of the EC05 values were not 

calculable because of the nature of the data. 

Findings: 

 Measured test concentrations ranged from 104 to 114 % of nominal. 

Survival in all control groups was 100 %. 

Canola: Plant survival was not significantly affected at the 176 g a.s./ha treatment 

level in comparison to the controls. Phytotoxicity was observed in one replicate on 

day 21 with minor observations of stunting. Plants were not significantly different 

for plant dry weight or length at the 176 g a.s./ha treatment by Dunnett’s Test as 

compared to the controls. 

Corn: Plant survival was not significantly affected at any treatment level in 

comparison to the controls. Phytotoxicity was observed throughout the study with 

observations of stunting, bleaching, leaf curling, and necrosis with severity of 

effects increasing with treatment concentration. Plants were significantly different 

for plant length and dry weight at the 88 and 176 g a.s./ha treatments by Dunnett’s 

Test as compared to the controls. The inhibition compared to the control for these 

two dosages were 18 % and 51 %, respectively for plant length, and 28 % and 

62 % for dry weight. The EC25 and EC50 dry weight values were determined as 76 

g a.s./ha and 134 g ai/ha (95 % cl: 117 to 155) by non-linear regression, 

respectively. The EC50 for corn length was determined as 172 g ai/ha (95 % cl: 
– 138 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

157 to 189). 

Oat: Plant survival was not significantly affected at any treatment level in 

comparison to the controls. Random phytotoxicity was observed throughout the 

study with observations of stunting, bleaching, leaf curling, and necrosis with 

severity of effects lessening by Day 21 in the higher treatment rates. Plants were 

not significantly different for plant length and dry weight for any treatment by 

Dunnett’s Test as compared to the controls. 

Onion: Plant survival was not significantly affected at any treatment level in 

comparison to the controls. Minor phytotoxicity was observed throughout the study 

with observations of stunting, leaf curling, and necrosis. Plants were not 

significantly different for plant length and dry weight for any treatment by Dunnett’s 

Test as compared to the controls. 

Ryegrass: Plant survival was not significantly affected at any treatment level in 

comparison to the controls. Phytotoxicity was observed throughout the study with 

observations of stunting, bleaching, leaf curling, and necrosis with severity of 

effects increasing with treatment concentration. Plants were significantly different 

for plant length and dry weight at the 176 g a.s./ha treatment by Dunnett’s Test as 

compared to the controls. The inhibition compared to the control was 14 % and 

35 %, respectively, for these two parameters. The EC25 dry weight values were 

determined as 168 g a.s./ha. 

Cucumber, soybean, sugarbeet, sunflower, tomato: Plant survival was not 

significantly affected at the 176 g a.s./ha treatment level in comparison to the 

controls. No phytotoxicity was observed in any treatment. Plants were not 

significantly different for plant dry weight or length at the 176 g a.s./ha treatment 

by Dunnett’s Test as compared to the controls. The EC25 plant length and dry 

weight values were determined as > 176 g a.s./ha. 

Endpoint results are summarised in table B.9.9.1.1-2. 

Table B.9.9.1.1-2a: Phytotoxic effects on ten plant species in a vegetative vigour test [11 – 176 g 

a.s./ha]. EC figures ± 95 % CI 

 Survival Plant length 

 NOEC LOEC EC25 NOEC LOEC EC05 EC25 EC50 

Canola 176 >176 >176 176 >176 >176 >176 >176 

Corn 176 >176 >176 44 88 53 

(38-72) 

106 

(92-122)

172 

(157-189)

Cucumber 176 >176 >176 176 >176 >176 >176 >176 

Oat 176 >176 >176 176 >176 >176 >176 >176 

Onion 176 >176 >176 176 >176 >176 >176 >176 

Ryegrass 176 >176 >176 88 176 >176 >176 >176 

Soybean 176 >176 >176 176 >176 >176 >176 >176 

Sugarbeet 176 >176 >176 176 >176 >176 >176 >176 

Sunflower 176 >176 >176 176 >176 >176 >176 >176 

Tomato 176 >176 >176 176 >176 >176 >176 >176 
– 139 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Plant dry weight 

 NOEC LOEC EC05 EC25 EC50 

Canola 176 >176 >176 >176 >176 

Corn 44 88 34 (22-54) 76 (60-96) 134 (117-155) 

Cucumber 176 >176 >176 >176 >176 

Oat 176 >176 >176 >176 >176 

Onion 176 >176 >176 >176 >176 

Ryegrass 88 176 176 168 (153-184) >176 

Soybean 176 >176 >176 >176 >176 

Sugarbeet 176 >176 >176 >176 >176 

Sunflower 176 >176 >176 >176 >176 

Tomato 176 >176 >176 >176 >176 

Table B.9.9.1.1-2b: Mean plant length and dry weight data of control plants 

Control Mean plant length (cm) Mean plant dry weight (g) 

Canola 35.7 8.652 

Corn 78.8 6.424 

Cucumber 34.4 9.132 

Oat 58.2 3.533 

Onion 31.7 0.628 

Ryegrass 51.9 3.433 

Soybean 27.6 3.857 

Sugarbeet 22.8 5.568 

Sunflower 70.6 14.429 

Tomato 33.5 4.908 

Conclusion: In a vegetative vigour study with 10 non-target terrestrial plant species, following a 

21 day post-emergent foliar application of BYI 08330 150 OD, the most sensitive 

endpoint was corn dry weight with an EC25 value of 76 g a.s./ha (95 % CI: 60-96) 

and an EC50 value of 134 g a.s./ha (95 % cl: 117 to 155). The NOEC for corn was 

44 g a.s./ha. 

Reference: Non-target terrestrial plants: an evaluation of the effects of BYI 08330 OD 

150 in the seedling emergence and growth test (Tier 1) 

Author(s), year: Gosch, H., Nguyen, D.H. (2006b) 

Report/Doc. number: SE06/03-Tier 1; M-273373-01-1 

Guideline(s): OECD 208 A (July 2000, draft) 

GLP: No 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030/0233(0152), Tox no.: 

TOX07087-00; analytical content: 148.28 g a.s./L 

Material and Methods: 

Test species: 2 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avena sativa, 

Gramineae), 4 dicotyledonous plants: cucumber (Cucumis sativus,

Cucurbitaceae), oilseed rape (Brassica napus, Brassicaceae), soybean (Glycine 

max.), sunflower (Helianthus annuus L., Asteraceae) 

Type of test: Seedling emergence and growth test 

Number of organisms: 20 plants per treatment group (4 pots with 5 seeds each) 
– 140 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Treatments: Control: deionised water; test item: 288 g a.s./ha in a spray volume of 200 L/ha. 

Calibration performed before application. 

Duration: 14 days following the emergence of 70 % of control seedlings 

Soil characteristic: Silty loam: 20.7 % clay, 65.1 % silt, 14.2 % sand, organic content: 1.19 %, pH: 7.4 

Test design: The day before application seeds were introduced manually in the soil. They were 

covered by about 2 to 5 mm with soil. Plant pots were sprayed. 

Test conditions: Temperature: 18°C at night, 23°C at day (short deviations); humidity: 28 - 98 %, 

16 h photoperiod of natural daylight supplemented by artificial lighting; Plants 

were initially top watered and then bottom watered. Commercial plastic flower pots 

of 10-cm diameter containing 420 g soil were used. 

Test parameters: Germination: Daily checks were made to identify the date when 70 % of the 

seedlings emerge in the control for each species. The number of plants was 

counted after 7 and 14 days. 

Mortality/Survival: Dead plants were removed after each assessment. The number 

of plants that survived after application was recorded at the final assessment. 

Phytotoxicity records: Visual phytotoxicity ratings were recorded (e.g. chlorosis, 

necrosis, stunting, abnormal growth) 7 and 14 days after the emergence of 70 % 

of seeds in the controls. Phytotoxicity was assessed according to EPPO Standard 

135. 

Growth stages: Growth stages were recorded at the final assessment according to 

BBCH-Monograph - Growth stages. 

Plant biomass: The dry weights were determined at the final assessment (day 14 

after 70 % emergence in the untreated control). The plants of one pot represent 

one replicate. 

Statistics: Statistical analysis was carried out using the Pairwise Mann-Whitney-U-test (one 

sided smaller). 

Findings: 

 The germination of the control plants was at least 95 % and all controls exhibited 

normal growth. 

There was no significant adverse effect of BYI 08330 OD 150 on the six species 

tested. 

Biomass was less compared to control in soybean, corn and oats by 0.2 %, 

10.6 % and 8.1 %, respectively. Biomass was greater compared to control in 

cucumber, oilseed rape and sunflower by 0.9 %, 8.1 %, and 1.1 % respectively. 

None of these differences were significant at the 95 % confidence limits and no 

adverse effect reached or exceeded the 50 % trigger for further testing. 

No phytotoxicity was observed in any of the tested species, except a marginal 

stunting in oats. There were no differences in growth stages, except a marginal 

difference in corn. 
– 141 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.9.1.1-3: Effects of Spirotetramat 150OD on six plant species in a seedling emergence and 

survival test [288 g a.s./ha] 

 14 d seedling emergence 14 d seedling survival 

Treatment Control 288 g a.s./ha Control 288 g a.s./ha 

Species % Emergence % Emergence % Survival % Survival 

Cucumber 90 100 100 100 

Oilseed rape 100 100 100 100 

Soybean 100 100 100 100 

Sunflower 95 100 100 100 

Corn 100 100 100 95 

Oat 95 95 100 100 

% Emergence = (No. plants emerged / No. seeds planted) x 100 

% Survival = (No. plants surviving / No. seeds emerged) x 100 

Conclusion: An application of 288 g a.s./ha BYI 08330 OD 150 showed no significant adverse 

effect (i.e. greater than 50 %) to representative non-target plants in the seedling 

emergence and growth test. 

The EC50 figures for all tested species were therefore > 288 g a.s./ha. 

Comments: Following deviations from US guideline requirements are notable: 

- The EPA recommends using at least 4 monocot species including corn, and at 

least six dicot species in four families including soybean and a root crop. 

- US EPA recommends that shoot length/plant height effects be measured and 

reported. 

- The EPA recommends 10 seeds/replicate and 4 replicates/dose; only 5 

seeds/replicate and 4 replicates/dose were used in this study. 

Reference: Tier I Seedling Emergence Non-Target Plant Study using BYI 08330 150OD 

Author(s), year: Christ, M.T., Lam, C.V. (2006b) 

Report/Doc. number: EBFNX017; M-276244-01-1 

Guideline(s): US-EPA 122-1 

GLP: Yes 

Deviations: Short deviation in the photoperiod from 13 to 15 h. The EPA recommends 10 

seeds/replicate and 4 replicates/dose; however, 5 seeds/replicate and 8 

replicates/dose were used in this study. Organic carbon content of the soil was 

slightly higher (2.4 %) than what EPA recommends (2 %). These deviations do not 

affect the validity of the study. 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030/0189(0152); analytical 

content: 15.1 % w/w; 

Material and Methods: 

Test species: 4 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avena sativa, 

Gramineae), ryegrass (Lolium perenne, Poaceae), onion (Allium cepa, Alliaceae) 

6 dicotyledonous plants: cucumber (Cucumis sativus, Cucurbitaceae), canola 

(Brassica napus, Brassicaceae), soybean (Glycine max.), sunflower (Helianthus 

annuus L., Asteraceae), sugarbeet (Beta vulgaris, Chenopodiaceae), tomato 

(Lycopersicon esculentum, Solanaceae) 

Type of test: Seedling emergence and growth test 

Number of organisms: 40 plants per treatment group (8 pots with 5 seeds each) 
– 142 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Treatments: Control: deionised water; test item: 176 g a.s./ha in a spray volume of 93.5 L/ha. 

Calibration performed before application. The nominal a.s. concentration in the 

spray solution was 1882 g a.s./L, the measured concentration was 1954 g a.s./L, 

representing 104 % of nominal. 

Duration: 21 days 

Soil characteristic: 22 % clay, 50 % silt, 28 % sand, organic content: 2.4 %, pH: 7.8 

Test design: The day before application seeds were introduced in the soil. They were covered 

by about 1.2 or 2.5 cm with soil. The spray solution was applied to the soil surface 

using an Allen Track Sprayer. 

Test conditions: Temperature: 18.6 - 24.4°C; humidity: 28 - 71 %, 16 h photoperiod of natural 

daylight supplemented by artificial lighting; Plants were bottom watered; 

commercial plastic flower pots were used. 

Test parameters: Samples of the spray stock solutions were collected at study initiation on day 0 

and analysed for the active substance. 

The endpoints evaluated on Day 7, Day 14, and Day 21 following spray 

application were the number of emerged seedlings, number of seedlings 

surviving, and phytotoxicity of each replicate and were evaluated by visual 

enumeration using the following rating system: 0 (no injury/effect), 10 to 20 % 

(indicates slight effect), 30 to 40 % (indicates moderate effect), 50 to 60 % 

(indicates severe effect), 70 to 80 % (indicates total plant effect), 90 % and above 

(plant death). 

Plants were excised at the soil surface on Day 21 for plant height and dry weight 

determination. Plant shoot height was measured by extending cut seedlings to 

their maximum length and recording. Plant dry weight was performed by placing 

all replicate plants within labelled paper bags. The plants were placed in a drying 

oven and allowed to dry for at least 48 hours. 

Statistics: All Day 21 data were subjected to a Shapiro-Wilk’s Test to assess departures 

from a normal distribution and a Levene’s Test to determine homogeneity of 

variance. To assess treatment effects between the control and treatment group, a 

Student’s t-test was performed. All statistical analyses were performed using the 

computer program SAS, descriptive statistics were performed using EXCEL. 

Findings: 

 The measured a.s. concentration was 104 % of nominal. 

Percent seedling emergence for the 10 plant species ranged from 88 to 100 % in 

the controls and from 80 to 100 % in the 176 g a.s./ha treatment. Percent seedling 

survival for the 10 plant species ranged from 91 to 100 % in the controls, and 84 

to 100 % in the 176 g a.s./ha treatment. 

Phytotoxicity ratings for the seedling emergence test ranged from 1 to 13 % for 

the 176 g a.s./ha treatment for all species. Similar phytotoxicity of minor necrosis 

and stunting was observed in the controls and treated levels of canola, the 

species most sensitive in this respect. The observed phytotoxicity was considered 

to be not treatment related as the level of incidence was similar in both the control 

and treated levels. 

Percent inhibition for plant length for the 10 plant species ranged from -4 to 8 % in 

comparison to the controls and the 176 g a.s./ha treatment. Percent inhibition for 

plant dry weight ranged from -13 to 19 % in comparison to the controls and the 

176 g a.s./ha treatment. 

The percent effect for each endpoint (i.e. emergence, survival, plant length, dry 

weight) was 19 % or less for each plant species. No significant effects were 

observed to any of the 10 species tested. 

Summarised results are given in table B.9.9.1.1-4. 
– 143 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.9.1.1-4: Phytotoxic effects of Spirotetramat 150OD on ten plant species in a seedling 

emergence and survival test [176 g a.s./ha] 

 21 d seedling emergence 21 d seedling survival 

Treatment Control 176 g a.s./ha Control 176 g a.s./ha 

Species % Emergence % Emergence % Survival % Survival 

Canola 88 80 91 84 

Corn 93 98 100 100 

Cucumber 95 98 97 100 

Oat 98 98 100 100 

Onion 100 98 100 100 

Ryegrass 100 95 100 100 

Soybean 98 93 100 100 

Sugarbeet 93 95 100 97 

Sunflower 95 100 100 100 

Tomato 90 90 100 100 

% Emergence = (No. plants emerged / No. seeds planted) x 100 

% Survival = (No. plants surviving / No. seeds emerged) x 100 

Conclusion: There were no significant adverse effects for any measured parameter at or above 

25 % as compared to the controls in a Tier I seedling emergence study at the 

single application rate of 176 g a.s./ha. Because there were no effects at or above 

25 %, a Tier 2 study is not required. 

The EC50 figures for all tested species were therefore > 176 g a.s./ha. 

Reference: Spirotetramat OD 150: Vegetative vigour test on non-target terrestrial plants 

Author(s), year: Friedrich, S. (2006) 

Report/Doc. number: M-279698-01-1 

Guideline(s): OECD 227 (Draft 2005) 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat OD 150; Batch no.: 08030/0189(0152), purity: 15.1 % w/w analysed 

Material and Methods: 

Test species: 2 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avena sativa, 

Gramineae), 1 dicotyledonous plant: canola (Brassica napus, Brassicaceae) 

Type of test: Vegetative vigour test 

Number of organisms: 20 / 10 / 8 replicates with 2 / 4 / 5 plants per pot for corn / rape / oats (40 plants 

per treatment) 

Treatments: Control: deionised water; test item (nominal): 0.17, 0.48, 0.96, 1.92 L product/ha 

in 400 L water; nominally equivalent to 26, 72, 144, 288 g a.s./ha; corresponding 

to actually applied 0.17, 0.47, 0.94, 1.86 L product/ha; Calibration had been 

performed before application. 

Duration: 21 days after treatment application 

Soil characteristic: Agricultural soil, sandy loam: 12.4 % clay, 48.8 % silt, 38.9 % sand, organic 

carbon: 0.9 %, pH: 6 

Test design: At the 2 leaf stage the test item was sprayed onto the leaves and the soil 

substrate using an automatic application cabin. 

Test conditions: Glasshouse conditions: temperature: 11 - 41°C; 17 - 99 % relative humidity; 16 h 

photoperiod; bottom watering. 
– 144 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Test parameter: Mortality and visual phytotoxicity were assessed at days 7, 14 and 21 after 

application. The severity of phytotoxic symptoms was described, e.g. chlorosis, 

necrosis, wilting (%/plant). Shoot fresh weight measurements were performed on 

day 21. Plant biomass was determined per replicate (pot). 

Statistics: Computation of EC50 (e.g. probit analysis). Software ToxRat Professional used. 

In Dec 2007 probit analysis using max. likelihood regression was conducted to 

derive EC05 and EC25 values with regard to shoot fresh weight. 

Findings: 

 Measured test concentrations ranged from to 82.1 to 90.9 % of nominal. 

Survival was 100 % in all treatment and the control group. 

B. napus: no phytotoxic symptoms. Growth inhibition effects in the two highest 

rates (11 and 35 %, respectively). 

A. sativa: no phytotoxic symptoms. Growth inhibition effects in the two highest 

rates (9 and 44 %, respectively). 

Z. mays: Necrotic, chlorotic and growth inhibitory effects in the two highest rates 

(with regard to the latter 7 and 41 %, respectively). 

Endpoint results are summarised in table B.9.9.1.1-5. 

Details of probit analysis of the shoot fresh weight: B. napus: Slope: 2.53929, 

Intercept: 4.04718, Variance of slope: 27.86593, Chi

2

: 0.00871, degrees of 

freedom: 2, p(Chi2): 0.99565, r2: 0.964 

A. sativa: Slope: 2.94907, Intercept: 3.88868, Variance of slope: 44.36333, Chi2: 

0.01456, degrees of freedom: 2, p(Chi2

): 0.99275, r2: 0.931 

Z. mays: Slope: 4.62209, Intercept: 3.49296, Variance of slope: 148.26663, Chi2: 

0.00001, degrees of freedom: 2, p(Chi2): 1.00000, r2: 1.000 

Table B.9.9.1.1-5: Phytotoxic effects on three plant species in a vegetative vigour test after treatment 

with Spirotetramat OD 150 [L product/ha] 

21 DAA Survival Shoot fresh weight 

species LC50 NOEC EC50 

BBCH 

B. napus > 1.92 0.48 > 1.92 15-17 

A. sativa > 1.92 0.96 > 1.92 16-21 

Z. mays > 1.92 0.96 > 1.92 16-18 

Table B.9.9.1.1-6: Effects on shoot fresh weight (g) of three plant species after 21 d exposure to 

Spirotetramat OD 150 [L product/ha, nominal] 

Treatment group Control 0.17 0.48 0.96 1.92 

B. napus

Mean ± SD 20.76 ± 2.19 20.41 ± 2.21 19.39 ± 2.97 17.87 ± 1.80 12.16 ± 1.95 

% reduction - 2 7 14* 41* 

A. sativa

Mean ± SD 15.22 ± 1.24 15.45 ± 1.52 14.25 ± 2.08 13.67 ± 1.42 9.18 ± 1.75 

% reduction - -2 6 10 40* 

Z. mays

Mean ± SD 34.76 ± 3.01 36.58 ± 3.57 35.13 ± 2.47 32.81 ± 2.76 20.10 ± 3.35 

% reduction - -5 -1 6 42* 

* statistically significant (Dunnett test, p ≤ 0.05), SD standard deviation 
– 145 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Tab. B.9.9.1.1-7: Results of the probit analysis of shoot fresh weight data: Selected effective 

concentrations (ECx, L product/ha) of the test item and their 95 %- and 99 %-confidence limits 

Parameter EC05 EC25 EC50 EC75 EC90 

B. napus

Value 

[L product/ha] 

0.534 1.290 2.373 4.363 7.575 

lower 95 %-cl 0.114 0.866 1.849 2.838 4.067 

upper 95 %-cl 0.818 1.614 4.907 20.750 78.794 

lower 99 %-cl 0.031 0.577 0.977 0.774 0.587 

upper 99 %-cl 2.970 2.423 9.284 76.109 546.253 

A. sativa

Value 

[L product/ha] 

0.659 1.409 2.381 4.024 6.470 

lower 95 %-cl 0.008 0.564 1.807 2.550 3.358 

upper 95 %-cl 1.039 1.880 16.834 342.214 5434.059 

lower 99 %-cl 0.000 0.257 0.421 0.104 0.027 

upper 99 %-cl 25.149 4.129 72.336 8399.393 678412.375 

Z. mays

Value 

[L product/ha] 

0.934 1.516 2.119 2.961 4.009 

lower 95 %-cl 0.920 1.508 2.108 2.929 3.942 

upper 95 %-cl 0.947 1.524 2.130 2.994 4.080 

lower 99 %-cl 0.902 1.497 2.094 2.887 3.854 

upper 99 %-cl 0.966 1.535 2.144 3.038 4.173 

Conclusion: In a vegetative vigour study with three non-target terrestrial plant species, 

following a 21 day post-emergent foliar application of Spirotetramat OD 150, the 

most sensitive endpoint was shoot fresh weight of oilseed rape with a NOEC 

value of 72 g a.s./ha. However, the EC50`s for all species were > 288 g a.s./ha. 

Comments: The study does not comply with EPA guidelines (e.g. EPA commented the 

variation in temperature and relative humidity to be too high). 

B.9.9.2 Risk assessment for terrestrial non-target plants 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 
– 146 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.10 Effects on biological methods of sewage treatment (OECD IIA 8.15) 

B.9.10.1 Activated sludge, respiration inhibition 

Active substance 

Reference: BYI 08330 - Toxicity to bacteria 

Author(s), year: Bruns; Caspers, N. 2004 

Report/Doc. number: 1312 N/04 B, Edition Number: M-275364-01-1, 05.10.2004 

Guideline(s): OECD 209 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 

Material and methods:

Test species: Activated sludge 

Source: Aeration tank of a domestic sewage treatment plant 

Type of test, duration: Laboratory aerobic activated sludge inhibition test, 3 hours 

Applied concentrations: 

 Nominal: 1000, 1800, 3200, 5600, 10000 mg/L 

 Toxic standard: 3,5-dichlorophenol (97 %): 2.5, 5.0, 10, 20, 40 mg/L 

Test conditions: 

 Substrate/test vessel: Activated sludge (solid concentration: 480 mg/L) treated with active substance, 

reference substance or water (control) 

 Incubation: Aerated for 3 hours at 20 ± 2°C 

Test parameters: Inhibition of respiration rate (rate of oxygen uptake) 

Statistics: None, because the inhibition at highest tested concentration was 22.2 mg/L 

Findings/conclusions: See table B.9.10.1-1 

Table B.9.10.1-1: Effect of spirotetramat on the respiration rate of activated sludge 

Test Substance 

Nominal 

concentration 

[mg/L] 

pH Respiration rate 

[mg O2/L/h] 

Inhibition 

[%] 

3,5-DCPa) 2.5 7.8 26.4 9.9 

3,5-DCP 5.0 7.7 22.8 22.2 

3,5-DCP 10 7.6 10.5 64.2 

3,5-DCP 20 7.6 4.0 86.3 

3,5-DCP 40 7.7 2.0 93.2 

Control 0 7.7 (mean) 29.3 (mean) -- 

BYI 08330 1000 7.6 30.0 0.0 

BYI 08330 1800 7.6 27.6 5.8 

BYI 08330 3200 7.6 27.0 7.9 

BYI 08330 5600 7.6 26.0 11.3 

BYI 08330 10000 7.6 22.8 22.2 

3 hours EC50 > 10000 mg/L 

a) 3,5-dichlorophenol

Conclusions: No inhibitory effects on aerobic waste water micro-organisms are expected if the 

test substance is correctly applied at the intended application rate. 
– 147 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.10.2 Risk assessment for biological methods of sewage treatment 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for the PMRA and the US-EPA is not included in this monograph. 

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian 

review and risk assessment, has been published by the PMRA and can be found at the PMRA’s web site 

www.pmra-arla.gc.ca. 

The registration decision made by the U.S. EPA's Office of Pesticide Program is supported by the risk 

assessments found at our Federal Docket website located at: 

http://www.regulations.gov/fdmspublic/component/main?main=AdvancedDocket

The Docket ID is "EPA-HQ-OPP-2007-0475". 
– 148 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.11 References relied on 

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

Annex II Data and Information 

IIA 8.1.1 Barfknecht, R. 2003 Acute oral toxicity for bobwhite quail 

(Colinus virginianus) for the test item BYI 

08330 (tech.) 

Bayer CropScience AG, 

Report No.: M-115911-02-2, 

Edition Number: M-115911-02-2 

Date: 29.10.2003, Amended: 13.01.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.1.2 Barfknecht, R. 2004 BYI 08330 techn. a.s.: 5-day-dietary LC50 

bobwhite quail (Colinus virginianus) 

Bayer CropScience AG, 

Report No.: BAR/LC017, 

Edition Number: M-084796-01-2 

Date: 18.08.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.1.3 Stoughton, T. L. 2006 Technical BYI 08330: A subacute dietary 

LC50 with mallards 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX012, 

Edition Number: M-274385-01-1 

Date: 03.05.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.1.4 Sabbert, T. J.; 

Nuessle, C. 

2006

a 

Effect of technical BYI 08330 on northern 

bobwhite reproduction 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNO220, 

Edition Number: M-277757-01-1 

Date: 21.09.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.1.4 Sabbert, T. J.; 

Nuessle, C. 

2006

b 

Effect of technical BYI 08330 on mallard 

reproduction 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX013, 

Edition Number: M-277794-01-2 

Date: 22.09.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.1.4 Stafford, J. M. 2007

a 

Long-Term Mallard Duck Dermal Exposure 

to BYI 08330 

Bayer CropScience AG, 

Report No.: EBFNP033 

Edition Number: M-290165-01-1 

Date: 05.07.2007 

GLP 

unpublished 

Yes BCS 
– 149 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.1.4 Stafford, J. M. 2007

b 

Semi-Field Study of the Effects of Dermal 

and Dietary Exposure to BYI 08330 in 

Mallard Ducks (Anas platyrhynchos) 

Bayer CropScience AG, 

Report No.: EBFNP034 

Edition Number: M-290000-01-1 

Date: 26.06.2007 

GLP 

unpublished 

Yes BCS 

IIA 8.1.4 /03 Temple, D.L; 

Martin, K.H.; 

Beavers, J.B.; 

Jaber, M. 

2006 BYI 08330 technical: A reproduction study 

with the mallard 

Wildlife International Ltd., Easton, MD, 

USA 

Bayer CropScience AG, 

Report No.: EBFNP024, 

Edition Number: M-278058-01-1 

Date: 29.09.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.2.1.1 Dorgerloh, M. 2004

a 

Acute toxicity of BYI 08330 (tech.) to fish 

(Oncorhynchus mykiss) 

Bayer CropScience AG, 

Report No.: DOM 24025, 

Edition Number: M-182649-01-2 

Date: 16.12.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.2.1.2 Dorgerloh, M. 2004

b 

Acute toxicity of BYI 08330 (tech.) to fish 

(Cyprinus carpio) 

Bayer CropScience AG, 

Report No.: DOM24022, 

Edition Number: M-128667-01-2 

Date: 25.11.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.2.1.2 Dorgerloh, M. 2005

a 

Acute toxicity of BYI 08330 (tech.) to fish 

(Lepomis macrochirus) 

Bayer CropScience AG, 

Report No.: DOM 24056, 

Edition Number: M-242689-01-2 

Date: 12.01.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.2.1.3 Nieden, D. 2005

a 

Acute toxicity of BYI 08330-enol to fish 

(Oncorhynchus mykiss) 

Bayer CropScience AG, 

Report No.: EBFNM013, 

Edition Number: M-251850-01-2 

Date: 18.05.2005 

GLP 

unpublished 

Yes BCS 
– 150 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.2.1.3 Neuhahn, A. 2006

a 

4-Methoxycyclohexanone - Acute fish 

toxicity 

Bayer Industry Services, Leverkusen 

Bayer CropScience AG, 

Report No.: 2006/0032/02, 

Edition Number: M-274305-01-1 

Date: 10.07.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.2.4 Dorgerloh, M. 2005

b 

Early-life stage toxicity of BYI 08330 tech. 

to fish (Pimephales promelas) 

Bayer CropScience AG, 

Report No.: EBFN0305, 

Edition Number: M-260676-01-2 

Date: 16.11.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.3.1.1 Dorgerloh, M. 2005

c 

Acute toxicity of BYI 08330 (tech.) to the 

waterflea Daphnia magna under static 

conditions 

Bayer CropScience AG, 

Report No.: DOM 24004, 

Edition Number: M-242683-01-2 

Date: 14.01.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.3.1.1 Dorgeloh, M. 2005

d 

Acute toxicity of BYI 08330-enol to the 

waterflea Daphnia magna in a static 

laboratory test system -limit test 

Bayer CropScience AG, 

Report No.: EBFNM012, 

Edition Number: M-254487-01-2 

Date: 14.07.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.3.1.1 Neuhahn, A. 2006

b 

4-Methoxycyclohexanone - Acute daphnia 

toxicity 

Bayer Industry Services, Leverkusen, 

Germany 

Bayer CropScience AG, 

Report No.: 2006/0032/01, 

Edition Number: M-274275-01-1 

Date: 10.07.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.3.2.1 Dorgerloh, M. 2005

e 

Influence of BYI 08330 (tech.) on 

development and reproductive output of the 

waterflea Daphnia magna in a static 

renewal laboratory test system 

Bayer CropScience AG, 

Report No.: EBFN0245, 

Edition Number: M-251843-01-2 

Date: 13.05.2005 

GLP 

unpublished 

Yes BCS 
– 151 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.4 Dorgerloh, M. 2004

c 

Pseudokirchneriella subcapitata - Growth 

inhibition test with BYI 08330 (tech.) 

Bayer CropScience AG, 

Report No.: DOM 23092, 

Edition Number: M-128874-01-2 

Date: 29.11.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.4 Kern, M.E.; 

Lam, C.V. 

2005 Toxicity of BYI 08330 to the freshwater 

diatom Navicula pelliculosa

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX008, 

Edition Number: M-252794-01-1 

Date: 15.06.2005 

Non GLP 

unpublished 

Yes BCS 

IIA 8.4 Kern, M. E.; 

Lam, C. V. 

2006 Toxicity of BYI 08330 to the blue-green 

alga Anabaena flos-aquae

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX007, 

Edition Number: M-264055-01-1 

Date: 12.01.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.4 Dorgerloh, M. 2005f Pseudokirchneriella subcapitata - Growth 

inhibition test with BYI 08330-enol 

Bayer CropScience AG, 

Report No.: EBFNM010, 

Edition Number: M-252531-01-2 

Date: 06.06.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.4 Weyers, A. 2006 4-Methoxycyclohexanone - Alga growth 

inhibition test 

Bayer Industry Services, Leverkusen, 

Germany 

Bayer CropScience AG, 

Report No.: 2006/0032/03, 

Edition Number: M-275954-01-1 

Date: 28.07.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.5.1 Dorgerloh, M. 2005

g 

Acute toxicity of BYI 08330 (tech.) to larvae 

of Chironomus riparius in a 48 h water-only 

study 

Bayer CropScience AG, 

Report No.: EBFNX072, 

Edition Number: M-262632-01-2 

Date: 08.12.2005 

GLP 

unpublished 

Yes BCS 
– 152 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.5.1 Dorgerloh, M. 2005

h 

Acute toxicity of BYI 08330-enol to larvae 

of Chironomus riparius in a 48 h water-only 

study 

Bayer CropScience AG, 

Report No.: EBFNX073, 

Edition Number: M-262638-01-2 

Date: 08.12.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.5.1 Dorgerloh, M. 2006

a 

Acute toxicity of BYI 08330-ketohydroxy to 

larvae of Chironomus riparius in a 48 h 

static laboratory test system 

Bayer CropScience AG, 

Report No.: EBFNO347, 

Edition Number: M-263956-01-2 

Date: 16.01.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.5.1 Dorgerloh, M. 2007

a 

Acute toxicity of 4-Methoxycyclohexanon to 

Larvae of Chironomus riparius in a 48 h 

static laboratory test system (Limit-Test) 

Bayer CropScience AG, 

Report No.: EBFX104, 

Edition Number: M-287218-01-1 

Date: 27.04.2007 

GLP 

unpublished 

Yes BCS 

IIA 8.5.1 Dorgerloh, M. 2007

b 

Acute toxicity of BYI 08330-cis-methoxycyclohexylamino carboxylic acid to Larvae 

of Chironomus riparius in a 48 h static 

laboratory test system (Limit-Test) 

Bayer CropScience AG, 

Report No.: EBFX105, 

Edition Number: M-287220-01-1 

Date: 27.04.2007 

GLP 

unpublished 

Yes BCS 

IIA 8.5.2 Dorgerloh, M. 2005i Chironomus riparius 28-day chronic toxicity 

test with BYI 08330 (tech.) in a watersediment system using spiked water 

Bayer CropScience AG, 

Report No.: EBFN0050, 

Edition Number: M-248099-02-2 

Date: 16.03.2005, Amended: 29.06.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.6 Dorgerloh, M. 2005j Lemna gibba G3 - Growth inhibition test 

with BYI 08330 (tech.) under static-renewal 

test conditions 

Bayer CropScience AG, 

Report No.: DOM 24019, 

Edition Number: M-255296-01-2 

Date: 29.07.2005 

GLP 

unpublished 

Yes BCS 
– 153 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.6 Dorgerloh, M. 2005

k 

Lemna gibba G3: Growth inhibition test 

with BYI 08330-enol under static conditions 

Bayer CropScience AG, 

Report No.: EBFNX065, 

Edition Number: M-252412-01-2 

Date: 07.06.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.7.1 Schmitzer, S. 2004 Effects of BYI 08330 (acute contact and 

oral) on honey bees (Apis mellifera L.) in 

the laboratory 

Ibcaon GmbH, Rossdorf, Germany 

Bayer CropScience AG, 

Report No.: 20091035, 

Edition Number: M-081227-01-2 

Date: 12.07.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.7.4 Schur, A. 2004

a 

Assessment of the side-effects of BYI 

08330 OD 100 on the honey bee (Apis 

mellifera L.) in a feeding test 

GAB Biotechnologie GmbH & IFU 

Umweltanalytik GmbH, NiefernOeschelbronn, Germany 

Bayer CropScience AG, 

Report No.: 20031168/01-BFEU, 

Edition Number: M-000345-01-2 

Date: 23.02.2004 

GLP 

unpublished 

 also filed: IIIA2 10.4.2.1 

 also filed: IIIA2 10.4.2.2 

Yes BCS 

IIA 8.7.4 Schur, A. 2004

b 

Assessment of the side-effects of BYI 

08330 SC 240 on the honey bee (Apis 

mellifera L.) in a feeding test 

GAB GmbH, Niefern-Oeschelbronn, 

Germany 

Bayer CropScience AG, 

Report No.: 20031166/01-BFEU, 

Edition Number: M-121877-01-2 

Date: 05.02.2004 

GLP 

unpublished 

 also filed: IIIA3 10.4.2.1 

Yes BCS 

IIA 8.8.1.1 Waltersdorfer, 

A. 

2005

a 

Toxicity to the parasitoid wasp Aphidius 

rhopalosiphi (DeStephani-Perez) 

(Hymenoptera: Bracconidae) in the 

laboratory; BYI 08330 150 OD; Code: AE 

1302943 00 OD15 A101 

Bayer CropScience AG, 

Report No.: CW05/023, 

Edition Number: M-254442-01-1 

Date: 15.07.2005 

GLP 

unpublished 

Yes BCS 
– 154 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.8.1.2 Waltersdorfer, 

A. 

2005

b 

Toxicity to the predatory mite 

Typhlodromus pyri Scheuten (Acari, 

Phytoseiidae) in the laboratory, BYI 08330 

150 OD; Code AE 1302943 00 OD15 

A101 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: CW05/024, 

Edition Number: M-254269-01-1 

Date: 12.07.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.8.2.1 Waltersdorfer, 

A. 

2006

a 

Toxicity to the parasitoid wasp Aphidius 

rhopalosiphi (DeStephani-Perez) 

(Hymenoptera: Braconidae) using an 

extended laboratory test with BYI 08330 

150 OD 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: CW05/071, 

Edition Number: M-264179-01-1 

Date: 18.01.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.8.2.2 Waltersdorfer, 

A. 

2005

c 

Toxicity to the predatory mite 

Typhlodromus pyri SCHEUTEN (Acari, 

Phytoseiidae) using an extended laboratory 

test BYI 08330 150 OD 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: CW05/030, 

Edition Number: M-256306-01-1 

Date: 25.08.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.8.2.4 Roehlig, U. 2005 Dose-response toxicity (LR50) of BYI 

08330 150 OD to the green lacewing 

Chrysoperla carnea (STEPH.) under 

extended laboratory conditions (including 

food-application) 

BioChem agrar, Gerichshain, Germany 

Bayer CropScience AG, 

Report No.: 051048082, 

Edition Number: M-262650-01-1 

Date: 14.12.2005 

GLP 

unpublished 

Yes BCS 
– 155 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.8.2.4 Waltersdorfer, 

A. 

2006

b 

Toxicity to the ladybird Coccinella 

septempunctata L. (Coleoptera, 

Coccinellidae) using an extended 

laboratory test including exposure to and 

oral uptake of BYI 08330 150 OD 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: CW05/053, 

Edition Number: M-264175-01-1 

Date: 18.01.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.9.1 Lechelt-Kunze, 

C. 

2004

a 

BYI 08830 (techn.): Acute toxicity to 

earthworms (Eisenia fetida) tested in 

artifical soil with 5 % peat 

Bayer CropScience AG, 

Report No.: LKC/RG-A-18/03, 

Edition Number: M-104105-01-2 

Date: 07.01.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.9.1 Lechelt-Kunze, 

C. 

2005 BYI 08330-cis-ketohydroxy: Acute toxicity 

to earthworms (Eisenia fetida) tested in 

artificial soil 

Bayer CropScience AG, 

Report No.: LKC/RG-A-47/05, 

Report includes Trial Nos.: Rg08/05 

Edition Number: M-258925-01-2 

Date: 17.10.2005 

GLP, unpublished 

Yes BCS 

IIA 8.9.1 Moser, T.; 

Scheffczyk, A. 

2005 BYI 08330-Enol: Reproduction toxicity to 

the earthworm Eisenida fetida in artificial 

soil 

ECT Oekotoxikologie GmbH, Floersheim, 

Germany 

Bayer CropScience AG, 

Report No.: P18RR, 

Edition Number: M-243305-01-2 

Date: 19.01.2005 

GLP, unpublished 

Yes BCS 

IIA 8.9.1 Leicher, T. 2008 Spirotetramat - 4-methoxycyclohexanon: 

Acute toxicity to earthworms (Eisenia 

fetida) tested in artificial soil with 5 percent 

peat 

Bayer CropScience AG, 

Report No.: LRT-RG-A-98/07, 

Edition Number: M-296072-01-1 

Date: 09.01.2008 

GLP, unpublished 

Yes BCS 

IIA 8.10.1 Lechelt-Kunze, 

C. 

2004

b 

BYI 08330 tech.: Determination of effects 

on nitrogen transformation in soil 

Bayer CropScience AG, 

Report No.: LKC-N-32/04, 

Edition Number: M-087831-01-1 

Date: 01.09.2004 

GLP, unpublished 

Yes BCS 
– 156 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.10.1 Lechelt-Kunze, 

C. 

2004

c 

BYI 08330 tech.: Determination of effects 

on carbon transformation in soil 

Bayer CropScience AG, 

Report No.: LKC-C-30/04, 

Edition Number: M-087840-01-1 

Date: 01.09.2004 

GLP, unpublished 

Yes BCS 

IIA 8.11.1 Banman, C. S.; 

Lam, C. V. 

2005 Acute toxicity of BYI 08330 technical to the 

sheepshead minnow (Cyprinodon 

variegatus) under flow-trough conditions 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX006, 

Edition Number: M-255363-01-1 

Date: 27.07.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.11.1 Cafarelle, M. A. 2005

a 

BYI 08330 - Acute toxicity to mysids 

(Americamysis bahia) under flow-through 

conditions 

Springborn Smithers Laboratories, 

Wareham, MA, USA 

Bayer CropScience AG, 

Report No.: EBFNX010, 

Edition Number: M-270200-01-1 

Date: 06.01.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.11.1 Cafarella, M. A. 2005

b 

BYI 08330 - Acute toxicity to eastern 

oysters (Crassostrea virginica) under flowthrough conditions 

Springborn Smithers Laboratories, Inc., 

Wareham, MA, USA 

Bayer CropScience AG, 

Report No.: EBFNX011, 

Edition Number: M-257677-01-1 

Date: 15.06.2005 

GLP 

unpublished 

Yes Springborn 

IIA 8.11.1 Banman, C. S.; 

Lam, C. V. 

2006 Toxicity of BYI 08330 technical to the 

saltwater diatom Skeletonema costatum

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX009-1, 

Edition Number: M-271037-02-1 

Date: 23.02.2006, Amended: 16.06.2006 

GLP 

unpublished 

Yes BCS 
– 157 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.12 Gosch, H.; 

Nguyen, D.H. 

2006

a 

Non-target terrestrial plants: an evaluation 

of the effects of BYI 08330 OD 150 in the 

vegetative vigour test (Tier 1) 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: VV06/03, 

Edition Number: M-273381-01-1 

Date: 20.06.2006 

Non GLP 

unpublished 

Yes BCS 

IIA 8.12 Christ, M. T.; 

Lam, C. V. 

2006

a 

Tier I and Tier II - Vegetative vigor nontarget plant study using BYI 08330 150 OD 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX016, 

Edition Number: M-276131-01-1 

Date: 09.08.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.12 Friedrich, S. 2006 Spirotetramat OD 150: Vegetative vigour 

test on non-target terrestrial plants 

BioChem agrar, Gerichshain, Germany 

Bayer CropScience AG, 

Report No.: 061048126, 

Edition Number: M-279698-01-1 

Date: 30.10.2006 

GLP 

unpublished 

Yes BCS 

IIA 8.12 Gosch, H.; 

Nguyen, D.H. 

2006

b 

Non-target terrestrial plants: an evaluation 

of the effects of BYI 08330 OD 150 in the 

seedling emergence and growth test 

(Tier 1). 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: SE06/03, 

Edition Number: M-273373-01-1 

Date: 20.06.2006 

Non GLP 

unpublished 

Yes BCS 

IIA 8.12 Christ, M. T.; 

Lam, C. V. 

2006

b 

Tier I seedling emergence - Non-target 

plant study using BYI 08330 150 OD 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: EBFNX017, 

Edition Number: M-276244-01-1 

Date: 09.08.2006 

GLP 

unpublished 

Yes BCS 
– 158 – 

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Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIA 8.14 Moser, T.; 

Scheffczyk, A. 

2005 BYI 08330-Enol: Effects on survival and 

reproduction of the predaceous mite 

Hypoaspis aculeifer Canestrini (Acari: 

Laelapidae) in standard soil (LUFA 2.1) 

ECT Oekotoxikologie GmbH, Floersheim, 

Germany 

Bayer CropScience AG, 

Report No.: P11HR, 

Edition Number: M-243307-01-1 

Date: 19.01.2005 

GLP 

unpublished 

Yes BCS 

IIA 8.15 Bruns; Caspers, 

N. 

2004 BYi 08330 - Toxicity to bacteria 

Bayer Industry Services GmbH, 

Leverkusen, Germany 

Bayer CropScience AG, 

Report No.: 1312 N/04 B, 

Edition Number: M-275364-01-1 

Date: 05.10.2004 

GLP 

unpublished 

Yes BCS 

IIA 8.16 Schneider, K. 2008 Residues of Spirotetramat in arthropods 

after spray application in citrus - magnitude 

and time course of residue decline 

RIFCON GmbH, Heidelberg, Germany 

Bayer CropScience AG, 

Report No.: RA07-031, 

Edition Number: M-296043-01-1 

Date: 09.01.2008 

GLP, unpublished 

Yes BCS 

Annex III Data and Information 

IIIA1 10.1.2 Wolf, C.; 

Gerlach, J. 

2006 Insectivorous birds in southern European 

citrus plantations focal species and their 

diet composition 

RIFCON GmbH, Hirschberg, Germany 

Bayer CropScience AG, 

Report No.: RC06-067, 

Edition Number: M-277503-01-1 

Date: 15.09.2006 

Non GLP 

unpublished 

Yes BCS 

IIIA1 10.1.2 Ebeling, M. 

Kyriazi, K. 

2008 Refinement for the risk assessment for the 

Great Tit in citrus orchards following 

treatment with Spirotetramat 

Bayer CropScience AG, 

Edition Number: not yet finalised 

Non GLP, unpublished 

Yes BCS 

IIIA1 10.1.2 Wikens, S., 

Schneider, K. 

2008 Residues of Spirotetramat in arthropods 

after spray application in citrus - magnitude 

and time course of residue decline 

RIFCON GmbH, Heidelberg, Germany 

Bayer CropScience AG, 

Report No.: RA07-031, 

Edition Number: M-296043-01-2 

Date: 09.01.2008 

GLP, unpublished 

Yes BCS 
– 159 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIIA1 10.1.7 Fischer, D. R., 

Ramanarayana

n, T. & Ortego, 

L. 

2006 BYI08330 150 OD - Magnitude of the 

residue in/on soybeans/wheat, potential 

wildlife feed items 

Bayer CropScience, Stilwell, KS, USA 

Bayer CropScience AG, 

Report No.: RAFNP002, 

Edition Number: M-277315-01-1 

Date: 06.09.2006 

GLP 

unpublished 

Yes BCS 

IIIA1 10.1.7 Wolf, C. 2004 Generic field monitoring of birds in potato 

cultivation in northern Germany 

Bayer CropScience AG, 

Report No.: WFC/FS 019, 

Edition Number: M-090336-02-1 

Date: 29.09.2004, Amended: 04.01.2005 

GLP 

unpublished 

Yes BCS 

IIIA 10.2.2.1 Nieden, D. 2005

d 

Acute toxicity of BYI 08330 OD 150 to fish 

(Oncorhynchus mykiss) under static 

conditions 

Bayer CropScience GmbH, Monheim, 

Germany 

Bayer CropScience AG, 

Report No.: EBFNM008, 

Edition Number: M-253916-01-2 

Date: 29.06.2005 

GLP, unpublished 

Yes BCS 

IIIA 10.2.2.2 Dorgerloh, M. 2006

d 

Acute toxicity of BYI 08330 OD 150 to 

larvae of Chironomus riparius in a 48 h 

static laboratory test system 

Bayer CropScience AG, 

Report No.: EBFNM007, 

Edition Number: M-264018-01-2 

Date: 19.01.2006 

GLP, unpublished 

Yes BCS 

IIIA 10.2.2.3 Dorgerloh, M. 2006

c 

Pseudokirchneriella subcapitata growth 

inhibition test with BYI 08330 OD 150 

Bayer CropScience AG, 

Report No.: EBFNM006, 

Edition Number: M-264263-01-2 

Date: 16.01.2006 

GLP 

unpublished 

Yes BCS 

IIIA1 10.3.1.3 G.D. Cappon, 

T.L. Fleeman, 

R.E. Chapin, 

and M.E. Hurtt 

2005 Effects of feed restriction during 

organogenesis on embryo-fetal 

development in rabbit 

Publisher:2005 Wiley-Liss, Inc., 

Journal:Birth Defects Research (Part B), 

Volume:74, 

Pages:424-430, 

Year:2005, 

Report No.: Lit. 8639, 

Edition Number: M-274544-01-1 

Non GLP 

published 

No 
– 160 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIIA1 10.4.2 Barth, M. 2005

a 

Acute toxicity of BYI 08330 150 OD to the 

honeybee Apis mellifera L. under 

laboratory conditions 

BioChem agrar, Gerichshain, Germany 

Bayer CropScience AG, 

Report No.: 051048032, 

Edition Number: M-259122-01-2 

Date: 18.10.2005 

GLP 

unpublished 

Yes BioChem 

IIIA1 10.4.2.1 Barth, M. 2005

b 

Acute toxicity of BYI 08330 150 OD to the 

honeybee Apis mellifera L. under 

laboratory conditions 

BioChem agrar, Gerichshain, Germany 

Bayer CropScience AG, 

Report No.: 051048032, 

Edition Number: M-259122-01-2 

Date: 18.10.2005 

GLP 

unpublished 

Yes BioChem 

IIIA1 10.4.2.2 Barth, M. 2005 Acute toxicity of BYI 08330 150 OD to the 

honeybee Apis mellifera L. under 

laboratory conditions 

BioChem agrar, Gerichshain, Germany 

Bayer CropScience AG, 

Report No.: 051048032, 

Edition Number: M-259122-01-2 

Date: 18.10.2005 

GLP 

unpublished 

Yes BioChem 

IIIA1 10.4.5 Schur, A. 2006 Assessment of side effects of Spirotetramat 

OD 150 on the honey bee (Apis mellifera

L.) in the field 

eurofins-GAB GmbH, Niefern-Öschelbronn, 

Germany 

Bayer CropScience AG, 

Report No.: 20061133/S1-BFEU, 

Edition Number: M-277194-02-2 

Date: 11.09.2006 

GLP 

unpublished 

Yes BCS 

IIIA1 10.4.7 Schur, A. 2005 Assessment of side effects of BYI 08330 

OD 100 on the honey bee (Apis mellifera

L.) in the semi-field 

GAB GmbH, Niefern-Oeschelbronn, 

Germany 

Bayer CropScience AG, 

Report No.: 20041144/01-BZEU, 

Edition Number: M-244490-01-2 

Date: 02.02.2005 

GLP 

unpublished 

Yes BCS 
– 161 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Annex point/ 

reference 

number 

Author(s) Year Title 

Source (where different from company) 

Company, Report No 

GLP or GEP status (where relevant) 

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

IIIA1 10.4.7 Koelzer, U. 2007 Summary of orientating research tunnel 

trials on effects of spirotetramat to honey 

bees according to EPPO 170 

RIFCon GmbH, Leichlingen, Germany 

Bayer CropScience AG, 

Report No.: PA07-174, 

Edition Number: M-294216-01-1 

Date: 17.10.2007 

Non GLP, unpublished 

Yes BCS 

IIIA1 10.5.2 Waltersdorfer, 

A. 

2006

c 

BYI 08330 150 OD; Toxicity to the 

predatory mite Typhlodromus pyri

SCHEUTEN (Acari, Phytoseiidae) using an 

extended laboratory test (under semi-field 

conditions aged residues on apple trees); 

AE 1302943 00 OD15 A101 

Bayer CropScience GmbH, Frankfurt am 

Main, Germany 

Bayer CropScience AG, 

Report No.: CW05/028, 

Edition Number: M-264153-01-1 

Date: 18.01.2006 

GLP 

unpublished 

Yes BCS 

IIIA1 10.5.4 Aldershof, S.A. 2006 Evaluating effects of BYI 08330 OD 150 

applications on mite fauna (Acari) in the 

field (grape vines, France) 

MITOX Consultants, Amsterdam, The 

Netherlands 

Bayer CropScience AG, 

Report No.: B127AFG, 

Edition Number: M-269107-01-1 

Date: 14.03.2006 

GLP 

unpublished 

Yes Mitox 
– 162 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Appendix 

Spirotetramat 

Environmental Risk Assessment (Ecotoxicology) 

for the EU (Annex III) 
– 163 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.1.8 Risk assessment for birds 

Birds may be exposed to spirotetramat by eating contaminated vegetation, seeds and fruits, invertebrate 

prey like arthropods (i.e. insects) or earthworms or vertebrate prey. 

Toxicity Exposure Ratios (TER) and Estimated Theoretical Exposure (ETE) were calculated based on the 

methods, i.e. equations and tabulated values described in the “Guidance Document on Risk Assessment for 

Birds and Mammals Under Council Directive 91/414/EEC” (Sanco/4145/2000 of 25. September 2002, 

http://ec.europa.eu/food/plant/protection/evaluation/guidance/wrkdoc19_en.pdf). 

The risk assessment is based on toxicity figures of the active substance. Data for the formulation are

considered not required, as results from mammalian testing with the formulation do not give an indication 

that the formulated active ingredient is significantly more toxic than the technical active ingredient. In 

addition, birds would typically be exposed to dry residues of spirotetramat on their food items following the 

dilution and spraying of the formulated product. 

The principles of ETE calculation according to the guidance document are presented below. The TER is 

calculated as toxicity endpoint/ETE. 

Maximum ETE values were estimated for use in acute and short-term risk assessments while time-weighted

average ETE values were calculated for use in assessing long-term risks. ETE values were calculated using 

the following equation: 

ETE (mg as/kg bw/day) = FIR/bw x RUD x Application rate x MAF x fTWA x PT x PD x AV 

where: FIR Food Intake Rate (g fresh weight per day) 

 bw Body weight (g) 

 RUD Residue per unit dose (mg/kg fresh weight) 

 MAF Multiple Application Factor 

 fTWA Time-weighted average factor (only used for calculating long-term ETE) 

 PT Proportion of diet obtained in treated area 

 PD Proportion of food-type in diet 

 AV Avoidance factor (no avoidance =1) 

As recommended by the EU Expert Group on Higher Tier Risk Assessment for Birds and Mammals, the 90th

percentile residues on food items were used for the acute risk assessment, and the 50th percentiles for the 

short and long-term risk assessments. The fTWA was calculated from the following equation: 

ftwa = (1-e-kt)/kt 

where: k = ln2/DT50 

 t = averaging time 

The relevant toxicity figures were taken from laboratory studies with spirotetramat. These endpoints are 

listed in table B.9.1.7-1 (above) for the different time scales. The reproductive NOAEL of 4.0 mg/kg bw is 

derived from a 2-generation reproduction study in the mallard. It is also the no effect level with regard to duck 

foot lesions. 

The risk assessment is based on the lowest toxicity endpoints (see table above), which were considered

ecotoxicologically relevant with regard to the risk assessment. 
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.1.8.1 First tier risk assessment (OECD IIIA 10.1.1; OECD IIIA 10.21.2) 

The calculations are based on the following representative application scenarios in the EU: 

• Lettuce: 2 x 0.072 kg a.s./ha (interval 14 d), Northern + Southern Europe 

• Citrus: 2 x 0.096 kg a.s./m canopy height/ha (interval 21 d), max. 0.288 kg a.s./ha, Southern Europe 

Assessment of exposure / Selection of scenarios:

The following relevant exposure scenarios were considered: 

Lettuce: 

- dietary exposure of a medium herbivorous bird feeding on treated herbage (category “leafy crops”) 

- dietary exposure of a small insectivorous bird feeding on contaminated arthropods 

Citrus: 

- dietary exposure of a small insectivorous bird feeding on contaminated arthropods 

In order to calculate the estimated theoretical exposure the respective figures (e.g. RUD, FIR/bw) were 

chosen from the guidance document to estimate food intake rates and concentrations in the food of concern 

(see Table 9.1.8.1-1). For both crops two applications are intended. However, as for citrus the focus is on 

insectivorous species and no residue accumulation is considered on insects, a MAF (multiple application 

factor) is included in the ETE estimations for lettuce only. The MAF is set at 1.2 for the acute and 1.4 for the 

short- and long-term scenario. A default half-life of 10 d is assumed for estimating residue decline on the 

crop. For acute exposure, the 90th percentile and for long-term exposure mean initial residue levels in the 

respective food items are applied. In the first tier worst case assessment it is assumed that the birds feed on 

a single food type, which is not avoided and that they satisfy their entire food demand in the treated area (PD 

and PT = 1). 

Possible food items like earthworms, for which no respective risk assessment was performed, are covered 

by the calculations presented because residues in these items are expected to be much lower. 

Discussion of foot lesions observed in chronic mallard studies:

During avian reproduction studies with the mallard duck dietarily exposed to spirotetramat the occurrence of 

dried and deeply cracked skin on the underside of the feet was observed. These dermal effects to the feet 

may have been exacerbated by an interaction between the wire mesh of the caging (possible physical 

damage) and food contact with excreta (possible chemical exposure) on the cage floors. As the causal 

mechanism was not fully understood in the standard reprotox studies, the notifier performed two further 

studies to gain more information. However, in these new, specially designed studies, the following results 

were obtained: 

1. Ducks only dermally exposed to spirotetramat experience much less severe and transient symptoms 

compared to dietarily exposed birds. 

2. Ducks kept in a semi-natural outdoor environment (turf ground) that were exposed to spirotetramat 

through contaminated diet and through contact with oversprayed turf, also exhibited severe foot 

symptoms. 

Thus, the hypothesis, that the foot lesions were partly caused by the contact with artificial pen floor, could not 

be verified. 

No foot effects were seen in bobwhite quails in the chronic reproduction study and in acute and subchronic 

studies with the bobwhite or the mallard. It can be assumed, that the specific pathology observed is 

somehow specific to duck feet, but as only two species were investigated chronically, this can not be 

concluded for sure. 

In conclusion, the effects on feet skin are considered as ecotoxicologically relevant, as they lead to a severe 

impairment of the bird`s general condition and behaviour. The NOAEL of 4 mg a.s./kg bw used in the 

chronic/reproductive risk assessment is also protective with regard to foot effects. 
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Effective application rate with regard to insectivorous birds in citrus = 0.096 kg/ha

The diet of canopy-dwelling insectivorous birds obtained from within the citrus orchard may contain residues 

from spray applications. Realistically, residues on foliage dwelling arthropods in the canopy should be 

calculated as product of the RUD (residue per unit dose) and that fraction of the application rate that can be 

expected to reach the respective compartment (“effective rate”) of the canopy, and not as product of the 

RUD and the maximum application rate for trees of 3 m height. The application rate is defined per m canopy 

height. Likewise, the maximum spray concentration and the predicted residues in the sprayed canopy (e.g. 

foliage, arthropods) are comparable regardless of whether a tree of 1, 2 or 3 m height has been sprayed. 

Thus, an application rate corresponding to 0.096 kg a.s./ha (= amount per 1 m tree height) is considered in 

the respective ETE calculations for citrus. 

 

Table B.9.1.8.1-1: Acute, short-term and long-term exposure of birds to spirotetramat and calculated 

toxicity exposure ratios, Tier 1 

Crop / Time 

horizon 

Indicator 

species 

Endpoint 

(mg/kg bw) FIR/bw RUD MAF ftwa ETE TER 

Lettuce, 72 g a.s./ha 

Medium 

herbivorous bird 0.76 87 1.2 / 5.7 > 350 Acute

Insectivorous 

bird 

> 2000 

1.04 52 / / 3.9 > 514 

Medium 

herbivorous bird 0.76 40 1.4 / 3.1 > 155 Short term

Insectivorous 

bird 

> 475 

1.04 29 / / 2.2 > 219 

Medium 

herbivorous bird 0.76 40 1.4 0.53 1.6 2.46 Long term

Insectivorous 

bird 

4 

1.04 29 / / 2.2 1.84 

Citrus, 96 g a.s./ha 

Acute Insectivorous 

bird > 2000 1.04 52 / / 5.2 > 385 

Short term Insectivorous 

bird > 475 1.04 29 / / 2.9 > 164 

Long term Insectivorous 

bird 4 1.04 29 / / 2.9 1.38 

FIR/bw...food intake rate in relation to body weight; RUD...residue per unit dose; MAF...multiple application factor; ftwa...time weighted 

average factor; ETE...estimated theoretical exposure; TER...toxicity exposure ratio 

The resulting acute and short-term TER`s are above the respective triggers of Annex VI of Directive 91/414 

and indicate a low risk to wild bird populations after oral intake of feed items contaminated with spirotetramat. 

The long-term TER`s, however, indicate potential risk at tier 1 level. Further refinement of the exposure is 

needed. 

B.9.1.8.2 Higher tier risk assessment 

The long-term risk is refined based on actual residue decline data of spirotetramat on plant foliage and by 

using information on focal species. 

Refinement of long-term risk to herbivorous birds in lettuce:

Focal species: Wood Pigeon (Columba palumbus), FIR/bw = 0.76 

The Wood Pigeon can be considered relevant as a model species for herbivorous birds in leafy crops. 

However, direct and quantitative reports about the extent of Wood Pigeons feeding on lettuce are not 
– 166 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

available. The notifier further argues: “This is not surprising, given the very limited nutritive value of lettuce on 

one hand, and the protective action vegetable farmers are prepared to take against herbivorous birds 

ravaging their crops. There are well known examples for Wood Pigeons feeding on vegetable seedlings, 

particularly on rape and cabbage. In northern France, Wood Pigeons are typically considered even as a pest 

on cabbage.” 

The FIR/bw figure is derived from Sanco/4145/2000. 

Refinement of PD for the Wood Pigeon: PD = 0.15

Overall, Wood Pigeons are herbivorous and feed mainly on grains, fruits, seeds, buds on trees, leaves, root 

crops and may also take invertebrate food, especially to feed their young (English Nature: Woodpigeon – 

Columba palumbus: http://www.plantpress.com/wildlife/o9- woodpigeon.php). They are known to be 

particularly fond of the leaves of cabbages and other brassica vegetables. 

In a study on the feeding biology of Wood Pigeons in the agricultural landscape, Murton et al. (1963) 

examined the crop contents of Wood pigeons over the year. During April and May, legume or brassica 

leaves made up less than about 15 % and 10 % of the diet of 56 and 90 Wood Pigeons, respectively. In the 

winter period (December - February), Wood Pigeons are forced to forage on leafy crops (particularly winter 

oil seed rape), since alternative food sources are at the minimum at this time of year (Inglis et al. 1989). 

However, no applications of spirotetramat are made on lettuce in the winter season. 

Very similar to the results of Murton et al., Colquhoun (1951) reported the share of cabbage to the crop 

content of shot Wood Pigeons at its maximum of around 25 % in January, but with only 13 % (% weight) in 

March and no more incidence until next winter. Thus, Wood Pigeons are unlikely to forage preferentially 

foliage treated with spirotetramat, particularly during the breeding season when plenty and more attractive 

alternative food is available. 

Despite considerable searching efforts, no single report was detected about wood pigeons - or another bird 

species - feeding on lettuce foliage. This lack of reports suggests that the occurrence of birds foraging on 

lettuce is probably not wide-spread, and certainly less important than that of birds foraging on cabbage. 

The PD-factor of 0.15 as identified for the Wood Pigeon and its wide-spread and well reported foraging

preference on cabbages can therefore be considered as appropriate surrogate PD-factor for the use in the 

exposure assessment of herbivorous birds on lettuce during spring/summer. 

It is thus considered conservative to use a PD-factor of 0.15 as worst-case estimation for the exposure of 

herbivorous birds to residues in young leafy crops treated with Spirotetramat. 

Refinement of the residue decline of spirotetramat on lettuce (MAF, fTWA):

In the study of Fischer et al. (2006), the half lives of spirotetramat on leaves of soybean, short grass (wheat), 

and tall grass (wheat) were determined. The mean half-life for soybean was 3.42 days and this figure has 

been proposed for the risk assessment by the notifier. However, when the half-life of one trial that received 

rain one day after application is excluded, the half-life is 4.45 d (see B.9.1.4.). This figure is used in the RMS 

risk assessment as representative worst-case. Soybean is considered as a model plant for the type of diet 

relevant for herbivorous birds in vegetable crops (non-grass herbs). 

A MAF of 1.1 and a 14-d twa factor of 0.41 can be derived according to the formulas given in 

SANCO/4145/2000-Guidance Document. 

To summarize, for the refined long-term risk assessment for herbivores the following assumptions are 

applied in the TER-calculation: Wood Pigeon: FIR/bw 0.76; DT50 4.45 d, MAF 1.1, 14d fTWA 0.41, PD 0.15 

Table B.9.1.8.2-1: Long-term exposure of herbivorous birds to spirotetramat and calculated toxicity 

exposure ratio, refined 

Crop / indicator 

species 

Endpoint 

(mg/kg bw) FIR/bw RUD MAF ftwa PD PT ETE TERlt 

Lettuce 

Wood Pigeon 4 0.76 40 1.1 0.41 0.15 1 0.15 27 
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

The resulting TER-value indicates a low risk to herbivorous birds after the use of spirotetramat in lettuce. 

If there is doubt on the use of the PD factor of 0.15 or on the relevance of the Wood Pigeon, it should be 

considered that under the unrealistic assumption that a medium sized bird would feed on 80 % treated 

lettuce, the TER would be equal to 5 and thus acceptable. 

Refinement of the long-term risk to insectivorous birds in lettuce:

Focal Species - Yellow wagtail (Motacilla flava) 

The default Tier 1 model species for insectivorous birds according to SANCO/4145/2000, the Wren 

(Troglodytes troglodytes) or the Blue Tit (Parus caeruleus) cannot be considered particularly relevant for the 

use of Spirotetramat in lettuce (“leafy crops”). 

Cramp (1998) described the habitat preference of the Wren to be within a predominantly moist mild climatic 

range, offering a wide variety of low cover and foraging opportunities, within or outside woodland, crops and 

aquatic vegetation, fallen trees and branches or heaps of brash, hedgerows, gardens, parks, and 

shrubberies. In a multi-annual study conducted in the years 1982-84 on the habitat requirements of birds on 

farmland in south-western Germany (Seitz 1989), Wrens were recorded 66 times in total per year from which 

21 records were made in spring and summer. From these 21 observations recorded during spring and 

summer, no Wrens were observed foraging in arable fields (average 0 %), but most of them foraging in 

forests (average 63.7 %), hedgerows (average 9.7 %), trees (average 9.7 %) and small forbs (average 

5.6 %). 

Likewise, the Blue Tit, the other insectivorous light weight bird proposed as theoretical worst case model is 

also of low relevance to vegetable/leafy crops. Rather, the Blue Tit is associated with the presence of trees 

(Cramp 1998). In the multi-annual study by Seitz (1989) referred to above, only 0.4 % of all Blue Tits were 

observed in agricultural fields. 

Based on its ecology, and the occurrence on arable land, the Yellow Wagtail (Motacilla flava) is considered 

more relevant for the refined risk assessment of spirotetramat in lettuce at the crop stage BBCH 42-43. This 

species is a summer visitor in Europe from April to September. Its nesting habitat is generally associated 

with areas of low dense herbage either natural or managed particularly along the fringes of wetlands and 

where low perches are available (Buxton et al., 1998). 

The preference of the Yellow Wagtail for fields cropped with spring sown vegetables/leafy crops has been 

reported by various authors: 

a) In a 3-year field study in agricultural land in UK, Mason and Macdonald (2000) monitored the 

territories of eight bird species in eastern England and reported a preference index. The land use in 

the study area was characterised by winter wheat (32 %), winter barley (22 %), potatoes (6.4 %), 

oilseed rape (5.2 %), salad crops (1.2 %), beans (0.8 %), grass (8.6 %), and set-aside (4.3 %). 

Highest densities of Yellow Wagtails were found in spring crops (potatoes, peas, beans and salad 

crops). Only a small fraction of the nests monitored (4 %) were found in grassland, with a similar 

proportion in set-aside. Potatoes held the greatest proportion of territories, although nests were 

found also in spring sown cereals, oil-seed rape, sugar beet, linseed and maize. 

b) The typical association of Yellow wagtails with spring-sown leafy row crops was also underlined by 

Anthes et al. (2002). The authors monitored territories and nests of Yellow wagtails during their 

breeding season from May to June 2002 on arable land in Southern Germany. Clearly most 

territories were counted in spring-sown row crops, (mainly sugar beet and potato but also 

strawberries) which were significantly preferred over their proportional share. 

c) The importance of spring sown row crops for the Yellow Wagtail was finally confirmed by a radiotracking study in potatoes (Wolf 2005, see above under B.9.1.4). 

This information suggests that the Yellow Wagtail is an appropriate focal species for the refined risk

assessment for insectivorous birds in lettuce. Although only limited information is directly available for lettuce 

fields, there are consistent reports of this species in other spring sown leafy row crops. As ecological habitat 

for insectivorous birds, spring crops like potatoes, sugar beet, and salad crops bear many similarities 
– 168 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

particularly in late spring / early summer, which is the season relevant for this refined risk assessment 

addressing the reproductive phase: the leafy crop is grown in rows between which birds can walk and forage, 

with only very limited growth of weeds. 

In consequence, the refined risk assessment for insectivorous birds in lettuce is based on the Yellow Wagtail 

as focal species. 

Yellow Wagtail: FIR/bw = 0.88

The food intake rate to body weight ratio for the Yellow Wagtail is calculated to be 0.88 according to

SANCO/4145/2000 (2002) based on an energy content for arthropods of 21.9 kJ/g dry weight, a water 

content of the food of 70.5 %, an assimilation efficiency of 76 % and a body weight of 17 g (Dittberner and 

Dittberner, 1984). The relationship between body weight and daily energy expenditure is described by the 

following equation according to Crocker et al. (2002): Log(DEE) = 1.0017 + 0.7034 x log bw. 

Refinement of diet composition: PD: 80 % large + 20 % small invertebrates.

The Yellow Wagtail is primarily an insectivorous bird but also feeds on other epigaeic invertebrates (e.g. 

spiders). Its foraging behaviour is well known and comprises mainly three techniques: picking (picks items 

from the ground while walking), run-picking (quick darting run at prey, picking it up either from the ground or 

as it takes off), and fly-catching (makes short flight from the ground or perch, catching the prey in mid-air). 

Based on these foraging techniques the Yellow Wagtail can be expected to feed on a mixed arthropod diet 

mainly comprising of insects that at least partly dwell on or visit the ground. 

A targeted study into the prey spectrum of Yellow Wagtails on arable land has been conducted by Wolf 

(2005). According to his results the Yellow Wagtails foraged on Diptera, Coleoptera, Aphidoidea, 

Hymenoptera and Araneae, and targeted specimen larger than the respective average size. 

Based on these results the notifier considered it to be conservative to assume that the typical diet of Yellow 

Wagtails from lettuce fields consists of 50 % large and 50 % small insects, taken equally from the foliage and 

from the ground. 

The diet composition Wolf (2005) found in his study is in accordance with the data of Davies (1977). In this 

study the feeding behaviour of Yellow Wagtails in flocks or solitary was examined on non-flooded areas of a 

meadow, where the birds fed predominantly on flies around dung pats. Five main prey items were identified. 

The smallest (Staphilinidae and small Spheroceridae) were rejected. The largest (Scatophagidae) were 

patchily distributed and difficult to capture when in swarms and so the next largest prey (large 

Spheroceridae) were also incorporated into the diet. The size of Scatophagidae eaten corresponds to the 

optimum in terms of energy intake per unit handling time. The birds’ behaviour in leaving patches could also 

be predicted by optimal foraging theory. According to these data Yellow Wagtails prefer insects of 3 - 4 mm 

and greater. The size of the insects and the definition of small and large feed items are essential since the 

estimation of the residues of plant protection products on insects as proposed by Kenaga (1973) is based on 

the residues on weed seeds and wheat seeds which are considered to be comparable to small or large 

insects. As wheat seeds are typically 4 - 5 mm in length, insects of ≥ 3 mm body length are defined as large. 

In consequence, the diet of yellow wagtails consists of 76.4 % large insects (Scatophagidae and large 

Spheroceridae) and 23.6 % small insects. With regard to the mass of insects it is reasonable to consider that 

these values are rather conservative as a given number of large insects would contribute more to the daily 

energy intake as the same number of small insects. 

As described above, the selection of the food items is probably determined by the rate of energy content to 

handling time and therefore not stringently a question of species composition. This can be supposed to be 

similar in different habitats/crops. 

In the table below, the diet composition of the Yellow Wagtail as found in Wolf (2005) is shown in detail. 
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.1.8.2-2: Composition of Yellow Wagtail food 

Based on 25 samples of faeces; size of individual arthropods estimated after the size of remains in faeces. 

 * values of stones estimated 

From these figures it can be derived that minimum 80 % of arthropods eaten have a size of 3 mm or more. 

These can be defined as large, as described above. 

For large, predominantly ground dwelling insects a mean RUD = 5.1 is recommended in SANCO/4145/2000. 

Based on these data the diet is considered to consist of 80 % large and 20 % small arthropods 

(corresponding to PD figures of 0.8 / 0.2). This is also in accordance with figures used for the refinement of 

other EU peer-reviewed substances. 

Refinement of the time spent in the treated area: PT = 0.4

The specific preference of the Yellow Wagtail for spring sown vegetable row crops has been confirmed by 

various authors. Mason & McDonald (2002) reported the highest preference of the Yellow Wagtail for 

potatoes, beans and salad crops, with very similar preference indices of 0.702, 0.693 and 0.752, 

respectively. Anthes et al (2002) confirmed the clear preference of Yellow Wagtails particularly for potatoes. 

No data on the specific use of lettuce fields are available but it is reasonable to assume that the use of 

lettuce fields is unlikely to be higher than that of the consistently most attractive crops, i.e. potatoes. As 

mentioned before, a radio-tracking study has been conducted on the foraging pattern of Yellow Wagtails

(Wolf 2005) in and around potato fields. On average, Yellow Wagtails spent 38.4 % of their time in potato 

fields, 39.8 % in cereal fields, 6.8 % in oil seed rape, 3.7 % on streets and field paths, and 11.3 % in other 

habitats. No data from lettuce fields are available from this study since this crop was not cultivated to a 

significant extent in the study area, but the results allow nevertheless to expect that Yellow Wagtails will not 

be exclusively foraging in any specific type of arable fields, including potato fields. 

Thus, for the refined risk assessment with the Yellow Wagtail as insectivorous species of concern, the mean 

portion of diet taken from treated lettuce fields (PT) is considered to be similar to the maximum PT reported 

from the most preferred crops (potatoes: PT = 0.38). Therefore a PT-value of 0.4 is considered a 

conservative estimation for the average portion of diet Yellow Wagtails may obtain from lettuce fields treated 

with Spirotetramat. 

The TER calculation is presented in table B.1.8.2-2. 

(Without refinement of PT the resulting TER is 6.3). 
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Refinement of long-term risk to insectivorous birds in citrus orchards:

The tier 1 model species for insectivorous birds in orchards, the Wren (Troglodytes troglodytes) or the Blue 

Tit (Parus caeruleus), are considered not relevant as typical birds inhabiting citrus orchards. 

Published literature (Barba & Gil-Degado 1990, Belda et al. 1998, Monros et al. 1999) provides evidence for 

the relevance of the Great Tit (Parus major) as typical insectivorous bird species in citrus plantations. 

Several ecological investigations show that Great Tits find favourable breeding habitats in Spanish citrus 

groves, that they occur there with relatively high population densities, and that, at least in certain areas of 

Spain, and at certain times of the year, citrus orchards are even more favourable habitats for Great Tits than 

the surrounding natural habitats (literature sources are summarised and evaluated in Wolf & Gerlach 2006, 

see RMS summary under B.9.1.4). 

In the first risk assessment provided by the notifier, the refinement of the exposure scenario was mainly 

based on the percentual diet composition of large and small arthropods and respective RUD figures as given 

in SANCO 4145/2000 (The derived TER was 6.6). However, in the meantime an arthropod residue study 

after spirotetramat application is available and it has also become scientifically more accepted that for the 

residue concentration in prey items, the location of the arthropod (on foliage or on the ground) is more 

decisive than its size. 

Refinement of spirotetramat residues on arthropods using values measured in the field:

The results of the insect residue study (Wilkens & Schneider 2008) were reported separately for foliage 

dwelling arthropods (21 d twa residue 1.9 mg/kg) and for ground dwelling arthropods (21 d twa residue 0.35 

mg/kg, see B.9.1.4). The 21-d time-weighted-average residue values for spirotetramat + -enol were used for 

the ETE refinement. The total measured residue, including the spirotetramat-ketohxdroxy-metabolite is not 

the ecotoxicollogically relevant figure, as the ketohydroxy metabolite can be considered as 

(eco)toxicologically inactive (see also B.9.1.8.3). The application rates in the field study were about 15 % 

higher than the GAP, however, the residues as derived in the study are included in the calculation. 

Refinement of PD-portion of arthropods taken from the canopy or from the ground:

Based on feeding observations reported in the literature an adapted exposure scenario for the great tit 

feeding in citrus groves on foliage and ground dwelling arthropods is developed in a second risk assessment 

provided by the notifier. 

Great tits mainly feed in the canopy. However, prey is also taken from the ground. In mixed woodlands in 

central Spain, the portion of ground foraging in April and May was reported to be 32 % (Atienza & Illera, 

1997). In this field work activities of the observed great tits in the microhabitat were recorded at 30-s 

intervals. From all foraging activities of the great tit (n=48), 32 % were performed on the ground and the 

remainder in tree- and bush canopies. 

For the long-term scenario an average portion of 32 % of ground dwelling arthropods is therefore considered 

as a realistic estimation of this part of the diet.

Proportion of caterpillars / other arthropods in the diet: 

Barba & Gil-Delgado (1990) investigated the seasonal variation of the nestling diet of the great tit in orange 

groves in eastern Spain. The proportion of caterpillars peaked in early May and declined to about 10 % in the 

period from May 26 - June 05, which fits very well the expected application timing of spirotetramat, and it 

declines even more in the following weeks (see figure below). 

This result confirms that caterpillars are not expected to be a significant part of the prey of great tits at the 

time when spirotetramat will be applied. However, because of the different energy content of caterpillars 

compared to other arthropods, and consequently FIR, the portion should be considered. 

This is also consistent with the observation that few caterpillars were sampled in the arthropod residue study 

conducted end of May / early June in a Spanish citrus orchard (Wilkens & Schneider 2008, see B.9.1.4). 

A PD value of 0.1 will therefore be included for caterpillars in the foliage dwelling arthropod part of the ETE 

calculation. 
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008
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Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Great Tit: FIR/bw = 0.85 for arthropods, 1.23 for caterpillars

Based on bodyweights, energetic requirement and energy values of respective feed items, the food intake 

per day in terms of fresh weight can be estimated according to SANCO/4145/2002. For the Great Tit 

(approximate weight 19 g, based on Buxton et al. 1998) this has been calculated to be 16.2 g of arthropods, 

giving an FIR/bw of 0.85 for arthropods, or 23.4 g and 1.23 for caterpillars. 

Refinement of PT - Proportion of diet obtained in the treated area

The results of the investigation of Crocker et al. (1998): “Contract PN0903: Improving the assessment of 

pesticide risk to birds in orchards” are reported in the EU Guidance Document SANCO/4145/2000. The data 

indicate that for blue tits - a common example of a small insectivore and closely related to the great tit – 95 % 

of the local population spent less than 61 % of potential foraging time in orchard trees. As this is a

conservative refinement approach (95th percentile, the median figure would be 20 %), and because of 

additional information presented below, it is considered acceptable to extrapolate this proportion from 

another tit species in orchards to great tits in citrus. 

This corresponds with a factor of 0.61 for refinement of PT. 

In a study conducted by Mols (2003) in the Netherlands on the foraging behaviour of great tits, the birds were 

attracted to the study orchards by providing nest boxes to compensate for missing nesting sites in modern 

orchards. This is similar to the situation in citrus groves studied in Spain (Barba & Gil-Delgado, see above). 

The birds were monitored with binoculars. An average proportion of foraging trips inside a one-ha orchard of 

48 % was observed for nine breeding pairs. This would correspond to a PT of 0.48. However, as that 

proportion is not verified by means of time-budget, it is considered as evidence only, that great tits strongly 

tend to spend foraging time outside orchards. 

The notifier also mentioned another new study with data from 25 radio-tracking sessions of 22 great tits in 

German pome fruit orchards, delivering a PT of 0.25. However, this study was not yet available to the RMS. 

Overall, considering all reported data on active foraging time of tits in- and outside orchards, the inclusion of 

a PT-value of 0.61 seems to represent a conservative estimation with regard to populations of great tits 

inhabiting monocultural orchards. 

Because of the statement that “Great Tits find favourable breeding habitats in Spanish citrus groves, that 

they occur there with relatively high population densities, and that, at least in certain areas of Spain, and at 

certain times of the year, citrus orchards are even more favourable habitats for Great Tits than the 

surrounding natural habitats” (reported in Wolf & Gerlach 2006, based on Belda et al. 1998), some more

justification on the refinement of the PT to < 1 with regard to the intended use of spirotetramat should be 

added. Taken on it`s own, this may appear to suggest a high habitat attractiveness. However, taking a closer 

look into the underlying study it becomes clear that there are several additional factors to be considered (e.g. 

altitude of the locations and time of observations). Belda et al. (1998) compared the laying date and the size 

of the first clutch of great tits in a Spanish orange grove with those in other, more natural habitats. Nest 

boxes were installed in the orange grove and two holm oak forests at different altitudes, a pine forest and a 

(semi-evergreen) zeen oak forest. Whilst the orange grove was close to the sea level, the forest sites were 

located at 500, 900-950, 1000-1050 and 900-1100 m altitude. The mean laying date of the first clutch in the 

orange plantation (21. April ± 6.2 d) was about 10-15 days earlier than in the forest habitats. However, this 

time point is significantly earlier than the first applications of spirotetramat (end of May / early June). The 

laying dates were also earlier at low compared to high altitudes. Overall, the earlier laying time and the 

greater size of the first clutch in the orange grove were linked to the earlier food availability in the evergreen 

habitats and particularly in the habitats with lower altitude. 

An earlier availability of food in the (evergreen) citrus grove cannot be translated into better food availability 

in May and June. In contrary, the typically preferred food of great tits (caterpillars) becomes rather scarce 

after the initial peak (Barba & Gil-Delgado 1990). Thus, an earlier peak of caterpillars is rather linked to an 

earlier disappearance than indicative of a good caterpillar supply later on. 

In conclusion, the results of Belda et al. (1998) only concerned the first clutch in April, whilst spirotetramat 

will typically be applied not before end of May / early June. Any early season effects which may favour 

evergreen sea-side level habitats like citrus groves in Spain in the early spring (e.g. early availability of 
– 173 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

caterpillars) may even have reversed at the spirotetramat application time, with metamorphosis of the 

caterpillars, increasing temperature and dryness. Therefore the results of Belda et al. (1998) do not put into 

question any proposal for a PT refinement for great tits nesting in citrus. The comparison of the breeding 

habitat quality of the citrus grove to not-nearby forests at about 1000 m altitude in early spring cannot be 

seen as indicative for the breeding habitat quality of the citrus grove compared to its surroundings in early 

summer. 

In conclusion, the following refinement factors are included in the revised ETE calculation for citrus: FIR/bw 

for the great tit: 0.85 for arthropods, 1.23 for caterpillars; PD 0.58 for foliage dwelling arthropods, 0.1 for foliar 

dwelling caterpillars, 0.32 for ground dwelling arthropods; PT 0.61; measured 21-d twa residues on foliage 

and ground dwelling arthropods: 1.9 and 0.35 mg/kg. 

All refined TERlt calculations are presented in the tables below: 

Table B.9.1.8.2-3: Long-term exposure of birds to spirotetramat and calculated toxicity exposure 

ratios, higher-tier refinement for lettuce 

Indicator 

species 

AR (kg 

a.s./ha) Food type FIR/bw RUD Ftwa MAF PT PD ETE Endpoint 

(mg/kg bw) TER trigger

Wood 

Pigeon 0.072 plants 0.76 40 0.41 1.1 1 0.15 0.15 4 27 5 

large 

arthropods 0.88 5.1 - - 0.4 0.8 0.10 

small 

arthropods 0.88 29 - - 0.4 0.2 0.15 

Yellow 

Wagtail 0.072 

sum: 0.25 

4 16 5 

Table B.9.1.8.2-4: Long-term exposure of birds to spirotetramat and calculated toxicity exposure 

ratios, higher-tier refinement for citrus 

Indicator 

species Food type FIR/bw C

1

 PT PD ETE Endpoint 

(mg/kg bw) TER trigger 

foliage-dwelling 

arthropods 0.85 1.9 0.61 0.58 0.571 

caterpillars 1.23 1.9 0.61 0.10 0.143 

ground dwelling 

arthropods 0.85 0.35 0.61 0.32 0.058 

Great Tit 

sum: 0.772 

4 5.2 5 

1

 21-d twa concentration of spirotetramat + -enol measured in arthropods in the field [mg/kg f.w.] 

Because the resulting TER for great tits in citrus is only marginally above the trigger, the following arguments 

could be additionally considered: 

- the residues in arthropods were derived after application rates 15 % higher than the GAP, a 15 % 

overestimation can therefore be assumed 

- The NOAEL of 4 mg/kg bw is the lowest figure from two mallard duck reproduction studies and one 

study with the bobwhite quail (NOAELrepro for the quail: 74 mg/kg bw, highest tested dose). The use 

of a geometric mean of the NOAELs for the two species might be a refinement option. 

Southern EU MS during their national risk evaluations should consider that the risk refinement for citrus as 

presented above does not cover a potential spirotetramat use during the period from March to mid of May 

(because a higher portion of caterpillars in the diet and potentially a higher PT fraction could be expected 

during this period). 
– 174 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Overall conclusion:

All resulting TER`s indicate a low risk to populations of birds after the use of spirotetramat according to GAP. 

References: 

Anthes, N.; Gastel, R.; Quetz, P.-Ch. (2002): Bestand und Habitatwahl einer Ackerpopulation der Schafstelze (Motacilla 

f. flava) im Landkreis Ludwigsburg, Nordwuerttemberg. Orn.Jh.Bad.-Württ, Vol. 18, p. 347-361. 

Atienza J.C. & J.C. Illera (1997): Tree species selection to perform singing and foraging behaviour by Great and Blue 

Tits: a trade-off between food gathering and territorial behaviour ? Bird study 44, p. 117-119. 

Barba & Gil-Delgado (1990): Seasonal variation in nestling diet of the Great Tit Parus major in orange groves in eastern 

Spain. Ornis scandinavica 21, p. 296-298. 

Belda et al. (1998): Laying date and clutch size of great tits Parus major in the Mediterranean region: a comparison of 

four habitat types. J. Ornithologie 139: 269-276. 

Colquhoun, M. K. (1951): The woodpigeon in Britain. Agricultural Research Council, London, The woodpigeon in Britain, 

Vol. 10, p. 1-74. 

Crocker et al. (1998): Contract PN0903: Improving the assessment of pesticide risk to birds in orchards. CSL report EH 

18/02, 38 pp. 

Dittberner, H. & Dittberner, W (1984): Die Schafstelze - Motacilla flava. Publisher:Ziemsen Verlag, Wittenberg, Die neue 

Brehm Bücherei, p. 72-79, 90-97, 128-131. 

Inglis, i. R.; Thearle, R. J. P. & Isaacson, A. J. (1989): Woodpigeon (Columba palumbus) damage to oilseed rape. Crop 

Protection, Vol. 8, p. 299-309. 

Mason, C.F. & Macdonald, S.M. (1999): Influence of landscape and land-use on the distribution of breeding birds in 

farmland in eastern England. The Zoological Society of London, J. Zool. Lond., Vol. 251, p. 339 - 348. 

Mols, Ch.M.H. (2003): Great Tits (Parus major) foraging for caterpillars contribute to biological control in apple orchards. 

Ph.D. Thesis, University of Utrecht, 104 pp. 

Murton, R.K.; Westwood, N.J. and Isaacson, A.J. (1963): The feeding habits of the woodpigeon Columba palumbus, 

stock dove C. oenas and turtel dove Streptopelia turtur. Ibis, Vol. 106, p. 174-188. 

Seitz, B.-J. (1989): Beziehungen zwischen Vogelwelt und Vegetation im Kulturland, Untersuchungen im 

suedwestdeutschen Huegelland - Beihefte zu den Veroeffentlichungen fuer Naturschutz und Landschaftspflege in 

Baden-Wuerttemberg. Landesanstalt für Umweltschutz Baden - Wuerttemberg, Beihefte zu den Veroeffentlichungen fuer 

Naturschutz und Landschaftspflege in Baden-Wuerttemberg, Volume 54, p.1-236. 

Wolf, C. & Gerlach, J. (2006): Insectivorous birds in southern European citrus plantations - focal species and their diet 

composition. Unpublished report. 

B.9.1.8.3 Risk to birds from plant metabolites 

The following metabolites of spirotetramat were found in lettuce metabolism at levels above 10 % of the total 

radioactive residue (TRR): BYI 08330-enol (17.8 %) and BYI 08330-enol glycoside (11.4 %). In smaller 

amounts the following metabolites were observed in plants metabolism studies at levels above 0.01 mg/kg: 

BYI 08330-ketohydroxy, BYI 08330- monohydroxy, BYI 08330-di-hydroxy, BYI 08330-desmethyl-enol, and 

BYI 08330-desmethyl-ketohydroxy. According to current guidance, only metabolites > 10 % have to be 

considered, however, as the notifier presented information also to minor metabolites, this is reported here. 

The enol metabolite is also the primary and main metabolite in the rat and hen metabolism study (see B.7 

part of this monograph). In so far, it can be considered to be toxicologically well characterised and to be 

covered by the respective avian toxicological studies which were conducted with spirotetramat. 

The enol glycoside metabolite is immediately and quantitatively metabolised to BYI 08330-enol after dietary 

uptake. Thus, this metabolite can be considered to be ecotoxicologically sufficiently covered. 

BYI 08330-desmethyl-enol was also observed in the rat and hen metabolism study and thus is covered by 

respective toxicological studies conducted with parent compound. 

Likewise, the ketohydroxy metabolite was detected in the rat, and furthermore, an acute toxicity study on rat 

is available for this metabolite (Schüngel, 2005) which reveals that this metabolite shows no toxicity to 

mammals (LD50 > 5000 mg a.s./kg bw). This is supported by the observation that the ketohydroxy 
– 175 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

metabolite proved less toxic than the parent compound and the enol metabolite in all ecotoxicological studies 

conducted. There is no reason to assume that these metabolites would be more toxic to birds. 

The metabolites BYI 08330-monohydroxy, BYI 08330-di-hydroxy and BYI 08330-desmethyl-ketohydroxy 

showed no toxicity in acute rat studies (LD50 > 5000 mg a.s./kg bw, Schüngel, 2005, Schüngel, M., 2006a 

and Schüngel, 2006b, respectively); it can thus be considered highly unlikely that exposure might result in 

unacceptable effects to terrestrial vertebrates. 

Hence, it is concluded that the plant metabolites of spirotetramat are covered by existing ecotoxicology or 

toxicology studies, or they were shown not to be toxic to terrestrial vertebrates. 

In conclusion, the risk to birds from plant metabolites appears low. 

B.9.1.8.4 Risk to birds from secondary poisoning 

The log POW of spirotetramat is 2.51 at pH 7 and hence it`s potential to bio-accumulate is considered low. 

An assessment of the risk through bioaccumulation is not triggered according to SANCO/4145/2002. 

This applies also to the main metabolites of spirotetramat as the log POW of BYI 08330-cis-ketohydroxy is 1.3 

and of BYI 08330-enol is 0.3 (at pH 7). 

B.9.1.8.5 Risk to birds from the consumption of contaminated drinking water 

The risk to birds from uptake of contaminated drinking water is calculated according to SANCO/4145/2000. 

As the exposure via drinking from surface water bodies is considered to be negligible, the exposure from 

puddles of spray liquid is assessed as a worst case. The calculations are based on the intended use in

lettuce (72 g a.s./ha), which covers also the citrus rate, because the concentration in the spray liquid is 

higher. Furthermore the formation of spray puddles in citrus orchards in Southern EU is highly unrealistic. 

TER = LD50/Dailydose 

Dailydose = (PEC drinking water x Totalwater ingestion rate)/W 

Total water ingestion rate = 0.059 x W0.67

W = bw in kg

Table B.9.1.8.5-1: Parameters and calculations for the assessment of the acute and short-term risk to 

birds from the uptake of contaminated drinking water from puddles and leaf axils 

Parameter Value 

Application rate [g ai/ha] 72 

Application volume [L/ha] 500 (- 1000) 

PECdrinking water [mg ai/L], assuming a dilution factor of 5 28.8 

W [kg], Body weight of a relevant indicator bird 0.015 

Total water ingestion rate [L/d] 0.0035 

Daily dose [mg ai/kg bw/d] 6.8 

LD50 acute [mg ai/kg bw] > 2000 

LD50 short-term [ mg ai/kg bw/d] > 475 

TER acute > 294 

TER short-term > 70 

The TER-values for acute and short-term exposure via contaminated drinking water indicate that the risk to 

birds via this route is low. 
– 176 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Overall conclusion 

After the use of spirotetramat according to the relevant GAP no unacceptable effects on populations of wild 

birds are expected. 

B.9.2.4 Risk assessment for aquatic organisms 

The risk assessment for aquatic organisms is based on the recommendations of the Guidance Document on 

Aquatic Ecotoxicology (http://ec.europa.eu/food/plant/protection/evaluation/guidance/wrkdoc10_en.pdf) and 

follows a tiered approach. 

The standard risk assessment (first tier) is based on the calculation of the toxicity/exposure ratios (TER) 

taking into consideration the most sensitive organism of each group. TER values will be estimated as the 

ratio of EC/LC50 or NOEC (toxicity, see table B.9.2.4-1) to PECsw (exposure, see table B.9.2.4-2 and B.9.2.4-

3) calculated with the FOCUS surface water model. This model based on scenarios considering a tiered 

sequence of exposure assessment steps (step 1, 2, 3 and 4). If trigger values for the acute risk (TERA) or the 

chronic risk (TERLT) stated in directive 91/414/EEC are failed and hence a potential risk is indicated 

according to the standard risk assessment, appropriate refinement steps can be considered (higher tier risk 

assessment). Usually, in the higher tier risk assessment higher tier data, e.g. additional single species tests 

and micro- and mesocosms can be considered. 

The aquatic risk assessment includes spirotetramat (as active substance and formulated in the product BYI 

08330 OD 150) and the environmentally relevant metabolites: 

• BYI 08330-enol (> 10 % in water and sediment, max 97.5 % in total water/sediment system at day 7) 

• BYI 08330-ketohydroxy (> 10 % in water and sediment, max 40.5 % in total water/sediment system at 

day 120) 

• 4-Methoxycyclohexanone (> 10 % in photolysis study) 

• 4-Methoxycyclohexylamin carboxylic acid (> 10 % in photolysis study). 

One metabolite, spirotetramat-MA-amide, is relevant for the groundwater risk assessment, but was not 

predicted to exceed 0.1 μg/L. Therefore aquatic tests are not required and an aquatic risk assessment is not 

triggered. 

Table B.9.2.4-1: Summary of acute and chronic toxicity data for aquatic organisms which are relevant 

for risk assessment (most sensitive species of each group) 

Group Test substance Time-scale 

(Test type) Endpoint Toxicity1(mg/L) 

Fish 

Cyprinodon variegatus Spirotetramat 96 h (flowthrough) 

Mortality, LC50 1.96 (mm)

Pimephales promelas Spirotetramat 33 d (flowthrough) 

Fry survival, NOEC 0.534 (mm)

Oncorhynchus mykiss BYI 08330 

OD 150 

(formulation) 

96 h (flowthrough) Mortality, LC50 9.48 (nom)

(1.41 mg as/L) 

Oncorhynchus mykiss BYI 08330-enol 96 h (static, 

limit) 

Mortality, LC50 > 100 (nom)

Danio rerio 4-Methoxycyclohexanone 

96 h (static, 

limit) 

Mortality, LC50 > 100 (nom)

Aquatic invertebrate 

Daphnia magna Spirotetramat 48 h (static) Mortality, EC50 > 42.7 (mm)

Crassostrea virginica Spirotetramat 96 h (static) Shell deposition, EC50 0.85 (mm)
– 177 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Group Test substance Time-scale 

(Test type) Endpoint Toxicity1(mg/L) 

Daphnia magna Spirotetramat 21 d (static) Mortality adults, NOEC 2.0 (nom)

Daphnia magna BYI 08330-enol 48 h (static, 

limit) 

Mortality, EC50 > 100 (nom)

Daphnia magna 4-Methoxycyclohexanone 

48 h (static, 

limit) 

Mortality, EC50 > 100 (nom)

Sediment dwelling organisms 

Chironomus riparius Spirotetramat 48 h (static, 

water only) 

Mortality, EC50 1.3 (nom)

Chironomus riparius Spirotetramat 21 d (static, 

spiked water) 

Emergence, NOEC 0.1 (nom)

Chironomus riparius BYI 08330 

OD 150 

(formulation) 

48 h (static, 

water only) 

Mortality, EC50 4.41 (mm)

(0.66 mg as/L) 

Chironomus riparius BYI 08330-enol 48 h (static, 

water only) 

Mortality, EC50 74.9 (nom)

Chironomus riparius BYI 08330-

ketohydroxy 

48 h (static, 

water only) 

Mortality, EC50 > 100 (nom)

Chironomus riparius 4-Methoxycyclohexanone 

48 h (static, 

water only) 

Mortality, EC50 > 100 (nom)

Chironomus riparius 4-Methoxycyclohexylamin 

carboxylic acid 

48 h (static, 

water only) 

Mortality, EC50 > 100 (nom)

Algae 

Skeletonema costatum Spirotetramat 72 h (static) Biomass: EbC50 

Growth rate: ErC50 

0.36 (mm)

0.96 (mm)

Pseudokirch subcap. BYI 08330 

OD 150 

(formulation) 

72 h (static) Biomass: EbC50 

Growth rate: ErC50 

43.4 (mm)

(6.56 mg as/L) 

> 54.3 (mm)

(> 8.2 mg as/L) 

Pseudokirch subcap. BYI 08330-enol 72 h (static) Biomass: EbC50 

Growth rate: ErC50 

> 100 (mm)

> 100 (mm)

Desmodesmus subcap. 4-Methoxycyclohexanone 

72 h (static, 

limit) 

Biomass: EbC50 

Growth rate: ErC50 

> 100 (nom)

> 100 (nom)

Higher plant 

Lemna gibba Spirotetramat 7 d (static) Yield: EyC50 

Growth rate: ErC50 

4.49 (mm)

6.21 (mm)

Lemna gibba BYI 08330-enol 7 d (static) Yield: EyC50 

Growth rate: ErC50 

5.4 (mm)

19.3 (mm)

1 Toxicity based on nominal (nom) or mean measured concentrations (mm) 

The risk to aquatic organisms from spirotetramat, its metabolites and the formulation was assessed for

applications in citrus (worst case) and lettuce. Global maximum PECSW values for the intended application 

scenarios were calculated with the FOCUS surface water model. For the active substance and the 

environmentally relevant metabolites, BYI 08330-enol and BYI 08330-ketohydroxy (both major in water and 

sediment), 4-methoxycyclohexylamino carboxylic acid and 4-methoxycyclohexanone (major metabolites in 

photolysis studies) the maximum PECSW values were calculated with FOCUS step 1 and step 2. 

In addition, FOCUS step 3 and step 4 calculations were performed for the application of spirotetramat in 

citrus (worst case). 

Details of PECSW calculations with the FOCUS surface water modelling are presented in the appendix to the 

fate section B.8 and the maximum observed PECSW values are summarised in table B.9.2.4-2 (citrus) and 

B.9.2.4-3 (lettuce). 
– 178 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.2.4-2: Citrus: Summary of maximum observed PECSW values of spirotetramat and 

metabolites using FOCUS step 1 – 4. 

max. PECSW [μg L-1] 

FOCUS 

STEP 1 

FOCUS 

STEP 2 

FOCUS 

STEP 3 

FOCUS 

STEP 4 Substance 

 South EU D6 – ditch* R4 – stream* D6 – ditch* R4 – stream*

Spirotetramat 84.9 11.6 8.43 6.51 5.86 nc 

BYI 08330-enol 168 15.6 nc nc nc nc 

BYI-ketohydroxy 46.5 8.59 nc nc nc nc 

4-Methoxycyclohexanone 8.38 1.39 nc nc nc nc 

4-Methoxycyclohexylaminocarboxylic acid 1.58 1.21 nc nc nc nc 

* worst case scenario 

nc...not calculated 

Table B.9.2.4-3: Lettuce: Summary of maximum observed PECSW values of spirotetramat and 

metabolites using FOCUS step 1 and 2. 

max. PECSW [μg L-1] 

FOCUS 

STEP 1 

FOCUS 

STEP 2 Substance 

 South EU (worst case) 

Spirotetramat 18.1 0.585 

BYI 08330-enol 37.1 0.950 

BYI-ketohydroxy 9.48 0.662 

4-Methoxycyclohexanone 1.72 0.071 

4-Methoxycyclohexylaminocarboxylic acid 0.069 0.061

Acute and chronic risk 

First a conservative risk assessment was performed and TERA and TERLT-values were estimated based on 

the maximum observed PECSW from FOCUS Step 1 and 2. If a TER value fails the relevant trigger value at 

the step 2 a refinement is required on the basis of FOCUS step 3 and step 4 calculations. 

Active substance, metabolites and formulation 

The TER-values were calculated for the most sensitive species of fish, invertebrates, algae and aquatic 

plants for the active substance, its metabolites and the formulation. 

For spirotetramat additionally endpoints from studies with marine test organisms were available and the 

results were considered for the acute risk assessment (see argumentation in the main section B.9 under

point B.9.2.1.5 Effects on marine or estuarine species). Thus the acute risk is based on the endpoint of 

marine fish species Cyprinodon variegates and the risk to aquatic invertebrates was assessed with the 

results of the eastern oyster. This endpoint (EC50: 0.85 mg a.s./L) is comparable with the endpoints from the 

most sensitive sediment dwelling species Chironomus riparius (EC50: 0.66 mg a.s./L, endpoint from test with 

the formulation). Furthermore the risk for algae was assessed by considering the endpoint from marine 

diatom Skeletonema costatum, the most sensitive algae species. 

A summary of toxicity (EC50/LC50 and NOEC), relevant global maximum PECSW and calculated TER values 

for spirotetramat, metabolites and the formulation are presented in tables B.9.2.4-4 until B.9.2.4-8: 
– 179 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.2.4-4: Citrus: Acute and chronic toxicity exposure ratios (TERA and TERLT) based on worst 

case PECSW from FOCUS STEP 1 

Organism Test substance 

Toxicity 

endpoint 

(mg a.i./L) 

Time 

scale 

PEC 

(mg/L) TER 

Annex 

VI 

Trigger 

Fish 

Cyprinodon variegatus Spirotetramat 1.96 96 h 0.0849 23 100 

Pimephales promelas Spirotetramat 0.534 33 d 0.0849 6 10 

Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.0849 17 100 

Oncorhynchus mykiss BYI 08330-enol > 100 96 h 0.1680 > 595 100 

Danio rerio 4-Methoxycyclohexanone > 100 96 h 0.0084 > 11933 100 

Aquatic invertebrates 

Daphnia magna Spirotetramat > 42.7 48 h 0.0849 > 503 100 

Crassostrea virginica Spirotetramat 0.85 48 h 0.0849 10 100 

Daphnia magna Spirotetramat 2.0 21 d 0.0849 24 10 

Daphnia magna BYI 08330-enol > 100 48 h 0.1680 > 595 100 

Daphnia magna 4-Methoxycyclohexanone > 100 48 h 0.0084 > 11933 100 

Sediment dwelling organisms 

Chironomus riparius Spirotetramat 1.3 48 h 0.0849 15 100 

Chironomus riparius Spirotetramat 0.1 21 d 0.0849 1 10 

Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0849 8 100 

Chironomus riparius BYI 08330-enol 74.9 48 h 0.1680 446 100 

Chironomus riparius BYI-ketohydroxy > 100 48 h 0.0465 > 2151 100 

Chironomus riparius 4-Methoxycyclohexanone > 100 48 h 0.0084 > 11933 100 

Chironomus riparius 4-Methoxycyclohexylamino-carboxylic acid > 100 48 h 0.0016 > 63291 100 

Algae 

Skeletonema costatum Spirotetramat 0.36/0.96 72 h 0.0849 4/11 10 

P. subcapitata BYI 08330 OD 150 6.56/>8.2 72 h 0.0849 77/>97 10 

P. subcapitata BYI 08330-enol > 100 72 h 0.1680 > 595 10 

Desmodesmus subcapitata 4-Methoxycyclohexanone > 100 72 h 0.0084 > 11933 10

Higher plant 

Lemna gibba Spirotetramat 4.49/6.21 7 d 0.0849 53/73 10 

Lemna gibba BYI 08330-enol 5.4/19.3 7 d 0.1680 32/115 10 

Table B.9.2.4-5: Lettuce: Acute and chronic toxicity exposure ratios (TERA and TERLT) based on worst 

case PECSW from FOCUS STEP 1 

Organism Test substance 

Toxicity 

endpoint 

(mg a.i./L) 

Time 

scale 

PEC 

(mg/L) TER 

Annex 

VI 

Trigger 

Fish 

Cyprinodon variegatus Spirotetramat 1.96 96 h 0.0181 108 100 

Pimephales promelas Spirotetramat 0.534 33 d 0.0181 30 10 

Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.0181 78 100 

Oncorhynchus mykiss BYI 08330-enol > 100 96 h 0.0371 > 2695 100 

Danio rerio 4-Methoxycyclohexanone > 100 96 h 0.0017 > 58140 100 

Aquatic invertebrates 

Daphnia magna Spirotetramat > 42.7 48 h 0.0181 > 2359 100 

Crassostrea virginica Spirotetramat 0.85 48 h 0.0181 47 100 

Daphnia magna Spirotetramat 2.0 21 d 0.0181 110 10 

Daphnia magna BYI 08330-enol > 100 48 h 0.0371 > 2695 100 

Daphnia magna 4-Methoxycyclohexanone > 100 48 h 0.0017 > 58140 100 

66 

Chironomus riparius Spirotetramat 1.3 48 h 0.0181 72 100 

Chironomus riparius Spirotetramat 0.1 21 d 0.0181 6 10 
– 180 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Organism Test substance 

Toxicity 

endpoint 

(mg a.i./L) 

Time 

scale 

PEC 

(mg/L) TER 

Annex 

VI 

Trigger 

Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0181 36 100 

Chironomus riparius BYI 08330-enol 74.9 48 h 0.0371 2019 100 

Chironomus riparius BYI-ketohydroxy > 100 48 h 0.0095 10549 100 

Chironomus riparius 4-Methoxycyclohexanone > 100 48 h 0.0017 > 58140 100 

Chironomus riparius 4-Methoxycyclohexylamino-carboxylic acid > 100 48 h 0.00007 > 14.4 x 105 100 

Algae 

Skeletonema costatum Spirotetramat 0.36/0.96 72 h 0.0181 20/53 10 

P. subcapitata BYI 08330 OD 150 6.56/>8.2 72 h 0.0181 362/>453 10 

P. subcapitata BYI 08330-enol > 100 72 h 0.0371 > 2695 10 

Desmodesmus subcapitata 4-Methoxycyclohexanone > 100 72 h 0.0017 > 58140 10 

Higher plant 

Lemna gibba Spirotetramat 4.49/6.21 7 d 0.0181 248/343 10 

Lemna gibba BYI 08330-enol 5.4/19.3 7 d 0.0371 146/520 10 

Table B.9.2.4-6: Citrus: Acute and chronic toxicity exposure ratios (TERA and TERLT) based on worst 

case PECSW from FOCUS STEP 2 – mentioned are only these organisms which failed the trigger at 

Step 1 

Organism Test substance 

Toxicity 

endpoint 

(mg a.s./L) 

Time 

scale 

PEC 

(mg/L) TER 

Annex 

VI 

Trigger 

Fish 

Oncorhynchus mykiss Spirotetramat 1.96 96 h 0.0116 169 100 

Oncorhynchus mykiss Spirotetramat 0.534 33 d 0.0116 46 10 

Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.0116 122 100 

Aquatic invertebrates 

Crassostrea virginica Spirotetramat 0.85 48 h 0.0116 73 100 

Sediment dwelling organisms 

Chironomus riparius Spirotetramat 1.3 48 h 0.0116 112 100 

Chironomus riparius Spirotetramat 0.1 21 d 0.0116 9 10 

Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0116 57 100 

Algae 

Skeletonema costatum Spirotetramat 0.36/0.96 72 h 0.0116 31/83 10 

Table B.9.2.4-7: Lettuce: Acute and chronic toxicity exposure ratios (TERA and TERLT) based on worst 

case PECSW from FOCUS STEP 2 – mentioned are only these organisms which failed the trigger at 

Step 1 

Organism Test substance 

Toxicity 

endpoint 

(mg a.s./L) 

Time 

scale 

PEC 

(mg/L) TER 

Annex 

VI 

Trigger 

Fish 

Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.00059 2410 100 

Aquatic invertebrates 

Crassostrea virginica Spirotetramat 0.85 48 h 0.00059 1453 100 

Sediment dwelling organisms 

Chironomus riparius Spirotetramat 1.3 48 h 0.00059 2222 100 

Chironomus riparius Spirotetramat 0.1 21 d 0.00059 171 10 

Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.00059 1128 100 
– 181 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.2.4-8: Citrus: Acute and chronic toxicity exposure ratios (TERA and TERLT) based on worst 

case PECSW from FOCUS STEP 3 – mentioned are only these organisms which failed the trigger at 

Step 2 

Organism Test substance 

Toxicity 

endpoint 

(mg a.s./L) 

Time 

scale 

PEC* 

(mg/L) TER 

Annex 

VI 

Trigger 

Aquatic invertebrates 

Crassostrea virginica Spirotetramat 0.85 48 h 0.0084 101 100 

Sediment dwelling organisms 

Chironomus riparius Spirotetramat 0.1 21 d 0.0084 12 10 

Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0084 78 100 

*highest PECsw from the worst case scenario D6 (Thiva) ditch 

Table B.9.2.4-9: Citrus: Acute toxicity exposure ratios (TERA ) based on worst case PECSW from 

FOCUS STEP 4 (5 m buffer zone) – mentioned are only these organisms which failed the trigger at 

Step 3 

Organism Test substance 

Toxicity 

endpoint 

(mg a.s./L) 

Time 

scale 

PEC* 

(mg/L) TER 

Annex 

VI 

Trigger 

Sediment dwelling organisms 

Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0059 113 100 

*highest PECsw from the worst case scenario D6 (Thiva) ditch 

Acute risk from active substance and formulation: 

Spirotetramat and the formulation BYI 08330 OD 150 are toxic to standard test species of fish, aquatic

invertebrates, algae and higher plants. The most sensitive species is the sediment dwelling organism 

Chironomus riparius with an LC50 of 0.66 mg a.s./L, based on a study performed with the formulation. In 

general the toxicity of the formulation is slightly higher when compared to the toxicity of the active substance 

alone. 

Citrus: All TERA values for fish, daphnids, algae and higher plants calculated with global maximum PECSW

from FOCUS step 2 (first tier risk assessment) meet the relevant triggers established in directive 

91/414/EEC. 

However, the calculated TER value for the most sensitive species Chironomus riparius (based on test with 

the formulation) is 8, 57 and 78 calculated with FOCUS STEP 1, 2 and 3 (worst case scenario D6 ditch),

respectively. Since all TER values are below the relevant trigger of 100 the TER is also calculated with 

PECsw from FOCUS STEP 4 considering a 5 m buffer zone to surface water. This calculation based on the 

PECsw of 0.00586 mg/L from the worst case scenario D6 (Thiva) ditch and resulted in a TER of 113. 

Lettuce: All TERA values for fish, aquatic invertebrates (daphnids and sediment dwelling organisms), algae 

and higher plants based on calculations with the global maximum PECSW from FOCUS STEP 2 are clearly 

above the relevant trigger values established in directive 91/414/EEC, thus the risk is considered acceptable. 

In conclusion, the acute risk can be considered as acceptable for applications in citrus provided that risk 

mitigation measures (at least a buffer zone of 5 m to surface water) are implemented. For applications in 

lettuce an acceptable risk is indicated without risk mitigation measures. 

Acute risk from metabolites: 

Aquatic toxicity studies with fish, aquatic invertebrate and algae have been conducted with the metabolites 

BYI 08330-enol and 4-methoxycyclohexanone. Tests with the metabolites BYI 08330-ketohydroxy and 4-

methoxycyclohexylamino carboxylic acid were performed with the most sensitive organism, Chironomus 

riparius, only. 
– 182 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

In conclusion all environmental relevant metabolites are of low acute toxicity to aquatic organisms and all 

calculated TER values for all intended uses are well above the relevant trigger values. Thus the risk from 

metabolites of spirotetramat to aquatic organisms is expected to be low. 

Chronic risk from active substance and formulation:

The assessment of the chronic toxicity is based on long term studies with fish (P. promelas) and aquatic 

invertebrates (C. riparius) with the active substance. 

Citrus: For fish the safety factor (TERLT) was calculated to be 46 based on PECsw from FOCUS step 2. For 

aquatic invertebrates a TERLT of 12 was estimated based on PECsw from FOCUS step 3. Both TERLT values 

are above the relevant trigger of 10. 

Lettuce: The safety factors for fish and aquatic invertebrates were calculated to be 30 (based on FOCUS 

step 1 calculation) and 171 (based on FOCUS step 2 calculation). Thus both TERLT values are clearly above 

the relevant trigger of 10. 

In conclusion for the intended uses in citrus and lettuce the chronic risk of spirotetramat for aquatic 

organisms is expected to be low. 

Bioaccumulation 

Spirotetramat has a logPOW of 2.51 and studies on bioconcentration are not triggered. Therefore the potential 

of bioconcentration of spirotetramat in aquatic organisms is expected to be low and the risk of 

bioaccumulation in aquatic ecosystems is considered acceptable. 

The bioaccumulation potential of all major metabolites in water and sediment (spirotetramat-enol, 

spirotetramat-ketohydroxy, 4-methoxycyclohexanone and 4-methoxycyclohexylamino carboxyl acid) is also 

assumed to be low, due to logPOW values clearly lower than 3. Thus, it can be concluded that the risk of 

bioaccumulation of the major metabolites in the aquatic ecosystem is acceptable. 

B.9.3.1 Risk assessment for mammals 

Wild mammals may be exposed to spirotetramat by eating contaminated vegetation, seeds and fruits, 

invertebrate prey like arthropods (i.e. insects) or earthworms or vertebrate prey. 

Toxicity Exposure Ratios (TER) and Estimated Theoretical Exposures (ETE) were calculated based on the 

methods, i.e. equations and tabulated values described in the “Guidance Document on Risk Assessment for 

Birds and Mammals Under Council Directive 91/414/EEC” (Sanco/4145/2000 of 25. September 2002). 

The risk assessment is based on toxicity values of the active substance and the acute oral LD50 for the EU 

lead formulation (Spirotetramat OD150). 

The relevant toxicity figures were taken from laboratory studies with spirotetramat. All of them were 

considered acceptable. A detailed evaluation of the respective mammalian toxicology studies can be found 

in the B.6 part of this monograph. These endpoints are listed in table B.9.3.3-1 (below) for the different time 

scales. 

Maximum ETE values were estimated for use in the acute risk assessment while time-weighted average ETE

values were calculated for use in assessing long-term risks. ETE values were calculated using the following 

equation: 

ETE (mg as/kg bw/day) = FIR/bw x RUD x Application rate x MAF x fTWA x PT x PD x AV 

where: FIR Food Intake Rate (g fresh weight per day) 
– 183 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

 bw Body weight (g) 

 RUD Residue per unit dose (mg/kg fresh weight) 

 MAF Multiple Application Factor 

 fTWA Time-weighted average factor (only used for calculating long-term ETE) 

 PT Proportion of diet obtained in treated area 

 PD Proportion of food-type in diet 

 AV Avoidance factor (no avoidance =1) 

As recommended by the EU Expert Group on Higher Tier Risk Assessment for Birds and Mammals, the 90th

percentile residues on food items were used for the acute risk assessment, and the 50th percentiles for the 

short and long-term risk assessments. The fTWA was calculated from the following equation: 

ftwa = (1-e-kt)/kt 

where: k = ln2/DT50 

 t = averaging time 

The risk assessment is based on the following toxicity endpoints, which were considered ecotoxicologically 

relevant with regard to risk assessment: 

Table B.9.3.1-1: Ecotoxicologically relevant mammalian toxicity endpoints for spirotetramat 

 Species mg/kg bw/day Reference 

Acute oral LD50 Rat, female > 2000 Eigenberg (2004) 

Acute oral LD50 formulation Rat, female > 2000* Schüngel (2005) 

Reproductive NOAEL Rat 70.7 / 82.5 (m / f) Young (2006) 

* equivalent to 306 g a.s. 

Choice of relevant NOAEL for long-term risk assessment:

The two-generation reproduction study in the rat, and the developmental toxicity studies in the rat and the 

rabbit have been evaluated in order to derive an appropriate endpoint for use in the long-term risk 

assessment. A clear NOAEL of 70.7 / 82.5 mg/kg bw/day in male / female rats is derived from the twogeneration study which is based on effects in the next higher dosage group on male reproductive 

performance, sperm cells and offspring body weight development. The value for males is used in risk 

assessment as this figure is considered as protective with regard to wild mammal populations. The NOAELs 

derived from developmental studies were higher. 

B.9.3.1.1 First tier risk assessment to mammals 

The calculations are based on the following representative application scenarios in the EU: 

• Lettuce: 2 x 0.072 kg a.s./ha (interval 14 d), Northern + Southern Europe 

• Citrus: 2 x 0.096 kg a.s./m canopy height/ha (interval 21 d), max. 0.288 kg a.s./ha used in risk 

assessment, Southern Europe 

Assessment of exposure / Selection of scenarios:

The default values for the acute and long-term exposure in the tier 1 risk assessment are selected according 

to recommendations of the “Guidance Document on Risk Assessment for Birds and Mammals Under Council 

Directive 91/414/EEC”. According to this guidance document, crop specific generic indicator species with 

specific daily food intake rates (FIR) related to the body weight (bw) should be used for the tier 1 risk 

assessment. Spirotetramat OD 150 is an insecticide that is applied by spray application in lettuce and citrus. 

For spray applications in leafy crops the risk assessment should be based on generic data for medium 

herbivorous mammals (e.g. hare). For spray application in orchards small herbivorous mammals are used as 

model organisms. 

To summarise, the following relevant exposure scenarios were considered: 
– 184 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

- dietary exposure of a medium herbivorous mammal feeding on treated herbage in lettuce (category 

“leafy crops”) 

- dietary exposure of a small herbivorous mammal feeding on short grass in citrus (category “orchard”)

In order to calculate the estimated theoretical exposure the respective figures were chosen to estimate food 

intake rates and concentrations in the food of concern (see Table B.9.3.3.1.-1). 

For both intended uses two applications are intended. Accordingly the MAF (multiple application factor) is set 

according to the recommendations of the guidance document at 1.2 for the acute scenarios and to 1.4 or 1.2 

for long-term in lettuce and citrus, respectively. A default half-life of 10 d is assumed for estimating residue 

decline on the crop. For acute exposure, the 90th percentile and for long-term exposure mean initial residue 

levels in the respective food items were applied. In the first tier worst case assessment it is assumed that the 

mammals feed on a single food type, which is not avoided and that they satisfy their entire food demand in 

the treated area. 

Possible food items like soil-dwelling arthropods or earthworms, for which no respective risk assessment was 

performed, are covered by the calculations presented because residues in these items are expected to be 

much lower. 

Table B.9.3.1.1-1: Acute and long-term exposure of mammals to spirotetramat and calculated toxicity 

exposure ratios, tier 1 

Crop / Time 

horizon 

Indicator species Endpoint (mg 

a.s./kg bw) FIR/bw RUD MAF ftwa ETE TER 

lettuce 

Acute Medium herbivorous 

mammal > 2000 0.28 87 1.2 / 2.1 > 950 

Acute 

formulation

Medium herbivorous 

mammal > 306 0.28 87 1.2 / 2.1 > 145 

Long term Medium herbivorous 

mammal 70.7 0.28 40 1.4 0.53 0.6 118 

citrus 

Acute Small herbivorous 

mammal > 2000 1.39 85 1.2 / 41 > 49 

Acute 

formulation

Small herbivorous 

mammal > 306 1.39 85 1.2 / 41 > 7.5

Long term Small herbivorous 

mammal 70.7 1.39 46 1.2 0.53 12 5.9 

FIR/bw...food intake rate in relation to body weight; RUD...residue per unit dose; MAF...multiple application factor; ftwa...time weighted 

average factor; ETE...estimated theoretical exposure; TER...toxicity exposure ratio 

The TERs except the TERacute for the formulation and the citrus application are above the respective trigger 

values of 10 (acute) or 5 (reproductive) and indicate a low risk to mammals after consumption of 

contaminated vegetation. 

As the TERacute is a “bigger than” value based on the usually highest tested rate of 2000 mg/kg bw in acute 

oral toxicity studies, and as no mortalities were observed at this dosage, this figure can be considered to 

exceed the trigger of 10. 

The risk to wild mammals after consumption of contaminated diet after the use of spirotetramat according to 

GAP is low. 

B.9.3.1.2 Higher tier risk assessment to mammals 

Not required. 
– 185 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.3.1.3 Risk to mammals from plant metabolites 

The following metabolites of spirotetramat were found in lettuce metabolism at levels above 10 % of the total 

radioactive residue (TRR): BYI 08330-enol (17.8 %) and BYI 08330-enol glycoside (11.4 %). In smaller 

amounts the following metabolites were observed in plants metabolism studies at levels above 0.01 mg/kg: 

BYI 08330-ketohydroxy, BYI 08330- monohydroxy, BYI 08330-di-hydroxy, BYI 08330-desmethyl-enol, and 

BYI 08330-desmethyl-ketohydroxy. According to current guidance, only metabolites > 10 % have to be 

considered, however, as the notifier presented information also to minor metabolites, this is reported here. 

The enol metabolite is also the primary and main metabolite in the rat and hen metabolism study (see B.7 

part of this monograph). In so far, it can be considered to be toxicologically well characterised and to be 

covered by the respective mammalian toxicological studies available which were conducted with the parent 

compound. 

The enol glycoside metabolite is immediately and quantitatively metabolised to BYI 08330-enol after dietary 

uptake. Thus, this metabolite can be considered to be ecotoxicologically sufficiently covered. 

BYI 08330-desmethyl-enol was also observed in the rat and hen metabolism study and thus is covered by 

respective toxicological studies conducted with the parent compound. 

Likewise, the ketohydroxy metabolite was detected in the rat, and furthermore, an acute toxicity study on rat 

is available for this metabolite (Schüngel, 2005) which reveals that this metabolite shows no toxicity to 

mammals (LD50 > 5000 mg a.s./kg bw). This is supported by the observation that the ketohydroxy 

metabolite proved less toxic than the parent compound and than the enol metabolite in all ecotoxicological 

studies conducted. 

The metabolites BYI 08330-monohydroxy, BYI 08330-di-hydroxy and BYI 08330-desmethyl-ketohydroxy 

showed no toxicity in acute rat studies (LD50 > 5000 mg a.s./kg bw, Schüngel, 2005, Schüngel, M., 2006

and Schüngel, 2006, respectively); it can thus be considered highly unlikely that exposure might result in 

unacceptable effects to terrestrial vertebrates. 

Hence, it is concluded that the plant metabolites of spirotetramat are covered by existing ecotoxicology or 

toxicology studies, or they were shown not to be toxic to terrestrial vertebrates. The risk to wild mammals 

from plant metabolites appears low. 

B.9.3.1.4 Risk to mammals from secondary poisoning 

The log POW of spirotetramat is 2.51 at pH 7 and hence it`s potential to bio-accumulate is considered low. 

An assessment of the risk through bioaccumulation is not triggered according to SANCO/4145/2002. 

This applies also to the main metabolites of spirotetramat as the log POW of BYI 08330-cis-ketohydroxy is 1.3 

and of BYI 08330-enol is 0.3 (at pH 7). 

B.9.3.1.5 Risk to mammals from the consumption of contaminated drinking water 

The risk to mammals from uptake of contaminated drinking water is calculated according to 

SANCO/4145/2000. As the exposure via drinking from surface water bodies is considered to be negligible, 

the exposure from puddles of spray liquid is assessed as a worst case. The calculations are based on the 

intended use in lettuce (72 g a.s./ha), which covers also the citrus rate, because the concentration in the 

spray liquid is higher. Furthermore the formation of spray puddles in citrus orchards in Southern EU is highly 

unrealistic. 

TER = LD50/Dailydose 

Dailydose = (PEC drinking water x Totalwater ingestion rate)/W 

Total water ingestion rate = 0.099 x W0.90 
– 186 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.3.3.5-1: Parameters and calculations for the assessment of the acute and short-term risk to 

mammals from the uptake of contaminated drinking water from puddles and leaf axils 

Parameter Value 

Application rate [g ai/ha] 72 

Application volume [L/ha] 500 (- 1000) 

PECdrinking water [mg ai/L], assuming a dilution factor of 5 28.8 

W [kg], Body weight of a relevant indicator mammal (pygmy 

shrew) 

0.006 

Total water ingestion rate [L/d] 0.001 

Daily dose [mg ai/kg bw/d] 4.8 

LD50 acute [mg ai/kg bw] technical substance / formulated s. > 2000 / > 306 

TER acute technical substance / formulation 421 / 64 

The TER-value for acute exposure via contaminated drinking water indicates that the risk to wild mammals 

via this route is negligible. 

Overall conclusions 

After the use of spirotetramat according to GAP no unacceptable effects on populations of wild mammals are 

expected. 

B.9.4.8 Risk assessment for honey bees 

Honey-bees may be exposed to formulated spirotetramat by direct spraying of the plant protection product 

while bees are foraging on flowers and weeds present in or adjacent to the crop treated. They may also be 

exposed through contact with fresh or dry residues or by oral uptake of contaminated pollen, nectar and 

honey dew. The LD50-values (48 h) for both oral and contact toxicity of the active substance are above 

100 μg/bee, indicating that spirotetramat is of low toxicity to adult bees. The acute toxicity of the lead 

formulation Spirotetramat OD150 is slightly higher by the oral route. 

The intended rates for spirotetramat are 2 x 72 g a.s./ha in lettuce and 2 x 96 g a.s./ m canopy height in 

citrus. With regard to citrus the rate per m canopy height is considered as effective application rate for bee 

exposure (see also B.9.5.4). In accordance with the Guidance Document on Terrestrial Ecotoxicology under 

Council Directive 91/414/EEC (SANCO/10329/2002) the acute risk for bees was expressed as a Hazard 

Quotient calculated by the following formula (single application rate in g/ha, LD50 in μg/bee): 

Hazard quotient (QH) = application rate/LD50
– 187 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.4.8-1: Risk to honey-bees from oral and contact exposure to technical and formulated 

Spirotetramat, expressed as Hazard Quotient QH

Test substance Exposure route Application rate 

[g a.s./ha] Endpoint Value [μg a.s./bee] QH

lettuce 

Spirotetramat 

techn. 

oral 

contact 

72 

72 

LD50 

LD50

> 107.3 

> 100 

< 0.67 

< 0.72 

Spirotetramat 

OD150 

oral 

contact 

72 

72 

LD50 

LD50

91.7 

162 

0.79 

0.44 

citrus 

Spirotetramat 

techn. 

oral 

contact 

96 

 96 

LD50 

LD50

> 107.3 

> 100 

< 0.89 

< 0.96 

Spirotetramat 

OD150 

oral 

contact 

96 

96 

LD50 

LD50

91.7 

162 

1.04 

0.59 

All resulting Hazard Quotients are significantly below the trigger of 50 and indicate a low risk to bees with 

respect to adult bee mortality after the use of spirotetramat according to GAP. 

In a brood feeding test, where bee colonies were fed a sugar solution containing 0.0144 % of Spirotetramat 

OD 100, brood effects, seen as a high termination rate, were detected (Schur 2004 a and b). 

However, according to its design, this test can be seen as a screening test with unrealistically severe 

exposure conditions, which is appropriate to evaluate an intrinsic hazard potential of a compound but is not 

necessarily indicative for a risk under realistic conditions (EPPO risk assessment scheme). 

In order to test for potential brood effects under more realistic exposure conditions, a semi-field brood test 

(Schur, 2005) was conducted. A worst-case application scenario was applied with three pre-flowering 

applications at 72 g a.s./ha followed by two applications at 96 g a.s./ha into the blooming crop during bee 

flight. In this study, an irritation of brood development in the earlier assessments in one of 3 colonies of the 

5x treatment was observed. However, there was no clear treatment relation and recovery took place. No 

effects to other endpoints, as adult mortality, foraging activity, or bee behavior were detected. 

An additional semi-field study with special focus on brood effects was conducted (Schur, 2006). Two 

application scenarios with applications into the blooming crop were tested, covering the lettuce scenario 

(even with additional pre-flowering applications) and the citrus scenario. The study was conducted in a model 

crop (Phacelia) which can be considered to represent worst-case conditions in terms of flower density and 

bee attractiveness. No adverse effects of the compound could be seen, neither in brood development or 

colony condition, nor on any other parameter assessed. 

A series of additional, non GLP-tunnel trials, which have not been conducted for regulatory purposes, was 

also reported by the notifier, because bee brood turned out to be negatively affected in these studies. After 

different application scenarios, the proportion of the larval cell comb area appeared to be reduced from about 

3 up to 14 days after application. In all cases the effects were transient; i.e. recovery was seen and the 

overall survival of the affected colonies was not endangered. 

At the time of writing this DAR (spring 2008), the notifier is conducting a field study to enable a reasonable 

conclusion with regard to the bee safety of Spirotetramat containing products under realistic exposure

conditions. As soon as these results are available, the RMS will report them in an addendum. 

Overall conclusion 

The hazard quotients for oral and contact toxicity of spirotetramat are below the trigger of 50, as specified in 

91/414/EEC, thus indicating a low risk for adult bees after application according to the intended use 

conditions. 
– 188 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Spirotetramat applications into blooming, bee-attractive crops may cause transient effects on honey-bee 

brood. However, many semi-field trials covering different application scenarios and worst case situations 

were conducted, all of them of good quality, and in no case a lasting effect on brood development occurred, 

and the overall condition and survival of colonies was not disturbed. 

As regards the potential risk to bees for the representative uses, the RMS recommends precautionary 

labelling as “Spe8: Dangerous to bees. To protect bees and other pollinating insects do not apply to crop 

plants when in flower or when flowering weeds are present. Do not use when bees are actively foraging.”. 

Especially for the citrus use, which possibly takes place also during blooming, EU Southern MS should apply 

their regional risk mitigation measures. 

B.9.5.4 Risk assessment for non-target arthropods 

Non-target arthropods may be exposed to formulated spirotetramat by direct spraying, contact with fresh or 

dry residues or by oral uptake of contaminated prey, nectar and pollen or via host organisms. 

A risk assessment for non-target arthropods was performed according to the recommendations of 

ESCORT II. In the first tier hazard quotients were calculated for exposure in in-field and off-field areas 

according to the following formulas: 

50

in field LR

application rate MAF HQ ×

− =

50

off -field LR

vegetation distribution factor

drift factor application rate MAF

HQ correction factor

















× ×

= ×

drift factor = % drift/100 (90 %ile drift according to Ganzelmeier et al. 1995) 

The correction factor and the vegetation distribution factor were set to 10. Drift figures were chosen

according to crop type in 1 m distance (lettuce) or 3 m distance (citrus). 

Effective application rate with regard to foliage dwelling arthropods in citrus = 0.096 kg/ha

Foliage dwelling arthropods in the canopy are exposed to that fraction of the application rate that can be 

expected to reach the respective compartment (“effective rate”) of the canopy, and not to the maximum 

application rate for trees of 3 m height. The application rate is defined per m canopy height. Likewise, the 

maximum spray concentration and the predicted residues in the sprayed canopy (e.g. foliage, arthropods) 

are comparable regardless of whether a tree of 1, 2 or 3 m height has been sprayed. Thus, an application 

rate corresponding to 0.096 kg a.s./ha (= amount per 1 m tree height) is considered in the non-target 

arthropods risk assessment for citrus. 

Multiple application factors (MAF) were calculated taking into account the measured half-life on plant surface 

of 4.45 days (for more details see B.9.1.8.2). For citrus as a fruit crop the respective spray drift for “late” use 

of 15.73 % was chosen, because the figure for “early” use is related to the early stage of orchard trees (i.e. 

apples) where only buds and no or very small leaves are present and the drift would be accordingly higher. 

In citrus leaves are fully developed during all stages, however. 
– 189 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.5.4-1: HQ calculations for Aphidius rhopalosiphi and Typhlodromus pyri

Crop 

Application rate 

[g a.s./ha] 

MAF Drift [%] 

LR50

[g a.s./ha] 

HQin-field HQoff-field

Aphidius rhopalosiphi

lettuce 72 1.2 2.77 114.7 0.75 0.02 

citrus 96 1.1 15.73 114.7 0.92 0.14 

Typhlodromus pyri

lettuce 72 1.2 2.77 0.333 259 7 

citrus 96 1.1 15.73 0.333 317 50 

Both HQ-values for Aphidius rhopalosiphi are below the trigger of 2 and indicate an acceptable risk. The 

triggers are definitely breached for Typhlodromus pyri for the in- and off-field area and indicate the need of 

further higher tiered data on this indicator species and additional species. In the following the in-crop- and 

off-crop risk to the four relevant functional groups of arthropods are assessed seperately. 

Larvicidal mode of action of spirotetramat:

In bees spirotetramat caused transient effects on bee larvae. With regard to non-target-arthropods few

guidance on the evaluation of larvicidal / “insect growth regulator” - effects is currently available. However, 

“worst-case” toxicity studies have been conducted with larvae of two different insect taxa: Coccinella 

septempunctata and Chrysoperla carnea, and the resulting effects at the intended rates were below 10 %. 

Hence it is considered that potential effects of spirotetramat on insect larvae / development is covered by the 

risk assessment presented below. 

In-crop risk assessment:

Parasitoid wasps (Aphidius rhopalosiphi): 

At the in-field rates of both intended uses spirotetramat effectuates only low mortality rates in this species in 

the laboratory. Under extended lab conditions effects are even lower. Hence the risk to parasitoids appears 

low. This is also indicated by the Hazard Quotients for this species (see table B.9.5.4-1). 

Predatory mites (Typhlodromus pyri): 

Very low doses of spirotetramat lead to complete mortality of T. pyri in the laboratory. 

If the mites are exposed to fresh residues on treated plants (extended lab), this still results in 100 % mortality 

at rates lower than intended. Even residues aged for up to 49 days yield in mortality > 50 %. However, after 

56 days of aging mortality was low and no effects on reproduction were seen in the extended lab. 

Based on the results from laboratory and extended laboratory testing, it is therefore considered that the incrop populations of predatory mites will have the possibility to recover after an approximate period of 8 

weeks or rather that mites from the off-crop area may immigrate. However, according to the results of the 

field study, it can be assumed that the initial toxicity under “real world” conditions will be lower. Southern EU 

member states during their national authorisations for citrus should evaluate the field study with respect to 

it`s representativeness for their purposes. 

Foliage-dwelling arthropods: 

In an extended laboratory study with Chrysoperla carnea, mortality of < 10 % was observed at rates up to 

288 g a.s./ha. In Coccinella septempunctata, no mortality occurred at the intended rates and at higher rates it 

stayed < 30 %. Hence the risk to foliage-dwelling arthropods can be considered low. 

Ground-dwelling arthropods: 

No studies were performed with ground-dwelling species, however, their risk is covered by two species of 

foliage-dwellers, which are well representing the arthropod fauna, especially with regard to the citrus use. 
– 190 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Off-crop risk assessment:

Parasitoid wasps (Aphidius rhopalosiphi): 

The risk to this species/group is low, as at drift rates no mortality or sublethal effects are expected. 

Predatory mites (Typhlodromus pyri): 

According to the laboratory and extended laboratory data high mortality may happen also at drift rates. 

However, in a field study in vineyards at rates approximately comparable to GAP no significant effects were 

detected on the acarine fauna. Hence under natural outdoor conditions off-crop mortality apparently is low 

and the risk to predatory mites after the use of spirotetramat according to GAP appears low. The potential for 

recolonisation of mites in the in-crop area is given. 

Foliage-dwelling arthropods: 

The risk to this species/group is low, as at drift rates no mortality or sublethal effects are expected according 

to the results of extended laboratory testing. 

Ground-dwelling arthropods: 

The risk to this group is covered by the data on foliage-dwelling species. 

Overall conclusion 

Both in- and off-field exposure to formulated spirotetramat according to the intended use conditions do not 

cause unacceptable effects to populations of terrestrial non-target arthropods. 

B.9.6.4 Risk assessment for earthworms 

The TER values for earthworms were calculated as the ratio between the acute effect concentrations (LC50) 

and the sublethal no observed effect concentration (NOEC), respectively, and the maximum PECs. The 

relevant endpoints (EC50 and NOEC) and overall worst case PECs values for the active substance and its 

metabolites are summarised in table B.9.6.4-1 and table B.9.6.4-2, respectively. Details for PECS calculation 

by FOCUS modelling are presented in the appendix to the fate section B.8. 

In the risk assessment for earthworms the active substance spirotetramat and its major metabolites in soil 

are considered: 

• BYI 08330-enol (100 % AR after 1 day, DT50 of 2.6 days in laboratory test) 

• BYI 08330-ketohydroxy (max. 24 % after 1 day, DT50 of 12.1 days in laboratory test) 

• 4-methoxycyclohexanone (max. 10 % after 2 days, DT50 of 0.6 days in soil photolysis study). 

Spirotetramat has a logPOW of > 2 and usually a correction of the relevant endpoints is necessary. However, 

in this case the study with the active substance was performed using an artificial soil containing 5 %

sphagnum peat and therefore a correction of the endpoints is not needed. 
– 191 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Table B.9.6.4-1: Summary of effects of spirotetramat and its metabolites on earthworms 

Substance Species Endpoint Time EC/LC50

[mg/kg dw] 

NOEC 

[mg/kg dw] Reference 

Study 

acceptable 

Spirotetramat 

(BYI 08330)) Eisenia fetida Mortality Body weight 14 d > 1000 > 1000 

1000 

1000 

LecheltKunze 2004 yes 

BYI 08330-

ketohydroxy 

(metabolite) 

Eisenia fetida Mortality 

Body weight 14 d > 1000 > 1000 

1000 

1000 

LecheltKunze 2005 yes 

4-Methoxycyclohexanone 

(metabolite) 

Eisenia fetida Mortality 

Body weight 14 d > 1000 > 1000 

1000 

125 

Leicher 

2008 yes 

BYI 08330-enol 

(metabolite) Eisenia fetida

Mortality 

Body weight 

Reproduction 

4 w 

4 w 

8 w 

> 1000 

> 1000 

1269 

1000 

1000 

32 

Moser & 

Scheffczyk 

2005 

yes 

Table B.9.6.4-2: Summary of maximum observed PECSoil values of spirotetramat and its metabolites 

max. PECS [mg kg-1] Substance 

Citrus Lettuce 

Spirotetramat 0.115 0.029 

BYI 08330-enol 0.093 0.024 

BYI-ketohydroxy 0.031 0.009 

4-Methoxycyclohexanone 0.004 0.001 

Acute effects 

Table B.9.6.4-3: Citrus: TER acute for earthworms 

Test substance 14 days LC50 

[mg/kg soil dw] 

max PECs

[mg/kg] TERA

Trigger 

value 

Refined risk 

assessment 

Spirotetramat 

(BYI 08330)) > 1000 0.115 > 8696 10 no refinement 

required 

BYI 08330-ketohydroxy 

(metabolite) > 1000 0.031 > 32258 10 no refinement 

required 

4-

Methoxycyclohexanone > 1000 0.004 > 250000 10 no refinement required 

Table B.9.6.4-4: Lettuce: TER acute for earthworms 

Test substance 14 days LC50 

[mg/kg soil dw] 

max PECs

[mg/kg] TERA

Trigger 

value 

Refined risk 

assessment 

Spirotetramat 

(BYI 08330)) > 1000 0.029 > 34483 10 no refinement 

required 

BYI 08330-ketohydroxy 

(metabolite) > 1000 0.009 > 111111 10 no refinement 

required 

4-

Methoxycyclohexanone > 1000 0.001 > 1000000 10 no refinement required 
– 192 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

Long term effects 

Table B.9.6.4-5: Citrus: TER long term for earthworms 

Test substance NOEC

[mg/kg soil dw] 

max PECs

[mg/kg] TERLT

Trigger 

value 

Refined risk 

assessment 

BYI 08330-enol 

(metabolite) 32 0.093 344 5 no refinement 

required 

Table B.9.6.4-6: Lettuce: TER long term for earthworms 

Test substance NOEC

[mg/kg soil dw] 

max PECs

[mg/kg] TERLT

Trigger 

value 

Refined risk 

assessment 

BYI 08330-enol 

(metabolite) 32 0.024 1333 5 no refinement 

required 

Active substance 

The calculated TERA value for spirotetramat is well above the trigger value of 10, thus the acute risk to 

earthworms after the application according to GAP can be considered as low. 

Since the representative use of spirotetramat included only a low number of applications in citrus and lettuce 

(maximum of 2 applications in both crops) and spirotetramat is not persistent a reproduction test was not 

required. 

Therefore the risk for earthworms after the application of spirotetramat is considered acceptable. 

Metabolites 

Three major soil metabolites were identified: BYI 08330-enol, BYI 08330-ketohydroxy and 4-

methoxycyclohexanone. Acute tests with BYI 08330-ketohydroxy and 4-methoxycyclohexanone are available 

and the toxicity of the metabolite BYI 08330-enol was assessed in a reproductive test. 

For the worst case application in citrus the TERA values for BYI 08330-ketohydroxy and 4-

methoxycyclohexanone were estimated to be > 32258 and > 250000, respectively. The acute risk of BYI 

08330-enol is covered with the study on the active substance due to the rapid and complete formation of this 

metabolite (100 % after 1 day). In addition an endpoint from reproductive study is available and the TERLT

value for this long term exposure of BYI 08330-enol was calculated to be 344. Thus, all calculated values 

meet the relevant trigger. 

Therefore risk for earthworms from the metabolites BYI 08330-enol, BYI 08330-ketohydroxy and 4-

methoxycyclohexanone is expected to be low. 

In conclusion, all calculated TER values for spirotetramat and its relevant metabolites (BYI 08330-enol, BYI 

08330-ketohydroxy and 4-methoxycyclohexynone) are well above the relevant trigger values, thus the risk to 

earthworms after the application according to GAP can be considered as low. 
– 193 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.7.4 Risk assessment for other soil non-target macro-organisms 

One study to assess the risk to gamasid mites was performed with the major metabolite BYI 08330-enol in 

soil. This major metabolite was used because the active substance spirotetramat will be rapidly converted to 

BYI 08330-enol (max. 100 % after 1 day) in soil. 

Table 9.7.4-1: Summary of effects on soil non-target macro-organisms 

Substance Species Endpoint Time EC/LC50

[mg/kg dw] 

NOEC 

[mg/kg dw] 

Reference/study 

acceptable 

BYI 08330-enol Gamasid mite 

(Hypoaspis aculeifer)

Mortality 

Reproduction 28 d > 1000 316 1000 

Moser & Scheffczyk 

2005/yes 

The TER value was calculated as the ratio between the NOEC and the maximum PECs. The worst case 

PECs value of BYI 08330-enol after applications in citrus and in lettuce was calculated to be 0.093 mg a.s./kg 

and 0.024 mg a.s./kg, respectively. For details of calculations see the appendix to the fate section B.8. 

A correction factor is not necessary because a natural soil with a low Corg content was used in the test. 

Table 9.7.4-2: Citrus: TER long term for gamasid mites 

Test substance NOEC 

[mg a.s./kg soil] 

maximum PECs 

[mg/kg] TER lt Trigger value 

BYI 08330-enol 316 0.093 3399 5 

Table 9.7.4-3: Lettuce: TER long term for gamasid mites 

Test substance NOEC 

[mg a.s./kg soil] 

maximum PECs 

[mg/kg] TER lt Trigger value 

BYI 08330-enol 316 0.024 13167 5 

The calculated TER values of 3399 (citrus) and 13167 (lettuce) for spirotetramat indicate an acceptable risk 

for soil non-target macro-organisms. The calculated TER values indicate an acceptable risk for soil nontarget macro-organisms. Furthermore it is indicated that spirotetramat, which will be converted in soil 

immediately to the BYI 08330-enol, has no adverse effects on juvenile life stages of soil dwelling arthropods. 

Thus the risk from spirotetramat for other non-target soil macro-organisms is expected to be low when 

applied according to GAP. 
– 194 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.8.4 Risk assessment for non-target micro-organisms 

Table 9.8.4-1: Summary of effects on non-target micro-organisms 

Test substance Test parameter Test concentration Time 

Effects (deviation from 

control) 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 3 % 

Spirotetramat Nitrogen mineralization 

1.3 mg a.s./kg 

(0.99 kg a.s./ha) 

28 days 

16 % 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 6 % 

Spirotetramat Carbon mineralization 

1.3 mg a.s./kg 

(0.99 kg a.s./ha) 

28 days 

7 % 

Active substance 

The effects of spirotetramat (tested at two doses: 0.13 mg/kg and 1.3 mg/kg) on the activity of the soil microflora were studied in standard laboratory tests. At both applied concentrations the impact on soil nitrogen 

transformation and soil respiration was < 25 %. The highest exposure concentration in the test was 

approximately 10 times higher than the expected PEC soil, which was calculated to be 0.115 mg/kg soil.

Thus, it can be concluded that spirotetramat, when applied according to GAP, will not significantly affect the 

soil micro-flora. 

Metabolites 

In the fate section for the compartment soil three major metabolites were identified: BYI 08330-enol 

BYI 08330-keto-hydroxyd and 4-methoxycyclohexanone. In soil BYI 08330-enol immediately occurred after 

application with a maximum of 100 % after 1 day, BYI 08330-keto-hydroxy reached the maximum on day 1 

with 16.3 %. Therefore, it can be concluded that the study performed with the active substance covered the 

possible effects on soil micro-organisms for these both metabolites. However, 4-methoxycyclohexanone 

(metabolite of photolysis) will not be formed under test condition without illumination. But toxicity test with 

aquatic organisms and earthworm indicate that 4- methoxycyclohexanone is not toxic to non-target 

organisms. In addition, under natural exposure condition in the field an exposure of non-target soil-organisms 

to relevant environmental concentrations of 4- methoxycyclohexanone is extremely unlikely. Because, the 

initial PECs is very low with 0.004 mg/kg at day 0 and from day 2 it can be expected that no measurable 

concentration of the metabolite will occur (PECs < 0.001 mg/kg). Thus, also a long term exposure can be 

excluded. 

In conclusion, the risk from soil degradation products for the activity of the soil micro-flora is considered to be 

low. 
– 195 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.9.2 Risk assessment for terrestrial non-target plants 

Terrestrial non-target plants may be exposed to formulated spirotetramat by spray drift in the vicinity of the 

treated area. 

The effects of Spirotetramat 150 OD on non-target plants were assessed in one vegetative vigour screening 

test, two dose-response vegetative vigour tests and two seedling emergence screening tests. The results are 

summarised in table 0.1-1 above. 

A quantitative risk assessment is performed which follows a deterministic TER approach. As specified in the 

“Guidance Document on Terrestrial Ecotoxicology under Council Directive 91/414/EEC” the risk for nontarget plants is considered acceptable if the TER for the most sensitive species is greater than 5. 

Spray drift is considered to be the relevant exposure route for terrestrial non-target plants. Consequently the 

PECs are calculated based on the drift models produced by the BBA (Ganzelmeier et al., 1995; updated by 

Rautmann et al., 2001). TERs were determined based on drift deposition at 1 respectively 3 m from the edge 

of the field considering a multiple application factor (MAF) (as for non-target arthropods, measured half-life 

on plants considered; guidance see the respective section B.9.5) and the application scenarios for lettuce 

and citrus. 

The most sensitive endpoint is the EC50 for corn dry weight of 134 g a.s./ha derived from a tier 2 vegetative 

vigour test; this figure is considered as relevant for the risk assessment. 

For citrus as a fruit crop the respective spray drift / ground deposition for “late” use of 15.73 % was chosen, 

because the figure for “early” use is related to the early stage of orchard trees (i.e. apples) where only buds 

and no or very small leaves are present and the drift would be accordingly higher. In citrus leaves are fully 

developed during all stages, however. 

The respective TER calculations are presented in table B.9.9.2-1 below. 

Table B.9.9.2-1: Risk assessment for non-target plants for Spirotetramat OD150 

TER values Application rate [g 

a.s./ha] x MAF 

Distance 

[m] 

Spray drift 

[%] 

Exposure1

[g a.s./ha]

EC50 

[g a.s./ha] No drift 

reduction 

50 % drift 

reduction 

lettuce

72 x 1.2 1 2.77 2.39 134 56 

citrus

3 15.73 50 2.7 5.4 

5 8.41 27 5 

288 x 1.1 

10 3.60 11.4 

134 

12 

MAF...Multiple application factor 

1 based on Ganzelmeier drift data 

The submitted studies on terrestrial plants and the calculated TER-values indicate that the risk for non-target 

plants is low at a distance of 1 m to the treated area for lettuce and at a distance of 5 m for citrus (3 m high 

trees as worst case) without drift reducing measures. When drift reducing nozzles are used in citrus, buffer 

zones can be reduced according to the table above (to 3 m with 50 % drift reduction). 

Overall conclusions 

The risk to non-target plants due to application of Spirotetramat OD150 at the intended representative uses 

is considered acceptable at 1 m (lettuce) and 5 m (citrus) distance to the field edge. The buffer zone for 

citrus may be reduced when drift reducing measures are applied. 
– 196 – 

Spirotetramat – Volume 3, Annex B.9 Ecotoxicology April 2008

B.9.10.2 Risk assessment for biological methods of sewage treatment 

For the effects of spirotetramat on the inhibition of the respiration rate of aerobic wastewater microorganisms an EC50 > 10000 mg/L was determined. Therefore it can be assumed that adverse effects on 

methods of sewage treatment are unlikely, when spirotetramat is applied according to GAP.