Appendix C. Ecolpgical Effects Data Summaries 

The summaries found in this appendix are based on the Austrian Agency for Health 

and Food Safety (AGES) review and are presented as they appear in "Volume 3, Annex 

B.9: Ecotoxicology" of the monograph prepared for registration of Spirotetramat in 

Europe. 

Ecotoxicology 

Acute oral toxicity (OECD IIA 8.1 .I) 

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

Sub-chronic toxicity and reproduction (OECD IIA 8.1.4) 

Supervised cage or field trials (OECD IIA 10.1.7) 

Acceptance of bait, granules or treated seeds by birds (OECD IIA 10.1.8) 

Effects of secondary poisoning (OECD IIA 10.1.9) 

Summary of effects on birds 

Effects on aquatic organisms (OECD IIA 8.2, lllA 10.2) 

Acute toxicity of active substance, metabolites and formulations to aquatic organisms 

Chronic toxicity of active substance, metabolites and formulations to aquatic 

organisms 

Bioaccumulation (OECD IIA 8.2.6, IIA 8.2.7) 

Effects on other terrestrial vertebrates (OECD lllA 10.3) 

Effects on bees (OECD IIA 8.7; OECD lllA 10.4.) 

Acute Toxicity (OECD IIA 8.7.1; OECD IIA 8.7.2; OECD 111 10.4.2 ) 

Residue test (OECD IIA 8.7.3) 

Bee brood feeding test (OECD IIA 8.7.4) 1 

Cage test (OECD lllA 10.4.4) 

Field tests (OECD lllA 10.4.5) Formulation: Spirotetramat OD 100 

Tunnel tests (OECD lllA 10.4.7) 

Summary of effects on honey-bees 

Effects on other arthropod species (OECD 11A 8.8, lllA 10.5) 

Laboratory, extended laboratory and semi-field tests (OECD IIA 8.8.1; OECD lllA 

10.5.1) 

Semi-field or field tests (OECD lllA 10.5.3) 

Summary 

Effects on earthworms (OECD IIA 8.9, lllA 10.6.3) 

Acute toxicity (OECD IIA 8.9.1, lllA 10.6.1.1) 

Sublethal effects (OECD IIA 8.9.2, lllA 10.6.4) 

Field studies 

Effects on soil non-target macro-organisms (OECD IIA 8.14, lllA 10.6.6) 

Effects on a species level (gamasid mites) 

Effects on a functional level (degradation in litter bags) (OECD lllA 10.6.7) 

Summary of effects on soil non-target macro-organisms 

Effects on soil non-target micro-organisms (OECD IIA 8.10, lllA 10.7) 

Nitrogen mineralization 

Carbon mineralization 

Summary of effects on non-target micro-organisms 

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

IIA 8.12, lllA 10.8) 

Effects on terrestrial non-target plants (OECD lllA 10.8.1) 

Effects on biological methods of sewage treatment (OECD IIA 8.1 5) 

Activated sludge, respiration inhibition 

TEXT SEARCHABLE DOCUMENT
Effects on birds (OECD IIA 8.1, lllA 10.1) 

B.9.1.1 Acute oral toxicity (OECD IIA 8.1 .l) 

Active substance - Spirotetramat (BY1 08330) 

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

Item BY1 08330 (techn.) 

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

ReportfDoc. number: BAR / LD 047 

Guideline(s): US EPA, subdiv. E, 5 71 -1 ; EPA Series 850- OPPTS No. 850.21 00 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: 

Material and methods: 

Test organism: 

Dosage levels: 

Number of birds and 

replicateslgroup: 

Administration: 

Duration: 

Test conditions: 

Test parameter: 

Observations: 

Body weight: 

Feed consumption: 

Necropsy: 

Statistics: 

Findinas: 

Regurgitation: 

Mortalities: 

Clinical signs: 

Body weight: 

Feed consumption: 

Gross post-mortem 

examination: 

Conclusion: 

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

purity 97.5 % 

Bobwhite quail (Colinus virginianus), sexually mature 

0, 500, 1000 and 2000 mglkg body weight (nominal) 

five males and five females /treatment group and control 

single oral administration in gelatine capsules 

14 d post-treatment observation period 

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 

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. 

recorded prior to test initiation and on days 7 and 14 

determined on days 3 and 7 

gross necropsy performed at termination 

none 

None. 

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

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. 

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

test termination. 

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

evidently in the highest dose group. 

no treatment-related findings at necropsy 

LD50 > 2000 mg a.s./kg bw, NOEL(morta1ity): 1000 mg a.s./kg bw, NOEL(subletha1 

effects) < 500 mg a.s./kg bw (lowest dose tested, effects: diarrhoea, soft 
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 (BY1 08330) 

Reference: BY1 08330 techn. as.: 5-Day-Dietary LC50 Bobwhite Quail (Colinus 

virginianus) 

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

ReporVDoc. number: BAR / LC01 7, 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: 

Material and methods: 

Test organism: 

Dosage levels: 

Number of birds and 

replicateslgroup: 

Administration: 

Duration: 

Test conditions: 

Analytics: 

Test parameter: 

Observations: 

Body weight: 

Feed consumption: 

Necropsy: 

Statistics: 

Findinas: 

Mortalities: 

Clinical signs: 

Spirotetramat (techn.), batch No.: MIX-Batch 0804510014, TOX-no. 6344-00; 

purity 97.5 % 

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

control (untreated diet); 31 3, 625, 1250,2500 and 5000 ppm a.s. (nominal) 

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

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

test substance mixed in diet 

5 days administration period and 3 days post-exposure period 

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 

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

sufficiently verified by analytical methods 

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

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

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

termination of the test on day 8 

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

Gross necropsy performed at termination 

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. 

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

of an injury. 

During the exposure period the chicks of the highest dose group showed signs of 
Body weight: 

intoxication like soft excrements (reversible). 

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

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

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

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 pprn and at 5000 pprn 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 pprn 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 Error! Style not defined.-la: Summary of effects of spirotetramat on Bobwhite Quail following 

short-term dietary exposure 

Table Error! Style not defined.9b: Body weight data 

* statistically significant 

Treatment 

(measured) 

[ppm as.] 

Control 

301 

624 

Mean bw 

gain d 0 - 5 

[Oh] 

46 

43 

43.4 

Cumulative 

mortality 

0120 

011 0 

011 0 

Mean bw 

gain d 6 - 8 

p.1 

23.5 

23.7 

26.2 

Mean bw 

per bird 

d 0 - 4 [g] 

21.4 

22.6 

23.0 

Mean feed 

consumption 

d 0 - 4 [glbirdld] 

3.1 

3.1 

3.1 

Mean feed 

consumption 

d 5 - 7 [g/bird/d] 

3.8 

3.9 

4.1 

Estimated 

intake d 0 - 4 

[mg askg bwld] 

0 

39.8 

82.6 
lble Error! Style not de 

Nominal dietary 

Concentration 

-- - 

0 mg a.s./kg diet 

313 mg a.s.lkg diet 

625 rng a.s.lkg diet 

1250 mg a.s.lkg diet 

2500 mg a.s.lkg diet 

5Q00 mg a.s.lkg diet 

led.&: Estimated daily food consumption per bird 

Individual Food Consumption in g I bird-I dav 11 

Exposure 1 Post Exposure 11 

Conclusion: LC50 > 4998 mg a.s.1kg food, equivalent to > 497 mg a.s./kg bwld, based on a 

mean feed consumption of 2.1 glbirdld, a mean body weight of 21 .I g and on 

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

food (measured); NOEC: 121 8 mg a.s./kg food (measuerd) based on food 

avoidance and reduced body weight gain. 

Reference: 

Author(s), year: 

ReporVDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test organism: 

Dosage levels: 

Number of birds and 

replicateslgroup: 

Administration: 

Duration: 

Test conditions: 

Analytics: 

Test parameter: 

Observations: 

Body weight: 

Feed consumption: 

Technical BY1 08330: A Subacute Dietary LCs0 with Mallards 

Stoughton, T.L.; 2005 

EBFNX012; M-274385-01-1 

EPA 71 -2, OPPTS No.: 850.2200; OECD No. 205 

Yes (certified laboratory) 

None 

Acceptable 

Spirotetramat (techn.), batch No.: 0804510014; purity 97.1 % 

Mallard Duck (Anas plafyrhynchos), 10 day-old hatchlings 

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 

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

test substance mixed in diet 

5 days administration period and 3 days post-exposure period 

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

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

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

sufficiently verified by analytical methods 

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

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

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

termination of the test on day 8 

daily feed consumption per group recorded for the pre-exposure, exposure and 
Necropsy: 

Statistics: 

Findinas: 

Mortalities: 

Clinical signs: 

Body weight: 

recovery periods 

Gross necropsy performed at termination 

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)) 

None 

None 

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 Error! Style not defined.9a: Summary of effects of spirotetramat on Mallard Duck following 

short-term dietary exposure 

Treatment 

(measured) 

[ppm as.] 

Control 

344 

708 

6050 I 0110 I 126.2 

6050 133.7 118.6* I 169.2* 1 

* statistically significantly different compared to control 

Cumulative 

mortality 

011 0 

011 0 

011 0 

Table Error! Style not defined.9b: Mean body weight data (g) 

I 

* 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. 

-15.1* 1 50.6* 1 9.ga 

Treatment (measured) 

[PPm a.s.1 

Control 

344 

708 

1436 

Mean bw 

d 0 - 5 

[gl 

199.2 

172.4 

162.8 

30.2 

Mean bw on day 0 

141.0 

137.3 

137.2 

136.6 

Mean bw 

gain d 0 - 5 

[a 

116.4 

70.1 * 

51.2* 

475 

Mean bw on day 5 

257.4 

207.5* 

188.4* 

162.2* 

Mean bw 

gain d 6 - 8 

[g] 

25.4 

53.9* 

64.1* 

Mean bw on day 8 

282.8 

261.3 

252.5* 

244.6* 

Mean feed 

consumption 

d 0 - 4 [g/bird/d] 

45.2 

32.6a 

27.7a 

Mean feed 

consumption 

d 5 -7 [glbirdldl 

41.5 

44.1 

41.9 

Estimated intake 

do-4 

[mg aslkg bwld] 

I 

65 

120 
Conclusion: LC5, > 6050 mg a.s./kg food, equivalent to > 475 mg a.s./kg bwld, based on a 

mean feed consumption of 9.9 glbirdld, a mean body weight of 126.2 g and on 

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

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

weight gain already at the lowest test concentration observed). 

6.9.1.3 Sub-chronic toxicity and reproduction (OECD IIA 8.1.4) 

Active substance - Spirotetramat 

Reference: Effect of Technical BY1 08330 on Northern Bobwhite Reproduction 

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

ReportIDoc. number: EBFN0220, M-277757-01-1 

Guideline(s): OPPTS Nr. 850.2300, FlFRA 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: 

Material and methods: 

Test organism: 

Dosage levels: 

Number of birds and 

replicateslgroup: 

Administration: 

Analytics: 

Study phases: 

Test conditions: 

Duration: 

Test parameter: 

Observations: 

Adult Body weight: 

Adult feed 

consumption: 

Spirotetramat (techn.), batch No.: 0804510014, purity 97.2 % 

Bobwhite quail (Colinus virginianus),l6 weeks of age 

Control (untreated diet); 80, 240 and 720 mg ailkg feed (nominal), corresponding 

to 0, 85, 264 and 802 mg ailkg 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 bwlday. 

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

Test substance mixed in the diet, prepared weekly 

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

sufficiently verified by analytical methods. 

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 

Feeding: standard commercial diet ad libitum; adult housings: average 22"C, 

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

17 h, wide spectrum illumination 

Approximately 22 weeks (pre-egg laying until termination) 

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 strengthlthickness. 

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

adult termination. 

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

Hatchling parameter 

Post-mortem 

examination: 

Statistics: 

Findinas: 

Mortalities: 

Clinical signs: 

Body weight: 

Feed consumption: 

Gross necropsy: . Reproductive results: 

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. 

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. 

All adult birds were subjected to a gross necropsy. 

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 an'd 

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. 

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

three at 85 ppm, two at 802 pprn). 

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

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

gain in the 802 pprn 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. 

No treatment-related effects upon feed consumption. 

No treatment-related findings. 

See Table Error! Style not defined.-I. 

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 pprn 

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 

pprn 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 Error! Style not defined.-I: Summary of reproductive results of spirotetramat in the bobwhite 

quail 

Reproductive parameter 

Overall mean food consumption (g/bird/day) 

Mean adult body weight total change (g) 

male 

female 

Total eggs laid 

Eggs set / Pen 

Test concentration (measured, pprn as.) 

Control 

20.6 

28 

62 

867 

44.61 

85 

20.2 

27 

54 

785 

39.17 

264 

---- 19.3 

23 

57 

805 

40.83 

802 

19.5 

25 

49* 

972 

48.94 
Reproductive parameter 

Test concentration (measured, ppm as.) 

Control 

Eggs set I Eggs laid ["%I 

Viable embryos 1 Eggs set [%I 

Viable embrvos 1 Pen 

Live 3-week embryos 1 Pen 

Hatchlings 1 Pen 

14-day old survivors 

Eaas laid I Hen 

85 I 264 

93.04 

95.59 

42.33 

Eggs cracked I Pen 

14-day survivors I Hen 

Mean egg shell thickness [mm] 

Mean hatchling body weight [g] 

802 

42.1 1 

40.1 1 

71 8 

48.17 

inhibition of above compared to control [%I 

Mean 1Cday survivor body weight [g] 

Live 3-week embryos I Viable embryos [%I 

Hatchlings I Live 3-week embryos [O/O] 

89.75* 

94.99 

37.44 

0.22 

42.24 

0.21 

7.2 

16day old survivors I Hatchlings [%I 

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.1kg bwld, 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 bwld, based on a mean body 

37.1 1 

34.61 

61 4 

43.61 

I 

35.5 

99.43 

92.73 

Hatchlings 1 Eggs laid [Oh] 

Hatchlings I Eggs set [%I 

1 Cday old survivors I Eggs set [O/O] 

Comments: 

86.4* 

94.44 

38.72 

0.67 

40.93 

0.20 

6.9 

I 99.43 

weight of 21 8 g and mean feed consumption of 20 glbirdld. 

US EPA recommends reporting the reproductive NOAEC as 264 ppm (23 mglkg 

bwld), based on a statistically significant effect on mean hatchling body weight 

and a reduction of the embryosleggs set (%) endpoint. 

90.38* 

93.13* 

45.72 

38.50 

35.06 

625 

44.72 

4.2 

35.6 

98.93 

93.03 

* Significantly different from the control, pc 0.05 

81.72 

88.01 

87.46 

Reference: Effect of Technical BY1 08330 on Mallard Reproduction 

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

ReporVDoc. number: EBFNX013, M-277794-01-2 

Guideline(s): USEPA FlFRA 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 

45.17 

42.28 

759 

54 

0.06 

36.76 

0.20 

7.0 

98.80 

Test substance: Spirotetramat (techn.), batch No.: 080451001 4, purity 97.2 % 

0.5 

42.1 7 

0.20 

6.8* 

2.8 

35.5 

99.57 

89.57 

79.26 

87.97 

86.87 

5.6 

34.5 

98.94 

93.64 

98.07 99.80 

73.1 1 

84.77 

83.72 

78.26 

86.54 

86.35 
Material and methods: 

Test organism: 

Dosage levels: 

Number of birds and 

replicateslgroup: 

Administration: 

Analytics: 

Study phases: 

Test conditions: 

Duration: 

Test parameter: 

Observations: 

Adult Body weight: 

Adult feed 

consumption: 

Egg parameter: 

Hatchling parameter 

Post-mortem 

examination: 

Statistics: 

Findinas: 

Mortalities: 

Clinical signs: 

Mallard duck (Anas platyrhynchos), 1 9 weeks of age 

Control (untreated diet); 80, 240 and 720 mg ailkg feed (nominal), corresponding 

to 0, 89, 269 and 869 mg ailkg 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.1kg bwlday (calculated). 

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

Test substance mechanically mixed in the diet, prepared weekly. 

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

sufficiently verified by analytical methods. 

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

photostimulation): 3 weeks; Egg-laying: approximately 1 1 weeks; Post-adult 

termination: 6 weeks 

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 

Approximately 21 weeks (pre-egg laying until termination) 

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, 1 Cday survival, 14-day 

survivor body weight, and eggshell strengthlthickness. 

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

adult termination. 

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

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. 

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. 

All adult birds were subjected to a gross necropsy. 

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. 

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. 

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 

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 pprn 

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 pprn 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 pprn treatment 

level. At the time of adult sacrifice (week 21) in the 89 pprn 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 pprn 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 pprn 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 Error! Style not defined.9. 

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

statistical difference from the control in the 269 pprn level. 
The Jonckheere's test indicated a statistical difference from the control in the 269 

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

viable embryos endpoints. 

Table Error! Style not defined.-2: Summary of reproductive results of spirotetramat in the mallard duck I I 

Test concentration (measured, ppm a.s.) Reproductive parameter Control 89 269 869 

Overall mean food consumption (glbirdlday) 113 11 8 86* 57 

Mean adult body weight total change (g) 17 5 -113* -31 5* 

male 

female 197 140 -8* -124* 

Total eaas laid 758 767 427* n.d. 

I Eaas laid I Hen 

1 Eaas set I Eaas laid T%l 1 88.4 1 92.2 1 87.1 1 n.d. I 

Eggs cracked 

Eggs cracked I Pen 

Eggs set 1 Pen 

2 

0.14 

48.1 

Viable embryos1 Eggs set [%I 

Viable embryos 1 Pen 

Live 3-week embryos 1 Pen 

Hatchlings1 Pen 

14-day old survivors 

1Cday survivors I Hen 

Mean egg shell thickness [mm] 

Mean hatchling body weight [g] 

1 

0.07 

50.5 

79.4 

40.0 

39.6 

Mean 14-day survivor body weight [g] 

Live $week embryos I Viable embryos [O/o] 

Hatchlings I Live 3-week embryos [%I 

14-day old survivors I Hatchlings p,] 

Hatchlings 1 Eggs laid ["h] 

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

27.8 

386 

29.7 

0.364 

37.2 

Hatchlings I Eggs set [%I 

14-day old survivors I Eggs set [%I 

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 ailkg bwld, based on 

a mean body weight of 11 22 g and mean feed consumption of 11 8 glbirdld. 

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. 

2 

0.13 

25.4* 

67.3 

25.2 

40.8 

260.6 

98.9 

69.5 

99.2 

48.5 

n.d. 

n.d. 

n.d. 

32.2 

386 

32.2 

0.364 

34.7* 

* Si~nificantly different from the control, uc 0.05 

54.5 

58.2 

54.1 

16.1* 

23.3* 

250.8 

99.2 

80.4 

100 

56.6 

n.d. 

n.d. 

n.d. 

8.1* 

80* 

8.9* 

0.344 

29.0' 

61.1 

61.1 

n.d. 

n.d. 

n.d. 

n.d. 

n.d. 

217.4* 

94.9* 

32.2* 

99.5 

20.7* 

n.d. 

n.d. 

n.d. 

n.d. 

n.d. 

23* 

25.3* 

n.d. 

n.d. 
Reference: 

Author(s), year: 

ReporVDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test organism: 

Dosage levels: 

Number of birds and 

replicateslgroup: 

Administration: 

Analytics: 

Study phases: 

Test conditions: 

Duration: 

Test parameter: 

Observations: 

Adult Body weight: 

Adult feed 

consumption: 

Egg parameter: 

Hatchling parameter 

Post-mortem 

examination: 

Statistics: 

BY1 08330 Technical: A Reproduction Study with the Mallard 

Temple, D.L.; Martin, K.H.; Beavers, J.B.; Jaber, M.; 2006 

EBFNP024, M-278058-01-1 

US EPA FlFRA 71 -4, OPPTS 850.2300 (Draft), OECD 206 

Yes 

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

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

recommended. 

Acceptable 

BY1 08330 (techn.), batch No.: 0804510014, purity 97.99 % 

Mallard duck (Anas platyrhynchos), 19 weeks of age 

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 bwlday. 

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

Test substance mechanically mixed in the diet, prepared weekly. 

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

sufficiently verified by analytical methods. 

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

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

termination; 6 weeks 

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 

Approximately 22 weeks (pre-egg laying until termination) 

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 weights were measured at study initiation, on weeks 2, 4, 6, 8, 9, and 

at adult termination. 

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

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. 

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. 

All adult birds were subjected to a gross necropsy. 

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

significant differences between groups. Dunnett's multiple comparison procedure 
Body weight: 

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

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. 

Findinas: 

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

Clinical signs: Birds in the 720 ppm as. 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 pprn 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. 

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

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

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

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

marked weight loss. 

Hatchlings: In the 80 pprn 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 pprn 

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 pprn 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 EPKs 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 andlor bleeding evident. These lesions were noted in four 

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

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

from the 720 pprn 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 pprn a.s. treatment groups. 

Reproductive results: See Table Error! Style not defined.-3. 
Table Error! Style not defined.-3: Summary of reproductive results of spirotetramat in the mallard duck 

Reproductive parameter 

Number of replicates 

Overall mean food consumption (glbirdlday) 

Mean adult body weight total change (g) 

male 

female 

Total eggs laid1 

Eggs laid / en' 

Eaas cracked' 

Eggs cracked / Eggs laid [%I 

Viable embryos I Eggs set [Oh] 

1 Cday old survivors ' 

14-dav survivors I Hen 

14-day old survivors / Hatchlings [Oh] 100 99 1 94 91 ' 1 

Test concentration (measured, ppm as.) 

15 

1 62 

-32 

58 

686 

46 

4 

Mean egg shell thickness [mm] 

Mean hatchling body weight [g] (sample size) 

Mean 14-day survivor body weight [g] 

Live 3-week embryos 1 Viable embryos [%I 

Hatchlinas I Live 3-week embrvos 1%1 

Control I 28.8 I 78.8 

2 

87 

426 

28 

1Cday old survivors / Eggs set [%I I 69 I 62 I 57 ' I 23*+ I 

* 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) 

no statistical analysis on this parameter 

201 

15 

159 

9 

84 

629 

42 

6 

0.389 

34 ( 12) 

274 

98 

79 

Hatchlings / Eggs set [Oh] 

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.1kg feed. This is equivalent to a NOAEL of 4.0 mg 

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

weight of 1147 g and mean feed consumption of 159 glbirdld. The statistically 

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

1 

83 

333 

22 

considered biologically not relevant. 

15 

142' 

4 

-2 

456 

30 

3 

0.382 

32+(13) 

266 

99 

77 

I I I I I 69 

Aw~endix: statistical analvsis of the re~roductive data bv the US EPA: 

14 

131 ' 

-34 

-73* 

276 

20 

3 

1 

91 

266 

18 

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 

2 

48* 

76 

5 

0.389 

31*'(14) 

257 

98 

63 + 

63 

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 

- 

0.373 

28* + (6) 

200* 

96 

41*+ 

60 25* 
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 I Live 3-week embryos [%],14- 

day old survivors 1 Hatchlings [%I and 1 Cday old survivors 1 Eggs set [%I. 

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 6.9.1.3-3). 

According to a published article, the Williams test maintains a predetermined a-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 IRMS): 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 
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 BY1 08330 

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

ReportlDoc. number: EBFNP033; M-290165-01-1 

Guideline(s): Not applicable 

GLP: Yes 

Deviations: Not applicable 

Validity: Acceptable 

Test substance: 

Material and methods: 

Test organism: 

Test design: 

Number of birds 

/group: 

Administration: 

Duration: 

Spirotetramat (BY1 08330), purity: 98.10 %, lot no. 08045-0014 

Mallard Duck (Anas platyrhynchos), age: 35 weeks 

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 

Twelve males and twelve femaleslgroup. One pairlcage. 

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

exposure group 

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

replacement with another group. 
Test conditions: 

Analytics: 

Test parameters: 

Observations: 

Feed consumption: 

Necropsy: 

Statistics: 

Findinas: 

Analytics: 

Mortalities: 

Clinical signs: 

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. 

Dietary analysis confirmed recoveries of 95 to 11 6 % 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 LCIMSIMS system. 

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. 

Not reported. 

None. 

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

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

0.1 1 (week 2) and 0.6 (week 8) mg a.s./kg were detected. The residue level of 

BY1 8330-enol was between 61.6 (week 2) and 97.6 (week 8) mgkg. Residues in 

the dietary control were < 0.01 mgkg. 

Severe foot symptoms progressed to warrant in extremis euthanisation of both 

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

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 
Body weight: 

Conclusion: 

Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test organism: 

Test design: 

Number of birds 

/group: 

Administration: 

Duration: 

Test conditions: 

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 weights of the dietary exposure group were significantly reduced compared 

to the control. 

Two separate sets of ducks exposed to Spirotetramat at 720 mglkg 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. 

Semi-Field Study of the Effects of Dermal and Dietary Exposure to BY1 08330 

in Mallard Ducks (Anas plafyrhynchos) 

Stafford, J. M.; 2007b 

EBFNP034; M-290000-01-1 

Not applicable 

Yes 

Not applicable 

Acceptable 

Spirotetramat (BY1 08330) techn., purity: 98.10 %, lot no. 08045-0014 

Movento formulation: BY1 08330 150 OD, lot no. 08030101 89(0125), purity: 14.7 % 

as. 

Mallard Duck (Anas platyrhynchos), age: 27 weeks 

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. 

Three treatment and control replicate pens. Six males and six femaleslpen (36 

birds / treatment or control group). 

Test substance mixed in diet and exposure to oversprayed turf. 

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

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 
Analytics: 

Test parameters: 

Observations: 

Feed consumption: 

Feet preservation: 

Statistics: 

Findinas: 

Mortalities: 

Clinical signs: 

Feed consumption: 

Body weight: 

Conclusion: 

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

15 m providing a total area of approx. 0.1 7 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. 

Dietary analysis confirmed recoveries of 82.7 to 98.9 5% of nominal. Homogeneity 

was demonstrated. 

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 was measured daily and average values were calculated per 

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

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. 

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

multiple comparison test. 

None. 

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. 

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. 

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

inconsistent and transient these effects were not considered treatment related. 

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. 
- Further studies for the refinement of the risk assessment 

Reference: BY108330 150 OD - Magnitude of the Residue inlon SoybeansNYheat, 

Potential Wildlife Feed Items 

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

ReportIDoc. number: RAFNP002; M-277315-01-1 

Guideline(s): Not applicable 

GLP: Yes (certified laboratory) 

Deviations: Not applicable 

Validity: Acceptable 

Test substance: Spirotetramat (BY1 08330) OD 150, 150 g a.s./L (nominal), batch no. 

08030101 89(0 1 52) 

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 kglhdapplication (0.1 57 Ib a.s./A) and with an 

application interval of 19 to 21 days. All applications were made using spray volumes of 11 0 to 167 Uha and 

used Dyne-Amic (0.25 % vlv) 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 BY1 08330 and its metabolites 

BY1 08330-enol, BY1 08330-ketohydroxy, BY1 08330-mono-hydroxy, and BY1 08330-enol-glucoside were 

quantified by high pressure liquid chromatographyltriple stage quadrupole mass spectrometry (Ic-mslms) 

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. 
Findinas: 

Table Error! Style not defined.-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 half-lives 

I (Tall ~rass)- I I I a Mean total spirotetramat residue immediately following the 2" application is the average from all trials of the total 

I 

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

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

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

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

Soybean ~ora~e 

Wheat Forage 

(Short Grass) 

Wheat Forage 

sample intervals following the second application of Spirotetramat 1500D. 

The mean half-life for total as. 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. 

Crop 

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. 

Maximum Total 

Spirotetramat Residue 

Following the Second 

Application [ppmlb 

Mean Total Spirotetramat 

Residue Immediately 

Following the Second 

Application [ppmIa 

8.32 

14.35 

13.04 

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 

ReportlDoc. number: RA07031 (Rifcon) 

Guideline(s): Not applicable 

GLP: Yes (certified test facility) 

Deviations: Not applicable 

Validity: Acceptable 

Mean Half-Life 

[days]c 

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 (BY108330) and its metabolites 

BY108330-enol and BY108330-ketohydroxy on foliage and ground dwelling arthropods in citrus orchards after 

spray application of Spirotetramat OD1 50B G. 

10.28 

20.35 

21.65 

- 

3.42 14.45 

2.13 

1.96 
Study-area: citrus orchards in the region ValencidSpain. 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.1ha) with a spray volume of 3000 Uha. 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 anabsjs: All samples were analysed for their content of Spirotetramat (BY108330) and its 

metabolites BY108330-enol and BY108330-ketohydroxy via HPLCIMS-MS. Residues are reported in terms of 

mg a.s./kg f.w. of matrix and mg metabolitekg f.w., respectively. The LOQ was 0.01 mglkg 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 011 -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 (DT,) 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-DT,,. 

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

Findinas: 

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 TWAsin arthropods are shown in the table below: 
Table Error! Style not defined.-2: Residue concentrations in ground and foliage dwelling arthropods 

after application of spirotetramat in the field b . 

Maximum measured 0.32 (DAT +2) 1 0.60 (DAT +2) I 0.62 (DAT +2) I 

Mean residue concentrations [mglkg f.w.1 in ground dwelling arthropods I 

Initial measured value 

IDAT+ll 

value (MMV) 

Parent 

0.21 

Parent+enol 

0.52 

I I 

Initial measured value 

(DAT+I\ 

1 DTso 1 2.8 d 1 4.7 d 1 5.1 d I 

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 

mglkg f.w. for parent, parent-kenol and total measured residues, respectively; and for foliage dwellers 4.0, 6.0 

and 6.2 mglkg 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 DTB~ 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 0.1 1 

mglkg 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 

understirnation of the residues, the study is considered acceptable. 

Total measured residue 

0.54 

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

Mean residue concentrations [mglkg f.w.1 in foliage dwelling arthropods 

Maximum measured 

value (MMV) 

MIA IDAT1 -21) 

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

and their diet composition (Position Paper) 

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

ReportIDoc. number: RC06-067; M-277503-01-1 

Guideline(s): Not applicable 

GLP: Yes (certified laboratory) 

Deviations: Not applicable 

Validity: Acceptable. 

Parent 

4.0 

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 

(Monros et al. 1999). 

4.0 (DAT +0) 

1.07 (27 % of MMW 

Parent+enol 

5.2 

Total measured residue 

5.3 

6.0 (DAT +1) 

1.90 (32 % of MMV) 

6.2 (DAT +I) 

2.03 (33 %of MMV) 
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 

(Monros 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 (Monros 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 Oh). For the size class of the remainder it is also 

reasonable to expect preferential feeding on larger prey (Monros 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 IRMSl: 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 10s 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. 

Monros et at. (1 999): Open nestings in Great Tits Parus major. Ardeola 46 (1): 89-91. 

Perrins (1979): British Tits. Collins, London. 
Reference: Generic field monitoring of birds in potato, cultivation in northern Germany 

Author(s), year: Wolf, Ch. 2004 

ReporVDoc. number: WFCIFS 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 Dotlingen 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, singinglbreedinglfeeding 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) I (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. 

Findinas: 

The results of the monitoring are shown in the table below: 
PORTION OF TIME (PT) in habitat of Yellow Wagtails after radio tracking 

potential foraging time I potato fields 1 38.4 1 % 1 (72.3) 

(90%ile) I I I 

other habitats (11.3 1% l(16.0) 

(sum of behaviour categories 

"foraging"+"active"+"unknown") 

spent per habitat; based on 20 individuals, 1 to 

5 24-hour sessions each [mean of individualsl, 

I I I . , 

FEEDING HABITAT of Yellow Waatails after transect Counts (based on DoDulation\ 

cereal fields 

Oil Seed rape 

street and field path 

I oil seed rape fields 1 0.22 

abundance of potentially foraging individuals of 

the population after 9 transect counts covering 

48.58 ha field crops each [individualslha] 

I I PREFERENCE OF POTATO FIELDS in Yellow Wagtails after radio tracking I I 

39.8 

6.8 

3.7 

I preference of potato fields as a feeding habitat 

[Jacobs' index (D). MCP (100%)1 

potato fields 

cereal fields 

sugar beet fields 

% 

% 

% 

0.60 

0.41 

0.32 

(44.1) 

(22.2) 

(10.7) 

DIET of Yellow Wagtails 

main arthropod orders actually eaten by 

Wagtails foraging in and around potato fields 

I (based on 25 samples of faeces) 

SIZE OF ARTHROPODS actually chosen by Yellow Wagtails 

Mean sizes of actually eaten individuals (after I taxonomic order 1 food 1 w~ulation I 

taxonomic order 1 steadiness' I mean numbe? 

Di~tera I 100"/0 1 3.96 

I 

Coleoptera 

A~hidoidea 

Hymenoptera 

Aranea 

I samples of faeces) compared to mean sizes of 

unselective samples of the population in potato 

fields 

I Hymenoptera 1 6.33 (20) 1 3.13 (25, f) I 

84% 

72% 

Coleoptera 

Aphidoidea 

1 Aranea 1 4.38 (16) 1 3.1 2 (58, g) I 

3.60 

5.28 

52% 

48% 

Di~tera 

MAIN BIRD SPECIES in ~otato fields ~otentiallv foraaina I 

0.80 

0.64 

. , 

7.22 (90) 

3.00 (132) 

[mml (n) 

7.49 (99) . ., 

5.85 (128, g) 

1.98 (350, f) 

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. 

. . [mm] (n, stratum3) 

2.78 (523. f) 

mean abundance of main species after 9 

transect counts covering 9.98 ha potato fields 

each 

' portion of samples containing this type 

mean number of individuals per sample 

f = foliage dwelling arthropods, obtained by inventory spraying; g = ground dwelling arthropods, obtained by pitfall 

trapping 

Yellow Wagtail 

pied wagtail 

Whitethroat 

0.60 individuals per ha 

0.29 individuals per ha 

0.12 individuals per ha 
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 (goth 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 

~~~~~ - - ~~~~~~ 

All values except for acute oral study based on measured figures; 

Table Error! 

Study type 

Acute oral 

Short-term 

dietary 

toxicity 

Subchronic l 

reproductive 

toxicity 

* 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 PMRA and US EPA is not included in this monograph. 

S'yle not defined.-1: Summary of avian toxicity endpoints for spirotetramat 

NOAEL 

(mgkg bw) 

c 500 

183 

< 65 

Adults: 23 

repro: 74 

Adults: < 9.4% 

repro: 9.4 

Adults+repro: 

4.0 

Test Wecies 

bobwhite quail 

bobwhite quail 

mallard duck 

bobwhite quail 

mallard duck 

mallard duck 

Reference 

Barf knecht, 

2003 

Barfknecht, 

2004 

Stoughton, 

2005 

Sabbert & 

Nuessle, 2006a 

Sabbert & 

Nuessle, 2006b 

Temple et al., 

2006 

LC50 

(mglkg feed) 

I 

> 5000 

> 6050 

I 

I 

I 

NOAEC 

(mgkg feed) 

I 

1250 

c 344 

Adults: 264 

repro: 802 

Adults: c 89* 

repro: 89 

Adults+repro: 

28.8 

LD5o 

(mglkg bw) 

> 2000 

>497 

> 475 

I 

I 

I 
6.9.2 Effects on aquatic organisms (OECD IIA 8.2, lllA 10.2) 

6.9.2.1 Acute toxicity of active substance, metabolites and formulations to aquatic organisms 

6.9.2.1.1 Acute toxicity to fish (OECD IIA 8.2.1, IIA 8.1 1 .I, lllA 10.2.2) 

Active substance 

Reference: Acute toxicity of BY1 08330 (tech.) to fish (Oncorhynchus mykiss) 

Author(s), year: Dorgerloh, M., 2004 a 

ReportIDoc. number: DOM 24025, Edition Number: M-182649-01-2, 16.12.2004 

Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92169lEEC C.l 

GLP: Yes 

Deviations: Measured test concentration dropped below 80 % of nominal concentration. 

Validity: Acceptable 

Test substance: Spirotetramat (BY1 08330), purity: 97.4 %, batch: 0804510014 

Material and methods: 

Test species: Rain bow trout (Oncorhynchus mykiss) 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature: 

pH: 

O2 content: 

Light regime: 

Test parameters: 

10 fish per concentration and control 

2.3 + 1.1 g (mean + SD) and 5.8 k 0.9 cm (mean + SD) 

0.58 g fishll test medium 

Semi static test (renewal of test media every 24 hours), 96 hours 

0 (control and solvent control), 0.5, 1 .O, 2.0, 4.0 and 8.0 mg/L 

-- (control and solvent control), 0.409,0.825, 1.5, 3.14 and 6.44 mg/L 

Aceton 0.2 mUL 

Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaC03 

11.3- 12.3 "C 

7.0 - 7.1 (0 h, new medium), 7.0 (96 h, aged medium) 

89 - 99 % saturation 

16 hours light I 8 hours darkness 

Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC-MSIMS and HPLC-UV) of BY1 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 

Findinas: 

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 mglL: No sublethal effects were 

reported over the whole test period. 

Mortality: 

At test concentration 2 1.5 rnglL: 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 mglL based on sublethal effects. 

See table 8.9.2.1 .I-1 
NOLC: 1.5 mg/L I LC50 (96 h): 2.54 ma/L (95 % C.I. 2.1 1 - 3.05 ma/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, Chi? 1.25 

Degrees of freedom: 3 

p (Chi2): 0.74 

Conclusion: LCs0 (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 

ReporVDoc. number: DOM24022, Edition Number: M-128667-01-2, 25.1 1.2004 

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: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature: 

pH: 

Spirotetramat (BY1 08330), purity: 97.4 %, batch: 08045/0014 

Common carp (Cyprinus carpio) 

10 fish per concentration and control 

0.9 * 0.3 g (mean k SD) and 4.0 k 0.5 cm (mean + SD) 

0.23 g fish/L test medium 

Semi static test (renewal of test media every 24 hours), 96 hours 

0 (control and solvent control), 1.25, 2.5, 5.0, 10.0 and 20.0 mg/L 

-- (control and solvent control), 1.02, 1.71, 3.65, 8.13 and 13.4 mg/L 

Aceton 0.2 mUL 

Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaC03 

20.5 - 23.2 "C 

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 BY1 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 

Findinas: 

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 r 1.71 mg1L: 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 

Mortality: 

position. 

Thus the NOEC was 1.02 mg/L based on sublethal effects. 

See table 8.9.2.1 .I -2 

Control I o o o I o 

Solvent control I 0 1 0 1 0 0 

Table Error! Style not defined.-2: Effects on common carp (C. carpio) exposed to spirotetramat 

I NOLC: 1.71 mgIL 

LCsn(96 hours): 2.59 maR (95 % C.I.: not determined due to mathematical reasons) I 

Spirotetramat 

[mg a.s JL] 

(mean measured) 

Details of the Probit: Slope b: 16.09 

95 OACI of b: not stated 

Variance of b: 29.9 

Intercept a: -1.66 

Goodness of fit, Chi': 17.69 

Degrees of freedom: 3 

p (Chi2): < 0.001 

LCSo (96 h): 2.59 mg/L 

NOEC (96 h): 1.02 mg/L 

based on mean measured concentration 

The US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. (95 %CI: not stated) 

Conclusion: 

Comments: 

. 

Cumulative mortality (Oh) 

24 hours 48 hours 72 hours 96 hours 
Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

-- 

Acute toxicity of BY1 08330 (tech.) to fish (Lepomis macrochirus) 

Dorgerloh, M., 2005 a 

DOM24056, Edition Number: M-242689-01-2, 12.01.2005 

OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92169lEEC C.l 

Yes 

None of relevance 

Acceptable 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature: 

pH: 

O2 content: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Behavioural effects: 

Mortality: 

Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 

Bluegill sunfish (Lepomis macrochirus) 

10 fish per concentration and control 

1.7 + 0.7 g (mean -c SD) and 4.9 + 0.6 cm (mean + SD) 

0.43 g fish/L test medium 

Semi static test (renewal of test media every 24 hours), 96 hours 

0 (control and solvent control), 0.5, 1 .O, 2.0, 4.0 and 8.0 mglL 

-- (control and solvent control), 0.48, 0.95, 1.90, 3.56 and 7.61 mgR 

Aceton 0.2 mUL 

Reconstituted water (according to ISO), hardness: 40 - 60 mglL as CaC03 

17.9 - 20.5 "C 

7.4 - 7.5 (0 h, new medium), 7.3 (96 h, aged medium) 

96 - 105 % saturation 

16 hours light 1 8 hours darkness 

Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC-MSIMS and HPLC-UV) of BY1 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. 

EC50: Probit analysis, NOEC: directly from raw data 

Over the whole test period the mean measured concentrations were in the range 

91 - 99 5% of nominal. 

Controls and at 0.5 mg1L: No sublethal effects were observed over the whole test 

period. 

At concentration levels 2 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. 

See table B.9.2.1.1-3 
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 

LC5, (96 h): 2.2 mg/L 

NOEC (96 h): 0.5 mg/L 

based on nominal concentration 

The US EPA has general concerns regarding uncertainties surrounding exposure 

Conclusion: 

Comments: 

concentrations in test media. 

Metabolites 

Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validiw: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Acute toxicity of BY1 08330-enol to fish (Oncorhynchus mykiss) 

Nieden, D., 2005 a 

Report No.: EBFNM013, Edition Number: M-251850-01-2, 18.05.2005 

OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92169lEEC C.l 

Yes 

None of relevance 

Acceptable 

BY1 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch:'692-101-09- 

0005 

Rainbow trout (Oncorhynchus mykiss) 

2 x 15 fish per concentration and control 

1.3 + 0.6 g (mean + SD) and 4.9 + 0.7 cm (mean + SD) 

0.49 g fish/L test medium 

Static limit test. 96 hours 

0 (control), 100 mg/L 

-- (control), 98 mg/L 

Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaC03. 
Temperature: 

pH: 

O2 content: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Behavioural effects: 

Mortality: 

Conclusion: 

Reference: 

Author(s), year: 

Report/Doc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Solubility: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Temperature: 

pH: 

O2 content: 

Light regime: 

Test parameters: 

After the addition of test substance the pH was adjusted to about 7 with NaOH. 

11.1 -13°C 

6.7 - 7.2 (0 h), 6.8 - 6.9 (96 h) 

86 - 102 % saturation 

16 hours light / 8 hours darkness 

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-MSIMS 

and HPLC-UV) of BY1 08330-enol in test solutions. 

None, because a limit test was performed 

Over the whole test period the mean measured concentration was 98.5 % 

(replicate A) and 97.7 % (replicate B) of nominal. 

No effects were observed in control and 100 mg/L treatment replicates. 

No mortality was reported in control and 100 mg/L treatment replicates. 

LC5() (96 h): > 1 00 mg/L 

NOEC (96 h): 100 mg/L 

based on nominal concentration 

4-Methoxycyclohexanone - Acute fish toxicity 

Neuhahn, A., 2006 a 

Report No.: 2006/0032/02, Edition Number: M-274305-01-1, 10.07.2006 

OECD 203, EU Directive 92/69/EEC C. 1 

Yes 

None of relevance 

Acceptable 

4-Methoxycyclohexanone (metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV5506-001 

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. 

Zebra fish (Danio rerio) 

10 fish per concentration and control 

Not reported, 3.4 + 0.26 cm (mean +. SD) 

0.64 g fish/L test medium 

Static limit test, 96 hours 

0 (control), 100 mg/L 

-- (control), 98.5 mg/L 

Reconstituted water (according to ISO), hardness: 246 mg/L as CaC03. 

20.5 - 21 .O "C 

7.6 - 7.9 

88 - 101 % saturation 

16 hours light 18 hours darkness 

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 
Statistics: 

Findinas: 

Analytical data: 

Behavioural effects: 

Mortality: 

Conclusion: 

Comments 

Comments: 

Formulation 

MS detection) of 4-Methoxycyclohexanone in test solutions. Test samples were 

analysed directly without centrifugation. 

None, because a limit test was performed 

The mean measured concentration was 105 % (0 hours) and 89.8 % (96 hours) of 

nominal. 

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. 

No mortality was reported in control and 100 mg/L treatment replicates. 

LC50 (96 h): > 100 mg/L 

NOEC (96 h): 100 mg/L 

based on nominal concentration 

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 ez2"C), 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 mg1L treatment level.. 

US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 

Reference: 

Author(s), year: 

Report/Doc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Control: 

Test conditions: 

Water quality: 

Temperature: 

pH: 

O2 content: 

Acute toxicity of BY1 08330 OD 150 to fish (Oncorhynchus mykiss) under 

static conditions 

Nieden, D., 2005 d 

EBFNM008, Edition Number: M-253916-01-2, 29.06.2005 

OECD 203, EPA 72-1, OPPTS 850.1075, EU Directive 92169lEEC C.l 

Yes 

None of relevance 

Acceptable 

BY1 08330 OD 150 (formulation), spirotetramat content: 148.89 g/L (1 5.1 %), 

batch: 08030101 89(0152) 

Rainbow trout (Oncorhynchus mykiss) 

10 fish per concentration and control 

0.8 2 0.2 g (mean + SD) and 4.6 & 0.3 cm (mean r SD) 

0.20 g fishll test medium 

Static test, 96 hours 

Formulation: 0 (control), 2.50, 5.00, 10.0, 20.0, 40.0 mg product1L 

Spirotetramat: 0 (control), 0.378, 0.755, 1.51, 3.02, 6.04 mg a.s./L 

Spirotetramat: -- (control), 0.394, 0.783, 1.57, 3.10, 6.45 mg a.s./L 

Untreated test water, no blank formulation control was used 

Reconstituted water (according to ISO), hardness: 40 - 60 mg/L as CaC03 

11.6 - 12.2 "C 

6.9 - 7.2 

95 - 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. 

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 

Findinas: 

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 productll concentration level: No sublethal effects were 

observed over the whole test period. 

At test concentrations of 5.0 and 10 mg productll: 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& based on sublethal effects. 

Mortality: See table Error! Style not defined.-4: 

Table Error! Style not defined.4: Effects on rainbow trout (0. mykiss) exposed to BY1 08330 OD 150 

(formulation\ 

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, Chi? 7.498 

Degrees of freedom: 3 

p (Chiz): 0.0576 

LC50 (96 h): 9.48 mg productll, 1.41 mg a.s./L 

NOEC (96 h): 2.5 mg productll, 0.378 mg a.s./L 

based on nominal concentration 

. - . . . - . . 

Conclusion: 

BY1 08330 OD 150 

[mg productll] 

(nominal) 

B.9.2.1.2 Acute toxicity to aquatic invertebrates (OECD IIA 8.3.1, IIA 8.5.1, lllA 10.2.2.2) 

Active substance 

Spirotetramat 

mg a.s./L 

(mean meas.) 

Cumulative mortality (%) 

Control 

NOLC: 2.5 rng product1L corresponding to 0.397 rng a.s./L 

LC50 (96 h): 9.48 mg/L (95 % C.I. 5.84 - 15.7 rng/L) corresponding to 1.41 rng 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 

24 hours 

0 

0 

0 

0 

70 

100 

2.5 

5.0 

10.0 

20.0 

40.0 

0.394 

0.783 

1.57 

3.1 

6.45 

48 hours 

0 

0 

0 

0 

80 

100 

72 hours 

0 

0 

0 

10 

90 

100 

96 hours 

0 

0 

10 

10 

100 

100 
Reference: Acute toxicity of BYI 08330 (tech.) to the waterflea Daphnia magna under 

static conditions 

Author(s), year: Dorgerloh, M., 2005 c 

ReportlDoc. number: DOM 24004, Edition Number: M-242683-01-2, 14.01.2005 

Guideline(s): OECD 202, EPA 72-2, OPPTS 850.101 0, 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: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Effects: 

Conclusion: 

Comments: 

Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 

Waterflea (Daphnia magna) 

6 replicates each with 5 daphnids per treatment and control 

First instar c 24 hours old 

Static test, 48 h 

0 (control and solvent control), 6.25, 12.5, 25.0, 50, 100 mg/L 

-- (control and solvent control), 4.7, 8.77, 20.3, 38.8, 42.7 mg/L 

0.4 mUL Aceton 

M7 medium (according to "original draft" of an EEC Daphnia magna pilot ring 

test), hardness: 196 mglL as CaC03 

20.5 - 20.6 "C 

6.3 - 6.4 (0 h), 6.6 - 6.7 (48 h) 

96 - 102 %, 8.5 - 9.0 mg O$L 

16 hours light / 8 hours darkness 

Immobility and sublethal effects were assessed after 24 and 48 hours. For 

chemical analysis (HPLC-UV) of BY1 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. 

EC50: None, because at highest test concentration (1 00 mg/L nominal) the 

immobilisation were c 50 %, NOEC: directly from the raw 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. 

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. 

EC50 (48 h): > 42.7 mg/L 

NOEC: 20.3 mglL 

based on mean measured concentration 

US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 
Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Feeding 

Test parameters: 

Statistics: 

Findinus: 

Analytical data: 

Effects: 

Acute toxicity of BY1 08330 (tech.) to larvae of Chironomus riparius in a 48 h 

water-only study 

Dorgerloh, M., 2005 g 

EBFNX072, Edition Number: M-262632-01-2, 08.1 2.2005 

No specified guideline, the study is following the procedure of OECD 202 

Yes 

None of relevance considering OECD 202 

Acceptable 

Spirotetramat (BY1 08330), purity: 97.4 %, batch: 0804510014 

Midge (Chironomus riparius) 

4 replicates each with 10 larvae per treatment and control 

L1 (first instar) larvae 

Static (water-only) test, 48 h 

0 (control and solvent control), 0.56, 1.00, 1.80, 3.20, 5.60, 10.0 mg/L 

-- (control and solvent control), 0.53, 0.96, 1.72, 3.04, 5.37, 9.47 mg/L 

0.1 mUL Aceton 

M7 medium (according to "original draft" of an EEC Daphnia magna pilot ring 

test), hardness: not stated 

20.4 - 21 "C 

8.2 - 8.6 (0 h), 8.2 - 8.9 (48 h) 

> 80 %, (8.6 - 8.9 mg O$L) 

16 hours light I 8 hours darkness 

Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MSIMS and HPLC-UV) of BY1 08330 in the test media samples 

were taken at test initiation (0 h) and termination (48 h). 

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. 

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. 

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 0.9.2.1.2-1 

Table Error! Style not defined.-1: Effects on midge (C. riparius) exposed to spirotetramat 

Spirotetramat [mg a.s./L] 

mean measured 

Control* 

Solvent control* 

Exposed chironomids 

(= 100 %) 

40 

40 

Mortality after 48 hours 

[nl 

3 

4 

[%I 

7.5 

10.0 
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 .I20 - 1.506 mg/L) I 

* Result of t-test is indicated no significant differences between control and solvent control, thus pooled control are used 

for statistical analysis. 

Spirotetramat [mg a.sJL] 

mean measured 

1.715 

3.035 

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: LClo (48 h): 0.699 mg/L 

LC5,) (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, 1 st instars were used here. 

-Test organisms should not be fed. 

Exposed chironomids 

(= 100 %) 

40 

40 

Mortality after 48 hours 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

In1 

29 

39 

-Mortality and sublethal effects should be measured every 24 hours. 

[%I 

72.5 

97.5 

Metabolites 

Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Acute toxicity of BY1 08330-enol to the waterflea Daphnia magna in a static 

laboratory test system - limit test 

Dorgerloh, M., 2005 d 

EBFNMOI 2, Edition Number: M-254487-01-2, 14.07.2005 

OECD 202, EPA 72-2, OPPTS 850.1 01 0, EU Directive 92/69/EEC C.2 

Yes 

None of relevance 

Acceptable 

BY1 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101 -09- 

0005, water solubility (at pH 7): 2.7gIL 

Waterflea (Daphnia magna) 

6 replicates each with 5 daphnids per treatment and control 

First instar < 24 hours old 

Static limit test, 48 h 

0 (control), 100 mg/L 

-- (control), 11 1.5 mg/L 
Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Effects: 

Conclusion: 

M7 medium (according to "original draft" of an EEC Daphnia magna pilot ring 

test), hardness: 196 mg/L as CaC03, adjusted to pH 7 before use for dilution 

procedure 

20.6 - 21.2 "C 

7.0 - 6.9 (0 h), 7.5 - 7.9 (48 h) 

89 - 96 %, 8.1 - 8.7 mg O$L 

16 hours light 18 hours darkness 

Immobility and sublethal effects were assessed after 24 and 48 hours. For 

chemical analysis (HPLC-UV) of BY1 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. 

EC50: None, because at the limit concentration (1 00 mg/L nominal) no 

immobilisations were observed, NOEC: directly from the raw data 

The measured concentrations at the start and end of the test were 1 13 and 1 10 % 

of nominal, overall mean measured concentration was 11 1.5 % of nominal. 

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. 

EC50 (48 h): > 100 mg/L 

NOEC: 100 mg/L 

based on nominal concentration 

Reference: Acute toxicity of BY1 08330-en01 to larvae of Chironomus riparius in a 48 h 

water-only study 

Author(s), year: Dorgerloh, M., 2005 h 

ReportIDoc. 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: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Temperature 

PH 

0, content: 

Light regime: 

Feeding 

BY1 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101 -09- 

0005, water solubility (at pH 7): 2.7g/L 

Midge (Chironomus riparius) 

4 replicates each with 10 larvae per treatment and control 

L1 (first instar) larvae 

Static (water-only) test, 48 h 

0 (control), 1 .O, 2.0, 4.0, 8.0, 16, 32, 64 mg/L 

-- (control), 0.95, 1.93, 4.06, 8.3, 15.65, 32.25, 66.45 mg/L 

M7 medium (according to OECD 202), hardness: not stated 

20.8 - 21 "C 

7.8 - 8.2 (0 h), 8.1 - 8.4 (48 h) 

> 90 %, (8.5 - 8.7 mg 02/L) 

16 hours light / 8 hours darkness 

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-MSIMS and HPLC-UV) of BY1 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: LClo and LCSo: Probit analysis, NOEC: not determined 

Findinas: 

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. 

Eifects: See table B.9.2.1.2-2 

Table Error! Style not defined.-2: Effects on midge (C. riparius) exposed to BY1 08330-enol 

I BY1 08330-enol rmaRl I .---*.-a 

I 1 11.62 mg/L (95% C.I. 4.74 - 28.48 mg/L) based on mean measured 

LC50 (48 h): I 74.85 mg/L (95% C.I. 48.86 - 114.64 mgR) based on nominal 

Exposed chironomids I 

(= 100 %) nominal I 

Control 

I 

I 1 97.78 mgR (95% C.I. 43.15 - 221.60 mg/L) based on mean measured ( 

Mortality after 48 hours 

mean measured [nl 1x1 I 

40 

1 .O 40 

64.0 

Conclusion: 

Comments: 

0.95 

67.59 I 40 I 16 I 40.0 

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 

LC50 (48 h): 74.9 mg1L 

LClo (48 h): 17.1 mglL 

NOEC (48 h): not determined 

0 

0 

LC1 0 (48 h): 17.07 mg/L (95% C.I. 12.15 - 23.98 mg/L) based on nominal I 

based on nominal concentration 

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, I st 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. 

0 

0 
Reference: Acute toxicity of BY1 08330-ketohydroxy to larvae of Chironomus riparius in 

a 48 h static laboratory test system 

Author(s), year: Dorgerloh, M., 2006 a 

ReportlDoc. number: EBFN0347, 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: BY1 08330-ketohydroxy (metabolite of spirotetramat), purity: 98.6 %, batch: 

NLL7549-7, water solubility (in distilled water): 0.228glL 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Feeding 

Midge (Chironomus riparius) 

4 replicates each with 10 larvae per treatment and control 

L1 (first instar) larvae 

Static (water-only) test, 48 h 

0 (control), 1 .O, 3.6, 10.0, 36, 100 mg/L 

-- (control), 0.94, 3.47, 10.01, 33.6, 93.2 mg/L 

M7 medium (according to OECD 202), hardness: not stated 

20.5 - 21.6 "C 

7.8 - 8.4 (0 h), 8.0 - 8.1 (48 h) 

> 80 %, (6.9 - 7.7 mg Oz/L) 

16 hours light / 8 hours darkness 

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-MSIMS and HPLC-UV) of BY1 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: LCio and LC5,: Probit analysis, NOEC: directly from raw data 

Findinas: 

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 

I I -- 

Control 40 1 2.5 I 

Table Error! Style not defined.4: Effects on midge (C. riparius) exposed to BY1 08330- ketohydroxy 

BY1 08330-enol 

[mgll] (nominal) 

Exposed chironomids 

(= 100 %) 

Mortality after 48 hours 

[nl ["A 
Conclusion: 

Comments: 

LCs0 (48 h): > 100 mg/L 

LClo (48 h): 23.9 mg/L 

NOEC (48 h): c 1 mg/L 

based on nominal concentration 

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, 1 st instars were used here. 

-Test organisms should not be fed. 

-For invertebrates other than Daphnia, the test duration should be 96-hours. 

-Moltality and sublethal effects should be measured every 24 hours. 

Reference: 4-Methoxycyclohexanone - Acute daphnia toxicity 

Author(s), year: Neuhahn, A. 2006 b 

ReportIDoc. number: 2006/0032/01, Edition Number: M-274275-01-1, 10.07.2006 

Guideline(s): OECD 202, EU Directive 92169lEEC C.2 

GLP: Yes 

Deviations: OECD: None of relevance 

US EPA: 

Validity: Acceptable 

Test substance: 

Solubility: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

4-Methoxycyclohexanone (metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV5506-001 

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. 

Waterflea (Daphnia magna) 

2 replicates each with 10 daphnids per treatment and control 

First instar < 24 hours old 

Static limit test, 48 h 

0 (control), 100 mg/L 

-- (control), 11 1.5 mg/L 

M4 medium, hardness: 273 mg/L as CaC03 

21.4 - 21.5 "C 

7.8 (control) and 9.2 (1 00 mglL) 

98 %,8.7 mg OdL 

16 hours light 18 hours darkness 

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. 

None, because at the limit concentration (100 mg/L nominal) no immobilisation 

were observed 

The measured concentrations at the start and end of the test were 103 and 90 % 
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 

ReportIDoc. 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: 

Solubility: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Feeding 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

4-Methoxycyclohexanon (photo-metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV55506-001 

> 100 mg (pH 8,20 "C) 

Midge (Chironomus riparius) 

6 replicates each with 10 larvae per treatment and control 

L1 (first instar) larvae, < 2 - 3 days old 

Static (water-only) limit-test, 48 h 

0 (control), 100 mg/L 

-- (control), 11 1 mg/L 

M7 medium (according to OECD 202), hardness: 231 mg/L CaC03 

20.3 - 20.9 "C 

8.0 (after 0 and 48 hours) 

z 90 %, (8.6 - 8.9 mg OdL) 

16 hours light / 8 hours darkness 

Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

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. 

A limit-test was performed, thus no statistic is applicable. 

The mean measured concentrations at the start and end of the test were 109 % 
Effects: 

Conclusion: 

Comments: 

Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Solubility: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Test conditions: 

Water quality: 

Temperature 

PH 

O2 content: 

Light regime: 

Feeding 

Test parameters: 

and 11 2 % of nominal, respectively. Overall mean measured concentrations is 

1 11 % of nominal. 

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 I00 mgR and the EC50 was > 100 mg/L. 

LC50 (48 h): > 100 mg/L 

NOEC (48 h): 100 mg/L 

based on nominal concentration 

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, 1 st 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. 

Acute toxicity of BY1 08330-cis-methoxy-cyclohexylamino carboxylic acid to 

Larvae of Chironomus riparius in a 48 h Static Laboratory Test System 

(Limit-Test) 

Dorgerloh, M., 2007 b 

EBFNXI 05, Edition Number: M-287220-01-1, 27.04.2007 

No specified guideline, the study is following the procedure of OECD 202 

Yes 

None of relevance 

Acceptable 

BY1 08330-cis-methoxy-cyclohexylamino acid (photo-metabolite of spirotetramat), 

purity: 93.7 %, batch: KTSI 0330-1 -4 

> 100 mg (pH 8,20 "C) 

Midge (Chironomus riparius) 

6 replicates each with 10 larvae per treatment and control 

L1 (first instar) larvae, < 2 - 3 days old 

Static (water-only) limit-test, 48 h 

0 (control), 100 mg/L 

-- (control), 88.6 mg1L 

M7 medium (according to OECD 202), hardness: 231 mg/L CaC03 

20.3 - 20.9 "C 

6.9 - 8.0 (0 h), 7.7 - 8. 0 (48 h) 

> 90%, (8.7 - 8.9 mg 02/L) 

16 hours light 18 hours darkness 

Only on time 0.01 mL aqueous fish food solution directly after insertion of the 

larvae 

Mortality and sublethal effects were assessed after 48 hours. For chemical 

analysis (HPLC-MSIMS) of BY1 08330-cis-methoxy-cyclohexylamino acid in the 

test media samples were taken at test initiation (0 h) and termination (48 h). Test 
Statistics: 

Findinas: 

Analytical data: 

Effects: 

Conclusion: 

Comments: 

Formulation 

Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Control: 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

samples were analysed by direct injection into a HPLC-UV instrument without a 

centrifugation. 

A limit-test was performed, thus no statistic is applicable. 

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 O/O of nominal. 

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. 

LC50 (48 h): > 100 mg/L 

NOEC (48 h): 100 mg/L 

based on nominal concentration 

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. 

Acute toxicity of BY1 08330 OD 150 to larvae of Chironomus riparius in a 

48 h static laboratory test system 

Dorgerloh, M., 2006 b 

EBFNM007, Edition Number: M-264018-01-2, 19.01 -2006 

No specified guideline, the study is following the procedure of OECD 202 

Yes 

None of relevance 

Acceptable 

BY1 08330 OD 150 (formulation), spirotetramat content: 148.89 g/L (14.89 % 

active ingredient), batch: 08030101 89(0152), density: 0.986 g/mL 

Midge ( Chironomus riparius) 

4 replicates each with 10 larvae per treatment and control 

L1 (first instar) larvae 

Static (water-only) test, 48 h 

Formulation: 0 (control), 2.12, 3.71, 6.62, 11.9, 21.2, 37.1, 66.2 mg productJL 

Spirotetramat: 0 (control), 0.32,0.56, 1 .O, 1.80, 3.20, 5.60, 10.0 mg a.s./L 

Spirotetramat: -- (control), 0.30, 0.51, 0.87, 1.38, 2.67, 4.83, 8.98 mg a.s./L 

Untreated test water. no blank formulation control was used 

M7 medium (according to OECD 202), hardness: not stated 

20.6 - 21.4 "C 

8.1 - 8.4 (0 h), 7.8 - 8.4 (48 h) 

> 90 %, (8.4 - 8.6 mg 02/L) 
Light regime: 16 hours light 1 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-MSIMS 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: LClo and LC5,: Probit analysis, NOEC: directly from raw data, correction of control 

mortality: ~bbott's formular 

Findinas: 

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. 

Effects: 

Overall mean measured concentrations ranged from 77 to 95 % of nominal. 

See table B.9.2.1.2-6 

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 

Table Error! Style not defined.-4: Effects on midge (C. riparius) exposed to BY1 08330 OD 150 

(formulation) 

Conclusion: 

Comments: 

BY1 08330-OD 150 

[mg productll] 

Degrees of freedom: 5 

p (Chi2): 0.971 

LC5, (48 h): 4.41 mg productlL, 0.66 mg a.s./L 

LClo (48 h): 2.39 mg productll, 0.36 mg a.s./L 

NOEC (48 h): c 2.0 mglproduct, c 0.30 mg a.s./L 

based on mean measured concentration 

While the study not deviated from recommendations given in OECD 202 

guideline, significant deviation from USEPA guidelines are notable: 

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 

LCsn I48 h)*: 4.41 ma ~roductlL (95% C.I. 3.45 - 7.68 ma/L). corres~ondina to 0.66 ma a.s./L 

Spirotetramat 

[mg a.s./L] 

(nominal) 

0.32 

0.56 

1 .OO 

1.80 

3.20 

5.60 

10.0 

(nominal) 

Control 

2.12, 

3.71 

6.62 

11.9 

21.2 

37.1 

66.2 

-Age of the test organisms is 2nd or 3rd instar, 1 st 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. 

(mean 

measured) 

Control 

2.00 

3.40 

5.75 

9.14 

17.6 

32.0 

59.3 

Exposed 

chironomids 

(= 100 %) 

40 

40 

40 

40 

40 

40 

40 

40 

Mortality after 48 hours 

In] 

4 

4 

13 

33 

39 

40 

40 

40 

[%I 

10 

10 

32.5 

82.5 

97.5 

100 

100 

100 
B.9.2.1.3 Effects on algal growth and growth rate (OECD IIA 8.4, lllA 10.2.2.3) 

Active substance 

Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent: 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Pseudokirchneriella subcapitata - Growth inhibition test with BY1 08330 

(tech.) 

Dorgerloh, M., 2004 c 

DOM 23092, Edition Number: M-128874-01-2, 29.1 1.2004 

Draft proposal for updating OECD 201, OPPTS 850.5400, JMAFF guideline (12 

Nousan No 8147) 

Yes 

EPA guideline: 

.The temperature is less than the EPA-specified temperatures (24-25") and the 

lighting intensity is higher than EPA specified lighting. 

*The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-1 20 hour tests. 

*Test concentrations dropped as low as 22% of the nominal, and test solutions 

were not centrifuged prior to analysis 

Acceptable 

Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 

Green alga (Pseudokirchneriella subcapitata) 

1 x lo4 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Static test, 72 h 

0 (medium control and solvent control), 0.31, 1 .O, 3.1, 10, 31, 100 mg/L 

-- (medium control and solvent control), 0.141, 0.471, 1.46, 5.38, 18.5, 63.2 mg/L 

0.5 mUL Aceton 

Nutrient medium (according to OECD guideline) 

22.5 - 23.1 "C 

7.8 - 8.0 (0 h), 7.5 - 8.3 (96 h) 

Continuous "cool white" illumination with fluorescent light (8000 lux & 15 %) 

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. 

LOEC: ANOVA Procedure, t-test; EC,: 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. 

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: 

Biomass & growth rate: 

None 

See table B.9.2.1.3-1 
Table Error! Style not defined.-1 : Effects of spirotetramat (BY1 08330) on the green alga P. subcapitata 

BY1 08330 [mgA] 

(mean measured) 

0 (pooled control) ') 

0.141 

0.471 

1.46 

5.38 

Conclusion: EbC50 (0-72 h): 6.58 mg1L 

E&50 (0-72 h): 8.1 5 mgIL, 

NOEC (0-72 h): 1.46 mg/L (biomass and growth rate) 

based on mean measured concentrations 

1 ,, 

Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Inhibition after 72 h 

ECso 

(95% C.I.) 

Validity: 

Biomass (AUC) 

-- 

-51.1 

-26.0 

-15.1 

40.9* 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

I 

Yield = biomass at the end of exposure minus the biomass at the start of exposure 

*) 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 

6.58 mg/L 

(6.29 - 6.91 mg/L) 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Initial measured: 

Solvent: 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

yield" 

-- 

-54.7 

-22.2 

-8.6 

46.7* 

Toxicity of BY1 08330 to the freshwater diatom Navicula pelliculosa 

Kern, M. E.; Lam, C.V., 2005 

EBFNX008, Edition Number: M-252794-01-1, 15.06.2005 

Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FlFRA 123-2 

Yes 

OECD: None of relevance 

US EPA: The number of replicates (3) were fewer than are accepted by EPA 

protocol (4 for Navicula sp.). 

Growth rate 

-- 

-1 1.3 

-5.3 

-2.2 

18.1* 

5.6 mglL 

(5.46 - 5.83 mg/L) 

Acceptable 

8.1 5 mglL 

(7.56 - 8.81 mg/L) 

Spirotetramat (BY1 08330), purity: 97.1 %, batch: 0804510014 

Freshwater diatom (Navicular pelliculosa) 

1 x lo4 cells1mL; 3 replicates for each concentration, medium control and solvent 

control 

Static test, 96 h 

0 (medium control and solvent control), 0.20, 0.51, 1.3, 3.2, 8.0, 20 mg1L 

-- (medium control and solvent control), 0.22, 0.47, 1.25, 3.2, 8.23, 20.4 mg1L 

0.5 mUL Aceton 

AAP Nutrient medium (according to OECD guideline) 

23.5-24.1 "C 

7.7 - 7.9 (0 h), 8.0 - 9.5 (96 h) 

Continuous illumination (4026 - 4693 lux), orbital shaking at 100 rpm 

Cell densities were determined using a 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 

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; ECS0: Regression analysis, 

comparison of control groups: T-test 

All statistical analyses are based on data of pooled control. 

Findinas: 

Analytical data: Mean measured concentrations were in the range of 92 - 11 0 % and 0 (c LOQ) - 

59 % of nominal concentrations on day 0 and day 4, respectively. 

Effects: 

Morphological effects: None 

Biomass & growth rate: See table 6.9.2.1.3-2 

Table Error! Style not defined.3: Effects of spirotetramat (BY1 08330) on the freshwater diatom N. 

I LOEC 1 0.55 mg/L 1 4.73 mg/L I 

pelliculosa 

BY1 08330 [mgR] 

(initial measured) 

0 (woled control) a' 

Conclusion: EbCSO (0-96 h): 4.05 mglL 

EG50 (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) 

I 

BY1 08330 [mgIL] 

(geomean day Wday 4) 

0 I~ooled control) a) 

EC5o 

(95% C.I.) 

Cumulative biomass 

Inhibition after 96 h ph] 

-- 

a' Result of t-test is indicated no significant differences between control and solvent control, thus pooled control are used 

I 

for statistical analysis. 

* Significant difference (alpha = 0.05) from the control 

** Statistically significant from control but not considered biologically significant 

4.05 mg/L 

(3.73 - 4.38 mg/L) 

Growth rate 

Inhibition after 96 h [Oh] 

-- 

15.0 mg/L 

(0 - 903 mg/L) 
Reference: Toxicity of BY1 08330 to the blue-green alga Anabaena flos-aqua 

Author(s), year: Kern, M.E.; Lam, C.V., 2006 

ReporVDoc. number: EBFNX007, Edition Number: M-264055-01-1, 12.01.2006 

Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FlFRA 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 (24-25"). 

Validity: Acceptable, even if the inhibiton for growth rate is lower than recommended the 

enpoints are acceptable for EU risk assessment. 

Test substance: Spirotetramat (BY1 08330), purity: 97.99 %, batch: 08045/0014 

Material and methods: 

Test species: Blue-green alga (Anabaena flos-aqua) 

Number of organisms: 1 x lo4 cells/mL; 3 replicates for each concentration, medium control and solvent 

control 

Type of test, duration: Static test, 96 h 

Applied concentrations: 

Nominal: 

Initial measured: 

Solvent: 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Effects: 

Morphological effects: 

Biomass & growth rate: 

0 (medium control and solvent control), 0.51, 1.3, 3.2, 8.0, 20 mglL 

-- (medium control and solvent control), 0.45, 1.29, 3.32, 9.05, 21.7 mg/L 

0.5 mUL Aceton 

AAP Nutrient medium (according to OECD guideline) 

23.3 - 23.8 "C 

7.7 - 7.9 (0 h), 8.1 - 8.3 (96 h) 

Continuous illumination with cool white fluorescents (2200 lux + 10 %), orbital 

shaking at 100 rpm 

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. 

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. 

Mean measured concentrations were in the range of 87 - 11 3 % and 31 - 52 % of 

nominal concentrations on day 0 and day 4, respectively. 

None 

See table B.9.2.1.3-3 

Table Error! Style not defined.4: Effects of spirotetramat (BY1 08330) on the blue-green algae 

Anabaena flos-aquae 

BY1 08330 [mgR] I BY1 08330 [mg/L] I Cumulative biomass I Growth rate I 

(initial measured) I (geomean day Olday 4) 1 Inhibition after 96 h [Oh] 1 Inhibition after 96 h [%I 

0 (pooled control) ") 

0.45 

1.29 

0 (pooled control) a' 

0.3 

0.71 8 

-- 

-43 

-15 

-- 

3 

-13 
for statistical analysis. 

** Biological significant based on % inhibition compared to the pooled controls 
Conclusion: EbCSO (0-96 h): 15.2 mg/L 

ErCSO (0-96 h): > 15.1 mg/L, 

NOEC (0-96 h): 5.68 mg/L (biomass); 15.1 mglL (growth rate) 

based on geometric mean measured concentrations (day 0 and day 4) 

Metabolites 

Reference: 

Author(s), year: 

ReporVDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent: 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Effects: 

Pseudokirchneriella subcapitata - Growth inhibition test with BY1 08330-enol 

Dorgerloh, M. 2005 f 

EBFNM010, Edition Number: M-252531-01-2, 06.06.2005 

Draft proposal for updating OECD 201, OPPTS 850.5400 

Yes 

US EPA: 

*The temperature is less than the EPA-specified temperatures (24-25%) and the 

lighting intensity is higher than EPA specified lighting. 

.The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-1 20 hour tests. 

Acceptable 

BY1 08330-enol (metabolite of spirotetramat), purity: 99.1 Oh, batch: 692-1 01 -09- 

0005 

Green alga (Pseudokirchneriella subcapitata) 

1 x 1 o4 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Static test, 72 h 

0 (medium control and solvent control), 1 .O, 3.1, 10, 31, 100 mg/L 

-- (medium control and solvent control), 1.02, 2.9, 10.1, 29.4, 102 mg/L 

0.5 mUL Dimethylformamide (DMF) 

Nutrient medium (according to OECD guideline) 

22.3 - 23.5 "C 

7.1 - 7.9 (0 h), 7.6 - 8.5 (96 h) 

Continuous "cool white" illumination with fluorescent light (5560 - 7350 lux), orbital 

shaking at 100 rpm 

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-MAIMS instrument without a centrifugation. 

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. 

Mean measured concentrations were in the range of 94 - 104 % and 94 - 102 % 

of nominal concentrations on day 0 and day 3, respectively. 
Morphological effects: None 

Biomass & growth rate: See table 8.9.2.1.3-4 

Table Error! Style not defined.-4: Effects of BY1 08330-enol (metabolite of spirotetramat) on the green 

alga P. subcapitata 

Inhibition after 72 hours 

BY1 08330 [mg/L] 

(nominal) Biomass (AUC) Growth rate 

0 (pooled control) ') -- .- 

for statistical analysis. 

* Significant difference (alpha = 0.05) from the control 

Conclusion: EbC5~ (0-72 h): 7 100 mg/L 

ErCSO (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 

ReportlDoc. 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 (24-25") 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: 

Solubility: 

Material and methods: 

Test species: 

Number of organisms: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

4-Methoxycyclohexanone (metabolite of spirotetramat), purity: 97.9 %, batch: 

SAV5506-001 

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 280% for all 

samples taken. 

Green alga (Desmodesmus subspicatus) 

5 x lo3 cells/mL; 3 replicates for each concentration, 6 replicates for the medium 

and solvent control 

Static limit test, 72 h 

0 (medium control), 100 mg/L 
Measured (mean): 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Effects: 

Morphological effects: 

Biomass & growth rate: 

Conclusion: 

Comments: 

Formulation 

-- (medium control), 92.7 mg/L 

Nutrient medium (according to OECD guideline) 

22.3 - 23.5 "C 

8.1 (0 h), 8.6 (72 h) 

Continuous illumination with a spectral range of 400 - 700 nm and an intensity of 

4000 - 8000 IUX 

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. 

None, limit test 

Mean measured concentration was 92.1 % on day 0 and 93.9 % day 3 of nominal 

concentration 

None 

No significant effects were observed at the limit concentration of 100 mg/L 

EbCS0 (0-72 h): > 100 mg/L 

E&,O (0-72 h): > 100 mg/L, 

NOEC (0-72 h): 100 mg/L (biomass and growth rate) 

based on nominal concentrations 

US EPA has general concerns regarding uncertainties surrounding exposure 

concentrations in test media. 

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BY1 08330 OD 

150 

Author(s), year: Dorgerloh, M., 2006 c 

ReportIDoc. number: EBFNMOO6, Edition Number: M-264263-01-2, 16.01.2006 

Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400, JMAFF guideline (1 2 

Nousan No 81 47) 

GLP: Yes 

Deviations: EPA guideline: 

*The temperature is less than the EPA-specified temperatures (24-25%) and the 

lighting intensity is higher than EPA specified lighting. 

*The test duration was shorter than is accepted by EPA protocol; the EPA 

requires 96-1 20 hour tests. 

Validity: Acceptable 

Test substance: BY1 08330 OD 150 (formulation), spirotetramat content: 148.89 g/L (1 5.1 Oh), 

batch: 08030/0189(0152) 

Material and methods: 

Test species: Green alga (Pseudokirchneriella subcapitata) 

Number of organisms: 1 x lo4 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: 

Measured (mean): 

Control: 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

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 

Spirotetramat: -- (control), 0.61, 1 .I 4, 2.02, 4.05, 8.2 mg a.s./L 

Untreated test medium, no blank formulation control was used 

Nutrient medium (according to OECD guideline) 

22.3 - 23.3 "C 

8.0 - 8.9 (0 h) 

Continuous "cool white" illumination with fluorescent light (8000 lux + 15 %) 

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; EC,: Probit analysis (for growth rate an EC50 could 

not be determined because the inhibition at highest test concentration was < 50%) 

Findinas: 

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 Error! Style not defined.-5 

Table Error! Style not defined.6: Effects of BY1 08330 OD 150 (formulation) on the green alga P. 

I EC5o 6.56 mg a.s.R 

(95% C.I.) (5.19 - 9.37 mg/L) > 8.2 mg a.s./L 

"Significant difference (alpha = 0.05) from the control 

" 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 productlL 

EF50 (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 
6.9.2.1 -4 Effects on aquatic plants (OECD IIA 8.6, lllA 10.8.2) 

Active substance 

Reference: Lemna gibba G3 - Growth inhibition test with BY1 08330 (tech.) under staticrenewal test conditions 

Author(s), year: Dorgerloh, M., 2005 j 

ReportlDoc. 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: 

Material and methods: 

Test species: 

Number of organisms: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 

Duckweed (Lemna gibba) 

12 fronds per vessel; 3 replicates for each concentration, control and solvent 

control 

Static renewal test (renewal of test medium on day 0, 3 and 5), 7 d 

0 (medium and solvent control), 0.30, 0.95, 3.05, 9.77, 31.3, 100 mglL 

-- (medium and solvent control), 0.1 4, 0.47, 1.54, 6.06, 20.5, 71.4 mglL 

0.5 mUL Aceton 

20X-AAP growth medium (according to OECD guideline), the pH was adjusted to 

7.5 & 0.1 

23.5 - 24.8 "C 

7.2 - 7.6 (new test media), 8.0 - 8.7 (aged test media); control: 7.4 (0 d), 8.7 (7 d) 

Continuous illumination with white fluorescent light (mean intensity: 8.04 kLux) in 

a growth incubator. 

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-MSIMS, HPLC-UV) of test the substance (BY1 

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. 

LOEC: ANOVA (a = 0.05, one sided) and Dunnett's multiple t-tests 

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 
Table Error! Stvle not defined.-1 : Analvtical results of mirotetramat 

~ctu~ncwrtra~on af 

Day Nominal ronce?ntra8m 1 Time 1 E 1 &BY( 08330 1 C4%tectian 11 Q&ection 21 Mean 1 %of / weishted 

3 a61& 

3 new 

5 w-J 

5 new 

7 

0 Sotvent control -=0.024B ~0.~326 eO.0245 - i 

3 aged c 0.0245 .E A0245 0.f3245 - I 

3 new 

sag& 1 

5 new 

5 aged 

5 Ftevu 

T 

0 

2- 

2 nw8 

5 aged 

5 new 

Effects: 

Morphological effects: None visual effects were observed in concentration levels up to 0.47 mg/L. At 1.54 

mgll slight chlorosis was observed at day 7. At 6.06 mg1L 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 

I IGIU 

D/, inhibition in 7 d I based 0" frond I based on total I 
* Significant difference (alpha = 0.05) from the control 

Table Error! Style not defined.4: Toxicity of spirotetramat on duckweed Lemna gibba based on time 

1 number) I I I 1 I 1 

Yield 

% inhibition in 7 d 

97.6' 

S~irotetramat ImgL1 

(nominal) 

100"' 

Growth rateyo inhibition in 7 d 

weighted mean measured concentrations 

Spirotetramat [mgR] 

time weighted mean 

measured 

71.4 

On frond 

number 

1 13.5* 

based on total 

frond area 

116.1' 

Growth rate (based on total area 

of plants 

Yield (based on frond number) 

- -- - - 

a)The 100 mg/L concentration level was excluded from EC50 calculations because of exceeded water solubility 

b)~he 100 mg/L concentration level was included into the EC50 calculations. 

LOEC 

ImNl 

6.06 

EC50 (Q5% C.I.) 

Img~l"' 

10.4 (7.96- 13.8) 

Endpoint 

Growth rate (based on frond 

Yield (based on biomass -dry 

weight) 

Visual effects on ~lants 

Conclusion: EyCSO (7 d): 4.62 mg1L 

ErC50 (7 d): 6.21 mg1L 

NOEC (7d): 1.54 mg/L (for biomasslyield and growth rate) 

based on time weighted mean measured concentrations 

EC~O (95% C.1.) 

[mslllb' 

9.96 (7.85 - 12.49) 

7 d 

7 d 

Comments: Spirotetramat is an insecticide and therefore a study on higher aquatic plants is 

not required according to directive 911414lEEC. However, in general an exposition 

of Lemna gibba is possible and therefore the study is considered in the aquatic 

risk assessment. 

Time 

scale 

7 d 

7 d 

7d 

Metabolites 

NOEC 

[mglLl 

1.54 

1.54 

1.54 

Reference: Lemna gibbaG3 - Growth inhibition test with BY1 08330-enol under static 

test conditions 

Author(s), year: Dorgerloh, M., 2005 k 

ReportIDoc. 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 

1.54 

0.47 

Test substance: 

6.06 

6.06 

Material and methods: 

Test species: 

Number of organisms: 

6.06 

1.54 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

6.21 (5.33 - 7.18) 

4.62 (2.04 - 10.3) 

BY1 08330-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09- 

0005 

6.21 (5.55 - 6.91) 

4.49 (2.70 - 7.20) 

6.96 (5.76 - 8.38) 

-- 

Duckweed (Lemna gibba) 

12 fronds per vessel; 3 replicates for each concentration, control and solvent 

control 

Static test, 7 d 

7.04 (6.07 - 8.12) 

-- 

0 (medium control), 0.09, 0.30, 0.95, 3.05, 9.77, 31.3, 100 mg/L 

-- (medium control), n.a., ma., 1.02, 3.41, 1 1.7, 37.6, 1 19 mg/L 
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 O), 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-MSIMS, 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 (a = 0.05, one sided) and Dunnett's multiple tests 

Findinas: 

Analytical data: Overall mean measured concentrations ranged from 1 14 % 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 

2 3.5 mgll 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 

* Significant difference (a=0.05) from the control 

Conclusion: EyCSO (7 d): 5.4 mg/L 

ErCSO (7 d): 19.3 mg/L 

NOEC (7d): 0.95 mg/L (yield); 3.05 mg/L (growth rate) 

based on nominal concentrations 

Comments: BY1 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 

911414lEEC. However, in general an exposition of Lemna gibba is possible and 

therefore the study is considered in the aquatic risk assessment. 
B.9.2.1.5 Effects on marine or estuarine organisms (OECD IIA 8.1 1, lllA 10.2.2.4, lllA 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 SANC0/3268/2001 rev.4 final it is stated 

"...in some cases, data are available on estuarinelmarine 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: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

-- - - - - 

Acute toxicity of BY1 08330 technical to the sheepshead minnow 

(Cyprinodon variegatus) under flow-trough conditions 

Banman, C. S.; Lam, C. V., 2005 

EBFNXOO6, Edition Number: M-255363-01-1, 27.07.2005 

OECD 203, US EPA OPPTS 850.1075, FlFRA 72-2 

Yes 

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: 

Test substance: 

Material and methods: 

Test species: I 

Number of organisms: 

Weight, length: 

Loading: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature: 

pH: 

O2 content: 

Light regime: 

Test parameters: 

Acceptable. 

Spirotetramat (BY1 08330), purity: 97.99 %, batch: 080451001 4 

Sheepshead minnow (Cyprinodon variegatus) 

20 fish per concentration and control 

0.1 3 r 0.03 g (mean k SD), 1.72 & 0.1 4 cm (mean + SD) 

0.03 g fish/L test material passing through tank each day 

Flow-through test (renewals approx. 5.3 x per day), 96 hours 

0 (control and solvent control), 0.63, 1.25, 2.50, 5.0, 10 mg/L 

-- (control and solvent control), 0.52, 1.17,2.52,4.54,9.77 mg/L 

0.1 mUL Aceton 

Artificial sea salts mixed with reverse osmosis water or blended soft water to 

produce a salinity of 17 %O 

21 .O - 22.5 "C 

8.1 - 8.4 (0 h), 7.7 - 8.1 (96 h) 

5.3 - 6.9 mg 02/L (67 - 87 %) 

16 hours light / 8 hours darkness, 30 min lightldark transition period 

Mortality and sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. 

For chemical analysis (HPLC method) of BY1 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. 
Statistics: LCs0: Moving average method, NOEC: directly from raw data 

Findinas: 

Analytical data: Mean measured concentrations were in the range of 92 - 11 5 % of nominal 

concentrations. 

Behavioural effects None in controls and at 0.52 mglL concentration level. 

After 96 hours at 2 1 .I7 mglL effects like loss of equilibrium and lying on the 

bottom of the aquarium were observed. Thus the NOEC is 0.52 mglL. 

Mortality: See table B.9.2.1.5-1 

Table Error! Style not defined.-I: Cumulative mortality of sheepshead minnow exposed to 

I NOEC = 0.52 mg/L 

LCso = 1.96 mglL (95%C.I. 1.64 - 2.37) based on moving average method I I LC50 = 2.10 mg/L (95%C.I. 1.73 - 2.52) based on Probit analysis I 

Details of the Probit: Slope b: 5.696 

95 O/o C.I. of b: 3.495 - 7.897 

Variance of b: 1 .I 2 

Intercept a: -1.838 

Goodness of fit, Chi2: 1.282 

Degrees of freedom: 3 

p (Chi2): 0.733 

Conclusion: LCs0 (96 h): 1.96 mg/L 

NOEC: 0.52 mglL 

based on mean measured concentration 

B.9.2.1.5.2 Effects on saltwater invertebrates 

Active substance 

Reference: BY1 08330 - Acute toxicity to mysids (Americamysis bahia) underflowthrough conditions 

Author(s), year: Cafarelle, M. A., 2005 a 

ReportIDoc. number: EBFNXOIO, Edition Number: M-270200-01-1, 06.01.2005 

Guideline(s): EPA OPPTS 850.1 035, FIFRA 72-3 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable. 

Test substance: Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 

Material and methods: 
Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature 

pH 

0, content: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Analytical data: 

Behavioural effects 

Mortality: 

Saltwater mysid (Americamysis bahia) 

2 replicates each with 10 mysids per treatment and control 

5- to 6-days old 

Flow-through test, 96 h 

0 (control and solvent control) 0.63, 1.3, 2.5, 5.0, 10.0 mg/L 

-- (control and solvent control), 0.73, 1.2, 2.6, 4.6, 9.0 mg/L 

0.1 mUL Dimethylformamide (DMF) 

Filtered natural seawater diluted with well water, salinity: 21 - 20 %o 

24 - 25 "C 

8.1 (0 h), 7.8 - 8.1 (96 h) 

> 60 % saturation, 4.9 - 7.4 mg 02/L 

16 hours light 18 hours darkness 

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. 

ECBO: Binominal probability and probit analysis, NOEC: directly from the raw data 

Overall mean measured concentrations were in the range of 90 - 120 % of 

nominal concentrations. 

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. 

See table 6.9.2.1.5-2 

I LCso (96 h) = 4.27 mg/L (95%C.I. not calculated due to mathematical reasons) based on probit analysis I 

Table Error! Style not defined.-2: Cumulative mortality of saltwater mysids exposed to spirotetramat 

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 

LC50 (96 h): 5.5 mg/L 

NOLC: c 0.73 mg/L 

based on mean measured concentration 

Conclusion: 

Spirotetramat (BY1 08330 

Nominal 

[mgnl 

Control 

Solvent control 

0.63 

1.3 

2.5 

5.0 

Cumulative mortality (%) 

Mean measured 

[mgn-I 

0.73 

1.2 

2.6 

4.6 

24 hours 

0 

0 

5 

0 

10 

15 

48 hours 

0 

0 

5 

0 

20 

20 

72 hours 

0 

0 

5 

5 

20 

25 

96 hours 

0 

0 

5 

5 

30 

25 
Reference: BY1 08330 - Acute toxicity to eastern oysters (Crassostrea virginica) under 

flow-through conditions 

Author(s), year: Cafarella, M. A., 2005 b 

ReportIDoc. number: EBFNXO11, Edition Number: M-257677-01-1, 15.06.2005 

Guideline(s): EPA OPPTS 850.1025, FlFRA 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: 

Material and methods: 

Test species: 

Number of organisms: 

Valve height: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Spirotetramat (BY1 08330), purity: 97.1 %, batch: 08045/0014 

Eastern Oyster (Crassostrea virginica) 

20 oysters per treatment and control 

37 + 5 mm (mean + SD) 

Flow-through test, 96 h 

0 (control and solvent control), 0.19, 0.38, 0.75, 1.5, 3.0 mg/L 

-- (control and solvent control), 0.20, 0.33, 0.55, 0.89, 2.1 mg/L 

0.1 mUL Dimethylformamide (DMF) 

Filtered natural seawater, salinity: 31 - 32 %O 

20 - 22 "C 

7.6 - 8.0 

3.8 - 7.3 mg 02/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. 

Findinas: 

Analytical data: Overall mean measured concentrations were in the range of 59 - 100 % of 

nominal concentrations. 

Mortality: None 

Shell Deposition: See table 6.9.2.1.5-3 

Table Error! Style not defined.-3: Mean shell deposition of eastern oysters (C. virginica) exposed to 

spirotetramat 

Spirotetramat [mglL] 

(mean measured) 

Pooled control a) 

0.20 

0.33 

0.55 . 

Mean shell deposition 

(mm + SD) 

3.5 + 1.2 

3.8 + 1.2 

3.4 + 1.1 

2.4 + 1.0 

Inhibition of shell growth [Oh] 

-- 

+7.9 

3 

31 * 
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 5 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) 

8.9.2.1.5.3 Effects on saltwater algae 

Reference: Toxicity of BY1 08330 technical to the saltwater diatom Skeletonema 

costatum 

Author(s), year: Banman, C. S. & Lam, C. V., 2006 

ReportlDoc. number: EBFNX009-1, Edition Number: M-271037-02-1, 16.06.2006 

Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FlFRA 123-2 

GLP: Yes 

Deviations: OECD: None of relevance 

USEPA: 

-The photoperiod for studies involving Skeletonema costatum is 14 hour light110 

hour dark cycle; however, continuous illumination was used in this study. 

-Test samples were not centrifugated prior to chemical analyses. 

Validity: Acceptable 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Initial measured: 

Solvent: 

Test conditions: 

Water quality: 

Temperature 

pH 

Incubation: 

Test parameters: 

Spirotetramat (BY1 08330), purity: 97.99 %, batch: 0804510014 

Marine diatom (Skeletonema costatum) 

1 x lo4 cells/mL; 3 replicates for each concentration, medium control and solvent 

control 

Static test, 96 h 

0 (medium control and solvent control), 0.256, 0.640, 1.60, 4.0, 10 mg/L 

-- (medium control and solvent control), 0.240, 0.622, 1.63, 4.15, 10.6 mg/L 

0.5 mUL Aceton 

ASTM saltwater media (slightly modified: 40 mg/L silicates instead of 20 mg/L 

silicates, salinity 27 %, 

19.4 - 21 .o "C 

8.0 - 8.4 

Photoperiod: 16.8 (lightldark), intensity (3897 - 4682 lux ), orbital shaking at 

100 rpm 

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 

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. 

Findinas: 

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 Error! Style not defined.-4: Effects of spirotetramat (BY1 08330) on the marine diatom S. 

costatum. 

BY1 08330 [mgR] 

(initial measured) 

0 (pooled control) a) 

0.24 

0.622 

1.63 

4.1 5 

10.6 

Conclusion: EbCSO (0-96 h): 0.36 mg/L 

ErCSO (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 

I 

BY1 08330 [mgR] 

(geomean) 

0 (pooled control) a) 

0.124 

0.394 

1.03 

2.7 

6.95 

ECso 

(95% C.I.) 

NOEC 

LOEC 

Cumulative biomass 

Inhibition after 96 h [Oh] 

-- 

-22 

0.1 24 mg/L 

0.394 ma/L 

57* 

87* 

105* 

104* 

a) no statistically significant difference between dilution control and solvent control 

1 

* Significant difference (alpha = 0.05) from the control 

0.36 mg/L 

(0.27 - 0.46 mg/L) 

Growth rate 

Inhibition after 96 h [%I -- 

-10 

27* 

61 * 

11 5* 

ill* 

0.98 mg/L 

(0.92 - 1.05 mg/L) 
B.9.2.1.6 Summary of acute toxicity 

Table Error! Style not defined.-1: Summary of acute toxicity data for aquatic organisms; values which 

are relevant for risk assessment are in bold 

Chironomus riparius 
. . , , 

a' LC10, NOEC was not determined 

"Limit test 

Test organism Test 1 condition 

4-Methoxycyclohexylamino caboxylic 

Test conc.: test concentration based on mean measured (mm) or nominal horn) concentration 

Time 

acid 

48hr 

96 hr 

48 hr 

72 hr 

Chironomus riparius 

Midge 

Formulation - BY1 08830 

Oncorhynchus mykiss 

Rainbow trout 

Chironomus riparius 

Midge 

P. subcapitafa 

Green alga 

static b, 

(wateronly) 

OD 150 

static 

static 

(wateronly) 

static 

mortality 

mortality 

mortality 

biomass 

growth rate 

Test NOEC 

conc. 1 [mg/L] 

EGdLCso 

> 100 

9.48 

(1.41 as.) 

4.41 

(0.66 as.) 

43.4 

(6.56 a.s.1 

> 54.3 

(> 8.2 as.) 

nom 

nom 

rnrn 

100 

2.5 

(0.38 a.s.) 

2.3ga) 

(0.36 a.s.) 

13.4 

(2.02 as.) 

Reference 

Dorgerloh, 

2007b 

Nieden, 

2005b 

Dorgerloh, 

2006b 

Dorgerloh, 

2006c 

study 

acceptable 

Yes 

Yes 

Yes 

Yes 
8.9.2.2 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, lllA 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, lllA 10.2.5.2) 

Active substance 

Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature: 

pH: 

O2 content: 

Light regime: 

Feeding 

Test parameters: 

Statistics: 

Early-life stage toxicity of BY1 08330 tech. to fish (Pimephales promelas) 

Dorgerloh, M., 2005 b 

EBFN0305, Edition Number: M-260676-01-2, 16.1 1.2005 

EPA 72-4a, EPA OPPTS 850.1 400, OECD 21 0 

Yes 

None of relevance 

Acceptable 

Spirotetramat (BY1 08330), purity: 97.1 %, batch: 0804510014 

Fathead minnow (Pimephales promelas) 

4 replicates x 25 eggs per test concentration and control, 

after completion of hatch larvae were thinned to 15 individuals per egg cup 

Freshly fertilized eggs < 24 hours old 

Flow-through test, 33 days (28 days post hatch) 

0 (control and solvent control), 0.0625, 0.125, 0.250, 0.500, 1 .OO mgR 

-- (control and solvent control), 0.0607, 0.121,2.243, 0.486, 0.971 mg/L 

Dimethylformamide (DMF) 0.1 mUL 

Reconstituted water (according to ISO), hardness: 45 - 52 mg/L as CaC03 

24.5 (23 - 26) "C 

7.0 - 7.3 during the total test period 

98 - I00 % saturation 

16 hours light / 8 hours darkness, 30 min transient period 

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. 

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-MSIMS and HPLC-UV) of BY1 08330 in test 

solutions samples were taken at -1, 0, 7, 14, 21, 28 and 33 days 

If control and solvent control can be pooled: T-test 

Testing for normal distribution: Ws procedure 

Time to hatch, hatching success, fry survival: Williams test with previous acrisine 
transformation 

Growth data: Williams test without previous data transformation 

Findinas: 

Analytical data: Overall mean measured concentrations in test media were 93 - 11 9 % 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 

hd - post hatch day 

g' no statistically significant difference between dilution control and solvent control 

* significant difference when compared to pooled control (alpha = 0.05) 

Table Error! Style not defined.9: Length and Weight 

* significant difference when compared to pooled control (alpha = 0.05) 

Spirotetramat [mgk] 

(mean measured) 

pooled control a) 

0.0566 

0.1 15 

Conclusion: Fry survival: NOEC: 0.534 mg/L, LOEC: 1.1 6 mg/L 

Hatchability and growth: NOEC : 1.16 mg/L, LOEC > 1.1 6 mg/L 

based on mean measured concentrations 

Length [mm] 

Test termination (phd 28) 

Mean (SD) 

18.0 (3.5) 

18.4(3.4) 

18.3 (3.4) 

B.9.2.2.3 Fish life cycle test (OECD IIA 8.2.5, lllA 10.2.5.3) 

Dry weight [mg] 

Test termination (phd 28) 

Mean (SD) - 

20.4 (7.4) 

21.8 (1 1.4) 

21.4 (1 1.3) 

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. 
6.9.2.2.4 Chronic toxicity to aquatic invertebrates (OECD IIA 8.3.2, lllA 10.2.6) 

Active substance 

-- - 

Reference: 

Author(s), year: 

Report/Doc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Measured (mean): 

Solvent 

Test conditions: 

Water quality: 

Temperature 

pH 

O2 content: 

Light regime: 

Feeding 

Test parameters: 

Statistics: 

- - 

Influence of BY1 08330 (tech.) on development and reproductive output of 

the waterflea Daphnia magna in a static renewal laboratory test system 

Dorgerloh, M., 2005 e 

EBFN0245, Edition Number: M-251843-01-2, 13.05.2005 

OECD 21 1, EEC Directive C.20, US EPA 72-4, OPPTS 850.1 300 

Yes 

The concentraton of the solvent (acetone) is slightly higher than recommended in 

OPPTS 850.1300 (0.2 ml1L instated of 0.1 ml1L). 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. 

Acceptable 

Spirotetramat (BY1 08330), purity: 97.1 %, batch: 0804510014 

Waterflea (Daphnia magna) 

12 replicates each with one daphnid per treatment and control 

First instar < 24 hours old 

Semi static test, 21 d, renewals of test solutions: 0, 2, 5, 7, 9, 12, 14, 16, 19 days 

0 (control and solvent control), 0.05, 0.1 3, 0.32, 0.8, 2.0, 5.0, 12.5 mg/L 

-- (control and solvent control), 0.055, 0.129, 0.322, 0.796, 1.84, 4.41, 10.9 mglL 

,0.2 mUL Aceton 

M7 medium (according to "original draff' of an EEC Daphnia magna pilot ring 

test), hardness: 196 mg1L as CaCO, 

20 - 20.9 "C 

7.0 - 7.3 (new test solution) 7.4 - 8.1 (aged test solution) 

6.3 - 10.8 mg OdL (> 60 % saturation) 

16 hours light I8 hours darkness 

Daily with Desmodesmus subspicatus suspension (1 x lo8 cellsIL), one exception 

was noted: On day 2 the three fold amount was fed for the first weekend 

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 (HPLCIUV) 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. 

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 
Statistical analyses concidering nominal concentrations are based on data of 

pooled controls. 

Statistical analyses concidering mean measured concentrations are using only 

negative control. 

Findinas: 

Analytical data: The mean measured concentrations ranged from 87.2 - 110.7 % 

Effects: See table B.9.2.2.4-1 

Table Error! Style not defined.-1: Summary of effects of long-term exposure of spirotetramat on 

Daphnia magna 

* Statistically significantly different from pooled control (alpha = 0.05) 

a' Statisics were performed using only negative control 

Conclusion: Mortality adult: NOEC 2.0 mgIL, LOEC 5.0 mglL 

Reproduction (offspring per surviving parental): NOEC: 5.0 mglL, 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 

ReportIDoc. 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 

mUL instead of 0.1 mUL), however no significant differences between solvent 

control and the water only control were noted. 

Validity: Acceptable 

Test substance: Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 
Material and methods: 

Test species: Midge (Chironomus riparius) 

Number of organisms: 4 replicates each with 20 larvae per treatment and control 

Age: First instar (LI) larvae 

Type of test, duration: Static test (spiked water exposure), 28 d 

Applied concentrations: 

Nominal: 0 (control and solvent control), 0.05, 0.1 0, 0.20, 0.40, 0.80, 1.60, 3.20 mgR 

Initial measured: -- (control and solvent control), 0.058, n.a., n.a., 0.401, n.a., ma., 2.95 mg1L 

Solvent: 0.2 mUL 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 % CaC03 to adjust the 

pH to 7 m 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 CaCOdL 

O2 content: 6.9 - 8.5 mg 02/L (> 60 % saturation) 

Light regime: 16 hours light I8 hours darkness, intensity: 500 - 1000 lux 

Feeding: 3 x weekly with a commercial ornamental fish food extract (0.5 - 1 mgllarvaelday) 

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 (HPLCIUV) 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 mg1L 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. 

Findinas: 

Analytical data: See table 8.9.2.2.5-1 

Table Error! Style not defined.-1: Measured concentration of spirotetramat in the overlaying water and 

the pore water 

Spirotetramat 

[mg a.sJL] 

Control 

Solvent control 

0.05 

0.40 

3.20 

Control 

Solvent control 

0.05 

1 hour 

Analysed 1 % of nominal conc. [mg/~lal 

7 days 

Analysed I x of nominal conc. [mg/~la) 

28 days 

Ana'ysed I % of nominal conc. [rngl~]~) 

Overlaying water 

< LOQ 

< LOQ 

0.0579 

0.401 

2.95 

-- 

-- 

115.8 

100.3 

92.2 

Pore water b' 

n.a. 

n.a. 

< LOQ 

< LOQ 

0.0529 

< LOQ 

< LOQ 

< LOQ 

-- 

-- 

0 

0 

1.7 

-- 

-- 

0 

n.a. 

n.a. 

< LOQ 

< LOQ 

< LOQ 

n.a. 

n.a. 

< LOQ 

-- 

-- 

0 

0 

0 

-- 

-- 

0 

n.a. 

n.a. 

< LOQ 

-- 

-- 

0 
a) means of two samples 

b' calculated to the real volume of pore water and the applied amount of a.s. 

Spirotetramat 

[mg a.s JL] 

0.40 

3.20 

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 mglL 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 8.9.2.2.5-2. 

LOQ ~0.00636 

NOEC I 1 0.8 mg/L 

LOEC I 0.2 mg/L I 1.6mgR 

(95% C.I.) I (n.d)" P u.0 UIYIL 

1 hour 

ER = midge emerged per vessellnumber of introduced larvae 

Analysed 

conc. [mg/~]~) 

< LOQ 

0.155 

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. 

% of nominal 

0 

0.3 

7 days 

* significant different from pooled control (alpha = 0.05) 

** slope b (probit analysis): 4.429 

cone. Ana'ysed [mgllla) 

< LOQ 

0.0639 

28 days 

Comments: 

% of nominal 

0 

0.1 

,o,cm AnalySed imslL1aj 

< LOQ 

< LOQ 

Conclusions: Emergence of pooled sexes: 

NOEC: 0.1 mgIL, LOEC: 0.2 mgIL, ECS0: 0.46 mg/L 

Development rate of pooled sexes, males and females: 

NOEC: 0.8 mgIL, LOEC: 1.6 mg/L, EC50: > 0.8 mg1L 

Endpoints based on initial nominal concentrations. 

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. 

% of nominal 

0 

0 
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. 

B.9.2.2.6 Microcosmos or mesocosmos study (OECD lllA 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 Error! Style not defined.-1: Summary of chronic toxicity data for aquatic organisms; values 

which are relevant for risk assessment are in bold 

6.9.2.3 Bioaccumulation (OECD IIA 8.2.6, IIA 8.2.7) 

Test organism 

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. 

Test 

condition 

B.9.2.3.2 Bioconcentration potential of metabolites in fish (OECD IIA 8.2.6.2) 

Environmental relevant metabolites are BY1 08330-enol, BY1 08330-ketohydroxy (both metabolites are major 

metabolites in water and sediment of waterlsediment study), 4-methoxycyclohexanone and 4- 

methoxycyclohexylamino carboxyl acid (both are major metabolites in photolysis study). 

The logpow of metabolites BY1 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 .O, respectively. 

Time 

Spirotetramat (BY1 08330) 

8.9.2.4 Risk assessment for aquatic organisms (OECD lllA 10.2, lllA 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 PMRA and US EPA is not included in this monograph. 

Test conc.: test concentration based on mean measured (mm) or nominal (nom) concentration 

Pimephales promelas 

Fathead minnow 

Daphnia magna 

Waterflea 

Chironomus riparius 

Midge (larvae) 

hatchability 

fry survival 

growth 

mortality adults 

reproduction 

growth 

emergence 

development 

Endpoint 

through 

semi 

static 

Static 

(Spiked 

water) 

mrn 

nom 

nOm 

Test 

conc. 

33 d 

21 d 

28 

1.16 

0.534 

1.16 

2.0 

5.0 

5.0 

0.1 

0.8 

NOEC 

[mg/L] 

> 

1.16 

> 1.16 

12.5 

12.5 

0.2 

1.6 

LOEC 

rmg//L1 

Dorgerloh 2005bl 

Yes 

Dorgerloh 2005e/ 

Yes 

Dorgerloh 2005il 

Yes 

Reference1 

study acceptable 
8.9.3 Effects on other terrestrial vertebrates (OECD lllA 10.3) 

Toxicity to mammals 

Table 8.9.3-1 : Ecotoxicologically relevant mammalian toxicity endpoints for spirotetramat 1 

" equivalent to 306 g a.s. 

Acute oral LDS0 

Acute oral LD50formulation 

Reproductive NOAEL 

6.9.3.1 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 PMRA and US EPA is not included in this monograph. 

Species 

Rat, female 

Rat, female 

Rat 

mglkg bwlday 

> 2000 

> 2000* 

70.7182.5 (mlf) 

Reference 

Eigenberg (2004) 

Schungel(2005) 

Young (2006) 
B.9.4 Effects on bees (OECD IIA 8.7; OECD lllA 10.4.) 

B.9.4.1 Acute Toxicity (OECD IIA 8.7.1 ; OECD IIA 8.7.2; OECD 111 10.4.2 ) 

Active Substance 

Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Dosage: 

Exposure route: 

Feeding: 

Test conditions: 

Test parameter: 

Statistics: 

Effects of BY1 08330 (Acute Contact and Oral) on Honey Bees (Apis mellifera 

L.) in the Laboratory 

Schmitzer, S.; 2004 

20091 035; M-081227-01-2 

OECD 21 3 & 21 4 (1 998) 

Yes 

ventral application in the contact test instead of dorsal as recommended in 

guideline 

Acceptable 

Spirotetramat (BY1 08330) tech., Batch no.: 0804510014 (mixed batch), 

Tox no.: 06695-00, purity: 97.2 % wlw 

Apis mellifera L. (Hymenoptera: Apidae), 4 - 6 week old adult worker honey bees 

Acute contact and oral limit toxicity test 

5 replicates with 10 bees for controls, toxic reference and the test item treatment 

groups 

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 pg a.s.lbee, based on the quantity of food actually consumed; 

contact test: 100 pg a.s.1bee (nominal); toxic reference: 0.04 - 0.3 pg 

dimethoatebee (Dimethoate 400 g/L) 

Application in the contact test: a 5 pL 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. 

Commercial ready-to-use syrup (30 % saccarose, 31 % glucose, 39 % fructose) 

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 

(0 1 mm), the inner walls were lined with filter paper. 

Bees were anaesthetised with C02 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 

Mortality and behavioural abnormalities were assessed 4, 24 and 48 h after 

treatment application. 

The contact and oral LDS0 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 (1 925). The software used to perform the statistical analysis was 

ToxRat Professional, Version 2.07. 

Findinas: 
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 pg 

a.s./bee and 0.17 pg a.s./bee, respectively. 

Table B.9.4.1-1: Mortality and behavioural abnormalities of the bees in the contact toxicity test .. - .. . . - .. 

Table B.9.4.1-2: Mortality and behavioural abnormalities of the bees in the oral toxicity test (results 

(resulrs are average Trom s repllcares per aosaguconrrol) 

Dosage after 4 hours after 24 hours after 48 hours 

[pg a.sJbee] 

Test item 

100 

Water 

Solvent 

Toxic standard 

0.30 

0.20 

0.15 

0.10 

Conclusion: 

Comments 

are average from 5 replicates per dosageJcontrol) 

48 h oral LD50 > 107.3 pg a.s./bee 

48 h contact LD50 > 100 pg a.s./bee 

As deviations to EPA OPPTS guidelines the test substance (oral) was 

offered in syrup instead of a 50 % sugar solution and ~dhasit~is notan 

EPA-recommended solvent. 

mortality 

mean 

0.0 

0.0 

0.0 

20.0 

28.0 

18.0 

0.0 

Dosage 

[pg a.sJbee] 

Test item 

107.3 

Water 

Solvent 

Toxic standard 

0.24 

0.12 

0.07 

0.04 

behav. 

abnorm. 

mean [%I 

0.0 

0.0 

0.0 

34.0 

12.0 

6.0 

0.0 

after 4 hours after 24 hours 

mortality 

mean [%I 

0.0 

0.0 

0.0 

94.0 

70.0 

46.0 

0.0 

mortality 

mean [%I 

0.0 

0.0 

0.0 

28.0 

0.0 

0.0 

0.0 

mortality 

mean [%I 

2.0 

0.0 

0.0 

94.0 

22.0 

14.0 

2.0 

after 48 hours 

behav. 

abnorm. 

mean [%I 

0.0 

0.0 

0.0 

24.0 

0.0 

0.0 

0.0 

behav. 

abnorm. 

mean [%] 

0.0 

2.0 

0.0 

0.0 

0.0 

0.0 

0.0 

mortality 

mean [%I 

4.0 

2.0 

0.0 

94.0 

26.0 

22.0 

2.0 

behav. 

abnorm. 

mean I%] 

0.0 

0.0 

0.0 

4.0 

2.0 

8.0 

0.0 

behav. 

abnorm. 

mean [%I 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

mortality 

mean [%] 

2.0 

2.0 

2.0 

96.0 

82.0 

62.0 

2.0 

behav. 

abnorm. 

mean [%I 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

I 0.0 
Formulation: Movento 150 OD 

Reference: 

Author(s), year: 

ReporVDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Dosage: 

- -- 

Acute toxicity of BY1 08330 150 OD to the honeybee Apis mellifera L. under 

laboratory conditions. 

Barth, M.; 2005a+b 

05 10 48 032; M-259122-01-2 

OECD 213 & 214 (1998) 

Yes (certified laboratory) 

None 

Acceptable 

Spirotetramat (BY1 08330) OD 150, Batch no.: 08030/0189(0152), TOX no.: 

07034-00; Analytical content of as.: 148.89 g/L 

Apis mellifera carnica P. (Hymenoptera: Apidae), 4 - 6 week old adult worker 

honey bees 

Acute contact and oral toxicity test 

3 replicates with 10 bees for controls, toxic reference and the test item treatment 

groups 

Control: oral test: 50 % sucrose solution, contact test: water and 0.1 % v/v Tween 

solution; test item: oral test: applied volume - 20 pL sucrose solution/bee; contact 

test: applied volume - 4 pUbee (0.1 % v/v Tween solution); toxic reference: 

0.0625 - 0.5 pg dimethoatelbee (Perfection EC 400) 

Application rates for contact and oral toxicity test (based on analysed content of 

as.): 

*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 pL 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 

Contact toxicity 

Test item BY1 08330 

[pg productlbee] [pg a.s.lbee] 

1324.5 200.0 

662.2 100.0 

331.1 50.0 

165.6 25.0 

82.8 12.5 

Oral toxicity* 

Test item BY1 08330 

[pg product/bee] [pg a.s./bee] 

131 7.0 198.9 

661.3 99.9 

330.7 49.9 

165.6 25.0 

82.7 12.5 
Statistics: 

Findinas: 

excretion. 

Mortality was corrected according to Abbott (1 925). For statistical calculation of 

the mortality results the Fisher's Exact Binomial test was used. The accepted 

significance level was p I 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* test. The calculation of statistical significance and the LD, was performed with 

the computer program ToxRat Professional 2.09. 

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 I 

10 % after 48 h and the contact and oral LD50 of the toxic reference were 0.263 pg 

a.s./bee and 0.1 94 pg 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 131 7 pg productlbee (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 pg productlbee. After consuming doses of 

661.3 and 131 7 pg productlbee 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 pg product per bee. Bees exposed to doses equal or greater 

than 662.2 pg productlbee 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 Analvses 

LDS0 = 607.1 8 uglbee 

95% Confidence Limits of LD5~ 506.17, 737.88 

Intercept = -1 0.21 

Slope = 3.67; Standard error of slope=0.55 

95 % Confidence Limits of Slope = 2.59, 4.74 

~oodn'ess-of- it 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 Analvses 

LD50 = 1073 uglbee 

95 % Confidence Limits of LD50 = 903.59, 1351 

Intercept=-1 3.83 

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 
Table B.9.4.1-3: Oral and contact toxicity LDS0 values of bees treated with BY1 08330 150 OD after 24 

and 48 h 

Test item 

BY1 08330 

150 OD 

Reference item 

Dimethoate 

EC 400 

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 [pg test 

itemlbee] 

82.78 

165.56 

331.12 

662.24 

1324.48 

Control (Tween) 

Toxic standard [pg a.s. 

hl 

0.0625 

0.125 

0.250 

0.500 

Control (Tween) 

cl: confidence limits 

treatment 

time 

24 h 

95%-cl lower 

Upper 

Slope b 

48 h 

95%-cl lower 

Upper 

Slope b 

24 h 

95%-cl lower 

Upper 

Slope b 

48 h 

95%-cl lower 

Upper 

Slope b 

Mortality [%I after 4 h 

(= corrected mortality) 

0.0 

0.0 

3.3 

10.0 

0.0 

0.0 

0.0 

0.0 

33.3 

73.3 

0.0 

contact toxicity test 

Mortality [Oh] after 24 h 

(= corrected mortality) 

0.0 

0.0 

3.3 

10.0 

63.3 

0.0 

0.0 

0.0 

63.3 

83.3 

0.0 

pg productlbee 

1151.0 

933.8 

1418.8 

4.1 

1072.9 

894.3 

1287.3 

4.6 

oral toxicity test 

Mortality [%I after 48 h 

(= corrected mortality) 

0.0 

0.0 

3.3 

10.0 

70.0 

0.0 

0.0 

3.3 

63.3 

90.0 

0.0 

pg a.sJbee 

0.263 

0.225 

0.308 

4.7 

0.243 

0.209 

0.283 

5.0 

pg productlbee 

607.1 

506.7 

727.4 

Slope b 

607.1 

506.7 

727.4 

Slope b 

pg a.sJbee 

3.7 

3.7 

0.194 

0.171 

0.220 

7.2 

0.189 

0.166 

0.21 4 

6.7 
Conclusion: 48 h oral LD5, = 607.1 productlbee equivalent to 91.7 pg a.s./bee 

48 h contact LD50 = 1072.9 pg productlbee equivalent to 162.0 pg a.s./bee 

Comments: As deviation to EPA guidelines, ~ween@is not an EPA-recommended 

solvent. 

Table B.9.4.1-5: Mortality of bees in the oral toxicity test (results are average from 3 replicates per 

dosage/control) 

6.9.4.2 Residue test (OECD IIA 8.7.3) 

Not submitted. 

Bee brood feeding test (OECD IIA 8.7.4) 

Studies with similar design are reported under B. 1.4.5. 

dosage 

Test item [pg consumed 

test item/bee] 

82.66 

165.56 

330.65 

661.30 

1316.99 

Control (sucrose solution) 

Toxic standard [pg 

consumed as. /bee] 

0.0625 

0.125 

0.249 

0.495 

Control (sucrose solution) 

6.9.4.4 Cage test (OECD lllA 10.4.4) 

Not required. 

Mortality [%] after 4 h 

(= corrected mortality) 

0.0 

0.0 

0.0 

50.0 

90.0 

0.0 

0.0 

0.0 

53.3 

96.7 

0.0 

Mortality [%I after 24 h 

(= corrected mortality) 

0.0 

0.0 

23.3 

50.0 

90.0 

0.0 

0.0 

3.3 

90.0 

96.7 

0.0 

Field tests (OECD lllA 10.4.5) 

Mortality PA] after 48 h 

(= corrected mortality) 

0.0 

0.0 

23.3 

50.0 

90.0 

0.0 

0.0 

6.7 

90.0 

96.7 

0.0 

Formulation: Spirotetramat OD 100 

Reference: Assessment of the Side-Effects of BY1 08330 OD 100 on the Honey Bee (Apis 

mellifera L.) in a Feeding Test 

Author(s), year: Schur, A.; 2004a 

ReportlDoc. number: 20031 168/01 -BFEU; M-000345-01-2 

Guideline(s): OEPPIEPPO Bulletin No. 22 (Oomen et al, 1992) 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 
Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Dosage: 

Experimental 

procedure: 

Feeding: 

Test conditions: 

Test parameters, 

observations: 

Statistics: 

Findinas: 

Spirotetramat OD 100 (a.s. BY1 08330), Batch no.: 08030/0038(0035), Tox no.: 

06293-00, content: 101.54 g1L 

Apis mellifera L. (Hymenoptera: Apidae) 

Feeding test, field study 

Method aimed at products with insect growth-regulating properties 

3 replicates (= bee colonies, approx. 12000 bees per colony) for control, toxic 

reference and the test item treatment group 

Control: Waterlsucrose solution, test item: dissolved in water and mixed in pure 

sucrose solution at a concentration of 0.01 44 % a.s. (analysed; 1371 mg product 

per colony); toxic reference: 300 mg lnsegar 25 WG per colony (a.s.: 25 % 

fenoxycarb) (dissolved in water and mixed in sucrose solution). 

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. 

A quantity of 1 kg sugar solution was offered per colony. 

Meteorological data (temperature, relative humidity, rainfall) were recorded. 

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 (Munster 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. 

None in original study report. In 2007 statistics was performed using t-test and 

Dunnett's t-test in an one-sided approach. 

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. 
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 lnsegar 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). 

Honev bee fliaht intensitv: 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 

Table Error! Style not defined.-I: Mortality and flight activity of honey bees in a feeding study with 

Spirotetramat OD 100 

Treatment group 

Feeding rate per colony 

(in pure sugar solution) 

Obf mortality on the day of start of feeding before feeding 

QM (average) 0 post-feeding mortality + 0 pre-feeding mortality 

Pre average value for the pre-feeding period DAF 3 to Obf 

post (DAF 1 to 21) average value for the post-feeding period DAFl to 21 

P - - dead pupae 

SD = Standard deviation 

rep. = replicate hive 

* mean number of bees leavinglentering the hive per minute 

Average Mortality 

[dead bees/ 

colony/dayl 

Test substance 

(BY1 08330 OD 100) 

0.0144% as. 

pre 

(DAF3to 

Obf): 

average 

of 3 rep. 

post + SD 

(DAF 1 to 

21) 

average 

of 3 rep. 

QQaverage): 

rep. 1 

6.824.1 

Control 

X dead adult bees and dead 

pupae (post-feeding, DAF 1 to 

21) 

rep. 2 

6.3~4.6 

rep. 1 

7.5k2.5 

Toxic standard 

300 mg product 

Average 

Flight activity* 

rep. 3 

7.325.2 

rep. 1 

5.0~3.1 

6.8 

Obf 

- DAF 1 

DAF 3 

rep. 2 

4.3&4.0 

rep. 1 

8.6k5.7 

DAF days after start 

rep. 3 

9.5k 

10.3 

rep. 2 

8.52 

14.4 

7.1 

rep. 3 

8.0*7.0 

10.4k 11.6 

1.5 

597 + 55P 

46.67 

57.00 

17.67 

of feeding 

rep. 2 

15*16.8 

rep. 1 

5.7k4.2 

7.2 

rep. 3 

7.4d.3 

rep. 1 

54.1&3 

4.9 + 4.0 

0.7 

282 + 29P 

28.67 

35.00 

23.33 

rep. 2 

6.3d.5 

rep. 3 

2.8k1.9 

91.2 + 106.5 

12.7 

321 + 5425P 

38.00 

57.00 

28.00 

rep. 2 

115.2~ 

129.9 

rep. 3 

104.a 

115.3 
Conclusion: 

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. 

Honev 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. 

Honev 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. 

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 BY1 08330 SC 240 on the Honey Bee (Apis 

mellifera L.) in a Feeding Test. 

Author(s), year: Schur, A.; 2004b 

RepotVDoc. number: 20031 166101 -BFEU; M-121877-01-2 

Guideline(s): OEPPIEPPO Bulletin No. 22 (Oomen et al, 1992) 

GLP: Yes (certified laboratory) 

Deviations: None 

Validity: Acceptable 

Test substance: Spirotetramat SC 240 (a.s. BY1 08330), Batch no.: 0721 81001 3, Tox no.: 06294- 

00, content: 243.85 glL (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 
Dosage: 

Statistics: 

Findinas: 

Control: Waterlsucrose 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 

lnsegar 25 WG per colony (a.s.: 25 % fenoxycarb) (dissolved in water and mixed 

in sucrose solution). 

Experimental The study was located in Southern Germany (location: Niefern). Bees from the 

procedure: 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, Mortality of adult bees, pupae and larvae, behaviour of the bees around the hives, 

observations: 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 (Munster 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. 

None. 

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. 
Table Error! Style not defined.9: Mortality and flight activity of honey bees in the feeding study with 

S~irotetramat SC 240 

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 lnsegar 25WG massively disturbed the brood 

development. 

Honev bee fliaht 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 

Treatment group 

Feeding rate per colony 

(in pure sugar solution) 

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. 

Honev 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 

DAF days after start of feeding 

Obf mortality on the day of start of feeding before feeding 

QM (average) 0 post-feeding mortality + 0 pre-feeding mortality 

Pre average value for the pre-feeding period DAF -3 to Obf 

post (DAF 1 to 21) average value for the post-feeding period DAFI to 2 

P - - dead pupae 

SD = Standard deviation 

* mean number of bees leavinglentering the hive per minute 

Test substance 

(BY1 08330 OD 100) 

0.0144 % a.s. 

+ 0.200 % adjuvant 

6.9 

15.8 + 24.1 

2.3 

988 + 1OP 

36.33 

45.00 

19.00 

Average Mortality 

[dead bees1 

colonylday] 

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 

pre 

(DAF -3 to Obf): 

post + SD 

(DAF 1 to 21) 

QM(average): 

Control 

7.1 

4.9 + 4.0 

0.7 

282 + 29P 

28.67 

35.00 

23.33 

Toxic standard 

300 mg product 

7.2 

91.2 i 106.5 

12.7 

321 + 5425P 

38.00 

57.00 

28.00 

C dead adult bees and dead pupae (postfeeding, DAF 1 to 21) 

Average 

Flight activity* 

Obf 

DAF 1 

DAF 3 
Conclusion: 

following assessment on BFD 11. In hive 3 of the BY1 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 BY1 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. 

Honev 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. 

It was concluded that the feeding of BY1 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 

ReportIDoc. number: 20061 133lS1 -BFEU; M-277194-02-1 

Guideline(s): OEPPIEPPO No. 170 (3) (2001) 

GLP: Yes (certified laboratory) 

Deviations: No 

Validity: Acceptable 

Test substance: Spirotetramat OD 150 (a.s. BY1 08330), Batch no.: 08030101 89(0152), content of 

a.s.: 15.1 % (wlw) (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 1 

colony. 

Treatment: Three test fields: 

- test item treated field TI (4 x 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 x 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 

waterlha. Calibrated commercial spraying equipment was used. 

Experimental The field test was located in Spain (region: Valencia). The crop Phacelia 
procedure: 

Statistics: 

Findinas: 

tanacetifolia was used in flowering stage. The field sizes were: test item field TI : 

2025 m2, test item field T2: 201 6 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 3'C' 

application in the test item field TI and before the 1' 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 (TI 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, Exposure of the bees was assessed by visual inspection of the P. tanacetifolia 

observations: 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 analysisldetermination 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. 

None. 

Data on mortality and flight activity are presented in the table below. 
Table Error! Style not defined.3: Mortality and flight activity of honey bees in the feeding'study with 

Movento 150 OD 

1 Treatment group 1 TI 1 TZ I Control I I 1" application date (01 MayO6, before flowering): application I I I I 

72.0 I I I 

rate g a.s./ha I I 1 

2"* application date (08May06, before flowering): application I 72.0 I 

application rate g a.s./ha I I I 

Mean mortalitv I Pre-appl. [DAA -2 to Obal: 43.4 135.4 87.7 I 

rate g a.s./ha 

3'(1 application date (17May06, during floweringlbee flight): 

application rate g a.s./ha 

4'h application date (24May06, during floweringlbee flight): 

Pre-appl. [DAA 7bal: I 22.0 I 17.8 I 2.8 

Post-aDDI. rDAA 7aal: 3.8 6.8 2.3 I 

72.0 

72.0 

[dead 

bees/colony/dayl 

96.0 

96.0 

I QM: I 0.1 I cO.1 I 0.1 

Mean number dead ouoaeharvae ~er colonvldav 0.3 0.1 <0.1 I I 

65.0 

29.0 

.. - 

,, . I I I 

Post-appl. [DAA Oaa to 281: 1 5.7 I 6.5 I 5.6 I 

Pre-appl. [DAA Oba]: 

Post-appl. [DAA Oaa]: 

I 1 Post-appl. [DAA Oaa to 101: I 11.9 I 16.3 I 12.2 I 

- - 

(post-application, DAA daa to 28) . 

bees/minute/m2] 

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 Oaa to 281 1 pre-application [DAA -2 to Oba] 

42.0 

15.0 . 

11.3 

15.0 

Mean flight intensity 

[foraging 

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 Oaa to DAA 10) was 11.9 foraging bees/minute/m2 in the test 

item group TI, 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 TI, 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. 

94.8 

23.0 

Pre-appl. [DAA -2 to Oba]: 

Pre-~DDI. rDAA Obal: .. - 

Post-appl. [DAA Oaa]: 

Pre-appl. [DAA 7bal: 

Post-appl. [DAA 7aal: 

Honev bee mortalitv 

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 (Tl: 15.0 dead beeslcolony, 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 TI and T2 compared to the mortality in the control group was observed at any 

11.5 

13.0 

13.7 

12.4 

11.9 

12.0 

14.6 

14.4 

15.0 

23.0 

14.4 

1 .O 

11.1 & 
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 TI of 5.7 dead honey bees/colony, 6.5 dead honey bees/colony in 

the test group T2 and 5.6 dead honey beeslcolony 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 TI, <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 TI, 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. 

Honev bee fliaht intensitv: 

The daily mean flight intensity (foraging bees/minute/m2) before the first application during exposure was 

15.0 in the test item treatment group TI, 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 TI, 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 TI and T2. The daily mean post-application (DAA Oaa to DAA 10) flight 

intensity was 1 1.9 foraging bees/minute/m2 in the test item group TI, 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 honev bee brood develo~ment 

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 TI 

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 TI showed a lack of eggs orland 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. 

Honev bee behaviour in front of the colonies and within the croD area 

No differences regarding the behaviour of the bees were observed between the test item treatment groups 

TI and T2 and the control group. 

Conclusion: The test item treatments (TI 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 TI 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 TI as well as 
in the test item group T2 and the control colonies. 

8.9.4.6 Tunnel tests (OECD lllA 10.4.7) 

Formulation: Spirotetramat OD 100 

Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Experimental 

procedure: 

Assessment of side effects of BY1 08330 OD 100 on the honey bee (Apis 

mellifera L.) in the semi-field. 

Schur, A.; 2005 

20041 144101 -BZEU; M-244490-01-2 

EPPO No. 170 (3) and Bulletin of lnsectology 56 (I), 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. 

Yes (certified laboratory) 

No 

Acceptable 

Spirotetramat OD 100 (a.s. BY1 08330), Batch no.: 08030101 lO(0073) 

Tox no.: 06593-00, content: 102.02 glL (analysed) 

Apis mellifera L. (Hymenoptera: Apidae) 

Semi-field test (tunnel) with special attention to the development of the bee brood 

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. 

Control: water, test item: treatment group TI : Test item applied 3 x on the crop 

before flowering at 72 g a.s. (692.33 g product)lha and 2 x after set-up of the 

colonies at 96 g a.s. (923.1 1 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: 

lnsegar WG 25 (a.s.: fenoxycarb, 25 %) 150 g a.s. per ha. All treatments were 

applied in 400 L waterha using a plot sprayer. 

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 TI (main application day). There 
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, Mortality, flight intensity, condition of the colonies and the development of the bee 

observations: 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). 

None in original study report. In 2007 statistics was performed using t-test and 

Dunnett's t-test in an one-sided approach.. 

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. 

Statistics: 

Findinas: 
Table Error! Style not defined.-1: Mortality and flight activity of honey bees in the feeding study with 

Spirotetramat OD 100 

I Exposure 1 TI: five spray treatments of BY1 08330 OD 100 three before I 

I - I and two during foraging activity in flowering oilseed I 

Treatment arou~ 

spring rape 

T2: one spray treatment of BY1 08330 OD 100 during 

foraging activity in flowering oil-seed spring rape 

Test substance I Control I Toxic standard 

I ". I (BY1 08330 OD 100) I (water) I (Insegar 25 WG) TI I T2 C I R 

Single application rate 

[in 400 L waterlha] 

post (BFD +2aa to 

+23) 

Regarding the total daily adult bee mortality observed during the exposure of the bees to the test item 

treated plants (TI 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 (TI: 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 lnsegar 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 

Tl, 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. 

Honev bee fliaht intensitv: 

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 

Mean Mortality 

[dead bees1 

replicatelday] 

3 x 72 g a.s./ha 

2 x 96 g a.s.1ha 

13.8 

5.0 

Pre 

post (BFD +2aa) 

5.5 

X dead pupae (post-application, 

BFD +2aa to +23) 

7.9 

2.7 

96 g a.s./ha 

12.9 

2.7 

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 

12 

10.3 

19.0 

14.5 

Daily mean flight 

intensity [foraging 

bees/m2/replicate] 

150 g a.s. 

11.2 

1.7 

4.5 

pre 

post (BFD +2aa) 

post (BFD +2aa to 

+9) 

29 

8.3 

12.2 

14.8 

4.1 8.1 

8 

8.4 

15.0 

14.1 

300 

10.7 

15.3 

13.8 
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. 

Honev 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 TI, 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 TI 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 .I .-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 lnsegar 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 Error! Style not defined.-2: Summary of the brood indices calculated based on individual brood 

assessments of single cells in the test treatment groups TI, T2, the control and toxic standard 

treatment 

(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) 

Treatment 

TI 

T2 

Control 

Toxic 

standard 

Honev 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 TI 

and T2, the toxic standard treatment and the control. 

Replicate 

IT1 

2T1 

3T1 

Mean 

1 T2 

2T2 

3T2 

Mean 

1 C 

2C 

3C 

Mean 

1 R 

2R 

3R 

Mean 

0 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1 .OO 

1.00 

Mean brood 

+4 

1.85 

2.03 

1.37 

1.75 

1.67 

1.85 

1.73 

1.75 

1.81 

1.72 

1.21 

1.58 

1.96 

1.76 

0.29 

1.34 

indices at x d 

+9 

3.57 

3.87 

2.56 

3.33 

3.16 

3.17 

3.24 

3.19 

3.60 

3.29 

2.20 

3.03 

3.36 

2.86 

0.41 

2.21 

after BFD 

+I 6 

3.70 

4.00 

3.21 

3.64 

3.78 

3.41 

3.52 

3.57 

3.98 

3.90 

3.79 

3.89 

3.71 

3.37 

2.13 

3.07 

+23 

2.23 

2.07 

3.42 

2.57 

2.40 

1.96 

2.01 

2.12 

2.96 

3.26 

1.80 

2.67 

1.45 

1.96 

3.81 

2.41 
Conclusion: It is concluded that the application of BY1 08330 OD 100 applied five times 

(treatment group TI) 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 

TI, 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 m2and 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 notifieis 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. 
Trials in Germanv or S~ain with different formulations of Spirotetramat and ~artlv with addition of 

RME 500 EW (rape oil methvl ester): 

Trial 1: Spirotetramat 240 SC as spray application, 300 Uha, additive RME 500 EW (rape oil methyl ester), 

2 replicates, Germany 

Results: 

Table Error! Style not defined.-3: Evaluation of Larval Cell Comb Area Proportion for Trial 1 

DAA: days after application 

* assessment on 0 DAA was done shortly before application 

Conclusion: 

The application of three different rates of Spirotetramat SC 240 in combination with the additive RME 

500 EW (0.2% a.i.1~) 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 Uha, additive RME 500 EW (rape oil methyl ester), 

2 replicates, Germany 

Results: 
I 1 ODAA* 1 3DAA 1 7DAA 1 15DAA 1 21 DAA I 

Table Error! Style not defined.-4: Evaluation of Larval Cell Comb Area Proportion for Trial 2 

I untreated (control) 1 6.7 / 7.5 1 3.3 1 2.5 1 1.7 1 

Treatment 

- 

lnsegar 25 WP, 100 g a ha 

(toxc reference) 5.8 'I 0.3 0.0 

% ." I 

Proportion of comb area covered by larval cells r SD [%I 

(at X days after application) 

* 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 Uha, additive RME 500 EW (rape oil 

methyl ester), 1 replicate, Germany 
I ODAA* 1 4DAA 1 7DAA I 14DAA I 22DAA I 28DAA 

Results: 

Table Error! Style not defined.-5: Evaluation of Larval Cell Comb Area Proportion for Trial 3a 

RME 500 EW 0.4% a.i./v 

Treatment 

I RME 500 EW. 0.4% a.i./v I 9.2 1 11.7 1 4.2 1 4.2 1 10.0 1 4.2 1 

Proportion of comb area covered by larval cells [%I 

(at X days after application) 

Table Error! Style not defined.4 (continued): Evaluation of Larval Cell Comb Area Proportion for Trial 

3b 

* 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.1~ 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.1~ 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.1~ 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.1~ 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. 

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 4: Spirotetramat 240 SC or OD 100 as spray application, 300 Uha, additive RME 500 EW (rape oil 

methyl ester), 1 replicate, Spain 

Results: 

Table Error! Style not defined.-6: Evaluation of Larval Cell Comb Area Proportion for Trial 4 

- Trial 5: Spirotetramat OD 150 as spray application, 300 - 400 Uha, no additive, 1 replicate, Germany 

Results: 

I Treatment 

Treatment I Proportion of comb area covered by larval cells ['%I 

(at X days after application) I 

Proportion of comb area covered by larval cells [Oh] 

(at X days after application) 

untreated (control) 

-1 DAA 

8.3 

3 DAA 

19.2 

7DAA 

10.8 

14DAA 

10.0 

20DAA 

15.8 

28 DAA 

6.7 
*. 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 Error! Style not defined.-8: Pupal Mortality Evaluation for Trial 5b 

lnsegar 25 WP, 150 g 

a.i./ha (toxic reference) 

Treatment 7 

Untreated (control). 

1 Treatment I Number of dead pupae in bee trap (at X days after application) I 

Number of dead pupae in bee trap (at X days after application) 

*. first observed dead pupae in bee trap 

DAA: days after application 

4 DAA* 

0 

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. 

7 DAA 

0 

10 DAA 

0 

14 DAA 

0 

15 DAA 

0 
- Trial 6: Spirotetramat OD 150 as spray application, 300 Uha, no additive, 1 replicate, Germany 

Results: 

Table Error! Style not defined.8: Evaluation of Larval Cell Comb Area Proportion for Trial 6 

Proportion of comb area covered by larval cells [%I 

Treatment (at X days after beginning of exposure) 

0 4 DAE 7DAE 15DAE 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 1 10.8 1 15.0 1 10.0 1 5.0 1 19.2 1 23.3 1 

150 g a.i.lha 

15 days prior to flowering 10.0 11.7 10.0 10.0 18.3 17.5 

150 g a.i./ha 

10 davs prior to flowerina 1 16.7 1 14.2 1 7.5 1 8.3 1 15.8 1 19.2 1 

150 g a.i./ha 

5 days prior to flowering 

150 g a.i./ha 

on flowering crop at bee flight 

150 g a.i./ha on flowering crop 

after daily bee flight 

lnsegar 25 WP, 150 g a.i./ha 

on flowering crop at bee flight 

(toxic reference) 

DAE: days after,beginning of exposure 

Conclusion: 

The application of 150 g a.i.ka 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 1 5th day. The application of 150 g Spirotetramatha 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 15'~ 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 Uha, no additive, 1 replicate, Germany 

Results: 

Table Error! Style not defined.-10: Evaluation of Larval Cell Comb Area Proportion for Trial 7 

Treatment Proportion of comb area covered by larval cells [%I 

(at X days after beginning of exposure) 
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 Uha, 1 replicate, Spain 

Results: 

Table Error! Style not defined.-1 1: Evaluation of Larval Cell Comb Area Proportion for Trial 8 

DAA: days after application 

Treatment 

untreated (control) 

*. dead bee hive 

Proportion of comb area covered by larval cells [%I 

(at X days after application) 

-3 DAA 

9.3 

7 DAA 

5.6 

14 DAA 

11.4 

21 DAA 

10.6 

31 DAA 

6.0 
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 

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 Lha, 1 replicate, Germany 

Results: 

Table Error! Style not defined.-12: Evaluation of Larval Cell Comb Area Proportion for Trial 9 

I 

lnsegar 25 W P, 150 g a.~./ha 11.7 8.8 

Treatment 

untreated 

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 lnsegar 

(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. 

Proportion of comb area covered by larval cells [Oh] 

(at X days after application) 

-1 DAA 

16.7 

4DAA 

20.0 

9DAA 

9.2 

16DAA 

11.2 

21 DAA 

21.7 

30 DAA 

11.7 
B.9.4.7 Summary of effects on honey-bees 

Table Error! Style not defined.9: Summary of semi-field and field data of Spirotetramat effects on 

Table Error! Style not defined.-1: Summary of acute effects of Spirotetramat (technical and formulated) 

on honey-bees 

honey-bees 

Test substance 

Spirotetramat OD1 00 

Test substance 

Spirotetramat techn. 

Spirotetramat OD1 50 

Spirotetramat SC240 

Spirotetramat OD1 00 

Exposure route 

Oral 

Contact 

Oral 

Contact 

Spirotetramat OD1 00 

Exposure route 

Endpoint 

48 h LD50 

48 h LDso 

48 h LD50 

48 h LD5n 

Feeding test, 

mixed in sucrose 

solution at 0.0144 % 

as. 

Value 

(pg a.sJbee) 

> 107.3 

> 100 

91.7 

162 

Observed effects 

Reference 

Schmitzer, 2004a 

Barth, 2005a 

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 I of three colonies. 

Feeding test, 1 Increased adult mortality. 

mixed h sucrose 

solution at 0.0144 % 

a.s. 

Brood development: termination of brood 

development with tendency to recover. 

No effects on flight activity and behaviour of 

bees. 

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 96a a.s./ha 

no adverse effects on adult and brood mortality, 

condition of the colonies, flight activity and 

behaviour of bees. 

Semi-field study I no adverse effects on adult and brood mortality, 

(tunnel design), 3 condition of the colonies, flight activity and 

Reference 

Schur, 2004a 

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 

Schur, 2004b 

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, 2006 

Schur, 2005 

Table Error! Style not defined.4: Summary of effects to honey bee brood (larvae) from additional 

non-GLP studies of Spirotetramat 

Trial 

1 

2 

Country 

Germany12 

002 

Germany1 

2002 

Treatment 

Spirotetrarnat 36 g a.i.lha 

RME 500 EW 0.2% a.iJv 

Spirotetrarnat 72 g a.i.lha 

RME 500 EW 0.2% a.iJv 

Spirotetrarnat 144 g a.i.lha 

RME 500 EW 0.2% a.iJv 

Spirotetrarnat 144 g a.i.lha 

Spirotetrarnat 36 g a.i.1ha 

RME 500 EW 0.2% a.iJv 

Spirotetramat 72 g a.i.lha 

RME 500 EW 0.2% a.iJv 

Formulation 

Type and 

content 

SC 

240 g a.i.1~ 

SC 

240 g a.i.lL 

Crop Effect 

3 DAA 

3 DAA 

3 DAA 

3DAA7 DAA 

3 DAA 

Unclear 

effect 

3 DAA 

15DAA21 DAA 

Recovery 

until 

7 DAA 

7 DAA 

7 DAA 

7 DAA 

7 DAA 
Formulation 

Type and 

content 

SC 

240 a.i./L 

OD 

100 g a.i.lL 

SC 

240 a,i.lL 

OD 

100 g a.i./L 

SC 

240 g a.i./L 

OD 

loo g 

OD 

150 g a.i.lL 

OD 

50 g 

OD 

150 a.i.~~ 

Treatment 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.i./v 

Spirotetramat 144 g a.i./ha 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.4% a.i./v 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.iJv 

RME 500 EW 0.4% a.iJv 

Spirotetramat 140 g a.i.lha 

Spirotetramat 144 g a.i.lha 

RME 500 EW 0.4% a.iJv 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.2% a.i./v 

RME 500 EW 0.4% a.iJv 

Spirotetramat 140 g a.i./ha 

Spirotetramat 144 g a.i./ha 

RME 500 EW 0.1% a.ih 

Spirotetramat 140 g a.i./ha 

Spirotetramat 100 g a.i./ha 

Spirotetramat 50 g a.iJha 

Spirotetramat 75 g a.iJha 

Spirotetramat 100 g a.i.lha 

Spirotetramat 150 g a.i./ha 

Spirotetramat 50 g a.iJha 

Spirotetramat 75 g a.iJha 

Spirotetramat 100 g a.i.lha 

Spirotetramat 150 g a.i.lha 

Spirotetramat 150 g a.i.lha 

20 days prior to flowering 

Spirotetramat 150 g a.i.lha 

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 

Spirotetramat 150 g a.i./ha 

on flowering crop at bee flight 

Spirotetramat 150 g a.i./ha 

on flowering crop after bee 

flight 

Trial 

3a 

3b 

4 

5a 

5b 

6 

Country 

Gennanyl 

2002 

Germany/ 

2003 

2003 

Germany/ 

2004 

Germany/ 

2004 

Germany/ 

2004 

Crop 

Phacelia 

Phacelia 

Phacelia 

Phacelia 

Phacelia 

Effect 

4 DAA - 14 DAA 

DAA 

4 DAA 

4DAADAA 

4 DAA 

3 DAA - 

8 DAA 

3 DAA - 

8 DAA 

3 DAA - 

15 DAA 

yz; 

3 DAA - 

14 DAA 

3 DAA - 

7 DAA 

3 DAA - 

7 DAA 

3 DAA - 

7 DAA 

4 DAE - 7 DAE 

4 DAE - 7 DAE 

4 DAE - 7 DAE 

Unclear 

effect 

3 DAA 

yDY*- 

14 DAA 

.,",":A 

15DAA22 DAA 

7 DAA 

7 DAA 

15 DAA 

15 DAA 

15 DAA 

1 

14 DAA 

14 DAA 

15 DAE 

15 DAE - 22 DAE 

Recovery 

until 

7 DAA 

22 DAA 

14 DAA 

22 DAA 

30DAA 

7 DAA 

14 DAA 

14 DAA 

21 DAA 

21 DAA 

21 DAA 

21 DAA 

1 

20 DAA 

14 DAA 

20 DAA 

22 DAE 

28 DAE 

15 DAE 
RME: rape oil methyl ester 

DAA: days after application 

DAE: days after beginning of exposure 

no influence on bee brood 0b~e~ed 

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 PMRA and US EPA is not included in this monograph. 

Effect 

3 DAE - 7 DAE 

3 DAE - 

15 DAE 

7 DAE 

7DAA14 DAA 

2 

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

Formulation 

Type and 

content 

OD 

150 a.i.1~ 

OD 

150 g a.i./L 

OD 

150 a.in 

Treatment 

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.ka 

5 days prior to flowering 

Spirotetramat 150 g a.i./ha 

on flowering crop at bee flight 

Spirdetramat 150 g a.i./ha 

on flowering crop after bee 

fl~ght 

Spirdetramat 75 g a.iJha 

Spirotetramat I00 g a.i.1ha 

Spirotetramat 150 g a.i./ha 

Spirotetramat 100 g a.i./ha 

Trial 

7 

8 

9 

8.9.5.1 Laboratory, extended laboratory and semi-field tests (OECD IIA 8.8.1 ; OECD lllA 

10.5.1) 

Unclear 

effect 

7 DAE 

7 DAE 

21 DAA31 DAA 

2 

9 DAA 

Crop 

Phacelia 

Phacelia 

Strawberry 

Country 

Germany1 

2004 

Spain/ 2004 

G~~~Y1 

Reference: Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) 

(Hymenoptera: Braconidae) in the laboratory; BY1 083301 50 OD 

Author(s), year: Waltersdorfer, A.; 2005a 

Report/Doc. number: CW051023; M-254442-01-1 

Guideline(s): Mead-Briggs et al. 2000 (IOBC) 

GLP: Yes (certified laboratory) 

Deviations: No reproduction assessment. 

Validity: Acceptable 

Recovery 

until 

15 DAE 

15 DAE 

15 DAE 

21 DAE 

15 DAE 

2 

16 DAA 

Test substance: Spirotetramat (BY1 08330) 150 OD; Batch no.: 08030101 89(0152), Tox no.: 

TOX07034-00, analytical content: 148.89 g a.s./L 
Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Exposure route, 

duration: 

Feeding: 

Test conditions: 

Test parameter: 

Statistics: 

Findinas: 

Adult wasps Aphidius rhopalosiphi (Hymenoptera: Braconidae), c 48 h old 

Acute contact laboratory test 

4 replicates with 15 wasps for each treatment group 

Control: deionised water; toxic standard: 0.1 g a.s.1ha (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 waterlha 

Adult wasps were exposed to dried residues on treated glass plates for 48 h. The 

test units consisted of two treated glass plates (1 00 x I00 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 pm) which was 

jammed in the orifices by pieces of Teflon tubing (8 mm outer diameter). 

Honeylwater solution 

Temperature: 19 - 22°C; relative humidity: 62 - 83 %, 16 h light, 570 - 1001 lux 

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. 

Mortality corrected according to Abbott. Fisheis Exact test, two-sided. 

Results are summarised in table 9.5.1 -1. 

Table Error! Style not defined.-1 : Toxicity of dried residues of Spirotetramat 150 OD on glass plates to 

Aphidius rhopalosiphi I I I Treatment 

I 

* statistically significant 

Mean mortality at 48 h [Oh] I Mean corrected mortality at 48 h [Oh] 

Control 

I 

Conclusion: 48 h LR50 = 114.676 g a.s.1ha; 95 % Confidence Interval: (86 - 145), based on the 

nominal content of the active ingredient 

8.3 

Toxic standard 

Reference: Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, 

Phytoseiidae) in the laboratory; BY1 083301 50 OD 

Author(s), year: Waltersdorfer A., 2005b 

ReportIDoc. number: CW051024; M-254269-01-1 

Guideline(s): Blumel et al. 2000 

GLP: Yes (certified laboratory) 

Deviations: Short deviation in relative humidity. No reproductive assessment. 

Validity: Acceptable 

2 a ailha I 5 -3.6 I 

100 I 100 

Test substance: Spirotetramat (BY1 08330) 150 OD; Batch no.: 08030/0189(0152), Tox no.: 

TOX07034-00, analytical content: 148.89 g a.s.1L 

Material and Methods: 

Test species: Typhlodromus pyri protonymphs 

I 
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 Lka 

on the glasses (after calibration). 

Exposure route, One exposure unit consisted of two cover slides (glass) which were put together 

duration: 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. 

Findinas: 

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 

was3.0%. - 

Table Error! Style not defined.-2: Toxicity of dried residues of Spirotetramat 150 OD on glass plates to 

Typhlodromus pyri 

I Reference item I 4 I 88.0 I 87.6 I I 

n.d. not determined 

Treatment 

Control 

Test item 

Test item 

Test item 

Test item 

Test item (DAA 1 ) 

Conclusion: 48 h LR,o = 0.333 g a.s./ha; 95 % Confidence Interval: (0.298 - 0.367) 

g a.sJ ha 

0 

0.19 

0.43 

Mortality [YO] 7 DAA 

Uncorr. 

3.0 

13.0 

72.0 

0.97 

2.21 

5 

Abbott 

0 

10.3 

71.1 

100 

100 

100 

P-Value(*) 

0.016 sign. 

~0.0001 sign. 

1 00 

1 00 

100 

~0.0001 sign. 

~0.0001 sign. 

~0.0001 sian. 
Extended laboratory studies 

Reference: 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Exposure route, 

procedure: 

Feeding: 

Test conditions: 

Test parameter: 

Statistics: 

Findinas: 

Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) 

(Hymenoptera: Braconidae) using an extended laboratory test; BY1 08330 150 

OD. 

Waltersdorfer A., 2006a 

CW051071; M-264179-01-1 

Mead-Briggs et al. 2000; Mead-Briggs et al. 2002 

Yes (certified laboratory) 

Increase in temperature due to technical reasons 

Acceptable 

Spirotetramat (BY1 08330) 150 OD; Batch no.: 08030101 89, Tox no.: TOX07034- 

00; (01 52), analytical content: 1 48.89 g a.s.R 

Adult wasps Aphidius rhopalosiphi (Hymenoptera: Braconidae) 

Acute contact extended laboratory test 

6 replicates with 5 females for each treatment group 

Control: deionised water; toxic standard: 2.5 g a.s.lha (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 Uha after calibration of the 

sprayer. 

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 padifor a period 

of 24 hours. The number of mummies was assessed 11 days later. 

Honeylwater solution 

Temperature: 19 - 23.5"C; relative humidity: 62 - 85 %, photo period 16 h, 688 - 

171 8 lux in the parasitation phase, 5590 - 19730 lux in the reproduction phase 

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. 

Mortality corrected according to Schneider-Orelli. One-way ANOVA, p-values are 

adjusted according to Dunnett. 

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. 
Table Error! Style not defined.4: Mortality and reproduction after exposure of Aphidius rhopalosiphi to 

freshly dried residues of Spirotetramat OD 150 applied onto barley plants 

n.s. not significant *:n.d. not detected, **:n.d. not detected due to capacity reasons 

80 g a.s./ha 

151 g a.s./ha 

288 g a.s./ha 

Reference 

Conclusion: 48 h LRS0 > 288 g a.s./ha. 

There was no reduction in reproductive success relative to the control at the 

tested treatment rates. 

Treatment 

Control 

Reference: 

Repellency 

0 

6.7 

0 

80 

Author(s), year: 

ReportlDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

%wasps 

on plant 

75.3 

Test substance: 

Mortality 

0 

6.7 

0 

80 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Reproduction 

Uncorr. 

0 

Rel. to 

control 

[%I 

Exposure route, 

duration: 

Feeding: 

Rate 

23 

Abbott 

0 

P-value 

29.5 

24.6 

16.6 

n.d.* 

Test conditions: 

Test parameters, 

procedure: 

Rel. to 

control 

["A 

0 

- 28.1 

- 7 

27.8 

n.d.* 

Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, 

Phytoseiidae) using an extended laboratory test BY1 08330150 OD. 

Waltersdorfer A., 2005c 

CW051030; M-256306-01-1 

Blumel et al. 2000 

Yes (certified laboratory) 

P-value * 

very short declines in rel. humidity due to opening of the door 

0.435 (n.s.) 

0.967 (n.s.) 

0.265 (n.s.) 

n.d.* 

Acceptable 

Spirotetramat (BY1 08330) 150 OD; Product code: AE 1302943 00 OD1 5 A1 01 ; 

Batch no.: 08030101 89, (01 52), Tox no.: TOX07034-00; 

analytical content: 148.89 g a.s./L 

56.7 

60.7 

60.6 

70.3 

Typhlodromus pyri protonymphs 

Acute contact extended laboratory test 

4 replicates with 20 individuals for each treatment group 

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 Uha using a sprayer after 

calibration. 

Protonymphs were exposed to freshly dried residues on leaves of Phaseolus 

vulgaris for 1 4 d. 

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. 

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 

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 (0 approx. 40 mm) was formed on the leaves. Sets of such 

24.8 

19.5 

19.5 

6.6 

0.071 (n.s.) 

0.195 (n.s.) 

0.244 (n.s.) 
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 

Findinas: 

Results are summarised in table 9.5.1 -4. All validity criteria were met. 

Table Error! Style not defined.4: Mortality and reproduction after exposure of Typhlodromus pyrito 

freshly dried residues of Spirotetramat OD 150 applied onto bean leaves 

I I orelli I 1x1 

Control 1 7.5 1 1 7.3 1 I I 

I Mortality 

n.s. not significant, sign. = significant, * Fisher's Exact test, ** one-way ANOVA 

Reproduction 

Conclusion: 7 d LRS0 1.588 g a.s./ha (95 % CI: 1.31 1 - 1.860) 

There was a significant reduction of reproduction at the low rates of 0.15 and 0.51 

g a.s./ha. 

Treatment I Uncorr. I Corr. Schn.- I P-value * I Rate I Rel. to control 1 P-value ** 

Reference: BY1 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 

ReporVDoc. number: CW05/028 

Guideline(s): Bliimel 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 (BY1 08330) 150 OD; Product code: AE 1302943 00 OD1 5 A1 01 ; 

Batch no.: 08030101 89(0152), Tox no.: TOX07034-00; product code: AE 1302943 
00 OD1 5 A1 01 ; analytical content: 148.89 g a.s./L 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Exposure route, 

duration: 

Feeding: 

Test conditions: 

Test parameters, 

procedure: 

Statistics: 

Findinas: 

Typhlodromus pyri protonymphs 

Acute + aged residue contact extended laboratory test 

100 protonymphs in each treatment group (5 replicates with 20 individuals) 

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 watedha using a 

plot sprayer after calibration. 

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. 

Pollen (birch, pine) 

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 

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. 

Mortality corrected according to Schneider-Orelli. 

The results are considered as valid since the mortalitylescaping value for the 

control stated in the laboratory method on glass was reached in this study 

(< 20 %) and the average number of eggslfemale (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: 
Table Error! Style not defined.4: Mortality of T. pyriafter exposure to freshly applied and aged 

residues . - - - - - - - of - - Soirotetramat OD 150 on a~~le leave discs -,--- ---~ - ~ - . . Days after last application 10 17 114121128142149156 

DAA: days after last application 

Treatment 

Control 

Test item 

Reference item (DAA1) 

Mortality after 7 days (Oh) 

51415117161413(7 

Corr. Mortality [%] 

93.7 1 100 1 90.5 1 92.8 1 100 1 58.3 1 50.5 1 10.8 

100 1 100 1 100 1 100 1 100 1 100 1 100 1 100 

n.d.: not determined 

Table Error! Style not defined.-6: Reproduction of T. pyriafter exposure to freshly applied and aged 

residues of Spirotetramat OD 150 on apple leave discs 

Conclusion: In this extended laboratory test the lethal and sublethal effects of Spirotetramat 

OD 150 residues (aged under semi-field conditions) on Typhlodromus pyriwere 

determined after application of 4 x 72 g a.s.1ha 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: 

Treatment 

DAA 0 to day 42 

DAA 49 

DAA 56 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Control 

Mean no. of eggs 

/female 

n.d. 

5.19 

6.88 

Test item 

Validity: 

Mean no. of eggs 

/female 

n.d. 

5.94 

7.87 

Test substance: 

Reduction rel. to control [%I 

n.d. 

-1 4.48 

-1 4.35 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Exposure route, 

Dose-response toxicity (LRS0) of BY1 08330 150 OD to the green lacewing 

Chrysoperla carnea (STEPH.) under extended laboratory conditions (including 

food-application). 

Rohlig, U.; 2005 

051 048082; M-262650-01-1 

Vogt et al. 2000 (IOBC), modified 

Yes (certified laboratory) 

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 

Acceptable 

Spirotetramat (BY1 08330) 150 OD; Product code: AE 1302943 00 OD1 5 A1 01 ; 

Batch no.: 08030101 89 (01 52), Tox no.: TOX07034-00; 

analytical content: 148.89 g a.s.lL 

Chrysoperla carnea, larvae, age 2-3 d (L,) 

Acute contact extended laboratory test 

40 replicates with 1 larvae for each treatment group 

Control: deionised water; toxic standard: 30 mL productlha (Dimethoate EC400); 

test item: 44, 72, 11 2, 184 and 288 g a.s./ha; deionised water as diluent. The test 

substance was applied in 200 Uha. 

Larvae were exposed to freshly dried residues on bean leaves and Sitotroga eggs 
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, The test consisted of two phases. In the exposure phase of the test, mortality of 

procedure: 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. 

Mortality corrected according to Abbott. Statistical analysis of mortality data 

(Fisher's Exact Binomial Test, 1 -sided, p I 0.05). The software ToxRat 

Professional, Version 2.09 (RATTE, 2004) was used. 

Statistics: 

Findinas: 

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 (2 15 

eggs/female/d) and therefore considered as not impacted by the treatment. 

Table Error! Style not defined.-7: Toxicity of dried residues of Spirotetramat 150 OD on Chrysoperla 

carnea in an extended laboratory test 

Reference item I 63.2 I I I 

Control 

Application rate 

la a.s./hal 

Conclusion: LRS0 > 288 g a.s./ha. 

No negative effects on reproduction after exposure to rates up to 288 g as. 

Spirotetramat 150 OD were observed. 

Treatment Mortality after 

24 days 

[%I 

Reproduction 

5.0 

Corrected mortality [%] 

Fecundity 

[mean number of eggs1 

female] 

Fertility 

mean hatching rate 

r%l 

18.9 81 
Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Exposure route, I 

duration: 

Feeding: 

Test conditions: 

Test parameters, 

procedure: 

Toxicity to the ladybird beetle Coccinella septempunctata L. (Coleoptera, 

Coccinellidae) using an extended laboratory test including exposure to and oral 

uptake of BY1 08330 150 OD. 

Waltersdorfer, A.; 2006b 

CW051053; M-264175-01-1 

Schmuck et al., 2000 (IOBC), modified 

Yes (certified laboratory) 

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. 

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. 

Spirotetramat (BY1 08330) 150 OD; Product code: AE 1302943 00 OD1 5 A1 01 ; 

Batch no.: 08030101 89 (01 52), Tox no.: TOX07034-00; 

analytical content: 148.89 g a.s./L 

Coccinella septempunctata, larvae, 4 d old 

Acute contact extended laboratory test 

40 replicates with 1 larvae for each treatment group 

Control: tap water; toxic standard: 80 mL productlha (Dimethoate EC37); test 

item: 33, 57, 97, 168 and 288 g a.s.1ha; tap water as diluent. The test substance 

was applied in 200 Uha. Application was done using a specially constructed 

sprayer, permitting application on a defined area. 

Larvae were exposed to freshly dried residues on bean leaves and Sitotroga eggs 

until pupation. 

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. 

Temperature: 23.6 - 26.6"C; relative humidity: 42 - 85 %, 16 h photo period, 1050 

- 1760 IUX 

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. 
Statistics: 

Findinas: 

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. 

Mortality corrected according to Schneider-Orelli. 

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 2 fertile eggs per female and day), this parameter is considered as not 

impacted by the test item rates. 

Table Error! Style not defined.4: Mortality and reproduction after exposure of Coccinella 

septempunctata to freshly dried residues of Spirotetramat OD 150 applied onto detached bean 

leaves: 

I Mortality [%I I Reproduction 

Treatment I [g a.sJha] I Uncorr. I corrected I Fertility (hatching I Fertile eggs per I I I I I rate] l%l I femaleand dav I 

Control (tap water) 

BY1 08330 150 OD 

I > 288 g a.s./ha I 

* n.d.: not determined 

BY1 08330 150 OD 

BY1 08330 150 OD 

BY1 08330 150 OD 

BY1 08330 150 OD 

Reference item 

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. 

33 

57 

97 

168 

288 

80 mL orod./ha 

32.5 

22.5 

17.5 

32.5 

47.5 

50.0 

100 

-14.8 

-22.2 

0 

22.2 

25.9 

100 

,- 

99.3 

88.9 

6.0 

3.8 

91.7 

89.3 

99.0 

86.7 

n.d.* 

1.8 

3.6 

2.0 

4.1 

n.d.* 
B.9.5.2 Semi-field or field tests (OECD lllA 10.5.3) 

Reference: Evaluating effects of BY1 08330 OD 150 applications on mite fauna (Acari) in 

the field (grape vines, France). 

Author(s), year: Aldershof, S.; 2006 

ReportlDoc. number: B127AFG; M-269107-01-1 

Guideline(s): Blumel et al., 2000; Hassan, 1985 ; Candolfi et al., 2000 

GLP: Yes (certified laboratory) 

Deviations: No 

Validity: Acceptable 

Test substance: Spirotetramat (BY1 08330) OD 150, purity: 148.89 g a.s./L (analysed), density 

0.986 g/mL, batch no. 08030101 89(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 productlha 

(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 Uha; 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. 

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 BY1 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 

Table Error! Style not defined.-1: Spray scenarios 

Scenario 

1 

2 

3 

4 

Description 

Full (2x) 

Drift 1 

(2x high) 

Drift 2 (2x low) 

Drift 3 (4x low) 

No of applications 

2. 

2 

2 

4 

Spray interval 

2 weeks 

2 weeks 

2 weeks 

1 week 

Nominal Rate 

96 g a.s./ha 

Application 1: 36.4 g a.s./ha 

Application 2: 35 g a.s./ha 

9.1 g a.s./ha 

4.8 g a.s./ha 
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. 

Findinas: 

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. , 

BY1 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 BY1 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. 
Table Error! Style not defined.9: Summary results 

I 1 08.06.05 1 16.06.05 ( 02.07.05 1 12.07.05 1 25.07.05 1 01.08.05 1 29.08.05 I Abbott values 

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 Error! Style not defined.-1 : Summary of effects of spirotetramat on non-target-arthropods (lab + 

ext lab) 

Test species 

Aphidius 

rhopalosiphi 

adults 

Typhodromus 

~~ri 

protonymphs 

Aphidius 

rhopalosiphi 

adults 

Exposure 

contact with 

dried residues 

on treated 

glass plates 

contact with 

dried residues 

on treated 

glass plates 

contact with 

dried residues 

on treated 

barley plants 

Test item 

Spirotetramat 

150 OD 

Spirotetramat 

150 OD 

Spirotetramat 

150 OD 

Rate 

[g a-slha] 

2 

9 

24 

70 

200 

0.19 

0.43 

0.97 

2.21 

5 

22 

42 

80 

151 

288 

Type of effect 

Corrected 

mortality 

Effect [%I 

-3.6 

-7.3 

0 

14.5 

85.5 

Reference 

Waltersdorfer 

2005a 

Waltersdorfer 

2005b 

Waltersdorfer 

2006a 

48 h LR50 = 114.7 g a.s./ha 

Corrected 

mortality 

10.3 

71.1 

100 

100 

100 

48 h LR50 = 0.333 g a.s./ha 

Corrected 

mortality / 

reproduction 

3.3 / n.p. 

3.3 / n.p. 

0 / -28.1 

6.7 / -7 

0 / 27.8 
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 PMRA and US EPA is not included in this monograph. 

Effect [%I Rate 

[g a.slha] 

Test species Exposure Test item Type of effect Reference 

Spirotetramat 

150 OD 

Spirotetramat 

150 OD 

Spirotetramat 

150 OD 

Spirotetramat 

150 OD 

Typhlodrornus 

p~ri 

protonymphs 

Typhlodrornus 

p~ri 

protonymphs 

Chrysoperla 

carnea 

larvae 

Coccinella 

septempunctata 

larvae 

Waltersdorfer 

2005c 

Waltersdorfer 

2006c 

Roehlig 2005 

Waltersdorfer 

2006b 

contact with 

dried residues 

on treated 

bean leaves 

contact with 

dried residues 

(acute and 

aged) on 

treated apple 

leaves 

contact with 

dried residues 

on treated 

bean leaves 

contact with 

dried residues 

on treated 

bean leaves, 

feeding with 

treated aphids 

48 h LR50 > 288 g a.s./ha 

0.15 

0.51 

1.7 

5.9 

20 

Corrected 

mortality / 

reproduction 

4.1 143.2 

1.4 / 48.7 

56.8 / n.d. 

98.6 / n.d. 

100 / n.d. 

aged Od: 93.7 

aged 7d: 100 

aged 14d: 90.5 

aged 21d: 92.8 

aged 28d: loo 

aged 42d: 58.3 

aged 49d: 50.5 

aged 56d: 10.8 

no reduction of 

reproduction on 

DAA 49+56 

5.3 

7.9 

2.6 

0 

5.3 

no effect on 

reproduction 

-14.8 

-22.2 

0 

22.2 

25.9 

no effect on 

reproduction 

4 x 72 (74 

'Orrected , 

mortality 

44 

72 

112 

184 

288 

Corrected 

mortality 

33 

57 

97 

168 

Corrected 

mortality 

288 
B.9.6 Effects on earthworms (OECD IIA 8.9, lllA 10.6.3) 

B.9.6.1 Acute toxicity (OECD IIA 8.9.1, lllA 10.6.1 .I) 

Active substance 

Reference: BY1 08830 (techn.): Acute toxicity to earthworms (Eisenia fetida) tested in 

artificial soil with 5 % peat 

Author(s), year: Lechelt-Kunze, C., 2004a 

ReporVDoc. number: LKCIRG-A-18/03, Edition Number: M-104105-01-2, Q7.01.2004 

Guideline(s): OECD 207 

GLP: Yes 

Deviations: None of relevance 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, age: 

Type of test, duration: 

Applied concentrations: 

Solventlvehicle: 

Toxic standard: 

Test conditions: 

Test substrate: 

Substrateltest vessel: 

Temperature: 

Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Water content: 

pH: 

Spirotetramat (BY1 08330), purity: 97.5 %, batch: 0804510014 

Earthworm (Eisenia fetida) 

4 replicates each with 10 individuals per treatment and control 

Average weight: 360 mglworm, clitellate adults z 2 months 

Laboratory acute test, 14 days 

Nominal: 0 (control, quartz sand), 32, 100, 178, 31 6, 562, 1000 mglkg dw soil 

Quartz sand 

2-Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mglkg dw soil 

5 % sphagnum peat, 20 % kaolinite clay, 74 % quartz sand, about 0.2 - 1 % 

CaC03 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 

625 g wet weight (500 g dw) in 1.5 L test containers 

Measured temperatures were not stated 

Continuous illumination (400 - 800 Lux) 

Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 14 

days) and mean body weight (measured at 0 and 14 days) 

NOEC: Dunnett's' Test (mortality, mean body weight) 

LC5, (toxic standard): Probit Analysis 

About 21 % (0 h), 19.6 - 20.1 % (14 d) 

6.4 (0 h), 6.4 (14 d) 

Mortality: Spirotetramat: No mortality was noticed in the control and all treatments up to 562 

mglkg dw soil. At 1000 mglkg dw soil the mortality was 3 % (do not differ 

statistically significantly in Dunnett test). 

Toxic standard: LC5,: 23 mglkg dw soil (95% C.I.: 21 - 24 mglkg dw soil) 

Behavioural effects: No effects were observed in the control and all treatments 

Body weight: See table B.9.6.1-1 
Table Error! Style not defined.-1 : Effects of BY1 08330 (spirotetramat) on the mean body weight of 

Conclusions: LC, > 1000 mglkg soil dw, NOEC (body weight, mortality): 1000 mglkg 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: BY1 08830 cis-ketohydroxy: Acute toxicity to earthworms (Eisenia fetida) 

tested in artificial soil 

Author(s), year: Lechelt-Kunze, C., 2005 

ReportlDoc. number: LKCIRG-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: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, age: 

Type of test, duration: 

Applied concentrations: 

Solventlvehicle: 

Toxic standard: 

Test conditions: 

Test substrate: 

Substrateltest vessel: 

Temperature: 

BY1 08830 cis-ketohydroxy (metabolite of spirotetramat), purity: 98.6 %, batch: 

NLL7549-7 

Earthworm (Eisenia fetida) 

4 replicates each with 10 individuals per treatment and control 

Average weight: 0.34 glworm, clitellate adults > 2 months 

Laboratory acute test, 14 days 

Nominal: 0 (control, quartz sand), 32, 100, 31 6, 1000 mglkg dw soil 

Quartz sand 

2-Chloroacetamide (purity > 97 %): 5.6, 10, 18,24,32 mglkg dw soil 

10 % sphagnum peat, 20 % kaolinite clay, 70 % quartz sand, about 0 % CaC03 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 

625 g wet weight (500 g dw) in 1.5 L test containers 

Measured temperatures were not stated 
Light regime: 

Test parameters: 

Statistics: 

Findinas: 

Water content: 

pH (mean): 

Mortality: 

Behavioural effects: 

Body weight: 

Continuous illumination (400 - 800 Lux) 

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) 

NOEC: Dunnett's' Test (mortality, mean body weight) 

LC5, (toxic standard): Probit Analysis 

23.3 % (0 h, mean), 24.7 % (14 d, mean) 

6.4 (0 h), 6.2 (14 d) 

No mortality was noticed in the control and all treatments. 

No effects were observed in the control and all treatments 

See table B.9.6.1-2 

Table Error! Style not defined.-2: Effects of BY1 08330-ketohydroxy on the mean body weight of Eisenia 

fotida 

weights at day 14, 

Toxic standard: LC5,: 23 mg/kg dw soil (95% C.I.: 21 - 24 mglkg dw soil) 

Conclusions: LC5, > 1000 mg/kg soil dw, NOEC (body weight, mortality): 1000 mglkg 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 

ReportIDoc. number: LRT-Rg-A-98/07, Edition Number: E310 341 1-3,09.01.2008 

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: 

Solventlvehicle: 

Toxic standard: 

Test conditions: 

Test substrate: 

Substrateltest vessel: 

Temperature: 

Light regime: 

Test parameters: 

Statistics: 

Nominal: 0 (control, quartz sand), 62.5, 125, 250, 500, 1000 mglkg dw soil 

Quartz sand 

Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mglkg dw soil 

5 % sphagnum peat, 20 % kaolinite clay, 74.8 % quartz sand, about 0.2 % CaC03 

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 

580 g wet weight (500 g dw) in 1.5 L test containers 

Measured temperatures were not reported 

Continuous illumination (400 - 800 Lux) 

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) 

NOEC: Dunnett's' Test (mortality, mean body weight) 

(toxic standard): Probit Analysis 

Findinas: 

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 ~.9.6.'1-3 

Table Error! Style not defined.-3: Effects of 4-methoxycyclohexanone on the mean body weight of 

Eisenia fetida L I 4-methoxycyclohexanone [mglkg dw soil] I Weight alteration of the surviviors 

Mean + standard devision I I 0 (control) + 8.93 + 6.07 

I EC50 > 1000 mglkg soil dw 

* significant difference when compared to control (Dunnett test, alpha = 0.05) 

Toxic standard: LC50: 11.1 mgkg dw soil (95% C.I.: 10.1 - 12.2 mglkg dw soil) 

Conclusions: LC50 > 1000 mglkg soil dw, NOEC (body weight, mortality): 125 mglkg 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, lllA 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. 
Metabolites 

Reference: BY1 08330-Enol: Reproduction toxicity to the earthworm Eisenida fetida in 

artificial soil 

Author(s), year: Moser, T.; Scheffczyk, A., 2005 

ReportIDoc. number: PI 8RR, Edition Number: M-243305-01-2, 19.01.2005 

Guideline(s): IS0 Guideline 11268-2 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: 

Material and methods: 

Test species: 

Number of organisms: 

Weight, age: 

Type of test, duration: 

Applied concentrations: 

Solventlvehicle: 

Toxic standard: 

Test conditions: 

Test substrate: 

Substrateltest vessel: 

Temperature: 

Light regime: 

Feeding: 

Test parameters: 

Statistics: 

Findinas: 

Water content: 

pH: 

Effects test item: 

BY1 08830-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101 -09- 

0005 

Earthworm (Eisenia .fetida) 

4 replicates each with 10 individuals per treatment and control 

Mean: 404 mglworm, clitellate adults 

Laboratory sublethal test, 8 weeks 

Nominal: 0 (control, quartz sand), 10, 32, 100, 31 6, 1000 mgkg dw soil 

Quartz sand 

Carbendazim (applied as SC formulation Derosal): 1, 3, 5 mg a.s.kg dw soil 

10 % sphagnum peat, 20 % kaolinite clay, 69.55 % quartz sand, 0.45 % CaC03 to 

adjust pH to 6.0 + 0.5, WHC,,: 50 + 10 %, pH and the water content were 

measured at the beginning and the end of the study 

500 g dry weightltest container 

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. 

16 hours light (485 - 558 Lux)/8hours dark 

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 

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) 

NOEC: Dunnett's multiple test, EC5, (reproduction): Probit analysis using max. 

likelihood regression 

Start: 32.1 - 35.4 % (53 - 58.6 % of WHC,,) 

End: 28.9 - 33.8 % (49 - 60.7 % of WHC,,) 

6.1 - 6.2 (start), 6.8 - 7.0 (end) 

See table B.9.6.2-1 
Table ~rror! Style not defined.-1: Effect of BY1 08330-enol (metabolite of spirotetramat) on mortality, 

growth and reproduction of Eisenia fetida 

BY1 08330-enol Mean mortality 

[mglkg dw soil] ["/.I 

Mean biomass increase Mean number of juveniles (SD) after 4 weeks [%I 

Control 0 34.7 148 (43.3) 

LOEC [mglkg dw] I > 1000 I 100 

Go [mg/kg dw] > 1000 1 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 (I 10 %): 0 % after 4 weeks 

Weight loss of adult worms (I 20 %): + 37.7 % after 4 weeks 

Number of juveniles/vessel (2 30 %): 148 + 43.3 % 

Coefficient of variation (I 30 %): 29.3 % 

Conclusions: ECS0 (reproduction): 1269 mg/kg soil dw 

NOEC (reproduction) 32 mglkg 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 lllA 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 PMRA and US EPA is not included in this monograph. 
B.9.7 Effects on soil non-target macro-organisms (OECD IIA 8.14, lllA 10.6.6) 

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

Metabolites 

Reference: BY1 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 

ReportIDoc. number: PI 1 HR, 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: 

Material and methods: 

Test species: 

Number of organisms: 

Age: 

Type of test, duration: 

Applied concentrations: 

Solvent: 

Toxic standard: 

Test conditions: 

Test substrate: 

Substratehest vessel: 

Temperature: 

Light regime: 

Feeding: 

Test procedure: 

Test parameters: 

Statistics: 

BY1 08830-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-1 01 -09- 

0005 

Gamasid mite (Hypoaspis aculeifeg 

4 replicates with 20 mites per treatment, 5 replicates with 20 mites per control and 

3 replicates with 20 mites per toxic reference 

Protonymphes 2 day old 

Laboratory reproduction test, 34 days 

Nominal: 0 (control, deionised water), 10, 32, 100, 316, 1000 mg /kg dw soil 

Deionised water 

Dimethoat (applied as EC formulation "Perfekthion"): 5 mg/kg soil dw 

LUFA 2.1 standard soil (type: sand): WHC,,: 34.6 %, pH 6.2 * 0.7, C,,,: 1.23 + 

0.3 % 

5.1 - 5.3 g wet weight (approx. 3 mm layer) / 30 mL glass container 

25 +2 "C 

Permanent dark 

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. 

0-day: Introduction of 20 individuals on the soil surface of each test unit randomly. 

1 4-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 c 50 % mortality (i.e. 100, 31 6, 1000 mglkg) 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.. 

Mortality of adults after 14 days, reproduction after a 7-day period 

NOEC (mortality adults): ANOVA followed by Dunnetts test, NOEC (reproduction): 
Welch-t-test with Bonferroni Adjustment 

Findinas: 

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 

a) corrected according to Abbott (1925) 

*Significant different by Dunnetts test (alpha = 0.05) 

Table Error! Style not defined.-1: Effects of BY1 08830-enol (metabolite of spirotetramat) on mortality 

and reproduction of gamasid mites 

Effects toxic standard: 88 % corrected mortality at 5 mglkg soil dw 

Validity criteria: Adult mortality in control after 14 days (5 25 %): 3% 

Mean reproduction rate in control (2 10 fertile eggs per female): 25.6 + 7.3 

Adult mortality in toxic standard after 14 days (50 - 99.5 %): 88 % 

NOEC (mortality): 31 6 mglkg soil dw 

LC/ECSO >1000 mglkg soil dw 

Conclusions: 

BY1 08330-enol 

[mg a.s.lkg dw] 

Control 

10 

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

Number of juveniles per female after 7 d 

Not submitted. Study is not required because spirotetramat is not persistent and the effects on soil microand macro-organisms are acceptable. 

Mean i SD 

25.6 & 7.3 

n.d. 

Mortality of parental mites 

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

Reduction [%] 

0.0 

n.d. 

Of dead 

mites at 14 days 

3 

8 

Corrected 

mortalitya)[%] 

0.0 

7.2 

Substance 

08330-en01 

Species 

Gamasid mite 

(Hypoaspis aculeifer) 

Endpoint 

Mortality 

Reproduction 

Time 

28 

EC/LC5~ 

[msn<g dwl 

> 1000 

NOEC 

[mglkg dwl 

31 6 

1000 

Referencelstudy 

acceptable 

Moser & Scheffczyk 

2005/yes 
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 PMRA and US EPA is not included in this monograph. 

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

B.9.8.1 Nitrogen mineralization 

Active Substance 

Reference: BY1 08330 tech.: Determination of effects on nitrogen transformation in soil 

Author(s), year: Lechelt-Kunze, C., 2004 b 

ReporVDoc. number: LKC-N-32/04, Edition Number: M-087831-01-1,01.09.2004 

Guideline(s): OECD 21 6 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: Spirotetramat (BY1 08330), purity: 97.4 %, batch: 080451001 4 

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 mglkg dry soil 

Solventlvehicle: Quartz sand 

Toxic standard: Sodium chloride: 16 glkg soil dw 

Test conditions: 

Test substrate: 

Substrateltest vessel: 

Incubation: 

Test parameters: 

Statistics: 

Findinas: 

pH: 

Effects test item: 

Natural Soil, collected on April 13~, 2004 at a depth of 0-20 cm, from 

experimental field on Lacherhof Germany: 

C,,: 0.7, pH: 5.6, 

texture (DIN): 6.3 % clay, 15.6 % silt, 78.1 % sand, 

microbial biomass: 393 mg microbial Clkg soil dw, 

additionally 5 glkg soil dw Lucerne-grass-green meal was added after the 

application. 

300 g soil dw1500 mL brown glass bottles 

20 & 2 "C in the dark, about 40 % MHC 

Samples (10 g dry soil) were extracted with 50 mL 1 M KC1 and soil particles were 

removed by filtration, NH4+ and NOi were determined using a "Bran & Liibbe 

Autoanalyzer 3. Samples were taken at 0, 7, 14 and 28 days after incubation. 

F-test (homogeneity of variance), T-test (significant differences) 

5.6 (start), 5.9 - 6.0 (end) 

See table B.9.8.1-1 
Table Error! Style not defined.-1 : Effects of spirotetramat on nitrogen transformation (mean values) d 

Validity criteria: Coefficient of variation in the control (5 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 mglkg soil and 1.32 mg/kg soil, 

corresponding to an application rate of 0.099 kg a.s.1ha and 0.99 kg a.s.1ha) was 

< 25 %, thus spirotetramat had no adverse effects on N-transformation of soil 

microorganisms. 

Treatment 

Control 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 

1.32 mg a.s./kg 

(0.99 kg a.s./ha) 

B.9.8.2 Carbon mineralization 

Active substance 

Time 

(days) 

0 

7 

14 

28 

0 

7 

14 

28 

0 

7 

14 

28 

Reference: BY1 08330 tech.: Determination of effects on carbon transformation in soil 

NH4-N 

(mg Nlkg dw) 

3.27 + 0.00 

1.93 + 0.08 

1.56 + 0.07 

1.48 + 0.14 

3.42 + 0.17 

1.95 + 0.07 

1.58 + 0.06 

1.57 + 0.02 

3.37 + 0.21 

1.93 + 0.13 

1.76 + 0.33 

1.71 k0.18 

Author(s), year: 

ReportfDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and methods: 

Test species: 

Type of test, duration: 

Applied concentrations: 

SolvenVvehicle: 

Toxic standard: 

Test conditions: 

Test substrate: 

NO3-N 

(mg Nlkg dw) 

28.34 + 0.41 

13.35 + 1.18 

13.46+ 1.47 

26.02 + 1.46 

30.07 + 0.55 

14.76 + 0.65 

13.77 + 1.24 

25.94 + 0.76 

30.00 + 0.22 

13.32 & 1.02 

13.60 + 0.54 

28.19+ 1.22 

Lechelt-Kunze, C., 2004 c 

LKC-C-30104, Edition Number: M-087840-01-1,01.09.2004 

OECD 21 7 

Yes 

None of relevance 

Acceptable 

Spirotetramat (BY1 08330), purity: 97.4 %, batch: 0804510014 

Nitrification rate 

(mg NOS-Nlkg soil 

dwld) 

-2.14 + 0.22 

0.02 + 0.37 

0.90 + 0.07 

-2.19 + 0.02 

-0.14 + 0.22 

0.87 + 0.08 

-2.38 + 0.12 

0.04 + 0.09 

1.04 + 0.05 

Soil microflora 

Carbon transformation test, 28 days 

0 (control, purified water), 0.13 and 1.32 mgkg dry soil 

Quartz sand 

Sodium chloride: 16 glkg soil dw 

% Deviation from 

control 

2 

980 

3 

11 

143 

16 

Natural Soil, collected on April 13~, 2004 at a depth of 0-20 cm, from 

experimental field on Lacherhof Germany: 

C,,: 0.7, pH: 5.6, 

texture (DIN): 6.3 % clay, 15.6 % silt, 78.1 % sand, 

microbial biomass: 393 mg microbial Clkg soil dw, 

in addition glucose was added to the soil samples (2 gkg soil dw) to induce 
maximum respiration rate. 

Substrateltest vessel: 350 g soil dw1500 mL brown glass bottles, three replicates per treatment 

Incubation: 20 + 2 "C in the dark, about 40 % MHC 

Test parameters: Evolved C02 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) 

Findinas: 

pH: 5.6 (start), 5.5 - 5.6 (end) 

Effects test item: See table B.9.8.2-1 

Validity criteria: Coefficient of variation in the control (5 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 mglkg 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. 

Table Error! Style not defined.-1 : Effects of NOA446510 on glucose-induced short-term respiration 

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

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

Treatment 

Control 

0.13 mg a.s./kg 

1.3 mg a.s./kg 

Test substance 

Spirotetramat 

Spirotetramat 

The risk assessment for the EU can be found in the Appendix at the end of section B.9. 

The risk assessment for PMRA and US EPA is not included in this monograph. 

Day 7 Day 14 Day 28 

Mean CO2 

(mgmg)) 

290.56.4 

267.85.9 

269.54.6 

Mean COl 

(mglhkg)) 

284.6k2.3 

256.9k3.1 

253.8k5.1 

Day 0 

Test parameter 

Nitrogen mineralization 

Carbon mineralization 

Mean 

Co2 

(mg'hn<g) 

264.5k1.7 

247.3k5.2 

245.922.6 

% 

deviation 

from 

control 

8 

7 

Yo 

deviation 

from 

control 

10 

11 

con 

Mean 

(mglhkg) 

268.3k4.2 

251.7k7.7 

248.7k0.6 

Yo 

deviation 

from 

control 

6 

7 

% 

variation 

from 

control 

6 

7 

Test concentration 

0.13 mg a.s./kg 

(0.099 kg a.s./ha) 

1.3 mg a.s./kg 

(0.99 kg a.s./ha) 

0.1 3 mg a.s./kg 

(0.099 kg a.s./ha) - 1.3 mg a.s./kg 

(0.99 kg a.s./ha) 

Time 

28 days 

28 days 

Effects (deviation from 

control) 

3% 

16 % 

6% 

7% 
B.9.9 Effects on other non-target organisms (flora and fauna) believed to be a risk (OECD 

IIA 8.1 2, lllA 10.8) 

6.9.9.1 Effects on terrestrial non-target plants (OECD lllA 10.8.1) 

B.9.9.1.1 Formulation 

Reference: Non-target terrestrial plants: an evaluation of the effects of BY1 08330 OD 

150 in the vegetative vigour test (Tier 1) 

Author(s), year: Gosch, H., Nguyen, D.H. (2006a) 

RepoNDoc. number: VV06103-Tier 1 ; M-273381-01-1 

Guideline(s): OECD 208 B (July 2000, draft) 

GLP: N o 

Deviations: None 

Validity: Acceptable 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Duration: 

Soil characteristic: 

Test design: 

Test conditions: 

Test parameter: 

Statistics: 

Findinas: 

Spirotetramat (BY1 08330) 150 OD, Batch no.: 08030/0233(0152), Tox no.: 

TOX07087-00, content: 148.28 g a.s./L 

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) 

Vegetative vigour test 

4 to 5 replicates with 4 to 5 plants per pot for each species (1 9 - 20 plants per 

treatment group) 

Control: deionised water; test item: 288 g a.s./ha in 200 Uha. The test item was 

sprayed on the leaves and above-ground portions of the plants. 

21 days after treatment application 

Standard soil: 20.7 % clay, 65.1 % silt, 14.2 % sand, organic carbon: 1.19 %, 

pH: 7.4 

At the 2 - 4 leaf stage the test item was sprayed on the leaves and above-ground 

portions of the plants. 

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. 

Visual phytotoxicity ratings were done at days 7, 14 and 21 after application. Plant 

biomass was determined per replicate (pot). 

Pairwise Mann-Whitney-U-test, one sided smaller 

There were no adverse effects on survival. 

There were visible signs of phytotoxicity such as chlorosis, necrosis, leaf 

deformation, bleaching, andlor 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 BY1 08330 OD 150 is shown in the table below. 
Table Error! Style not defined.-1 : Phytotoxicity assessments for the day 21 of the vegetative vigour test 

-- 

* compared to the control, where survival was 100 % (20 plants) in all cases 

(Tier 1) for the effects of BY1 08330 OD 150 

** 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: 

Corn 

20 (1 00) 

0 

C 

56.9 

Sunflower 

20 (1 00) 

0 

B-C 

43.1 

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 

Oats 

20 (1 00) 

0 

B 

56.6 

Soybean 

20 (1 00) 

0 

A 

40.2 

C = severe symptom(s) throughout the whole plant with younger or newly developed leaves growing normally 

Oilseed rape 

20 (1 00) 

0 

D 

83.3 

Survival in number 

(out of 20) and (%) 

Inhibition of 

survival * (%) 

Phytotoxicity 

(see below) 

Dry Weight ** 

(% inhibition) 

D =total plant symptom(s) with the plant showing poor vigour. 

Cucumber 

19 (95) 

5 

B-C 

41.3 

E = moribund 

Conclusion: An application rate of 288 g a.s.1ha BY1 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 lengthlplant height effects be measured and 

reported. 

Reference: Tier I and Tier II Vegetative Vigor Non-Target Plant Study using BY1 08330 

150 OD 

Author(s), year: Christ, M.T., Lam, C.V. (2006a) 

ReporVDoc. number: EBFNXOI 6; 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: BY1 08330 150 OD, Batch no.: 0803010189(0152), Tox no.: TOX07087-00, purity: 

15.1 % wlw 

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: Vegetative vigour test 

Number of organisms: 8 replicates with 5 plants per pot for each species (40 seeds per treatment) 
Treatments: 

Statistics: 

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 (mg1L): 0, 

118, 235, 471, 941, 1882; measured test concentrations (mg1L): 0. 123, 252, 527, 

1072, 21 13; 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 

green house. 

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 injuryleffect), 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). 

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, ECoS 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 ECo, values were not 

calculable because of the nature of the data. 

Findinas: 

Measured test concentrations ranged from 104 to 11 4 % 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 5% for dry weight. The EC25 and EC50 dry weight values were determined as 76 

g a.s./ha and 134 g ailha (95 % cl: 1 17 to 155) by non-linear regression, 

respectively. The EC50 for corn length was determined as 172 g ailha (95 % cl: 

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. 

Rvearass: 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, sovbean, suaarbeet. 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 .I -2. 

Table Error! Style not defined.-2a: Phytotoxic effects on ten plant species in a vegetative vigour test 

Canola 

Corn 

Cucumber 

Oat 

Onion 

Ryegrass 

Soybean 

Sugarbeet 

Sunflower 

Tomato 

Plant dry weight 

NOEC 

176 

44 

176 

176 

176 

88 

176 

176 

176 

176 

LOEC 

>I76 

88 

>I76 

>I76 

>I76 

176 

>I76 

>I76 

>I76 

>I76 

EC05 

>I76 

34 (22-54) 

>I76 

>I76 

>I76 

176 

>I76 

>I76 

>I76 

>I76 

EC25 

>I76 

76 (60-96) 

>I76 

>I76 

>I76 

168 (1 53- 1 84) 

>I76 

>I76 

>I76 

>I76 

EC50 

>I76 

134 (117-155) 

>I76 

>I76 

>I76 

>I76 

>I76 

>I76 

2176 

>I76 
Table Error! Style not defined.-2b: Mean plant length and dry weight data of control plants 

I Tomato I 33.5 I 4.908 

I I I 

- 

Sugarbeet 

U 

Conclusion: In a vegetative vigour study with 10 non-target terrestrial plant species, following a 

21 day post-emergent foliar application of BY1 08330 150 OD, the most sensitive 

endpoint was corn dry weight with an ECZ5 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.1ha. 

Mean plant dry weight (g) 

8.652 

6.424 

9.132 

3.533 

0.628 

3.433 

3.857 

Control 

Canola 

Corn 

Cucumber 

Oat 

Onion 

Ryegrass 

Sovbean 

Reference: Non-target terrestrial plants: an evaluation of the effects of BY1 08330 OD 

Mean plant length (cm) 

35.7 

78.8 

34.4 

58.2 

31.7 

51.9 

27.6 

Sunflower 70.6 1 14.429 

22.8 

150 in the seedling emergence and growth test (Tier 1) 

Author(s), year: Gosch, H., Nguyen, D.H. (2006b) 

ReportlDoc. number: SE06103-Tier 1 ; M-273373-01-1 

Guideline(s): OECD 208 A (July 2000, draft) 

GLP: No 

Deviations: None 

5.568 

Validity: Acceptable 

Test substance: Spirotetramat (BY1 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) 

Treatments: Control: deionised water; test item: 288 g a.s./ha in a spray volume of 200 Uha. 

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. 
Statistics: 

Findinas: 

Mortalitv/Survival: Dead plants were removed after each assessment. The number 

of plants that survived after application was recorded at the final assessment. 

Phvtotoxicitv 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 staaes: 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. 

Statistical analysis was carried out using the Paimise Mann-Whitney-U-test (one 

sided smaller). 

The germination of the control plants was at least 95 % and all controls exhibited 

normal growth. 

There was no significant adverse effect of BY1 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. 

Table Error! Style not defined.4: Effects of Spirotetramat 1500D on six plant species in a seedling 

emergence and survival test [288 g a.slha] 

I 14 d seedling emergence 

I I 

14 d seedling survival 

I I I I 

I 

Treatment 

Species 

Cucumber 

I Corn I 100 I 100 I 100 I 95 I 

I Control 

Soybean 

1 oat I 95 I 95 I 100 I 100 

% Emergence = (No. plants emerged / No. seeds planted) x 100 

Control 288 g a.sJha 

I % Emergence 

Oilseed raoe 100 100 100 100 1 90 I 100 

% Survival = (No. plants surviving /No. seeds emerged) x 100 

288 g a.sJha 

100 I 100 

Sunflower I 95 100 100 I 100 I 100 I 100 I 100 

Conclusion: An application of 288 g a.s./ha BY1 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. 

% Emergence 

100 

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 lengthlplant height effects be measured and 

reported. 

% Survival % Survival 
- The EPA recommends 10 seedslreplicate and 4 replicates/dose; only 5 

seedslreplicate and 4 replicatesldose were used in this study. 

Reference: 

Author(s), year: 

ReportIDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Duration: 

Soil characteristic: 

Test design: 

Test conditions: 

Test parameters: 

Tier I Seedling Emergence Non-Target Plant Study using BY1 08330 1500D 

Christ, M.T., Lam, C.V. (2006b) 

EBFNX017; M-276244-01-1 

US-EPA 122-1 

Yes 

Short deviation in the photoperiod from 13 to 15 h. The EPA recommends 10 

seedslreplicate and 4 replicatesldose; however, 5 seedslreplicate and 8 

replicatesldose 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. 

Acceptable 

Spirotetramat (BY1 08330) 150 OD; Batch no.: 08030101 89(0152); analytical 

content: 15.1 % wlw; 

4 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avena sativa, 

G ramineae), ryegrass (Lolium perenne, Poaceae), onion (Allium cepa, All iaceae) 

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) 

Seedling emergence and growth test 

40 plants per treatment group (8 pots with 5 seeds each) 

Control: deionised water; test item: 176 g a.s./ha in a spray volume of 93.5 Llha. 

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. 

21 days 

22 % clay, 50 % silt, 28 5% sand, organic content: 2.4 %, pH: 7.8 

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. 

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. 

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 injury1effect);lO 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 
Statistics: 

Findinas: 

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. 

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. 

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 -1 3 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 .l-4. 

Table Error! Style not defined.-4: Phytotoxic effects of Spirotetramat 1500D on ten plant species in a 

seedling emergence and survival test [I76 g a.sJha] 

21 d seedling emergence I I 

21 d seedling survival 

I 

% 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 EC5, figures for all tested species were therefore > 176 g a.s./ha. 

I 

Canola 

Corn 

Cucumber 

Oat 

Onion 

Ryegrass 

Soybean 

Sugarbeet 

Sunflower 

Tomato 

I Treatment Control 176 g a.sJha Control 176 g a.sJha 

I Species % Emergence % Survival 

% Emergence = (No. plants emerged / No. seeds planted) x 100 

88 

93 

95 

98 

100 

100 

98 

93 

95 

90 

% Emergence % Survival 

80 

98 

98 

98 

98 

95 

93 

95 

100 

90 

91 

100 

97 

100 

100 

100 

100 

100 

100 

100 

84 

100 

100 

100 

100 

100 

100 

97 

100 

100 
Reference: 

Author(s), year: 

ReporUDoc. number: 

Guideline(s): 

GLP: 

Deviations: 

Validity: 

Test substance: 

Material and Methods: 

Test species: 

Type of test: 

Number of organisms: 

Treatments: 

Duration: 

Soil characteristic: 

Test design: 

Test conditions: 

Test parameter: 

Statistics: 

Findinas: 

Spirotetramat OD 150: Vegetative vigour test on non-target terrestrial plants 

Friedrich, S. (2006) 

M-279698-01-1 

OECD 227 (Draft 2005) 

Yes (certified laboratory) 

None 

Acceptable 

Spirotetramat OD 150; Batch no.: 08030/01 89(0152), purity: 15.1 % w/w analysed 

2 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avena sativa, 

Gramineae), 1 dicotyledonous plant: canola (Brassica napus, Brassicaceae) 

Vegetative vigour test 

20 / 10 / 8 replicates with 2 14 / 5 plants per pot for corn / rape / oats (40 plants 

per treatment) 

Control: deionised water; test item (nominal): 0.17, 0.48, 0.96, 1.92 L producffha 

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 productlha; Calibration had been 

performed before application. 

21 days after treatment application 

Agricultural soil, sandy loam: 12.4 % clay, 48.8 % silt, 38.9 % sand, organic 

carbon: 0.9 %, pH: 6 

At the 2 leaf stage the test item was sprayed onto the leaves and the soil 

substrate using an automatic application cabin. 

Glasshouse conditions: temperature: 1 1 - 41 "C; 17 - 99 % relative humidity; 16 h 

photoperiod; bottom watering. 

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 (Wplant). Shoot fresh weight measurements were performed on 

day 21. Plant biomass was determined per replicate (pot). 

Computation of EC50 (e.g. probit analysis). Software ToxRat Professional used. 

In Dec 2007 probit analysis using max. likelihood regression was conducted to 

derive ECo5 and ECZ5 values with regard to shoot fresh weight. 

Measured test concentrations ranged from to 82.1 to 90.9 % of nominal. 

Survival was 100 % in all treatment and the control group. 

6. napus: no phytotoxic symptoms. Growth inhibition effects in the two highest 

rates (1 1 and 35 %, respectively). 

A. sativa: no phytotoxic symptoms. Growth inhibition effects in the two highest 

rates (9 and 44 %, respectively). 

Z. mavs: 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: 4.0471 8, Variance of slope: 27.86593, chi2: 0.00871, degrees of 

freedom: 2, p(chi2): 0.99565, 12: 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, ?: 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, 1': 1.000 

Table Error! Style not defined.-5: Phytotoxic effects on three plant species in a vegetative vigour test 

after treatment with Spirotetramat OD 150 [L productlha] L i 

1 Z. mays I > 1.92 I 0.96 I > 1.92 I 16-18 I 

BBCH 21 DAA I Survival I Shoot fresh weight 

6. napus I > 1.92 I 0.48 I > 1.92 I 15-17 

Table Error! Style not defined.-6: Effects on shoot fresh weight (g) of three plant species after 21 d 

species LC50 

A. sativa > 1.92 0.96 > 1.92 16-21 

NOEC 

exposure to Spirotetramat OD 150 [L productlha, nominal] 

I A. sativa I 

EC50 

Mean + SD 1 20.76k2.19 1 20.41k2.21 1 19.39_+2.97 1 17.87k1.80 1 12.16+1.95 

Mean + SD 1 15.22 k 1.24 1 15.45 + 1.52 1 14.25 k2.08 1 13.67 + 1.42 1 9.18 + 1.75 

% reduction I I -2 I 6 I 10 I 40* 

Treatment group 

% reduction I 

Z. mays 

Mean + SD 1 34.76k3.01 1 36.5823.57 1 35.13i2.47 1 32.81+2.76 1 20.10+3.35 

8. napus 

Control 0.1 7 

I %reduction I -5 I - 1 I 6 I 42* I 

* statistically significant (Dunnett test, p C 0.05), SD standard deviation 

2 I 7 

Tab. Error! Style not defined.-7: Results of the probit analysis of shoot fresh weight data: Selected 

0.48 

14* I 41* 

lower 95 %-cl I 0.114 I 0.866 I 1.849 I 2.838 I 4.067 

upper 95 %-cl I 0.818 1.614 4.907 20.750 78.794 I 

0.96 

effective concentrations (ECx, L productlha) of the test item and their 95 %- and 99 confidence 

limits 

Parameter I EC05 I EC25 I EC50 I EC75 I EC90 

6. napus 

1.92 

Value 

IL ~roductlhal 

lower 99 %-cl I 0.031 

0.534 

Value 

[L productlha] 

lower 95 %-cl 

upper 95 %-cl 

lower 99 %-cl 

upper 99 %-cl 

1 upper 99 %-cl I 0.966 I 1.535 I 2.144 I 3.038 4.173 I 

0.577 I 0.977 I 0.774 

[L productlha] 

lower 95 %-cl 

upper 95 %-cl 

lower 99 %-cl 

1.290 

0.587 

upper 99 %-cl I 2.970 

Z. mays 

Value I 0.934 I 1 -51 6 I 2.119 I 2.961 I 4.009 

0.659 

0.008 

1.039 

0.000 

25.149 

0.920 

0.947 

0.902 

2.373 

A. sativa 

2.423 9.284 76.1 09 

1.409 

0.564 

1.880 

0.257 

4.129 

546.253 

1.508 

1.524 

1.497 

4.363 

2.381 

1.807 

16.834 

0.421 

72.336 

7.575 

2.108 

2.130 

2.094 

4.024 

2.550 

342.214 

0.104 

8399.393 

6.470 

3.358 

5434.059 

0.027 

678412.375 

2.929 

2.994 

2.887 

3.942 

4.080 

3.854 
/ 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 PMRA and US EPA is not included in this monograph. 

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: BY1 08330 - Toxicity to bacteria 

Author(s), year: Bruns; Caspers, N. 2004 

ReportlDoc. number: 131 2 Nl04 B, Edition Number: M-275364-01-1, 05.10.2004 

Guideline(s): OECD 209 

GLP: Yes 

Deviations: None of relevance 

Validity: Acceptable 

Test substance: 

Material and methods: 

Test species: 

Type of test, duration: 

Applied concentrations: 

Nominal: 

Toxic standard: 

Test conditions: 

Substrateltest vessel: 

Incubation: 

Test parameters: 

Statistics: 

Findinas/conclusions: 

Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 

Activated sludge 

Source: Aeration tank of a domestic sewage treatment plant 

Laboratory aerobic activated sludge inhibition test, 3 hours 

1000,1800,3200,5600,10000 mglL 

3,5-dichlorophenol (97 %): 2.5, 5.0, 10, 20, 40 mg/L 

Activated sludge (solid concentration: 480 mglL) treated with active substance, 

reference substance or water (control) 

Aerated for 3 hours at 20 + 2°C 

Inhibition of respiration rate (rate of oxygen uptake) 

None, because the inhibition at highest tested concentration was 22.2 mglL 

See table B.9.10.1-1 
Conclusions: No inhibitory effects on aerobic waste water micro-organisms are expected if the 

test substance is correctly applied at the intended application rate.