### Appendix 

### C. Ecolpgical EffectsData Summaries The summaries found in this appendix are based on the AustrianAgency for Health and Food Safety (AGES) review and are presented as they appear in "Volume3, 

### 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, metabolitesand formulationsto aquatic organisms Chronic toxicity of active substance, metabolites and formulationsto 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) Fieldtests (OECD lllA 10.4.5) Formulation: SpirotetramatOD 100 Tunnel tests (OECD lllA 10.4.7) Summary of effects on honey-bees Effectson 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) Effectson a species level (gamasid mites) Effectson 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) Effectson terrestrial non-target plants (OECD lllA 10.8.1) Effects on biological methodsof sewage treatment (OECD IIA 8.15) Activated sludge, respiration inhibition

### 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 (Colinusvirginianus) 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. 850.2100 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-Batch08045/0014, TOX-no. 6344-00; purity 97.5 %
Bobwhitequail (Colinus virginianus), sexually mature 0, 500, 1000 and 2000 mglkg bodyweight (nominal) five males and five females/treatment group and control single oral administration in gelatine capsules 14 d post-treatmentobservation period acclimation 6 weeks; Feeding: 15 h starvation priorto 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 mortalityand 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 initiationand 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 birdsthat died suffered from diarrhoea from day 1 after administrationon. Before death they showed reduced activity, fluffed feathers, ptosis and apathy. Diarrhoea and soft excrementswere 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. Birdsof all test doses reduced their food consumption from day 0 to 3, most evidently in the highest dose group. no treatment-relatedfindings 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, softexcrement, reduced feed consumption)



### B.9.1.2 Short-term dietarytoxicity (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 periodwas only 3 d and not 7 days as recommendedby 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:
Feedconsumption: Necropsy: Statistics:

Findinas: Mortalities:
Clinical signs: Spirotetramat (techn.), batch No.: MIX-Batch0804510014,TOX-no. 6344-00; purity 97.5 % Bobwhite quail 

### (Colinus virginianus), 10days old and weighing 15

### - 20.9 g control (untreateddiet); 31 3, 625, 1250,2500 and 5000 ppm a.s. (nominal) correspondingto 301,624, 1218,2552and 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 

### adlibitum; housing in stainless steel wire cages; temperature: 32.6 

### - 38.3 "C, relative humidity: not reported, photoperiod 14 h Concentration, homogeneity and stabilityof 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 untiltermination. Individualbody weights were measured at test initiation, on day 5 and at terminationof 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 Statisticswas 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 programwas ToxRat Professional Version 2.09 Software.
None treatment related. One chick of the 1250 ppm group was sacrificed because of an injury. Duringthe exposure periodthe chicks of the highest dose group showed signs of

### Bodyweight: intoxication like soft excrements (reversible). Body weights were statistically analysedfor day 0, +5 and +8. At the beginning the birdsat 625 pprn and at 5000 pprn had a slightly higher bodyweight, 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 itwas concludedthat there was no impact on body weight. A 

### significantly reduced bodyweight change during the exposure periodfor the birds at 2500 pprn and at 5000 pprnwas detected. This finding is is in good compliance with the reduced food consumption at these concentrations. Inthe recovery period, the birds of the two highest concentrations grew more than the control or the lower concentrationgroups. At 5000 pprnthe increased growth rate was even statistically significant. These findings indicated a severe starvation effect at the high test concentration. These effectswere compensated as soon as the birdswere switchedto normal diet. Feedconsumption: 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 BobwhiteQuail following short-term dietaryexposure

Table Error! Style not defined.9b: Bodyweight data

* statisticallysignificant Treatment (measured) [ppm as.]

Control

301 624 Mean bw gain d 0

## - 5 [Oh] 46 43 43.4 Cumulative

mortality

0120 0110 0110 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 Meanfeed consumption d 0

# - 4 [glbirdld] 3.1 3.1 3.1 Meanfeed consumption d 5

# - 7 [g/bird/d] 3.8 3.9 4.1 Estimated intake d 0

# - 4 [mg askg bwld] 0 39.8 82.6lble Error! Style not de

Nominal dietary Concentration

-- - 0 mga.s./kg diet 313 mga.s.lkg diet 625 rng a.s.lkg diet 1250 mga.s.lkg diet 2500 mg a.s.lkg diet 5Q00mga.s.lkg diet led.&: Estimated daily food consumption per bird Individual Food Consumption ing I bird-Idav 

# 11 Exposure 

# 1 Post Exposure 

# 11

Conclusion: LC50 

### > 4998 mg a.s.1kg food, equivalentto > 497 mg a.s./kg bwld, based on a meanfeed 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 BY108330: A Subacute Dietary 

### LCs0with 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 (Anasplafyrhynchos), 10day-old hatchlings control (untreated diet); 313, 625, 1250, 2500 and 5000 ppm a.s. (nominal) correspondingto 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 sufficientlyverified by analytical methods Observations for signs of intoxicationwere 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 performedat termination Body weight and growth datawere analysed using TOXSTAT (if normally distributed, data were subjected to parametric analysis (one-wayANOVA); if not normally distributedafter transformation, datawere subjectedto nonparametric analysis (Kruskal-WallisANOVA))
None None Body weights for Day 5 

### and body weight change from Day 

### 0to Day 5 

### exhibited a statistical difference at all treatment levels relative to the control. Bodyweight change from Day 5 

### to Day 8 

### exhibited a statistical difference at all treatment levels relativeto the control. However, these were not an adverse effect since the increase in bodyweightat the treatment levels was greater than inthe control. Feed consumption: Dueto 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 infeed consumption was observed in all treatment groups during the exposure period as comparedto the control. During the recovery period the groups treated with up to 2843 

### ppm showed a significant increase in food consumption and returnedto normal or higher levels as comparedto the controls. Necropsy: Nofindings.

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 significantlydifferent compared to control Cumulative

mortality

0110 0110 0110



### Table Error! Style notdefined.9b: Mean bodyweight data 

### (g) 

# I * 

### statistically significantlydifferent

a 

### Apparent significant reduction infeed consumption based on empirical analysis as comparedto the control group. Since there were no replicates forfeed consumption foreseen inthe usedtest 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 Meanfeed consumption d 5 -7 [glbirdldl 41.5 44.1 41.9 Estimated intake do-4 [mg aslkg bwld] I 65 120Conclusion: 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: Effectof 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). Eggswere not candled on day 

### 0 (deviationto 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 (untreateddiet); 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) 

### 1treatment group
Test substance mixed in the diet, prepared weekly Concentration, homogeneityand stabilityof the test substance in the diet were sufficientlyverified by analytical methods. Acclimation: 23 days; Pre-photostimulation:

### 7 weeks; 11weeks 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, 13air changes per hour, photoperiod 7 h increasingto 17 h, wide spectrum illumination Approximately 22 weeks (pre-egg laying untiltermination)
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 bodyweight, 14-daysurvival, 14- day survivor bodyweight, and eggshell strengthlthickness. Adult body weights were measuredat 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. Eggswere candled laterto determine embryo viability and to determine embryo survival. Egg shell thicknesswas measured. The group bodyweight of the surviving hatchlingsper penwas determined. At 14 days of age, the average bodyweight by parental pen of all surviving chicks was determined. All adult birdswere subjected to a gross necropsy.
A SAS based statisticalprogramwas usedfor data analysis (USEPA, 2002). Data from treatment groupswere comparedto controls using the Shapiro-Wilk's an'd Levene's test for equalvariance to determine if dose groups had unequal variances. If normality and variances were equal, then parametric analyseswere conducted usingANOVA followed by Dunnett's or William's test. If variances were unequal, then the nonparametric analyses were conducted usingthe Jonckheere or Mann-Whitney procedures.
No treatment-related mortalities. Seven incidental mortalitiesoccurred (2 controls, three at 85 ppm, two at 802 pprn). Notreatment-related signs of toxicitywere 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 approximate5 % 

### 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. Notreatment-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 statisticaldifference 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 biologicallysignificant. The Jonckheere's test indicated a statisticaldifference from the control in the 802 pprn level for the percent viable embryos of eggs set endpoint. The approximate 3 % 

### reduction in means betweenthe high treatment level as compared to the control, however, is considered not biologicallysignificant. 93 % 

### of this parameter is acceptable even for control levels.
Table 

### Error! Style not defined.-I: Summary of reproductive resultsof spirotetramat in the bobwhite quail Reproductiveparameter
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 48.94Reproductiveparameter Test concentration(measured, ppm as.) Control Eggs set I Eggs laid 

### ["%I Viable embryos 1Eggs set [%I Viable embrvos 1Pen 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-daysurvivors I Hen Mean egg shell thickness [mm] Mean hatchlingbodyweight [g] 802

42.1 1 40.1 1 71 8 48.17

inhibition of above compared to control [%I Mean 1Cdaysurvivor body weight [g] Live 3-week embryos IViable embryos [%I Hatchlings I Live 3-weekembryos [O/O] 89.75* 94.99 37.44

0.22 42.24 0.21 7.2

16dayold survivors I Hatchlings [%I

Conclusion: Based on all parameters, and the considerationof 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 (equivalentto 23 mg a.s.1kg bwld, based on measured concentrations). The NOAEC for reproductive parameterswas 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 34.61 614 43.61

I 35.5 99.43 92.73
Hatchlings 1Eggs laid [Oh] Hatchlings I Eggs set [%I 1Cday 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 reproductiveNOAEC as 264 ppm (23 mglkg bwld), based on a statistically significant effect on mean hatchling body weight and a reductionof 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

* Significantlydifferentfromthe 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 73.11 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 Bodyweight:
Adult feed consumption: Egg parameter:

Hatchling parameter

Post-mortem examination: Statistics:

Findinas: Mortalities:

Clinical signs: Mallard duck (Anasplatyrhynchos), 1 9 weeks of age Control (untreateddiet); 80, 240 and 720 mg ailkgfeed (nominal), corresponding to 0, 89, 269 and 869 mg ailkgfeed (measured). The amount of a.s. technical was adjustedto reflect 100 % 

### a.s.. The daily dietary doseswere 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 1weeks; 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 increasingto 17 h, wide spectrum illumination Approximately 21 weeks (pre-egglaying untiltermination)
Adult birdswere observed at least once dailyfor signs of toxicity, injuries, illness or mortality. Reproductive parameters includedegg production, egg fertility, embryo survival, hatchability, hatchling body weight, 1 Cday survival, 14-day survivor bodyweight, and eggshell strengthlthickness. Adult bodyweights were measuredat study initiation,on weeks 3, 5, 7, 9, and at adult termination. Feed consumptionfor each pen was measured weekly throughout the test. Eggs were collectedtwice 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 measuredon the day they were removed from the hatcher and on day 14. Hatchlingswere observed dailyfor signs of toxicity, injuries, illnessor mortality. All adult birdswere subjected to a gross necropsy.
A SAS based statistical programwas used for data analysis (USEPA, 2002). Data from treatment groups were comparedto 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 usingANOVA followed by Dunnett's or William's test. If 

### variances were unequal, then the nonparametric analyses were conducted using the Jonckheere or Mann-Whitneyprocedures.
A total of five adult mallards died during the exposure period. One in the control group, one inthe 269 mg a.s./kg treatment level and three in the 869 mg a.s./kg treatment level (priorto week 12 adult sacrifice). The mortalities upto 269 ppm were considered nottreatment-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 inthe 869 mg a.s./kg treatment level began to express signs of ataxia (loss of balance). The birds in this treatment levelwere also notedto have severe peeling and

### crackingof the foot webbing. Duringstudy week 7, one henwas found dead. By study week 8, nineteen of the remainingtwenty 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 notfeed or drink and by week 9, bodyweights of these adult birds showed a marked decrease from week 1 

### body weights. The foot symptoms were originallydiagnosed as "bumblefoot" by the attending veterinarian although they were notedto be atypicalof thisfoot conditionwhich 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 birdsto reduce the foot symptoms prior to reproductive phase. At week 12, two more adult mortalities occurred and approximately 79 % 

### of the remaining 24 birdswere observed with moderateto severe foot symptoms. All of the adult mallard in the 869 mg a.s./kg treatment levelwere sacrificed at the end of week 12 post study initiationfor humane reasons due to the condition of the feet resulting in emaciation. The severe peelingand 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 mortalityoccurred during this time period. Adults were able to feed and drink, did not show the same signs of ataxia as seen inthe 869 pprntreatment level and were able to complete the reproductive phase of the study. A lightto moderately severe form of the peeling and cracking foot symptoms were observed in one bird at week 14 post study initiation at the 89 pprntreatment level. At the time of adult sacrifice (week 21) inthe 89 pprn treatment level, approximately 27 % 

### of the adult birds showed foot symptoms. No bird mortalities occurred at thistreatment level and the adult body weights at termination were not statisticallydifferent 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 symptomswere indistinguishable from foot cage wear that is typically seen due to housing conditions. Excludingthe foot symptomsthat were indistinguishable from cage wear or "typical" bumblefoot,the foot symptoms appeared relatedto dose. However, it was unclearwhether 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 statisticaldifference was determined at the 269 ppm treatment levelfor both female and male body weight gain. Hatchlings: Inthe 89 pprn treatment level a statistical difference was indicatedfor the body weight (approximate 6.5 % 

### reduction). For 14-d-oldhatchlingsthe difference was not significantat 89 ppm, but at 269 pprn it was significant. Feed consumption: A significant statisticaldifference 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. Birdsof the highesttreatment levelwere emaciated. Reproductive results: See Table Error! Style not defined.9. 

### For the eggs laid and eggs set per pen endpoints, the respectivetests indicated a statistical difference from the control in the 269 pprn level.The Jonckheere's test indicated a statisticaldifference from the control in the 269 ppm levelfor 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 inthe mallard duck I I Test concentration (measured, ppma.s.) Reproductive parameter Control 89 269 869 Overall mean food consumption (glbirdlday) 113 118 86* 57 Mean adult bodyweight total change (g) 17 5 -113* -315* 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 1Pen 2 0.14 48.1

Viable embryos1Eggs set 

### [%I Viable embryos 1Pen Live 3-week embryos 1Pen Hatchlings1Pen 14-day old survivors 1Cdaysurvivors 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-daysurvivor bodyweight [g] Live $week embryos I Viable embryos [O/o] Hatchlings I Live 3-week embryos 

### [%I 14-dayold survivors I Hatchlings p,] Hatchlings 1Eggs laid ["h]

n.d.not determined for the 869 ppm treatment level due to adult sacrifice 12weeks 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 hatchlingsbody weight as biologically adverse and propose a NOAEC of 89 mg a.s./kg feed, based on several statisticallysignificant reproductive parametersand 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 inthe hatchlings bodyweight in the lowest dose group as biologically not adverse, therefore the reproductive NOAEC is 89 ppm. However, no NOEC based on foot symptomscould be determined andthe 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 differentfrom 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 

### 08330Technical: 

### A Reproduction Studywith 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 accordingto guidelines. Minor deviation to EPA guideline only: age of birds lowerthan recommended. Acceptable
BY1 08330 (techn.), batch 

### No.: 0804510014, purity 97.99 %
Mallardduck (Anasplatyrhynchos), 19weeks 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 debilitatedcondition of the birds, the 720 ppm a.s. treatment group was terminated in week 5. The amount of a.s. technical was adjustedto 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 stabilityof 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 10weeks; 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 increasingto 17 h, wide spectrum illumination Approximately 22 weeks (pre-egg laying untiltermination)
Adult birdswere observed daily for signs of toxicity and abnormal behaviour. Photographsof the duck's feet were taken at every body weight intervalfor potentialcomparison of foot lesions. Reproductive parameters included egg production, egg fertility, hatchability, hatchling body weight, 14-daysurvival, 14- day survivor bodyweight. Adult bodyweights 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. Eggswere collected twice daily and candledweekly 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 measuredon the day they were removed from the hatcher and on day 14. Hatchlingswere observed daily for signs of toxicity. All adult birdswere subjected to a gross necropsy.
An analysis of variance (ANOVA) was performedto determine statistically significantdifferences 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 methodfollowing arcsine square root transformation. See also "Appendix": US EPA re-analyzedthe data applying different test procedures. Findinas: Mortalities: A single mortalityoccurred 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 limbweakness and crackedand bleeding lesions on the bottoms of their feet, on Day 3 

### of Week 3. 

### Marked, continued loss of bodyweight 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 includedthose that normally are associatedwith injuries and penwear. Such signs included bumblefoot (plantar pododermatitis), lameness, feather loss, molting and neck, back and shoulder lesions. Treatment-related findings inthe 80 

### pprn treatment and above groups included lesions on the bottoms of the feet characterised bythickened areas that appeared rough, dry and hard, with cracking,fissuring and/or bleeding evident. Parents: Slight and transient statistically significant bodyweight changes inthe 30 

### and 80 

### pprn treatment groupswere considered nottreatment related. A significant statisticaldifference in body weight and bw changeswas determined in hens of the 200 

### pprn treatment group. Birds in the 720 

### pprn treatment group exhibited marked weight loss. Hatchlings: Inthe 80 

### pprn treatment level a slight but statistically significant reduction was seen in the hatchling's body weight. Feedconsumption: 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 11through 18

### that were statistically significant.At the 720 

### pprn test concentration there were statisticallysignificant treatmentrelated reductions in feed consumptionfrom Week 1to Week 5 (terminationof the adult birdsfor the high dose group). According to the 

### US EPKs re-analysis(see Appendix), the mean overallfeed 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 lesionswere 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 henswith regressed ovaries and the numbers of drakes with reducedtesteswere 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 bodyweight 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-davsurvivors I Hen

14-day old survivors/ Hatchlings[Oh] 100 99 

# 1 94 91 

### ' 

# 1 Test concentration (measured, ppmas.)

15 1 62 -32

58 686 46 4

Mean egg shell thickness [mm] Mean hatchling body weight [g] (samplesize) Mean 14-day survivor body weight [g] Live 3-week embryos 1Viable embryos [%I Hatchlinas I Live 3-weekembrvos 1%1 Control 

# I 28.8 

# I 78.8

2 87 426 28

1Cdayold survivors / Eggs set [%I 

# I 69 

# I 62 

# I 57 

### ' 

# I 23*+ 

# I * Significantly differentfrom 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, accordingto EPA analysis (seeAppendix)

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 productionand hatchabilityat 78.8 mg a.s.1kg feed. This is equivalentto a NOAELof 4.0 mg a.s./kg bwld, based on a measured feed concentrationof 28.8 ppm, a mean body weight of 1147 g and mean feed consumptionof 159glbirdld. 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 accordingto 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 convertedto proportions (e.g., by dividing the counts by the number of eggs set from a pen). All data that were inherently proportionsor were convertedto a proportion were arcsine-transformedto meet assumptionsof normality. However, notests 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 performedto determine statistically significant differences between groups. Then Dunnett's multiple comparison procedure was usedto compare the - 0.373 28*

+ (6) 200*

96 41*+

60 25*

### treatment meanswith the control group mean andassess the statistical significanceof the observed differences. In general, the EPA prefers notto transform data, but rathertests the assumptions of normality and homogeneity priorto analysisto determine whether parametric or non-parametrictests should be performed. Therefore the US EPA developed a SAS programfor 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 parametersfor 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 

### 1Hatchlings[%Iand 1 Cday old survivors 

### 1Eggs set 

## [%I. The EPA notes the following: First, according to the resultsof Levene'stest (equalityof 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 convertedto proportions and arcsinetransformed by the study authors, did not violatethe basic assumptionsof 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-Whitneytest with a Bonferroni adjustment. However, the EPA's statistical analyses did result in lower NOAECvalues in a number of cases, primarily resultingfrom the EPA's application of the more-sensitive Williams' (parametric) and Jonckheere's (non-parametric)tests when the assumptionof a negative dose-response relationship could reasonably be supported, as comparedto the study authors' relianceon Dunnett's multiple comparison procedure. Most importantly it should be notedthat for the endpoint "mean hatchlingbodyweight" there was a significant reductionat 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 identifiedfor this endpoint, the EPA recommendsthat a requestfor another avian reproduction study with mallardsthattests at lower levels should be considered.
In a responseto the EPA re-analysisBayer 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 positivedifference) when usedto make multiple comparisons and when sample sizes are unequal. For the hatchlingweight parameter, sample sizeswere unequal (see table 6.9.1.3-3). Accordingto a publishedarticle, the Williams test maintains a predetermineda-level less and less as the imbalance increases. Another publication recommends applying the Williams test at 2.5 %

### to quantitative parametersof repeated toxicity studies. If this recommendationisfollowed the p-value calculated for the 30 ppm dose level (p=0.031) of the hatchlingweight parameter is higher than the recommendedstatistical level (p=0.025).
Comment IRMS): A statisticianof AGES (RMS authority) looked at the issue and, overall, agreed with the EPA's position. Hence the parameter "hatchling body weight" is consideredto show a statistically significant difference at 30 ppm comparedto the control. Hovever, in the RMS opinion the question of statisticalsignificance of this difference in hatchlingweight is a borderline case anyway, if the p-valuesof both tests applied are looked at. While the outcome of the statistical analysis is importantfor the interpretationof the study, for the derivationof a "No observedadverse effect level" the magnitude and biological relevance of an observed effect should also be considered. The bodyweight reduction comparedto the control is 6 %. 

### BCS comparedthe hatchlingbodyweight data obtained at the 30 ppm levelwith historical control bodyweightdata 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 hatchlingbodyweights (mean of 20 studies = 

### 33 

## +3 g). Further, it is importantto note that the NOEC for the 14-day old duckling weight is 80 ppm. This supports that any reduction in the hatchling bodyweightwas temporary and recovery took place in the ducklings of the lowest

### dose level. Hence, the dose levelof 30 ppm, correspondingto 4 

### mg/kg bw/d, is considered as NOAELwith regardto reproductive effects inthe mallardduck. During joint review discussions between the authorities, PMRA Canada agreed with the RMS positionto consider the 30 ppm level as NOAELfor 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 reproductionstudies with the mallard duck dietarily exposed to spirotetramat the ducks experiencedsevere foot problems, expressed as dried and deeply cracked skin on the underside of the feet. These dermal effectsto the feet may have been exacerbated by an interactionbetweenthe wire mesh of the caging (possible physicaldamage) 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 Exposureto 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 (Anasplatyrhynchos), age: 35 weeks The objective of the study was to determine if dermal exposureto treatment excrement alone is sufficient to elicit a dermal response in the feet of ducks not exposedvia diet. Four groups were incorporated intothis study: 1. 

### Dietarycontrol: 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 nominaldietary concentration: 720 mg a.s./kg feed, correspondingto 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 excrementsfrom group 3 to the floor of their cages Twelve males and twelve femaleslgroup. One pairlcage.
Test substance mixed in diet 

### / dermal exposureto 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 andtop of the cages were made of polycarbonate-coated welded-wire mesh, the sides of galvanised steel. Isolatedenvironmental conditions were provided by establishingone room for the two control groups and one room each for dermal and dietary exposure group. Temperature: 14

### - 33 "C, relative humidity: 45 

## - 94 %, 

### photoperiod7 h. Duringthe experimental period, excrementswere catched in plastic trays. Every other day, all cageswere scrubbed with brushes dipped in water. Separate equipment were used in each room. The excrement collectedfrom the dietary control and exposure cage was spreadwith spatulas evenly and throroughly across the entire floor of the dermal group cages. Dietary analysisconfirmed recoveries of 95 to 116 % 

### of nominal. Excrementsample analysis: Composite excrement samples were collectedfrom 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 analysedto 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 birdswere captured and their feed closely examined. Notations were made in greater detail regarding unique conditionsof 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 dietarytreatment group residuesof 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 (week2) and 97.6 (week 8) mgkg. Residues in the dietary control were < 

### 0.01 mgkg. Severefoot symptoms progressedto 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 intheir feet: From week 3 on, limpingwas observed. Fromweek 4 

### on additional symptoms appeared: usingwings 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 unusualfor ducksto experience temporary cracking and healing of the skin of their feet from time to time. Inthe dermal exposure group four occurrences of minor cracking along the digitswere noted at week 4, 

### and three occurrences of moderate cracking during the middletwo weeks. Effects seemedto have beentransient. Duringweek four 10cases of inflamed webbing (transient) were noted. Dietary exposure group: Moderateto 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 crackingobserved 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 reducedcompared to the control. Two separate sets of ducks exposedto Spirotetramat at 720 mglkgfeed 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 exposedto the excrement of dietarily exposed ducks experienced fewer cases of minor to moderate cracking and drying of the feet during 8 weeks of repeated exposureto the excrement, and symptoms were transient, less severe and did not leadto behavioural symptoms.

Semi-Field Study of the Effects of Dermal and Dietary Exposureto BY108330 

### in Mallard Ducks 

### (Anasplafyrhynchos) 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 150OD, lot no. 08030101 89(0125), purity: 14.7 % 

### as.
Mallard Duck (Anasplatyrhynchos), age: 27 weeks The objectiveof 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 inthe 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 (event2) and 140 g a.s./ha (event 3). Additionally, treatedfeed was placed in the treatment pens immediately after turf application. At the end of 10weeks, all treatedfeed was replaced with clean feed and remaining birdswere 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 exposureto oversprayedturf. Ten weeks of treatment followed by a 4-week recovery period. Acclimation: 2 weeks and 4 days; feeding: basal diet and water adlibitum; 

### 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:

Bodyweight:
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. 0.17 ac. each. At the opposite end of the penswhere the feed was placed a 9.14 m untreated zone was maintainedto simulate edge or adjacent habitat. At the center of the untreatedzone a small pond was constructed. The ducks hadto cross 36.6 m (120ft) 

### of treated turf from the pondto obtain feed. Natural temperature and humidity conditions, natural photoperiod. Weather data as well as rain events were recorded. Dietaryanalysis confirmed recoveries of 82.7 to 98.9 

### 5% of nominal. Homogeneity was demonstrated. Daily observationsfor mortality and behavioural abnormalities. All birdswere monitored every two weeks for changes inthe condition of the feet and body weight measurement. Notationswere made in great detail regarding unique conditionsof the feet and digital photoswere taken. Notations were also madeto document bird moving across the treated field and two sessions on birds behaviour and movements were captured by digitalvideo. Feed consumption was measured daily and averagevalueswere 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 tendencyto fly rather than walk increased as treatment progressed. Limping had entirely ceased one week post-treatment. Foot condition: Abnormalities were noted byweek 2 of treatment including cracking of the skin along the underside of the digits. Though mildcracks 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 abnormalfoot conditionsamong treated birds ranged from 29.2 (week 14) 

### to 63.9 % 

### (week 8). The number of severe cases increased untilweek 10. Duringthe recovery period, significant improvements were made in the condition of the feet. Byweek 14, only 7 

### minor cases of feet crackingwere recorded. Duringthe entire study, no infection was noted in crackedfeet. Food consumptionfollowedthe same general pattern in all pens during the course of the study, though average weekly food consumption inthe control pens was slightly higherthan in treatment pens. Variations of group body weights betweendifferent pens occurred, but being inconsistent and transient these effects were not considered treatment related. Ducks that received spirotetramat-treated feed andwere 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 reluctanceto 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 riskassessment

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. 0803010189(01 52) Material and methods: Four soybean field trials and four wheat fieldtrials were conducted in NAFTA Growing Regions 2 (one soybean trial), 3 (onewheat 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 150OD were made to established soybeans or wheat at a target application rate of 0.176 kglhdapplication(0.1 57 Ib a.s./A) andwith 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. Forthe soybean trials, the first application was made either at BBCH growth stage 15to 16or at stage 69to 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 priorto the applicationsand 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 intervalsfrom the soybean and wheat trials. The forage samples were collected prior to the first application (control samples) and at various time intervalsafter 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 individualanalyte residueswere 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 residuesfrom 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 insoybean forage (broad leaf plants) and wheat forage (short and tall grass) followingthe second application of Spirotetramat 150

### OD and calculated half-lives



# I

(Tall ~rass)- 

# I I 

# I

a Mean total spirotetramat residue immediatelyfollowing the 2" applicationis the average from all trials of the total 

# I spirotetramat residue from the sample interval immediatelyfollowing the 2" application of Spirotetramat 150 OD.

Maximumtotal Spirotetramat residue following the second application is the maximumtotal as. residue from all of the trials for the sample intervals followingthe second applicationof Spirotetramat 150OD.

Mean half-life is the average of the half-lives calculatedfor each trial from the total Spirotetramat residue at each of the Soybean ~ora~e

Wheat Forage (Short Grass) Wheat Forage

sample intervals followingthe second applicationof Spirotetramat 1500D.

The mean half-life for total as. residue in soybean forage is 4.45 days without inclusionof the half-lifefrom trial FN333- 05W which received 1.3 inches of rain 1 day following the second application of Spirotetramat 150OD. Crop

The highest peak residue level detected was in unmownwheat (21.65 ppm) which was slightly higher as comparedto 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 150OD was in trials FN331-05Wand FN333-05W which received rain (0.02 inches and 1.3 inches, respectively) one day following the second application. Conclusions: Total Spirotetramat residuesdissipated with half-livesof between2 and 4.5 days in soybean forage (broad leaf plants) andwheat forage (short andtall grass) after 

### two foliar applications of Spirotetramat 150OD at the highest proposedseasonal use rate. Mean and maximum residue values determined immediately after the second application as well as the calculated half-livesare shown in the table above. Maximum Total Spirotetramat Residue Followingthe Second

Application [ppmlb MeanTotal Spirotetramat

Residue Immediately Followingthe Second

Application [ppmIa 8.32 14.35
13.04

Reference: Residues of Spirotetramat inarthropods after spray application incitrus 

## - 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 (certifiedtest 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-enoland BY108330-ketohydroxyon 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 inthe 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 

### .- -------*-.----.-- (equivalentto nominal 270 g a.s.1ha) with a spray volume of 3000 Uha. The BBCH principlegrowth stage of the trees was 7. Arthropod sampling: Ground dwellingarthropods were sampled using pitfall traps with a diameter of approx. 

### .--------- 

## ---------- -.-- 12.5 cm, which were dug at surface level and protectedwith a cover. 86 to 90traps 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. Samplingwas carried out also before application of the test substance, samplingtook placefor ground dwellers: DAT -1, DAT 0 (before application), DAT +1, +2, +3, +4, 

### +5, +7, +9, +11, +14 and +21; andfor foliage dwellers: DAT -1, DAT 

### 0 (after application), DAT +1, +2, +3, +4, +5, +7, 

### +9, +11, +14 and +21. After swift identificationand 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 metabolitekgf.w., respectively.The LOQ was 0.01 mglkgfor 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 usedto calculate the TWAs of daily intervals between DAT 011-21, expressed as percentage of the maximum measured residuevalue. TWAs of residue values were calculated by interpolating valuesfor 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 determinedto assess the potentialexposure 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, Dermapteraand Lepidoptera larvaewere the most abundant groups in the pitfalltraps, whereas Ephemeroptera, Diptera, Coleoptera, Neuroptera (adults and larvae) and Hymenoptera (without Formicidae)were most abundant in the samples from inventoryspraying. 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 concentrationsandTWAsin 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 [mglkgf.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 applicationof 99.6 or 121.3 g spirotetramat per ha and meter canopy height (equivalentto nominal 270 g a.s./ha) to citrus orchards, maximum measured residues in ground dwellers were 0.32, 0.60 and 0.62 mglkgf.w. for parent, parent-kenoland total measured residues, respectively; and for foliage dwellers 4.0, 6.0 and 6.2 mglkgf.w. for parent, parent+enol and total measured residues, respectively. The decline of residue levels infoliage 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 mglkgf.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. Becausethis shortcoming of the study does not leadto 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 Ohof MMV) 0.39 (63 %of MMV) Mean residue concentrations [mglkgf.w.1 in foliage dwelling arthropods

Maximum measured value (MMV) MIAIDAT1 -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 (Parusmajor) 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 (Parusmajor) 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 constellationscan be observed in orange groves in Spain (Gil-Delgado & Barba 1987). Two investigationson breeding biology and success have been made regarding both the occurrence in tree holesand 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 publicationsdeliver 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 biologyof great tits revealed an earlier onset of breeding and a larger clutch size (mean 7.7 eggs) in orange groves, comparedto 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 (Quercusfaginea) at high altitude) (Belda et al. 1998). The populationof greattits breeding in tree holes inthe same study area (Sagunto, Spain) reached a density of 5 

## - 8 pairs per 10 haand average clutch size is 6.4 eggs (Gil-Delgado & Barba 1987). Comparable to the resultsfrom 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 citrustrees, wall crevices). Therefore, based on the resultsof the above cited papers, great tits obviously inhabit citrus orchards and can serve as a representative for a small insectivorous birdfor pesticide risk assessment in citrus.
Datafor refinement of the portion of diet (PD) are evaluated in the position paper. In Spanish citrusorchards,the diet of Great Tits largely consistsof arthropods.The mean size of the prey broughtto the nestlingswas 15.5 mm on the average (Monros et al. 1997). Another study on the diet compositionof 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 1988were predominantlyfed with Lepidopterato about 87.8 % in numbers of 526 prey items (Barba and Gil-Delgado 1990). Among the lepidopterans49.8 %were imagines, almost exclusively Noctuidae, 23.6 %were lepidopteran caterpillars, 14.3 % lepidopteranpupae (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). Caterpillarswere the most abundant prey deliveredto the young only during 6-15 May, while Lepidoptera imagineswere more abundant during the rest of the season and thus were considered as an important resource for the greattits 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). Forthe size class of the remainder it isalso reasonable to expect preferentialfeeding on larger prey (Monros et al. 1997)
Conclusion: Based on the resultsof the above cited papers, great tits obviously inhabit citrus orchards and can serve as a representativefor a small insectivorous birdfor 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 Scandinavica21: 296-298. Belda et al. (1998): Laying date and cluth size of great tits Parus major inthe Mediterranean region: a comparisonof four habitat types. J. Ornithologie 139: 269-276. Gil-Delgado& Barba (1987): Aves nidificantes en huecosde 10s naranjos. Mediterranea Ser. Biol. 9: 29-40. La Manita (1982): Dati quantitativesull' 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): BritishTits. 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 knownto 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 relevancyof potato fields and other habitatsas feeding habitats for birds, census counts were carried out along differenttransects, representing all main agrarian habitats within the study area. Birdswere counted and observed during a slow-goingwalk 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, singinglbreedinglfeedingyoung, locomotion, resting, others) per passedfield were recorded. 20 

### Yellow Wagtails were trapped on and in the margin of potato fields, tagged with radio transmittersand 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 behaviourwas recorded continuously to get informationof the home range, habitat selection and time budget of birds using potato fields living in a cultivable areafor potatoes. Birdswere followed continuously by car. Emphasiswas 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 indicatesthe relation between the portion of time a given habitat was used and the spatial portion of this habitatwithin the birds' homerange: D = 

### (r 

### - p) 

### I (r 

## + p

## - 2rp), where r isthe portion the habitatwas used by the bird and p isthe portion of the same habitatwithin the homerangeof the bird. To get informationabout 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 unselectivelywere obtained from another GLP study, conducted at the same time in the same fields. At two fields, rainfallwas recorded daily. A logger for temperature and humidity measurementwas installed. Findinas: The results of the monitoringare shown inthe table below:PORTIONOF 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; basedon 20 individuals, 1to 5 24-hour sessions each [mean of individualsl,

I I I 

### . , FEEDING HABITAT of Yellow Waatails after transect Counts (based on DoDulation\ cerealfields Oil Seed rape street and field path



## I oil seed rape fields 

## 1 0.22 abundanceof potentially foraging individualsof 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



# Ipreference of potato fields as a feeding habitat

[Jacobs' index (D). MCP (100%)1 potato fields cerealfields 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 potatofields 

# 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 

# Isamples of faeces) comparedto meansizes 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 3.12 (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-trackingof 20 individualYellow Wagtails (eachfor a minimumof 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 provisioningor 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 proportionfor foraging as to be derived from the available proportion intheir home range [Jacobs' index (D)].Thus Yellow Wagtails positivelyselected potatoes as a foraging habitat, but only to a little extent. . 

### . [mm] (n, stratum3) 2.78 (523.f)

mean abundanceof main species after 9 transect counts covering 9.98 ha potatofields each 

# ' portion of samples containing this type

mean number of individuals per sample f =foliage dwelling arthropods, obtained by inventoryspraying; 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 individualYellow Wagtails were confirmed by census data of the populationof 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 PiedWagtail 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 

### percentile72.3 %). 

### Food of Yellow Wagtails in and around potato fieldswas dominated by Diptera, Coleoptera and Aphidoidea. In all taxonomic arthropod orders large individualswere 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 

### Effectsof 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 measuredfigures; 

### Table Error!

Study type Acute oral
Short-term dietary toxicity

Subchronicl reproductive toxicity

* foot lesions in all treatment groups

B.9.1.8 

### Risk assessment for birds The risk assessment for the EU can befound inthe Appendix at the end of section B.9. 

### The risk assessmentfor PMRA and US EPA is not includedin 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 (mglkgfeed) I

> 5000

> 6050 I
I
I 

### NOAEC (mgkgfeed) 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


### I6.9.2 Effects on aquatic organisms(OECD IIA 8.2, lllA 10.2)
6.9.2.1 Acute toxicity of active substance, metabolites and formulationsto aquatic organisms
6.9.2.1.1 Acute toxicity to fish (OECD IIA 8.2.1, IIA 8.11.I, lllA 10.2.2)
Active substance
Reference: Acute toxicity of BY1 08330 (tech.) to fish 

### (Oncorhynchusmykiss) 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: Measuredtest 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: O2content: Light regime: Test parameters: 10fish per concentration and control 2.3 

## + 1.1 g (mean 

## + SD) and 5.8 k 0.9 cm (mean 

## + SD) 0.58 g fishlltest medium Semi static test (renewal of test media every 24 hours), 96 hours
0 (controland solventcontrol), 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 Mortalityand sublethal effects were assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MSIMSand HPLC-UV) of BY1 08330 intest solutions sampleswere taken daily from new and aged test media. Test samples were directly injected into the HPLC-UVwithout 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. Behaviouraleffects: Controlsand concentration levels up to 0.825 mglL: No sublethal effectswere reportedover the whole test period.

Mortality: At test concentration 2 1.5 rnglL: After 96 hours following symptoms were noted: Remainedon 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-1NOLC: 1.5 mg/L I LC50 (96 h): 2.54 ma/L (95 % C.I. 2.11

### -3.05 ma/L)

Detailsof 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 Degreesof 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 

### (Cyprinuscarpio) 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 Directive92/69/EEC C.1 GLP: Yes Deviations: Measured test concentrationdropped 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) 10fish 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
Reconstitutedwater (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)

### O2content: 

### 97

### - 103 % 

### saturation Light regime: 

### 16 hours light

### / 

### 8 hoursdarkness 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 solutionssamples were taken dailyfrom new and aged test media. Test samples were directly injected into the HPLC-UV without centrifugation. Statistics: EC50: Probitanalysis, NOEC: directly from raw data Findinas: Analytical data: Overthe whole test periodthe mean measured concentrations were in the range 

### 69

## - 83 % 

### of nominal. Behaviouraleffects: 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: Remainedon the bottom of aquarium, loss of equilibrium, labored respiration, dark coloration, layingon the sides or backs,turned in a vertical

Mortality: position. Thus the NOEC was 

### 1.02mg/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.sJL] (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 

### (96h): 

### 2.59 mg/L NOEC 

### (96h): 

### 1.02mg/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 72 hours 48 hours 96 hours

### Reference: Author(s), year: ReportlDoc. number: Guideline(s): GLP: Deviations: Validity: -- 

### Acute toxicity of 

### BY1 08330 

### (tech.) to fish 

### (Lepomismacrochirus) 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: O2content: Light regime: Test parameters:

Statistics: Findinas: Analytical data:
Behaviouraleffects:

Mortality: Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014 Bluegillsunfish (Lepomismacrochirus) 10fish 

### 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



### -- (controland solvent control), 0.48, 0.95, 1.90, 3.56 and 7.61 mgR Aceton 0.2 mUL Reconstituted water (accordingto 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 

### 18 hours darkness Mortalityand sublethaleffects were assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MSIMS and HPLC-UV) of BY1 08330 in test solutionssamples were taken daily from new and aged test media. Test samples were directly injected into the HPLC-UVwithout centrifugation. EC50: Probit analysis, NOEC: directly from raw data
Overthe whole test period the mean measured concentrations were inthe 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 effectslike 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.9.2.1.1-3Details 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 Directive92169lEEC 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 15fish 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 (accordingto ISO), hardness: 40 

### - 60 mg/L as CaC03.Temperature: pH: O2content: Light regime: Test parameters:

Statistics: Findinas: Analytical data:
Behaviouraleffects: 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: O2content: Light regime: Test parameters: After the addition of test substancethe 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 sampleswere taken for chemical analysis (HPLC-MSIMS and HPLC-UV)of BY1 08330-enol in test solutions. None, because a limittest was performed
Over the whole test period the mean measured concentration was 98.5 % (replicateA) 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/Ltreatment 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 ultraturrax treatment. There was no indicationfor any unsolved materialafter mixing. Substance specific GC analysisshowed recoveries of >80% for all samples taken.
Zebra fish (Danio rerio) 10fish per concentration and control Not reported, 3.4 

## + 0.26 cm (mean 

### +. SD) 0.64 g fish/L test medium Static limittest, 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 18hours darkness Mortality and sublethal effects were assessed after 2, 24, 48, 72 and 96 hours. At 0, 24 and 96 hourssamples were takenfor chemical analysis (GS analysis andStatistics: Findinas: Analytical data:
Behavioural effects: Mortality: Conclusion:

Comments

Comments:



### Formulation MS detection) of 4-Methoxycyclohexanone in test solutions. Test sampleswere analysed directly without centrifugation. None, because a limit test was performed
The mean measured concentrationwas 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 effectswere observed at later time points. No mortalitywas 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 deviatedfrom recommendations given in OECD 202 and EU guideline, deviationfrom USEPA guidelinesare notable: -Minor: The temperaturewas 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 150to fish 

### (Oncorhynchusmykiss)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), spirotetramatcontent: 148.89 g/L (1 5.1 %), batch: 0803010189(0152) Rainbow trout (Oncorhynchusmykiss) 10fish per concentration and control 0.8 2 0.2 g (mean 

## + SD) and 4.6 & 0.3 cm (mean r SD) 0.20 g fishlltest 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 controlwas used
Reconstitutedwater (according to ISO), hardness: 40 

## - 60 mg/L as CaC03 11.6 

### - 12.2 "C 6.9 

### - 7.2 95 

### - 102 % saturationLight regime: 16 hours light

### / 8 hours darkness Test parameters: Mortality and sublethaleffects were assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MS/MSand HPLC-UV) of spirotetramat in test solutionssamples were taken at 0, 48 and 96 hoursfrom each concentration. Statistics: EC50: Logit and Probit analysis, NOEC: directlyfrom raw data Findinas: Analytical data: Overthe whole test periodthe mean measured concentrations of active substance were in the rangefrom 103to 107 % of nominal. Behaviouraleffects: Controls and 2.5 mg productllconcentration level: No sublethal effectswere observed over the whole test period. At test concentrations of 5.0 and 10 mg productll: After 96 hours following symptoms were noted: Remainedon 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 BY108330 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 calculatedby 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 

### Daphniamagna 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 850.1010, EU Directive 92/69/EEC C.2 GLP: Yes Deviations: Minor deviation from EPA guideline: The pH is lower and the hardness is higher than EPA-recommendedrange. 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 O2content: 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 mULAceton
M7 medium (according to "original draft" of an EEC Daphnia magna pilot ring test), hardness: 196mglL 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 16hours 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 (100 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 rangedfrom 42.7 

### - 81.0 % of nominal. After 48 hours no immobilitywas observed in the control, solvent control and in test concentrations upto 20.3 mg/L. At 38.8 and 42.7 mg/L the immobilisationwas 7 and 23 %. No sublethal effects were noted during the whole study. Thus the NOEC was determinedto 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 O2content: Light regime: Feeding
Test parameters:

Statistics:

Findinus: Analytical data:

Effects: 

### Acute toxicity of BY1 08330 

### (tech.) to larvaeof Chironomusriparius

### in a 48 

### h water-only study Dorgerloh, M., 2005 g EBFNX072, Edition Number: M-262632-01-2,08.1 2.2005 No specifiedguideline, 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 mULAceton
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 hoursdarkness Only on time 0.01 mL aqueous fish food solution directly after insertionof 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 Mortalityafter 48 hours [nl 3 4 

## [%I 7.5 10.0LC10 (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

* Resultof t-test is indicated no significant differencesbetweencontrol 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 instarswere 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 EBFNMOI2, Edition Number: M-254487-01-2, 14.07.2005 OECD 202, EPA 72-2, OPPTS 850.1010, 

### EU Directive92/69/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 limittest, 48 h
0 (control), 100 mg/L



## -- (control), 111.5 mg/LWater quality:

Temperature pH O2content: Light regime: Test parameters:

Statistics:
Findinas: Analytical data:
Effects:

Conclusion: 

### M7 medium (according to "originaldraft" 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 18hours darkness Immobilityand 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 immobilisationswere observed, NOEC: directlyfrom 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 determinedto 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 

### Chironomusriparius 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 isfollowing 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 (accordingto 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 insertionof thelarvae Test parameters: Mortality and sublethal effects were assessed after 48 hours. For chemical analysis (HPLC-MSIMS and HPLC-UV) of BY1 08330-enol inthe 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 

# I Exposed chironomids (= 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 [nl 

## 1x1 

# I mean measured 40 40 1

.O

64.0

Conclusion:

Comments: 0.95

67.59 

# I 40 

# I 16 

# I 40.0

Detailsof 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 is2nd or 3rd instar, I st instarswere 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 0Reference: Acute toxicity of BY1 08330-ketohydroxyto larvae of 

### Chironomusriparius 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 specifiedguideline, the study isfollowing 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 distilledwater): 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 O2content: 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 insertionof 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: directlyfrom 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)exposedto BY1 08330- ketohydroxy BY1 08330-enol [mgll] (nominal) Exposed chironomids (= 100 %) Mortalityafter 48 hours [nl 

## ["AConclusion:

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 deviatedfrom recommendations given in OECD 202 guideline, significant deviationfrom USEPA guidelinesare notable: -Age of the test organisms is2nd or 3rd instar, 1 st instarswere used here. -Test organisms should not be fed. -For invertebratesother than Daphnia,the test duration should be 96-hours. -Moltalityand sublethal effects should be measured every 24 hours.



### Reference: 4-Methoxycyclohexanone

# - Acute daphniatoxicity 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 Directive92169lEEC 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 O2content: Light regime: Test parameters:

Statistics:
Findinas: Analytical data: 4-Methoxycyclohexanone (metaboliteof spirotetramat), purity: 97.9 %, batch: SAV5506-001 Within the test, the item was soluble at the limit concentrationof 100 mg/L in test medium after ultraturrax treatment.There was no indicationfor any unsolved material after mixing. Substance specific GC analysis showed recoveriesof >80% for all samples taken. Waterflea (Daphnia magna) 2 replicates each with 10 daphnids per treatment and control First instar 

### < 24 hours old Static limittest, 48 h
0 (control), 100 mg/L



### -- (control), 111.5 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 effectswere assessedafter 24 and 48 hours. For chemical analysis (GC analysis and MS detection) of 4-methoxycyclohexanonein the test media sampleswere taken at test initiation (0 h) and termination (48 h). Test sampleswere analysed directlywithout centrifugation. None, because at the limit concentration (100 mg/L nominal) no immobilisation were observed
The measured concentrationsat the start and end of the test were 103and 90 %of nominal, overall mean measured concentration was 97 % of nominal. Effects: After 48 hours no immobility and no sublethal effectswere observed in the control and inthe limittest concentrations of 100 mg/L. Thus the NOEC was determinedto 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-Methoxycyclohexanonto Larvae of 

### Chironomusriparius 

### 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 specifiedguideline, 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 O2content: Light regime: Feeding
Test parameters:

Statistics: Findinas: Analytical data: 4-Methoxycyclohexanon (photo-metaboliteof spirotetramat), purity: 97.9 %, batch: SAV55506-001
> 100 mg (pH 8,20 

### "C) Midge (Chironomusriparius) 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), 111 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 sublethaleffects were assessed after 48 hours. For chemical analysis (HPLC-MS/MS) of 4-Methoxycyclohexanonin 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-testwas 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 O2content: Light regime: Feeding Test parameters: and 11 2 % of nominal, respectively. Overall mean measured concentrationsis 111 % of nominal. After 48 hours no mortality and no sublethal effectswere observed in the control and in the limit test concentrations of 100 mg/L. Thus the NOEC was determinedto be I00mgR 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 is2nd or 3rd instar, 1 st instarswere 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-cyclohexylaminocarboxylicacid to Larvaeof 

### Chironomusriparius in a 48 

### h Static Laboratory Test System (Limit-Test) Dorgerloh, M., 2007 b EBFNXI05, Edition Number: M-287220-01-1,27.04.2007 No specifiedguideline, 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: KTSI0330-1 -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 18hours darkness Only on time 0.01 mL aqueousfish food solution directly after insertionof the larvae Mortality and sublethaleffects 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). TestStatistics: 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 O2content: samples were analysed by direct injection into a HPLC-UV instrument without a centrifugation. 

### A limit-testwas 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 inthe control and inthe limit test concentrations of 100 mg/L. Thus the NOEC was determinedto 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 deviationfrom USEPA guidelinesare notable: -Age of the test organisms is 2nd or 3rd instar, 1st instarswere used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test duration should be 96-hours. -Mortalityand 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 150to larvae of 

### Chironomusriparius in a 48 h 

### static laboratory test system Dorgerloh, M., 2006 

### b EBFNM007, Edition Number: M-264018-01-2, 19.01-2006 No specifiedguideline, 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: 0803010189(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 18 hoursdarkness 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-MSIMSand 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,: Probitanalysis, 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 77to 95 %of nominal. See table B.9.2.1.2-6

Detailsof 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: 

### Effectson midge 

### (C. 

### riparius)exposed to BY1 08330 OD 150 (formulation)

Conclusion:

Comments: BY108330-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), correspondingto 0.36mg 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 instarswere 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 100B.9.2.1.3 Effectsonalgal 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: 

### Pseudokirchneriellasubcapitata

# - Growth inhibition test with BY108330 (tech.) Dorgerloh, M., 2004 c DOM 23092, Edition Number: M-128874-01-2,29.1 1.2004 Draft proposal for updatingOECD 201, OPPTS 850.5400, JMAFF guideline (12 Nousan No 8147) Yes EPA guideline: .The temperature is lessthan 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 centrifugedpriorto 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 replicatesfor 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 mULAceton 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-photometerat 578 nm) at 24, 48 and 72 hours. Morphological examinationswere 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 pooledcontrol.
Mean measured concentrations were in the range of 84 

### - 94 % and 22 

### -48 % of nominal concentrations on day 0 and day 3, respectively. Effects: Morphologicaleffects: Biomass & growth rate: None See table B.9.2.1.3-1Table Error! Style not defined.-1: Effects of spirotetramat (BY108330) onthe 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): 8.15 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: Inhibitionafter 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 significantdifferences, thus pooled control are usedfor

statisticalanalysis. * 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: Initialmeasured: Solvent: Test conditions:

Water quality: Temperature pH Incubation: Test parameters: yield"


### -- -54.7 -22.2 -8.6 46.7*

Toxicity of BY108330 tothe freshwater diatom 

### Naviculapelliculosa 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 (4for Navicula sp.). Growth rate


### -- -11.3 -5.3 -2.2 18.1*

5.6 mglL (5.46 -5.83 mg/L)

Acceptable 8.15 mglL (7.56

## -8.81 mg/L)

Spirotetramat (BY108330), purity: 97.1 %, batch: 0804510014
Freshwater diatom (Navicularpelliculosa) 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 mULAceton
AAP Nutrient medium (accordingto 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 densitieswere determined using a electronic particlecounter after 24, 48, 72and 96 hours. In addition the cell counts using a haemocytometerand a microscope were performed in order to confirmed measured cell densities. Morphological examinationswere made under a microscope on each study day. For chemical analysis (HPLC method) of test the substance, samples of test solutionwere 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 statisticalanalyses 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: Morphologicaleffects: None Biomass & growth rate: See table 6.9.2.1.3-2
Table Error! Style not defined.3: Effects of spirotetramat (BY1 08330) 

### on thefreshwater diatom N.



# I LOEC 

# 1 0.55 mg/L 

# 1 4.73 mg/L 

# I

pelliculosa BY1 08330 [mgR] (initial measured) 0 (woledcontrol) 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 Inhibitionafter 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 statisticalanalysis. * Significant difference (alpha = 0.05) from the control ** Statistically significantfrom control but not considered biologically significant 4.05 mg/L (3.73 -4.38 mg/L) Growth rate Inhibitionafter 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% inhibitionat the highest test concentration for growth rate. US EPA: The temperature is lessthan the EPA-specified temperatures (24-25"). Validity: Acceptable, even if the inhibitonfor growth rate is lower than recommendedthe 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: Morphologicaleffects: 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 mULAceton
AAP Nutrient medium (accordingto 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 densitieswere determined using a electronic particle counter after 24, 48, 72 and 96 hours. In addition the cell counts using a hemocytometerand a microscopewere 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 statisticalanalyses are based on data of pooled control.
Mean measured concentrationswere 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: Effectsof spirotetramat (BY1 08330) on the blue-greenalgae 

### 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 Inhibitionafter 96 h [Oh] 

# 1 Inhibition after 96 h [%I 0 (pooled control) ") 0.45 1.29 0 (pooled control)a' 0.3 0.718 

### -- -43 -15 

### -- 3 -13for statistical analysis. ** Biologicalsignificant based on % inhibition comparedto the pooledcontrolsConclusion: 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: 692-101-09- 0005
Green alga (Pseudokirchneriella subcapitata) 1 x 1 

## o4cells/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 (measuringthe extinction in a single-beam-photometerat 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 solutionwere 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 pooledcontrol.
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: Effectsof BY108330-enol(metabolite of spirotetramat) onthe 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 durationwas shorterthan 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 (metaboliteof spirotetramat), purity: 97.9 %, batch: SAV5506-001 Within the test, the item was added to the limit concentrationof 100 mg/L intest medium by weighing-in. There was no indicationfor any unsolved material after' mixing. Substance specific GC analysis showed recoveriesof 280%for all samples taken.
Green alga (Desmodesmussubspicatus) 5 x 

## lo3 cells/mL; 3 replicatesfor each concentration, 6 replicates for the medium and solvent control Static limittest, 72 h
0 (medium control), 100 mg/LMeasured (mean): Test conditions:

Water quality: Temperature pH Incubation:
Test parameters:

Statistics: Findinas: Analytical data:
Effects: Morphologicaleffects: 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 intensityof 4000 

### - 8000 IUX Cell densitieswere measured in a microcell counter or alternatively, were determined by means of a microscopiccountingchamber at 24, 48 and 72 hours. For chemical analysis (GC analysis and MS detection) of test the substance, samples of test solutionwere taken on day 0 and day 3. The test samples were directly analysed without a centrifugation. None, limittest
Mean measured concentration was 92.1 % on day 0 and 93.9 % day 3 of nominal concentration
None No significanteffects were observed at the limit concentration of 100 mg/L EbCS0 (0-72 h): 

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

### > 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 8147) GLP: Yes Deviations: EPA guideline: *The temperature is lessthan 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 replicatesfor each concentration, 6 replicatesfor 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 Untreatedtest medium, no blank formulation controlwas 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 inhibitionat highesttest 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 periodwere 42 

### -54 % of nominal. Effects: Morphologicaleffects: 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) onthe 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 (0-72h): 6.56 mg a.s./L correspondingto 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 substance6.9.2.1 -4 Effects on aquatic plants

### (OECD IIA8.6, lllA 10.8.2) Active substance
Reference: 

### Lemna gibbaG3

# - Growth inhibitiontest with BY1 08330 (tech.) under staticrenewaltest 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 intensitywas higher than the EPA recommended light intensity. -All test concentrations have not been centrifuged priorto analysis. -Precipitatewas 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 

### (Lemnagibba) 12fronds per vessel; 3 replicatesfor 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), 0.14, 0.47, 1.54, 6.06, 20.5, 71.4 mglL 0.5 mULAceton 20X-AAP growth medium (according to OECD guideline), the pHwas adjustedto 7.5 & 0.1 23.5 

### -24.8 "C 7.2 

## - 7.6 (new test media), 8.0 

### - 8.7 (agedtest 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 areawere determined using LemnaTec Scanalyzer machine on day 0, 3, 5 and 7, respectively. Dry weights of plants (neededfor yield calculations)were measuredat the end of the study (final biomass). For chemical analysis (HPLC-MSIMS, HPLC-UV) of test the substance (BY1 08330) samples of test solutionwere taken from freshly prepared test media (0 d, 3 d, 5 d) and from aged test media (3 d, 5 d, 7 d). Test sampleswere analysed by direct injection into an HPLC instrument without centrifugation. LOEC: ANOVA (a 

### = 0.05, one sided) and Dunnett's multiplet-tests
Overall mean measured concentrations rangedfrom 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 resultsof mirotetramat ~ctu~ncwrtra~on af Day Nominalronce?ntra8m 

### 1 Time 

### 1 E 1 &BY( 08330 

### 1 C4%tectian 11 Q&ection 21 Mean 

### 1 %of 

### / weishted

3a61& 3new 5 

### w-J 5 new

7 0 Sotvent control -=0.024B ~0.~326 eO.0245 

### - 

### i

3aged c 0.0245 .E A0245 0.f3245 

### - I 3new sag& 

# 1

5new

5 aged 5 Ftevu

T 0 2- 2 nw8 5aged 5 new

Effects: Morphological effects: None visual effectswere observed in concentration levels up to 0.47 mg/L. At 1.54 mgll slight chlorosiswas observed at day 7. At 6.06 mg1L and higher concentration levels strong chlorosis and necrosis were noted. Biomass & growth rate: Doublingtime Td of the control = 2.2 d (fulfilledthe 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 

### Lemnagibba based on time



# 1 number) 

# I I I 1 

# I 

# 1 Yield % inhibition in 7 d 97.6' S~irotetramat 

### ImgL1 (nominal) 100"' Growth rateyoinhibitionin7 d



### weighted mean measured concentrations Spirotetramat [mgR] timeweighted mean measured 71.4 On frond number 113.5* based on total frond area 116.1'

Growth rate (based on total area of plants Yield (based on frond number)

- -- - - a)The 100 mg/Lconcentration levelwas 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 biomasslyieldand 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: 

### LemnagibbaG3

# - 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) 12fronds 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, 11.7, 37.6, 119 mg/LTest conditions: Water quality: 20X-AAP growth medium (according to OECD guideline), the pH was adjustedto 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 illuminationwith white fluorescent light (mean intensity: 8.96 kLux) in a growth incubator. Test parameters: Biomass: Frond numbers and total frond areawere 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 solutionwere 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 114 % to 120 % on day 0 and from 97 %to 120 % on day 7 of nominal. Effects: Morphologicaleffects: None visual effects were observed in concentration levels upto 0.95 mg.L.At 2 3.5 mgll chlorosiswas noted. Biomass & growth rate: Doublingtime Tdof the control = 1.9 d (fulfilledthe validity criterion), see also table B.9.2.1.4-3

* Significantdifference 

### (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-enolis 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 possibleand 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/EECand the EU Aquatic Ecotoxicology Guidance Document. However inthe 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 inthe 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

### technicalto the sheepshead minnow (Cyprinodonvariegatus)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'sthe recommendationof 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: O2content: Light regime: Test parameters: Acceptable. Spirotetramat (BY1 08330), purity: 97.99 %, batch: 080451001 4 Sheepshead minnow (Cyprinodon variegatus) 20 fish per concentration and control 0.13 r 0.03 g (mean k SD), 1.72 & 0.14 cm (mean 

# + SD) 0.03 g fish/L test materialpassing through tank each day Flow-throughtest (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 mULAceton
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 lightldarktransition period Mortality and sublethal effectswere 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: directlyfrom 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 mglLconcentration level. After 96 hours at 2 1.I7 mglLeffects like loss of equilibrium and lyingon 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 mortalityof sheepshead minnow exposed to



# I NOEC = 0.52 mg/L LCso = 1.96 mglL (95%C.I. 1.64

### -2.37) basedon moving average method 

# I I LC50 = 2.10 mg/L (95%C.I. 1.73

### -2.52) based on Probitanalysis 

# 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 Intercepta: -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 

### (Americamysisbahia) 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 850.1035, 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 (Americamysisbahia) 2 replicates each with 10 mysids pertreatment 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 dilutedwith 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 18hours darkness Mortality and sublethal effects were assessed after 0, 24,48, 72 and 96 hours. For chemical analysis (HPLC method) of spirotetramat inthe test solution samples were taken at 0 and 96 hours. Test sampleswere analysed by direct injection into an HPLC instrument without centrifugation. ECBO: Binominal probabilityand probit analysis, NOEC: directlyfrom 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) = 4.27mg/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 Goodnessof 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 % (rangedfrom 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 O2content: Spirotetramat (BY108330), purity: 97.1 %, batch: 08045/0014 Eastern Oyster 

### (Crassostreavirginica) 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



## -- (controland 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 mortalitiesand sublethal effectswere 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: Williamstest, comparison of control groups: T-test. All statistical analyses are based on data of pooled control. Findinas: Analytical data: Overall mean measured concentrationswere 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) exposedto spirotetramat Spirotetramat[mglL]

(mean measured)

Pooledcontrol a) 0.20 0.33 0.55

. Meanshell deposition (mm 

### + SD)

3.5

## + 1.2 3.8

## + 1.2 3.4

## + 1.1 2.4

## + 1.0 Inhibitionof shell growth [Oh]



## -- +7.9

3 31*NOEC = 0.33 mg/L EC50 (96 h)= 0.85 mg/L (95%C.I. 0.59 - 1.3mg/L) a' no statistically significant difference between dilution control and solvent control * statistically significant differencefromthe 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 updatingOECD 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 priorto chemical analyses. Validity: Acceptable
Test substance: Material and methods: Test species:

Number of organisms: Type of test, duration: Applied concentrations:

Nominal: Initialmeasured: Solvent: Test conditions:

Water quality: Temperature pH Incubation:
Test parameters: Spirotetramat (BY1 08330), purity: 97.99 %, batch: 0804510014 Marine diatom (Skeletonema costatum) 1x 

## lo4 cells/mL; 3 replicates for each concentration, medium control and solvent control Static test, 96 h
0 (mediumcontrol 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 mULAceton
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 densitieswere determined using an electronic particle counter after 24, 48, 72 and 96 hours. In addition the cell counts using a haemocytometerand a microscopewere 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 concentrationswere inthe range of 94 

### - 106 % 

### and 25 

### -46 % 

### of nominal concentrations on day 0 and day 4, respectively. Effects: Morphologicaleffects: None Biomass& 

### growth rate: See table B.9.2.1.5-4
Table Error! Style not defined.-4: Effectsof spirotetramat (BY1 08330) 

### on the marine diatom 

### S. costatum.

BY1 08330 [mgR] (initial measured) 0 (pooled control) a) 0.24 0.622 1.63 4.15 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 Cumulativebiomass Inhibition after 96 h [Oh]


### -- -22

0.124 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 Inhibitionafter 96 h 

### [%I

-- -10 27* 61

* 115* 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

Chironomusriparius. . , , a' LC10, NOECwas not determined "Limit test Test organism Test 

# 1 condition 4-Methoxycyclohexylaminocaboxylic

Test conc.: test concentrationbased on mean measured (mm) or nominal horn)concentration Time acid

48hr

96 hr

48 hr

72 hr Chironomus riparius Midge Formulation

## - BY108830 Oncorhynchus mykiss Rainbowtrout Chironomus riparius Midge

P. subcapitafa Green alga staticb,(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

Yes8.9.2.2 Chronictoxicity of active substance, metabolites and formulationsto aquatic organisms
B.9.2.2.1 Chronictoxicityto 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 toxicitytest 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: O2content: Light regime: Feeding

Test parameters:

Statistics: Early-lifestage toxicity of BY1 08330 tech. to fish (Pimephalespromelas) Dorgerloh, M., 2005 b EBFN0305, Edition Number: M-260676-01-2, 16.11.2005 EPA 72-4a, EPA OPPTS 850.1 400, OECD 210 Yes None of relevance Acceptable Spirotetramat (BY108330), 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 individualsper 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 (accordingto ISO), hardness: 45 

### -52 mg/L as CaC03 24.5 (23 

### -26) "C 7.0 

### - 7.3 during the totaltest 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 hatchedshrimp nauplii adlibidum. Feeding was started shortly after hatching. Uneaten food an faeces were siphoned in order to minimise microbiological growth and biodegradationof test substance. Embryo mortality, hatched larvae, fry mortality and abnormal appearance or behaviour were assessed weekdays. At test termination the length andthe weight were determined. Determined endpoints were: Time to hatch, hatching success, overallfry survival (fry survival before and after thinning), standard length and dry weight. For chemical analysis (HPLC-MSIMS and HPLC-UV) of BY1 08330 intest solutions samples were taken at -1, 0, 7, 14, 21, 28 and 33 days If control and solvent control can be pooled: T-test Testingfor normal distribution: Ws procedure Time to hatch, hatching success, fry survival: Williams test with previous acrisinetransformation Growth data: Williams test without previous data transformation Findinas: Analytical data: Overall mean measured concentrations in test mediawere 93 

## - 11 9 % of nominal. Biologicalobservation: 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 effectswere 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 differencewhen compared to pooled control (alpha= 0.05)
Table Error! Style not defined.9: Length and Weight

* significant differencewhen 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: 1.16 mg/L Hatchability and growth: NOEC : 1.16 mg/L, LOEC > 1.16 mg/L based on mean measured concentrations Length [mm] Test termination (phd28) Mean (SD) 18.0 (3.5) 18.4(3.4) 18.3 (3.4)

B.9.2.2.3 Fish life cycle test (OECD IIA8.2.5, lllA 10.2.5.3) Dry weight [mg] Test termination (phd 28) Mean (SD)

- 20.4 (7.4) 21.8 (11.4) 21.4 (11.3)

No study submitted. The logpowof spirotetramat is 2.51 and the potentialof bioaccumulation is expectedto 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 toxicityto aquatic invertebrates(OECD IIA8.3.2, 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 O2content: Light regime: Feeding
Test parameters:

Statistics: - - 

### Influenceof BY1 08330 (tech.) on development and reproductive output of thewaterflea 

### Daphnia magna in a static renewal laboratory test system Dorgerloh, M., 2005 e EBFN0245, Edition Number: M-251843-01-2, 13.05.2005 OECD 211, EEC Directive C.20, US EPA 72-4, OPPTS 850.1300 Yes The concentraton of the solvent (acetone) is slightly higher than recommended in OPPTS 850.1300 (0.2 ml1L instatedof 0.1 ml1L). Howeveran additional solvent control was addedto make sure that the solvent was not initiating any toxic response, thus no negative impact on the results can be concluded. Acceptable Spirotetramat (BY108330), 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, 19days
0 (control and solvent control), 0.05, 0.13, 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 mULAceton
M7 medium (according to "originaldraff' of an EEC Daphniamagna pilot ring test), hardness: 196 mg1L as CaCO, 20

## -20.9 "C 7.0 

## - 7.3 (newtest 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 (dryweight) 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 Bartlettstest and Kolmogorroff-Smirnovtest. 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 BinominalTest with Bonferroni Correction Reproduction:T-test Body length: Dunnett's Test Parental body mass (dry weight): U-test after Bonferroni-HolmStatistical analyses concidering nominal concentrationsare 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 

### Daphniamagna

* Statistically significantlydifferentfrom pooled control (alpha= 0.05) a' Statisicswere performed using only negativecontrol Conclusion: Mortalityadult: 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 nominalconcentrations.

B.9.2.2.5 Chronictoxicity to sediment dwelling organisms 

### (OECD IIA 8.5.2)

- - -- Reference: 

### Chironomusriparius28-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 concentrationof the used solvent control was higher than recommended(0.2 mUL instead of 0.1 mUL), however no significant differences betweensolvent control and the water only controlwere noted. Validity: Acceptable
Test substance: Spirotetramat (BY1 08330), purity: 97.2 %, batch: 0804510014Material and methods: Test species: Midge (Chironomusriparius)

Number of organisms: 4 replicates each with 20 larvae per treatment and control Age: First instar (LI) larvae Type of test, duration: Static test (spikedwater exposure), 28 d Applied concentrations:

Nominal: 0 (control and solvent control), 0.05, 0.10, 0.20, 0.40, 0.80, 1.60, 3.20 mgR Initial measured: 

### -- (control and solvent control), 0.058, n.a., n.a., 0.401, n.a., ma., 2.95 mg1L Solvent: 0.2 mULAceton 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 % CaC03to adjust the pHto 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 biologicalevaluation and 1for 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 O2content: 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 ornamentalfish 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 differencescomparedto controlwere observed 3 times per week. For chemical analysis (HPLCIUV) of spirotetramat in the overlayingwater column and the pore water of sediment samples were taken from fresh after 1 hours, 7 and 28 daysfrom 0.05, 0.4 and 3.2 mg1Ltreatment. Statistics: Sex ratio: chi2-test EC50:Probitanalysis LOEC: ANOVA and multiple t-tests Comparison of control groups: Student-t test for homogeneous variances All statisticalanalyses 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 inthe overlaying water and the porewater

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 

# Ix 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 Porewater 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 -- 

### -- 0a) means of two samples b' calculated to the realvolume of porewater and the applied amount of a.s.

Spirotetramat

[mg a.sJL]
0.40 3.20

Effects: Sex ratio: No relationshipbetween treatment and sex ratio was found, therefore number of males and females midgeswas pooledfor further endpoint calculations. Start of emergence: Incontrols the emergence started on day 14; at concentration levels up to 0.2 mglLfirst midges emerged on day 15; at 0.4 and 0.8 mg/l emergence started on day 17and 18, respectively Forfurther 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 significantdifferences between control and solvent control, thus pooled control are usedfor statisticalanalysis. %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 nominalconcentrations. While no major deviationsare notedfrom guidline OECD 219 significant deviation from USEPA recommendations are notable: The EPA requiresthat the sediment be spiked with the test compound and allowed to equilibrate with overlyingwater priorto test initiation; the overlying water was spiked in this test and equilibriumwas not achieved. EPA also requires that mean-measured, rather than initial measuredvalues, be usedto perform statistical analyses and report endpoints. % of nominal 0 0EPA also recommends having endpoints calculated based on measured concentrations in overlyingwater, bulk sediment, and interstitialwater. EPA recommendsa pH in the range of 6 to 7, the pHvalues measured in the test were outside this range.

B.9.2.2.6 Microcosmos or mesocosmosstudy (OECD lllA 10.2.3) No study submitted. Not required, becausethe 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 relevantfor riskassessment 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 potentialof active substance in fish (OECD IIA 8.2.6.1) No study submitted. Not required, because spirotetramat has a logpow2.51 and a bioaccumulation study is not triggered. Test condition

B.9.2.3.2 Bioconcentration potentialof metabolites in fish (OECD IIA 8.2.6.2) Environmentalrelevant metabolites are BY1 08330-enol, BY1 08330-ketohydroxy (both metabolites are major metabolites in water and sediment of waterlsediment study), 4-methoxycyclohexanoneand 4- methoxycyclohexylamino carboxyl acid (both are major metabolites in photolysis study). The logpowof 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 logpowwas not provided. Howeverfor these metabolitesthe logpowwas estimated by RMS using the program ALOGPS 2.1. The logpowfor 4-methoxycyclohexanone and 4-methoxycyclohexylamino carboxyl acid was calculatedto be in the rangefrom -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 inthe Appendix at the end of section B.9. The risk assessment for PMRA and US EPA is not included inthis monograph. Test conc.: test concentrationbased on mean measured (mm) or nominal (nom) concentration Pimephalespromelas Fathead minnow
Daphnia magna Waterflea Chironomusriparius Midge (larvae) hatchability fry survival

growth mortalityadults

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

Dorgerloh2005bl Yes Dorgerloh 2005e/ Yes Dorgerloh 2005il Yes Reference1

study acceptable8.9.3 Effects on other terrestrial vertebrates 

### (OECD lllA 10.3) Toxicity to mammals
Table 8.9.3-1 : Ecotoxicologically relevant mammaliantoxicity endpointsfor spirotetramat 1

" equivalentto 306 g a.s. Acute oral LDS0 Acute oral LD50formulation Reproductive NOAEL

6.9.3.1 Risk assessmentfor mammals The risk assessmentfor 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 Effectson bees (OECD IIA 8.7; OECD lllA 10.4.) B.9.4.1 Acute Toxicity (OECD IIA 8.7.1 

### ;OECD IIA8.7.2; 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: Effectsof BY1 08330 (Acute Contact and Oral) on Honey Bees 

### (Apis mellifera L.) inthe Laboratory Schmitzer, S.; 2004 20091 035; M-081227-01-2 OECD 213 & 214 (1998) Yes ventral application inthe contact test insteadof 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 itemtreatment groups Control: oral test: sugar solution with tap water and sugar solution with solvent acetone, contacttest: 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; contacttest: 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 offeredto the bees in syringes which were weighted before and after introduction inthe cages. Commercial ready-to-usesyrup (30 % saccarose, 31 % glucose, 39 %fructose) Test unitswere stainless steel cages of 10cm x 8.5 cm x 5.5 cm, the front side was a removable glass sheet, the bottomwas perforatedwith 98 ventilation holes 

### (0 1 mm), the inner walls were linedwith filter paper. Bees were anaesthetised with C02in the contacttest. Exposuretime: 48 h. Temperature: 25"C, relative humidity: 58 

### - 72 %, darkness (except during observation), ventilation to avoid possibleaccumulation of pesticide vapour Mortality and behavioural abnormalitieswere assessed4, 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 calculationwas carried out taking into account the mortality data corrected by control mortality using Abbott's formula (1 925). The software usedto perform the statistical analysiswas ToxRat Professional, Version 2.07. Findinas:The resultsof the mortality and behavioural assessment are presented inTable 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: Mortalityand behaviouralabnormalities of the bees in the contacttoxicitytest



## .. 

## - .. . 

# . - 

## ..

Table B.9.4.1-2: Mortality and behaviouralabnormalities of the bees inthe oral toxicity test (results (resulrs are averageTrom 

### 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 averagefrom 5 

### replicates per dosageJcontrol)

48 h oral LD50

### > 107.3 pg a.s./bee 48 h contact LD50

### > 100 pg a.s./bee As deviationsto EPA 

### OPPTS guidelines the test substance (oral) was offered in syrup instead of a 50 % sugar solution and ~dhasit~is not an 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 laboratoryconditions. Barth, M.; 2005a+b 05 1048 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 beesfor controls, toxic reference and the test item treatment groups Control: oral test: 50 % sucrose solution, contacttest: water and 0.1 %v/v Tween solution; test item: oral test: applied volume 

## - 20 pLsucrose 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 micropipetteto the dorsal thorax. Application in the oral test: Bees were fed with a defined quantity of sucrose solution containingthe 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 beeswas counted at 4,24 and 48 h. During assessmentstimes any behavioural abnormalities of the beeswere recorded, considering the following parameters comparedto control: healthy or affected (paralysis, lateral position, lying on the back), any differences in activity, in positionwithin 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 12.5Statistics:

Findinas: excretion. Mortalitywas corrected according to Abbott (1 925). For statistical calculationof the mortality resultsthe Fisher's Exact Binomialtest was used. The accepted significance levelwas p 

### I 0.05 (one-sidedgreater). The median lethal dose (LD50) along with the 95 % confidence limitswas 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 modelwas evaluated by Pearson's X* test. The calculation of statisticalsignificance and the LD, was performed with the computer programToxRat Professional2.09.
The resultsof the mortalityand behavioural assessmentare presentedinTables B.9.4.1-3 and -4. The validity criteriawere met as mortality in the controlwas I 10 % after 48 h and the contact and oral LD50of the toxic reference were 0.263 pg a.s./bee and 0.194 pg a.s./bee, respectively. Inthe oral toxicitytest statisticallysignificant 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 productlbeemost bees were affected at the 4 and 24 h assessment. Bees had recovered at the 48 h assessment. Inthe contact toxicity test no effects on behaviourwere 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 ProbitAnalvses LDS0 = 607.18 uglbee 95% Confidence Limits of LD5~ 506.17, 737.88 Intercept = -10.21 Slope 

### = 3.67; Standard error of slope=0.55 95 % Confidence Limitsof Slope = 2.59, 4.74 

# ~oodn'ess-of- it Test: Pearson Chi-Square value= 1.95; Degreesof 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-FitTest: Pearson Chi-Square value= 2.90; Degreesof freedom= 3; p(Chi-Square)=0.41

### Table B.9.4.1-3: Oral and contact toxicity LDS0 values of beestreated with BY1 08330 150 OD after 24 and 48 h Test item
BY108330 150 OD

Referenceitem
Dimethoate EC 400



### Table B.9.4.1-4: Mortalityof 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%-cllower 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 contacttoxicity 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 0.214

6.7Conclusion: 48 h oral LD5, = 607.1 productlbeeequivalent to 91.7 pg a.s./bee 48 h contact LD50 = 1072.9 pg productlbee equivalentto 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 inthe oraltoxicity test (resultsare averagefrom 3 replicatesper dosage/control)

6.9.4.2 Residuetest (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 consumedas./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) MortalityPA]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: SpirotetramatOD 100

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

### (Apis 

### melliferaL.) in a Feeding Test Author(s), year: Schur, A.; 2004a ReportlDoc. number: 20031168/01 -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:
Materialand 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 0.0144 % 

### a.s. (analysed; 1371 mg product per colony); toxic reference: 300 mg lnsegar 25 

### WG per colony (a.s.: 25 % 

### fenoxycarb) (dissolved inwater 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 nectarwere found in the near surroundings. All colonies were in good conditionat 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. Mortalityof adult bees, pupae and larvae, behaviour of the bees aroundthe hives, conditionof the colonies and the developmentof the bee broodwere 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 mortalitytook 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 influenceof the test substance was evaluated by comparingthe 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 pupaewere examined for specific effects of IGR's. None in original study report. In2007 statistics was performed using t-test and Dunnett's t-test in an one-sided approach.
The test was considered to be valid becausethe following parameterswere met: A detectable effect of the toxic standardwas 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 betweenthe colonies inthe control and the colonies in the toxic standard treatment.Regardingthe total daily mortality in the test substance treatment, a slightly increased mean mortalitywas noticed in the test substance group on DAF 6, 13 and 15. Throughout the entire post-feeding period a sum of 55 dead pupaewere counted in the test substance treatment comparedto 29 dead pupae in the control group. A sum of 5425 dead pupaewere counted duringthe entire post-feeding period in the toxic standard which is evidence that lnsegar 25 

### WG massively disturbed the brood developmentof these test colonies. The sum of dead pupae and adultswere analysed seperately over the postfeeding period (controlvs treatment and control vs toxic standard). As result, no statistically significant effect on pupae mortalitywas detected, but there was a statisticallysignificant difference in adult bee mortality between control and treatment (t-test, pooled, one-sided, p=0.0447). Honev bee fliaht intensitv: Regarding the flight intensityobserved in front of the hives no remarkable differenceswere observed at both post-feedingassessment 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 regardingthe 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 studywith 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-feedingmortality Pre average value for the pre-feeding period DAF 3to 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 numberof bees leavinglenteringthe 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 1to 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 controltreatment. 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 developmentcycle. 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 noticedfrom BFD (brood fixing day) to BFD 5. 

### The cellswhich showed 

### aterminated brooddevelopment 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 refilledagain with new brood. Hive 2 and 3 of the test substance treatment were not able to compensatethe effect onthe brood development caused by the test substance untilthe end of the observation period. No clear effect which could be attributedto the test substancewas observed at hive 1. Honev bee behaviour in front of the colonies: No differences regarding the behaviour of the beeswere noticed betweenthe test substance treatment, the toxic standard treatment and the control. Itwas concluded that the feeding of Spirotetramat OD 100 had a slight but in the context not biologicallysignificant effect on adult and pupae mortalityof 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 hightermination rate was noticed 4 days after start of feeding in two of the three test substance colonies. Inthese colonies the further brood development was massively disturbed at the following assessments andthey were not able to compensate the effect on the brooddevelopment until the end of the observation period. No effectwas detected by the regular broodassessments.

Formulation: SpirotetramatSC 240

- - 

### Reference: Assessment of the Side-Effectsof 

### BY1 08330 

### SC 240 

### on the Honey Bee (Apis mellifera L.) 

### in a Feeding Test. Author(s), year: Schur, A.; 2004b RepotVDoc. number: 20031166101-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.: 0721810013, 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 accessto 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 beeswere 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, pupaeand larvae, behaviour of the bees around the hives, observations: conditionof the colonies andthe developmentof the bee broodwere assessed before and after the feeding of the test substance. Wooden bee traps (Munsterdesign) with gauze on bottom and on 100 % 

### of the top were attached to the entrance of the colonies in order to register those dead beeswhich 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 inthe 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 influenceof the test substance was evaluated by comparingthe results in the test substance treatment to the control data and those from the toxic standardtreatment and furthermore by comparing the pre- and post-feeding results. Dead larvae and pupaewere examined for specific effects of IGR's. None.
The test was consideredto 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 inthe broodtermination rate in cells between the colonies inthe control and the colonies in the toxic standard treatment.

### Table Error! Style not defined.9: 

### Mortality and flight activityof honey bees in the feeding study with S~irotetramat SC 240

Regardingthe total daily mortalityinthe test substance treatment an increased daily mean mortality was noticed on DAF 6 as well as on DAF 15to 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 periodcompared to 29 dead pupae inthe control group. A sum of 5425 dead pupae were counted during the entire post-feeding period, mainly between DAF 10and 21, inthe toxic standardwhich is evidence that lnsegar25WG massively disturbed the brood development. Honev bee fliaht intensity: Regardingthe flight intensity observed infront of the hives no remarkable differenceswere observed at both post-feedingassessment dates DAF 1 and 3. Condition of the colonies: During the assessmentsof 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 controltreatment. 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 comparingthe individual brood assessments of single cells, the indices (thevalues of the different brood stages of all cells in DAF days after start of feeding Obf mortalityon the day of start of feeding beforefeeding QM (average) 0 post-feeding mortality + 0 pre-feeding mortality Pre average value for the pre-feeding period DAF-3 to Obf post (DAF 1to 21) average value for the post-feedingperiod DAFI to 2 P - - dead pupae SD = Standard deviation * mean numberof bees leavinglenteringthe hive per minute Test substance 

### (BY108330 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 bythe number of observed cells) of the control group showedthe course to be expected in a natural bee development cycle. In all coloniesof the test substance treatment a termination of the brood in almost all cellswas noticed from BFD (Brood area fixing day) to BFD 5. Hive 1 and 2 were able to compensatethis 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 1to 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 1to 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 situationof the marked brood areawas similar on BFD 11 comparedtothe previousassessment because the cells remained empty until this assessment day and even at BFD 15the brood index was still below the level of 1 (egg stage). These observations indicatean effect of BY1 08330 SC 240 to the brood development in the first 5 to 15 days after start of feeding. Neverthelessthe brood indicescontinued to rise from BFD 11 to 22 in all test substance colonies and at the final brood assessmentthe big part of the cells contained brood. Honev bee behaviour infront of the colonies: No differences regardingthe behaviour of the beeswere noticed betweentest substance treatment, toxic standard treatment and control. Regardingthe flight intensityobserved in front of the hives no remarkable differences were observed at both post-feeding assessment dates DAF 1 and 3. Itwas concluded that the feeding of BY1 08330 SC 240 caused an increase of adult honey bee mortality 6 and 15to 17 days after start of feeding but did not result in an adverse effect on the flight activity andthe behaviour of the bees in front of the hives. An almost total termination of the brooddevelopment 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 broodat 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: 20061133lS1 -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.: 0803010189(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 brooddevelopment Number of organisms: Four normally developed, healthy and queen-right colonies were used per test field. Each colony contained one bodywith 12frames and approx. 20000 bees 

### 1 colony. Treatment: Three test fields:



### - test item treated field TI (4x 

### 72 g a.s./ha, two times before flowering (no bees in the field) and 2 x during floweringof the crop and foraging activity of the bees)


## - test item field T2 (2 x 

### 96 g a.s./ha during floweringof the crop and foraging activity of the bees)



### - untreatedcontrol field The applicationswere carried out in approximately7-day intervals in 200 L 

### waterlha. Calibrated commercial spraying equipmentwas used. Experimental The field test was located in Spain (region: Valencia). The crop Phacelia

### procedure:

Statistics: Findinas: tanacetifoliawas used in flowering stage. The field sizeswere: test item field TI: 

### 2025 m2,test item field T2: 2016 m2 and the control field C: 3306 m2.The test fields were not close to other flowering crops or extensive bloomingweeds, which may be attractive to bees andwere separated by a distance of at least 2 km. Commercial bee colonies were placed near the test fields 3 days beforethe 3'C' application in the test item fieldTI and before the 1' application in the test item fieldT2, 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 governmentalweather station in Almansa, approximately20 km (T1 andT2 field) and 25 km (controlfield) away fromthe field site. The degree of cloudcoverage and wind speed during applications was assessed at the test fields. Test parameters, Exposure of the beeswas assessed byvisual inspection of the P. 

### tanacetifolia observations: pollen on combs entered inthe hives during the study. The influence of the test item on the honey bees was evaluated by comparingthe resultsof the test item treatments to those of the controltreatment. The following pointswere assessed:

• 

### Condition of the colonies (strength) and development of the bee brood Mortality inthe field and in the bee traps in front of the hives

• 

### Foragingactivity (number of forager bees/m2/minute flowering crop)

• 

### Behaviourof the bees on the crop and around the hive Flight intensity and foraging activitywere assessed in 5 

### marked squares (1 m2) that were regularlydistributed. Mortalityand foraging activity of the beeswere checked priorto first applications in the test fields (over 2 days and shortly before first application) and followed up after application (foragingactivity: over 10days after first application during exposure, mortality: over 28 days after first application during exposure). In order to recordthe number of dead bees water-permeable linen sheets were spread out infront of the hives. Dead bee traps were attached to the entrance of the hives in order to register those dead beeswhich were carried out of the hives. Furthermore, mortality was recorded at three impartially selected places in each test field using linen sheets. Dead beeswere 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). Collectionof flowers: Forthe possible analysisldetermination of residuesof 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 (01MayO6, beforeflowering): application 

# I I I I 72.0 

# I I 

# I rate g a.s./ha 

## I 

## I 1 2"* application date (08May06, beforeflowering): 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 floweringlbeeflight): application rate g a.s./ha 4'h application date (24May06, during floweringlbeeflight):

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 Oaato 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 daato 28) 

### .

bees/minute/m2]

ba = before application inthe test item fields during the exposure period aa = after application in the test itemfields during the exposure period DAA = days afterfirst applications inthe test itemfields during the exposure period QM = post-application[DAA Oaa to 281 1pre-application[DAA -2 to Oba] 42.0 15.0


### .

11.3 15.0 Meanflight 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 inthe test item group TI, 16.3 foraging bees/minute/m2in the test item group T2 comparedto 12.2 foraging bees/minute/m2in the control group. The degree of P. tanacetifolia pollen from the total amount of pollen per colony rangedfrom 10 % up to 33 % during time of exposure (brood assessments on 19May06and 25May06) at the test field in the test item group TI, from 8 % upto 40 % at the test field T2 and from 6 % up to 85 % in the control field. The resultsof the pollen assessments in the colonies confirm that the beeswere actively foraging on the test fields. A quantitative comparison between the resultsof the treatments is not possible, because the foraging and storage of pollen in a bee colony depends on outside conditionsas 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 beforethe 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 inwhich this slightly increased mortality has been observed, no preexposure applicationwas 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 beeswas 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 inthe number of dead bees of the treatment groupsTI and T2 comparedto 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 applicationto 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-applicationduring 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 inthe 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 inthe treatment group TI, 0.1 dead pupae and larvae/colony/day inthe treatment group T2 and ~0.1 dead pupae and larvae/colony/day in the control group. Since larval and pupae mortalitywas at a comparable level in all treatment groups, there were no treatment-relatedeffects. Honev bee fliaht intensitv: The daily mean flight intensity (foraging bees/minute/m2)beforethe 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 inthe treatment groupT2 as well as inthe control group. On the second application date (DAA 7) no reduction inthe 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 intensitywas 11.9 foraging bees/minute/m2inthe test item group TI, 16.3 foraging bees/minute/m2in the test item group T2 comparedto 12.2 foraging bees/minute/m2 inthe control group. The intense flight andforaging activity of the bees on the test fields was also supported by the fact that the bees stored actively P. tanacetifolia pollen inthe combs of the colonies of all treatment groups. Condition of the colonies and honev bee brood develo~ment Assessments of the colony strength asjudged by number of bee ways between combs filledwith bees and the brood nest size (number of brood combs per colony) did not indicate significant differencesbetween treatment groups T1, T2 and the control colonies. On the frequent assessments during exposure in the test fields and afterwards (upto 29 days after the first brood assessment) 3 colonies of the test item treatment TI and all colonies inthe test item group T2 and in the control group showed all broodstages and a similar development. One colony in the test item treatment TI showed a lack of eggs orland larvaeon the last 2 assessment dates. Inthat colony a tendency to swarm or to remove the old queen was recorded (queen cells on combs) startingfrom the first brood assessment after set-up of the colonies at the test fields. It is very likelythat this was caused by a lack of space for the colonyto 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 conditionover the entire test period it isvery unlikelythat the lack of brood was caused by the applicationof the test item. Honev bee behaviour in front of the colonies and within the croD area No differences regardingthe behaviour of the beeswere 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. Differencesof bee behaviour between control and treatment groupswere not observed. The conditionof the colonies as assessed by colony strength and size of the brood nest was not affected by any of the treatments. Fromthe detailedassessment of the honey bee brood status one colony inthe test item treatment groupTI 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) startingfrom the first brood assessment after set-up of the colonies at the test fields. It isvery likelythat 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 satisfiedwith the old queen. No evidence of an irritationor termination of the development based upon exposure to treated crops was obtained in the other colonies of the test group TI as well asin 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 20041144101-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-fieldconditions. Yes (certified laboratory) No Acceptable Spirotetramat OD 100(a.s. BY1 08330), Batch no.: 08030101lO(0073) Tox no.: 06593-00, content: 102.02 glL (analysed) Apis mellifera L. (Hymenoptera:Apidae) Semi-field test (tunnel) with special attention to the developmentof 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)lhaand 2 x after set-up of the colonies at 96 g a.s. (923.11 g product)/ha. Treatment group T2: test item applied once, 3 d after set-up of the colonies at a rate of 96 g a.s.ha; toxic reference: lnsegar

### WG 25 (a.s.: fenoxycarb, 25 %) 150g a.s. per ha. All treatmentswere applied in 400 Lwaterha using a plot sprayer. The semi-fieldtest 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-exposuretunnel tents were set-up in the test field. The area covered with rape was 47.52 m2per tunnel. The tunnels hada 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). Beforethe start of bee-exposure inthe tunnels a path was created along the tunnelwalls at both front sides and through the middle of each tunnel by removing the plants and levellingthe ground. Subsequently, the path was covered with linen sheets. For the test, small healthy colonieswith 12 combs were used. All nucleiwere 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 bodieswith 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). Therewere 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 attentionwas directed to the brood development of the colonies. The influenceof 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 Foragingactivity (number of forager bees/m2 flowering rape) Behaviourof 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 mortalitywas recorded inthe crop. Therefore the oilseed spring rape (Brassica napus)was removed priorto the set-up of the hives and water-permeable linen sheets (covered area approximately 15 m2)were spread out inthe 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 periodof 10 days the bee colonies were taken out of the tunnels and transferredto an areawithout flowering main crops and where no pesticideswere applied during the time of assessments. Mortality of adult honey bees inthe 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 andfive times afterwards (4, 9, 16, 23 and 29 days after BFD). Assessment of the development of the bee broodtook place at the BFD (egg), 4 days (youngto 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 transferredto 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 statisticswas performed using t-test and Dunnett's t-test in an one-sided approach..
The test was consideredto be valid because a detectable effect of the toxic standard was found (brood termination before a successful hatch, increased



### . mortalityof 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 studywith Spirotetramat 

### OD 100 

# I Exposure 

# 1 TI: five spray treatments of BY108330 OD 100three before 

# I I

- 

## I and two during foraging activity inflowering oilseed 

## I

Treatment arou~ spring rape T2: one spray treatment of BY108330 OD 100during foraging activity inflowering oil-seedspring 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 Lwaterlha]

post (BFD +2aato +23)

Regarding the total daily adult bee mortality observed during the exposureof the beesto 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 standardtreatment. 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). Inthe 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 12dead pupae in the test item treatment Tl, 29 dead pupae in treatment T2,8 dead pupae in the control treatment comparedto 300 dead pupae in the toxic standard treatment. The sum of dead pupae and adults were analysed separatelyover 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 comparedto 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 assessmentsof the condition of the colonies throughout the study no remarkable observations were made regarding the strength of the colonies and the brood nest size inthe 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 BFDto BFD +2ba (before application) post (BFD +2aa) main application day after application post (BFD +2aato +9) mean value for BFD +2aato BFD +9 post (BFD +2aato +23) mean value for BFD +2aato BFD +23 QM(average) mean mortalityper day after application divided by mean mortalityper day before appl. BFD brood area fixing day 12
10.3 19.0 14.5 Daily mean flight intensity [foraging bees/m2/replicate] 150g a.s. 11.2
1.7
4.5

pre post (BFD +2aa) post (BFD +2aato +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 comparedto the controltreatment. 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 brooddevelopment: 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 periodwhich resulted in a retarded increase of the brood index from BFD to BFD +9 comparedto 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 duringthe test. However, since one control colony showed a high termination rate between BFD and BFD +4 and the other 

### two coloniesof the control showed a termination of about 20 % 

### duringthe test, the effects noticed inthe 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 lnsegarWG 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 inthe 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 (cappedcell), 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 groupsTI and T2, the toxic standard treatment and the control. Replicate
IT1 2T1 3T1 

### Mean 1 T2 2T2 3T2 

### Mean

1 C 2C 3C 

### Mean

1R 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 concludedthat the application of BY1 08330 OD 100applied 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 mortalityof adult honey bees and brood, flight intensityof the bees on the crop andthe behaviour of the bees in the crop area or in front of the hive. An irritationof the brooddevelopment at the earlier assessmentswas noticed in one colony of the test item treatment group TI, and about one quarter of the marked cells showed a terminationof bee brood in the colonies of treatment group T2. However, since one control colony showed a high termination rate between BFD and BFD 

### +4 andthe 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 ascribedto the application of the test item. The colonieswere in good condition during the observation period of 29 days.

Additional non-GLP studies onthe effects of spirotetramat onthe bee brood: Introduction: For research and product positioning purposes Bayer CropscienceAG performed orientating honey bee semi-field (tunnel) studieswith Spirotetramat. The objectives of these studies were to investigate potential side effects of different formulationsand rates of spirotetramat, and different pre-application intervals, to honey bees in flowering crops under tunnel conditions. Nine honey beetunnel studies were performed between 2002 and 2007, seven in Germany,two in Spain. For all studies Phaceliatanacetifolia 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 170guideline under non-GLP conditions but according to GEP (good experimental practice). The general test performancewas as follows: Honeybeecoloniesof an appropriate size (approx. 3000 

### - 3500 bees) were confined intunnels (44 

### m2and60 m2in Spain and 50 m2(100 m2for the strawberry trial, due to lower density of the flowering crop) in Germany) with a flowering crop on a field. Forthe reported studies one or two replicateswere set up for each treatment group. Each study comprised one untreated control, one toxic reference (fenoxycarb) and one to six differenttest item treatment groups. Honey bees were introduced into the tunnels some days priorto application to get familiar with the new environmental conditions (unless preflowering applicationswere tested). The bee colonieswere 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 removalfrom the tunnels. In particular, the endpointsadult, larval and pupal mortality, flight intensityand brood development were assessed, furthermore honey and pollen storage, egg laying and breeding activity, colony strength, hive weight and behaviouralanomalies of the honey bees were recorded. The obtained resultsfor each endpoint andtreatment group from the post-applicationperiod were comparedto those of the pre-applicationphase. 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 resultsof the endpoints related to bee brood development with regardto the larval development and mortalityof 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 thereforewere 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 reportedstudies, a statistical evaluationwas notfeasible. Hence, the determination of effects was performed on the basis of expertjudgment.

### 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: 

### Spirotetramat240 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 Proportionfor 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 combinationwith 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 reductionof larvalcomb area proportion. The reduction of larvalcomb area proportionwas 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 groupswere not impacted and the survival of the colonies was not endangered.

Trial 2: 

### Spirotetramat240 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 Proportionfor Trial 2



# I untreated (control) 

# 1 6.7 

# / 7.5 

# 1 3.3 

# 1 2.5 

# 1 1.7 

# 1 Treatment



## - lnsegar25 WP, 100 g a ha (toxc reference) 5.8 'I 0.3 0.0

% ." I Proportionof comb area covered by larval cells r SD 

## [%I (at X days after application)

* assessment on 0 DAA was done shortly beforeapplication
Conclusion: SpirotetramatSC 240 in combination with 0.2 % a.i./v RME 500 EW caused transient effects on honey bee larvaewhen applied to flowering 

### Phacelia at 36 and 72 g a.i./ha. The reduction of larvalcomb 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 seemedto last until 7 to 21 days after application. After the application of SpirotetramatSC 240 at a rate of 144 g a.i./ha with the additive RME 500 EW an unclear effectwas observed 7 days after application, and the application at the same rate without the additive RME 500 

### EW caused no reductionof larval cell comb area proportion. The survival of the colonies was not endangered by the effects observed. Due to unfavourableweather conditions and therefore disadvantageousconditionsfor appropriate foraging, the breeding activitywas 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 pupaewas found in front of the bee hives from 4 days after application to 7 days after application.The effectswere less pronounced comparedto the toxic standard. All other (brood and nonbrood related) endpoints were not impacted.



### Trial 3: Spirotetramat240 SC or 100OD 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 LarvalCell Comb Area Proportionfor Trial 3a



### RME 500 EW 0.4% a.i./v Treatment



# I RME500 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 Proportionof comb area covered by larval cells 

### [%I (at X days after application)

Table Error! Style not defined.4 (continued): Evaluationof Larval Cell Comb Area Proportionfor Trial 3b

* assessment on 0 DAA was done shortly before application
Conclusion: The application of 144g a.i./ha Spirotetramat SC 240 as a tank mixture with 0.4 % a.i.1~ RME 500 EW to flowering Phaceliacaused 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 pronounced4 days after application, but unclear at the following assessment days. Forthe 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 upto 22 DAA, more pronounced from 4 days until 7 days after application; in the first trial effects were unclear. Spirotetramat 

### OD 100applied 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 observedwhen 0.4 % a.i.1~ RME 500 EW was applied alone. In all Spirotetramattreatment groups the survival of all colonies was not endangered. Inthe second trial a slight increase in dead pupae found in front of the bee hive 15 days after applicationwas 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 recoverywas 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 controlwas very low during the exposure phase in the tunnel and the toxic reference did not cause a distinct effect on the larvae. Inthe two Spirotetramat OD 100treatment groups, the figures for 7 

### days after application may indicate a slight reductionof larval cells, however,this cannot be diagnosedwith certainty. Trial 4: 

### Spirotetramat 240 SC or OD 100as 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 Proportionfor Trial 4



### Trial 5: 

### SpirotetramatOD 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 mortalitywere 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 mortalitywas recorded, are listed in the following table.


### Table Error! Style not defined.-8: 

### Pupal Mortality Evaluation for Trial 5b

lnsegar25 WP, 150g a.i./ha (toxic reference) Treatment

7Untreated(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 trialsthe application of Spirotetramat OD 150to flowering Phacelia at four different rates (50 g, 75 g, 100 g and 150 g a.i./ha) causedtransient effects to honey bee larvae. The reduction of larval comb area proportionwas 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 14days after application on. All other brood and non-brood related endpointswere not impactedand 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 Proportionfor 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 priorto flowering 150 g a.i./ha on flowering crop at bee flight 150g 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,beginningof exposure Conclusion: The application of 150 g a.i.ka of SpirotetramatOD 150five days priorto flowering (start of exposure) and on the flowering crop during and after bee flight causedtransient reductionsof larval cell comb area proportion. The effects were different regarding the time of application. When applied 5 days before floweringthe effects were obvious on the fourth and seventh days of exposure and less clear on the 1 5th day. The application of 150 g Spirotetramathato 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 reductionof 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 relatedendpoints were not impacted. Spray intervalsof 10, 15 and 

### 20 days before flowering of 

### Phacelia did not cause effectsto 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: 

### Evaluationof 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 beginningof exposure Conclusion: Application of Spirotetramat OD 150at 100 g a.s./ha 5 days priorto 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 effectsto honey bee brood starting at three days and upto 15 days after start of exposure. A full recovery was observed 21 days after start of exposure in this treatment group. Spray intervalsof 10days before flowering of Phaceliadid 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 difficultto unambiguously assess whether these observation have to be consideredto 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 endpointswere not impacted.



### Trial 8: SpirotetramatOD 150 as spray application, 300 Uha, 1 replicate, Spain



# - Results: 

### Table Error! Style not defined.-1 1: 

### Evaluationof Larval Cell Comb Area Proportionfor 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 

### Phaceliacaused transient effects on honey bee larvae. No larval comb area was recorded on 7 

### and 14days after application. 21 and 31 days after applicationthe areawith larval cells was still rather low, but due to the lack of replication it is unclearwhether this has to be attributedto natural variability. No evaluation of potentialeffects of the test item on the larvalcomb 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 highestapplication rate had nearly no larval cells shortly before the application and diedthereafter, which is uncommonfor 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 haveto be interpreted with care. Additionally,the overall brooddevelopmentwas weak in all hives (see e.g. bee brood evaluationat -3 

### days after application). Therefore and due to lack of replication, it is difficultto attribute changes in larval abundances as seen in this study, to potential treatment effectsor ratherto naturalvariation, 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 100g a.i./ha to flowering strawberries undertunnel 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 doubtfulwhether this finding is indicating a treatment-related effect. The survivals of the colonieswere 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 mortalitieswere less pronounced comparedto 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 endpointswere not impacted. Proportionof 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 11.7B.9.4.7 

### Summary of effects on honey-bees

Table Error! Style not defined.9: Summary of semi-fieldand field data of Spirotetramat effects on Table Error! Style not defined.-1: Summary of acute effectsof Spirotetramat (technicalandformulated) on honey-bees

honey-bees Test substance SpirotetramatOD100 Test substance Spirotetramattechn. Spirotetramat OD1 50

Spirotetramat SC240

Spirotetramat OD100 Exposure route Oral Contact

Oral Contact

Spirotetramat OD100 Exposure route Endpoint 48 h LD50 48 h LDso 48 h LD50 48 h LD5n

Feedingtest, 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 increasedmortality of adults and pupae during 21 d after start of feeding No effect on flight acivity, bee behaviour. Brood development: Hightermination rate intwo 

# I of three colonies. Feedingtest, 

## 1 Increased adult mortality. mixed 

## hsucrose solution at 0.0144 % a.s. Brood development:terminationof brood developmentwith tendency to recover. No effects on flight activity and behaviour of bees. Semi-fieldstudy, 2 appl. on crop before flowering at 72 g a.s./ha 

## + 2 x 72 g a.s./ha after flow. or 1x 96a a.s./ha no adverse effects on adult and brood mortality, conditionof the colonies, flight activity and behaviour of bees.

Semi-field study 

# I no adverse effects on adult and brood mortality, (tunnel design), 3 conditionof 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 afterflow. or 1 x 96g a.s./ha Schur, 2004b

behaviour of bees. Irritationof brood development inthe 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 36g a.i.lha RME 500 EW 0.2% a.iJv Spirotetrarnat 72 g a.i.lha RME 500 EW 0.2% a.iJv Spirotetrarnat 144g a.i.lha RME 500 EW 0.2% a.iJv Spirotetrarnat 144g a.i.lha Spirotetrarnat 36g a.i.1ha RME 500 EW 0.2% a.iJv Spirotetramat72 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 DAA15DAA21 DAA Recovery until

7 DAA

7 DAA

7 DAA
7 DAA

7 DAAFormulation Type and content

SC 240 a.i./L

OD 100 g a.i.lL

SC 240 a,i.lL

OD 100g a.i./L SC 240 g a.i./L OD loo g

OD 150g a.i.lL

OD 50 g

OD 150 a.i.~~ Treatment Spirotetramat 144g a.i./ha RME 500 EW 0.2% a.i./v Spirotetramat 144g a.i./ha Spirotetramat 144g a.i./ha RME 500 EW 0.4% a.i./v Spirotetramat 144g a.i./ha RME 500 EW 0.2% a.iJv RME 500 EW 0.4%a.iJv Spirotetramat 140g a.i.lha Spirotetramat 144g a.i.lha RME 500 EW 0.4% a.iJv Spirotetramat 144g a.i./ha RME 500 EW 0.2%a.i./v RME 500 EW 0.4%a.iJv Spirotetramat 140g a.i./ha Spirotetramat 144g a.i./ha RME 500 EW 0.1%a.ih Spirotetramat 140g a.i./ha Spirotetramat 100g a.i./ha Spirotetramat 50 g a.iJha
Spirotetramat 75 g a.iJha
Spirotetramat 100g a.i.lha
Spirotetramat 150g a.i./ha Spirotetramat 50 g a.iJha
Spirotetramat 75 g a.iJha
Spirotetramat 100g a.i.lha
Spirotetramat 150g a.i.lha
Spirotetramat 150g a.i.lha 20 days priorto flowering Spirotetramat 150g a.i.lha 15days priorto flowering Spirotetramat 150g a.i./ha 10 days priorto flowering Spirotetramat 150g a.i./ha

5 days priorto flowering Spirotetramat 150g a.i./ha on flowering crop at bee flight

Spirotetramat 150g 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 DAERME: rapeoil methyl ester DAA: days after application DAE: days after beginning of exposure no influenceon bee brood0b~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 befound inthe 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 arthropodspecies (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 priorto flowering Spirotetramat 150 g a.i./ha 15 days priorto flowering Spirotetramat 150 g a.i./ha 10 days priorto flowering Spirotetramat 150 g a.i.ka

5 days priorto flowering Spirotetramat 150 g a.i./ha on flowering crop at beeflight

Spirdetramat 150 g a.i./ha on flowering crop after bee fl~ght Spirdetramat75 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 laboratoryand 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 parasitoidwasp Aphidius rhopalosiphi(DeStephani-Perez) (Hymenoptera: Braconidae) inthe laboratory; BY1 08330150 OD Author(s), year: Waltersdorfer,A.; 2005a Report/Doc. number: CW051023; M-254442-01-1 Guideline(s): Mead-Briggset al. 2000 (IOBC) GLP: Yes (certified laboratory) Deviations: No reproductionassessment. 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.: 0803010189(0152),Tox no.: TOX07034-00, analytical content: 148.89 g a.s./LMaterial 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: deionisedwater; 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 Lwaterlha Adult wasps were exposed to dried residues on treated glass plates for 48 h. The test units consisted of two treated glass plates (100 x I00x 3 mm) and an untreated acryl frame (inner size 92 x 92 x 14 mm) with 3 ventilation holes on each side. These holeswere covered with gauze (mesh size 80 pm) which was jammed in the orifices by pieces of Teflon tubing (8 mm outer diameter). Honeylwatersolution Temperature: 19

## - 22°C; relative humidity: 62 

### - 83 %, 16 h light, 570 

### - 1001 lux Observationsof mortalitywere performed approximately 24 and 48 h after start of exposure. The number of alive, affected, moribund and deadwasps was recorded. No reproductionassessment. Mortality corrected accordingto 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 platesto Aphidius rhopalosiphi
I I 

# I Treatment 

# I

* statisticallysignificant Mean mortalityat 48 h [Oh] 

# I Mean corrected mortalityat 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 Typhlodromuspyri SCHEUTEN (Acari, Phytoseiidae) in the laboratory; BY1 083301 50 OD Author(s), year: WaltersdorferA., 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) 150OD; 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 

# IType 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 consistedof two cover slides (glass) which were put together duration: at the longer sides with a narrow gap between the slides. Cover slideswere placed on tissue paperwhich 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 transferredto a plastic tray filled with deionised water. The narrow gap betweenthe two cover slides was filled with water by capillaryforces to provide the miteswith water. The test unitswere prepared one day before application to make sure that the foam rubber was saturatedwith water. Protonymphs were exposedto dried residues for 14 d. Feeding: Pollen (birch 

## + pine) Test conditions: Temperature: 24 

### - 25.5"C; relative humidity: 63 

### - 71 % (very short deviationto 35°C due to opening of the door), 16 h photo period, 795 - 1060 lux Test parameter: The number of dead and living miteswas counted on days 1,3, and 7. The number of escaped miteswas calculated as the difference from the total number exposed. On day 7 the number of females, males, eggs andjuveniles was counted. Statistics: Mortalitycorrected accordingto 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 inthe control chambers upto day 7 after treatment was3.0%. 

### - Table Error! Style not defined.-2: Toxicity of dried residues 

### of Spirotetramat 150 

### OD on glass plates to 

### Typhlodromuspyri



# 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(*)

0.016sign. ~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: Toxicityto 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-Briggset al. 2002 Yes (certified laboratory) Increaseintemperature due to technical reasons Acceptable Spirotetramat (BY1 08330) 150OD; Batch no.: 0803010189, Tox no.: TOX07034- 00; (0152), analytical content: 1 48.89 g a.s.R Adult wasps Aphidius rhopalosiphi(Hymenoptera: Braconidae) Acute contact extended laboratorytest 6 replicates with 5 females for each treatment group Control: deionisedwater; 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 calibrationof 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 consistedof a pot with treated barley plants which were enclosed within a clear polyacrylic cylinderwith a hole for the introduction of the parasitoids. After introductionthe holewas closed by a stopper. The top of the cylinderwas 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. Duringthe exposure phase of 2 days the test animals hadaccess to the sugar solution. From the water control and the dose rates 80, 151 and 288 g a.s./ha, 15 impartiallychosen females per treatment were each transferredto a cylinder containing untreated cereal plants infestedwith Rhopalosiphumpadifor 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 Mortalitywas assessed after 2, 24 and 48 h.The number of alive, affected, moribund and deadwasps was recorded. Repellenceof 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 accordingto Schneider-Orelli.One-wayANOVA, p-valuesare adjustedaccording to Dunnett. All validity criteriawere 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 detecteddue 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 relativeto 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 laboratorytest BY1 08330150OD. Waltersdorfer

### A., 2005c CW051030; M-256306-01-1 Blumel et al. 2000 Yes (certified laboratory) P-value 

### *

very short declines in rel. humiditydue 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 A101

### ; Batch no.: 0803010189, (0152), 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 individualsfor each treatment group Control: deionisedwater; 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 suspensionfor the reference item were preparedstepwise on the day of application. Deionised water was used as diluent. The itemswere applied to Phaseolus vulgarisleaves inthe equivalent of 200 Uha using a sprayer after calibration. Protonymphswere exposed to freshly dried residues on leaves of Phaseolus vulgarisfor 1 4 d. Pollen (birch, pine). The water supply for the miteswas 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 declinesto 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 laidafter 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.)cylinderswere placed on a plastic tray such that the filter paper was constantly provided with deionised water. After the test unitswere 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 examinedto ensure they were undamaged and in good health. Mortality of 80 protonymphswas assessed 1, 3,7, 10, 12 and 14 days after exposure by counting the number of living and dead mites. The number of escaped miteswas calculated as the difference from the total number exposed. Additionally on days 7, 10, 12 and 14the number of females, males, eggs and juveniles was counted. Eggs and juveniles were removed. The reproduction rate of surviving miteswas evaluated over the period of 7-14 days after treatment by counting the total number of females and eggs. Statistics: Mortality corrected accordingto Schneider-Orelli. Fisher's Exact test, two-sided, p-valuesare adjusted accordingto Bonferroni-Holm;one-wayANOVA, p-values adjusted according to Dunnett Findinas: Results are summarised in table 9.5.1 -4. All validity criteriawere met.

Table Error! Style not defined.4: Mortality and reproductionafter exposure of Typhlodromus pyrito freshly dried residues of Spirotetramat 

### OD 150applied 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 Exacttest, ** one-wayANOVA 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; miteswere exposedto freshly applied and under semi-field conditions aged residues on excised apple leaf discs Validity: Acceptable
Test substance: Spirotetramat (BY108330) 150 OD; Product code: AE 130294300 OD15 A101

### ; Batch no.: 08030101 89(0152), Tox no.: TOX07034-00; product code: AE 130294300 OD15 A101 

### ; 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: Typhlodromuspyriprotonymphs Acute 

## + aged residue contact extended laboratorytest 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. Protonymphswere exposedto 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-fieldconditions with rain protection during the whole study. Pollen (birch, pine) Weather data (temperature and rel. humidity) in the outdoor areawere recorded. Lab conditions: Temperature: 24.5 

## - 28°C; relative humidity: 61 

### - 73 %, 990 

### - 1567 lux Bioassayswere initiatedon 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 miteswere exposed on these leaves. In each bioassay, mortalitywas 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 plastictray such that the filter paper was constantly provided with deionisedwater. The number of escaped miteswas calculatedas 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 miteswas 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. Mortalitycorrected accordingto Schneider-Orelli.
The resultsare 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) inthe control group exceeded 4 eggs per female. The mean corrected mortalityof 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: 

### Mortalityof 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: 

### Reproductionof 

### T. pyriafter exposure to freshly applied and aged residues of Spirotetramat OD 150 on apple leave discs

Conclusion: Inthis 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. mortalitywere found on DAA 42,49 and DAA56. After 7 weeks of aging, no effects > 11 % on survival or reproductionwere 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 modifiedfor the extended laboratory test (exposureon natural substrate) in such a way that bean leaveswere used instead of glass plates and that treated food was given Acceptable Spirotetramat (BY1 08330) 150OD; Product code: AE 1302943 00 OD15 A101

### ; Batch no.: 0803010189 (01 52), Tox no.: TOX07034-00; analytical content: 148.89 g a.s.lL Chrysoperlacarnea, larvae, age 2-3 d (L,) Acute contact extended laboratory test 40 replicates with 1 larvae for each treatment group Control: deionisedwater; toxic standard: 30 mL productlha (Dimethoate EC400); test item: 44, 72, 11 2, 184 and 288 g a.s./ha; deionisedwater as diluent. The test substance was applied in 200 Uha. Larvaewere exposed to freshly dried residueson bean leaves and Sitotrogaeggs

### 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 assessmentday. 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 exposureto dried spray deposits on bean leaves and feeding with treated Sitotroga eggs. In the reproductionphase, 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. Cageswere fixed to a glass plate and an acrylic plate. Oviposition cages consistedof glass beakers covered with cotton gauze during egg laying. Hatching cagesfor F1 larvae consisted of plastic cages with clear cover. Ovipositionwas started one week after the first egg laying was observed (pre-ovipositionperiod). Test plantswere kidney beans (Phaseolusvulgaris), grown in the laboratory of the test facility under controlledconditions. Primary leaveswere cut shortly beforethe application,when the plantswere 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 fecunditywere calculated. Mortality corrected according to Abbott. Statistical analysis of mortality data (Fisher's Exact BinomialTest, 1 -sided, p I 

### 0.05). The softwareToxRat Professional, Version 2.09 (RATTE, 2004) was used. Statistics:

Findinas: All validity criteria were met. Resultsare summarised intable 9.5.1-7. 

### Statistical analysis revealed no significant differences concerning mortality between control andtest item treatment groups. Also the reproductive output was above the lower limitgiven as validity criterion for the glass plate method (2 

### 15 eggs/female/d) and therefore consideredas not impacted by the treatment.
Table Error! Style not defined.-7: Toxicity of dried residues of Spirotetramat 150 

### OD on 

### Chrysoperla carnea inan extended laboratorytest

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 150OD 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 Coccinellaseptempunctata L. (Coleoptera, Coccinellidae) using an extended laboratory test including exposureto 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 modifiedfor the extended laboratory test in such a way that bean leaves were usedfor exposure insteadof 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 offeredto larvae which had not yet pupated. Acceptable. The validity criterionfor control mortalitywas slightly not met. However, validity criteria have been definedfor glass plate studies only and consideringthe special design the study can be consideredvalid. Spirotetramat (BY108330) 150 OD; Product code: AE 1302943 00 OD15 A101

### ; Batch no.: 0803010189 (0152), Tox no.: TOX07034-00; analytical content: 148.89 g a.s./L
Coccinellaseptempunctata, larvae, 4 d old Acute contact extended laboratory test 40 replicates with 1 larvaefor 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 applicationon a defined area. Larvaewere exposed to freshly dried residueson bean leaves and Sitotroga eggs until pupation. Larvaewere fed with aphids (Acyrthosiphonpisum) ad libitum which had been treated for four days and afterwardsfedwith untreatedaphids. 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 Phaseolusvulgaris, which was laid on a wet cotton wool pad in a petri dish (9 cm diameter) after application. A constant supply of deionisedwater 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 ringswas coated with a lubricant (Hostaflon) in order to preventthe larvae from escaping. Once dry, the steel ringswere placed onto them into the petri dishes and one larvawas added to each test unit. Additionally potted bean plants with aphids (Acyrthosiphonpisum on Vicia faba) were sprayed on the day of application with the corresponding rates of the test item. The aphidswere 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 offeredto those larvae which had not yet pupated. After 3 days the remaining larvae and the adults were fed with untreatedfood.

### Statistics: Findinas: All test unitswere 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 andthe treated surface. Once the larvae had pupated, the emerged beetles were transferredto plastic boxes at the day of their individual hatching. After this feeding period all beetleswere transferred to glassjars. Their sex was determined and fresh stems of bean plants with aphids and untreated pollen were introduced as required. Sheets of black paperwere offeredto the beetlesfor egglaying, which was checked daily. Pieces of paperwith freshly laid eggswere 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. Mortalitycorrected according to Schneider-Orelli.
The mortalityvalue 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 parameterscan only be evaluated qualitatively, due to the very high species-inherentvariability in egg-laying performance.As all valuesfor the three highestand the lowest dose rate were within the historical data base for control beetles (22 

### fertile eggs perfemale and day), this parameter is considered as not impacted by the test item rates. Table Error! Style not defined.4: Mortality and reproductionafter exposure of Coccinella septempunctatatofreshly 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 exposureto rates up to 288 g a.s. Spirotetramat 150OD 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 150applications on mitefauna (Acari) in thefield (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 (3week 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 m2surface. Plots consistedof 20 adjacent vine positions. Plotswere 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 localweather station. Sampling: The effects of BY1 08330 OD 150were expressed interms of population changes relativeto the water control. Population samples were taken shortly before each application, 1week after the first application and approximately 1, 4 and 8 or 9 weeks after the last application. Fortest item scenario 4 and water an additional sample was taken 11 weeks after the last treatment. The evaluationwas based on time to recovery (= population density similar to control). Three leavesof 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 pooledfor 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 2weeks 2 weeks
2weeks 1week Nominal Rate 96 g a.s./ha Application 1: 36.4 g a.s./ha Application2: 35 g a.s./ha 9.1 g a.s./ha 4.8 g a.s./haarthropodswere inspected under a binocular microscope. The first 16 phytoseiid mites observed in each plot were mounted for species identification.
Statistics: Effectvalues were calculatedfrom total phytoseiid mite numbers according to Abbott (1925) and Henderson-Tilton(1955). Inaddition,total phytoseiid mite numbers were analysed statistically usingthe non-parametric Man-Whitney 

### Utest. Findinas: Test conditions: Weather parameters were in an acceptable range; there was no rain on the application days. Predatory mites: Several acarine taxawere identified. The most abundanttaxon prevailing in the vineyardwas the predatory mite family Phytoseiidae. Phytoseiid populations almost exclusively consistedof the species Typhlodromus pyri (98 % 

### - 100% throughout the study period). Other mitetaxa 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 scenariostested. No statistically significant differences with the water control were found for any of the BY1 08330 OD 150treatments on all sampling dates, except on 16June, 5 days after the first treatment, when the 3rddrift 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 %) throughoutthe study. This finding was probablydue to natural variation of mitefield populations, rather than to an actual treatment effect. Population dynamics observed in other test itemtreatment plots closely resembled fluctuations detected in water control plots. The different spray scenariosof the test item neither induced effects on Eriopheiid mites. The reference item severely affected Phytoseiid mites in a negativeway, with a statistically significant reduction in phytoseiid numbers of 43 %to 96 %. Thus, the trial is considered valid for the purpose of evaluating potential consequencesof test item treatmentsto 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 statisticallysignificant (P < 0.05)


### Conclusion: It is concluded that SpirotetramatOD 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 150OD 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/ 27.8n.d. not determined, n.p. no assessment performed negative figures indicate a positiveeffect compared to the control


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


### The risk assessmentfor 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] Reference Test item Test species Type of effect Exposure

Spirotetramat 150OD

Spirotetramat 150OD

Spirotetramat 150OD

Spirotetramat 150OD 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 reductionof 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 288B.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 toxicityto 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 replicateseach with 10 individuals per treatment and control Average weight: 360 mglworm, clitellate adults z 2 months Laboratoryacute test, 14 days Nominal: 0 (control, quartz sand), 32, 100, 178, 316, 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 contentwere measured at the beginning and the end of the study 625 g wet weight (500 g dw) in 1.5 Ltest containers Measuredtemperatures were not stated Continuous illumination (400 

### - 800 Lux) Mortality (assessedafter 7 and 14 days), condition and behaviour (observedat 14 days) and mean body weight (measured at 0 and 14days) NOEC: Dunnett's' Test (mortality, mean bodyweight) LC5, (toxic standard): ProbitAnalysis
About 21 % (0 h), 19.6 

### -20.1 % (14 d) 6.4 (0 h), 6.4 (14 d) Mortality: Spirotetramat: No mortalitywas 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 effectswere observed in the control and all treatments Body weight: See table B.9.6.1-1

### Table Error! Style not defined.-1 : 

### Effectsof BY1 08330 (spirotetramat) onthe 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 guideline207 are notable, deviationsto 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 14days and measured concentrationsare not reported



### Metabolites
Reference: BY1 08830 cis-ketohydroxy: Acute toxicity to earthworms (Eiseniafetida) 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 (Eiseniafetida) 4 replicates each with 10 individualsper treatment and control Average weight: 0.34 glworm, clitellate adults > 2 months Laboratoryacute test, 14days Nominal: 0 (control, quartz sand), 32, 100, 316, 1000 mglkg dw soil Quartz sand 2-Chloroacetamide (purity > 97 %): 5.6, 10, 18,24,32 mglkgdw soil
10 % sphagnum peat,20 % kaoliniteclay, 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 measuredat the beginning and the end of the study 625 g wet weight (500 g dw) in 1.5 L test containers Measured temperatureswere not statedLight 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 14days) and mean bodyweight (measuredat 0 and 14days) NOEC: Dunnett's' Test (mortality, mean bodyweight) LC5, (toxic standard): ProbitAnalysis
23.3 % (0 h, mean), 24.7 % (14 d, mean) 6.4 (0 h), 6.2 (14 d) No mortalitywas noticed in the control and all treatments. No effectswere observed in the control and all treatments See table B.9.6.1-2


### Table Error! Style not defined.-2: 

### Effectsof 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 mglkgdw soil) Conclusions: LC5, > 1000 mg/kg soil dw, NOEC (body weight, mortality): 1000 mglkg soil dw Comments: While no deviations to the OECD guideline207 are notable, deviationsto the OPPTS guideline are remarkeable: The recommendedtest 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 concentrationsare not reported



### Reference: Spirotetramat

# -4-methoxycyclohexanone:Acute toxicity to earthworms (Eiseniafetida)tested in artificial soil with 5 % 

### peat Author(s), year: Leicher, T., 2008 ReportIDoc. number: LRT-Rg-A-98/07, Edition Number: E310 3411-3,09.01.2008 Guideline(s): OECD 207 GLP: Yes Deviations: None of relevance Validity: Acceptable
Test substance: 4-methoxycyclohexanon (metabolite of spirotetramat), purity: 97.9 %, batch: SVA 5506-001 Material and methods: Test species: Earthworm (Eisenia fetida)

Number of organisms: 4 replicateseach with 10 individuals per treatment and control Weight, age: Average weight: 0.29 g/worm, clitellate adults > 2 months Type of test, duration: Laboratoryacute test, 14daysApplied 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 contentwere measured at the'beginning and the end of the study 580 g wet weight (500 g dw) in 1.5 L test containers Measured temperatureswere not reported Continuous illumination (400 - 800 Lux) Mortality (assessedafter 7 and 14days), condition and behaviour (observedat 7 and 14 days) and mean body weight (measuredat 0 and 14 days) NOEC: Dunnett's' Test (mortality, mean bodyweight) (toxic standard): ProbitAnalysis 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 mortalitywas noticed in the control and all treatments. Behavioural effects: No effects were observed inthe control and all treatments Body weight: See table ~.9.6.'1-3


### Table Error! Style not defined.-3: Effects

### of 4-methoxycyclohexanoneon 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 > 1000mglkgsoil dw

* significant differencewhen comparedto control (Dunnetttest, 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 (bodyweight, 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 concentrationsare 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: BY108330-Enol: Reproductiontoxicitytothe 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): IS0Guideline 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:

Substrateltestvessel: 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, clitellateadults Laboratory sublethal test, 8 weeks Nominal: 0 (control, quartz sand), 10, 32, 100, 316, 1000 mgkgdw soil Quartzsand 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 contentwere measured at the beginning and the end of the study 500 g dry weightltest container 17-22 "C Thetemperaturewas only for few and short time period belowthe recommended 18 "C, thus this did not affected the results of the test. 16 hours light (485 

### -558 Lux)/8hoursdark 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 (countedafter 8 weeks) and condition and behaviourof juveniles (observed after 8 weeks) NOEC: Dunnett's multipletest, 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/kgdw] > 1000 

# 1 1269 (95% C.I. 1007 - 1896) * significantlydifferent to control (alpha = 0.05)

Effects toxic standard: Reproduction: LOEC = 1 mg a.s./kg dw soil Validity criteria: Adult mortality (I10 %): 0 % after 4 weeks Weight lossof adult worms (I 20 %): 

## + 37.7 %

### after 4weeks Number of juveniles/vessel (2 30 %): 148

## +43.3 % Coefficientof 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) indicatedan acceptable risk for the intended uses and no refinement is necessary.


### B.9.6.4 Risk assessmentfor 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 assessmentfor PMRA and 

### US EPA is not included in this monograph.B.9.7 Effectson soil non-target macro-organisms (OECD IIA 8.14, lllA 10.6.6)
B.9.7.1 Effectsona species level (gamasid mites)
Metabolites
Reference: BY108330-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: PI1

### HR, Edition Number: M-243307-01-1, 19.01.2005 Guideline(s): SECOFASE, final report (Lokke & von Gestel 1996), Guidance document on regulatorytesting proceduresfor 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:
Substratehestvessel: Temperature: Light regime: Feeding:

Test procedure:

Test parameters: Statistics: BY1 08830-enol (metabolite of spirotetramat), purity: 99.1 %, batch: 692-101-09- 0005
Gamasid mite (Hypoaspisaculeifeg 4 replicates with 20 mites per treatment, 5 replicates with 20 mites per control and 3 replicates with 20 mites per toxic reference Protonymphes2 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 miteswere 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 miteswere 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 levelswhich caused c 50 % mortality (i.e. 100, 316, 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 unitswere transferred individuallyto 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 (mortalityadults): 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) *Significantdifferent by Dunnetts test (alpha= 0.05) Table Error! Style not defined.-1: Effects of BY108830-enol (metabolite of spirotetramat) on mortality and reproduction of gamasid mites

Effects toxic standard: 88 % corrected mortalityat 5 mglkg soil dw Validity criteria: Adult mortality in control after 14 days (525 %): 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): 316 mglkg soil dw LC/ECSO 

### > 1000 mglkg soil dw Conclusions: BY108330-enol [mg a.s.lkg dw] Control 10

B.9.7.2 Effects ona 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 (Hypoaspisaculeifer) Endpoint Mortality Reproduction Time

28 EC/LC5~ [msn<gdwl

> 1000 NOEC [mglkg dwl 316 1000 Referencelstudy acceptable Moser & Scheffczyk 2005/yesB.9.7.4 Risk assessment for other soil non-target macro-organisms

The risk assessmentfor the EU can befound 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 Effectsonsoil non-target micro-organisms 

### (OECD IIA8.10, lllA 10.7)
B.9.8.1 Nitrogen mineralization Active Substance
Reference: BY108330tech.: Determination of effects on nitrogen transformation insoil 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 216 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: 16glkg soil dw Test conditions:

Test substrate:

Substrateltestvessel: Incubation: Test parameters:

Statistics: Findinas:

pH: Effects test item: Natural Soil, collected on April 13~, 2004 at a depth of 0-20 cm, from experimentalfield 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 mealwas 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 particleswere removed by filtration, NH4+ and NOiwere determined using a "Bran & Liibbe Autoanalyzer 3.Sampleswere taken at 0, 7, 14and 28 days after incubation. F-test (homogeneityof variance), T-test (significant differences)
5.6 (start), 5.9 

### - 6.0 (end) See table B.9.8.1-1Table Error! Style not defined.-1: Effectsof spirotetramat on nitrogentransformation (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, correspondingto 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-transformationof 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 217 Yes None of relevance Acceptable Spirotetramat (BY1 08330), purity: 97.4 %, batch: 0804510014 Nitrificationrate (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 transformationtest, 28 days 0 (control, purifiedwater), 0.13 and 1.32 mgkgdry soil Quartz sand Sodium chloride: 16glkg 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 experimentalfield on Lacherhof Germany: C,,: 0.7, pH: 5.6,

texture (DIN): 6.3 % clay, 15.6% silt, 78.1 % sand, microbial biomass: 393 mg microbialClkg soil dw, in addition glucosewas added to the soil samples (2 gkgsoil dw) to inducemaximum respiration rate. Substrateltestvessel: 350 g soil dw1500 mL brown glass bottles, three replicates per treatment Incubation: 20 

## +2 "C in the dark, about 40 % MHC Test parameters: EvolvedC02 was measuredwith a gas analyserfor at least 12 hours. Samples were taken at 0, 7, 14 and 28 days after incubation. Statistics: F-test (homogeneityof variance), T-test (significant differences) Findinas:



### pH: 5.6 (start), 5.5 

### - 5.6 (end) Effectstest 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, correspondingto 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-transformationof soil micro-organisms. 

### Table Error! Style not defined.-1 :

### Effectsof 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 befound in the Appendix at the end of section B.9. The risk assessmentfor PMRA and 

### US EPA is not included in this monograph. Day 14 Day 7 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) 0.13 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) believedto be a risk (OECD IIA 8.12, 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 effectsof 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: No 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) 150OD, 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 Helianthusannuus (Dicotyledonae: Asteraceae) Vegetativevigour test 4 to 5 replicates with 4 to 5 plants per pot for each species (19 

### - 20 plants per treatment group) Control: deionisedwater; test item: 288 g a.s./ha in 200 Uha. The test item was sprayed on the leaves and above-ground portionsof 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 portionsof the plants. Glasshouse conditions: temperature: 18°C at night, 23°C at day; natural daylight supplemented by artificial lighting, 16 h photoperiod; Plantswere watered from the bottom. Visual phytotoxicity ratingswere done at days 7, 14and 21 after application. Plant biomasswas determined per replicate (pot). Pairwise Mann-Whitney-U-test, one sided smaller
There were no adverse effects on survival. There were visible signs of phytotoxicitysuch as chlorosis, necrosis, leaf deformation, bleaching, andlor stunting in all crops tested. There were differences in growth stages in corn and oats. Biomasswas 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 :

### Phytotoxicityassessmentsfor the day 21 

### of the vegetativevigour test

-- * comparedto the control,where survivalwas 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 comparedto the control) phytotoxicity ratings: Corn 20 (100)
0
C
56.9 Sunflower

20 (100)
0
B-C
43.1

0 = no injuryor effect A = slight symptom(s) throughout the whole plant or more moderatesymptom(s) on a small area, i.e. one leaf B = moderate symptom(s) throughout the whole plant or severe symptom(s) on a limitedarea, i.e. one-two leaves Oats 20 (100)
0
B
56.6 Soybean 20 (100)
0
A
40.2

C = severe symptom(s) throughout the whole plant with younger or newly developed leaves growing normally Oilseed rape

20 (100)
0
D
83.3 Survival in number (out of 20) and (%) Inhibitionof survival 

### * (%) Phytotoxicity (see below) DryWeight 

### ** (% 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 150showed several significant adverse effects (i.e. greaterthan 50 %) to representativenon-targetplants in the vegetativevigour test. Oilseed rape was the most sensitive species tested. These results trigger further testing. Comments: Following deviationsfrom 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 IIVegetative Vigor Non-Target Plant Study using BY1 08330 150 OD Author(s), year: Christ, M.T., Lam, C.V. (2006a) ReporVDoc. number: EBFNXOI6; 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 150OD, 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 (Loliumperenne, Poaceae), onion (Alliumcepa, Alliaceae) 6 dicotyledonousplants: cucumber (Cucumis sativus, Cucurbitaceae), canola (Brassica napus, Brassicaceae), soybean (Glycine max.), sunflower (Helianthus annuus L., Asteraceae), sugarbeet (Beta vulgaris, Chenopodiaceae), tomato (Lycopersiconesculentum, Solanaceae) Type of test: Vegetative vigourtest Number of organisms: 8 replicates with 5 plants per pot for each species (40 seeds per treatment)Treatments:

Statistics: Control: deionisedwater; 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; measuredtest concentrations (mg1L): 0. 123, 252, 527, 1072, 2113; Calibration had been performed before application. Duration: 21 days after treatment application Soil characteristic: Silt loam: 22 % clay, 50 % silt, 28 % sand, organic matter: 2.4 % (organic carbon 1.2 %), pH: 7.8 Test design: At the 2 

## - 4 leaf stage the test item was sprayed using an Allen Track Sprayer from above the plant canopy. The test unitswere then transferredto 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 ratingswere done at days 7, 14and 21 after application using the following rating system: 0 (no injuryleffect), 10to 20 % (indicates slight effect), 30 to 40 % (indicates moderateeffect), 50 to 60 % (indicates severe effect), 70 to 80 % (indicatestotal plant effect), 90 % and above (plant death). Shoot height measurements were performedon day 21. Plant biomasswas determined per replicate (pot). Descriptive statisticswere performed using EXCEL. One-way-ANOVA and Dunett'stest were applied. Statistical analyseswere performed using SAS procedure. In Dec 2007 the applicant was askedto 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 becauseof the nature of the data. Findinas: Measured test concentrations ranged from 104to 11 4 % of nominal. Survival in all control groups was 100 %. Canola: Plant survivalwas 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. Plantswere not significantly different for plantdry weight or length at the 176 g a.s./ha treatment by Dunnett's Test as compared to the controls. 

### Corn: Plant survivalwas not significantly affected at any treatment level in comparison to the controls. Phytotoxicitywas observed throughoutthe study with observationsof 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 comparedto the controls. The inhibition comparedto the controlfor these two dosages were 18 % and 51 %, respectivelyfor plant length, and 28 % and 62 

### 5% for dry weight. The EC25and EC50dry weight values were determined as 76 g a.s./ha and 134 g ailha (95 % cl: 117 to 155) by non-linear regression, respectively. The EC50 for corn lengthwas determined as 172 g ailha (95 % cl: 157to 189). Oat: Plant survivalwas not significantly affected at any treatment level in 

## - comparisonto the controls. Random phytotoxicitywas observed throughoutthe study with observationsof stunting, bleaching, leaf curling, and necrosis with severity of effects lessening by Day 21 inthe higher treatment rates. Plantswere not significantly differentfor plant length and dry weight for any treatment by Dunnett's Test as comparedto the controls. Onion: Plant survival was not significantly affected at any treatment level in

### comparisonto the controls. Minor phytotoxicity was observed throughoutthe study with observationsof stunting, leaf curling, and necrosis. Plantswere not significantlydifferentfor plant length and dry weight for any treatment by Dunnett's Test as compared to the controls. Rvearass: Plant survivalwas not significantly affected at any treatment level in comparison to the controls. Phytotoxicitywas observed throughoutthe study with observationsof stunting, bleaching, leaf curling, and necrosis with severity of effects increasing with treatment concentration. Plantswere 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 inhibitioncompared to the control was 14 % 

### and 35 %, 

### respectively,for these two parameters. The EC25dry weight values were determinedas 168 g a.s./ha. Cucumber, sovbean, suaarbeet. sunflower, tomato: Plant survivalwas not significantly affected at the 176 g a.s./ha treatment level in comparisonto the controls. No phytotoxicitywas observed in any treatment. Plantswere 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 EC25plant 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 vegetativevigour 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 >I76Table Error! Style not defined.-2b: Mean plant length and dryweight data of control plants



# ITomato 

## I 33.5 

## I 4.908

# I I 

# I -

Sugarbeet

U Conclusion: In a vegetative vigour study with 10 non-targetterrestrial plant species, following a 21 day post-emergent foliar application of BY1 08330 150OD, 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: 117to 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: anevaluation of the effects of BY108330 

### 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 inthe seedling emergence and growthtest (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 (July2000, 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 (Brassicanapus, Brassicaceae), soybean (Glycine max.), sunflower (Helianthusannuus 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: 14days followingthe 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 beforeapplication 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 initiallytop watered and then bottom watered. Commercial plastic flower pots of 10-cm diameter containing420 g soil were used. Test parameters: Germination: Daily checks were made to identifythe date when 70 % of the seedlings emerge in the control for each species. The number of plantswas / counted after 

### 7 and 14 days.Statistics:
Findinas: Mortalitv/Survival: Dead plants were removed after each assessment. The number of plantsthat 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. Phytotoxicitywas assessed according to EPPO Standard 135. Growth staaes: Growth stages were recordedat 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 inthe untreated control). The plantsof one pot represent one replicate. Statisticalanalysis 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. Biomasswas less compared to control in soybean, corn and oats by 0.2 %, 10.6 % and 8.1 %, respectively. Biomass was greater comparedto 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 exceededthe 50 % trigger for further testing. No phytotoxicitywas 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 onsix plant species ina seedling emergenceand 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 150showed no significant adverse effect (i.e. greater than 50 %) to representative non-target plants in the seedling emergenceand growth test. % Emergence

100

The EC50 figuresfor 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 includingcorn, 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 seedslreplicateand 4 replicates/dose;only 5 seedslreplicate and 4 replicatesldosewere 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 photoperiodfrom 13to 15 h. The EPA recommends 10 seedslreplicate and 4 replicatesldose; however, 5 seedslreplicate and 8 replicatesldosewere used inthis 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) 150OD; Batch no.: 0803010189(0152); analytical content: 15.1 % wlw; 4 monocotyledonousplants: corn (Zea mays, Poaceae), oat (Avena sativa, Gramineae), ryegrass (Loliumperenne, Poaceae), onion (Alliumcepa, Alliaceae) 6 dicotyledonous plants: cucumber (Cucumissativus, Cucurbitaceae), canola (Brassica napus, Brassicaceae), soybean (Glycine max.), sunflower (Helianthus annuus L., Asteraceae), sugarbeet (Beta vulgaris, Chenopodiaceae), tomato (Lycopersiconesculentum, Solanaceae) Seedling emergence and growthtest 40 plants per treatment group (8 pots with 5 seeds each) Control: deionisedwater; test item: 176 g a.s./ha in a spray volume of 93.5 Llha. Calibration performed before application. The nominala.s. concentration in the spray solution was 1882 g a.s./L, the measured concentrationwas 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 applicationseeds 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 photoperiodof natural daylight supplemented by artificial lighting; Plantswere bottomwatered; commercial plastic flower potswere used. Samples of the spray stock solutionswere collected at study initiation on day 0 and analysedfor the active substance. The endpoints evaluatedon Day 7, Day 14, and Day 21 following spray applicationwere the number of emerged seedlings, number of seedlings surviving, and phytotoxicityof 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). Plantswere excised at the soil surface on Day 21 for plant height and dry weight determination. Plant shoot height was measured by extending cut seedlingstoStatistics:

Findinas: their maximum length and recording. Plant dry weight was performed by placing all replicate plantswithin labelled paper bags. The plantswere placed in a drying oven and allowedto dry for at least48 hours. All Day 21 data were subjectedto a Shapiro-Wilk's Test to assess departures from a normal distribution and a Levene's Test to determine homogeneityof variance. To assess treatment effects betweenthe control and treatment group, a Student's t-testwas performed. All statistical analyses were performed usingthe computer program SAS, descriptive statistics were performed using EXCEL.
The measured a.s. concentrationwas 104 % of nominal. Percent seedling emergence for the 10 plant species rangedfrom 88 to 100 % in the controls and from 80 to 100 % in the 176 g a.s./ha treatment. Percent seedling survivalfor the 10 plantspecies ranged from 91 to 100 % in the controls, and 84 to 100 % inthe 176 g a.s./ha treatment. Phytotoxicity ratings for the seedling emergence test rangedfrom 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 inthis respect. The observed phytotoxicity was considered to be not treatment related as the levelof incidence was similar in both the control and treated levels. Percent inhibitionfor plant length for the 10 plant species rangedfrom -4 to 8 % in comparisonto the controls and the 176 g a.s./ha treatment. Percent inhibitionfor plant dry weight ranged from -1 3 to 19% in comparisonto the controls and the 176 g a.s./ha treatment. The percent effectfor each endpoint (i.e. emergence, survival, plant length, dry weight) was 19 % or lessfor each plant species. No significant effects were observedto any of the 10 species tested. Summarised results are given intable B.9.9.1 .l-4. Table Error! Style not defined.-4: Phytotoxic effects of Spirotetramat 1500Donten 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 comparedto the controls in a Tier Iseedling emergence study at the single application rate of 176 g a.s./ha. Becausethere were no effects at or above 25 %, a Tier 2 study is not required. The EC5, figuresfor all tested species were therefore > 176g a.s./ha. 

# I

Canola Corn Cucumber Oat Onion Ryegrass Soybean Sugarbeet Sunflower Tomato 

# I Control 176 g a.sJha Treatment 176 g a.sJha Control 

# I % Survival % Emergence Species

% Emergence = (No. plants emerged/ No. seeds planted) x 100 88 93 95 98 100 100 98 93 95 

### 90 % Survival % Emergence 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.: 08030/0189(0152), purity: 15.1 % w/w analysed
2 monocotyledonous plants: corn (Zea mays, Poaceae), oat (Avenasativa, Gramineae), 1 dicotyledonous plant: canola (Brassicanapus, Brassicaceae) Vegetative vigour test 20

### / 10

### / 8 replicates with 2 14

### / 5 plants per potfor 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 actuallyapplied 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 leavesandthe 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 biomasswas determined per replicate (pot). Computation of EC50(e.g. probit analysis). Software ToxRat Professional used. In Dec 2007 probit analysis using max. likelihood regressionwas conductedto derive ECo5and ECZ5 values with regardto shoot fresh weight.
Measuredtest 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 inthe two highest rates (11 and 35 %, respectively). A. sativa: no phytotoxic symptoms. Growth inhibitioneffects in the two highest rates (9 and 44 %, respectively). Z. mavs: Necrotic, chlorotic and growth inhibitoryeffects in the two highest rates (with regardto the latter 

### 7 and 41 %, respectively). Endpoint results are summarised in table B.9.9.1.1-5. Detailsof probit analysis of the shoot fresh weight: B. napus: Slope: 2.53929, Intercept: 4.04718, 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, 

## ?: 0.931Z. 

### 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 Shootfresh weight

6. napus 

# I > 1.92 

# I 0.48 

# I > 1.92 

# I 15-17

Table Error! Style not defined.-6: Effects onshoot 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 0.17 Control



# 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 itemand 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 vegetativevigour study with three non-target terrestrial plant species, following a 21 day post-emergentfoliar application of SpirotetramatOD 150, the most sensitive endpointwas shoot fresh weight of oilseed rape with a NOEC value of 72 g a.s./ha. However,the EC50'sfor all specieswere > 288 g a.s./ha. Comments: The study does not comply with EPA guidelines (e.g. EPA commented the variation in temperature and relative humidityto be too high).

B.9.9.2 Risk assessment for terrestrial non-target plants
The risk assessment for the EU can befound inthe 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 Laboratoryaerobic activatedsludge inhibitiontest, 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, referencesubstance or water (control) Aerated for 3 hours at 20

## + 2°C Inhibitionof respiration rate (rate of oxygen uptake) None, becausethe inhibitionat highest tested concentrationwas 22.2 mglL See table B.9.10.1-1Conclusions: No inhibitoryeffects on aerobic waste water micro-organisms are expected 

### if the test substance is correctly applied at the intended application rate.