# Annex B



# Spirotetramat

B.9 Ecotoxicology

Reviewed by: AGES, Austria

Peer – reviewed by: PMRA, Canada US EPA, USA– 2 –

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

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

................................................... ................................1
B.9.1 Effects on birds (OECD IIA 8.1, IIIA 10.1)...

................................................... ........................4

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

................................................... .......................4 B.9.1.2 Short-term dietary toxicity (OECD IIA 8.1.2; OECD I IA 8.1.3).......................................... ........5 B.9.1.3 Sub-chronic toxicity and reproduction (OECD IIA 8.1 .4) ................................................ ..........9 B.9.1.4 Supervised cage or field trials (OECD IIA 10.1.7) ..

................................................... .............19 B.9.1.5 Acceptance of bait, granules or treated seeds by bi rds (OECD IIA 10.1.8) ...........................30 B.9.1.6 Effects of secondary poisoning (OECD IIA 10.1.9) ...

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

................................................... ........................30
B.9.2 Effects on aquatic organisms (OECD IIA 8.2, I IIA 10.2) .......................................... ..........31

B.9.2.1 Acute toxicity of active substance, metabolites and formulations to aquatic organisms........31 B.9.2.2 Chronic toxicity of active substance, metabolites a nd formulations to aquatic

organisms ..........................................

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

................................................... ...............76
B.9.3 Effects on other terrestrial vertebrates (OEC D IIIA 10.3)....................................... ...........77
B.9.4 Effects on bees (OECD IIA 8.7; OECD IIIA 10.4 .) ................................................. ..............78

B.9.4.1 Acute Toxicity (OECD IIA 8.7.1; OECD IIA 8.7.2; OEC D III 10.4.2 )..................................... .78 B.9.4.2 Residue test (OECD IIA 8.7.3)......................

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

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

................................................... .......................83 B.9.4.5 Field tests (OECD IIIA 10.4.5) Formulation: Spiro tetramat OD 100.................................... .83 B.9.4.6 Tunnel tests (OECD IIIA 10.4.7) ....................

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

................................................... ................106
B.9.5 Effects on other arthropod species (OECD IIA 8.8, IIIA 10.5).................................... .....109

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

10.5.1)............................................

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

................................................... ................120 B.9.5.3 Summary............................................

................................................... ................................122
B.9.6 Effects on earthworms (OECD IIA 8.9, IIIA 10. 6.3)............................................... ............124

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

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

................................................... .............127 B.9.6.3 Field studies......................................

................................................... .................................129
B.9.7 Effects on soil non-target macro-organisms (O ECD IIA 8.14, IIIA 10.6.6).....................130

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

................................................... ...............130 B.9.7.2 Effects on a functional level (degradation in litte r bags) (OECD IIIA 10.6.7)........................1 31 B.9.7.3 Summary of effects on soil non-target macro-organis ms................................................. ....131
B.9.8 Effects on soil non-target micro-organisms (O ECD IIA 8.10, IIIA 10.7).........................13 2

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

................................................... ..........................132 B.9.8.2 Carbon mineralization..............................

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

................................................... .......134

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

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

................................................... ........................135

B.9.9.1 Effects on terrestrial non-target plants (OECD IIIA 10.8.1) ........................................... .......135– 3 –

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

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

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

................................................... ...................146 B.9.11 References relied on...............................

................................................... ...........................148
Appendix

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

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

................................................... ..............176 B.9.3.1 Risk assessment for mammals........................

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

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

................................................... .............188 B.9.6.4 Risk assessment for earthworms.....................

................................................... ..................190 B.9.7.4 Risk assessment for other soil non-target macro-org anisms ............................................. ..193 B.9.8.4 Risk assessment for non-target micro-organisms .....

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

................................................... ............195 B.9.10.2 Risk assessment for biological methods of sewage tr eatment............................................ .196– 4 –

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

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

B.9.1.1 Acute oral toxicity (OECD IIA 8.1.1)

Active substance – Spirotetramat (BYI 08330)

Reference: Acute Oral Toxicity for Bobwhite Quail ( Colinus virginianus ) for the Test Item BYI 08330 (techn.) Author(s), year: Barfknecht, R. (2003) Report/Doc. number: BAR / LD 047 Guideline(s): US EPA, subdiv. E, § 71-1; EPA Series 850- OPPTS No. 850.2100 GLP: Yes (certified laboratory) Deviations: None Validity: Acceptable

Test substance : Spirotetramat (techn.), batch No.: MIX-Batch 0804 5/0014, TOX-no. 6344-00; purity 97.5 % Material and methods: Test organism: Bobwhite quail (Colinus virginianus), sexually mature Dosage levels: 0, 500, 1000 and 2000 mg/kg body wei ght (nominal) Number of birds and replicates/group: five males and five females / treatment group and c ontrol

Administration: single oral administration in gelat ine capsules Duration: 14 d post-treatment observation period Test conditions: acclimation 6 weeks; Feeding: 15 h starvation prior to dosing, standard diet ad libitum ; housing singly in pens with air-condition; 10 h p hotoperiod, daylight visual spectrum, 18.6 - 21.1 o C, 46.6 - 80.8 % relative humidity Test parameter: Observations: Observations on mortality and signs of intoxication were made hourly on the day of dosing and at least once work-daily throughout t he observation period. Body weight: recorded prior to test initiation and on days 7 and 14 Feed consumption: determined on days 3 and 7 Necropsy: gross necropsy performed at termination Statistics: none Findings: Regurgitation: None. Mortalities: A female of the highest dose died on d ay 6 and a male on day 10. Clinical signs: The birds that died suffered from d iarrhoea from day 1 after administration on. Before death they showed reduced activity, fluffed feathers, ptosis and apathy. Diarrhoea and soft excrements were also observed at 500 and 1000 mg a.s./kg bw and ceased on day 3 and 10, respectively. Body weight: Birds at 2000 mg a.s./kg bw lost body weight from day 0 to 7, but recovered until test termination. Feed consumption: Birds of all test doses reduced t heir food consumption from day 0 to 3, most evidently in the highest dose group. Gross post-mortem examination: no treatment-related findings at necropsy

Conclusion: LD 50 > 2000 mg a.s./kg bw, NOEL(mortality): 1000 mg a.s ./kg bw, NOEL(sublethal effects) < 500 mg a.s./kg bw (lowest dose tested, e ffects: diarrhoea, soft– 5 –

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

excrement, reduced feed consumption)

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

Active substance - Spirotetramat (BYI 08330)

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

) Author(s), year: Barfknecht, R. (2004) Report/Doc. number: BAR / LC017, M-084796-01-2 Guideline(s): EPA, OPPTS No.: 850.2200; OECD No. 20 5 GLP: Yes (certified laboratory) Deviations: None relevant; relative humidity not re ported, temperatures were warmer (32.6 – 38.3 °C) than EPA-recommended (22 - 27 °C), acclima tion period was only 3 d and not 7 days as recommended by EPA guidelines. Validity: Acceptable

Test substance: Spirotetramat (techn.), batch No.: MIX-Batch 08045 /0014, TOX-no. 6344-00; purity 97.5 % Material and methods: Test organism: Bobwhite quail (Colinus virginianus), 10 days old and weighing 15 - 20.9 g Dosage levels: control (untreated diet); 313, 625, 1250, 2500 and 5000 ppm a.s. (nominal) corresponding to 301, 624, 1218, 2552 and 4998 mg m easured a.s./kg feed Number of birds and replicates/group: 10 birds per diet group, 2 control groups, sex unde termined

Administration: test substance mixed in diet Duration: 5 days administration period and 3 days p ost-exposure period Test conditions: acclimation: 3 d; feeding: standard rearing diet fo r quails ad libitum ; housing in stainless steel wire cages; temperature: 32.6 - 38. 3 °C, relative humidity: not reported, photoperiod 14 h Analytics: Concentration, homogeneity and stability of the test substance in the diet were sufficiently verified by analytical methods Test parameter: Observations: Observations for signs of intoxicati on were made daily during acclimation, twice on the first exposure day and continued at least on ce daily until termination. Body weight: Individual body weights were measured at test initiation, on day 5 and at termination of the test on day 8 Feed consumption: daily feed consumption per group for the periods day 0-4 and day 5-7 determined Necropsy: Gross necropsy performed at termination Statistics: Statistics was performed in 2008 upon r equest of US EPA (not included in study report): The data were subjected to an analysis of equal variances (Bartlett‘s test). In case of equal variances (which was the case for all tests) subsequent analyses were conducted using parametric techniques (Dunnett ’s test). The program was ToxRat Professional Version 2.09 So ftware. Findings: Mortalities: None treatment related. One chick of t he 1250 ppm group was sacrificed because of an injury. Clinical signs: During the exposure period the chic ks of the highest dose group showed signs of– 6 –

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

intoxication like soft excrements (reversible). Body weight: Body weights were statistically analys ed for day 0, +5 and +8. At the beginning the birds at 625 ppm and at 5000 ppm had a slightly higher body weight, which was statistically significant. After the exposure ( day +5) only the birds at 2500 ppm showed significantly lower body weights while at da y +8 (end of recovery) no differences between control and treatment groups we re found. From these results it was concluded that there was no impact on body w eight. A significantly reduced body weight change during t he exposure period for the birds at 2500 ppm and at 5000 ppm was detected. Thi s finding is is in good compliance with the reduced food consumption at the se concentrations. In the recovery period, the birds of the two highes t concentrations grew more than the control or the lower concentration groups. At 5 000 ppm the increased growth rate was even statistically significant. These findings indicated a severe starvation effect at the high test concentration. These effects were compensated as soon as the birds were switched to normal diet. Feed consumption: Food avoidance in the 2500 ppm a. s. group and above. Chicks consumed 30 - 50 % less of the amount of the control groups. No d ifferences in the postexposure period. Necropsy: No findings except the injured bird (not treatment related). Table B.9.1.2-1a: Summary of effects of spirotetram at on Bobwhite Quail following short-term dietary exposure

Treatment (measured) [ppm a.s.] Cumulative

mortality Mean bw

per bird d 0 – 4 [g] Mean bw

gain d 0 - 5 [%] Mean bw

gain d 6 - 8 [%] Mean feed

consumption d 0 - 4 [g/bird/d] Mean feed

consumption d 5 - 7 [g/bird/d] Estimated

intake d 0 - 4 [mg as/kg bw/d]

Control 0/20 21.4 46 23.5 3.1 3.8 0

301 0/10 22.6 43 23.7 3.1 3.9 39.8

624 0/10 23.0 43.4 26.2 3.1 4.1 82.6

1218 1/10 20.8 41.3 25.5 3.1 3.6 182.7

2552 0/10 19.6 28.7 28.2 1.8 3.4 234.7

4998 0/10 21.1 24.5 37.2 2.1 4.3 497.4

Table B.9.1.2-2b: Body weight data Nominal dietary Body Weights (g) Concentration Day 0 ⇐ ∆ ⇒ Day 5 ⇐ ∆ ⇒ Day 8 0 mg ai./kg diet mean 17.4 25.4 31.4 SD 1.5 (n=20) +46.0 %
2.5 (n=20) +23.5 %
4.3 (n=20)

313 mg ai./kg diet mean 18.6 26.6 32.9 SD 1.3 (n=10) +43.0 %
2.7 (n=10) +23.5 %
3.7 (n=10)

625 mg ai./kg diet mean 18.9* 27.1 34.2 SD 1.4 (n=10) +43.3 %
2.3 (n=10) +26.1 %
2.9 (n=10)

1250 mg ai./kg diet mean 17.2 24.3 30.5 SD 0.9 (n=10) +41.6 %
1.5 (n=9) +25.5 %
1.4 (n=9)

2500 mg ai./kg diet mean 17.1 22.0* 28.2 SD 1.2 (n=10) +28.6 %*
3.4 (n=10) +28.1 %
4.5 (n=10)

5000 mg ai./kg diet mean 18.8* 23.4 32.1 SD 1.3 (n=10) +24.5 %*
1.8 (n=10) +37.1 %*
2.6 (n=10)

* statistically significant– 7 –

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

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

Conclusion: LC 50 > 4998 mg a.s./kg food, equivalent to > 497 mg a.s ./kg bw/d, based on a mean feed consumption of 2.1 g/bird/d, a mean body weight of 21.1 g and on measured test substance concentrations; no mortalit y level = 4998 mg a.s./kg food (measured); NOEC: 1218 mg a.s./kg food (measue rd) based on food avoidance and reduced body weight gain.

Reference: Technical BYI 08330: A Subacute Dietary LC 50 with Mallards Author(s), year: Stoughton, T.L.; 2005 Report/Doc. number: EBFNX012; M-274385-01-1 Guideline(s): EPA 71-2, OPPTS No.: 850.2200; OECD N o. 205 GLP: Yes (certified laboratory) Deviations: None Validity: Acceptable

Test substance: Spirotetramat (techn.), batch No.: 08045/0014; pur ity 97.1 % Material and methods: Test organism: Mallard Duck ( Anas platyrhynchos )

, 10 day-old hatchlings Dosage levels: control (untreated diet); 313, 625, 1250, 2500 and 5000 ppm a.s. (nominal) corresponding to 344, 708, 1436, 2843 and 6050 mg m easured a.s./kg feed Number of birds and replicates/group: 10 birds per diet group, one control group, sex und etermined

Administration: test substance mixed in diet Duration: 5 days administration period and 3 days p ost-exposure period Test conditions: acclimation: 5 d; feeding: starter ration ad libitum ; housing in stainless steel wire cages; average temperature: 22 °C, relative humidit y: 56 %, photoperiod 14 h Analytics: Concentration, homogeneity and stability of the test substance in the diet were sufficiently verified by analytical methods Test parameter: Observations: Observations for signs of intoxicati on were made daily during acclimation, twice on the first exposure day and continued at least on ce daily until termination. Body weight: individual body weights were measured at test initiation, on day 5 and at termination of the test on day 8 Feed consumption: daily feed consumption per group recorded for the pre-exposure, exposure and– 8 –

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

recovery periods Necropsy: Gross necropsy performed at termination Statistics: Body weight and growth data were analys ed using TOXSTAT (if normally distributed, data were subjected to parametric anal ysis (one-way ANOVA); if not normally distributed after transformation, data wer e subjected to nonparametric analysis (Kruskal-Wallis ANOVA)) Findings: Mortalities: None Clinical signs: None Body weight: Body weights for Day 5 and body weight change from Day 0 to Day 5 exhibited a statistical difference at all treatment levels rela tive to the control. Body weight change from Day 5 to Day 8 exhibited a statistical difference at all treatment levels relative to the control. However, these were not an adverse effect since the increase in bodyweight at the treatment l evels was greater than in the control. Feed consumption: Due to the fact that there were n o replicates for feed consumption (based on guideline study design) no statistical analysis cou ld be performed on this parameter. A reduction in feed consumption was obse rved in all treatment groups during the exposure period as compared to the contr ol. During the recovery period the groups treated with up to 2843 ppm showed a sig nificant increase in food consumption and returned to normal or higher levels as compared to the controls. Necropsy: No findings.

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

Treatment (measured) [ppm a.s.] Cumulative

mortality Mean bw

d 0 – 5 [g] Mean bw gain d 0 - 5 [g] Mean bw

gain d 6 - 8 [g] Mean feed

consumption d 0 - 4 [g/bird/d] Mean feed

consumption d 5 -7 [g/bird/d] Estimated intake d 0 - 4 [mg as/kg bw/d]

Control 0/10 199.2 116.4 25.4 45.2 41.5 /

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

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

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

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

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

a Apparent significant reduction in feed consumption based on empirical analysis as compared to the cont rol group. Since there were no replicates for feed consumption foreseen in the used test guideline no statistical analysis could be performed on this parameter.

Table B.9.1.2-2b: Mean body weight data (g) Treatment (measured) [ppm a.s.] Mean bw on day 0 Mean bw on day 5 Mean bw on day 8

Control 141.0 257.4 282.8

344 137.3 207.5* 261.3

708 137.2 188.4* 252.5*

1436 136.6 162.2* 244.6*

2843 143.4 151.0* 222.1*

6050 133.7 118.6* 169.2* * statistically significantly different compared to control– 9 –

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

Conclusion: LC 50 > 6050 mg a.s./kg food, equivalent to > 475 mg a.s ./kg bw/d, based on a mean feed consumption of 9.9 g/bird/d, a mean body weight of 126.2 g and on measured test substance concentrations; no mortalit y level = 6050 mg a.s./kg feed; NOEC < 344 mg a.s./kg feed (food avoidance an d resulting reduced body weight gain already at the lowest test concentratio n observed).

B.9.1.3 Sub-chronic toxicity and reproduction (OECD IIA 8.1.4)
Active substance – Spirotetramat

Reference: Effect of Technical BYI 08330 on Northern Bobwhite Reproduction Author(s), year: Sabbert, T.J. and Nuessle C.; 2006 a Report/Doc. number: EBFNO220, M-277757-01-1 Guideline(s): OPPTS Nr. 850.2300, FIFRA Guideline 7 1-4, OECD 206 GLP: Yes Deviations: Minor deviation: age of birds lower tha n recommended in guidelines (16 weeks instead 20 w according to OECD 206 and 7 months acc ording to EPA). Eggs were not candled on day 0 (deviation to all gu idelines). Validity: Acceptable

Test substance: Spirotetramat (techn.), batch No.: 08045/0014, pur ity 97.2 % Material and methods: Test organism: Bobwhite quail ( Colinus virginianus ),16 weeks of age Dosage levels: Control (untreated diet); 80, 240 an d 720 mg ai/kg feed (nominal), corresponding to 0, 85, 264 and 802 mg ai/kg feed (measured). The amount of a.s. technical was adjusted to reflect 100 % a.s.. The daily dietary d oses were 0, 8, 23 and 74 mg a.s./kg bw/day. Number of birds and replicates/group: 18 pairs (1 pair in one pen) / treatment group

Administration: Test substance mixed in the diet, p repared weekly Analytics: Concentration, homogeneity and stability of the test substance in the diet were sufficiently verified by analytical methods. Study phases: Acclimation: 23 days; Pre-photostimul ation: 7 weeks; 11 weeks prior to egg-laying and for 11 weeks during the reproductive phase of t he study Pre-egg laying (with photostimulation): 3 weeks; Egg-laying: approximate ly 11 weeks; Post-adult termination: 6 weeks Test conditions: Feeding: standard commercial diet ad libitum ; adult housings: average 22°C, relative humidity: 56 %, 13 air changes per hour, p hotoperiod 7 h increasing to 17 h, wide spectrum illumination Duration: Approximately 22 weeks (pre-egg laying un til termination) Test parameter: Observations: Clinical appearance and mortality of the parental generation were monitored daily throughout the study. Reproductive parameters inclu ded egg production, egg fertility, embryo survival, hatchability, hatchling body weight, 14-day survival, 14- day survivor body weight, and eggshell strength/thi ckness. Adult Body weight: Adult body weights were measure d at study initiation, on weeks 3, 5, 7, 9, and at adult termination. Adult feed consumption: Feed consumption for each pen was measured weekly t hroughout the test.– 10 –

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

Egg parameter: Eggs were collected daily and candl ed weekly to detect egg shell cracks or abnormal eggs. Eggs were candled later to determine embryo viability and to determine embryo survival. Egg shell thickness was measured. Hatchling parameter The group body weight of the s urviving hatchlings per pen was determined. At 14 days of age, the average body weight by parental pe n of all surviving chicks was determined. Post-mortem examination: All adult birds were subjected to a gross necropsy.

Statistics: A SAS based statistical program was use d for data analysis (USEPA, 2002). Data from treatment groups were compared to controls usi ng the Shapiro-Wilk`s and Levene`s test for equal variance to determine if do se groups had unequal variances. If normality and variances were equal, t hen parametric analyses were conducted using ANOVA followed by Dunnett`s or Will iam`s test. If variances were unequal, then the nonparametric analyses were conducted using the Jonckheere or Mann-Whitney procedures. Findings: Mortalities: No treatment-related mortalities. Seve n incidental mortalities occurred (2 controls, three at 85 ppm, two at 802 ppm). Clinical signs: No treatment-related signs of toxic ity were observed in any test group. Body weight: Parents: There was an approximate 14 % reduction in the mean female weight gain in the 802 ppm group as compared to the contro l (statistically significant, Jonckheere`s test). Hatchlings: An approximate 5 % reduction in the med ian body weights was noted in the highest treatment level as compared to the c ontrol (statistically significant, Dunnett`s test). This small reduction is considered to be not biologically relevant. Feed consumption: No treatment-related effects upon feed consumption. Gross necropsy: No treatment-related findings. Reproductive results: See Table B.9.1.3-1. For the percent eggs set of eggs laid, the Jonckhee re and Mann-Whitney`s test indicated a statistical difference from the control in the 85, 264 and 802 ppm levels. However, the difference in medians between the treatment levels and the control was 1.85 % at maximum. This difference is c onsidered to be not biologically significant. The Jonckheere`s test indicated a statistical diffe rence from the control in the 802 ppm level for the percent viable embryos of eggs se t endpoint. The approximate 3 % reduction in means between the high treatment l evel as compared to the control, however, is considered not biologically si gnificant. 93 % of this parameter is acceptable even for control levels.

Table B.9.1.3-1: Summary of reproductive results of spirotetramat in the bobwhite quail
Test concentration (measured, ppm a.s.) Reproductive parameter Control 85 264 802
Overall mean food consumption (g/bird/day) 20.6 20 .2 19.3 19.5 Mean adult body weight total change (g)
male 28 27 23 25

female 62 54 57 49* Total eggs laid 867 785 805 972 Eggs set / Pen 44.61 39.17 40.83 48.94 Eggs set / Eggs laid [%] 93.04 89.75* 86.4* 90.38*– 11 –

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

Test concentration (measured, ppm a.s.) Reproductive parameter Control 85 264 802
Viable embryos / Eggs set [%] 95.59 94.99 94.44 93.13* Viable embryos / Pen 42.33 37.44 38.72 45.72 Live 3-week embryos / Pen 42.11 37.11 38.50 45.17 Hatchlings / Pen 40.11 34.61 35.06 42.28 14-day old survivors 718 614 625 759 Eggs laid / Hen 48.17 43.61 44.72 54 Eggs cracked / Pen 0.22 0.67 0.06 0.5 14-day survivors / Hen 42.24 40.93 36.76 42.17 Mean egg shell thickness [mm] 0.21 0.20 0.20 0.20 Mean hatchling body weight [g] 7.2 6.9 7.0 6.8* inhibition of above compared to control [%] / 4.2 2 .8 5.6 Mean 14-day survivor body weight [g] 35.5 35.6 35.5 34.5 Live 3-week embryos / Viable embryos [%] 99.43 98.93 99.57 98.94 Hatchlings / Live 3-week embryos [%] 92.73 93.03 89 .57 93.64 14-day old survivors / Hatchlings [%] 99.43 98.80 9 8.07 99.80 Hatchlings / Eggs laid [%] 81.72 79.26 73.11 78.26 Hatchlings / Eggs set [%] 88.01 87.97 84.77 86.54 14-day old survivors / Eggs set [%] 87.46 86.87 83. 72 86.35 * Significantly different from the control, p< 0.05

Conclusion: Based on all parameters, and the consideration of biological relevance, the NOAEC for adult bobwhite quails was 264 mg a.s./kg feed, based on statistically significant reductions in adult female body weight gain (equivalent to 23 mg a.s./kg bw/d, based on measured concentrations). The NOAEC for reproductive parameters was the highe st dosage tested, 802 mg a.s./kg feed. This is equivalent to 74 mg a.s./kg b w/d, based on a mean body weight of 218 g and mean feed consumption of 20 g/b ird/d. Comments: US EPA recommends reporting the reproductive NOAEC as 264 ppm (23 mg/kg bw/d), based on a statistically significant effect on mean hatchling body weight and a reduction of the embryos/eggs set (%) endpoin t.

Reference: Effect of Technical BYI 08330 on Mallard Reproducti on Author(s), year: Sabbert, T.J. and Nuessle C.; 2006 b Report/Doc. number: EBFNX013, M-277794-01-2 Guideline(s): USEPA FIFRA 71-4, OPPTS 850.2300 (Dra ft), OECD 206, ASTM standard E 1062-86 GLP: Yes Deviations: None relevant, minor deviation to EPA g uideline: age of birds lower than recommended Validity: Acceptable

Test substance: Spirotetramat (techn.), batch No.: 08045/0014, pur ity 97.2 % Material and methods:– 12 –

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

Test organism: Mallard duck ( Anas platyrhynchos ), 19 weeks of age Dosage levels: Control (untreated diet); 80, 240 an d 720 mg ai/kg feed (nominal), corresponding to 0, 89, 269 and 869 mg ai/kg feed (measured). The amount of a.s. technical was adjusted to reflect 100 % a.s.. The daily dietary d oses were 0, 9, 22 and 50 mg a.s./kg bw/day (calculated). Number of birds and replicates/group: 15 pairs (1 pair in one pen) / treatment group

Administration: Test substance mechanically mixed i n the diet, prepared weekly. Analytics: Concentration, homogeneity and stability of the test substance in the diet were sufficiently verified by analytical methods. Study phases: Acclimation: 3 weeks; Pre-photostimul ation: 7 weeks; Pre-egg laying (with photostimulation): 3 weeks; Egg-laying: approximate ly 11 weeks; Post-adult termination: 6 weeks Test conditions: Feeding: standard commercial diet ad libitum ; adult housings: average 22°C, relative humidity: 55 %, 13 air changes per hour, p hotoperiod 7 h increasing to 17 h, wide spectrum illumination Duration: Approximately 21 weeks (pre-egg laying un til termination) Test parameter: Observations: Adult birds were observed at least o nce daily for signs of toxicity, injuries, illness or mortality. Reproductive parameters included egg production, egg fertility, embryo survival, hatchability, hatchling body weigh t, 14-day survival, 14-day survivor body weight, and eggshell strength/thickne ss. Adult Body weight: Adult body weights were measure d at study initiation, on weeks 3, 5, 7, 9, and at adult termination. Adult feed consumption: Feed consumption for each pen was measured weekly t hroughout the test.

Egg parameter: Eggs were collected twice daily and candled weekly to detect egg shell cracks or abnormal eggs. Eggs were candled later to determine embryo viability and to determine embryo survival. Egg shell thickness and strength were measured. Hatchling parameter Body weights were measured on the day they were removed from the hatcher and on day 14. Hatchlings were observed daily for signs of toxicity, injuries, illness or mortality. Post-mortem examination: All adult birds were subjected to a gross necropsy.

Statistics: A SAS based statistical program was use d for data analysis (USEPA, 2002). Data from treatment groups were compared to controls usi ng the Shapiro-Wilk`s and Levene`s test for equal variance to determine if do se groups had unequal variances. If normality and variances were equal, t hen parametric analyses were conducted using ANOVA followed by Dunnett`s or Will iam`s test. If variances were unequal, then the nonparametric analyses were conducted using the Jonckheere or Mann-Whitney procedures. Findings: Mortalities: A total of five adult mallards died du ring the exposure period. One in the control group, one in the 269 mg a.s./kg treatment level an d three in the 869 mg a.s./kg treatment level (prior to week 12 adult sacrifice). The mortalities up to 269 ppm were considered not treatment-related. The birds at the highest dose level died as a result of emaciation. Clinical signs: Approximately week 7 post study ini tiation, nine of the thirty adult mallard in the 869 mg a.s./kg treatment level began to express sig ns of ataxia (loss of balance). The birds in this treatment level were also noted t o have severe peeling and cracking of the foot webbing. During study week 7, one hen was found dead. By– 13 –

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

study week 8, nineteen of the remaining twenty eigh t birds in the 869 mg a.s./kg treatment level had severe peeling and cracking on the bottoms of their feet. The birds could not feed or drink and by week 9, body w eights of these adult birds showed a marked decrease from week 1 body weights. The foot symptoms were originally diagnosed as “bumblefoot” by the attendi ng veterinarian although they were noted to be atypical of this foot condition wh ich 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 bird s to reduce the foot symptoms prior to reproductive phase. At week 12, two more a dult mortalities occurred and approximately 79 % of the remaining 24 birds were o bserved with moderate to severe foot symptoms. All of the adult mallard in t he 869 mg a.s./kg treatment level were sacrificed at the end of week 12 post st udy initiation for humane reasons due to the condition of the feet resulting in emaciation. The severe peeling and cracking of feet were observ ed in one bird at the 269 ppm treatment level beginning week 9 post study initiat ion. By adult sacrifice (week 21), approximately 54 % of the 269 mg a.s./kg study birds had the foot condition. One mortality occurred during this time period. Adu lts were able to feed and drink, did not show the same signs of ataxia as seen in th e 869 ppm treatment level and were able to complete the reproductive phase of the study. A light to moderately severe form of the peeling an d cracking foot symptoms were observed in one bird at week 14 post study initiati on at the 89 ppm treatment level. At the time of adult sacrifice (week 21) in the 89 ppm treatment level, approximately 27 % of the adult birds showed foot s ymptoms. No bird mortalities occurred at this treatment level and the adult body weights at termination were not statistically different from the control birds. At week 16 post study initiation in the control gro up, one adult mallard was observed to have a bumblefoot-like condition. At ad ult sacrifice (week 21), six of twenty eight (one mortality occurred) control birds were indicated as having bumblefoot-like foot symptoms. These symptoms, howe ver, were not the peeling and cracking foot symptoms that was seen in the 269 and 869 mg a.s./kg treatment levels. These symptoms were indistinguish able from foot cage wear that is typically seen due to housing conditions. Excluding the foot symptoms that were indistinguish able from cage wear or “typical” bumblefoot, the foot symptoms appeared re lated to dose. However, it was unclear whether the foot symptoms had an effect on the outcome of the other endpoints analysed for this study. The NOEC based o n foot symptoms was < 89 mg a.s./kg and the LOEC was 89 mg a.s./kg. No clinical signs were observed in hatchlings. Body weight: Parents: A significant statistical dif ference was determined at the 269 ppm treatment level for both female and male body weigh t gain. Hatchlings: In the 89 ppm treatment level a statist ical difference was indicated for the body weight (approximate 6.5 % reduction). For 14-d-old hatchlings the difference was not significant at 89 ppm, but at 26 9 ppm it was significant. Feed consumption: A significant statistical differe nce was determined at the 269 ppm treatment level for adult feed consumption as compared to the contr ol. Gross necropsy: No apparent treatment-related findi ngs except for the foot lesions. Birds of the highest treatment level were emaciated. Reproductive results: See Table B.9.1.3-2. For the eggs laid and eggs set per pen endpoints, t he respective tests indicated a statistical difference from the control in the 269 ppm level. The Jonckheere`s test indicated a statistical diffe rence from the control in the 269– 14 –

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

Table B.9.1.3-2: Summary of reproductive results of spirotetramat in the mallard duck
Test concentration (measured, ppm a.s.) Reproductive parameter Control 89 269 869
Overall mean food consumption (g/bird/day) 113 118 86* 57*

Mean adult body weight total change (g)
male 17 5 -113* -315*

female 197 140 -8* -124*

Total eggs laid 758 767 427* n.d.

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

Eggs cracked 2 1 2 n.d.

Eggs cracked / Pen 0.14 0.07 0.13 n.d.

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

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

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

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

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

Hatchlings / Pen 27.8 32.2 8.1* n.d.

14-day old survivors 386 386 80* n.d. 14-day survivors / Hen 29.7 32.2 8.9* n.d.

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

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

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

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

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

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

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

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

14-day old survivors / Eggs set [%] 58.2 61.1 25.3* n.d.
* Significantly different from the control, p< 0.05 n.d. not determined for the 869 ppm treatment level due to adult sacrifice 12 weeks post study initiat ion

Conclusion: The study authors consider neither the foot effect s nor the slightly reduced hatchlings body weight as biologically adverse and propose a NOAEC of 89 mg a.s./kg feed, based on several statistically signif icant reproductive parameters and reduced adult body weight gain. This is equivalent to 9.4 mg ai/kg bw/d, based on a mean body weight of 1122 g and mean feed consumpt ion of 118 g/bird/d. The RMS considers the slight reduction in the hatch lings body weight in the lowest dose group as biologically not adverse, therefore t he reproductive NOAEC is 89 ppm. However, no NOEC based on foot symptoms could be determined and the LOEC was 89 mg a.s./kg.– 15 –

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

Reference: BYI 08330 Technical: A Reproduction Study with the Mallard Author(s), year: Temple, D.L.; Martin, K.H.; Beaver s, J.B.; Jaber, M.; 2006 Report/Doc. number: EBFNP024, M-278058-01-1 Guideline(s): US EPA FIFRA 71-4, OPPTS 850.2300 (Dr aft), OECD 206 GLP: Yes Deviations: None relevant. Minor deviation: relativ e humidity lower than required according to guidelines. Minor deviation to EPA guideline only: age of birds lower than recommended. Validity: Acceptable

Test substance: BYI 08330 (techn.), batch No.: 08045/0014, purity 97.99 % Material and methods: Test organism: Mallard duck ( Anas platyrhynchos ), 19 weeks of age Dosage levels: Control (untreated diet); 30, 80, 20 0 and 720 mg a.s./kg feed (nominal), equivalent to measured concentrations of 28.8, 78.8, 201 and 7 27 mg a.s./kg feed. Due to the debilitated condition of the birds, the 720 ppm a.s. treatment group was terminated in week 5. The amount of a.s. technical was adjusted to reflect 100 % a.s.. The daily dietary doses were 0, 4.2, 10 and 2 3.6 mg a.s./kg bw/day. Number of birds and replicates/group: 15 pairs (1 pair in one pen) / treatment group

Administration: Test substance mechanically mixed i n the diet, prepared weekly. Analytics: Concentration, homogeneity and stability of the test substance in the diet were sufficiently verified by analytical methods. Study phases: Acclimation: 4 weeks; Pre-photostimul ation: 9 weeks; Pre-egg laying (with photostimulation): 3 weeks; Egg-laying: approximate ly 10 weeks; Post-adult termination: 6 weeks Test conditions: Feeding: standard commercial diet ad libitum ; adult housings: average 21.5 - 23.3°C, relative humidity: 27.3 - 64.5 %, 15 air ch anges per hour, photoperiod 7 h increasing to 17 h, wide spectrum illumination Duration: Approximately 22 weeks (pre-egg laying un til termination) Test parameter: Observations: Adult birds were observed daily for signs of toxicity and abnormal behaviour. Photographs of the duck`s feet were taken at every body weight interval for potential comparison of foot lesions. Reproductive parameters included egg production, egg fertility, hatchability, hatchling body weight, 14-day survival, 14- day survivor body weight. Adult Body weight: Adult body weights were measure d at study initiation, on weeks 2, 4, 6, 8, 9, and at adult termination. Adult feed consumption: Feed consumption for each pen was measured weekly t hroughout the test.

Egg parameter: Eggs were collected twice daily and candled weekly to detect egg shell cracks or abnormal eggs. Eggs were candled later to determine embryo viability and to determine embryo survival. Egg shell thickness and strength were measured. Hatchling parameter Body weights were measured on the day they were removed from the hatcher and on day 14. Hatchlings were observed daily for signs of toxicity. Post-mortem examination: All adult birds were subjected to a gross necropsy.

Statistics: An analysis of variance (ANOVA) was per formed to determine statistically significant differences between groups. Dunnett`s m ultiple comparison procedure was used to compare the treatment means with the co ntrol group mean.– 16 –

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Percentage data were examined using Dunnett`s metho d following arcsine square root transformation. See also “Appendix”: US EPA re -analyzed the data applying different test procedures. Findings: Mortalities: A single mortality occurred in the 200 ppm a.s. treatment group. Clinical signs: Birds in the 720 ppm a.s. treatment group were first noted exhibiting clinical signs of toxicity, which ultimately included loss of coor dination, lower limb weakness and cracked and bleeding lesions on the bottoms of thei r feet, on Day 3 of Week 3. Marked, continued loss of body weight was also appa rent 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 eu thanized. Incidental clinical observations noted during the t est included those that normally are associated with injuries and penwear. Such sign s included bumblefoot (plantar pododermatitis), lameness, feather loss, molting an d neck, back and shoulder lesions. Treatment-related findings in the 80 ppm treatment and above groups included lesions on the bottoms of the feet characterised by thickened areas that appeared rough, dry and hard, with cracking, fissuring and/o r bleeding evident. Body weight: Parents: Slight and transient statisti cally significant body weight changes in the 30 and 80 ppm treatment groups were considered not tre atment related. A significant statistical difference in body weight and bw change s was determined in hens of the 200 ppm treatment group. Birds in the 720 ppm t reatment group exhibited marked weight loss. Hatchlings: In the 80 ppm treatment level a slight but statistically significant reduction was seen in the hatchling`s body weight. Feed consumption: At the 80 ppm test concentration, there was a slight and transient, statistically significant reduction in mean feed consumption duri ng Week 15. At the 200 ppm test concentration, there were treatment-related re ductions in feed consumption during Week 4 and from Week 11 through 18 that were statistically significant. At the 720 ppm test concentration there were statistic ally significant treatmentrelated reductions in feed consumption from Week 1 to Week 5 (termination of the adult birds for the high dose group). According to the US EPA`s re-analysis (see Appendix), the mean overall feed consumption was st atistically 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 roug h, dry and hard, with cracking, fissuring and/or bleeding evident. These lesions were noted in four males and six females from the 80 ppm a.s. treatmen t group, 13 males and 14 females from the 200 ppm a.s. treatment group and 1 4 males and 12 females from the 720 ppm a.s. treatment group. The numbers of hens with regressed ovaries and the numbers of drakes with reduced testes were increased in both the 80 and 20 0 ppm a.s. treatment groups. Reproductive results: See Table B.9.1.3-3.– 17 –

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

Table B.9.1.3-3: Summary of reproductive results of spirotetramat in the mallard duck
Test concentration (measured, ppm a.s.) Reproductive parameter Control 28.8 78.8 201
Number of replicates 15 15 15 14 Overall mean food consumption (g/bird/day) 162 159 142 + 131 +
Mean adult body weight total change (g)
male -32 9 4 -34

female 58 84 -2 -73* Total eggs laid 1 686 629 456 276 Eggs laid / Hen 1 46 42 30 20 Eggs cracked 1 4 6 3 3 Eggs cracked / Eggs laid [%] 2 1 1 2 Viable embryos / Eggs set [%] 87 83 91 48* 14-day old survivors 1 426 333 266 76 14-day survivors / Hen 1 28 22 18 5 Mean egg shell thickness [mm] 0.389 0.382 0.389 0.3 73 Mean hatchling body weight [g] ( sample size ) 34 ( 12 ) 32 + ( 13

) 31* + ( 14 ) 28* + ( 6 ) Mean 14-day survivor body weight [g] 274 266 257 20 0* Live 3-week embryos / Viable embryos [%] 98 99 98 96 Hatchlings / Live 3-week embryos [%] 79 77 63 + 41* +
14-day old survivors / Hatchlings [%] 100 99 94 91 +
Hatchlings / Eggs set [%] 69 63 60 25* 14-day old survivors / Eggs set [%] 69 62 57 + 23* + * Significantly different from the control, p< 0.05 , Dunnett`s test, study author`s analysis

+ Significantly different from the control, p< 0.05, Williams`s test or Jonckheere`s test, according to EPA analysis (see Appendix)
1 no statistical analysis on this parameter

Conclusion: The NOAEC was 28.8 mg a.s./kg feed (measured), bas ed on treatment related foot lesions, effects upon offspring body weights a nd on egg production and hatchability at 78.8 mg a.s./kg feed. This is equiv alent to a NOAEL of 4.0 mg a.s./kg bw/d, based on a measured feed concentratio n of 28.8 ppm, a mean body weight of 1147 g and mean feed consumption of 159 g /bird/d. The statistically significant reduction of the hatchlings body weight at the 28.8 ppm dose level is considered biologically not relevant.

Appendix: statistical analysis of the reproductive data by the US EPA: The reproductive data of this study were re-analyze d according to US EPA Environmental Fate and Effect s Division’s (EFED) standard protocol, since not all endpoints typically considered by the EPA were init ially statistically analyzed and included in the study re port. The study authors have explained that paramet ers that were raw count data endpoints were not statisticall y analyzed because they were inherently non-normall y distributed. Instead, these raw count data were con verted to proportions (e.g., by dividing the counts by the number of eggs set from a pen). All data that were inherently proportions or were converted to a propo rtion were arcsine-transformed to meet assumptions of nor mality. However, no tests of normality or homogenei ty of variance were performed on any endpoint paramete r even after these transformations. Once the data were transformed, an analysis of variance (ANOVA) w as performed to determine statistically significant differences between groups. Then Dunnett's multiple comparison procedure was used to compare the– 18 –

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treatment means with the control group mean and ass ess the statistical significance of the observed differences. In general, the EPA prefers not to transform data, but rather tests the assumptions of normality and homogeneity prior to analysis to determine whether parametric or non-parametric tests should be perfor med. Therefore the US EPA developed a SAS program for an alyzing data from avian reproduction studies, Chick s version 3.1, which was employed for the re-analysis . 22 variables were analyzed on a ‘per pen’ basis. The reproductive parameters for which the EPA’s and study authors’ conclusions diverged are the follow ing: Overall mean food consumption, Mean hatchling body weight [g], Hatchlings / Live 3-week embryos [%],14

- day old survivors / Hatchlings [%] and 14-day old s urvivors / Eggs set [%]. The EPA notes the following: First, according to th e results of Levene’s test (equality of variance) a nd Shapiro-Wilks’ test (normality), most of the count data (# of eggs set, # of eggs laid, # of viable em bryos, # of live embryos, # of eggs hatched, and hatchling surv ival) that were converted to proportions and arcsin etransformed by the study authors, did not violate t he basic assumptions of normality and homogeneity a nd could be analyzed using parametric tests without tr ansformations. Second, all of the study authors’ findings could be reproduced by the EPA when strictly employing Dunn ett’s test or its non-parametric analog, the Mann-Whitney test with a Bonferroni adjustment. However, the EP A’s statistical analyses did result in lower NOAEC valu es in a number of cases, primarily resulting from t he EPA’s application of the more-sensitive Williams’ ( parametric) and Jonckheere’s (non-parametric) tests when the assumption of a negative dose-response relation ship could reasonably be supported, as compared to the study authors’ reliance on Dunnett's multiple compa rison procedure. Most importantly it should be noted that for the en dpoint “mean hatchling bodyweight” there was a sign ificant reduction at all test levels and a NOAEC could not be determined (based on a ~6 % reduction at the low est test level). Since this study results in the most s ensitive NOAEC of all the available avian reproduct ion studies for spirotetramat, and a definitive NOAEC c ould not be identified for this endpoint, the EPA recommends that a request for another avian reprodu ction study with mallards that tests at lower level s should be considered.

In a response to the EPA re-analysis Bayer Crop Sci ence express their concern on the use of the Willia ms` test in the evaluation of avian reproduction data. They argue that the Williams test is susceptible to increasing type 1 error (false positive difference) when used to make multiple comparisons and when sa mple sizes are unequal. For the hatchling weight paramet er, sample sizes were unequal (see table B.9.1.3-3)

. According to a published article, the Williams test maintains a predetermined α -level less and less as the imbalance increases. Another publication recommends applying the Williams test at 2.5 % to quantitativ e parameters of repeated toxicity studies. If this re commendation is followed the p-value calculated for the 30 ppm dose level (p=0.031) of the hatchling weight pa rameter is higher than the recommended statistical level (p=0.025).

Comment (RMS) : A statistician of AGES (RMS authority) looked at the issue and, overall, agreed with the EPA`s position. Hence the parameter “hatchling body weight” is considered to show a statistically sign ificant difference at 30 ppm compared to the control. Hovever, in the RMS opinion the question of statist ical significance of this difference in hatchling w eight is a borderline case anyway, if the p-values of both tes ts applied are looked at. While the outcome of the statistical analysis is important for the interpret ation of the study, for the derivation of a “No obs erved adverse effect level” the magnitude and biological relevanc e of an observed effect should also be considered. The bodyweight reduction compared to the control is 6 % . BCS compared the hatchling bodyweight data obtained at the 30 ppm level with historical contro l bodyweight data from 20 duck reproduction studies provided by Wildlife International. This suggests t hat the hatchling bodyweights were not adverse, sin ce they were in the range of historical control hatchling b odyweights (mean of 20 studies = 33 ± 3 g). Further , it is important to note that the NOEC for the 14-day old duckling weight is 80 ppm. This supports that any reduction in the hatchling bodyweight was temporary and recovery took place in the ducklings of the lo west– 19 –

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

dose level. Hence, the dose level of 30 ppm, corres ponding to 4 mg/kg bw/d, is considered as NOAEL wit h regard to reproductive effects in the mallard duck. During joint review discussions between the authori ties, PMRA Canada agreed with the RMS position to consider the 30 ppm level as NOAEL for the purpose of risk assessment.

B.9.1.4 Supervised cage or field trials (OECD IIA 1 0.1.7)

- Further studies on the Mallard Duck

Introduction: During avian reproduction studies with the mallard duck dietarily exposed to spirotetramat the ducks experienced severe foot problems, expressed as drie d and deeply cracked skin on the underside of the f eet. These dermal effects to the feet may have been exac erbated by an interaction between the wire mesh of the caging (possible physical damage) and food contact with excreta (possible chemical exposure) on the ca ge floors. However, the causal mechanism is not fully understood, and the notifier performed two further studies to gain more information.

Reference: Long-Term Mallard Duck Dermal Exposure to BYI 08330 Author(s), year: Stafford, J. M.; 2007a Report/Doc. number: EBFNP033; M-290165-01-1 Guideline(s): Not applicable GLP: Yes Deviations: Not applicable Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 98.10 %, lot no . 08045-0014 Material and methods: Test organism: Mallard Duck ( Anas platyrhynchos )

, age: 35 weeks Test design: The objective of the study was to dete rmine if dermal exposure to treatment excrement alone is sufficient to elicit a dermal re sponse in the feet of ducks not exposed via diet. Four groups were incorporated int o this study: 1. Dietary control: received untreated diet, excrem ents collected every other day for transfer to dermal control cages; 2. Dermal control: received untreated diet, applica tion of excrements from group 1 to the floor of their cages 3. Dietary exposure: received nominal dietary conce ntration: 720 mg a.s./kg feed, corresponding to 760 mg a.s./kg feed (measured), ho used in isolated room, excrements collected every other day for transfer t o dermal exposure cages. Two sets of ducks were sequentially exposed due to seve re reactions necessitating euthanisation of the first set of ducks at week 4 o f the study. 4. Dermal exposure: housed in isolated room, receiv ed untreated diet, application of excrements from group 3 to the floor of their ca ges Number of birds /group: Twelve males and twelve females/group. One pair/cag e.

Administration: Test substance mixed in diet / derm al exposure to excrements from dietary
exposure group Duration: Eight weeks of treatment. Dietary exposur e group: 4 weeks exposure, then replacement with another group.– 20 –

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Test conditions: Acclimation: 2 weeks; feeding: basal diet and water ad libitum ; housing in cages with the dimensions 76 x 83 x 44 cm. The floor, bac k wall and top of the cages were made of polycarbonate-coated welded-wire mesh, the sides of galvanised steel. Isolated environmental conditions were provi ded by establishing one room for the two control groups and one room each for de rmal and dietary exposure group. Temperature: 14 - 33 °C, relative humidity: 45 - 94 %, photoperiod 7 h. During the experimental period, excrements were cat ched in plastic trays. Every other day, all cages were scrubbed with brushes dip ped in water. Separate equipment were used in each room. The excrement col lected from the dietary control and exposure cage was spread with spatulas evenly and throroughly across the entire floor of the dermal group cages. Analytics: Dietary analysis confirmed recoveries of 95 to 116 % of nominal. Excrement sample analysis: Composite excrement samp les were collected from the dietary groups every two weeks following treatm ent initiation. These samples were analysed to provide a general idea of the degr ee of exposure, not for precise quantification. Excreta were analysed to determine concentrations of Spirotetramat and its enol metabolite using an LC/M S/MS system. Test parameters: Observations: All birds were monitored every two w eeks 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 in terval all birds were captured and their feed closely examined. Notations were mad e in greater detail regarding unique conditions of the feet and digital photos we re taken. Feed consumption: Not reported. Necropsy: None. Statistics: Body weights were compared using one-wa y analysis of variance. Findings: Analytics: As result of the excreta sample analysis in the dietary treatment group residues of 0.11 (week 2) and 0.6 (week 8) mg a.s./kg were dete cted. The residue level of BYI 8330-enol was between 61.6 (week 2) and 97.6 (w eek 8) mg/kg. Residues in the dietary control were < 0.01 mg/kg. Mortalities: Severe foot symptoms progressed to war rant in extremis euthanisation of both sets of ducks of the dietary exposure group after 4 weeks of exposure. Clinical signs: Behaviour: One female in the dermal exposure group appeared emaciated and weak and showed diminished appetite from week 5 on, however, the condition of her feet was normal and this observation was consid ered not treatment-related. Ducks in both dietary exposure sets displayed abnor mal behaviour and adverse reactions in their feet: From week 3 on, limping wa s observed. From week 4 on additional symptoms appeared: using wings to aid lo comotion, decreased ambulation, piloerection, lacrimation, cracking on feet (cracks wrap around edges of feet). After 4 weeks of exposure, ducks were eut hanised. Detailed foot observations: Dietary control ducks d id not experience any cracking or drying. In the dermal control group two notation s were made of minor cuts along the foot digits. The study author states it i s not unusual for ducks to experience temporary cracking and healing of the sk in of their feet from time to time. In the dermal exposure group four occurrences of minor cracking along the digits were noted at week 4, and three occurrences of moderate cracking during the middle two weeks. Effects seemed to have been t ransient. During week four 10 cases of inflamed webbing (transient) were noted . Dietary exposure group: Moderate to extreme dermal cracking along the digit s, drying of the skin and impaired mobility and perceived discomfort due to t he sores of the feet were– 21 –

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observed. This severe cracking was distinctly diffe rent from transient minor cracking observed in other groups. A transient effe ct of inflamed webbing during week 4 was also seen. Some birds became reluctant t o feed and became emaciated and weak. Body weight: Body weights of the dietary exposure g roup were significantly reduced compared to the control. Conclusion: Two separate sets of ducks exposed to Spirotetrama t at 720 mg/kg feed exhibited symptoms of progressively worsening of cracking and drying of the feet, and impaired mobility within 2 weeks. The symptoms prog ressed to warrant in extremis euthanisation of both sets of ducks after 4 weeks of exposure. Ducks dermally exposed to the excrement of dietarily expo sed ducks experienced fewer cases of minor to moderate cracking and drying of t he feet during 8 weeks of repeated exposure to the excrement, and symptoms we re transient, less severe and did not lead to behavioural symptoms.

Reference: Semi-Field Study of the Effects of Dermal and Dieta ry Exposure to BYI 08330 in Mallard Ducks ( Anas platyrhynchos ) Author(s), year: Stafford, J. M.; 2007b Report/Doc. number: EBFNP034; M-290000-01-1 Guideline(s): Not applicable GLP: Yes Deviations: Not applicable Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) techn., purity: 98.10 %, lot no. 08045-0014 Movento formulation: BYI 08330 150 OD, lot no. 0803 0/0189(0125), purity: 14.7 %

a.s. Material and methods: Test organism: Mallard Duck ( Anas platyrhynchos )

, age: 27 weeks Test design: The objective of the study was to dete rmine 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 ex posure by walking on turf (mowed grass) treated with the test substance as we ll as by dietary exposure from feed mixed with the test substance. Birds in the treatment group received a dietary con centration of 200 mg a.s./kg feed. Formulated spirotetramat was applied in spray solution to the turf in the pens. The birds were placed in holding cages before the application and the sides of the pens were covered with plastic cover. Turf a pplications took place on three occasions spaced 14 d apart and at a rate of 158 g a.s./ha (event 1), 86.8 g a.s./ha (event 2) and 140 g a.s./ha (event 3). Addi tionally, treated feed was placed in the treatment pens immediately after turf applic ation. At the end of 10 weeks, all treated feed was replac ed with clean feed and remaining birds were left for observation through a 4-week recovery period. Number of birds /group: Three treatment and control replicate pens. Six mal es and six females/pen (36 birds / treatment or control group). Administration: Test substance mixed in diet and ex posure to oversprayed turf. Duration: Ten weeks of treatment followed by a 4-we ek recovery period. Test conditions: Acclimation: 2 weeks and 4 days; feeding: basal die t and water ad libitum ; housing in predator-proof aviaries enclosing a tota l area of approximately 1.03 ac– 22 –

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(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. eac h. At the opposite end of the pens where the feed was placed a 9.14 m untreated z one was maintained to simulate edge or adjacent habitat. At the center of the untreated zone a small pond was constructed. The ducks had to cross 36.6 m (120 ft) of treated turf from the pond to obtain feed. Natural temperature and hu midity conditions, natural photoperiod. Weather data as well as rain events we re recorded. Analytics: Dietary analysis confirmed recoveries of 82.7 to 98.9 % of nominal. Homogeneity was demonstrated. Test parameters: Observations: Daily observations for mortality and behavioural abnormalities. All birds were monitored every two weeks for changes in the condit ion of the feet and body weight measurement. Notations were made in great de tail regarding unique conditions of the feet and digital photos were take n. Notations were also made to document bird moving across the treated field and t wo sessions on birds behaviour and movements were captured by digital vi deo. Feed consumption: Feed consumption was measured da ily and average values were calculated per bird and day for the exposure and post-exposure per iods. Feet preservation: At the end of 10 weeks, the birds in Control pen 2 and Treatment pen 4 were euthanised and their feet were removed and preserve d. After the recovery period the same was done with birds of other treatment and control groups. Statistics: Body weights were compared using one-wa y analysis of variance followed by a multiple comparison test. Findings: Mortalities: None. Clinical signs: Behavioural observations: Minor abn ormal behaviours were first observed in week 6 in 2 birds and in most affected treatment birds b y week 8. These included limping, reluctance to walk, birds sitting down imm ediately upon landing and stumbling while walking. The tendency to fly rather than walk increased as treatment progressed. Limping had entirely ceased o ne week post-treatment. Foot condition: Abnormalities were noted by week 2 of treatment including cracking of the skin along the underside of the dig its. Though mild cracks are apparently normal, as supported by the occurrence a mong control birds, the treated birds experienced more minor cracking than the controls, and also showed moderate and severe cracking. The incidence of abnormal foot conditions among tre ated birds ranged from 29.2 (week 14) to 63.9 % (week 8). The number of severe cases increased until week 10. During the recovery period, significant improve ments were made in the condition of the feet. By week 14, only 7 minor cas es of feet cracking were recorded. During the entire study, no infection was noted in cracked feet. Feed consumption: Food consumption followed the sam e general pattern in all pens during the course of the study, though average weekly food consumptio n in the control pens was slightly higher than in treatment pens. Body weight: Variations of group body weights betwe en different pens occurred, but being inconsistent and transient these effects were not c onsidered treatment related. Conclusion: Ducks that received spirotetramat-treated feed and were exposed to oversprayed turf, showed minor, moderate and severe cracking of the skin of the feet from the second week on. Behavioural effects as limping and the reluctance to walk were seen in affected birds. During a 4 week recovery pe riod, significant improvements were made in the condition of the feet.– 23 –

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

- Further studies for the refinement of the risk asse ssment

Reference: BYI08330 150 OD - Magnitude of the Residue in/on So ybeans/Wheat, Potential Wildlife Feed Items Author(s), year: Fischer, D. R., Ramanarayanan, T. & Ortego, L.; 2006 Report/Doc. number: RAFNP002; M-277315-01-1 Guideline(s): Not applicable GLP: Yes (certified laboratory) Deviations: Not applicable Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) OD 150, 150 g a.s./L (no minal), batch no. 08030/0189(0152) Material and methods: Four soybean field trials and four wheat field tria ls were conducted in NAFTA Growing Regions 2 (one soybean trial), 3 (one wheat trial), 4 (one soybean trial and one wheat trial), and 5 (two soybean tri als and two wheat trials) in multiple regions througout the US. The locations of the soybean trials were in Ge orgia, Mississippi, Kansas and Nebraska, the locations of the wheat trials in Florida, Mississippi, Kansas an d Nebraska. At each test site, two foliar application s of Spirotetramat 150 OD were made to established soybeans or wheat at a target application rate of 0 .176 kg/ha/application (0.157 Ib a.s./A) and with a n application interval of 19 to 21 days. All applicat ions were made using spray volumes of 110 to 167 L/ ha and used Dyne-Amic (0.25 % v/v) as an additive. For the soybean trials, the first application was made eit her at BBCH growth stage 15 to 16 or at stage 69 to 74. Fo r the wheat trials, the first application was made at BBCH growth stage 25 to 32. The wheat trials includ ed two treated plots; one plot was mowed one to eig ht days prior to the applications and represented shor t grass; the other plot was not mowed and represent ed tall grass. Duplicate forage samples were collected from each of the treated plots at each of the samp ling intervals from the soybean and wheat trials. The fo rage samples were collected prior to the first appl ication (control samples) and at various time intervals aft er the first and second applications, up to 35 days following the second application. The samples were homogenize d, and the residues of BYI 08330 and its metabolite s BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-mo no-hydroxy, and BYI 08330-enol-glucoside were quantified by high pressure liquid chromatography/t riple stage quadrupole mass spectrometry (Ic-ms/ms) using the stable isotopically labelled analytes as internal standards and the method of external stand ard quantitation. The individual analyte residues were converted to Spirotetramat molar equivalents and su mmed to give a total Spirotetramat residue. For each trial, the total residue was measured for each sample collected at each sampling interval. Th e mean total Spirotetramat residue values at each sam ple interval were calculated from the measured tota l residues from the duplicate samples collected at th at interval. The foliar dissipation half-life of the total Spiro tetramat residue in soybean forage (broad leaf plan ts) and wheat forage (short and tall grass) following the s econd application of Spirotetramat 150 OD was calcu lated using an MS Excel 2002 electronic speadsheet.– 24 –

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

Findings:
Table B.9.1.4-1: Residues of spirotetramat in soybe an forage (broad leaf plants) and wheat forage (short and tall grass) following the second applica tion of Spirotetramat 150 OD and calculated halflives Crop Mean Total Spirotetramat Residue Immediately Following the Second Application [ppm] a Maximum Total Spirotetramat Residue Following the Second Application [ppm] b Mean Half-Life [days] c

Soybean Forage 8.32 10.28 3.42 / 4.45 d Wheat Forage (Short Grass) 14.35 20.35 2.13

Wheat Forage (Tall Grass) 13.04 21.65 1.96

a Mean total spirotetramat residue immediately following the 2 nd application is the average from all trials of the total spirotetramat residue from the sample interval immediately follow ing the 2 nd application of Spirotetramat 150 OD. b Maximum total Spirotetramat residue following the second application is the max imum total a.s. residue from all of the trials for the sample intervals following the secon d application of Spirotetramat 150 OD.

c Mean half-life is the average of the half-lives ca lculated for each trial from the total Spirotetrama t residue at each of the sample intervals following the second application o f Spirotetramat 150OD. d The mean half-life for total a.s. residue in soybe an forage is 4.45 days without inclusion of the hal f-life from trial FN333- 05W which received 1.3 inches of rain 1 day followi ng the second application of Spirotetramat 150 OD.
The highest peak residue level detected was in unmo wn wheat (21.65 ppm) which was slightly higher as compared to mown wheat (20.35 ppm) and soybean foli age (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 a pplication for any of the trials. For any of the tr ials, the earliest time that rainfall occurred following appl ication of Spirotetramat 150 OD was in trials FN331 -05W and FN333-05W which received rain (0.02 inches and 1.3 inches, respectively) one day following the second application. Conclusions: Total Spirotetramat residues dissipated with half-l ives of between 2 and 4.5 days in soybean forage (b road leaf plants) and wheat forage (short and tall grass ) after two foliar applications of Spirotetramat 15 0 OD at the highest proposed seasonal use rate. Mean and ma ximum residue values determined immediately after the second application as well as the calculated ha lf-lives are shown in the table above.

Reference: Residues of Spirotetramat in arthropods after spray application in citrus – magnitude and time course of decline Author(s), year: Wilkens, S. and Schneider, K.; 200 8 Report/Doc. number: RA07031 (Rifcon) Guideline(s): Not applicable GLP: Yes (certified test facility) Deviations: Not applicable Validity: Acceptable

Test substance: Spirotetramat OD150B G formulation, 150 g a.s./L ( nominal), 152.8 g a.s./L (analytical), batch no. 2007-0002474 Material and methods: The aim of this field study was to determine residu e levels of Spirotetramat (BYI08330) and its metabo lites BYI08330-enol and BYI08330-ketohydroxy on foliage a nd ground dwelling arthropods in citrus orchards af ter spray application of Spirotetramat OD150B G.– 25 –

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

Study area: citrus orchards in the region Valencia/Spain. Two orchards (1.2 and 1.3 ha) with two plots each (a total of 4 replicates, approx. 0.6 ha each). Application rate : One application on each plot at a rate of 99.6 an d 121.3 g a.s. per ha and canopy height (equivalent to nominal 270 g a.s./ha) with a spray volume of 3000 L/ha. The BBCH principle growth stag e of the trees was 7. Arthropod sampling : Ground dwelling arthropods were sampled using pit fall traps with a diameter of approx. 12.5 cm, which were dug at surface level and protec ted with a cover. 86 to 90 traps were installed per plot within the orchard rows. The traps were activated a pprox. 24 h before sampling. Foliage dwelling arthropods were collected by inven tory spray sampling (full cover application of a “knockdown” insecticide containing natural pyrethru m). Trees were sprayed until shortly before run-off

. Sampling was carried out also before application of the test substance, sampling took place for ground dwellers: DAT -1, DAT 0 (before application), DAT + 1, +2, +3, +4, +5, +7, +9, +11, +14 and +21; and fo r foliage dwellers: DAT -1, DAT 0 (after application) , DAT +1, +2, +3, +4, +5, +7, +9, +11, +14 and +21. After swift identification and quantification of the main taxonomic groups, the samples were stored deep fro zen until residue analysis. Residue analysis : All samples were analysed for their content of Sp irotetramat (BYI08330) and its metabolites BYI08330-enol and BYI08330-ketohydroxy via HPLC/MS-MS. Residues are reported in terms of mg a.s./kg f.w. of matrix and mg metabolite/kg f.w. , respectively. The LOQ was 0.01 mg/kg for both a.s . and metabolites. Calculation and statistics : Initial and maximum measured values of the mean r esidues (n=4) were expressed for parent, parent+enol and parent+enol+ketohydroxy (total residue). The mean residue values were used to calculate the TWAs of daily intervals between DAT 0 /1-21, expressed as percentage of the maximum measured residue value. TWAs of residue values were calculated by interpolating values for days for wh ich no experimental data were available (linear interpo lation) and by calculating the mean over a given pe riod. The residue decline (DT 50 ) of parent, parent+enol and total residue in folia ge dwelling arthropods was determined to assess the potential exposure of foli age dwellers to the a.s.. The data used are based o n the arithmetic mean of the residue values of 4 replicat es. First order kinetic was assumed. In ground dwelling arthropods residues were low and the kinetic profile was more complex and did not a llow the calculation of a SFO-DT 50 . Climate data (precipitation, temperature) were reco rded during the study. Findings: Abundance of arthropod groups: Arachnida, Isopoda, Myriapoda, Coleoptera, Dermaptera and Lepidoptera larvae were the most abundant groups in the pitfall traps, whereas Ephemeroptera, Diptera, Coleoptera, Neuroptera (adults and larvae) and Hymenoptera (wit hout Formicidae) were most abundant in the samples from inventory spraying. Precipitation: Between DAT 8 and 20, three slight r ainfalls (0.5-1 mm) occurred, and on day 15 one med ium rainfall (6 mm) took place. Validation of the analytical method: The individual recovery values for Spirotetramat for arthropods r anged from 78 to 97 %, with an overall recovery of 92 ± 3 .2 %. For Spirotetramat-enol recovery was 99 ± 3.5 %, and for Spirotetramat-ketohydroxy 79 ± 8.6 %. Residue concentrations and TWAs in arthropods are s hown in the table below:– 26 –

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

Table B.9.1.4-2: Residue concentrations in ground a nd foliage dwelling arthropods after application of spirotetramat in the field Mean residue concentrations [mg/kg f.w.] in ground dwelling arthropods Parent Parent+enol Total measured residue Initial measured value (DAT+1) 0.21 0.52 0.54

Maximum measured value (MMV) 0.32 (DAT +2) 0.60 (DAT +2) 0.62 (DAT +2)

TWA (DAT1-21) 0.15 (46 % of MMV) 0.35 (59 % of MMV) 0.39 (63 % of M MV) Mean residue concentrations [mg/kg f.w.] in foliage dwelling arthropods Parent Parent+enol Total measured residue Initial measured value (DAT+1) 4.0 5.2 5.3

Maximum measured value (MMV) 4.0 (DAT +0) 6.0 (DAT +1) 6.2 (DAT +1)

TWA (DAT1-21) 1.07 (27 % of MMV) 1.90 (32 % of MMV) 2.03 (33 % of M MV) DT 50 2.8 d 4.7 d 5.1 d

Conclusion: After application of 99.6 or 121.3 g spirotetramat per ha and meter canopy height (equivalent to nomin al 270 g a.s./ha) to citrus orchards, maximum measured res idues in ground dwellers were 0.32, 0.60 and 0.62 mg/kg f.w. for parent, parent+enol and total measur ed residues, respectively; and for foliage dwellers 4.0, 6.0 and 6.2 mg/kg f.w. for parent, parent+enol and tota l measured residues, respectively. The decline of r esidue levels in foliage dwellers followed a first-order k inetic, the DT 50 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 preva lent in Spain. Comment (RMS) : In several instances residues exceeding the LOQ w ere found in arthropods collected before application of spirotetramat (both ground an d foliage dwelling). These residues amounted up to 0.11 mg/kg f.w. in the case of ground dwellers. Upon req uest the notfier tried to find an explanation and a dmitted that some kind of contamination of samples must hav e taken place, however they were unable to identify the situation or location of contamination. Because thi s shortcoming of the study does not lead to an understimation of the residues, the study is consid ered acceptable.

Reference: Insectivorous birds in southern European citrus pla ntations - focal species and their diet composition (Position Paper) Author(s), year: Wolf, Ch. & Gerlach, J.; 2006 Report/Doc. number: RC06-067; M-277503-01-1 Guideline(s): Not applicable GLP: Yes (certified laboratory) Deviations: Not applicable Validity: Acceptable.

The position paper gives a summary and evaluation o f literature sources on the Great Tit ( Parus major ) as focal species in citrus orchards. As a typical insectivorous bird in Europe inhabitin g a wide range of habitats with trees, like woodlan ds, gardens and orchards, the great tit ( Parus major ) can be regarded as a representative focal species also in citrus plantations. Great tits usually breed in hol es (Perrins 1979). Tree holes, as well as nestboxes are accepted, and both constellations can be observed i n orange groves in Spain (Gil-Delgado & Barba 1987)

. Two investigations on breeding biology and success have been made regarding both the occurrence in tre e holes and in nestboxes. Moreover observations of op en nesting in this species have also been reported (Monrós et al. 1999).– 27 –

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

Some publications deliver information on the occurr ence of great tits in citrus plantations: The popul ation density of great tits in the orange grove was rangi ng between 13.0 and 13.6 pairs per 10 ha, respectiv ely (Monrós et al. 1999). A comparative study on breedi ng biology of great tits revealed an earlier onset of breeding and a larger clutch size (mean 7.7 eggs) i n orange groves, compared to four natural habitats (two holm oak forests ( Quercus ilex ) at low and high altitudes, a pine forest ( Pinus sylvestris ) at high altitude and a zeen oak forest ( Quercus faginea ) at high altitude) (Belda et al. 1998). The population of great tits breeding in tree holes in the same study area (Sagunto, Spain) reached a density of 5 - 8 pairs per 10 ha and average clutch size is 6.4 eggs (Gil-Delgado & Barba 1987). Comparable to the results from Spain, a study of La Manita (1982) on the breeding bird community in a suburban area around Palermo, Italy, revealed a sim ilar population density of great tits (5 - 6 pairs per 10 ha) nesting in similar sites (holes in citrus trees, wa ll crevices). Therefore, based on the results of the above cited papers, great tits obviously inhabit citrus orchard s and can serve as a representative for a small insectivorous bird for pesticide risk assessment in citrus.

Data for refinement of the portion of diet (PD) are evaluated in the position paper. In Spanish citrus orchards, the diet of Great Tits largely consists of arthropods. The mean size of th e prey brought to the nestlings was 15.5 mm on the average (Monrós et al. 1997). Another study on the diet composition of great tits foraging in modern citrus orchards in Spain revealed that great tit nestling s in a modern orange grove near Sagunto, East Spain, from April to August 1988 were predominantly fed with Lepidoptera to about 87.8 % in numbers of 526 prey items (Barba and Gil-Delgado 1990). Among the lepidopterans 49.8 % were imagines, almost exclusiv ely Noctuidae, 23.6 % were lepidopteran caterpillar s, 14.3 % lepidopteran pupae (Barba & Gil-Delgado 1990 ). Furthermore the nestlings obtained 5.7 % spiders and 6.5 % other prey items including Hymenoptera, C oleoptera (Curculionidae), Orthoptera (grass-hopper s), egg cocoons and orange pieces (Barba & Gil-Delgado 1990). Caterpillars were the most abundant prey delivered to the young only during 6-15 May, while Lepidopter a imagines were more abundant during the rest of the season and thus were considered as an important resource for the great tits breeding in orange grov es (Barba & Gil-Delgado 1990). Thus, the diet of Great tits in Spanish citrus orch ards consisted at ca. 88 % of large insects (lepido pteran caterpillars 23.6 %, pupae 14.3 %, and imagines 49. 8 %). For the size class of the remainder it is als o reasonable to expect preferential feeding on larger prey (Monrós et al. 1997)

Conclusion: Based on the results of the above cited papers, gre at tits obviously inhabit citrus orchards and can s erve as a representative for a small insectivorous bird for pesticide risk assessment in citrus. Remark (RMS) : The diet composition of great tits, as discussed in this position paper, was not used in the final risk assessment. This is discussed under B. 9 .1.8.2.

References: Barba & Gil-Delgado (1990): Seasonal variation in nes tling diet of the great tit Parus major in orange groves in eastern Spain. Ornis Scandinavica 21: 296-298. Belda et al. (1998): Laying date and cluth size of g reat tits Parus major in the Mediterranean region: a comparison of four habitat types. J. Ornithologie 139: 269-276. Gil-Delgado & Barba (1987): Aves nidificantes en huec os de los naranjos. Mediterranea Ser. Biol. 9: 29-40. La Manita (1982): Dati quantitative sull` avifauna nidificante in una zona suburbana di Palermo. Avocet ta 6: 41-46. Monrós et al. (1999): Open nestings in Great Tits Parus major . Ardeola 46 (1): 89-91. Perrins (1979): British Tits. Collins, London.– 28 –

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

Reference: Generic field monitoring of birds in potato, cultiv ation in northern Germany Author(s), year: Wolf, Ch. 2004 Report/Doc. number: WFC/FS 019; M-090336-02-1 Guideline(s): Not applicable. The test was especial ly 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 diff erent potato fields in the “Wildeshauser Geest” reg ion near the village Dötlingen between the towns of Wil deshausen and Oldenburg, in Lower Saxony, Germany. This region is a typical area of potato cultivation in Europe and known to hold a population of Yellow Wagtails ( Motacilla flava ). To identify bird species of concern for potato fiel ds (besides the Yellow Wagtail) and to appraise the relevancy of potato fields and other habitats as fe eding habitats for birds, census counts were carrie d out along different transects, representing all main ag rarian habitats within the study area. Birds were c ounted and observed during a slow-going walk on 4 defined transects within and in the vicinities of all 6 mon itored fields at least once a week during the whole study period. In summary the transects had a length of 63 00 m and covered a crop area of 48.58 ha. The species, t he number of individuals, the location (e.g. flying over, vegetation, ground) and the behaviour (e.g. foragin g, singing/breeding/feeding young, locomotion, rest ing, others) per passed field were recorded. 20 Yellow Wagtails were trapped on and in the margi n of potato fields, tagged with radio transmitters and tracked for one to five daylight periods each. The transmitters, weighting 0.7 g, are known not to inf luence bird`s behaviour significantly. The location, habit at and behaviour was recorded continuously to get information of the home range, habitat selection an d time budget of birds using potato fields living i n a cultivable area for potatoes. Birds were followed c ontinuously by car. Emphasis was placed on spatial and temporal feeding activities and habitat used. To analyse the habitat selection of the Yellow Wagt ails while “potentially foraging” (categories “acti ve”, “foraging” and “unknown” combined) the Jacob`s pref erence index (D) (Jacobs 1974) for each habitat and tracking session was calculated based on the minimu m convex polygon (MCP) home range. The Jacob`s index indicates the relation between the portion of time a given habitat was used and the spatial port ion of this habitat within the birds` homerange: D = (r - p) / (r + p - 2rp), where r is the portion the habitat was used by the bird and p is the port ion of the same habitat within the homerange of the bird. To get information about the food items, actually s elected by the Yellow Wagtails, samples of faeces w ere analysed quantitatively for composition (taxonomic orders of arthropods, other items). Reference sampl es of arthropods living in potato fields and sampled unse lectively were obtained from another GLP study, conducted at the same time in the same fields. At two fields, rainfall was recorded daily. A logge r for temperature and humidity measurement was inst alled. Findings: The results of the monitoring are shown in the tabl e below:– 29 –

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PORTION OF TIME (PT) in habitat of Yellow Wagtails aft er radio tracking potato fields 38.4 % (72.3) cereal fields 39.8 % (44.1) oil seed rape fields 6.8 % (22.2) street and field path 3.7 % (10.7) potential foraging time (sum of behaviour categories “foraging”+“active”+“unknown”) spent per habitat; based on 20 individuals, 1 to 5 24-hour sessions each [mean of individuals], (90%ile) other habitats 11.3 % (16.0) FEEDING HABITAT of Yellow Wagtails after transect coun ts (based on population) potato fields 0.60
cereal fields 0.41 sugar beet fields 0.32 abundance of potentially foraging individuals of the population after 9 transect counts covering 48.58 ha field crops each [individuals/ha]

oil seed rape fields 0.22 PREFERENCE OF POTATO FIELDS in Yellow Wagtails after radio t racking preference of potato fields as a feeding habitat [Jacobs’ index (D), MCP (100%)] 0.16

DIET of Yellow Wagtails taxonomic order steadiness 1 mean number 2 Diptera 100% 3.96 Coleoptera 84% 3.60 Aphidoidea 72% 5.28 Hymenoptera 52% 0.80 main arthropod orders actually eaten by Wagtails foraging in and around potato fields (based on 25 samples of faeces)

Aranea 48% 0.64 SIZE OF ARTHROPODS actually chosen by Yellow Wagtails taxonomic order food [mm] (n) population [mm] (n, stratum 3 )

Diptera 7.49 (99) 2.78 (523, f) Coleoptera 7.22 (90) 5.85 (128, g) Aphidoidea 3.00 (132) 1.98 (350, f) Hymenoptera 6.33 (20) 3.13 (25, f) Mean sizes of actually eaten individuals (after samples of faeces) compared to mean sizes of unselective samples of the population in potato fields

Aranea 4.38 (16) 3.12 (58, g)

MAIN BIRD SPECIES in potato fields potentially foraging Yellow Wagtail 0.60 individuals per ha Pied Wagtail 0.29 individuals per ha mean abundance of main species after 9 transect counts covering 9.98 ha potato fields each Whitethroat 0.12 individuals per ha

1 portion of samples containing this type 2 mean number of individuals per sample 3 f = foliage dwelling arthropods, obtained by inven tory spraying; g = ground dwelling arthropods, obta ined by pitfall trapping

Conclusion Radio-tracking of 20 individual Yellow Wagtails (ea ch for a minimum of 24 and a maximum of 120 hours) in an agrarian landscape with a high number of potato fields in the north-western part of Lower Saxony sh owed that this field type was used as a main feeding hab itat by Yellow Wagtails. However, cereal fields (ba rley, wheat and rye) have been used as well to a (in summ ary) similar high proportion by Yellow Wagtails, wh ich foraged for chick provisioning or the bird’s own us e. Only one individual fed almost exclusively on po tato fields while tracking, while one individual did not use potato fields as feeding habitat at all. Potato fields were on average selected to a slightl y higher proportion for foraging as to be derived f rom the available proportion in their home range [Jacobs’ i ndex (D)]. Thus Yellow Wagtails positively selected potatoes as a foraging habitat, but only to a littl e extent.– 30 –

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Tracking data of individual Yellow Wagtails were co nfirmed by census data of the population of this sp ecies within the study area. Moreover census data confirm ed the Yellow Wagtail to be the most relevant bird species in potato, followed by the Pied Wagtail and the Whitethroat. To sum up, it can be ascertained, that potato field s offered a significant but not exclusive feeding h abitat for the tracked Yellow Wagtails. For risk assessment purposes a value for portion of time spent foraging in potato fields (PT) can be d erived 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 foragin g time in potato fields (90 th percentile 72.3 %). Food of Yellow Wagtails in and around potato fields was dominated by Diptera, Coleoptera and Aphidoide a. In all taxonomic arthropod orders large individuals were selected distinctly.

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

Not required.

B.9.1.6 Effects of secondary poisoning (OECD IIA 10 .1.9)

Not required. See B.9.1.8.4.

B.9.1.7 Summary of effects on birds

Table B.9.1.7-1: Summary of avian toxicity endpoint s for spirotetramat

Study type Test species NOAEC (mg/kg feed) NOAEL

(mg/kg bw) LC 50

(mg/kg feed) LD 50

(mg/kg bw) Reference

Acute oral bobwhite quail / < 500 / > 2000 Barfknecht, 2003
bobwhite quail 1250 183 > 5000 > 497 Barfknecht, 2004 Short-term dietary toxicity mallard duck < 344 < 65 > 6050 > 475 Stoughton, 2005
bobwhite quail Adults: 264

repro: 802 Adults: 23

repro: 74 / / Sabbert & Nuessle, 2006a

mallard duck Adults: < 89*

repro: 89 Adults: < 9.4*

repro: 9.4 / / Sabbert & Nuessle, 2006b Subchronic / reproductive toxicity
mallard duck Adults+repro: 28.8 Adults+repro:

4.0 / / Temple et al., 2006 All values except for acute oral study based on meas ured figures; * foot lesions in all treatment groups

B.9.1.8 Risk assessment for birds

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.– 31 –

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

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".

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

B.9.2.1 Acute toxicity of active substance, metabol ites and formulations to aquatic organisms

B.9.2.1.1 Acute toxicity to fish (OECD IIA 8.2.1, I IA 8.11.1, IIIA 10.2.2)

Active substance

Reference: Acute toxicity of BYI 08330 (tech.) to fish ( Oncorhynchus mykiss ) Author(s), year: Dorgerloh, M., 2004 a Report/Doc. number: DOM 24025, Edition Number: M-18 2649-01-2, 16.12.2004 Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, E U Directive 92/69/EEC C.1 GLP: Yes Deviations: Measured test concentration dropped bel ow 80 % of nominal concentration. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 Material and methods: Test species: Rainbow trout ( Oncorhynchus mykiss ) Number of organisms: 10 fish per concentration and control Weight, length: 2.3 ± 1.1 g (mean ± SD) and 5.8 ± 0.9 cm (mean ± SD) Loading: 0.58 g fish/L test medium Type of test, duration: Semi static test (renewal o f test media every 24 hours), 96 hours Applied concentrations: Nominal: 0 (control and solvent control), 0.5, 1.0 , 2.0, 4.0 and 8.0 mg/L Measured (mean): -- (control and solvent control), 0.409,0.825, 1.5, 3.14 and 6.44 mg/L Solvent Aceton 0.2 mL/L Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 40 - 60 mg/L as CaCO 3 Temperature: 11.3 – 12.3 °C pH: 7.0 – 7.1 (0 h, new medium), 7.0 (96 h, aged m edium) O 2 content: 89 – 99 % saturation Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of B YI 08330 in test solutions samples were taken daily from new and age d test media. Test samples were directly injected into the HPLC-UV without cen trifugation. Statistics: EC50: Probit analysis, NOEC: directly f rom raw data Findings: Analytical data: Over the whole test period the mea n measured concentrations were in the range from 77 to 85 % of nominal. Behavioural effects: Controls and concentration lev els up to 0.825 mg/L: No sublethal effects were reported over the whole test period. At test concentration ≥ 1.5 mg/L: After 96 hours following symptoms were n oted: Remained on the bottom of aquarium, loss of equilib rium, labored respiration, remained at water surface, turned in a vertical pos ition. Thus the NOEC was 0.825 mg/L based on sublethal eff ects.– 32 –

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

Mortality: See table B.9.2.1.1-1

Table B.9.2.1.1-1: Effects on rainbow trout ( O. mykiss ) exposed to spirotetramat Cumulative mortality (%) Spirotetramat [mg a.s./L] (mean measured) 24 hours 48 hours 72 hours 96 hours

Control 0 0 0 0 Solvent control 0 0 0 0
0.409 0 0 0 0 0.825 0 0 0 0 1.5 0 0 0 0 3.14 0 30 30 80 6.44 60 100 100 100 NOLC: 1.5 mg/L LC 50 (96 h): 2.54 mg/L (95 % C.I. 2.11 – 3.05 mg/L)

Details of the Probit: Slope b: 8.4 95 %CI of b: not stated Variance of b: 3.85 Intercept a: 1.6 Goodness of fit, Chi2: 1.25 Degrees of freedom: 3 p (Chi2): 0.74 Conclusion: LC 50 (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 uncertai nties surrounding exposure concentrations in test media.

Reference: Acute toxicity of BYI 08330 (tech.) to fish ( Cyprinus carpio ) Author(s), year: Dorgerloh, M., 2004 b Report/Doc. number: DOM24022, Edition Number: M-128 667-01-2, 25.11.2004 Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, E U Directive 92/69/EEC C.1 GLP: Yes Deviations: Measured test concentration dropped bel ow 80 % of nominal concentration. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 Material and methods: Test species: Common carp ( Cyprinus carpio ) Number of organisms: 10 fish per concentration and control Weight, length: 0.9 ± 0.3 g (mean ± SD) and 4.0 ± 0.5 cm (mean ± SD) Loading: 0.23 g fish/L test medium Type of test, duration: Semi static test (renewal o f test media every 24 hours), 96 hours Applied concentrations: Nominal: 0 (control and solvent control), 1.25, 2. 5, 5.0, 10.0 and 20.0 mg/L Measured (mean): -- (control and solvent control), 1.02, 1.71, 3.65, 8.13 and 13.4 mg/L Solvent Aceton 0.2 mL/L Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 40 - 60 mg/L as CaCO 3– 33 –

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

Temperature: 20.5 – 23.2 °C pH: 7.1 – 7.2 (0 h, new medium), 7.1 (96 h, aged m edium) O 2 content: 97 – 103 % saturation Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of B YI 08330 in test solutions samples were taken daily from new and age d test media. Test samples were directly injected into the HPLC-UV without cen trifugation. Statistics: EC50: Probit analysis, NOEC: directly f rom raw data Findings: Analytical data: Over the whole test period the mea n measured concentrations were in the range 69 – 83 % of nominal. Behavioural effects: Controls and at 1.02 mg/L: No sublethal effects were observed over the whole test period. At concentration levels ≥ 1.71 mg/L: After 96 hours following symptoms were noted: Remained on the bottom of aquarium, loss of equilibrium, labored respiration, dark coloration, laying on the sides o r backs, turned in a vertical position. Thus the NOEC was 1.02 mg/L based on sublethal effe cts. Mortality: See table B.9.2.1.1-2

Table B.9.2.1.1-2: Effects on common carp ( C. carpio ) exposed to spirotetramat Cumulative mortality (%) Spirotetramat [mg a.s./L] (mean measured) 24 hours 48 hours 72 hours 96 hours

Control 0 0 0 0 Solvent control 0 0 0 0
1.02 0 0 0 0 1.71 0 0 0 0
3.65 0 10 50 100 8.13 100 100 100 100 13.4 100 100 100 100 NOLC: 1.71 mg/L LC 50 (96 hours): 2.59 mg/L (95 % C.I.: not determined du e to mathematical reasons)

Details of the Probit: Slope b: 16.09 95 %CI of b: not stated Variance of b: 29.9 Intercept a: -1.66 Goodness of fit, Chi2: 17.69 Degrees of freedom: 3 p (Chi2): < 0.001 Conclusion: LC 50 (96 h): 2.59 mg/L NOEC (96 h): 1.02 mg/L based on mean measured concentration Comments: The US EPA has general concerns regarding uncertai nties surrounding exposure concentrations in test media. (95 %CI: not stated)– 34 –

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

Reference: Acute toxicity of BYI 08330 (tech.) to fish ( Lepomis macrochirus ) Author(s), year: Dorgerloh, M., 2005 a Report/Doc. number: DOM24056, Edition Number: M-242 689-01-2, 12.01.2005 Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, E U Directive 92/69/EEC C.1 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods: Test species: Bluegill sunfish ( Lepomis macrochirus ) Number of organisms: 10 fish per concentration and control Weight, length: 1.7 ± 0.7 g (mean ± SD) and 4.9 ± 0.6 cm (mean ± SD) Loading: 0.43 g fish/L test medium Type of test, duration: Semi static test (renewal o f test media every 24 hours), 96 hours Applied concentrations: Nominal: 0 (control and solvent control), 0.5, 1.0 , 2.0, 4.0 and 8.0 mg/L Measured (mean): -- (control and solvent control), 0.48, 0.95, 1.90, 3.56 and 7.61 mg/L Solvent Aceton 0.2 mL/L Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 40 – 60 mg/L as CaCO 3 Temperature: 17.9 – 20.5 °C pH: 7.4 – 7.5 (0 h, new medium), 7.3 (96 h, aged m edium) O 2 content: 96 – 105 % saturation Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of B YI 08330 in test solutions samples were taken daily from new and age d test media. Test samples were directly injected into the HPLC-UV without cen trifugation. Statistics: EC50: Probit analysis, NOEC: directly f rom raw data Findings: Analytical data: Over the whole test period the mea n measured concentrations were in the range 91 – 99 % of nominal. Behavioural effects: Controls and at 0.5 mg/L: No s ublethal effects were observed over the whole test period. At concentration levels ≥ 1.0 mg/L: Transient effects like remaining on the bottom of aquarium, laying on the sides or backs, labored respiration, hyperactivity, inactivity, convulsions and remaining on the water surface were observed. Thus the NOEC was 0.5 mg/L based on sublethal effec ts. Mortality: See table B.9.2.1.1-3– 35 –

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

Table B.9.2.1.1-3: Effects on bluegill sunfish ( L. macrochirus ) exposed to spirotetramat Cumulative mortality (%) Spirotetramat [mg a.s./L] (nominal) 24 hours 48 hours 72 hours 96 hours

Control 0 0 0 0 Solvent control 0 0 0 0
0.5 0 0 0 0 1.0 0 20 20 20
2.0 0 10 10 10 4.0 20 50 100 100 8.0 80 100 100 100 NOLC: 0.5 mg/L LC 50 (96 hours): 2.2 mg/L (95 % C.I.: not determined due to mathematical reasons)

Details of the Probit: Slope b: 4.44 95 %CI of b: not stated Variance of b: 1.21 Intercept a: 3.48 Goodness of fit, Chi2: 8.93 Degrees of freedom: 3 p (Chi2): 0.0302 Conclusion: LC 50 (96 h): 2.2 mg/L NOEC (96 h): 0.5 mg/L based on nominal concentration Comments: The US EPA has general concerns regarding uncertai nties surrounding exposure concentrations in test media.

Metabolites

Reference: Acute toxicity of BYI 08330-enol to fish ( Oncorhynchus mykiss ) Author(s), year: Nieden, D., 2005 a Report/Doc. number: Report No.: EBFNM013, Edition N umber: M-251850-01-2, 18.05.2005 Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, E U Directive 92/69/EEC C.1 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005 Material and methods: Test species: Rainbow trout ( Oncorhynchus mykiss ) Number of organisms: 2 x 15 fish per concentration and control Weight, length: 1.3 ± 0.6 g (mean ± SD) and 4.9 ± 0.7 cm (mean ± SD) Loading: 0.49 g fish/L test medium Type of test, duration: Static limit test, 96 hours Applied concentrations: Nominal: 0 (control), 100 mg/L Measured (mean): -- (control), 98 mg/L Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 40 - 60 mg/L as CaCO 3 .– 36 –

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

After the addition of test substance the pH was adj usted to about 7 with NaOH. Temperature: 11.1 – 13 °C pH: 6.7 – 7.2 (0 h), 6.8 – 6.9 (96 h) O 2 content: 86 – 102 % saturation Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 4, 24, 48, 72 and 96 hours. At 0, 48 and 96 hours samples were taken for chemic al analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330-enol in test solutions. Statistics: None, because a limit test was performe d Findings: Analytical data: Over the whole test period the mea n measured concentration was 98.5 % (replicate A) and 97.7 % (replicate B) of nominal. Behavioural effects: No effects were observed in co ntrol and 100 mg/L treatment replicates. Mortality: No mortality was reported in control and 100 mg/L treatment replicates. Conclusion: LC 50 (96 h): > 100 mg/L NOEC (96 h): 100 mg/L based on nominal concentration

Reference: 4-Methoxycyclohexanone - Acute fish toxicity Author(s), year: Neuhahn, A., 2006 a Report/Doc. number: Report No.: 2006/0032/02, Editi on Number: M-274305-01-1, 10.07.2006 Guideline(s): OECD 203, EU Directive 92/69/EEC C.1 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: 4-Methoxycyclohexanone (metabolite of spirotetrama t), purity: 97.9 %, batch: SAV5506-001 Solubility: Within the test, the item was soluble at the limit concentration of 100 mg/L in test medium after ultra turrax treatment. There was no i ndication for any unsolved material after mixing. Substance specific GC analys is showed recoveries of >80% for all samples taken. Material and methods: Test species: Zebra fish ( Danio rerio ) Number of organisms: 10 fish per concentration and control Weight, length: Not reported, 3.4 ± 0.26 cm (mean ± SD) Loading: 0.64 g fish/L test medium Type of test, duration: Static limit test, 96 hours Applied concentrations: Nominal: 0 (control), 100 mg/L Measured (mean): -- (control), 98.5 mg/L Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 246 mg/L as CaCO 3 . Temperature: 20.5 – 21.0 °C pH: 7.6 – 7.9 O 2 content: 88 – 101 % saturation Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 2, 24, 48, 72 and 96 hours. At 0, 24 and 96 hours samples were taken for chemic al analysis (GS analysis and– 37 –

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

MS detection) of 4-Methoxycyclohexanone in test sol utions. Test samples were analysed directly without centrifugation. Statistics: None, because a limit test was performe d Findings: Analytical data: The mean measured concentration wa s 105 % (0 hours) and 89.8 % (96 hours) of nominal. Behavioural effects: After 2 hours abnormalities in swimming were noted for 50 % of fish. However this effect was transient and no effects were observed a t later time points. Mortality: No mortality was reported in control and 100 mg/L treatment replicates. Conclusion: LC 50 (96 h): > 100 mg/L NOEC (96 h): 100 mg/L based on nominal concentration Comments While the study not deviated from recommendations given in OECD 202 and EU guideline, deviation from USEPA guidelines are nota ble: -Minor: The temperature was slightly lower than the recommended levels for zebra fish (23 ±2oC), and the weight of fish wasn’t reported. -Major: The guidelines require at least 30 fish to be tested, and only ten were tested at the 100 mg/L treatment level.

. Comments: US EPA has general concerns regarding uncertaintie s surrounding exposure concentrations in test media.

Formulation

Reference: Acute toxicity of BYI 08330 OD 150 to fish ( Oncorhynchus mykiss ) under static conditions Author(s), year: Nieden, D., 2005 d Report/Doc. number: EBFNM008, Edition Number: M-253 916-01-2, 29.06.2005 Guideline(s): OECD 203, EPA 72-1, OPPTS 850.1075, E U Directive 92/69/EEC C.1 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330 OD 150 (formulation), spirotetramat cont ent: 148.89 g/L (15.1 %), batch: 08030/0189(0152) Material and methods: Test species: Rainbow trout ( Oncorhynchus mykiss ) Number of organisms: 10 fish per concentration and control Weight, length: 0.8 ± 0.2 g (mean ± SD) and 4.6 ± 0.3 cm (mean ± SD) Loading: 0.20 g fish/L test medium Type of test, duration: Static test, 96 hours Applied concentrations: Nominal: Formulation: 0 (control), 2.50, 5.00, 10. 0, 20.0, 40.0 mg product/L Spirotetramat: 0 (control), 0.378, 0.755, 1.51, 3. 02, 6.04 mg a.s./L Measured (mean): Spirotetramat: -- (control), 0.39 4, 0.783, 1.57, 3.10, 6.45 mg a.s./L Control: Untreated test water, no blank formulation control was used Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 40 - 60 mg/L as CaCO 3 Temperature: 11.6 – 12.2 °C pH: 6.9 – 7.2 O 2 content: 95 – 102 % saturation– 38 –

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

Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of s pirotetramat in test solutions samples were taken at 0, 48 and 96 hours from each concentration. Statistics: EC50: Logit and Probit analysis, NOEC: directly from raw data Findings: Analytical data: Over the whole test period the mea n measured concentrations of active substance were in the range from 103 to 107 % of no minal. Behavioural effects: Controls and 2.5 mg product/L concentration level: No sublethal effects were observed over the whole test period. At test concentrations of 5.0 and 10 mg product/L: After 96 hours following symptoms were noted: Remained on the bottom of aqua rium, dark coloration, loss of equilibrium, labored respiration, hyperactivity, laying on sides or backs. Thus the NOEC was 2.5 mg product/L based on subleth al effects. Mortality: See table B.9.2.1.1-4:

Table B.9.2.1.1-5: Effects on rainbow trout ( O. mykiss ) exposed to BYI 08330 OD 150 (formulation) Cumulative mortality (%) BYI 08330 OD 150 [mg product/L] (nominal) Spirotetramat

mg a.s./L (mean meas.) 24 hours 48 hours 72 hours 96 hours

Control 0 0 0 0
2.5 0.394 0 0 0 0
5.0 0.783 0 0 0 10 10.0 1.57 0 0 10 10 20.0 3.1 70 80 90 100 40.0 6.45 100 100 100 100 NOLC: 2.5 mg product/L corresponding to 0.397 mg a. s./L LC 50 (96 h): 9.48 mg/L (95 % C.I. 5.84 – 15.7 mg/L) cor responding to 1.41 mg a.s./L calculated by Logit LC 50 (96 h): 11.8 mg/L (95 % C.I. n.d.) corresponding t o 1.76 mg a.s./L calculated by Probit

Details of the Probit: Slope b: 5.607 95 % C.I. of b: 0.994 – 10.221 Variance of b: 2.27 Intercept a: -0.898 Goodness of fit, Chi2: 7.498 Degrees of freedom: 3 p (Chi2): 0.0576 Conclusion: LC 50 (96 h): 9.48 mg product/L, 1.41 mg a.s./L NOEC (96 h): 2.5 mg product/L, 0.378 mg a.s./L based on nominal concentration

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

Active substance– 39 –

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

Reference: Acute toxicity of BYI 08330 (tech.) to the waterfle a Daphnia magna under static conditions Author(s), year: Dorgerloh, M., 2005 c Report/Doc. number: DOM 24004, Edition Number: M-24 2683-01-2, 14.01.2005 Guideline(s): OECD 202, EPA 72-2, OPPTS 850.1010, E U Directive 92/69/EEC C.2 GLP: Yes Deviations: Minor deviation from EPA guideline: The pH is lower and the hardness is higher than EPA-recommended range. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods: Test species: Waterflea ( Daphnia magna ) Number of organisms: 6 replicates each with 5 daph nids per treatment and control Age: First instar < 24 hours old Type of test, duration: Static test, 48 h Applied concentrations: Nominal: 0 (control and solvent control), 6.25, 12 .5, 25.0, 50, 100 mg/L Measured (mean): -- (control and solvent control), 4.7, 8.77, 20.3, 38.8, 42.7 mg/L Solvent 0.4 mL/L Aceton Test conditions: Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring test), hardness: 196 mg/L as CaCO 3 Temperature 20.5 – 20.6 °C pH 6.3 – 6.4 (0 h), 6.6 – 6.7 (48 h) O 2 content: 96 – 102 %, 8.5 – 9.0 mg O 2 /L Light regime: 16 hours light / 8 hours darkness Test parameters: Immobility and sublethal effects w ere assessed after 24 and 48 hours. For chemical analysis (HPLC-UV) of BYI 08330 in the tes t media samples were taken at test initiation (0 h) and termination (48 h). Th e water samples were analysed by direct injection into the HPLC-UV instrument withou t centrifugation. Statistics: EC50: None, because at highest test con centration (100 mg/L nominal) the immobilisation were < 50 %, NOEC: directly from the raw data Findings: Analytical data: The mean measured concentrations a t the start and end of the test were in the range of 22 – 90 %, overall mean measured concentra tion ranged from 42.7 – 81.0 % of nominal. Effects: After 48 hours no immobility was observed in the control, solvent control and in test concentrations up to 20.3 mg/L. At 38.8 and 42 .7 mg/L the immobilisation was 7 and 23 %. No sublethal effects were noted during the whole study. Thus the NOEC was determined to be 20.3 mg/L and th e EC50 was > 42.7 mg/L. Conclusion: EC50 (48 h): > 42.7 mg/L NOEC: 20.3 mg/L based on mean measured concentration Comments: US EPA has general concerns regarding uncertaintie s surrounding exposure concentrations in test media.– 40 –

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

Reference: Acute toxicity of BYI 08330 (tech.) to larvae of Chironomus riparius in a 48 h water-only study Author(s), year: Dorgerloh, M., 2005 g Report/Doc. number: EBFNX072, Edition Number: M-262 632-01-2, 08.12.2005 Guideline(s): No specified guideline, the study is following the procedure of OECD 202 GLP: Yes Deviations: None of relevance considering OECD 202 Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 Material and methods: Test species: Midge ( Chironomus riparius ) Number of organisms: 4 replicates each with 10 lar vae per treatment and control Age: L1 (first instar) larvae Type of test, duration: Static (water-only) test, 48 h Applied concentrations: Nominal: 0 (control and solvent control), 0.56, 1. 00, 1.80, 3.20, 5.60, 10.0 mg/L Measured (mean): -- (control and solvent control), 0.53, 0.96, 1.72, 3.04, 5.37, 9.47 mg/L Solvent 0.1 mL/L Aceton Test conditions: Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring test), hardness: not stated Temperature 20.4 – 21 °C pH 8.2 – 8.6 (0 h), 8.2 – 8.9 (48 h) O 2 content: > 80 %, (8.6 – 8.9 mg O 2 /L) Light regime: 16 hours light / 8 hours darkness Feeding Only on time 0.01 mL aqueous fish food sol ution directly after insertion of the larvae Test parameters: Mortality and sublethal effects we re assessed after 48 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330 in t he test media samples were taken at test initiation (0 h) and termination (48 h). Statistics: Correction of mortality: Abbott’s formu la, LC10 and LC50: Probit analysis, NOEC: not determined, comparison of controls: Student-t t est for homogeneous variances. All statistical analyses are based on data of poole d control. Findings: Analytical data: The mean measured concentrations a t the start and end of the test were in the range of 106 – 109 % and 82 – 83 % of nominal, resp ectively. Overall mean measured concentrations ranged from 95 to 96 % of n ominal. Effects: After 48 hours the mortality in pooled con trols was 8.7 %, thus the validity criteria of maximum control mortality of 10 % is met, further information see table B.9.2.1.2-1

Table B.9.2.1.2-1: Effects on midge ( C. riparius ) exposed to spirotetramat Mortality after 48 hours Spirotetramat [mg a.s./L] mean measured Exposed chironomids (= 100 %) [n] [%]
Control* 40 3 7.5 Solvent control* 40 4 10.0
0.528 40 4 10.0 0.958 40 13 32.5– 41 –

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

Mortality after 48 hours Spirotetramat [mg a.s./L] mean measured Exposed chironomids (= 100 %) [n] [%]

1.715 40 29 72.5 3.035 40 39 97.5 5.365 40 40 100 9.465 40 40 100

LC10 (48 h): 0.699 mg/L (95% C.I. 0.536 – 0.913 mg/ L) LC50 (48 h): 1.299 mg/L (95% C.I. 1.120 – 1.506 mg/ L) * Result of t-test is indicated no significant diff erences between control and solvent control, thus p ooled control are used for statistical analysis.

Details of the Probit: Slope b: 4.76 95 % CI of b: not stated Variance of b: 0.54 Intercept a: 4.46 Goodness of fit, Chi2: 6.62 Degrees of freedom: 4 p (Chi2): 0.158 Conclusion: LC 10 (48 h): 0.699 mg/L LC 50 (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 guideli nes are notable: -Age of the test organisms is 2nd or 3rd instar, 1s t instars were used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test dur ation should be 96-hours. -Mortality and sublethal effects should be measured every 24 hours

.

Metabolites

Reference: Acute toxicity of BYI 08330-enol to the waterflea Daphnia magna in a static laboratory test system - limit test Author(s), year: Dorgerloh, M., 2005 d Report/Doc. number: EBFNM012, Edition Number: M-254 487-01-2, 14.07.2005 Guideline(s): OECD 202, EPA 72-2, OPPTS 850.1010, E U Directive 92/69/EEC C.2 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005, water solubility (at pH 7): 2.7g/L Material and methods: Test species: Waterflea ( Daphnia magna ) Number of organisms: 6 replicates each with 5 daph nids per treatment and control Age: First instar < 24 hours old Type of test, duration: Static limit test, 48 h Applied concentrations: Nominal: 0 (control), 100 mg/L Measured (mean): -- (control), 111.5 mg/L Test conditions:– 42 –

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

Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring test), hardness: 196 mg/L as CaCO 3 , adjusted to pH 7 before use for dilution procedure Temperature 20.6 – 21.2 °C pH 7.0 – 6.9 (0 h), 7.5 – 7.9 (48 h) O 2 content: 89 – 96 %, 8.1 – 8.7 mg O 2 /L Light regime: 16 hours light / 8 hours darkness Test parameters: Immobility and sublethal effects w ere assessed after 24 and 48 hours. For chemical analysis (HPLC-UV) of BYI 08330-enol in th e test media samples were taken at test initiation (0 h) and termination (48 h). Test samples were analysed by direct injection into a HPLC-UV instrument without a centrifugation. Statistics: EC50: None, because at the limit concen tration (100 mg/L nominal) no immobilisations were observed, NOEC: directly from the raw data Findings: Analytical data: The measured concentrations at the start and end of the test were 113 and 110 % of nominal, overall mean measured concentration was 111.5 % of nominal. Effects: After 48 hours no immobility and no sublet hal effects were observed in the control, and in the limit test concentrations of 100 mg/L. Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. Conclusion: EC50 (48 h): > 100 mg/L NOEC: 100 mg/L based on nominal concentration

Reference: Acute toxicity of BYI 08330-enol to larvae of Chironomus riparius in a 48 h water-only study Author(s), year: Dorgerloh, M., 2005 h Report/Doc. number: EBFNX073, Edition Number: M-262 638-01-2, 08.12.2005 Guideline(s): No specified guideline, the study is following the procedure of OECD 202 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005, water solubility (at pH 7): 2.7g/L Material and methods: Test species: Midge ( Chironomus riparius ) Number of organisms: 4 replicates each with 10 lar vae per treatment and control Age: L1 (first instar) larvae Type of test, duration: Static (water-only) test, 48 h Applied concentrations: Nominal: 0 (control), 1.0, 2.0, 4.0, 8.0, 16, 32, 64 mg/L Measured (mean): -- (control), 0.95, 1.93, 4.06, 8 .3, 15.65, 32.25, 66.45 mg/L Test conditions: Water quality: M7 medium (according to OECD 202), hardness: not stated Temperature 20.8 – 21 °C pH 7.8 – 8.2 (0 h), 8.1 – 8.4 (48 h) O 2 content: > 90 %, (8.5 – 8.7 mg O 2 /L) Light regime: 16 hours light / 8 hours darkness Feeding Only on time 0.01 mL aqueous fish food sol ution directly after insertion of the– 43 –

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

larvae Test parameters: Mortality and sublethal effects we re assessed after 48 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330-enol in the test media samples were taken at test initiation (0 h) and ter mination (48 h). Test samples were analysed by direct injection into a HPLC-UV in strument without a centrifugation. Statistics: LC 10 and LC 50 : Probit analysis, NOEC: not determined Findings: Analytical data: The mean measured concentrations a t the start and end of the test were in the range of 98 – 105 % and 88 – 103 % of nominal, resp ectively. Overall mean measured concentrations ranged from 95 to 104 % of nominal. Effects: See table B.9.2.1.2-2

Table B.9.2.1.2-2: Effects on midge ( C. riparius ) exposed to BYI 08330-enol
BYI 08330-enol [mg/L] Mortality after 48 hours

nominal mean measured Exposed chironomids (= 100 %) [n] [%]

Control 40 0 0
1.0 0.95 40 0 0 2.0 1.93 40 0 0 4.0 4.06 40 0 0 8.0 8.28 40 0 0
16.0 15.68 40 5 12.5 32.0 32.16 40 11 27.5 64.0 67.59 40 16 40.0 LC10 (48 h): 17.07 mg/L (95% C.I. 12.15 – 23.98 mg/ L) based on nominal 11.62 mg/L (95% C.I. 4.74 – 28.48 mg/L) based on me an measured LC50 (48 h): 74.85 mg/L (95% C.I. 48.86 – 114.64 mg /L) based on nominal 97.78 mg/L (95% C.I. 43.15 – 221.60 mg/L) based on mean measured

Details of the Probit: Slope b: 1.38 95 % CI of b: not stated Variance of b: 0.248 Intercept a: 2.246 Goodness of fit, Chi2: 0.175 Degrees of freedom: 1 p (Chi2): 0.676 Conclusion: LC 50 (48 h): 74.9 mg/L LC 10 (48 h): 17.1 mg/L NOEC (48 h): not determined based on nominal concentration Comments: While the study not deviated from recommendations given in OECD 202 guideline, significant deviation from USEPA guideli nes are notable: -Age of the test organisms is 2nd or 3rd instar, 1s t instars were used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test dur ation should be 96-hours. -Mortality and sublethal effects should be measured every 24 hours

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Reference: Acute toxicity of BYI 08330-ketohydroxy to larvae o f Chironomus riparius in a 48 h static laboratory test system Author(s), year: Dorgerloh, M., 2006 a Report/Doc. number: EBFNO347, Edition Number: M-263 956-01-2, 16.01.2006 Guideline(s): No specified guideline, the study is following the procedure of OECD 202 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330-ketohydroxy (metabolite of spirotetramat ), purity: 98.6 %, batch: NLL7549-7, water solubility (in distilled water): 0 .228g/L Material and methods: Test species: Midge ( Chironomus riparius ) Number of organisms: 4 replicates each with 10 lar vae per treatment and control Age: L1 (first instar) larvae Type of test, duration: Static (water-only) test, 48 h Applied concentrations: Nominal: 0 (control), 1.0, 3.6, 10.0, 36, 100 mg/L Measured (mean): -- (control), 0.94, 3.47, 10.01, 33.6, 93.2 mg/L Test conditions: Water quality: M7 medium (according to OECD 202), hardness: not stated Temperature 20.5 – 21.6 °C pH 7.8 – 8.4 (0 h), 8.0 – 8.1 (48 h) O 2 content: > 80 %, (6.9 – 7.7 mg O 2 /L) Light regime: 16 hours light / 8 hours darkness Feeding Only on time 0.01 mL aqueous fish food sol ution directly after insertion of the larvae Test parameters: Mortality and sublethal effects we re assessed after 48 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of BYI 08330-keto hydroxy in the test media samples were taken at test initiation (0 h) a nd termination (48 h). Test samples were analysed by direct injection into a HP LC-UV instrument without a centrifugation. Statistics: LC 10 and LC 50 : Probit analysis, NOEC: directly from raw data Findings: Analytical data: The mean measured concentrations a t the start and end of the test were in the range of 93 – 105 % and 92 – 96 % of nominal, respe ctively. Overall mean measured concentrations ranged from 93 to 100 % of nominal. Effects: See table B.9.2.1.2-3

Table B.9.2.1.2-3: Effects on midge ( C. riparius ) exposed to BYI 08330- ketohydroxy Mortality after 48 hours BYI 08330-enol [mg/L] (nominal) Exposed chironomids (= 100 %) [n] [%]

Control 40 1 2.5
1.0 40 2 5.0 3.6 40 1 2.5 10 40 1 2.5 36 40 2 5.0
100 40 11 27.5

LC10: 23.9 mg/L (95% C.I.: not determined due to ma thematical reasons) LC50: > 100 mg/L (95% C.I.: not determined due to m athematical reasons)– 45 –

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Conclusion: LC 50 (48 h): > 100 mg/L LC 10 (48 h): 23.9 mg/L NOEC (48 h): < 1 mg/L based on nominal concentration Comments: While the study not deviated from recommendations given in OECD 202 guideline, significant deviation from USEPA guideli nes are notable: -Age of the test organisms is 2nd or 3rd instar, 1s t instars were used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test dur ation should be 96-hours. -Mortality and sublethal effects should be measured every 24 hours

.

Reference: 4-Methoxycyclohexanone - Acute daphnia toxicity Author(s), year: Neuhahn, A. 2006 b Report/Doc. number: 2006/0032/01, Edition Number: M -274275-01-1, 10.07.2006 Guideline(s): OECD 202, EU Directive 92/69/EEC C.2 GLP: Yes Deviations: OECD: None of relevance US EPA: Validity: Acceptable

Test substance: 4-Methoxycyclohexanone (metabolite of spirotetrama t), purity: 97.9 %, batch: SAV5506-001 Solubility: Within the test, the item was soluble at the limit concentration of 100 mg/L in test medium after ultra turrax treatment. There was no i ndication for any unsolved material after mixing. Substance specific GC analys is showed recoveries of >80% for all samples taken. Material and methods: Test species: Waterflea ( Daphnia magna ) Number of organisms: 2 replicates each with 10 dap hnids per treatment and control Age: First instar < 24 hours old Type of test, duration: Static limit test, 48 h Applied concentrations: Nominal: 0 (control), 100 mg/L Measured (mean): -- (control), 111.5 mg/L Test conditions: Water quality: M4 medium, hardness: 273 mg/L as Ca CO 3 Temperature 21.4 – 21.5 °C pH 7.8 (control) and 9.2 (100 mg/L) O 2 content: 98 %,8.7 mg O 2 /L Light regime: 16 hours light / 8 hours darkness Test parameters: Immobility and sublethal effects w ere assessed after 24 and 48 hours. For chemical analysis (GC analysis and MS detection) of 4-methoxycyclohexanone in the test media samples were taken at test initiatio n (0 h) and termination (48 h). Test samples were analysed directly without centrif ugation. Statistics: None, because at the limit concentratio n (100 mg/L nominal) no immobilisation were observed Findings: Analytical data: The measured concentrations at the start and end of the test were 103 and 90 %– 46 –

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of nominal, overall mean measured concentration was 97 % of nominal. Effects: After 48 hours no immobility and no sublet hal effects were observed in the control and in the limit test concentrations of 100 mg/L. Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. Conclusion: EC50 (48 h): > 100 mg/L NOEC: 100 mg/L based on nominal concentration Comments: While the study not deviated from recommendations given in OECD 202 and EU guideline, significant deviation from USEPA guideli nes 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 u ncertainties surrounding exposure concentrations in test media.

Reference: Acute toxicity of 4-Methoxycyclohexanon to Larvae o f Chironomus riparius in a 48 h Static Laboratory Test System (Limit-Test ) Author(s), year: Dorgerloh, M., 2007 a Report/Doc. number: EBFNX104, Edition Number: M-287 218-01-1, 27.04.2007 Guideline(s): No specified guideline, the study is following the procedure of OECD 202 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: 4-Methoxycyclohexanon (photo-metabolite of spirote tramat), purity: 97.9 %, batch: SAV55506-001 Solubility: > 100 mg (pH 8, 20 °C) Material and methods: Test species: Midge ( Chironomus riparius ) Number of organisms: 6 replicates each with 10 lar vae per treatment and control Age: L1 (first instar) larvae, < 2 – 3 days old Type of test, duration: Static (water-only) limit- test, 48 h Applied concentrations: Nominal: 0 (control), 100 mg/L Measured (mean): -- (control), 111 mg/L Test conditions: Water quality: M7 medium (according to OECD 202), hardness: 231 mg/L CaCO 3 Temperature 20.3 – 20.9 °C pH 8.0 (after 0 and 48 hours) O 2 content: > 90 %, (8.6 – 8.9 mg O 2 /L) Light regime: 16 hours light / 8 hours darkness Feeding Only on time 0.01 mL aqueous fish food sol ution directly after insertion of the larvae Test parameters: Mortality and sublethal effects we re assessed after 48 hours. For chemical analysis (HPLC-MS/MS) of 4-Methoxycyclohexanon in t he 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 instrum ent without a centrifugation. Statistics: A limit-test was performed, thus no sta tistic is applicable. Findings: Analytical data: The mean measured concentrations a t the start and end of the test were 109 %– 47 –

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and 112 % of nominal, respectively. Overall mean me asured concentrations is 111 % of nominal. Effects: After 48 hours no mortality and no subleth al effects were observed in the control and in the limit test concentrations of 100 mg/L. Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. Conclusion: LC 50 (48 h): > 100 mg/L NOEC (48 h): 100 mg/L based on nominal concentration Comments: While the study not deviated from recommendations given in OECD 202 guideline, significant deviation from USEPA guideli nes are notable: -Age of the test organisms is 2nd or 3rd instar, 1s t instars were used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test dur ation should be 96-hours. -Mortality and sublethal effects should be measured every 24 hours

. Furthermore US EPA has general concerns regarding u ncertainties surrounding exposure concentrations in test media.

Reference: Acute toxicity of BYI 08330-cis-methoxy-cyclohexyla mino carboxylic acid to Larvae of Chironomus riparius in a 48 h Static Laboratory Test System (Limit-Test) Author(s), year: Dorgerloh, M., 2007 b Report/Doc. number: EBFNX105, Edition Number: M-287 220-01-1, 27.04.2007 Guideline(s): No specified guideline, the study is following the procedure of OECD 202 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330-cis-methoxy-cyclohexylamino acid (photo-metabolite of spirotetramat), purity: 93.7 %, batch: KTS10330-1-4 Solubility: > 100 mg (pH 8, 20 °C) Material and methods: Test species: Midge ( Chironomus riparius ) Number of organisms: 6 replicates each with 10 lar vae per treatment and control Age: L1 (first instar) larvae, < 2 – 3 days old Type of test, duration: Static (water-only) limit- test, 48 h Applied concentrations: Nominal: 0 (control), 100 mg/L Measured (mean): -- (control), 88.6 mg/L Test conditions: Water quality: M7 medium (according to OECD 202), hardness: 231 mg/L CaCO 3 Temperature 20.3 – 20.9 °C pH 6.9 – 8.0 (0 h), 7.7 – 8. 0 (48 h) O 2 content: > 90%, (8.7 – 8.9 mg O 2 /L) Light regime: 16 hours light / 8 hours darkness Feeding Only on time 0.01 mL aqueous fish food sol ution directly after insertion of the larvae Test parameters: Mortality and sublethal effects we re assessed after 48 hours. For chemical analysis (HPLC-MS/MS) of BYI 08330-cis-methoxy-cycl ohexylamino acid in the test media samples were taken at test initiation (0 h) and termination (48 h). Test– 48 –

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samples were analysed by direct injection into a HP LC-UV instrument without a centrifugation. Statistics: A limit-test was performed, thus no sta tistic is applicable. Findings: Analytical data: The mean measured concentrations a t the start and end of the test were 87.8 % and 89.4 % of nominal, respectively. Overall mean m easured concentrations is 88.6 % of nominal. Effects: After 48 hours no mortality and no subleth al effects were observed in the control and in the limit test concentrations of 100 mg/L. Thus the NOEC was determined to be 100 mg/L and the EC50 was > 100 mg/L. Conclusion: LC 50 (48 h): > 100 mg/L NOEC (48 h): 100 mg/L based on nominal concentration Comments: While the study not deviated from recommendations given in OECD 202 guideline, significant deviation from USEPA guideli nes are notable: -Age of the test organisms is 2nd or 3rd instar, 1s t instars were used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test dur ation should be 96-hours. -Mortality and sublethal effects should be measured every 24 hours

. Furthermore US EPA has general concerns regarding u ncertainties surrounding exposure concentrations in test media.

Formulation

Reference: Acute toxicity of BYI 08330 OD 150 to larvae of Chironomus riparius in a 48 h static laboratory test system Author(s), year: Dorgerloh, M., 2006 b Report/Doc. number: EBFNM007, Edition Number: M-264 018-01-2, 19.01.2006 Guideline(s): No specified guideline, the study is following the procedure of OECD 202 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08330 OD 150 (formulation), spirotetramat cont ent: 148.89 g/L (14.89 % active ingredient), batch: 08030/0189(0152), densit y: 0.986 g/mL Material and methods: Test species: Midge ( Chironomus riparius ) Number of organisms: 4 replicates each with 10 lar vae per treatment and control Age: L1 (first instar) larvae Type of test, duration: Static (water-only) test, 48 h Applied concentrations: Nominal: Formulation: 0 (control), 2.12, 3.71, 6.6 2, 11.9, 21.2, 37.1, 66.2 mg product/L Spirotetramat: 0 (control), 0.32, 0.56, 1.0, 1.80, 3.20, 5.60, 10.0 mg a.s./L Measured (mean): Spirotetramat: -- (control), 0.30 , 0.51, 0.87, 1.38, 2.67, 4.83, 8.98 mg a.s./L Control: Untreated test water, no blank formulation control was used Test conditions: Water quality: M7 medium (according to OECD 202), hardness: not stated Temperature 20.6 – 21.4 °C pH 8.1 – 8.4 (0 h), 7.8 – 8.4 (48 h) O 2 content: > 90 %, (8.4 – 8.6 mg O 2 /L)– 49 –

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Light regime: 16 hours light / 8 hours darkness Feeding Only on time 0.01 mL aqueous fish food sol ution directly after insertion of the larvae Test parameters: Mortality and sublethal effects we re assessed after 48 hours. For chemical analysis (HPLC-MS/MS and HPLC-UV) of t he actual concentration of spirotetramat in the test media samples were tak en at test initiation (0 h) and termination (48 h). Statistics: LC 10 and LC 50 : Probit analysis, NOEC: directly from raw data, co rrection of control mortality: Abbott’s formular Findings: Analytical data: The mean measured concentrations o f spirotetramat at the start and end of the test were in the range of 86 – 109 % and 68 – 80 % of nominal, respectively. Overall mean measured concentrations ranged from 77 to 95 % of nominal. Effects: See table B.9.2.1.2-6

Table B.9.2.1.2-4: Effects on midge ( C. riparius ) exposed to BYI 08330 OD 150 (formulation)
BYI 08330-OD 150 [mg product/L] Mortality after 48 hours

(nominal) (mean

measured) Spirotetramat

[mg a.s./L] (nominal) Exposed

chironomids (= 100 %) [n] [%]

Control Control 40 4 10
2.12 2.00 0.32 40 4 10 3.71 3.40 0.56 40 13 32.5 6.62 5.75 1.00 40 33 82.5 11.9 9.14 1.80 40 39 97.5 21.2 17.6 3.20 40 40 100 37.1 32.0 5.60 40 40 100
66.2 59.3 10.0 40 40 100 LC 10 (48 h)*: 2.39 mg product /L (95% C.I. 1.83 – 3.1 m g/L), corresponding to 0.36 mg a.s./L LC 50 (48 h)*: 4.41 mg product/L (95% C.I. 3.45 – 7.68 m g/L), corresponding to 0.66 mg a.s./L

Details of the Probit: Slope b: 6.735 95 % CI of b: 4.925 – 8.544 Variance of b: 0.37 Intercept a: 1.91 Goodness of fit, Chi2: 6.10 Degrees of freedom: 5 p (Chi2): 0.971 Conclusion: LC 50 (48 h): 4.41 mg product/L, 0.66 mg a.s./L LC 10 (48 h): 2.39 mg product/L, 0.36 mg a.s./L NOEC (48 h): < 2.0 mg/product, < 0.30 mg a.s./L based on mean measured concentration Comments: While the study not deviated from recommendations given in OECD 202 guideline, significant deviation from USEPA guideli nes are notable: -Age of the test organisms is 2nd or 3rd instar, 1s t instars were used here. -Test organisms should not be fed. -For invertebrates other than Daphnia, the test dur ation should be 96-hours. -Mortality and sublethal effects should be measured every 24 hours

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

Active substance

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330 (tech.) Author(s), year: Dorgerloh, M., 2004 c Report/Doc. number: DOM 23092, Edition Number: M-12 8874-01-2, 29.11.2004 Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400, JMAFF guideline (12 Nousan No 8147) GLP: Yes Deviations: EPA guideline:

• The temperature is less than the EPA-specified tem peratures (24-25oC) and the lighting intensity is higher than EPA specified lig hting.

• The test duration was shorter than is accepted by EPA protocol; the EPA requires 96-120 hour tests.

• Test concentrations dropped as low as 22% of the n ominal, and test solutions were not centrifuged prior to analysis Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods: Test species: Green alga ( Pseudokirchneriella subcapitata) Number of organisms: 1 x 10 4 cells/mL; 3 replicates for each concentration, 6 r eplicates for the medium and solvent control Type of test, duration: Static test, 72 h Applied concentrations: Nominal: 0 (medium control and solvent control), 0 .31, 1.0, 3.1, 10, 31, 100 mg/L Measured (mean): -- (medium control and solvent co ntrol), 0.141, 0.471, 1.46, 5.38, 18.5, 63.2 mg/L Solvent: 0.5 mL/L Aceton Test conditions: Water quality: Nutrient medium (according to OECD guideline) Temperature 22.5 – 23.1 °C pH 7.8 – 8.0 (0 h), 7.5 – 8.3 (96 h) Incubation: Continuous “cool white” illumination w ith fluorescent light (8000 lux ± 15 %) Test parameters: Cell numbers per volume were estim ated photometrically (measuring the extinction in a single-beam-photometer at 578 nm) a t 24, 48 and 72 hours. Morphological examinations were made under a micros cope on each study day. For chemical analysis (HPLC method) of test the sub stance, samples of test solution were taken on day 0 and day 3. Test sample s were analysed by direct injection into a HPLC-UV instrument without a centr ifugation. Statistics: LOEC: ANOVA Procedure, t-test; EC x : Probit analysis, comparison of control groups: Kolmogorov-Smirnov-test (normal distributio n) and Cochran’s test procedure on variance homogeneity All statistical analyses are based on data of poole d control. Findings: Analytical data: Mean measured concentrations were in the range of 84 – 94 % and 22 – 48 % of nominal concentrations on day 0 and day 3, respecti vely. Effects: Morphological effects: None Biomass & growth rate: See table B.9.2.1.3-1– 51 –

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

Table B.9.2.1.3-1: Effects of spirotetramat (BYI 08 330) on the green alga P. subcapitata
Inhibition after 72 h BYI 08330 [mg/L] (mean measured) Biomass (AUC) Yield 1) Growth rate 0 (pooled control) 2)
-- -- -- 0.141 -51.1 -54.7 -11.3 0.471 -26.0 -22.2 -5.3 1.46 -15.1 -8.6 -2.2 5.38 40.9* 46.7* 18.1* 18.5 93.9* 101.2* 122.7* 63.2 98.5* 101.5* 130.7* NOEC 1.46 mg/L 1.46 mg/L 1.46 mg/L LOEC 5.38 mg/L 5.38 mg/L 5.38 mg/L EC 50 (95% C.I.) 6.58 mg/L (6.29 – 6.91 mg/L) 5.6 mg/L (5.46 – 5.83 mg/L) 8.15 mg/L (7.56 – 8.81 mg/L) 1) Yield = biomass at the end of exposure minus the b iomass at the start of exposure 2) Results statistical analyses of control groups ind icated no significant differences, thus pooled cont rol are used for statistical analysis. * Significant difference (alpha = 0.05) from the con trol

. Conclusion: E b C 50 (0-72 h): 6.58 mg/L E r C 50 (0-72 h): 8.15 mg/L, NOEC (0-72 h): 1.46 mg/L (biomass and growth rate) based on mean measured concentrations

Reference: Toxicity of BYI 08330 to the freshwater diatom Navicula pelliculosa Author(s), year: Kern, M.E.; Lam, C.V., 2005 Report/Doc. number: EBFNX008, Edition Number: M-252 794-01-1, 15.06.2005 Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FIFRA 123-2 GLP: Yes Deviations: OECD: None of relevance US EPA: The number of replicates (3) were fewer tha n are accepted by EPA protocol (4 for Navicula sp.). Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 Material and methods: Test species: Freshwater diatom ( Navicular pelliculosa) Number of organisms: 1 x 10 4 cells/mL; 3 replicates for each concentration, med ium control and solvent control Type of test, duration: Static test, 96 h Applied concentrations: Nominal: 0 (medium control and solvent control), 0 .20, 0.51, 1.3, 3.2, 8.0, 20 mg/L Initial measured: -- (medium control and solvent c ontrol), 0.22, 0.47, 1.25, 3.2, 8.23, 20.4 mg/L Solvent: 0.5 mL/L Aceton Test conditions: Water quality: AAP Nutrient medium (according to O ECD guideline) Temperature 23.5 – 24.1 °C pH 7.7 – 7.9 (0 h), 8.0 – 9.5 (96 h) Incubation: Continuous illumination (4026 – 4693 l ux), orbital shaking at 100 rpm Test parameters: Cell densities were determined usi ng a electronic particle counter after 24, 48, 72– 52 –

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

and 96 hours. In addition the cell counts using a h aemocytometer and a microscope were performed in order to confirmed mea sured cell densities. Morphological examinations were made under a micros cope on each study day. For chemical analysis (HPLC method) of test the sub stance, samples of test solution were taken on day 0 and day 4. The test sa mples were directly analysed without a centrifugation. Statistics: NOEC: ANOVA followed by Dunnett’s test; EC 50 : Regression analysis, comparison of control groups: T-test All statistical analyses are based on data of poole d control. Findings: Analytical data: Mean measured concentrations were in the range of 92 – 110 % and 0 (< LOQ) – 59 % of nominal concentrations on day 0 and day 4, respectively. Effects: Morphological effects: None Biomass & growth rate: See table B.9.2.1.3-2

Table B.9.2.1.3-2: Effects of spirotetramat (BYI 08 330) on the freshwater diatom N. pelliculosa BYI 08330 [mg/L] (initial measured) BYI 08330 [mg/L] (geomean day 0/day 4) Cumulative biomass Inhibition after 96 h [%] Growth rate Inhibition after 96 h [%] 0 (pooled control) a) 0 (pooled control) a)
-- -- 0.22 0.03 11 3.2 0.47 0.193 10 2.4 1.25 0.55 15* 4.4** 3.2 1.00 16* 3.5 8.23 4.73 62* 13* 20.4 15.4 99* 74*

NOEC 0.19 mg/L 1.0 mg/L LOEC 0.55 mg/L 4.73 mg/L EC 50 (95% C.I.) 4.05 mg/L (3.73 – 4.38 mg/L) 15.0 mg/L (0 – 903 mg/L) a) Result of t-test is indicated no significant diffe rences between control and solvent control, thus po oled control are used for statistical analysis. * Significant difference (alpha = 0.05) from the con trol ** Statistically significant from control but not co nsidered biologically significant

Conclusion: E b C 50 (0-96 h): 4.05 mg/L E r C 50 (0-96 h): 15.0 mg/L, NOEC (0-96 h): 0.19 mg/L (biomass); 1.0 mg/L (growt h rate) based on geometric mean measured concentrations (da y 0 and day 4)

Reference: Toxicity of BYI 08330 to the blue-green alga Anabaena flos-aqua Author(s), year: Kern, M.E.; Lam, C.V., 2006 Report/Doc. number: EBFNX007, Edition Number: M-264 055-01-1, 12.01.2006 Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FIFRA 123-2 GLP: Yes Deviations: All guidelines: The study results did n ot exhibit 50% inhibition at the highest test concentration for growth rate. US EPA: The temperature is less than the EPA-specif ied temperatures (24-25oC). Validity: Acceptable, even if the inhibiton for gro wth rate is lower than recommended the enpoints are acceptable for EU risk assessment.– 53 –

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

Test substance: Spirotetramat (BYI 08330), purity: 97.99 %, batch: 08045/0014 Material and methods: Test species: Blue-green alga ( Anabaena flos-aqua) Number of organisms: 1 x 10 4 cells/mL; 3 replicates for each concentration, med ium control and solvent control Type of test, duration: Static test, 96 h Applied concentrations: Nominal: 0 (medium control and solvent control), 0 .51, 1.3, 3.2, 8.0, 20 mg/L Initial measured: -- (medium control and solvent c ontrol), 0.45, 1.29, 3.32, 9.05, 21.7 mg/L Solvent: 0.5 mL/L Aceton Test conditions: Water quality: AAP Nutrient medium (according to O ECD guideline) Temperature 23.3 – 23.8 °C pH 7.7 – 7.9 (0 h), 8.1 – 8.3 (96 h) Incubation: Continuous illumination with cool whit e fluorescents (2200 lux ± 10 %), orbital shaking at 100 rpm Test parameters: Cell densities were determined usi ng a electronic particle counter after 24, 48, 72 and 96 hours. In addition the cell counts using a h emocytometer and a microscope were performed in order to confirmed mea sured cell densities. Morphological examinations were made under a micros cope on each study day. For chemical analysis (HPLC method) of test the sub stance, samples of test solution were taken on day 0 and day 4. The test sa mples were directly analysed without a centrifugation. Statistics: NOEC: ANOVA followed by Dunnett’s test; EC 50 : Regression analysis, comparison of control groups: T-test All statistical analyses are based on data of poole d control. Findings: Analytical data: Mean measured concentrations were in the range of 87 – 113 % and 31 – 52 % of nominal concentrations on day 0 and day 4, respecti vely. Effects: Morphological effects: None Biomass & growth rate: See table B.9.2.1.3-3

Table B.9.2.1.3-3: Effects of spirotetramat (BYI 08 330) on the blue-green algae Anabaena flos-aquae BYI 08330 [mg/L] (initial measured) BYI 08330 [mg/L] (geomean day 0/day 4) Cumulative biomass Inhibition after 96 h [%] Growth rate Inhibition after 96 h [%] 0 (pooled control) a) 0 (pooled control) a)
-- -- 0.45 0.3 -43 3
1.29 0.718 -15 -13 3.32 2.08 -51 -13 9.05 5.68 -6 -3 21.7 15.1 48** 14

NOEC 5.68 mg/L 15.1 mg/L LOEC 15.1 mg/L > 15.1 mg/L EC 50 (95% C.I.) 15.2 mg/L (14.9 – 15.5 mg/L) > 15.1 mg/L

a) Result of t-test is indicated no significant diffe rences between control and solvent control, thus po oled control are used for statistical analysis. ** Biological significant based on % inhibition comp ared to the pooled controls– 54 –

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

Conclusion: E b C 50 (0-96 h): 15.2 mg/L E r C 50 (0-96 h): > 15.1 mg/L, NOEC (0-96 h): 5.68 mg/L (biomass); 15.1 mg/L (grow th rate) based on geometric mean measured concentrations (da y 0 and day 4)

Metabolites

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330-enol Author(s), year: Dorgerloh, M. 2005 f Report/Doc. number: EBFNM010, Edition Number: M-252 531-01-2, 06.06.2005 Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400 GLP: Yes Deviations: US EPA:

• The temperature is less than the EPA-specified tem peratures (24-25oC) and the lighting intensity is higher than EPA specified lig hting.

• The test duration was shorter than is accepted by EPA protocol; the EPA requires 96-120 hour tests. Validity: Acceptable

Test substance: BYI 08330-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005 Material and methods: Test species: Green alga ( Pseudokirchneriella subcapitata) Number of organisms: 1 x 10 4 cells/mL; 3 replicates for each concentration, 6 r eplicates for the medium and solvent control Type of test, duration: Static test, 72 h Applied concentrations: Nominal: 0 (medium control and solvent control), 1 .0, 3.1, 10, 31, 100 mg/L Measured (mean): -- (medium control and solvent co ntrol), 1.02, 2.9, 10.1, 29.4, 102 mg/L Solvent: 0.5 mL/L Dimethylformamide (DMF) Test conditions: Water quality: Nutrient medium (according to OECD guideline) Temperature 22.3 – 23.5 °C pH 7.1 – 7.9 (0 h), 7.6 – 8.5 (96 h) Incubation: Continuous “cool white” illumination w ith fluorescent light (5560 - 7350 lux), orbital shaking at 100 rpm Test parameters: Cell numbers per volume were estim ated photometrically (measuring the extinction in a single-beam-photometer at 578 nm) a t 24, 48 and 72 hours. Morphological examinations were made under a micros cope on each study day. For chemical analysis (HPLC method) of test the sub stance, samples of test solution were taken on day 0 and day 3. The test sa mples were analysed by direct injection into an HPLC-MA/MS instrument without a c entrifugation. Statistics: LOEC: ANOVA Procedure, t-test; EC 50 : 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 poole d control. Findings: Analytical data: Mean measured concentrations were in the range of 94 – 104 % and 94 – 102 % of nominal concentrations on day 0 and day 3, respe ctively. Effects:– 55 –

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

Morphological effects: None Biomass & growth rate: See table B.9.2.1.3-4

Table B.9.2.1.3-4: Effects of BYI 08330-enol (metab olite of spirotetramat) on the green alga P. subcapitata
Inhibition after 72 hours BYI 08330 [mg/L] (nominal) Biomass (AUC) Growth rate

0 (pooled control) 1)
-- -- 1.0 -9.2 -1.0 3.1 -15.1 -3.4 10 0.2 0.5 31 1.9 1.5 100 32.9* 13.0* NOEC 31 mg/L 31 mg/L LOEC 100 mg/L 100 mg/L EC 50 > 100 mg/L) > 100 mg/L) 1) Result of t-test is indicated no significant diffe rences between control and solvent control, thus po oled control are used for statistical analysis. * Significant difference (alpha = 0.05) from the co ntrol

. Conclusion: E b C 50 (0-72 h): > 100 mg/L E r C 50 (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 tes t Author(s), year: Weyers, A., 2006 Report/Doc. number: 2006/0032/03, Edition Number: M -275954-01-1, 28.07.2006 Guideline(s): EU Directive 92/69/EEC C.3 GLP: Yes Deviations: EPA guideline:

• The temperature is less than the EPA-specified tem peratures (24-25oC) and the lighting intensity is higher than EPA specified lig hting.

• The test duration was shorter than is accepted by EPA protocol; the EPA requires 96-120 hour tests. Validity: Acceptable

Test substance: 4-Methoxycyclohexanone (metabolite of spirotetrama t), purity: 97.9 %, batch: SAV5506-001 Solubility: Within the test, the item was added to the limit concentration of 100 mg/L in test medium by weighing-in. There was no indication for any unsolved material after mixing. Substance specific GC analysis showed recov eries of >80% for all samples taken. Material and methods: Test species: Green alga ( Desmodesmus subspicatus) Number of organisms: 5 x 10 3 cells/mL; 3 replicates for each concentration, 6 r eplicates for the medium and solvent control Type of test, duration: Static limit test, 72 h Applied concentrations: Nominal: 0 (medium control), 100 mg/L– 56 –

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

Measured (mean): -- (medium control), 92.7 mg/L Test conditions: Water quality: Nutrient medium (according to OECD guideline) Temperature 22.3 – 23.5 °C pH 8.1 (0 h), 8.6 (72 h) Incubation: Continuous illumination with a spectra l range of 400 – 700 nm and an intensity of 4000 - 8000 lux Test parameters: Cell densities were measured in a microcell counter or alternatively, were determined by means of a microscopic counting chamb er at 24, 48 and 72 hours. For chemical analysis (GC analysis and MS detection ) of test the substance, samples of test solution were taken on day 0 and da y 3. The test samples were directly analysed without a centrifugation. Statistics: None, limit test Findings: Analytical data: Mean measured concentration was 92 .1 % on day 0 and 93.9 % day 3 of nominal concentration Effects: Morphological effects: None Biomass & growth rate: No significant effects were observed at the limit concentration of 100 mg/L Conclusion: E b C 50 (0-72 h): > 100 mg/L E r C 50 (0-72 h): > 100 mg/L, NOEC (0-72 h): 100 mg/L (biomass and growth rate) based on nominal concentrations Comments: US EPA has general concerns regarding uncertaintie s surrounding exposure concentrations in test media.

Formulation

Reference: Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330 OD 150 Author(s), year: Dorgerloh, M., 2006 c Report/Doc. number: EBFNM006, Edition Number: M-264 263-01-2, 16.01.2006 Guideline(s): Draft proposal for updating OECD 201, OPPTS 850.5400, JMAFF guideline (12 Nousan No 8147) GLP: Yes Deviations: EPA guideline:

• The temperature is less than the EPA-specified tem peratures (24-25oC) and the lighting intensity is higher than EPA specified lig hting.

• The test duration was shorter than is accepted by EPA protocol; the EPA requires 96-120 hour tests. Validity: Acceptable

Test substance: BYI 08330 OD 150 (formulation), spirotetramat cont ent: 148.89 g/L (15.1 %), batch: 08030/0189(0152) Material and methods: Test species: Green alga ( Pseudokirchneriella subcapitata) Number of organisms: 1 x 10 4 cells/mL; 3 replicates for each concentration, 6 r eplicates for the medium and solvent control Type of test, duration: Static test, 72 h Applied concentrations:– 57 –

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

Nominal: Formulation: 0 (control), 9.53, 17.1, 30. 9, 55.6, 100 mg product/L Spirotetramat: 0 (control), 1,44, 2,58, 4.67, 8.4, 15.1 mg a.s./L Measured (mean): Spirotetramat: -- (control), 0.61 , 1.14, 2.02, 4.05, 8.2 mg a.s./L Control: Untreated test medium, no blank formulatio n control was used Test conditions: Water quality: Nutrient medium (according to OECD guideline) Temperature 22.3 – 23.3 °C pH 8.0 – 8.9 (0 h) Incubation: Continuous “cool white” illumination w ith fluorescent light (8000 lux ± 15 %) Test parameters: Cell numbers per volume were estim ated by direct cell counting under a microscope at 24, 48 and 72 hours. In addition morp hological examinations were performed. For chemical analysis (HPLC method) of test the sub stance, samples of test solution were taken on day 0 and day 3. The test sa mples were analysed by direct injection into an HPLC-UV instrument without a cent rifugation. Statistics: LOEC: ANOVA procedure; EC x : Probit analysis (for growth rate an EC50 could not be determined because the inhibition at highest test concentration was < 50%) Findings: Analytical data: Mean measured concentrations of th e 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 sub stance over the whole test period were 42 – 54 % of nominal. Effects: Morphological effects: At 100 mg/L (nominal) some c ells were enlarged. Biomass & growth rate: See table B.9.2.1.3-5

Table B.9.2.1.3-6: Effects of BYI 08330 OD 150 (for mulation) on the green alga P. subcapitata
Inhibition after 72 hours BYI 08330 OD 150 [mg product/L] (nominal) BYI 08330 [mg a.s./L] (mean measured) Biomass (AUC) Growth rate

9.53 0.61 3.6 -0.2 17.1 1.14 8.0 2.4
30.9 2.02 14.1 3.7 55.6 4.05 29.8* 10.8* 100 8.2 60.2* 27.1*

NOEC 2.02 mg a.s./L 2.02 mg a.s./L LOEC 4.05 mg a.s./L 4.05 mg a.s./L EC 50 (95% C.I.) 6.56 mg a.s./L (5.19 – 9.37 mg/L) > 8.2 mg a.s./L
* Significant difference (alpha = 0.05) from the con trol 1) Yield = biomass at the end of exposure minus the b iomass at the start of exposure

. Conclusion: E b C 50 (0-72 h): 6.56 mg a.s./L corresponding to 43.4 mg product/L E r C 50 (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 r ate) corresponding to 13.4 mg product/L based on geometric mean measured concentrations of active substance– 58 –

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

B.9.2.1.4 Effects on aquatic plants (OECD IIA 8.6, IIIA 10.8.2)

Active substance

Reference: Lemna gibba G3 - Growth inhibition test with BYI 08330 (tech.) under staticrenewal test conditions Author(s), year: Dorgerloh, M., 2005 j Report/Doc. number: DOM 24019, Edition Number: M-25 5296-01-2, 29.07.2005 Guideline(s): OECD 221, EPA OPPTS 850.4400 GLP: Yes Deviations: EPA guideline: -The light intensity was higher than the EPA recomm ended light intensity. -All test concentrations have not been centrifuged prior to analysis. -Precipitate was observed in the highest test level

. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods: Test species: Duckweed ( Lemna gibba ) Number of organisms: 12 fronds per vessel; 3 repli cates for each concentration, control and solvent control Type of test, duration: Static renewal test (renew al of test medium on day 0, 3 and 5), 7 d Applied concentrations: Nominal: 0 (medium and solvent control), 0.30, 0.9 5, 3.05, 9.77, 31.3, 100 mg/L Measured (mean): -- (medium and solvent control), 0.14, 0.47, 1.54, 6.06, 20.5, 71.4 mg/L Solvent 0.5 mL/L Aceton Test conditions: Water quality: 20X-AAP growth medium (according to OECD guideline), the pH was adjusted to 7.5 ± 0.1 Temperature 23.5 – 24.8 °C pH 7.2 – 7.6 (new test media), 8.0 – 8.7 (aged tes t media); control: 7.4 (0 d), 8.7 (7 d) Incubation: Continuous illumination with white flu orescent light (mean intensity: 8.04 kLux) in a growth incubator. Test parameters: Biomass: Frond numbers and total f rond area were determined using LemnaTec Scanalyzer machine on day 0, 3, 5 and 7, respective ly. Dry weights of plants (needed for yield calculation s) were measured at the end of the study (final biomass). For chemical analysis (HPLC-MS/MS, HPLC-UV) of test the substance (BYI 08330) s amples of test solution were taken from freshly pre pared test media (0 d, 3 d, 5 d) and from aged test media (3 d, 5 d, 7 d). Test samples were analysed by direct injection into an HPLC instrument without ce ntrifugation. Statistics: LOEC: ANOVA ( α = 0.05, one sided) and Dunnett’s multiple t-tests Findings: Analytical data: Overall mean measured concentratio ns ranged from 47 % to 71 % of nominal. An overview of all measured concentrations is given in table B.9.2.1.4-1– 59 –

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

Table B.9.2.1.4-1: Analytical results of spirotetra mat

Effects: Morphological effects: None visual effects were obs erved in concentration levels up to 0.47 mg/L. At 1 .54 mg/l slight chlorosis was observed at day 7. At 6.0 6 mg/L and higher concentration levels strong chlorosis and necrosis were noted. Biomass & growth rate: Doubling time T d of the control = 2.2 d (fulfilled the validity crit erion), see also table B.9.2.1.4-2

Table B.9.2.1.4-2: Effects of spirotetramat on duck weed Lemna gibba Growth rate% inhibition in 7 d Spirotetramat [mg/L] (nominal) Spirotetramat [mg/L]

time weighted mean measured Yield

% inhibition in 7 d based on frond number based on total

frond area
0.30 0.14 -3.5 2.4 -3.6 0.95 0.47 -5.5 5.8 -4.0 3.05 1.54 -2.3 5.7 -2.7 9.77 6.06 45.7* 34* 50.1* 31.3 20.5 87.4* 70.2* 91.8*– 60 –

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

Growth rate% inhibition in 7 d Spirotetramat [mg/L] (nominal) Spirotetramat [mg/L]

time weighted mean measured Yield

% inhibition in 7 d based on frond number based on total

frond area
100 a ) 71.4 97.6* 113.5* 116.1* * Significant difference (alpha = 0.05) from the con trol

Table B.9.2.1.4-3: Toxicity of spirotetramat on duc kweed Lemna gibba based on time weighted mean measured concentrations

Endpoint Time scale NOEC

[mg/L] LOEC

[mg/L] EC 50 (95% C.I.)

[mg/L] a) EC 50 (95% C.I.) [mg/L] b) Growth rate (based on frond number) 7 d 1.54 6.06 10.4 (7.96 – 13.8) 9.96 (7.85 – 12.49 )

Growth rate (based on total area of plants 7 d 1.54 6.06 6.21 (5.33 – 7.18) 6.21 (5.55 – 6.91)

Yield (based on frond number) 7 d 1.54 6.06 4.62 (2 .04 – 10.3) 4.49 (2.70 – 7.20) Yield (based on biomass – dry weight) 7 d 1.54 6.06 6.96 (5.76 – 8.38) 7.04 (6.07 – 8.12)

Visual effects on plants 7 d 0.47 1.54 -- --
a) The 100 mg/L concentration level was excluded from EC50 calculations because of exceeded water solubili ty b) The 100 mg/L concentration level was included into the EC50 calculations.

Conclusion: E y C 50 (7 d): 4.62 mg/L E r C 50 (7 d): 6.21 mg/L NOEC (7d): 1.54 mg/L (for biomass/yield and growth rate) based on time weighted mean measured concentrations Comments: Spirotetramat is an insecticide and therefore a st udy on higher aquatic plants is not required according to directive 91/414/EEC. How ever, in general an exposition of Lemna gibba

is possible and therefore the study is considered in the aquatic risk assessment.

Metabolites

Reference: Lemna gibba G3 - Growth inhibition test with BYI 08330-enol un der static test conditions Author(s), year: Dorgerloh, M., 2005 k Report/Doc. number: EBFNX065, Edition Number: M-252 412-01-2, 07.06.2005 Guideline(s): OECD 221, OPPTS 850.4400 GLP: Yes Deviations: OECD: None of relevance US EPA: The light intensity was higher than the EPA recommended light intensity. Validity: Acceptable

Test substance: BYI 08330-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005 Material and methods: Test species: Duckweed ( Lemna gibba ) Number of organisms: 12 fronds per vessel; 3 repli cates for each concentration, control and solvent control Type of test, duration: Static test, 7 d Applied concentrations: Nominal: 0 (medium control), 0.09, 0.30, 0.95, 3.0 5, 9.77, 31.3, 100 mg/L Measured (mean): -- (medium control), n.a., n.a., 1.02, 3.41, 11.7, 37.6, 119 mg/L– 61 –

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

Test conditions: Water quality: 20X-AAP growth medium (according to OECD guideline), the pH was adjusted to 7.5 ± 0.1 Temperature 22.3 – 24.0 °C pH 7.2 – 7.8 (day 0), 8.2 – 8.8 (day 7) Incubation: Continuous illumination with white flu orescent light (mean intensity: 8.96 kLux) in a growth incubator. Test parameters: Biomass: Frond numbers and total f rond area were determined using LemnaTec Scanalyzer machine on day 0, 3, 5 and 7, respective ly. Dry weight of plants (needed for yield calculations ) was measured at the end of the study (final biomass). For chemical analysis (HPLC-MS/MS, HPLC-UV) of test the substance s amples of test solution were taken on day 0 and on day 7 (tes t termination). Test samples were analysed by direct injection into an HPLC inst rument without centrifugation. Statistics: LOEC: ANOVA ( α = 0.05, one sided) and Dunnett’s multiple tests Findings: Analytical data: Overall mean measured concentratio ns ranged from 114 % to 120 % on day 0 and from 97 % to 120 % on day 7 of nominal. Effects: Morphological effects: None visual effects were obs erved in concentration levels up to 0.95 mg.L. At

≥ 3.5 mg/l chlorosis was noted. Biomass & growth rate: Doubling time T d of the control = 1.9 d (fulfilled the validity crit erion), see also table B.9.2.1.4-3

Table B.9.2.1.4-4: Effects of BYI 08330-enol on duc kweed Lemna gibba Growth rate inhibition [%] BYI 08330-enol [mg/L] (nominal) Yield inhibition [%] based on frond number based on total frond area 0.09 0.1 -0,9 -2,9 0.30 0.1 -2,7 -2,4 0.95 3.1 0,1 0,1 3.05 27.8* 8,3 9,5 9.77 80.8* 38,5* 55,2* 31.3 84.4* 50,3* 60,8* 100 88.6* 45,8* 67,0* NOEC 0.95 mg/L 3.05 mg/L 3.05 mg/L LOEC 3.05 mg/L 9:77 mg/L 9:77 mg/L EC 50 (95% C.I.) 5.4 (3.3 – 9.1) mg/L 60.8 (21.6 - 1643) mg/L 19.3 (6.9 – 74.2) mg/L * Significant difference ( α =0.05) from the control

Conclusion: E y C 50 (7 d): 5.4 mg/L E r C 50 (7 d): 19.3 mg/L NOEC (7d): 0.95 mg/L (yield); 3.05 mg/L (growth rat e) based on nominal concentrations Comments: BYI 08330-enol is a metabolite of spirotetramat wh ich is an insecticide and therefore a study on higher aquatic plants is not r equired according to directive 91/414/EEC. However, in general an exposition of Lemna gibba is possible and therefore the study is considered in the aquatic ri sk assessment.– 62 –

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

B.9.2.1.5 Effects on marine or estuarine organisms (OECD IIA 8.11, IIIA 10.2.2.4, IIIA 10.2.2.5)

General comment: Studies on four marine species (fish, shrimp, oyste r, algae) with the active substance are available. Usually these studies are no required according to directive 91/414/EEC and the EU Aquatic Ecotoxicolo gy Guidance Document. However in the aquatic Guidance document SANCO/3268/2001 rev.4 final it is stated “...in some cases, data are available on estuarine/ma rine species. At present, there is no requirement u nder directive 91/414/EEC to perform these studies, but if data are available, they must be submitted and s hould be considered in the risk assessment.” In general m arine organisms are regarded as representative for freshwater ecosystem. Thus the RMS has evaluated th e studies and the results have been considered in t he risk assessment.

B.9.2.1.5.1 Effects on saltwater fish

Active Substance

Reference: Acute toxicity of BYI 08330 technical to the sheeps head minnow ( Cyprinodon variegatus ) under flow-trough conditions Author(s), year: Banman, C. S.; Lam, C. V., 2005 Report/Doc. number: EBFNX006, Edition Number: M-255 363-01-1, 27.07.2005 Guideline(s): OECD 203, US EPA OPPTS 850.1075, FIFR A 72-2 GLP: Yes Deviations: OECD: None of relevance USEPA: Based on EFED’s guidance document, “Estuarin e Fish 96-Hour Acute Toxicity Standard Evaluation Procedure’s the recomm endation of fish weight is between 0.5 and 5 g. Validity: Acceptable.

Test substance: Spirotetramat (BYI 08330), purity: 97.99 %, batch: 08045/0014 Material and methods: Test species: Sheepshead minnow ( Cyprinodon variegatus ) Number of organisms: 20 fish per concentration and control Weight, length: 0.13 ± 0.03 g (mean ± SD), 1.72 ± 0.14 cm (mean ± SD) Loading: 0.03 g fish/L test material passing throu gh tank each day Type of test, duration: Flow-through test (renewals approx. 5.3 x per day), 96 hours Applied concentrations: Nominal: 0 (control and solvent control), 0.63, 1. 25, 2.50, 5.0, 10 mg/L Measured (mean): -- (control and solvent control), 0.52, 1.17, 2.52, 4.54, 9.77 mg/L Solvent 0.1 mL/L Aceton Test conditions: Water quality: Artificial sea salts mixed with rev erse osmosis water or blended soft water to produce a salinity of 17 ‰ Temperature: 21.0 – 22.5 °C pH: 8.1 – 8.4 (0 h), 7.7 - 8.1 (96 h) O 2 content: 5.3 – 6.9 mg O 2 /L (67 – 87 %) Light regime: 16 hours light / 8 hours darkness, 3 0 min light/dark transition period Test parameters: Mortality and sublethal effects we re assessed after 4, 24, 48, 72 and 96 hours. For chemical analysis (HPLC method) of BYI 08330 in test solutions samples were taken at 0, 24 and 96 hours. Test samples were analysed by direct injection into an HPLC instrument without centrifugation.– 63 –

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

Statistics: LC 50 : Moving average method, NOEC: directly from raw da ta Findings: Analytical data: Mean measured concentrations were in the range of 92 – 115 % of nominal concentrations. Behavioural effects None in controls and at 0.52 mg /L concentration level. After 96 hours at ≥ 1.17 mg/L effects like loss of equilibrium and lyi ng on the bottom of the aquarium were observed. Thus the NOEC is 0.52 mg/L. Mortality: See table B.9.2.1.5-1

Table B.9.2.1.5-1: Cumulative mortality of sheepshe ad minnow exposed to spirotetramat Cumulative mortality (%) Spirotetramat (mean measured) [mg/L] 4 hours 24 hours 48 hours 72 hours 96 hours

Blank control 0 0 0 0 0 Solvent control 0 0 0 0 0
0.52 0 0 0 0 0 1.17 0 0 0 0 10 2.52 0 0 0 35 60 4.54 0 10 100 100 100
9.77 0 100 100 100 100 NOEC = 0.52 mg/L LC 50 = 1.96 mg/L (95%C.I. 1.64 – 2.37) based on moving average method LC 50 = 2.10 mg/L (95%C.I. 1.73 – 2.52) based on Probit a nalysis

Details of the Probit: Slope b: 5.696 95 % C.I. of b: 3.495 – 7.897 Variance of b: 1.12 Intercept a: -1.838 Goodness of fit, Chi2: 1.282 Degrees of freedom: 3 p (Chi2): 0.733 Conclusion: LC 50 (96 h): 1.96 mg/L NOEC: 0.52 mg/L based on mean measured concentration

B.9.2.1.5.2 Effects on saltwater invertebrates

Active substance

Reference: BYI 08330 - Acute toxicity to mysids ( Americamysis bahia) under flowthrough conditions Author(s), year: Cafarelle, M. A., 2005 a Report/Doc. number: EBFNX010, Edition Number: M-270 200-01-1, 06.01.2005 Guideline(s): EPA OPPTS 850.1035, FIFRA 72-3 GLP: Yes Deviations: None of relevance Validity: Acceptable.

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods: Test species: Saltwater mysid ( Americamysis bahia )– 64 –

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

Number of organisms: 2 replicates each with 10 mys ids per treatment and control Age: 5- to 6-days old Type of test, duration: Flow-through test, 96 h Applied concentrations: Nominal: 0 (control and solvent control) 0.63, 1.3 , 2.5, 5.0, 10.0 mg/L Measured (mean): -- (control and solvent control), 0.73, 1.2, 2.6, 4.6, 9.0 mg/L Solvent 0.1 mL/L Dimethylformamide (DMF) Test conditions: Water quality: Filtered natural seawater diluted w ith well water, salinity: 21 – 20 ‰ Temperature 24 – 25 °C pH 8.1 (0 h), 7.8 – 8.1 (96 h) O 2 content: > 60 % saturation, 4.9 – 7.4 mg O 2 /L Light regime: 16 hours light / 8 hours darkness Test parameters: Mortality and sublethal effects we re assessed after 0, 24, 48, 72 and 96 hours. For chemical analysis (HPLC method) of spirotetrama t in the test solution samples were taken at 0 and 96 hours. Test samples were ana lysed by direct injection into an HPLC instrument without centrifugation. Statistics: EC 50 : Binominal probability and probit analysis, NOEC: directly from the raw data Findings: Analytical data: Overall mean measured concentratio ns were in the range of 90 – 120 % of nominal concentrations. Behavioural effects At 2.6 mg/L and higher concentr ations sublethal effects like erratic swimming, lethargy and laying on the bottom of test vessel we re observed. Thus the NOEC was determined to be 1.2 mg/L. Mortality: See table B.9.2.1.5-2

Table B.9.2.1.5-2: Cumulative mortality of saltwate r mysids exposed to spirotetramat Spirotetramat (BYI 08330 Cumulative mortality (%) Nominal [mg/L] Mean measured

[mg/L] 24 hours 48 hours 72 hours 96 hours

Control 0 0 0 0 Solvent control 0 0 0 0
0.63 0.73 5 5 5 5 1.3 1.2 0 0 5 5 2.5 2.6 10 20 20 30
5.0 4.6 15 20 25 25 10 9.0 60 100 100 100 NOLC (96 hours) < 0.73 mg/L LC 50 (96 h) = 5.5 mg/L (95%C.I. 1.2 – 9.0 mg/L) based o n binominal probability LC 50 (96 h) = 4.27 mg/L (95%C.I. not calculated due to mathematical reasons) based on probit analysis

Details of the Probit: Slope b: 2.926 95%C.I. of b: 0.793 and 5.059 Intercept a: -1.845 Goodness of fit, Chi2: 13.25 Degrees of freedom: 3 p (Chi2): 0.0041 Conclusion: LC 50 (96 h): 5.5 mg/L NOLC: < 0.73 mg/L based on mean measured concentration– 65 –

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

Reference: BYI 08330 - Acute toxicity to eastern oy sters ( Crassostrea virginica ) under flow-through conditions Author(s), year: Cafarella, M. A., 2005 b Report/Doc. number: EBFNX011, Edition Number: M-257 677-01-1, 15.06.2005 Guideline(s): EPA OPPTS 850.1025, FIFRA 72-3 GLP: Yes Deviations: • During the final 24 hours of exposure the oxygen s aturation drop below 60 % (ranged from 52 – 96 %).

• The measured concentrations drop below 59% of nomi nal concentration. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 Material and methods: Test species: Eastern Oyster ( Crassostrea virginica ) Number of organisms: 20 oysters per treatment and control Valve height: 37 ± 5 mm (mean ± SD) Type of test, duration: Flow-through test, 96 h Applied concentrations: Nominal: 0 (control and solvent control), 0.19, 0. 38, 0.75, 1.5, 3.0 mg/L Measured (mean): -- (control and solvent control), 0.20, 0.33, 0.55, 0.89, 2.1 mg/L Solvent 0.1 mL/L Dimethylformamide (DMF) Test conditions: Water quality: Filtered natural seawater, salinity : 31 – 32 ‰ Temperature 20 – 22 °C pH 7.6 – 8.0 O 2 content: 3.8 – 7.3 mg O 2 /L (52 – 96 % saturation) Light regime: 16 hours light / 8 hours darkness Test parameters: Observations of mortalities and su blethal effects were made at 0, 24, 48, 72 and 96 hours and the shell growth was determined at tes t 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 s amples were analysed by direct injection into an HPLC instrument without centrifug ation. Statistics: EC 50 : Linear regression analysis, NOEC: Williams test, comparison of control groups: T-test. All statistical analyses are based on data of poole d control. Findings: Analytical data: Overall mean measured concentratio ns were in the range of 59 – 100 % of nominal concentrations. Mortality: None Shell Deposition: See table B.9.2.1.5-3

Table B.9.2.1.5-3: Mean shell deposition of eastern oysters ( C. virginica ) exposed to spirotetramat Spirotetramat [mg/L] (mean measured) Mean shell deposition (mm ± SD) Inhibition of shell growth [%]

Pooled control a) 3.5 ± 1.2 -- 0.20 3.8 ± 1.2 +7.9 0.33 3.4 ± 1.1 3 0.55 2.4 ± 1.0 31* 0.89 1.6 ± 0.9 54* 2.1 0.4 ± 0.7 89*– 66 –

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

Spirotetramat [mg/L] (mean measured) Mean shell deposition (mm ± SD) Inhibition of shell growth [%]

NOEC = 0.33 mg/L EC 50 (96 h)= 0.85 mg/L (95%C.I. 0.59 – 1.3 mg/L) a) no statistically significant difference between di lution control and solvent control * statistically significant difference from the poo led control (p ≤ 0.05)

Conclusion: EC 50 (48 h): 0.85 mg/L NOEC: 0.33 mg/L based on mean measured concentration Comments: US EPA has general concerns regarding uncertaintie s surrounding exposure concentrations in test media. (95 %CI: not stated)

B.9.2.1.5.3 Effects on saltwater algae

Reference: Toxicity of BYI 08330 technical to the saltwater di atom Skeletonema
costatum Author(s), year: Banman, C. S. & Lam, C. V., 2006 Report/Doc. number: EBFNX009-1, Edition Number: M-2 71037-02-1, 16.06.2006 Guideline(s): Draft proposal for updating OECD 201 (2004), OPPTS 850.5400, FIFRA 123-2 GLP: Yes Deviations: OECD: None of relevance USEPA: -The photoperiod for studies involving Skeletonema costatum is 14 hour light/10 hour dark cycle; however, continuous illumination w as used in this study. -Test samples were not centrifugated prior to chemi cal analyses. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.99 %, batch: 08045/0014 Material and methods: Test species: Marine diatom ( Skeletonema costatum) Number of organisms: 1 x 10 4 cells/mL; 3 replicates for each concentration, med ium control and solvent control Type of test, duration: Static test, 96 h Applied concentrations: Nominal: 0 (medium control and solvent control), 0 .256, 0.640, 1.60, 4.0, 10 mg/L Initial measured: -- (medium control and solvent c ontrol), 0.240, 0.622, 1.63, 4.15, 10.6 mg/L Solvent: 0.5 mL/L Aceton Test conditions: Water quality: ASTM saltwater media (slightly modi fied: 40 mg/L silicates instead of 20 mg/L silicates, salinity 27 ‰ Temperature 19.4 – 21.0 °C pH 8.0 – 8.4 Incubation: Photoperiod: 16:8 (light/dark), intens ity (3897 - 4682 lux ), orbital shaking at 100 rpm Test parameters: Cell densities were determined usi ng an electronic particle counter after 24, 48, 72 and 96 hours. In addition the cell counts using a h aemocytometer and a microscope were performed in order to confirmed mea sured cell densities. Morphological examinations were made under a micros cope on each study day. For chemical analysis (HPLC method) of test the sub stance, samples of test– 67 –

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

solution were taken on day 0 and day 4. . Test samp les were analysed by direct injection into an HPLC instrument without centrifug ation. Statistics: NOEC: ANOVA followed by Dunnett’s test; EC 50 : Linear regression analysis, comparison of control groups: T-test All statistical analyses are based on data of poole d controls. Findings: Analytical data: Mean measured concentrations were in the range of 94 – 106 % and 25 – 46 % of nominal concentrations on day 0 and day 4, respecti vely. Effects: Morphological effects: None Biomass & growth rate: See table B.9.2.1.5-4

Table B.9.2.1.5-4: Effects of spirotetramat (BYI 08 330) on the marine diatom S. costatum. BYI 08330 [mg/L] (initial measured) BYI 08330 [mg/L] (geomean) Cumulative biomass Inhibition after 96 h [%] Growth rate Inhibition after 96 h [%] 0 (pooled control) a) 0 (pooled control) a)
-- -- 0.24 0.124 -22 -10 0.622 0.394 57* 27* 1.63 1.03 87* 61* 4.15 2.7 105* 115* 10.6 6.95 104* 111*

NOEC 0.124 mg/L LOEC 0.394 mg/L EC 50 (95% C.I.) 0.36 mg/L (0.27 – 0.46 mg/L) 0.98 mg/L (0.92 – 1.05 mg/L) a) no statistically significant difference between di lution control and solvent control * Significant difference (alpha = 0.05) from the con trol

Conclusion: E b C 50 (0-96 h): 0.36 mg/L E r C 50 (0-96 h): 0.98 mg/L NOEC (0-72 h): 0.12 mg/L (biomass and growth rate) based on geometric mean measured concentrations– 68 –

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

B.9.2.1.6 Summary of acute toxicity

Table B.9.2.1.6-1: Summary of acute toxicity data f or aquatic organisms; values which are relevant for risk assessment are in bold

Test organism Test condition Time Endpoint Test

conc. NOEC

[mg/L] EC 50 /LC 50

[mg/L] Reference study acceptable Spirotetramat Oncorhynchus mykiss Rainbow trout semi static 96 hr mortality mm 0.825 2.54 Dorgerloh, 2004a yes Cyprinus carpio Common carp semi static 96 hr mortality mm 1.02 2.59 Dorgerloh, 2004b yes Lepomis macrochirus Bluegill sunfish semi static 96 hr mortality nom 0.5 2.2 Dorgerloh, 2005a yes Cyprinodon variegatus Sheephead minnow flow through 96 hr mortality mm 0.52 1.96 Banman& Lam, 2005 yes Daphnia magna Waterflea static 48 hr immobility mm 20.3 > 42.7 Dorgerloh, 2005c yes

Chironomus riparius Midge static(wateronly) 48 hr mortality mm <0.53 1.30 Dorgerloh, 2005g yes

Americamysis bahia Saltwater mysid flow through 96 hr mortality mm < 0.73 4.27 Cafarelle, 2005a yes Crassostrea virginica Eastern oyster flow through 96 hr shell deposition mm 0.33 0.85 Cafarelle, 2005b yes
P. subcapitata Green alga static 72 hr biomass growth rate mm 1.46 6.58 8.15 Dorgerloh,

2004c yes
Navicular pellicullosa Diatom (freshwater) static 96 hr biomass growth rate mm 0.19 1.00 4.05

15.0 Kern&Lam,

2005 yes
Anabaena flos-aqua Blue-green alga static 96 hr biomass growth rate mm 5.68 15.1 15.2

> 15.1 Kern&Lam,

2006 yes

Skeletonema costatum Diatom (marine) static 96 hr biomass growth rate mm 0.124 0.36 0.96 Banman&

Lam, 2006 yes
Lemna gibba Duckweed semi static 7 d yield growth rate mm 1.54 4.49 6.21 Dorgerloh,

2005j yes

BYI 08330-enol Oncorhynchus mykiss Rainbow trout static b) 96 hr mortality nom 100 > 100 Nieden, 2005a yes Daphnia magna Waterflea static b) 48 hr immobility nom 100 > 100 Dorgerloh, 2005d yes

Chironomus riparius Midge static(wateronly) 48 hr mortality nom 17.1 a) 74.9 Dorgerloh, 2005h yes

P. subcapitata Green alga static 72 hr biomass growth rate nom 31 > 100 Dorgerloh, 2005f yes
Lemna gibba Duckweed static 7 d yield growth rate mm 0.95 3.05 5.4

19.3 Dorgerloh,

2005k yes
BYI 08330-ketohydroxy
Chironomus riparius Midge static(wateronly) 48 hr mortality nom 23.9 a) > 100 Dorgerloh, 2006a yes

4-Methoxycyclohexanone Danio rerio Zebra fish static b) 96 hr mortality nom 100 > 100 Neuhahn, 2006a yes Daphnia magna Waterflea static b) 48 hr immobility nom 100 > 100 Neuhahn, 2006b yes

Chironomus riparius Midge static b)(wateronly) 48 hr mortality nom 100 > 100 Dorgerloh, 2007a yes

D. subspicatus Green alga static b) 72 hr biomass growth rate nom 100 > 100 Weyers, 2006 yes– 69 –

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

Test organism Test condition Time Endpoint Test

conc. NOEC

[mg/L] EC 50 /LC 50

[mg/L] Reference study acceptable 4-Methoxycyclohexylamino caboxylic acid
Chironomus riparius Midge static b)(wateronly) 48 hr mortality nom 100 > 100 Dorgerloh, 2007b yes

Formulation - BYI 08830 OD 150 Oncorhynchus mykiss Rainbow trout static 96 hr mortality nom 2.5 (0.38 a.s.) 9.48

(1.41 a.s.) Nieden,

2005b yes

Chironomus riparius Midge static(wateronly) 48 hr mortality mm 2.39 a) (0.36 a.s.) 4.41

(0.66 a.s.) Dorgerloh,

2006b yes

P. subcapitata Green alga static 72 hr biomass

growth rate nom 13.4 (2.02 a.s.) 43.4

(6.56 a.s.) > 54.3 (> 8.2 a.s.) Dorgerloh,

2006c yes

Test conc.: test concentration based on mean measur ed (mm) or nominal (nom) concentration a) LC10, NOEC was not determined b) Limit test– 70 –

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

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

organisms

B.9.2.2.1 Chronic toxicity to fish to fish (OECD II A 8.2.2., IIA 8.2.3, IIIA 10.2.5.1)

No study submitted. Not required, because a fish ea rly life stage toxicity test has been carried out.

B.9.2.2.2 Fish early life stage toxicity test (OECD IIA 8.2.4, IIIA 10.2.5.2)

Active substance

Reference: Early-life stage toxicity of BYI 08330 tech. to fis h ( Pimephales promelas ) Author(s), year: Dorgerloh, M., 2005 b Report/Doc. number: EBFN0305, Edition Number: M-260 676-01-2, 16.11.2005 Guideline(s): EPA 72-4a, EPA OPPTS 850.1400, OECD 2 10 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 Material and methods: Test species: Fathead minnow ( Pimephales promelas ) Number of organisms: 4 replicates x 25 eggs per te st concentration and control, after completion of hatch larvae were thinned to 15 individuals per egg cup Age: Freshly fertilized eggs < 24 hours old Type of test, duration: Flow-through test, 33 days (28 days post hatch) Applied concentrations: Nominal: 0 (control and solvent control), 0.0625, 0.125, 0.250, 0.500, 1.00 mg/L Measured (mean): -- (control and solvent control), 0.0607, 0.121, 2.243, 0.486, 0.971 mg/L Solvent Dimethylformamide (DMF) 0.1 mL/L Test conditions: Water quality: Reconstituted water (according to I SO), hardness: 45 – 52 mg/L as CaCO 3 Temperature: 24.5 (23 – 26) °C pH: 7.0 – 7.3 during the total test period O 2 content: 98 – 100 % saturation Light regime: 16 hours light / 8 hours darkness, 3 0 min transient period Feeding Larvae were fed 5 x daily (except on weeke nds/holidays: 2 x daily) with live newly hatched shrimp nauplii ad libidum . Feeding was started shortly after hatching. Uneaten food an faeces were siphoned in order to mi nimise microbiological growth and biodegradation of test substance. Test parameters: Embryo mortality, hatched larvae, fry mortality and abnormal appearance or behaviour were assessed weekdays. At test terminati on the length and the weight were determined. Determined endpoints were: Time to hatch, hatching success, overall fry survival (fry survival before and after thinning), standard length and dry weight. For chemical analysis (HPLC-MS/MS and HPLC-UV) of B YI 08330 in test solutions samples were taken at -1, 0, 7, 14, 21, 2 8 and 33 days Statistics: If control and solvent control can be p ooled: T-test Testing for normal distribution: R/s procedure Time to hatch, hatching success, fry survival: Will iams test with previous acrisine– 71 –

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

transformation Growth data: Williams test without previous data tr ansformation Findings: Analytical data: Overall mean measured concentratio ns in test media were 93 – 119 % of nominal. Biological observation: Time to hatch: In control a nd all treatment levels hatching began on day 4 and continued until day 5. Over the total test period no morphological and beh avioural effects were observed. Effects: See table B.9.2.2.2-1 and table B.9.2.2.2- 2 Table B.9.2.2.2-1: Hatching success and fry surviva l
Spirotetramat [mg/L] (mean measured) Egg hatch [%] phd 0 Mean (SD) Fry survival [%] phd 0 Mean (SD) Fry survival [%] phd 28 Mean (SD) Overall fry survival [%] a) Mean (SD) pooled control b) 85 (8.8) 98 (2.6) 88 (11.1) 87 (11.1) 0.0566 85 (11.5) 94 (9.0) 87 (16.3) 82 (18.4) 0.115 84 (8.0) 95 (5.9) 85 (6.4) 81 (6.0) 0.247 88 (5.7) 100 (0) 85 (17.5) 85 (17.5) 0.534 91 (5.0) 92 (4.7) 78 (19.9) 73 (21.7) 1.16 84 (8.0) 83 (7.1) 78 (14.8) 65 (11.5)*
NOEC 1.16 mg/l 0.534 mg/L LOEC > 1.16 mg/L 1.16 mg/l a) calculated as follows: (fry survival before thinni ng/number of fry hatched)*(fry survival/number of f ry after thinning)*100 phd – post hatch day b) no statistically significant difference between di lution control and solvent control * significant difference when compared to pooled co ntrol (alpha = 0.05)

Table B.9.2.2.2-2: Length and Weight

Spirotetramat [mg/L] (mean measured) Length [mm] Test termination (phd 28) Mean (SD) Dry weight [mg] Test termination (phd 28) Mean (SD) pooled control a) 18.0 (3.5) 20.4 (7.4) 0.0566 18.4(3.4) 21.8 (11.4) 0.115 18.3 (3.4) 21.4 (11.3) 0.247 18.3 (3.9) 21.5 (11.4) 0.534 19.1 (2.2) 22.1 (7.5) 1.16 18.6 (3.4) 21.9 (10.4)
NOEC 1.16 mg/l LOEC > 1.16 mg/L a) no statistically significant difference between di lution control and solvent control * significant difference when compared to pooled co ntrol (alpha = 0.05)

Conclusion: Fry survival: NOEC: 0.534 mg/L, LOEC: 1.16 mg/L Hatchability and growth: NOEC : 1.16 mg/L, LOEC > 1 .16 mg/L based on mean measured concentrations

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

No study submitted. The logP OW of spirotetramat is 2.51 and the potential of bioa ccumulation is expected to be low. Furthermore spirotetramat is not persistent in water or sediment of aquatic systems and theref ore a fish life-cycle test is not required.– 72 –

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

B.9.2.2.4 Chronic toxicity to aquatic invertebrates (OECD IIA 8.3.2, IIIA 10.2.6)

Active substance

Reference: Influence of BYI 08330 (tech.) on develo pment and reproductive output of the waterflea Daphnia magna in a static renewal laboratory test system Author(s), year: Dorgerloh, M., 2005 e Report/Doc. number: EBFN0245, Edition Number: M-251 843-01-2, 13.05.2005 Guideline(s): OECD 211, EEC Directive C.20, US EPA 72-4, OPPTS 850.1300 GLP: Yes Deviations: The concentraton of the solvent (aceton e) is slightly higher than recommended in OPPTS 850.1300 (0.2 ml/L instated of 0.1 ml/L). However an additional solvent control was added to make sure that the solvent was not initiating any toxic response, thus no negative impact on the results ca n be concluded. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.1 %, batch: 08045/0014 Material and methods: Test species: Waterflea ( Daphnia magna ) Number of organisms: 12 replicates each with one d aphnid per treatment and control Age: First instar < 24 hours old Type of test, duration: Semi static test, 21 d, re newals of test solutions: 0, 2, 5, 7, 9, 12, 14, 16 , 19 days Applied concentrations: Nominal: 0 (control and solvent control), 0.05, 0. 13, 0.32, 0.8, 2.0, 5.0, 12.5 mg/L Measured (mean): -- (control and solvent control), 0.055, 0.129, 0.322, 0.796, 1.84, 4.41, 10.9 mg/L Solvent 0.2 mL/L Aceton Test conditions: Water quality: M7 medium (according to “original draft” of an EEC Daphnia magna pilot ring test), hardness: 196 mg/L as CaCO 3 Temperature 20 – 20.9 °C pH 7.0 – 7.3 (new test solution) 7.4 – 8.1(aged te st solution) O 2 content: 6.3 – 10.8 mg O 2 /L (> 60 % saturation) Light regime: 16 hours light / 8 hours darkness Feeding Daily with Desmodesmus subspicatus suspension (1 x 10 8 cells/L), one exception was noted: On day 2 the three fold amount was fed f or the first weekend Test parameters: Parent mobility, reproduction (mea n time to first brood, age at first brood, offsprin g per surviving parental) and sublethal effects were observed daily (with exception of the first weekend after test initiation), at stu dy termination body length and parental body mass (dry weight) were reported. For chemical analysis (HPLC/UV) of spirotetramat in test media duplicate samples were taken from fresh (on days 0, 9, 19) and old so lutions (on days 2, 12, 21) from each test concentration. Statistics: In general data for parent mobility wer e acrisine transformed before further evaluation. Variance homogeneity and normal distrib ution were analysed by Bartletts test and Kolmogorroff-Smirnov test. If control groups can be pooled a t- test was perfo rmed. All NOEC were derived by comparing each treatment g roup with pooled controls: Parent mortality: Fishers Exact Binominal Test with Bonferroni Correction Reproduction: T-test Body length: Dunnett’s Test Parental body mass (dry weight): U-test after Bonfe rroni-Holm– 73 –

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

Statistical analyses concidering nominal concentrat ions are based on data of pooled controls. Statistical analyses concidering mean measured conc entrations are using only negative control. Findings: Analytical data: The mean measured concentrations r anged from 87.2 – 110.7 % Effects: See table B.9.2.2.4-1

Table B.9.2.2.4-1: Summary of effects of long-term exposure of spirotetramat on Daphnia magna

Spirotetramat [mg/L] (nominal) Spirotetramat

[mg/L] (mean measured) Parent

mortality at day 21 [%] Mean time to

first brood [days] Offspring

per surviving parental Mean dry

weight of parent after 21 d [mg] Mean length

of parent after 21d [mm]

Blank control Blank control 0 8.7 ± 1.85 117.3 ± 27.6 1.07 ± 0.14 4.50 ± 0.13 Solvent control Solvent control 0 8.7 ± 1.19 115.2 ± 16.4 0.98 ± 0.15 4.42 ± 0.17 Pooled control Pooled control 0 8.7 ± 1.52 116.3 ± 22 .1 1.02 ± 0.15 4.46 ± 0.15 0.05 0.055 0 9.9 ± 0.92 127.3 ± 16.6 1.09 ± 0.17 4. 55 ± 0.07 0.13 0.012 0 9.1 ± 1.10 127.4 ± 17.6 0.89 ± 0.27 4. 56 ± 0.15 0.32 0.322 0 8.7 ± 1.78 113.0 ± 25.5 0.90 ± 0.34 4. 55 ± 0.06 0.8 0.796 0 9.1 ± 1.11 118.7 ± 14.0 1.00 ± 0.12 4.5 8 ± 0.09
2.0 1.84 0 9.4 ± 1.09 122.4 ± 16.8 1.05 ± 0.12 4.61 ± 0.11 5.0 4.41 10 7.9 ± 3.40 115. ± 25.0 1.14 ± 0.32 4.61 ± 0.11 12.5 10.9 20 7.2 ± 2.00 93.5 ± 30.5* 0.81 ± 0.12* 4 .21 ± 0.13* NOEC (based on nominal) 2.0 mg/L 12.5 mg/L 5.0 mg/L 5.0 mg/L 5.0 mg/L LOEC (based on nominal) 5.0 mg/L > 12.5 mg/L 12.5 mg /L 12.5 mg/L 12.5 mg/L NOEC (based on mean measured) a) 10.9 mg/L 10.9 mg/L 4.41 mg/L 4.41 mg/L 4.41 mg/L LOEC (based on mean measured) a) > 10.9 mg/L > 10.9 mg/L 10.9 mg/L 10.9 mg/L 10.9 m g/L * Statistically significantly different from pooled control (alpha = 0.05) a) Statisics were performed using only negative contro l

Conclusion: Mortality adult: NOEC 2.0 mg/L, LOEC 5.0 mg/L Reproduction (offspring per surviving parental): NO EC: 5.0 mg/L, LOEC: 12.5 mg/L Growth (weight and length): NOEC: 5.0 mg/L, LOEC: 1 2.5 mg/L All endpoints are based on nominal concentrations.

B.9.2.2.5 Chronic toxicity to sediment dwelling org anisms (OECD IIA 8.5.2)

Reference: Chironomus riparius 28-day chronic toxicity test with BYI 08330 (tech. ) in a water-sediment system using spiked water Author(s), year: Dorgerloh, M., 2005 i Report/Doc. number: M-248099-02-2, 16.03.2005, Amen ded: 29.06.2006 Guideline(s): OECD 219 GLP: Yes Deviations: The concentration of the used solvent c ontrol was higher than recommended (0.2 mL/L instead of 0.1 mL/L), however no significant d ifferences between solvent control and the water only control were noted. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods:– 74 –

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

Test species: Midge ( Chironomus riparius ) Number of organisms: 4 replicates each with 20 lar vae per treatment and control Age: First instar (L1) larvae Type of test, duration: Static test (spiked water exposure), 28 d Applied concentrations: Nominal: 0 (control and solvent control), 0.05, 0. 10, 0.20, 0.40, 0.80, 1.60, 3.20 mg/L Initial measured: -- (control and solvent control) , 0.058, n.a., n.a., 0.401, n.a., n.a., 2.95 mg/L Solvent: 0.2 mL/L Aceton Test system: Water quality: M7 medium (according to OECD), the height of water column was 6.0 cm Sediment: 74 % quartz sand, 4 – 5 % sphagnum peat, 20 % Kaolin, 1 % CaCO 3 to adjust the pH to 7 ± 0.5; the wet sediment layer was 1.5 cm Size of test vessels: 0.6 L glass beakers (9.5 cm diameter) No. of replicates: 4 for biological evaluation and 1 for chemical analysis per each concentration and control Test condition: Temperature: 19.8 – 20.1 °C pH: 8.3 – 8.7 Hardness: 320 – 338 mg CaCO 3 /L O 2 content: 6.9 – 8.5 mg O 2 /L (> 60 % saturation) Light regime: 16 hours light / 8 hours darkness, i ntensity: 500 – 1000 lux Feeding: 3 x weekly with a commercial ornamental f ish food extract (0.5 – 1 mg/larvae/day) Test parameters: The sex, the time point of emergen ce and the number of emerged midges were recorded daily. Behavioural differences compared to control were observed 3 times per week. For chemical analysis (HPLC/UV) of spirotetramat in the overlaying water column and the pore water of sediment samples were taken f rom fresh after 1 hours, 7 and 28 days from 0.05, 0.4 and 3.2 mg/L treatment. Statistics: Sex ratio: Chi 2 -test EC 50 : Probit analysis LOEC: ANOVA and multiple t-tests Comparison of control groups: Student-t test for ho mogeneous variances All statistical analyses are based on data of poole d controls. Findings: Analytical data: See table B.9.2.2.5-1 Table B.9.2.2.5-1: Measured concentration of spirot etramat in the overlaying water and the pore water 1 hour 7 days 28 days Spirotetramat [mg a.s./L] Analysed

conc. [mg/L] a) % of nominal Analysed conc. [mg/L] a) % of nominal Analysed conc. [mg/L] a) % of nominal
Overlaying water Control < LOQ -- n.a. -- n.a. -- Solvent control < LOQ -- n.a. -- n.a. -- 0.05 0.0579 115.8 < LOQ 0 < LOQ 0 0.40 0.401 100.3 < LOQ 0 < LOQ 0 3.20 2.95 92.2 0.0529 1.7 < LOQ 0 Pore water b)
Control < LOQ -- n.a. -- n.a. -- Solvent control < LOQ -- n.a. -- n.a. -- 0.05 < LOQ 0 < LOQ 0 < LOQ 0 0.40 < LOQ 0 < LOQ 0 < LOQ 0 3.20 0.155 0.3 0.0639 0.1 < LOQ 0 LOQ <0.00636– 75 –

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

a) means of two samples b) calculated to the real volume of pore water and th e applied amount of a.s.

Effects: Sex ratio: No relationship between treatme nt and sex ratio was found, therefore number of males and females midges was pooled for f urther endpoint calculations. Start of emergence: In controls the emergence start ed on day 14; at concentration levels up to 0.2 mg/L first midges emerged on day 1 5; at 0.4 and 0.8 mg/l emergence started on day 17 and 18, respectively For further details see table B.9.2.2.5-2.

Table B.9.2.2.5-2: Effects of spirotetramat on midg e ( C. riparius ) in a water-spiked test Emergence of inserted larvae Spiroteramat [mg a.s./L] initial nominal concentration (overlying water) Number of

emerged midges (out of 80) total [%] male [%] female [%] Emergence

rate (ER) Development

rate (1/d) pooled sex

controls (pooled) a) 144 90.0 43.1 46.9 0.90 0.061 0.05 65 72.3 42.50 38.75 0.81 0.063 0.10 72 90.0 40.00 50.00 0.90 0.062 0.20 59 74.8 42.50 31.25 0.74* 0.062 0.40 54 67.5 36.25 31.25 0.68* 0.062 0.80 6 7.5 3.75 3.75 0.08* 0.061
1.60 0 0 - - - - 3.20 0 0 - - - - NOEC 0.1 mg/L 0.8 mg/L LOEC 0.2 mg/L 1.6 mg/L EC 50 (95% C.I.) 0.46 mg/L (n.d)** > 0.8 mg/L

ER = midge emerged per vessel/number of introduced larvae n.d. = not determined due to mathematical reasons a) Result of t-test indicated no significant differen ces between control and solvent control, thus poole d control are used for statistical analysis. * significant different from pooled control (alpha = 0.05) ** slope b (probit analysis): 4.429

Conclusions: Emergence of pooled sexes: NOEC: 0.1 mg/L, LOEC: 0.2 mg/L, EC 50 : 0.46 mg/L Development rate of pooled sexes, males and females : NOEC: 0.8 mg/L, LOEC: 1.6 mg/L, EC 50 : > 0.8 mg/L Endpoints based on initial nominal concentrations. Comments: While no major deviations are noted from guidline OECD 219 significant deviation from USEPA recommendations are notable: The EPA requires that the sediment be spiked with t he test compound and allowed to equilibrate with overlying water prior t o test initiation; the overlying water was spiked in this test and equilibrium was n ot achieved. EPA also requires that mean-measured, rather than i nitial measured values, be used to perform statistical analyses and report end points. EPA also recommends having endpoints calculated bas ed on measured concentrations in overlying water, bulk sediment, a nd interstitial water. EPA recommends a pH in the range of 6 to 7, the pH values measured in the test were outside this range.– 76 –

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

B.9.2.2.6 Microcosmos or mesocosmos study (OECD III A 10.2.3)

No study submitted. Not required, because the risk assessment for intended uses (see B.9.2.4) indicate d a low risk for aquatic organisms and no further refin ement is necessary.

B.9.2.2.7 Summary of chronic toxicity

Table B.9.2.2.7-1: Summary of chronic toxicity data for aquatic organisms; values which are relevant for risk assessment are in bold

Test organism Test condition Time Endpoint Test

conc. NOEC

[mg/L] LOEC

[mg//L] Reference/

study acceptable Spirotetramat (BYI 08330) Pimephales promelas Fathead minnow flowthrough 33 d hatchability fry survival growth mm 1.16 0.534 1.16 > 1.16

1.16 > 1.16 Dorgerloh 2005b/

yes

Daphnia magna Waterflea semi static 21 d mortality adults reproduction growth nom 2.0 5.0 5.0 5.0

12.5 12.5 Dorgerloh 2005e/

yes
Chironomus riparius Midge (larvae) Static (spiked water) 28 d emergence development nom 0.1 0.8 0.2

1.6 Dorgerloh 2005i/

yes

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

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

B.9.2.3.1 Bioconcentration potential of active subs tance in fish (OECD IIA 8.2.6.1)

No study submitted. Not required, because spirotetr amat has a logP OW 2.51 and a bioaccumulation study is not triggered.

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

Environmental relevant metabolites are BYI 08330-en ol, BYI 08330-ketohydroxy (both metabolites are maj or metabolites in water and sediment of water/sediment study), 4-methoxycyclohexanone and 4- methoxycyclohexylamino carboxyl acid (both are majo r metabolites in photolysis study). The logP OW of metabolites BYI 08330-enol and BYI-ketohydroxy is 0.3 (at pH 7) and 1.3 (at pH 7), respectively. For the two major photolysis metaboli tes information about the logP OW was not provided. However for these metabolites the logP OW was estimated by RMS using the program ALOGPS 2.1. The logP OW for 4-methoxycyclohexanone and 4-methoxycyclohexyl amino carboxyl acid was calculated to be in the range from -0.04 to 0.74 and -2.49 to 1.0, resp ectively.

B.9.2.4 Risk assessment for aquatic organisms (OECD IIIA 10.2, IIIA 10.2.1)

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.– 77 –

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

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".

B.9.3 Effects on other terrestrial vertebrates (OEC D IIIA 10.3)

Toxicity to mammals

Table B.9.3-1: Ecotoxicologically relevant mammalia n toxicity endpoints for spirotetramat

Species mg/kg bw/day Reference
Acute oral LD 50 Rat, female > 2000 Eigenberg (2004) Acute oral LD 50 formulation Rat, female > 2000* Schüngel (2005) Reproductive NOAEL Rat 70.7 / 82.5 (m / f) Young ( 2006) * equivalent to 306 g a.s.

B.9.3.1 Risk assessment for mammals

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".– 78 –

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

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

B.9.4.1 Acute Toxicity (OECD IIA 8.7.1; OECD IIA 8. 7.2; OECD III 10.4.2 )

Active Substance

Reference: Effects of BYI 08330 (Acute Contact and Oral) on Ho ney Bees ( Apis mellifera L.) in the Laboratory Author(s), year: Schmitzer, S.; 2004 Report/Doc. number: 20091035; M-081227-01-2 Guideline(s): OECD 213 & 214 (1998) GLP: Yes Deviations: ventral application in the contact test instead of dorsal as recommended in guideline Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) tech., Batch no.: 08045/ 0014 (mixed batch), Tox no.: 06695-00, purity: 97.2 % w/w Material and Methods: Test species: Apis mellifera L. (Hymenoptera: Apidae), 4 - 6 week old adult wor ker honey bees Type of test: Acute contact and oral limit toxicity test Number of organisms: 5 replicates with 10 bees for controls, toxic reference and the test item treatme nt
groups Dosage: Control: oral test: sugar solution with tap water and sugar solution with solvent acetone, contact test: tap water with 1 % Adhaesit and solvent acetone; test item: oral test: 107.3 μg a.s./bee, based on the quantity of food actually consumed; contact test: 100 μg a.s./bee (nominal); toxic refe rence: 0.04 - 0.3 μg dimethoate/bee (Dimethoate 400 g/L) Exposure route: Application in the contact test: a 5 μL droplet of a.s. in solvent and Adhaesit was placed on the ventral bee thorax. Application in th e oral test: Syrup was mixed with the test substance, tap water and acetone and offered to the bees in syringes which were weighted before and after introduction i n the cages. Feeding: Commercial ready-to-use syrup (30 % saccar ose, 31 % glucose, 39 % fructose) Test conditions: Test units were stainless steel ca ges of 10 cm x 8.5 cm x 5.5 cm, the front side was a removable glass sheet, the bottom was perfora ted with 98 ventilation holes (Ø 1 mm), the inner walls were lined with filter pa per. Bees were anaesthetised with CO 2 in the contact test. Exposure time: 48 h. Temperature: 25°C, relative hu midity: 58 - 72 %, darkness (except during observation), ventilation to avoid p ossible accumulation of pesticide
vapour Test parameter: Mortality and behavioural abnormali ties were assessed 4, 24 and 48 h after treatment application. Statistics: The contact and oral LD 50 of the toxic standard was estimated with Moving Average Computations (after Thompson 1947). The LD5 0 calculation was carried out taking into account the mortality data correcte d by control mortality using Abbott's formula (1925). The software used to perfo rm the statistical analysis was ToxRat Professional, Version 2.07. Findings:– 79 –

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

The results of the mortality and behavioural asses sment are presented in Table B.9.4.1-1 and -2. The oral and contact LD 50 of the toxic reference were 0.15 μg a.s./bee and 0.17 μg a.s./bee, respectively.

Table B.9.4.1-1: Mortality and behavioural abnormal ities of the bees in the contact toxicity test (results are average from 5 replicates per dosage/c ontrol)
after 4 hours after 24 hours after 48 hours mortality behav.

abnorm. mortality behav. abnorm. mortality behav. abnorm. Dosage [μg a.s./bee]

mean [%] mean [%] mean [%] mean [%] mean [%] mean [ %] Test item 100 0.0 0.0 0.0 0.0 2.0 0.0

Water 0.0 0.0 0.0 0.0 2.0 0.0 Solvent 0.0 0.0 0.0 0.0 2.0 0.0 Toxic standard 0.30 20.0 34.0 94.0 4.0 96.0 0.0

0.20 28.0 12.0 70.0 2.0 82.0 0.0 0.15 18.0 6.0 46.0 8.0 62.0 0.0 0.10 0.0 0.0 0.0 0.0 2.0 0.0

Table B.9.4.1-2: Mortality and behavioural abnormal ities of the bees in the oral toxicity test (result s are average from 5 replicates per dosage/control)
after 4 hours after 24 hours after 48 hours mortality behav.

abnorm. mortality behav. abnorm. mortality behav. abnorm. Dosage [μg a.s./bee]

mean [%] mean [%] mean [%] mean [%] mean [%] mean [ %] Test item 107.3 0.0 0.0 2.0 0.0 4.0 0.0

Water 0.0 0.0 0.0 2.0 2.0 0.0 Solvent 0.0 0.0 0.0 0.0 0.0 0.0 Toxic standard 0.24 28.0 24.0 94.0 0.0 94.0 0.0

0.12 0.0 0.0 22.0 0.0 26.0 0.0 0.07 0.0 0.0 14.0 0.0 22.0 0.0 0.04 0.0 0.0 2.0 0.0 2.0 0.0

Conclusion: 48 h oral LD 50 > 107.3 μg a.s./bee 48 h contact LD 50 > 100 μg a.s./bee Comments As deviations to EPA OPPTS guidelines the test sub stance (oral) was offered in syrup instead of a 50 % sugar solution a nd Adhäsit ® is not an EPA-recommended solvent.– 80 –

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

Formulation: Movento 150 OD

Reference: Acute toxicity of BYI 08330 150 OD to the honeybee Apis mellifera L. under laboratory conditions. Author(s), year: Barth, M.; 2005a+b Report/Doc. number: 05 10 48 032; M-259122-01-2 Guideline(s): OECD 213 & 214 (1998) GLP: Yes (certified laboratory) Deviations: None Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) OD 150, Batch no.: 08030 /0189(0152), TOX no.: 07034-00; Analytical content of a.s.: 148.89 g/L Material and Methods: Test species: Apis mellifera carnica P. (Hymenoptera: Apidae), 4 - 6 week old adult wor ker honey bees Type of test: Acute contact and oral toxicity test Number of organisms: 3 replicates with 10 bees for controls, toxic reference and the test item treatme nt
groups Dosage: Control: oral test: 50 % sucrose solution, contact test: water and 0.1 % v/v Tween solution; test item: oral test: applied volume - 20 μL sucrose solution/bee; contact test: applied volume - 4 μL/bee (0.1 % v/v Tween so lution); toxic reference: 0.0625 - 0.5 μg dimethoate/bee (Perfection EC 400) Application rates for contact and oral toxicity tes t (based on analysed content of a.s.): Contact toxicity Oral toxicity* Test item [μg product/bee] BYI 08330 [μg a.s./bee] Test item

[μg product/bee] BYI 08330

[μg a.s./bee]
1324.5 200.0 1317.0 198.9 662.2 100.0 661.3 99.9 331.1 50.0 330.7 49.9 165.6 25.0 165.6 25.0 82.8 12.5 82.7 12.5 *based on actual intake Exposure route: Application in the contact test: An aesthetised bees were treated individually by topical application with a micropipette to the dors al thorax. Application in the oral test: Bees were fed with a defined quantity of sucr ose solution containing the test item. Groups of 10 bees were provided with 200 μL o f the test solution in a glass ampoule. Feeding: 50 % aqueous sucrose solution; continuous feeding. Test conditions: Test units were disposable cages o f cardboard with holes in the bottom for ventilation and a glass plate in front for observat ion of the bees (dimensions inside: 80 mm x 45 mm x 65 mm). Exposure time: 48 h. Temperature: 25 - 26°C, relati ve humidity: 59 - 61 %, darkness (except during observation), ventilation Test parameter: The number of dead and affected bee s was counted at 4, 24 and 48 h. During assessments times any behavioural abnormalities of the bees were recorded, considering the following parameters compared to co ntrol: healthy or affected (paralysis, lateral position, lying on the back), a ny differences in activity, in position within the cage (all on the bottom), abnor mal amount and colour of– 81 –

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

excretion. Statistics: Mortality was corrected according to Ab bott (1925). For statistical calculation of the mortality results the Fisher’s Exact Binomial t est was used. The accepted significance level was p ≤ 0.05 (one-sided greater). The median lethal dose ( LD 50 ) along with the 95 % confidence limits was calculate d by Probit analysis according to the maximum likelihood method (FINNEY 1971) or a ccording to the linear weighed regression. The goodness-of-fit of the mode l was evaluated by Pearson’s X 2 test. The calculation of statistical significance and the LD 50 was performed with the computer program ToxRat Professional 2.09. Findings: The results of the mortality and behavioural asses sment are presented in Tables B.9.4.1-3 and -4. The validity criteria were met as mortality in the control was ≤ 10 % after 48 h and the contact and oral LD 50 of the toxic reference were 0.263 μg a.s./bee and 0.194 μg a.s./bee, respectively. In the oral toxicity test statistically significant effects on the survival were found at consumed doses of 330.7, 661.3 and 1317 μg product/ bee (23.3, 50.0 and 90.0 % mortality, respectively) during 48 h. No effects on behaviour were observed in bees consuming doses up to 330.7 μg product/bee. Af ter consuming doses of 661.3 and 1317 μg product/bee most bees were affect ed at the 4 and 24 h assessment. Bees had recovered at the 48 h assessme nt. In the contact toxicity test no effects on behaviou r were observed after exposure to doses up to 331.3 μg product per bee. Bees expos ed to doses equal or greater than 662.2 μg product/bee were affected at the 4 an d 24 h assessment. Bees had recovered at the 48 h assessment. Acute Oral Test: Results Based on EPA’s Probit Anal yses LD 50 = 607.18 ug/bee 95% Confidence Limits of LD 50 = 506.17, 737.88 Intercept = -10.21 Slope = 3.67; Standard error of slope=0.55 95 % Confidence Limits of Slope = 2.59, 4.74 Goodness-of-Fit Test: Pearson Chi-Square value= 1.95; Degrees of freedom= 3; p(Chi-Square)=0.58

Acute Contact Test: Results Based on EPA’s Probit A nalyses LD 50 = 1073 ug/bee 95 % Confidence Limits of LD 50 = 903.59, 1351 Intercept=-13.83 Slope = 4.56; Standard error of slope =0.89 95 % Confidence Limits of Slope = 2.82, 6.30 Goodness-of-Fit Test: Pearson Chi-Square value= 2.90; Degrees of freedom= 3; p(Chi-Square)=0.41– 82 –

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

Table B.9.4.1-3: Oral and contact toxicity LD 50 values of bees treated with BYI 08330 150 OD after 24 and 48 h contact toxicity test oral toxicity test treatment time μg product/bee μg a.s./bee μg product/bee μg a.s./b ee 24 h 95%-cl lower Upper Slope b 1151.0 933.8 1418.8 4.1 - 607.1 506.7 727.4 Slope b -

3.7 Test item

BYI 08330 150 OD

48 h 95%-cl lower Upper Slope b 1072.9 894.3 1287.3 4.6 - 607.1 506.7 727.4 Slope b -

3.7 24 h 95%-cl lower Upper Slope b - 0.263 0.225 0.308 4.7 - 0.194 0.171 0.220 7.2 Reference item

Dimethoate EC 400
48 h 95%-cl lower Upper Slope b - 0.243 0.209 0.283 5.0 - 0.189 0.166 0.214 6.7
cl: confidence limits

Table B.9.4.1-4: Mortality of bees in the contact t oxicity test (results are average from 3 replicates per dosage/control) dosage Test item [μg test item/bee] Mortality [%] after 4 h (= corrected mortality) Mortality [%] after 24 h (= corrected mortality) Mortality [%] after 48 h (= corrected mortality)

82.78 0.0 0.0 0.0 165.56 0.0 0.0 0.0 331.12 3.3 3.3 3.3 662.24 10.0 10.0 10.0 1324.48 0.0 63.3 70.0 Control (Tween) 0.0 0.0 0.0 Toxic standard [μg a.s. /bee]
0.0625 0.0 0.0 0.0 0.125 0.0 0.0 3.3
0.250 33.3 63.3 63.3 0.500 73.3 83.3 90.0 Control (Tween) 0.0 0.0 0.0– 83 –

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

Table B.9.4.1-5: Mortality of bees in the oral toxi city test (results are average from 3 replicates pe r dosage/control) dosage Test item [μg consumed test item/bee] Mortality [%] after 4 h (= corrected mortality) Mortality [%] after 24 h (= corrected mortality) Mortality [%] after 48 h (= corrected mortality)

82.66 0.0 0.0 0.0 165.56 0.0 0.0 0.0 330.65 0.0 23.3 23.3 661.30 50.0 50.0 50.0 1316.99 90.0 90.0 90.0 Control (sucrose solution) 0.0 0.0 0.0 Toxic standard [μg consumed a.s. /bee]
0.0625 0.0 0.0 0.0 0.125 0.0 3.3 6.7 0.249 53.3 90.0 90.0
0.495 96.7 96.7 96.7 Control (sucrose solution) 0.0 0.0 0.0

Conclusion: 48 h oral LD 50 = 607.1 product/bee equivalent to 91.7 μg a.s./bee 48 h contact LD 50 = 1072.9 μg product/bee equivalent to 162.0 μg a.s ./bee Comments: As deviation to EPA guidelines, Tween ® is not an EPA-recommended solvent.

B.9.4.2 Residue test (OECD IIA 8.7.3)

Not submitted.

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

Studies with similar design are reported under B. 1 .4.5.

B.9.4.4 Cage test (OECD IIIA 10.4.4)

Not required.

B.9.4.5 Field tests (OECD IIIA 10.4.5)

Formulation: Spirotetramat OD 100

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

L.) in a Feeding Test Author(s), year: Schur, A.; 2004a Report/Doc. number: 20031168/01-BFEU; M-000345-01-2 Guideline(s): OEPP/EPPO Bulletin No. 22 (Oomen et a l, 1992) GLP: Yes (certified laboratory) Deviations: None Validity: Acceptable– 84 –

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

Test substance: Spirotetramat OD 100 (a.s. BYI 08330), Batch no.: 08030/0038(0035), Tox no.: 06293-00, content: 101.54 g/L Material and Methods: Test species: Apis mellifera L. (Hymenoptera: Apidae) Type of test: Feeding test, field study Method aimed at products with insect growth-regulat ing properties Number of organisms: 3 replicates (= bee colonies, approx. 12000 bees per colony) for control, toxic reference and the test item treatment group Dosage: Control: Water/sucrose solution, test item: dissolved in water and mixed in pure sucrose solution at a concentration of 0.0144 % a.s . (analysed; 1371 mg product per colony); toxic reference: 300 mg Insegar 25 WG per colony (a.s.: 25 % fenoxycarb) (dissolved in water and mixed in sucros e solution). Experimental procedure: The study was located in Southern Germany (location : Niefern). Bees from the test colonies were free-flying, with access to natu ral nectar sources. No main sources of nectar were found in the near surroundin gs. All colonies were in good condition at start of the test. Colonies of honey b ees were fed with the test substance. Feeding: A quantity of 1 kg sugar solution was offe red per colony. Test conditions: Meteorological data (temperature, relative humidity, rainfall) were recorded. Test parameters, observations: Mortality of adult bees, pupae and larvae, behaviou r of the bees around the hives, condition of the colonies and the development of th e bee brood were assessed before and after the feeding of the test substance. Wooden bee traps (Münster design) with gauze on bot tom and on 100 % of the top were attached to the entrance of the colonies i n order to register those dead bees which were carried out of the hives. Evaluatio n of mortality took place over three days before start of feeding (once a day at t he 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 morn ing). The influence of the test substance was evaluated by comparing the results in the test substance treatment to the control data and those from the toxic standa rd treatment and furthermore by comparing the pre- and post-feeding results. Dead l arvae and pupae were examined for specific effects of IGR`s. Statistics: None in original study report. In 2007 statistics was performed using t-test and Dunnett`s t-test in an one-sided approach. Findings: The test was considered to be valid because the fo llowing parameters were met: A detectable effect of the toxic standard was found (brood termination before a successful hatch, mortality of pupae). After 7 days at the latest all colonies had taken up the whole test solution. A clear difference was found in the brood terminati on rate in cells between the colonies in the control and the colonies in the tox ic standard treatment.– 85 –

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

Table B.9.4.5-1: Mortality and flight activity of h oney bees in a feeding study with Spirotetramat OD 100

Treatment group Test substance (BYI 08330 OD 100) Control Toxic standard

Feeding rate per colony (in pure sugar solution) 0.0144% a.s. - 300 mg product

pre (DAF –3 to 0bf): rep. 1

6.8±4.1 rep. 2

6.3±4.6 rep. 3

7.3±5.2 rep. 1 7.5±2.5 rep. 2 4.3±4.0 rep. 3

9.5± 10.3 rep. 1 5.0±6.1 rep. 2

8.5± 14.4 rep. 3 8.0±7.0

average of 3 rep. 6.8 7.1 7.2

post ± SD (DAF 1 to 21) rep. 1

8.6±5.7 rep. 2

15±16.8 rep. 3

7.4±8.3 rep. 1 5.7±4.2 rep. 2 6.3±4.5 rep. 3 2.8±1.9 rep. 1

54.1±53 rep. 2

115.2± 129.9 rep. 3

104.2± 115.3
average of 3 rep. 10.4 ± 11.6 4.9 ± 4.0 91.2 ± 106.5 Average Mortality [ dead bees/ colony/day ]

QM (average) : 1.5 0.7 12.7

∑ dead adult bees and dead pupae (post-feeding, DAF 1 to 21) 597 + 55P 282 + 29P 321 + 5425P

0bf 46.67 28.67 38.00

DAF 1 57.00 35.00 57.00 Average Flight activity*

DAF 3 17.67 23.33 28.00
DAF days after start of feeding 0bf mortality on the day of start of feeding befor e feeding QM (average) ∅ post-feeding mortality ÷ ∅ pre-feeding mortality Pre average value for the pre-feeding period DAF –3 to 0bf post (DAF 1 to 21) average value for the post-feedi ng period DAF1 to 21 P = dead pupae SD = Standard deviation rep. = replicate hive * mean number of bees leaving/entering the hive per minute

Regarding the total daily mortality in the test su bstance treatment, a slightly increased mean mortality was noticed in the test su bstance group on DAF 6, 13 and 15. Throughout the entire post-feeding period a sum of 55 dead pupae were counted in the test substance treatment compared to 29 dead pupae in the control group. A sum of 5425 dead pupae were counted during the entire post-feeding period in the toxic standard which is evidence that Insegar 25 WG massively disturbed the brood development of these test colon ies. The sum of dead pupae and adults were analysed sepe rately over the postfeeding period (control vs treatment and control vs toxic standard). As result, no statistically significant effect on pupae mortality was detected, but there was a statistically significant difference in adult bee m ortality between control and treatment (t-test, pooled, one-sided, p=0.0447). Honey bee flight intensity: Regarding the flight intensity observed in front o f the hives no remarkable differences were observed at bo th post-feeding assessment dates DAF 1 and 3 between the control, the test sub stance and the toxic standard
treatment. Condition of the colonies: During the assessments of the condition of the col onies throughout the study no remarkable observations wer e made regarding the strength of the colonies and the brood nest size in the test substance treatment– 86 –

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compared to the control treatment. The colonies of the test substance treatment, control and toxic standard treatment showed all bro od stages at the assessment dates during the experimental phase of the study. Honey bee brood development: By comparing the individual brood assessments of single cells, the indices (the values of the dif ferent brood stages of all cells in each treatment group, assessed at the same date, su mmed up and divided by the number of observed cells) of the control group show ed the course to be expected in a natural bee development cycle. In hive 2 and 3 of the test substance treatment a termination of the bee brood in more th an three quarters of the cells observed was noticed from BFD (brood fixing day) to BFD 5. The cells which showed a terminated brood development were empty at the first assessment after start of feeding of the test substance. Even at the following assessments a massively disturbed brood development was observed with empty cells, cells refilled or refilled again with new brood. Hive 2 a nd 3 of the test substance treatment were not able to compensate the effect on the brood development caused by the test substance until the end of the o bservation period. No clear effect which could be attributed to the test substa nce was observed at hive 1. Honey bee behaviour in front of the colonies : No differences regarding the behaviour of the bees were noticed between the test substance treatment, the toxic standard treatment and the control. Conclusion: It was concluded that the feeding of Spirotetramat OD 100 had a slight but in the context not biologically significant effect on adul t and pupae mortality of the bees at the dosage tested and did not result in an adver se effect on the flight activity and the behaviour of the bees in front of the hives . Regarding the brood development a high termination rate was noticed 4 d ays after start of feeding in two of the three test substance colonies. In these colonies the further brood development was massively disturbed at the followin g assessments and they were not able to compensate the effect on the brood development until the end of the observation period. No effect was detected by t he regular brood assessments.

Formulation: Spirotetramat SC 240

Reference: Assessment of the Side-Effects of BYI 08330 SC 240 on the Honey Bee ( Apis mellifera

L.) in a Feeding Test. Author(s), year: Schur, A.; 2004b Report/Doc. number: 20031166/01-BFEU; M-121877-01-2 Guideline(s): OEPP/EPPO Bulletin No. 22 (Oomen et a l, 1992) GLP: Yes (certified laboratory) Deviations: None Validity: Acceptable

Test substance: Spirotetramat SC 240 (a.s. BYI 08330), Batch no.: 07218/0013, Tox no.: 06294- 00, content: 243.85 g/L (analysed); Adjuvant: RME E W 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-regulat ing properties Number of organisms: 3 replicates (= bee colonies, approx. 12000 bees per colony) for control, toxic reference and the test item treatment group– 87 –

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Dosage: Control: Water/sucrose solution, test item: dissolved in water and mixed in pure sucrose solution at a concentration of 0.0144 % a.s . plus 0.200 % of the adjuvant RME EW 500 (analysed; 637 mg product per colony); t oxic reference: 300 mg Insegar 25 WG per colony (a.s.: 25 % fenoxycarb) (d issolved in water and mixed in sucrose solution). Experimental procedure: The study was located in Southern Germany (location : Niefern). Bees from the test colonies were free-flying, with access to natu ral nectar sources. No main sources of nectar were found in the near surroundin gs. All colonies were in good condition at start of the test. Colonies of honey b ees were fed with the test substance. Feeding: A quantity of 1 kg sugar solution was offe red per colony. Test conditions: Meteorological data (temperature, relative humidity, rainfall) were recorded. Test parameters, observations: Mortality of adult bees, pupae and larvae, behaviou r of the bees around the hives, condition of the colonies and the development of th e bee brood were assessed before and after the feeding of the test substance. Wooden bee traps (Münster design) with gauze on bot tom and on 100 % of the top were attached to the entrance of the colonies i n order to register those dead bees which were carried out of the hives. Evaluatio n of mortality took place over three days before start of feeding (once a day at t he 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 morn ing). The influence of the test substance was evaluated by comparing the results in the test substance treatment to the control data and those from the toxic standa rd treatment and furthermore by comparing the pre- and post-feeding results. Dead l arvae and pupae were examined for specific effects of IGR`s. Statistics: None. Findings: The test was considered to be valid because the fo llowing parameters were met: A detectable effect of the toxic standard was found (brood termination before a successful hatch, mortality of pupae). After 7 days at the latest all colonies had taken up the whole test solution. A clear difference was found in the brood terminati on rate in cells between the colonies in the control and the colonies in the tox ic standard treatment.– 88 –

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Table B.9.4.5-2: Mortality and flight activity of h oney bees in the feeding study with Spirotetramat SC 240

Treatment group Test substance (BYI 08330 OD 100) Control Toxic standard

Feeding rate per colony (in pure sugar solution) 0.0144 % a.s. + 0.200 % adjuvant - 300 mg product

pre (DAF –3 to 0bf): 6.9 7.1 7.2

post ± SD (DAF 1 to 21) 15.8 ± 24.1 4.9 ± 4.0 91.2 ± 106.5 Average Mortality [ dead bees/ colony/day ]

QM (average) : 2.3 0.7 12.7

∑ dead adult bees and dead pupae (postfeeding, DAF 1 to 21) 988 + 10P 282 + 29P 321 + 5425P

0bf 36.33 28.67 38.00

DAF 1 45.00 35.00 57.00 Average Flight activity*

DAF 3 19.00 23.33 28.00 DAF days after start of feeding 0bf mortality on the day of start of feeding befor e feeding QM (average) ∅ post-feeding mortality ÷ ∅ pre-feeding mortality Pre average value for the pre-feeding period DAF –3 to 0bf post (DAF 1 to 21) average value for the post-feedi ng period DAF1 to 2 P = dead pupae SD = Standard deviation * mean number of bees leaving/entering the hive per minute

Regarding the total daily mortality in the test su bstance treatment an increased daily mean mortality was noticed on DAF 6 as well a s on DAF 15 to 17 with 23.3 to 71.0 dead bees/colony. Only 10 dead pupae were c ounted in the test substance treatment throughout the entire post-feed ing period compared to 29 dead pupae in the control group. A sum of 5425 dead pupae were counted during the entire post-feeding period, mainly between DAF 10 and 21, in the toxic standard which is evidence that Insegar 25WG massiv ely disturbed the brood development. Honey bee flight intensity : Regarding the flight intensity observed in front of the hives no remarkable differences were observed at bo th post-feeding assessment dates DAF 1 and 3. Condition of the colonies : During the assessments of the condition of the co lonies throughout the study no remarkable observations wer e made regarding the strength of the colonies and the brood nest size in the test substance treatment compared to the control treatment. The colonies of the test substance treatment, control and toxic standard treatment showed all bro od stages at the assessment dates during the experimental phase of the study. Honey bee brood development : By comparing the individual brood assessments of single cells, the indices (the values of the dif ferent brood stages of all cells in each treatment group, assessed at the same date, su mmed up and divided by the number of observed cells) of the control group show ed the course to be expected in a natural bee development cycle. In all colonies of the test substance treatment a termination of the brood in almost all cells was noticed from BFD (Brood area fixing day) to BFD 5. Hive 1 and 2 were able to com pensate this lack of brood and the main part of the empty cells was filled again w ith eggs and larvae at the– 89 –

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following assessment on BFD 11. In hive 3 of the BY I 08330 SC 240 treatment the situation of the marked brood area was similar on B FD 11 compared to the previous assessment because the cells remained empt y until this assessment day and even at BFD 15 the brood index was still below the level of 1 (egg stage). These observations indicate an effect of BYI 08330 SC 240 to the brood development in the first 5 to 15 days after start o f feeding. Nevertheless the brood indices continued to rise from BFD 11 to 22 in all test substance colonies and at the final brood assessment the big part of the cell s contained brood. Honey bee behaviour in front of the colonies : No differences regarding the behaviour of the bees were noticed between test sub stance treatment, toxic standard treatment and control. Regarding the fligh t intensity observed in front of the hives no remarkable differences were observed a t both post-feeding assessment dates DAF 1 and 3. Conclusion: It was concluded that the feeding of BYI 08330 SC 240 caused an increase of adult honey bee mortality 6 and 15 to 17 days after start of feeding but did not result in an adverse effect on the flight activity and the behaviour of the bees in front of the hives. An almost total termination of the brood development was noticed 4 days after start of feeding in all coloni es but the colonies showed the tendency to recover until the end of the observatio n period since most of the cells observed contained developing brood at the final br ood assessment and no effect was detected by the regular brood assessments.

Formulation: Movento 150 OD

Reference: Assessment of Side Effects of Spirotetramat OD 150 on the Honey Bee ( Apis mellifera L.) in the Field. Author(s), year: Schur, A.; 2006 Report/Doc. number: 20061133/S1-BFEU; M-277194-02-1 Guideline(s): OEPP/EPPO No. 170 (3) (2001) GLP: Yes (certified laboratory) Deviations: No Validity: Acceptable

Test substance: Spirotetramat OD 150 (a.s. BYI 08330), Batch no.: 08030/0189(0152), content of a.s.: 15.1 % (w/w) (analysed) Material and Methods: Test species: Apis mellifera mellifera (Hymenoptera: Apidae) Type of test: field study with special attention to bee brood development Number of organisms: Four normally developed, healt hy and queen-right colonies were used per test field. Each colony contained one body with 12 frame s and approx. 20000 bees / colony. Treatment: Three test fields: - test item treated field T1 (4 × 72 g a.s./ha, two times before flowering (no bees in the field) and 2 x during flowering of the crop and foraging activity of the bees) - test item field T2 (2 × 96 g a.s./ha during flowe ring of the crop and foraging activity of the bees) - untreated control field The applications were carried out in approximately 7-day intervals in 200 L water/ha. Calibrated commercial spraying equipment was used. Experimental The field test was located in Spain (region: Valenc ia). The crop Phacelia– 90 –

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procedure: tanacetifolia was used in flowering stage. The field sizes were: test item field T1: 2025 m 2 , test item field T2: 2016 m 2 and the control field C: 3306 m 2 . The test fields were not close to other flowering crops or e xtensive blooming weeds, which may be attractive to bees and were separated by a d istance of at least 2 km. Commercial bee colonies were placed near the test f ields 3 days before the 3 rd application in the test item field T1 and before th e 1 st application in the test item field T2, respectively. To ensure that the bees are exposed to the test field detailed assessments of foraging activity were done before as well as after the application. Test conditions: Temperature and rainfall data were recorded at the governmental weather station in Jalance, approximately 15 km (T1 field), 17 km ( T2 field) and 18 km (control field) away from the field site. The humidity data were recorded at the governmental weather station in Almansa, approximat ely 20 km (T1 and T2 field) and 25 km (control field) away from the field site. The degree of cloud coverage and wind speed during applications was assessed at the test fields. Test parameters, observations: Exposure of the bees was assessed by visual inspect ion of the P. tanacetifolia pollen on combs entered in the hives during the stu dy. The influence of the test item on the honey bees wa s evaluated by comparing the results of the test item treatments to those of the control treatment. The following points were assessed: • Condition of the colonies (strength) and developm ent of the bee brood • Mortality in the field and in the bee traps in fr ont of the hives • Foraging activity (number of forager bees/m2/minu te flowering crop) • Behaviour of the bees on the crop and around the hive Flight intensity and foraging activity were assesse d in 5 marked squares (1 m 2 ) that were regularly distributed. Mortality and fora ging activity of the bees were checked prior to first applications in the test fie lds (over 2 days and shortly before first application) and followed up after applicatio n (foraging activity: over 10 days after first application during exposure, mortality: over 28 days after first application during exposure). In order to record the number of dead bees water-permeable linen sheets were spread out in front of the hives. Dead bee traps were attached to the entrance of the hives in order to register t hose dead bees which were carried out of the hives. Furthermore, mortality wa s recorded at three impartially selected places in each test field using linen shee ts. Dead bees were differentiated in adult worker bees, males, freshly emerged bees, pupae and larvae during each assessment. The condition of the colonies and the bee brood dev elopment were checked once before first application during exposure and five t imes afterwards (up to 26 days after first application during exposure). Collection of flowers: For the possible analysis/determination of residue s of the test item in flower clusters of P. tanacetifolia samples were taken during the trial. The residue analysis was not part of the present st udy. Statistics: None. Findings: Data on mortality and flight activity are presente d in the table below.– 91 –

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Table B.9.4.5-3: Mortality and flight activity of h oney bees in the feeding study with Movento 150 OD

Treatment group T1 T2 Control

1 st application date (01May06, before flowering): appl ication rate g a.s./ha 72.0 - -

2 nd application date (08May06, before flowering): appl ication rate g a.s./ha 72.0 - -

3 rd application date (17May06, during flowering/bee fl ight): application rate g a.s./ha 72.0 96.0 -

4 th application date (24May06, during flowering/bee fl ight): application rate g a.s./ha 72.0 96.0 -

Pre-appl. [DAA -2 to 0ba]: 43.4 135.4 87.7 Pre-appl. [DAA 0ba]: 42.0 94.8 65.0 Post-appl. [DAA 0aa]: 15.0 23.0 29.0 Pre-appl. [DAA 7ba]: 22.0 17.8 2.8 Post-appl. [DAA 7aa]: 3.8 6.8 2.3 Post-appl. [DAA 0aa to 28]: 5.7 6.5 5.6 Mean mortality [ dead bees/colony/day ]

Q M : 0.1 <0.1 0.1 Mean number dead pupae/larvae per colony/day (post-application, DAA 0aa to 28) 0.3 0.1 <0.1

Pre-appl. [DAA -2 to 0ba]: 11.3 11.5 13.7 Pre-appl. [DAA 0ba]: 15.0 13.0 12.4 Post-appl. [DAA 0aa]: 11.9 14.4 14.4 Pre-appl. [DAA 7ba]: 12.0 15.0 1.0 Post-appl. [DAA 7aa]: 14.6 23.0 11.1 Mean flight intensity [ foraging bees/minute/m 2 ]

Post-appl. [DAA 0aa to 10]: 11.9 16.3 12.2 ba = before application in the test item fields dur ing the exposure period aa = after application in the test item fields duri ng the exposure period DAA = days after first applications in the test item fields during the exposure period QM = post-application [DAA 0aa to 28] / pre-applicati on [DAA –2 to 0ba]

To insure that the bees were exposed to the test fi eld and the test item detailed assessments of forag ing activity were done before as well as after the appl ication during the exposure period. The daily mean foraging activity after the applications (DAA 0aa t o DAA 10) was 11.9 foraging bees/minute/m 2 in the test item group T1, 16.3 foraging bees/minute/m 2 in the test item group T2 compared to 12.2 foragin g bees/minute/m 2 in the control group. The degree of P. tanacetifolia pollen from the total amount of pollen per colony ranged from 10 % up to 33 % during time of e xposure (brood assessments on 19May06 and 25May06) at the test field in the test item group T 1, from 8 % up to 40 % at the test field T2 and fro m 6 % up to 85 % in the control field. The results of the po llen assessments in the colonies confirm that the b ees were actively foraging on the test fields. A quantitativ e comparison between the results of the treatments is not possible, because the foraging and storage of polle n in a bee colony depends on outside conditions as well as on the individual need of pollen in the bee colo ny. Honey bee mortality The number of dead bees on the day before as well a s shortly before the first application during expos ure in the test fields was increased in single colonies of the test item treatment group T2 as well as in the control group. Since in both of the test fields in which th is slightly increased mortality has been observed, no preexposure application was carried out, the temporari ly increased mortality in those colonies was clearl y not caused by the test item. However, on the assessment s after first application during exposure (day 0 af ter application) the mean mortality in all treatment gr oups was on a low level and no test item related in crease in the number of dead bees was observed (T1: 15.0 dead bees/colony, T2: 23.0, control: 29.0). On the seco nd application date during exposure (7 days after the first one) the mortality in all treatment groups wa s on a similar low level at the assessments after applicat ions. No test item related difference in the number of dead bees of the treatment groups T1 and T2 compared to the mortality in the control group was observed at any– 92 –

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time after the applications. This resulted in a dai ly mean post-application mortality (day 0 after app lication to day 28) in the test item treatment group T1 of 5.7 dead honey bees/colony, 6.5 dead honey bees/colony in the test group T2 and 5.6 dead honey bees/colony in the control group. The value for QM (mean mortality post-application d uring exposure divided by the mean mortality preapplication during exposure) was calculated as 0.1 in the test item treatment group T1, <0.1 in the te st item group T2 and 0.1 in the control group. The mean number of dead pupae and larvae from day 0 to 28 after first application during exposure was on a low level in all treatments with 0.3 dead pupae a nd larvae/colony/day in the treatment group T1, 0.1 dead pupae and larvae/colony/day in the treatment group T2 and <0.1 dead pupae and larvae/colony/day in the control group. Since larval and pupae mortality was at a comparable level in all treatment groups, the re were no treatment-related effects. Honey bee flight intensity: The daily mean flight intensity (foraging bees/minu te/m 2 ) before the first application during exposure was 15.0 in the test item treatment group T1, 13.0 in t he test item treatment group T2 and 12.4 in the con trol group. The mean flight intensity after the applicat ions on DAA 0 was 11.9 foraging bees/minute/m 2 in the treatment group T1, 14.4 foraging bees/minute/m 2 in the treatment group T2 as well as in the contro l group. On the second application date (DAA 7) no reduction in the mean flight intensity was noticed after applications in the test item group T1 and T2. The daily mean post-application (DAA 0aa to DAA 10) fli ght intensity was 11.9 foraging bees/minute/m 2 in the test item group T1, 16.3 foraging bees/minu te/m 2 in the test item group T2 compared to 12.2 foraging bees/m inute/m 2 in the control group. The intense flight and foraging activity of the bee s on the test fields was also supported by the fact that the bees stored actively P. tanacetifolia pollen in the combs of the colonies of all treatme nt groups. Condition of the colonies and honey bee brood devel opment Assessments of the colony strength as judged by num ber of bee ways between combs filled with bees and the brood nest size (number of brood combs per colo ny) did not indicate significant differences betwee n treatment groups T1, T2 and the control colonies. O n the frequent assessments during exposure in the t est fields and afterwards (up to 29 days after the firs t brood assessment) 3 colonies of the test item tre atment T1 and all colonies in the test item group T2 and in t he control group showed all brood stages and a simi lar development. One colony in the test item treatment T1 showed a lack of eggs or/and larvae on the last 2 assessment dates. In that colony a tendency to swar m or to remove the old queen was recorded (queen cells on combs) starting from the first brood asses sment after set-up of the colonies at the test fiel ds. It is very likely that this was caused by a lack of space for the colony to grow, or that the colony tried t o breed a new queen because they were not satisfied with the old queen. Since the other 3 colonies of the treatm ent group T1 were in good condition over the entire tes t period it is very unlikely that the lack of brood was caused by the application of the test item. Honey bee behaviour in front of the colonies and wi thin the crop area No differences regarding the behaviour of the bees were observed between the test item treatment group s T1 and T2 and the control group. Conclusion: The test item treatments (T1 and T2) did not resul t in an adverse effect on honey bees as determined by mortality and flight intensit y. Differences of bee behaviour between control and treatment groups were not obser ved. The condition of the colonies as assessed by colony strength and size of the brood nest was not affected by any of the treatments. From the detaile d assessment of the honey bee brood status one colony in the test item treatment group T1 showed a lack of brood on the last assessments. In that colony a ten dency to swarm or to remove the old queen was recorded (queen cells on combs) s tarting from the first brood assessment after set-up of the colonies at the test fields. It is very likely that this was caused by a lack of space for the colony to gro w, or that the colony tried to breed a new queen because they were not satisfied w ith the old queen. No evidence of an irritation or termination of the dev elopment based upon exposure to treated crops was obtained in the other colonies of the test group T1 as well as– 93 –

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in the test item group T2 and the control colonies.

B.9.4.6 Tunnel tests (OECD IIIA 10.4.7)

Formulation: Spirotetramat OD 100

Reference: Assessment of side effects of BYI 08330 OD 100 on t he honey bee ( Apis mellifera L.) in the semi-field. Author(s), year: Schur, A.; 2005 Report/Doc. number: 20041144/01-BZEU; M-244490-01-2 Guideline(s): EPPO No. 170 (3) and Bulletin of Inse ctology 56 (1), 91 – 96; 2003: Honey bee brood ring-test in 2002: method for the assessment of side effects of plant protection products on the honey bee brood under se mi-field conditions. GLP: Yes (certified laboratory) Deviations: No Validity: Acceptable

Test substance: Spirotetramat OD 100 (a.s. BYI 08330), Batch no.: 08030/0110(0073) Tox no.: 06593-00, content: 102.02 g/L (analysed) Material and Methods: Test species: Apis mellifera L. (Hymenoptera: Apidae) Type of test: Semi-field test (tunnel) with special attention to the development of the bee brood Number of organisms: Small healthy colonies ["Mini- Plus-Beuten"; 1 queen and approximately 1 kg bees (6000 - 8000 bees per colony)] with 12 combs were u sed. 3 replicates (= bee colonies) for control, toxic reference and the test item treatment group. Treatments: Control: water, test item: treatment gr oup T1: Test item applied 3 x on the crop before flowering at 72 g a.s. (692.33 g product)/ha and 2 x after set-up of the colonies at 96 g a.s. (923.11 g product)/ha. Treatm ent group T2: test item applied once, 3 d after set-up of the colonies at a rate of 96 g a.s./ha; toxic reference: Insegar WG 25 (a.s.: fenoxycarb, 25 %) 150 g a.s. p er ha. All treatments were applied in 400 L water/ha using a plot sprayer. Experimental procedure: The semi-field test was located in the south of Ger many at Niefern near Pforzheim. The crop used was oil-seed spring rape (Brassica napus) . Before full flowering and start of bee-exposure tunnel tents we re set-up in the test field. The area covered with rape was 47.52 m 2 per tunnel. The tunnels had a size of 5.0 x 12 m and a height of about 3.5 m. The tunnel frames were covered with light plastic gauze (mesh size: 1.5 mm). Before the start of bee-exposure in the tunnels a path was created along the tunnel walls at both f ront sides and through the middle of each tunnel by removing the plants and le velling the ground. Subsequently, the path was covered with linen sheet s. For the test, small healthy colonies with 12 combs were used. All nuclei were p roduced at the same time. The corresponding queens originated from one breedi ng line in order to guarantee uniform bee material in all treatment groups. Each bee hive consisted of two bodies with one bottom and one lid (height: 40 mm). The outer dimensions of this hive were 300 mm x 300 mm, the inner dimensions are 230 mm x 230 mm and the height is 170 mm. Each body contained six frame s (130 mm x 200 mm). The control and toxic standard applications were pe rformed on the same day as the forth test item application in treatment group T1 (main application day). There– 94 –

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were three tunnels (one hive per tunnel) per treatm ent. Feeding: No supplementary feeding. Test conditions: Meteorological data (temperature, relative humidity, rainfall) were recorded. Test parameters, observations: Mortality, flight intensity, condition of the colon ies and the development of the bee brood were assessed before and after the main appli cation day. Particular attention was directed to the brood development of the colonies. The influence of the test item was evaluated by com paring the results in the tunnel tents of the test item treatments to the control da ta and those of the toxic standard treatment. The following points were assessed: • Condition of the colonies (strength) and developm ent of the bee brood • Mortality at the edge of the treated area and in the bee traps • Foraging activity (number of forager bees/m 2 flowering rape) • Behaviour of the bees on the crop and around the hive Wooden bee traps with gauze on bottom and on the to p were attached to the entrance of the hives in order to register those de ad bees which were carried out of the hives. Furthermore, the mortality was record ed in the crop. Therefore the oilseed spring rape (Brassica napus) was removed prior to the set-up of the hives and water-permeable linen sheets (covered area appr oximately 15 m 2 ) were spread out in the array. The mortality of adult hon ey bees found on the linen sheets and in the bee trap at the entrance of the c olonies was assessed. After an exposure period of 10 days the bee colonies were ta ken out of the tunnels and transferred to an area without flowering main crops and where no pesticides were applied during the time of assessments. Mortality o f adult honey bees in the bee trap at the entrance of the colonies was assessed f or another 13 days [until day +23 after BFD (brood area fixing day)]. The condition of all colonies was checked two days before the main application day and five t imes afterwards (4, 9, 16, 23 and 29 days after BFD). Assessment of the development of the bee brood took place at the BFD (egg), 4 days (young to old larvae ), 9 days (capped cells), 16 days (capped cells shortly before hatch) and 23 day s after BFD (empty cells or cells containing eggs) using the acetate sheet meth od. After an exposure period of 10 d, the bee colonies were taken out of the tun nels and transferred to an area without flowering main crops and pesticide applicat ion. Mortality at the entrance of the colonies was assessed for another 13 d (BFD +23 ). Statistics: None in original study report. In 2007 statistics was performed using t-test and Dunnett`s t-test in an one-sided approach.. Findings: The test was considered to be valid because a dete ctable effect of the toxic standard was found (brood termination before a succ essful hatch, increased mortality of pupae). Results are presented in the t able below.– 95 –

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Table B.9.4.6-1: Mortality and flight activity of h oney bees in the feeding study with Spirotetramat OD 100 Exposure T1: five spray treatments of BYI 08330 OD 1 00 three before and two during foraging activity in flowering oils eed spring rape T2: one spray treatment of BYI 08330 OD 100 during foraging activity in flowering oil-seed spring rap e Treatment group Test substance (BYI 08330 OD 100) Control (water) Toxic standard

(Insegar 25 WG) T1 T2 C R Single application rate [in 400 L water/ha] 3 × 72 g a.s./ha 2 × 96 g a.s./ha 96 g a.s./ha - 150 g a.s.

pre 13.8 12.9 7.9 11.2

post (BFD +2aa) 5.0 2.7 2.7 1.7

post (BFD +2aa to +23) 5.5 4.5 4.1 8.1 Mean Mortality [ dead bees/ replicate/day ]

Q M(average) : 0.4 0.3 0.5 0.7

∑ dead pupae (post-application, BFD +2aa to +23) 12 29 8 300

pre 10.3 8.3 8.4 10.7
post (BFD +2aa) 19.0 12.2 15.0 15.3 Daily mean flight intensity [foraging bees/m 2 /replicate] post (BFD +2aa to +9) 14.5 14.8 14.1 13.8

aa after application pre average values for BFD to BFD +2ba (before appl ication) post (BFD +2aa) main application day after applica tion post (BFD +2aa to +9) mean value for BFD +2aa to BFD +9 post (BFD +2aa to +23) mean value for BFD +2aa to BF D +23 QM(average) mean mortality per day after applicati on divided by mean mortality per day before appl. BFD brood area fixing day

Regarding the total daily adult bee mortality obser ved during the exposure of the bees to the test ite m treated plants (T1 and T2) in the tunnels and in th e time afterwards no remarkable observations were m ade compared to the control or toxic standard treatment . The mean post-application adult bee mortality was comparable in all treatment groups (T1: 5.5 dead be es/replicate/day, T2: 4.5, control: 4.1, toxic stan dard: 8.1). In the toxic standard, all three colonies sho wed an increased number of dead pupae in approximat ely the same period of time from BFD +12 to BFD +19 whi ch is a typical effect of Insegar WG 25. The total number of dead pupae per colony from BFD +2aa to BF D +23 was 12 dead pupae in the test item treatment T1, 29 dead pupae in treatment T2, 8 dead pupae in the control treatment compared to 300 dead pupae in the toxic standard treatment. The sum of dead pupae and adults were analysed sepa rately over the post-treatment period (control vs treatment and control vs toxic standard). As result , no statistically significant effect on pupae mort ality nor adult bee mortality between control and treatment ( Dunnett`s test, test) was found, whereas pupae mort ality was significantly higher in the toxic standard grou p. Honey bee flight intensity: The application(s) of the test item and the toxic s tandard had no effects on flight intensity compared to the control treatment. The daily mean post-application flight intensity was on a similar level in all trea tment groups (see table above). Condition of the colonies: During the assessments of the condition of the colo nies throughout the study no remarkable observation s were made regarding the strength of the colonies an d the brood nest size in the test item treatment gr oups– 96 –

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

T1 and T2 compared to the control treatment. The co lonies of the test item treatment groups and contro l showed all brood stages at the assessment dates dur ing the experimental phase of the study. In one col ony of the toxic standard no eggs were observed and que en cells were noticed at the last brood assessment which is the evidence that the queen of this colony died. Honey bee brood development: By comparing the individual brood assessments of si ngle cells, the indices (the values of the differen t brood stages of all cells in each treatment group, assess ed at the same date, summed up and divided by the number of observed cells) showed the course to be e xpected in a natural bee development cycle in two colonies of the test item treatment group T1, in th e colonies of treatment group T2, in two colonies o f the control group and in one colony of the toxic standa rd. In one control colony and in one colony of T1 a termination of brood in single cells was observed d uring the observation period which resulted in a re tarded increase of the brood index from BFD to BFD +9 comp ared to the other test hives of the same treatment (for details see table B.1.4.1.1.-4). In the colonies of T2 about 25 % of the marked cells showed a termina tion of the bee brood during the test. However, since one c ontrol colony showed a high termination rate betwee n BFD and BFD +4 and the other two colonies of the co ntrol showed a termination of about 20 % during the test, the effects noticed in the test item treatmen ts can not be clearly ascribed to the application o f the test item. One colony of the toxic standard treatment wa s obviously affected by the treatment with Insegar WG 25 as can be seen in a decreasing index between the fi rst assessment and BFD +4, as well as in a very low index on the following assessment on BFD +9.
Table B.9.4.6-2: Summary of the brood indices calcu lated based on individual brood assessments of single cells in the test treatment groups T1, T2, t he control and toxic standard treatment

Mean brood indices at x d after BFD Treatment Replicate 0 +4 +9 +16 +23

1T1 1.00 1.85 3.57 3.70 2.23

2T1 1.00 2.03 3.87 4.00 2.07

3T1 1.00 1.37 2.56 3.21 3.42 T1

Mean 1.00 1.75 3.33 3.64 2.57

1T2 1.00 1.67 3.16 3.78 2.40

2T2 1.00 1.85 3.17 3.41 1.96

3T2 1.00 1.73 3.24 3.52 2.01 T2

Mean 1.00 1.75 3.19 3.57 2.12

1C 1.00 1.81 3.60 3.98 2.96

2C 1.00 1.72 3.29 3.90 3.26

3C 1.00 1.21 2.20 3.79 1.80 Control

Mean 1.00 1.58 3.03 3.89 2.67

1R 1.00 1.96 3.36 3.71 1.45

2R 1.00 1.76 2.86 3.37 1.96

3R 1.00 0.29 0.41 2.13 3.81 Toxic
standard

Mean 1.00 1.34 2.21 3.07 2.41 (categories of brood indices: 0 - termination of de velopment, 1 - egg stage, 2 - young larvae, 3 - old larvae, 4 - pupae stage (capped cell), 5 - empty after hatch)

Honey bee behaviour in front of the colonies and wi thin the crop area: No differences regarding the behaviour of the bees were noticed between the test item treatment groups T1 and T2, the toxic standard treatment and the contro l.– 97 –

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

Conclusion: It is concluded that the application of BYI 08330 OD 100 applied five times (treatment group T1) and once (treatment group T2) on a bee-attractive flowering crop did not result in an adverse effect on the mor tality of adult honey bees and brood, flight intensity of the bees on the crop and the behaviour of the bees in the crop area or in front of the hive. An irritation of the brood development at the earlier assessments was noticed in one colony of th e test item treatment group T1, and about one quarter of the marked cells showe d a termination of bee brood in the colonies of treatment group T2. However, sin ce one control colony showed a high termination rate between BFD and BFD +4 and the other two colonies of the control showed a termination of about 20 % duri ng the test, the effects noticed in the test item treatments can not be ascribed to the application of the test item. The colonies were in good condition during the obse rvation period of 29 days.

Additional non-GLP studies on the effects of spirot etramat on the bee brood:

Introduction: For research and product positioning purposes Bayer CropScience AG performed orientating honey bee semi-field (tunnel) studies with Spirotetramat. The objectives of these studies were to investigate po tential side effects of different formulations and rates of spirotetramat, and different pre-application inter vals, to honey bees in flowering crops under tunnel conditio ns. Nine honey bee tunnel studies were performed betwee n 2002 and 2007, seven in Germany, two in Spain. For all studies Phacelia tanacetifolia was used as flowering crop with the exception of o ne study performed in 2007 on strawberry. All studies were carried out fo llowing the guidance of the EPPO 170 guideline unde r non-GLP conditions but according to GEP (good exper imental practice). The general test performance was as follows: Honeyb ee colonies of an appropriate size (approx. 3000 - 3500 bees) were confined in tunnels (44 m 2 and 60 m 2 in Spain and 50 m2 (100 m 2 for the strawberry trial, due to lower density of the flowering crop) in Germany) wi th a flowering crop on a field. For the reported st udies one or two replicates were set up for each treatment group . Each study comprised one untreated control, one t oxic reference (fenoxycarb) and one to six different tes t item treatment groups. Honey bees were introduced into the tunnels some days prior to application to get famil iar with the new environmental conditions (unless p reflowering applications were tested). The bee coloni es were examined for potential test item related ef fects for at least 7 days after the application inside the tunne ls and for at least additional 14 days after remova l from the tunnels. In particular, the endpoints adult, larval and pupal mortality, flight intensity and brood de velopment were assessed, furthermore honey and pollen storage , egg laying and breeding activity, colony strength , hive weight and behavioural anomalies of the honey bees were recorded. The obtained results for each endpoi nt and treatment group from the post-application perio d were compared to those of the pre-application pha se. 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 resul ts of the endpoints related to bee brood developmen t with regard to the larval development and mortality of p upae (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 wei ght) were not impacted and therefore were not repor ted in the notifier`s summary, which is focussing on the p otential of brood effects. Due to the fact that only one or two replicates per treatment group were used in the reported studies, a statistical evaluation was not feasible. Hence, the determination of effects was performed on the basi s of expert judgment.– 98 –

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

Trials in Germany or Spain with different formulati ons of Spirotetramat and partly with addition of RME 500 EW (rape oil methyl ester):

Trial 1 : Spirotetramat 240 SC as spray application, 300 L/h a, additive RME 500 EW (rape oil methyl ester), 2 replicates, Germany Results :

Table B.9.4.6-3: Evaluation of Larval Cell Comb Are a Proportion for Trial 1

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

0 DAA* 3 DAA 7 DAA 15 DAA 21 DAA
untreated (control) 18.3 9.2 6.7 30.8 14.2 36 g a.i./ha + RME 500 EW, 0.2 % a.i./v 17.5 2.5 5.0 17.5 12.5

72 g a.i./ha + RME 500 EW, 0.2 % a.i./v 21.7 1.7 5.0 20.8 20.0

144 g a.i./ha + RME 500 EW, 0.2 % a.i./v 17.5 1.7 6.7 7.5 16.7

144 g a.i./ha 23.3 5.8 3.3 20.8 17.5 Insegar 25 WP, 100 g a.i./ha (toxic reference) 18.3 0.8 0.0 18.3 10.0

DAA: days after application * assessment on 0 DAA was done shortly before appli cation Effect

Unclear effect

Conclusion: The application of three different rates of Spirote tramat SC 240 in combination with the additive RME 500 EW (0.2% a.i./v) to flowering Phacelia caused transient effects on honey bee larval abund ance. The application of Spirotetramat at a rate of 144 g a.i ./ha without the additive RME 500 EW caused a less pronounced reduction of larval comb area proportion . The reduction of larval comb area proportion was visible 3 days after application. Seven days after application no more differences compared with the c ontrol were observed. The toxic standard showed a clear ef fect on brood development also 7 DAA. All other bro od and non-brood endpoints in Spirotetramat treated gr oups were not impacted and the survival of the colo nies was not endangered.

Trial 2 : Spirotetramat 240 SC as spray application, 300 L/h a, additive RME 500 EW (rape oil methyl ester), 2 replicates, Germany Results :– 99 –

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

Table B.9.4.6-4: Evaluation of Larval Cell Comb Are a Proportion for Trial 2

Treatment Proportion of comb area covered by larval cells ± SD [%] (at X days after application)

0 DAA* 3 DAA 7 DAA 15 DAA 21 DAA
untreated (control) 6.7 7.5 3.3 2.5 1.7 36 g a.i./ha + RME 500 EW, 0.2% a.i./v 10.0 2.7 0.0 0.2 0.0

72 g a.i./ha + RME 500 EW, 0.2% a.i./v 3.3 0.8 2.5 3.7 3.3

144 g a.i./ha + RME 500 EW 0.2% a.i./v 6.0 3.3 4.2 3.3 1.7

144 g a.i./ha 9.2 7.5 7.5 5.8 5.8 Insegar 25 WP, 100 g a.i./ha (toxic reference) 5.8 1.0 0.0 0.3 0.0

* assessment on 0 DAA was done shortly before appli cation

Conclusion: Spirotetramat SC 240 in combination with 0.2 % a.i. /v RME 500 EW caused transient effects on honey bee larvae when applied to flowering Phacelia at 36 and 72 g a.i./ha. The reduction of larval co mb 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 applicatio n rate of 36 g a.i./ha the effect seemed to last until 7 t o 21 days after application. After the application of Spirotetramat SC 240 at a rate of 144 g a.i./ha wit h the additive RME 500 EW an unclear effect was observed 7 days after application, and the applicat ion at the same rate without the additive RME 500 E W caused no reduction of larval cell comb area propor tion. The survival of the colonies was not endanger ed by the effects observed. Due to unfavourable weather conditions and therefor e disadvantageous conditions for appropriate foragi ng, the breeding activity was low and thus the total br ood area of the bee hives during the monitoring per iod until the end of the study was quite low, as well, with a n overall decreasing tendency in all colonies. For spirotetramat SC 240 in combination with 0.2 % a.i./v RME 500 EW at 36 g a.i./ha and 144 g a.i./ha , a slight increase in dead pupae was found in front of the bee hives from 4 days after application to 7 d ays after application. The effects were less pronounced compa red to the toxic standard. All other (brood and non

brood related) endpoints were not impacted.

Trial 3 : Spirotetramat 240 SC or 100 OD as spray applicatio n, 300 L/ha, additive RME 500 EW (rape oil methyl ester), 1 replicate, Germany– 100 –

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

Results : Table B.9.4.6-5: Evaluation of Larval Cell Comb Are a Proportion for Trial 3a

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

0 DAA* 4 DAA 7 DAA 14 DAA 22 DAA 28 DAA

untreated (control) 10.0 6.7 5.0 9.2 8.3 4.2

240 SC, 144 g a.i./ha + RME 500 EW 0.4% a.i./v 8.3 1.0 0.0 0.8 4.2 3.3

240 SC, 144 g a.i./ha + RME 500 EW 0.2% a.i./v 11.7 2.5 2.5 4.2 6.7 4.2

RME 500 EW, 0.4% a.i./v 9.2 11.7 4.2 4.2 10.0 4.2

100 OD, 140 g a.i./ha 10.8 2.5 0.5 2.5 5.8 4.2

Insegar 25 WP, 100 g a.i./ha (toxic reference) 10.8 0.8 0.0 2.5 6.7 2.5

Table B.9.4.6-5 (continued): Evaluation of Larval C ell Comb Area Proportion for Trial 3b

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

0 DAA* 4 DAA 7 DAA 15 DAA 22 DAA 30 DAA

untreated (control) 5.0 7.5 6.7 6.7 4.2 2.5

240 SC, 144 g a.i./ha + RME 500 EW 0.4% a.i./v 7.5 0.0 2.5 2.5 2.5 1.7

240 SC, 144 g a.i./ha + RME 500 EW 0.2% a.i./v 5.8 0.0 0.0 1.7 0.8 2.5

RME 500 EW, 0.4% a.i./v 6.7 3.3 5.0 5.8 3.3 2.5

100 OD, 140 g a.i./ha 7.5 0.0 5.0 5.0 4.2 1.7

Insegar 25 WP, 100 g a.i./ha (toxic reference) 9.2 0.0 0.0 2.8 2.5 0.8

* assessment on 0 DAA was done shortly before appli cation

Conclusion: The application of 144 g a.i./ha Spirotetramat SC 2 40 as a tank mixture with 0.4 % a.i./v RME 500 EW t o flowering Phacelia

caused transient effects on honey bee larvae. The reduction of larval comb area proportion was visible from 4 days until 14 days af ter application in the first trial, whereas in the second trial these transient effects were pronounced 4 days afte r application, but unclear at the following assessm ent days. For the second tank mixture of 144 g a.i./ha Spirotetramat SC 240 plus 0.2 % a.i./v RME 500 EW, transient effects on honey bee larvae were observed in one trial up to 22 DAA, more pronounced from 4 days until 7 days after application; in the first t rial effects were unclear. Spirotetramat OD 100 app lied at 140 g a.i./ha to flowering Phacelia led to a short and transient depression in larval abundance in both trials. No effect on the larval comb area was observed when 0. 4 % a.i./v RME 500 EW was applied alone. In all Spirotetramat treatment groups the survival of all colonies was not endangered. In the second trial a slight increase in dead pupae found in front of the bee hive 15 days after appli cation was found for 144 g a.i./ha Spirotetramat SC 240 as a t ank mixture with 0.4 % a.i./v RME 500 EW. The effec ts were less pronounced compared to the toxic referenc e Insegar. A full recovery was recorded at 22 days after application at the latest. All other brood and non- brood related endpoints were not impacted.– 101 –

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

Trial 4 : Spirotetramat 240 SC or OD 100 as spray applicatio n, 300 L/ha, additive RME 500 EW (rape oil methyl ester), 1 replicate, Spain Results: Table B.9.4.6-6: Evaluation of Larval Cell Comb Are a Proportion for Trial 4

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

-1 DAA 2 DAA 7 DAA 14 DAA 21 DAA

untreated (control) 3.1 1.0 1.8 5.0 5.6

240 SC, 144 g a.i./ha + RME 500 EW, 0.1% a.i./v 5.0 5.0 1.9 3.4 6.3

100 OD, 140 g a.i./ha 5.0 2.1 0.5 1.9 9.4

100 OD, 100 g a.i./ha 8.1 2.8 1.4 6.1 7.5

Insegar 25 WP, 100 g a.i./ha (toxic reference) 3.8 6.3 0.9 2.1 7.5

Conclusion: The evaluation of potential effects of the test ite ms on the larval cell comb area proportions is prob lematic in this case and results cannot be unambiguously inter preted, since the larval abundance in the control w as very low during the exposure phase in the tunnel an d the toxic reference did not cause a distinct effe ct on the larvae. In the two Spirotetramat OD 100 treatme nt groups, the figures for 7 days after application may indicate a slight reduction of larval cells, howeve r, this cannot be diagnosed with certainty.

Trial 5 : Spirotetramat OD 150 as spray application, 300 - 4 00 L/ha, no additive, 1 replicate, Germany Results : Table B.9.4.6-7: Evaluation of Larval Cell Comb Are a Proportion for Trial 5a

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

0 DAA* 3 DAA 8 DAA 15 DAA 21 DAA 29 DAA
Untreated (control) 16.7 6.7 9.2 23.3 12.5 6.7 50 g a.i./ha 19.2 0.0 0.0 11.7 15.0 5.0 75 g a.i./ha 18.3 0.0 0.0 11.7 15.0 5.8 100 g a.i./ha 19.2 0.0 0.0 7.5 16.7 5.0 150 g a.i./ha 20.8 0.0 0.0 13.3 20.8 5.0 Insegar, 150 g a.i./ha (toxic reference) 18.3 0.0 0.0 10.0 13.3 5.0

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

-1 DAA 3 DAA 7 DAA 14 DAA 20 DAA 28 DAA
untreated (control) 8.3 19.2 10.8 10.0 15.8 6.7 50 g a.i./ha 10.0 2.5 0.0 1.7 -** -** 75 g a.i./ha 12.5 5.8 0.0 5.8 16.7 10.0 100 g a.i./ha 13.3 7.5 0.0 11.7 23.3 7.5– 102 –

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

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

0 DAA* 3 DAA 8 DAA 15 DAA 21 DAA 29 DAA 150 g a.i./ha 10.0 7.5 0.0 5.8 20.0 13.3 Insegar 25 WP, 150 g a.i./ha (toxic reference) 14.2 0.0 0.0 5.0 20.0 5.8

* : assessment on 0 DAA was done shortly before app lication -**: no egg-laying activity after loss of the queen (not treatment related)

Assessments of pupal mortality were performed on -3 , -1, 0, 1, 2, 3, 6, 7, 8, 9, 13, 14, 17, 20, 23 an d 28 DAA. Only assessment dates during which increased p upal mortality was recorded, are listed in the foll owing table.

Table B.9.4.6-8: Pupal Mortality Evaluation for Tri al 5b Treatment Number of dead pupae in bee trap (at X da ys after application)

4 DAA* 7 DAA 10 DAA 14 DAA 15 DAA
Untreated (control) 0 0 0 0 0 50 g a.i./ha 1 12 34 0 1 75 g a.i./ha 0 15 35 2 0 100 g a.i./ha 6 48 47 2 0 150 g a.i./ha 5 43 33 1 1 Insegar 25 WP, 150 g a.i./ha (toxic reference) 2 16 12 27 46

Treatment Number of dead pupae in bee trap (at X da ys after application)

3

DAA* 6

DAA 7

DAA 8

DAA 9

DAA 13

DAA 14

DAA 17

DAA 20

DAA
Untreated (control) 1 0 0 0 1 0 0 0 0 50 g a.i./ha 0 12 13 18 14 2 1 16 0 75 g a.i./ha 0 19 15 15 18 4 2 0 0 100 g a.i./ha 2 25 20 19 20 3 2 0 0 150 g a.i./ha 1 21 25 21 21 3 1 0 0 Insegar 25 WP,150 g a.i./ha (toxic reference) 1 27 21 22 14 0 8 8 2

*: first observed dead pupae in bee trap DAA: days after application

Conclusion: In both trials the application of Spirotetramat OD 150 to flowering Phacelia at four different rates (50 g, 75 g, 100 g and 150 g a.i./ha) caused transient effects t o honey bee larvae. The reduction of larval comb ar ea proportion was visible from 3 days after applicatio n to 8 to 15 days after application. The depression was transient and full recovery of the bee colony and b rood status was recorded at 20 days after applicati on at the latest. There was an increase in numbers of dead pupae foun d in front of the bee hive from 7 days and 6 days a fter application (trial 1, 2) which lasted until 10 and 9 days after application, respectively. A normal pu pal mortality level was recorded from 14 days after app lication on. All other brood and non-brood related endpoints were not impacted and the survival of all colonies was not endangered.– 103 –

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

Trial 6 : Spirotetramat OD 150 as spray application, 300 L/h a, no additive, 1 replicate, Germany Results : Table B.9.4.6-9: Evaluation of Larval Cell Comb Are a Proportion for Trial 6

Treatment Proportion of comb area covered by larval cells [%] (at X days after beginning of exposure)

0 4 DAE 7 DAE 15 DAE 22 DAE 28 DAE
untreated (control) 13.3 21.7 16.7 8.3 19.2 13.3 150 g a.i./ha 20 days prior to flowering 10.8 15.0 10.0 5.0 19.2 23.3

150 g a.i./ha 15 days prior to flowering 10.0 11.7 10.0 10.0 18.3 17.5

150 g a.i./ha 10 days prior to flowering 16.7 14.2 7.5 8.3 15.8 19.2

150 g a.i./ha 5 days prior to flowering 12.5 0.8 1.7 3.5 12.5 12.5

150 g a.i./ha on flowering crop at bee flight 16.7 2.5 5.0 4.2 7.5 11.7

150 g a.i./ha on flowering crop after daily bee flight 11.7 7.5 3.3 5.8 16.7 12.5

Insegar 25 WP, 150 g a.i./ha on flowering crop at bee flight (toxic reference) 12.5 1.7 0.0 0.0 16.7 7.5

DAE: days after beginning of exposure

Conclusion: The application of 150 g a.i./ha of Spirotetramat O D 150 five days prior to flowering (start of exposu re) and on the flowering crop during and after bee flight c aused transient reductions of larval cell comb area proportion. The effects were different regarding th e time of application. When applied 5 days before flowering the effects we re obvious on the fourth and seventh days of exposu re and less clear on the 15 th day. The application of 150 g Spirotetramat/ha to flowering crop after bee flight caused effects on the fourth and the seventh day af ter beginning of exposure. When Spirotetramat OD 15 0 was applied at the same rate during bee flight to t he flowering crop, a clear reduction of the larval comb area was observed on the fourth and seventh day and less clear effects on the 15 th and 22 nd day. All other brood and non-brood related endpoints were not impacted. Spray intervals of 10, 15 and 20 days before flower ing of Phacelia did not cause effects to the honey bee brood or any other endpoint.

Trial 7 : Spirotetramat OD 150 as spray application, 300 L/h a, no additive, 1 replicate, Germany Results : Table B.9.4.6-10: Evaluation of Larval Cell Comb Ar ea Proportion for Trial 7

Treatment Proportion of comb area covered by larval cells [%] (at X days after beginning of exposure)

0 DAE 3 DAE 7 DAE 15 DAE 21 DAE 28 DAE

untreated (control) 20.0 10.8 16.7 21.7 11.7 6.7

100 g a.i./ha 20 days prior to flowering 20.8 12.5 4.2 9.2 8.3 7.5

100 g a.i./ha 15 days prior to flowering 25.0 10.0 5.0 20.0 15.0 11.7

100 g a.i./ha 32.5 10.0 10.0 10.8 14.2 15.0– 104 –

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

Treatment Proportion of comb area covered by larval cells [%] (at X days after beginning of exposure)

0 DAE 3 DAE 7 DAE 15 DAE 21 DAE 28 DAE 10 days prior to flowering 100 g a.i./ha 5 days prior to flowering 16.7 0.8 4.2 16.7 13.3 8.3

100 g a.i./ha on flowering crop at bee flight 27.5 2.5 0.0 3.3 5.0 4.2

100 g a.i./ha on flowering crop after daily bee flight 22.5 14.2 3.3 10.0 15.8 8.3

Insegar 25 WP, 150 g a.i./ha on flowering crop at bee flight (toxic reference) 17.5 0.8 0.0 10.8 9.2 8.3

OD: oil dispersion DAE: days after beginning of exposure

Conclusion: Application of Spirotetramat OD 150 at 100 g a.s./h a 5 days prior to flowering, and application to the flowering crop after daily bee flight caused transi ent effects to honey bee brood in terms of reductio n of larval abundance at 3 and 7 days, or at 7 days after start of exposure, respectively. Spirotetramat OD 150 ap plied at the same rate to the flowering crop during bee f light showed likewise transient effects to honey be e brood starting at three days and up to 15 days after star t of exposure. A full recovery was observed 21 days after start of exposure in this treatment group. Spray in tervals of 10 days before flowering of Phacelia did not cause effects to the honey bee brood. After applica tion 15 and 20 days before flowering, abundance of larvae 7 days after start of exposure was distinctl y 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 - respo nse relationship (no effects after application 10 days before flowering) and under consideration of the in trinsic variation of this endpoint, it is difficult to unam biguously assess whether these observation have to be considered to be treatment effects, in particular s ince the study was not replicated. Therefore, the f igures here are classified as “unclear effects”. All other brood or non-brood related endpoints were not impa cted.

Trial 8 : Spirotetramat OD 150 as spray application, 300 L/h a, 1 replicate, Spain Results : Table B.9.4.6-11: Evaluation of Larval Cell Comb Ar ea Proportion for Trial 8

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

-3 DAA 7 DAA 14 DAA 21 DAA 31 DAA
untreated (control) 9.3 5.6 11.4 10.6 6.0 75 g a.i./ha 6.3 0.0 0.0 2.4 1.3 100 g a.i./ha 0.0 0.0 15.0 10.5 6.3 150 g a.i./ha 0.2 0.0* -* -* -* Insegar 25 WP, 150 g a.i./ha (toxic reference) 5.0 0.0 0.0 -* -*

DAA: days after application

*: dead bee hive

Conclusion: The application of Spirotetramat OD 150 at a rate o f 75 g a.i./ha to flowering Phacelia caused transient effects on honey bee larvae. No larval comb area wa s recorded on 7 and 14 days after application. 21 a nd– 105 –

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

31 days after application the area with larval cell s was still rather low, but due to the lack of repl ication it is unclear whether this has to be attributed to natura l variability. No evaluation of potential effects of the test item on the larval comb area proportions can be given f or the application rates of 100 and 150 g a.i./ha. At the colony of the lower rate no larvae were found short ly before and 7 days after application. This might be explain ed by a re-queening of the colony since at 14 days after application larvae in sufficient proportions were r ecorded. The colony of the highest application rate had nearly no larval cells shortly before the applicati on and died thereafter, which is uncommon for a bee trial and might be explained by the presence of a disease (A treatment effect is not to be assumed here, since t he affected colony did lack any larval stages from the beginning of the study). Presumably, the same happ ened for the colony of the toxic reference, which died 2 1 days after application. Due to the above mentioned observations, the result s of this trial have to be interpreted with care. Additionally, the overall brood development was wea k in all hives (see e.g. bee brood evaluation at -3 days after application). Therefore and due to lack of re plication, it is difficult to attribute changes in larval abundances as seen in this study, to potential trea tment effects or rather to natural variation, respe ctively.

Trial 9 : strawberry; Spirotetramat OD 150 as spray applicat ion, 400 L/ha, 1 replicate, Germany Results : Table B.9.4.6-12: Evaluation of Larval Cell Comb Ar ea Proportion for Trial 9

Treatment Proportion of comb area covered by larval cells [%] (at X days after application)

-1 DAA 4 DAA 9 DAA 16 DAA 21 DAA 30 DAA

untreated 16.7 20.0 9.2 11.2 21.7 11.7 100 g a.i./ha 9.6 14.6 4.2 7.1 22.9 13.3 Insegar 25 WP, 150 g a.i./ha 11.7 4.2 1.4 5.2 12.0 8.8

Conclusion: After application of Spirotetramat OD 150 at a rate of 100 g a.i./ha to flowering strawberries under t unnel conditions, a transient decline of honey bee larval abundance was observed. However, since there was a lso a significant decrease of larval abundance seen in the control group at the same assessment date, it i s doubtful whether this finding is indicating a treat ment-related effect. The survivals of the colonies were not endangered at any time. There was a slight increase in dead pupae found in front of the test item bee hives at 9 days after application. But the mortalities we re less pronounced compared to the toxic reference Insegar (1 dead pupa in the control, 11 dead pupae in the t est item and 14 in the toxic reference). All other brood and non-brood related endpoints were not impacted.– 106 –

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

B.9.4.7 Summary of effects on honey-bees

Table B.9.4.7-1: Summary of acute effects of Spirot etramat (technical and formulated) on honey-bees
Test substance Exposure route Endpoint Value (μg a.s./bee) Reference
Spirotetramat techn. Oral Contact 48 h LD 50

48 h LD 50 > 107.3 > 100 Schmitzer, 2004a
Spirotetramat OD150 Oral Contact 48 h LD 50

48 h LD 50 91.7 162 Barth, 2005a

Table B.9.4.7-2: Summary of semi-field and field da ta of Spirotetramat effects on honey-bees Test substance Exposure route Observed effects Refer ence Spirotetramat OD100 Feeding test, mixed in sucrose solution at 0.0144 %

a.s. Slightly increased mortality of adults and pupae during 21 d after start of feeding No effect on flight acivity, bee behaviour. Brood development: High termination rate in two of three colonies. Schur, 2004a

Spirotetramat SC240 Feeding test, mixed in sucrose solution at 0.0144 %

a.s. Increased adult mortality. Brood development: termination of brood development with tendency to recover. No effects on flight activity and behaviour of bees. Schur, 2004b

Spirotetramat OD100 Semi-field study, 2 appl. on crop before flowering at 72 g a.s./ha + 2 x 72 g a.s./ha after flow. or 1 x 96g a.s./ha no adverse effects on adult and brood mortality, condition of the colonies, flight activity and behaviour of bees. Schur, 2006

Spirotetramat OD100 Semi-field study (tunnel design), 3 appl. on crop before flowering at 72 g a.s./ha + 2 x 96 g a.s./ha after flow. or 1 x 96g a.s./ha no adverse effects on adult and brood mortality, condition of the colonies, flight activity and behaviour of bees. Irritation of brood development in the earlier assessments in one of 3 colonies of the 5x treatment, no clear treatment relation, recovery. Schur, 2005

Table B.9.4.7-3: Summary of effects to honey bee br ood (larvae) from additional non-GLP studies of Spirotetramat

Trial Country Treatment Formulation Type and content Crop Effect Unclear effect Recovery

until

Spirotetramat 36 g a.i./ha RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA

Spirotetramat 72 g a.i./ha RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA

Spirotetramat 144 g a.i./ha RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 1 Germany/2

002

Spirotetramat 144 g a.i./ha SC 240 g a.i./L Phacelia

- 3 DAA 7 DAA
Spirotetramat 36 g a.i./ha RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 15 DAA -

21 DAA -
Spirotetramat 72 g a.i./ha RME 500 EW 0.2% a.i./v 3 DAA - 7 DAA 2 Germany/

2002

Spirotetramat 144 g a.i./ha

RME 500 EW 0.2% a.i./v SC 240 g a.i./L Phacelia

- 3 DAA 7 DAA– 107 –

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

Trial Country Treatment Formulation Type and content Crop Effect Unclear effect Recovery

until

Spirotetramat 144 g a.i./ha - - - Spirotetramat 144 g a.i./ha RME 500 EW 0.4% a.i./v 4 DAA - 14 DAA - 22 DAA

Spirotetramat 144 g a.i./ha RME 500 EW 0.2% a.i./v - 4 DAA - 7 DAA 14 DAA

RME 500 EW 0.4% a.i./v SC 240 g a.i./L

- - - 3a Germany/

2002

Spirotetramat 140 g a.i./ha OD 100 g a.i./L Phacelia

7 DAA 4 DAA/ 14 DAA 22 DAA

Spirotetramat 144 g a.i./ha RME 500 EW 0.4% a.i./v 4 DAA 7 DAA - 30 DAA -
Spirotetramat 144 g a.i./ha RME 500 EW 0.2% a.i./v 4 DAA - 7 DAA 15 DAA -

22 DAA 30 DAA

RME 500 EW 0.4% a.i./v SC 240 g a.i./L

- - - 3b Germany/

2003

Spirotetramat 140 g a.i./ha OD 100 g a.i./L Phacelia

4 DAA - 7 DAA

Spirotetramat 144 g a.i./ha RME 500 EW 0.1% a.i./v SC 240 g a.i./L - 7 DAA 14 DAA

Spirotetramat 140 g a.i./ha - 7 DAA 14 DAA 4 Spain/ 2003

Spirotetramat 100 g a.i./ha OD 100 g a.i./L Phacelia

- - - Spirotetramat 50 g a.i./ha 3 DAA -
8 DAA 15 DAA 21 DAA

Spirotetramat 75 g a.i./ha 3 DAA -
8 DAA 15 DAA 21 DAA

Spirotetramat 100 g a.i./ha 3 DAA -
15 DAA - 21 DAA 5a Germany/

2004

Spirotetramat 150 g a.i./ha OD 150 g a.i./L Phacelia

3 DAA - 8 DAA 15 DAA 21 DAA

Spirotetramat 50 g a.i./ha 3 DAA -
14 DAA - 1

- 1

Spirotetramat 75 g a.i./ha 3 DAA -
7 DAA 14 DAA 20 DAA

Spirotetramat 100 g a.i./ha 3 DAA -
7 DAA - 14 DAA 5b Germany/

2004

Spirotetramat 150 g a.i./ha OD 150 g a.i./L Phacelia

3 DAA -
7 DAA 14 DAA 20 DAA

Spirotetramat 150 g a.i./ha 20 days prior to flowering - - -
Spirotetramat 150 g a.i./ha 15 days prior to flowering - - -
Spirotetramat 150 g a.i./ha 10 days prior to flowering - - -
Spirotetramat 150 g a.i./ha 5 days prior to flowering 4 DAE -
7 DAE 15 DAE 22 DAE

Spirotetramat 150 g a.i./ha on flowering crop at bee flight 4 DAE - 7 DAE 15 DAE -

22 DAE 28 DAE 6 Germany/

2004

Spirotetramat 150 g a.i./ha

on flowering crop after bee

flight OD

150 g a.i./L Phacelia

4 DAE - 7 DAE - 15 DAE

7 Germany/

2004 Spirotetramat 150 g a.i./ha

20 days prior to flowering OD 150 g a.i./L Phacelia - 7 DAE 15 DAE– 108 –

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

Trial Country Treatment Formulation Type and content Crop Effect Unclear effect Recovery

until

Spirotetramat 150 g a.i./ha 15 days prior to flowering - 7 DAE 15 DAE

Spirotetramat 150 g a.i./ha 10 days prior to flowering - - -
Spirotetramat 150 g a.i./ha 5 days prior to flowering 3 DAE - 7 DAE - 15 DAE

Spirotetramat 150 g a.i./ha on flowering crop at bee flight 3 DAE -
15 DAE - 21 DAE

Spirotetramat 150 g a.i./ha on flowering crop after bee

flight 7 DAE - 15 DAE

Spirotetramat 75 g a.i./ha 7 DAA -
14 DAA 21 DAA -

31 DAA -

Spirotetramat 100 g a.i./ha

- - - 8 Spain/ 2004

Spirotetramat 150 g a.i./ha OD 150 g a.i./L Phacelia

- 2

- 2

- 2

9 Germany/

2007 Spirotetramat 100 g a.i./ha OD 150 g a.i./L Strawberry - 9 DAA 16 DAA

RME: rape oil methyl ester DAA: days after application DAE: days after beginning of exposure

-: no influence on bee brood observed

1 No egg laying activity after loss of the queen (n ot treatment related)

2 bee in hive died presumably not due to treatment related effects

B.9.4.8 Risk assessment for honey bees

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".– 109 –

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

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

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

10.5.1)

Reference: Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera: Braconidae) in the laboratory; BYI 08 330150 OD Author(s), year: Waltersdorfer, A.; 2005a Report/Doc. number: CW05/023; M-254442-01-1 Guideline(s): Mead-Briggs et al. 2000 (IOBC) GLP: Yes (certified laboratory) Deviations: No reproduction assessment. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030 /0189(0152), Tox no.: TOX07034-00, analytical content: 148.89 g a.s./L Material and Methods: Test species: Adult wasps Aphidius rhopalosiphi (Hymenoptera: Braconidae), < 48 h old Type of test: Acute contact laboratory test Number of organisms: 4 replicates with 15 wasps for each treatment group Treatments: Control: deionised water; toxic standar d: 0.1 g a.s./ha (37 % Dimethoate, EC); test item: 2, 9, 24, 70 and 200 g a.s./ha; deionised wat er as diluent; treatments applied with a calibrated sprayer in 200 L water/ha Exposure route, duration: Adult wasps were exposed to dried residues on treat ed glass plates for 48 h. The test units consisted of two treated glass plates (1 00 x 100 x 3 mm) and an untreated acryl frame (inner size 92 x 92 x 14 mm) with 3 ventilation holes on each side. These holes were covered with gauze (mes h size 80 μm) which was jammed in the orifices by pieces of Teflon tubing ( 8 mm outer diameter). Feeding: Honey/water solution Test conditions: Temperature: 19 - 22°C; relative h umidity: 62 - 83 %, 16 h light, 570 – 1001 lux Test parameter: Observations of mortality were perf ormed approximately 24 and 48 h after start of exposure. The number of alive, affected, moribund a nd dead wasps was recorded. No reproduction assessment. Statistics: Mortality corrected according to Abbott . Fisher`s Exact test, two-sided. Findings: Results are summarised in table 9.5.1-1.

Table B.9.5.1-1: Toxicity of dried residues of Spir otetramat 150 OD on glass plates to Aphidius rhopalosiphi

Treatment Mean mortality at 48 h [%] Mean corrected mortality at 48 h [%]
Control 8.3 - 2 g ai/ha 5 -3.6 9 g ai/ha 1.7 -7.3 24 g ai/ha 8.3 0 70 g ai/ha 21.7 14.5 200 g ai/ha 86.7 85.5* Toxic standard 100 100 * statistically significant

Conclusion: 48 h LR 50 = 114.676 g a.s./ha; 95 % Confidence Interval: (86 – 145), based on the– 110 –

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

nominal content of the active ingredient

Reference: Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, Phytoseiidae) in the laboratory; BYI 08330150 OD Author(s), year: Waltersdorfer A., 2005b Report/Doc. number: CW05/024; M-254269-01-1 Guideline(s): Blümel et al. 2000 GLP: Yes (certified laboratory) Deviations: Short deviation in relative humidity. N o reproductive assessment. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030 /0189(0152), Tox no.: TOX07034-00, analytical content: 148.89 g a.s./L Material and Methods: Test species: Typhlodromus pyri protonymphs Type of test: Acute contact laboratory test Number of organisms: 4 replicates with 20 individua ls for each treatment group Treatments: Control: deionised water; toxic standar d: 4 g a.s./ha (37 % Dimethoate); test item: 0.19, 0.43, 0.97, 2.21 and 5.00 g a.s./ha. The test item was sprayed in 200 L/ha on the glasses (after calibration). Exposure route, duration: One exposure unit consisted of two cover slides (gl ass) which were put together at the longer sides with a narrow gap between the s lides. Cover slides were placed on tissue paper which again was placed on to p of a wet foam rubber. To avoid the escape of the mites a barrier of sticky m aterial was added. This test arena was transferred to a plastic tray filled with deionised water. The narrow gap between the two cover slides was filled with water by capillary forces to provide the mites with water. The test units were prepared one day before application to make sure that the foam rubber was saturated with w ater. Protonymphs were exposed to dried residues for 14 d

. Feeding: Pollen (birch + pine) Test conditions: Temperature: 24 - 25.5°C; relative humidity: 63 - 71 % (very short deviation to 35°C due to opening of the door), 16 h photo period , 795 – 1060 lux Test parameter: The number of dead and living mites was counted on days 1, 3, and 7. The number of escaped mites was calculated as the diffe rence from the total number exposed. On day 7 the number of females, males, egg s and juveniles was counted. Statistics: Mortality corrected according to Abbott . Fisher`s Exact test, two-sided. Findings: Results are summarised in table 9.5.1-2. The resul ts can be considered as valid as the mortality/escaping rate in the control chamb ers up to day 7 after treatment was 3.0%.– 111 –

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

Table B.9.5.1-2: Toxicity of dried residues of Spir otetramat 150 OD on glass plates to Typhlodromus pyri Mortality [%] 7 DAA Treatment g a.s./ ha Uncorr. Abbott P-Value(*) Control 0 3.0 0 - Test item 0.19 13.0 10.3 0.016 sign. Test item 0.43 72.0 71.1 <0.0001 sign. Test item 0.97 100 100 <0.0001 sign. Test item 2.21 100 100 <0.0001 sign. Test item (DAA 1) 5 100 100 <0.0001 sign. Reference item 4 88.0 87.6 -
n.d. not determined

Conclusion: 48 h LR 50 = 0.333 g a.s./ha; 95 % Confidence Interval: (0.29 8 – 0.367)– 112 –

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

Extended laboratory studies

Reference: Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera: Braconidae) using an extended laborat ory test; BYI 08330 150 OD. Author(s), year: Waltersdorfer A., 2006a Report/Doc. number: CW05/071; M-264179-01-1 Guideline(s): Mead-Briggs et al. 2000; Mead-Briggs et al. 2002 GLP: Yes (certified laboratory) Deviations: Increase in temperature due to technica l reasons Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030 /0189, Tox no.: TOX07034- 00; (0152), analytical content: 148.89 g a.s./L Material and Methods: Test species: Adult wasps Aphidius rhopalosiphi (Hymenoptera: Braconidae) Type of test: Acute contact extended laboratory tes t Number of organisms: 6 replicates with 5 females fo r each treatment group Treatments: Control: deionised water; toxic standar d: 2.5 g a.s./ha (37 % Dimethoate); test item: 22, 42, 80, 151 and 288 g a.s./ha, deionised water as diluent. The substances were applied in the equivalent of 400 L/ ha after calibration of the
sprayer. Exposure route, procedure: Adult wasps were exposed to freshly dried residues on barley plants for 48 h. One day after the start of the study, oat plants we re infected with Rhopalosiphum padi

. The test units consisted of a pot with treated barl ey plants which were enclosed within a clear polyacrylic cylinder with a hole for the introduction of the parasitoids. After introduction the hole was closed by a stopper . The top of the cylinder was closed with fine mesh gauze. The test animals were introduced from prepared test tubes by slightly blowing them. When all wasps were in the test unit, the glass tube was removed and the orifice closed with a feed ing stopper. During the exposure phase of 2 days the test animals had acces s to the sugar solution. From the water control and the dose rates 80, 151 a nd 288 g a.s./ha, 15 impartially chosen females per treatment were each transferred to a cylinder containing untreated cereal plants infested with Rhopalosiphum padi for a period of 24 hours. The number of mummies was assessed 11 days later. Feeding: Honey/water solution Test conditions: Temperature: 19 – 23.5°C; relative humidity: 62 - 85 %, photo period 16 h, 688 – 1718 lux in the parasitation phase, 5590 – 19730 lu x in the reproduction phase Test parameter: Mortality was assessed after 2, 24 and 48 h. The number of alive, affected, moribund and dead wasps was recorded. Repellence of the test item was determined during the initial 3 h after the release of the females. Five separate observations were made at 30-minute intervals start ing approximately 15 - 30 min after the introduction of all wasps. Reproduction assessment: number of mummies. Statistics: Mortality corrected according to Schnei der-Orelli. One-way ANOVA, p-values are adjusted according to Dunnett. Findings: All validity criteria were met. No dose related re pellent effect of the test item was observed. Results are summarised in table 9.5.1-3.– 113 –

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

Table B.9.5.1-3: Mortality and reproduction after e xposure of Aphidius rhopalosiphi to freshly dried residues of Spirotetramat OD 150 applied onto barle y plants Mortality Reproduction Repellency Treatment Uncorr. Abbott Rate Rel. to control [%] P-value * % wasps on plant Rel. to

control [%] P-value

Control 0 0 23 0 - 75.3 - - 22 g a.s./ha 3.3 3.3 n.d.** n.d.** - 75.1 0.4 1 (n. s.) 42 g a.s./ha 3.3 3.3 n.d.** n.d.** - 69.0 8.4 0.892 (n.s.) 80 g a.s./ha 0 0 29.5 - 28.1 0.435 (n.s.) 56.7 24.8 0.071 (n.s.) 151 g a.s./ha 6.7 6.7 24.6 - 7 0.967 (n.s.) 60.7 19 .5 0.195 (n.s.) 288 g a.s./ha 0 0 16.6 27.8 0.265 (n.s.) 60.6 19.5 0.244 (n.s.) Reference 80 80 n.d.* n.d.* n.d.* 70.3 6.6 - n.s. not significant *:n.d. not detected, **:n.d. not detected due to capacity reasons Conclusion: 48 h LR 50 > 288 g a.s./ha. There was no reduction in reproductive success rela tive to the control at the tested treatment rates.

Reference: Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, Phytoseiidae) using an extended laboratory test BYI 08330150 OD. Author(s), year: Waltersdorfer A., 2005c Report/Doc. number: CW05/030; M-256306-01-1 Guideline(s): Blümel et al. 2000 GLP: Yes (certified laboratory) Deviations: very short declines in rel. humidity du e to opening of the door Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; Batch no.: 08030/0189, (0152), Tox no.: TOX07034-00 ; analytical content: 148.89 g a.s./L Material and Methods: Test species: Typhlodromus pyri protonymphs Type of test: Acute contact extended laboratory tes t Number of organisms: 4 replicates with 20 individua ls for each treatment group Treatments: Control: deionised water; toxic standar d: 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. T he test concentrations and the suspension for the reference item were prepared ste pwise on the day of application. Deionised water was used as diluent. T he items were applied to Phaseolus vulgaris

leaves in the equivalent of 200 L/ha using a spray er after calibration. Exposure route, duration: Protonymphs were exposed to freshly dried residues on leaves of Phaseolus vulgaris for 14 d. Feeding: Pollen (birch, pine). The water supply for the mites was insured sticking a pin into each of the leaves. Additional food was added on da ys 3, 7 and 12. Test conditions: Temperature: 24 - 25°C; relative h umidity: 67 - 72 % (with short declines to 40 % due to opening of the door), 16 h photo period, 968 – 1026 lux Test parameters, procedure: One exposure unit consisted of an intact leaf of Phaseolus vulgaris , which was laid after application on a layer of wet filter pap er on top of a water soaked foam. A circle of insect glue (ø approx. 40 mm) was forme d on the leaves. Sets of such– 114 –

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

cylinders were placed on a plastic tray such that t he filter paper was constantly provided with deionised water. After the test units were set up (day 0) the proton ymphs were placed onto the plant surface. The mites were transferred with a fi ne brush under a stereomicroscope and immediately afterwards examine d to ensure they were undamaged and in good health. Mortality of 80 protonymphs was assessed 1, 3, 7, 1 0, 12 and 14 days after exposure by counting the number of living and dead mites. The number of escaped mites was calculated as the difference from the total number exposed. Additionally on days 7, 10, 12 and 14 the number of females, males, eggs and juveniles was counted. Eggs and juveniles were remo ved. The reproduction rate of surviving mites was evaluated over the period of 7-14 days after treatment by counting the total number of females and eggs. Statistics: Mortality corrected according to Schnei der-Orelli. Fisher`s Exact test, two-sided, p-values are adjusted according to Bonferroni-Holm; one-way ANOVA, p-values adjusted according to Dunnett Findings: Results are summarised in table 9.5.1-4. All valid ity criteria were met.

Table B.9.5.1-4: Mortality and reproduction after e xposure of Typhlodromus pyri to freshly dried residues of Spirotetramat OD 150 applied onto bean leaves Mortality Reproduction Treatment Uncorr. Corr. Schn.- Orelli P-value * Rate Rel. to control [%] P-value **

Control 7.5 - - 7.3 - 0.15 g a.s./ha 11.3 4.1 n.s. 4.1 43.2 0.081 (n.s.) 0.51 g a.s./ha 8.8 1.4 n.s. 3.7 48.7 0.060 (n.s.) 1.7 g a.s./ha 60.0 56.8 <0.0001 sign. n.d. / / 5.9 g a.s./ha 98.8 98.6 <0.0001 sign. n.d. / / 20 g a.s./ha 100 100 <0.0001 sign. n.d. / / Reference 100 100 / / n.s. not significant, sign. = significant, * Fishe r`s Exact test, ** one-way ANOVA Conclusion: 7 d LR 50 1.588 g a.s./ha (95 % CI: 1.311 – 1.860) There was a significant reduction of reproduction a t the low rates of 0.15 and 0.51 g a.s./ha.

Reference: BYI 08330 150 OD; Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, Phytoseiidae) using an extended laboratory test (under semi-field conditions aged residues on apple trees) Author(s), year: Waltersdorfer A., 2006c Report/Doc. number: CW05/028 Guideline(s): Blümel et al. 2000 GLP: Yes (certified laboratory) Deviations: The test design was modified - potted a pple trees were treated instead of glass plates; mites were exposed to freshly applied and u nder semi-field conditions aged residues on excised apple leaf discs Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; Batch no.: 08030/0189(0152), Tox no.: TOX07034-00; product code: AE 1302943– 115 –

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

00 OD15 A101; analytical content: 148.89 g a.s./L Material and Methods: Test species: Typhlodromus pyri protonymphs Type of test: Acute + aged residue contact extended laboratory test Number of organisms: 100 protonymphs in each treatm ent group (5 replicates with 20 individuals) Treatments: Control: deionised water; toxic standar d: 40 g a.s./ha (37 % Dimethoate EC) in 200 L water; test item: 4 x 72 g a.s./ha applied to potted apple trees (7 days interval) ( Malus sylvestris ) under semi-field conditions in 600 L water/ha usi ng a plot sprayer after calibration. Exposure route, duration: Protonymphs were exposed to aged residues on apple leaves under laboratory conditions for 7 d. Aging of the spray residues on the potted apple trees took place under natural semi-field conditions with rain protection during the whole study. Feeding: Pollen (birch, pine) Test conditions: Weather data (temperature and rel. humidity) in the outdoor area were recorded. Lab conditions: Temperature: 24.5 - 28°C; relative humidity: 61 - 73 %, 990 – 1567 lux Test parameters, procedure: Bioassays were initiated on DAA (days after last te st substance application) 0; 7; 14; 21; 28; 42; 49 and 56: leaves were taken from t he apple trees, and predatory mites were exposed on these leaves. In each bioassa y, mortality was assessed over a period of 7 days after exposure on excised l eaf discs by counting the number of living and dead mites for each bioassay o n 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 circl e of insect glue was formed on the leaves. These sets were placed on a plastic tra y such that the filter paper was constantly provided with deionised water. The numbe r of escaped mites was calculated as the difference from the total number exposed. At day +7 of each test, percentage mortality (including escapers) was calculated as mean of the 4 replicates. The reproduction rate of surviving mites was then e valuated over the period of 7- 14 days by counting the total number of offspring ( eggs and larvae) produced. From these data the endpoints mortality and effects on reproduction were calculated. Statistics: Mortality corrected according to Schnei der-Orelli. Findings: The results are considered as valid since the mort ality/escaping value for the control stated in the laboratory method on glass wa s reached in this study (< 20 %) and the average number of eggs/female (cal culated as sum of 4 assessment dates – from day 7 on) in the control gr oup exceeded 4 eggs per female. The mean corrected mortality of the nymphs, and the mean reproduction rate of the surviving females exposed to the test item and the toxic reference are given in the tables below:– 116 –

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

Table B.9.5.1-5: Mortality of T. pyri after exposure to freshly applied and aged residue s of Spirotetramat OD 150 on apple leave discs Days after last application 0 7 14 21 28 42 49 56 Treatment Mortality after 7 days (%) Control 5 4 5 17 6 4 3 7 Corr. Mortality [%] Test item 93.7 100 90.5 92.8 100 58.3 50.5 10.8 Reference item (DAA1) 100 100 100 100 100 100 100 100 DAA: days after last application

Table B.9.5.1-6: Reproduction of T. pyri after exposure to freshly applied and aged residue s of Spirotetramat OD 150 on apple leave discs

Treatment Control Test item Mean no. of eggs /female Mean no. of eggs /female Reduction rel. to control [%]

DAA 0 to day 42 n.d. n.d. n.d. DAA 49 5.19 5.94 -14.48 DAA 56 6.88 7.87 -14.35 n.d.: not determined

Conclusion: In this extended laboratory test the lethal and su blethal effects of Spirotetramat OD 150 residues (aged under semi-field conditions) on Typhlodromus pyri were determined after application of 4 x 72 g a.s./ha on to apple trees. 58.3, 50.5 and 10.8 % corr. mortality were found on DAA 42, 49 and DAA56. After 7 weeks of aging, no effects > 11 % on survival or reproductio n were observed any longer.

Reference: Dose-response toxicity (LR 50 ) of BYI 08330 150 OD to the green lacewing Chrysoperla carnea

(STEPH.) under extended laboratory conditions (inc luding food-application). Author(s), year: Röhlig, U.; 2005 Report/Doc. number: 051048082; M-262650-01-1 Guideline(s): Vogt et al. 2000 (IOBC), modified GLP: Yes (certified laboratory) Deviations: Guideline modified for the extended lab oratory test (exposure on natural substrate) in such a way that bean leaves were used instead of glass plates and that treated food was given Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; Batch no.: 08030/0189 (0152), Tox no.: TOX07034-00; analytical content: 148.89 g a.s./L Material and Methods: Test species: Chrysoperla carnea , larvae, age 2-3 d (L 1 ) Type of test: Acute contact extended laboratory tes t Number of organisms: 40 replicates with 1 larvae fo r each treatment group Treatments: Control: deionised water; toxic standar d: 30 mL product/ha (Dimethoate EC400); test item: 44, 72, 112, 184 and 288 g a.s./ha; deio nised water as diluent. The test substance was applied in 200 L/ha. Exposure route, Larvae were exposed to freshly dried residues on be an leaves and Sitotroga eggs– 117 –

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

duration: until pupation. Feeding: Larvae were fed with treated eggs of Sitotroga cerealella 3 times per week, adults with artificial food (diet according to the guideli ne) at each assessment day. Test conditions: Temperature: 23 - 26°C; relative h umidity: 66 - 77 %, 16 h photo period, 2100 lux Test parameters, procedure: The test consisted of two phases. In the exposure p hase of the test, mortality of the larvae and pupae and adult emergence was determ ined following exposure to dried spray deposits on bean leaves and feeding wit h treated Sitotroga eggs. In the reproduction phase, the sublethal effects on re productive performance (egg laying and hatching success) were assessed. Exposure cages consisted of glass cylinders with ga uze cover and a treated bean leaf on moistened filter paper as bottom. Cages wer e fixed to a glass plate and an acrylic plate. Oviposition cages consisted of glass beakers covered with cotton gauze during egg laying. Hatching cages for F1 larv ae consisted of plastic cages with clear cover. Oviposition was started one week after the first egg laying was observed (pre-oviposition period). Test plants were kidney beans ( Phaseolus vulgaris ), grown in the laboratory of the test facility under controlled conditions. Primary leaves were cut shortly before the application, when the plants were 3 weeks old. The number of surviving larvae and hatched adults, as well as the number of eggs laid and larvae hatched (F1) were recorded over a p eriod of 43 days. From these data the endpoints mortality and fecundity were cal culated. Statistics: Mortality corrected according to Abbott . Statistical analysis of mortality data (Fisher`s Exact Binomial Test, 1-sided, p ≤ 0.05). The software ToxRat Professional, Version 2.09 (RATTE, 2004) was used. Findings: All validity criteria were met. Results are summar ised in table 9.5.1-7. Statistical analysis revealed no significant differ ences concerning mortality between control and test item treatment groups. Als o the reproductive output was above the lower limit given as validity criterion f or the glass plate method ( ≥ 15 eggs/female/d) and therefore considered as not impa cted by the treatment.

Table B.9.5.1-7: Toxicity of dried residues of Spir otetramat 150 OD on Chrysoperla carnea in an extended laboratory test Reproduction Fecundity Fertility Treatment Mortality after 24 days [ % ] [mean number of eggs/ female] mean hatching rate [ % ] Control 5.0 18.9 81 Application rate [ g a.s./ha ] Corrected mortality [ % ]

44 5.3 19.5 80 72 7.9 18.7 81 112 2.6 20.2 81 184 0 18.6 80
288 5.3 19.0 80 Reference item 63.2 - - LR 50 > 288 g a.s./ha

Conclusion: LR 50 > 288 g a.s./ha. No negative effects on reproduction after exposure to rates up to 288 g a.s. Spirotetramat 150 OD were observed.– 118 –

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

Reference: Toxicity to the ladybird beetle Coccinella septempunctata L. (Coleoptera, Coccinellidae) using an extended laboratory test in cluding exposure to and oral uptake of BYI 08330 150 OD. Author(s), year: Waltersdorfer, A.; 2006b Report/Doc. number: CW05/053; M-264175-01-1 Guideline(s): Schmuck et al., 2000 (IOBC), modified GLP: Yes (certified laboratory) Deviations: Guideline modified for the extended lab oratory test in such a way that bean leaves were used for exposure instead of glass plates; in addition, the larvae got treated food (aphids) for 4 days of the study to include a possible oral uptake of the test item and afterwards untreated food was given; on da y 7 aphids on new plants were sprayed and this food was offered to larvae wh ich had not yet pupated. Validity: Acceptable. The validity criterion for co ntrol mortality was slightly not met. However, validity criteria have been defined for gl ass plate studies only and considering the special design the study can be con sidered valid.

Test substance: Spirotetramat (BYI 08330) 150 OD; Product code: AE 1302943 00 OD15 A101; Batch no.: 08030/0189 (0152), Tox no.: TOX07034-00; analytical content: 148.89 g a.s./L Material and Methods: Test species: Coccinella septempunctata , larvae, 4 d old Type of test: Acute contact extended laboratory tes t Number of organisms: 40 replicates with 1 larvae fo r each treatment group Treatments: Control: tap water; toxic standard: 80 mL product/ha (Dimethoate EC37); test item: 33, 57, 97, 168 and 288 g a.s./ha; tap water as diluent. The test substance was applied in 200 L/ha. Application was done using a specially constructed sprayer, permitting application on a defined area. Exposure route, duration: Larvae were exposed to freshly dried residues on be an leaves and Sitotroga eggs until pupation. Feeding: Larvae were fed with aphids ( Acyrthosiphon pisum ) ad libitum which had been treated for four days and afterwards fed with untre ated aphids. Adults were fed with aphids and pollen. Test conditions: Temperature: 23.6 – 26.6°C; relati ve humidity: 42 - 85 %, 16 h photo period, 1050 - 1760 lux Test parameters, procedure: Exposure units consisted of a detached leaf of Phaseolus vulgaris , which was laid on a wet cotton wool pad in a petri dish (9 cm diam eter) after application. A constant supply of deionised water from the bottom was assured. Per individual test unit one steel ring (about 3 cm diameter and 3 cm high) was placed on the leaf. The top inner edge of the rings was coated wi th a lubricant (Hostaflon) in order to prevent the larvae from escaping. Once dry, the steel rings were placed onto them int o the petri dishes and one larva was added to each test unit. Additionally potted bean plants with aphids ( Acyrthosiphon pisum on Vicia faba ) were sprayed on the day of application with the cor responding rates of the test item. The aphids were brushed from the leaves and w ere used as food for 4 days. Afterwards untreated aphids were fed until day 7. O n day 7 new food was sprayed and offered to those larvae which had not yet pupat ed. After 3 days the remaining larvae and the adults were fed with untreated food.– 119 –

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

All test units were assessed daily and the conditio n of the beetles recorded. At every feeding session, dead aphids and exuviae from earlier feeding sessions were removed, in order to maintain a constant conta ct between the larvae and the treated surface. Once the larvae had pupated, the e merged beetles were transferred to plastic boxes at the day of their in dividual hatching. After this feeding period all beetles were transfer red to glass jars. Their sex was determined and fresh stems of bean plants with aphi ds and untreated pollen were introduced as required. Sheets of black paper were offered to the beetles for egglaying, which was checked daily. Pieces of paper wi th freshly laid eggs were cut out of the paper and stored in petri dishes. Mortal ity during the egg laying period was recorded and the sex of the dead animals was de termined. Statistics: Mortality corrected according to Schnei der-Orelli. Findings: The mortality value for the control was slightly a bove that mentioned in the guideline for glass plate studies (32.5 % mortality instead of max. 30 %). However, validity criteria have been defined for gl ass plate studies only and considering the special design the study can be con sidered valid. As recommended by the guideline (for glass plate st udies) the reproductive performance parameters can only be evaluated qualit atively, due to the very high species-inherent variability in egg-laying performa nce. As all values for the three highest and the lowest dose rate were within the hi storical data base for control beetles ( ≥ 2 fertile eggs per female and day), this parameter is considered as not impacted by the test item rates. Table B.9.5.1-8: Mortality and reproduction after e xposure of Coccinella septempunctata to freshly dried residues of Spirotetramat OD 150 applied onto detached bean leaves: Mortality [%] Reproduction Treatment [g a.s./ha] Uncorr. corrected Fertility (hatching rate) [%] Fertile eggs per

female and day Control (tap water) - 32.5 - 99.3 6.0 BYI 08330 150 OD 33 22.5 -14.8 88.9 3.8 BYI 08330 150 OD 57 17.5 -22.2 91.7 1.8 BYI 08330 150 OD 97 32.5 0 89.3 3.6 BYI 08330 150 OD 168 47.5 22.2 99.0 2.0 BYI 08330 150 OD 288 50.0 25.9 86.7 4.1 Reference item 80 mL prod./ha 100 100 n.d.* n.d.* LR 50 > 288 g a.s./ha * n.d.: not determined Conclusion: LR 50 > 288 g a.s./ha. No negative effects on reproduction after exposure to rates up to 288 g a.s. Spirotetramat 150 OD were observed.– 120 –

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

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

Reference: Evaluating effects of BYI 08330 OD 150 applications on mite fauna (Acari) in the field (grape vines, France). Author(s), year: Aldershof, S.; 2006 Report/Doc. number: B127AFG; M-269107-01-1 Guideline(s): Blümel et al., 2000; Hassan, 1985 ; C andolfi et al., 2000 GLP: Yes (certified laboratory) Deviations: No Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) OD 150, purity: 148.89 g a.s./L (analysed), density 0.986 g/mL, batch no. 08030/0189(0152); sample no. TOX07034-00, development no. 30-00364846 Material and Methods: Test species: Predatory mites in grapes Type of test: Field study Location: Commercial vineyard in Fources in the Arm agnac region, South-West France Test design Treatments: water control; reference it em: Dimethoate applied at 1 L product/ha (400 g a.s./L EC) applied during the first and the last application of the fourth test scenario (3 week interval); test item: randomised d esign with 5 replicates per treatment. Application volumes were 500 L/ha; spray ing equipment was calibrated before use. Plot layout: size: 20 m length, 40 m 2 surface. Plots consisted of 20 adjacent vine positions. Plots were separated by un treated 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 applicat ion rates is provided in table B.9.5.2-1. Table B.9.5.2-1: Spray scenarios Scenario Description No of applications Spray interva l Nominal Rate 1 Full (2x) 2 2 weeks 96 g a.s./ha 2 Drift 1

(2x high) 2 2 weeks Application 1: 36.4 g a.s./ha Application 2: 35 g a.s./ha 3 Drift 2 (2x low) 2 2 weeks 9.1 g a.s./ha 4 Drift 3 (4x low) 4 1 week 4.8 g a.s./ha

Test conditions: Throughout the experiment the clim atic conditions (rel. humidity, temperature) were measured at the test site with a portable devi ce. Rainfall data were obtained from a local weather station. Sampling: The effects of BYI 08330 OD 150 were expr essed in terms of population changes relative to the water control. Population samples w ere taken shortly before each application, 1 week after the first application and approximately 1, 4 and 8 or 9 weeks after the last application. For test item sce nario 4 and water an additional sample was taken 11 weeks after the last treatment. The evaluation was based on time to recovery (= population density similar to c ontrol). Three leaves of average size were collected from ea ch of the 8 innermost vines of a plot ( 24 leaves per plot). The material collected from on e plot was pooled for each sampling point. The leaf washing method according to Boller (1984) was applied. The collected– 121 –

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

arthropods were inspected under a binocular microsc ope. The first 16 phytoseiid mites observed in each plot were mounted for specie s identification.

Statistics: Effect values were calculated from tota l phytoseiid mite numbers according to Abbott (1925) and Henderson-Tilton (1955). In addit ion, total phytoseiid mite numbers were analysed statistically using the non-p arametric Man-Whitney U test. Findings: Test conditions: Weather parameters were in an acce ptable range; there was no rain on the application days. Predatory mites: Several acarine taxa were identifi ed. The most abundant taxon prevailing in the vineyard was the predatory mite family Phytoseiidae . Phytoseiid populations almost exclusively consisted of the species Typhlodromus pyri (98 % - 100 % throughout the study period). Other mite taxa encou ntered were Eriophyoidea (rust mites), Tydeidae, Tarsonemidae and Oribatida (fungivorous mites), however, their numbers were too low for evaluation. BYI 08330 OD 150 had no effect on any of the taxa f ound, in any of the spray scenarios tested. No statistically significant diff erences with the water control were found for any of the BYI 08330 OD 150 treatments on all sampling dates, except on 16 June, 5 days after the first treatment, when the 3 rd drift rate (4 applications at 4.8 g a.s./ha) showed an effect value of 41 % (A bbott) or 31 % (HendersonTilton). However, on later sampling dates this trea tment no longer showed statistically significant effects. Moreover, Abbott effects remained below 50 % (Henderson-Tilton effects below 40 %) throughout th e study. This finding was probably due to natural variation of mite field pop ulations, rather than to an actual treatment effect. Population dynamics observed in o ther test item treatment plots closely resembled fluctuations detected in water co ntrol plots. The different spray scenarios of the test item neither induced effects on Eriopheiid mites. The reference item severely affected Phytoseiid mit es in a negative way, with a statistically significant reduction in phytoseiid n umbers of 43 % to 96 %. Thus, the trial is considered valid for the purpose of evalua ting potential consequences of test item treatments to predatory mites. Population s of Eriophydoidea could however increase, probably due to reduced predation by phytoseiids. All effects in the reference item group were statis tically significantly different to the control.– 122 –

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

Table B.9.5.2-2: Summary results
08.06.05 16.06.05 02.07.05 12.07.05 25.07.05 01.08. 05 29.08.05 Abbott values drift 1 (high 2x) -1.5 % -11.4 % -5.8 % 2.1 % drift 2 (low 2x) 17.3 % 27.6 % 14.2 % -1.0 % drift 3 (low 4x) 13.9 % 41.1 % 43.8 % 31.9 % -17.2 % full (2x) 4.1 % 16.3 % 36.2 % 13.9 % 12.8 % reference 18.9 % 43.1 % 94.1 % 95.6 % Henderson-Tilton values drift 1 (high 2x) -10.1 % -4.4 % 3.5 % drift 2 (low 2x) 12.5 % -3.8 % -18.2 % drift 3 (low 4x) 31.7 % 34.7 % 21.0 % -28.7 % full (2x) 12.7 % 33.4 % 10.3 % 94.6 % 9.0 % reference 29.8 % 92.7 % P-values (Mann-Whitney U test, comparison to water) drift 1 (high 2x) 0.834 0.965 0.917 0.917 drift 2 (low 2x) 0.175 0.076 0.465 0.917 drift 3 (low 4x) 0.465 0.028 0.327 0.117 0.754 full (2x) 0.754 0.347 0.175 0.465 0.754 reference 0.347 0.009 0.014 0.009 P-values in bold italics are statistically significant (P < 0.05)

Conclusion: It is concluded that Spirotetramat OD 150, applied 2 times at a nominal rate of 96 g a.s./ha or lower, or 4 times at a nominal rate of 4.8 g a.s./ha, has no effects on the acarine mite fauna detected with leaf sampling in vineyards in France.

B.9.5.3 Summary

Table B.9.5.3-1: Summary of effects of spirotetrama t on non-target-arthropods (lab + ext lab)

Test species Exposure Test item Rate [g a.s./ha] Type of effect Effect [%] Reference

Corrected mortality -3.6

-7.3
0
14.5
85.5 Aphidius rhopalosiphi
adults contact with

dried residues
on treated glass plates Spirotetramat

150 OD 2

9
24
70
200

48 h LR 50 = 114.7 g a.s./ha Waltersdorfer
2005a

Corrected mortality 10.3

71.1
100
100
100 Typhlodromus pyri protonymphs contact with

dried residues
on treated glass plates Spirotetramat

150 OD 0.19

0.43
0.97
2.21
5

48 h LR 50 = 0.333 g a.s./ha Waltersdorfer
2005b

Corrected mortality / reproduction 3.3 / n.p.

3.3 / n.p.
0 / -28.1
6.7 / -7
0 / 27.8 Aphidius rhopalosiphi
adults contact with

dried residues
on treated barley plants Spirotetramat

150 OD 22

42
80
151
288

48 h LR 50 > 288 g a.s./ha Waltersdorfer
2006a– 123 –

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

Test species Exposure Test item Rate [g a.s./ha] Type of effect Effect [%] Reference

Typhlodromus pyri protonymphs contact with

dried residues
on treated
bean leaves Spirotetramat

150 OD 0.15

0.51
1.7
5.9
20 Corrected

mortality / reproduction 4.1 / 43.2

1.4 / 48.7
56.8 / n.d.
98.6 / n.d.
100 / n.d. Waltersdorfer

2005c

Typhlodromus pyri protonymphs contact with

dried residues (acute and aged) on treated apple
leaves Spirotetramat

150 OD 4 x 72 (7d) Corrected mortality aged 0d: 93.7

aged 7d: 100 aged 14d: 90.5 aged 21d: 92.8 aged 28d: 100 aged 42d: 58.3 aged 49d: 50.5 aged 56d: 10.8 no reduction of reproduction on DAA 49+56 Waltersdorfer

2006c

Chrysoperla

carnea larvae contact with

dried residues
on treated
bean leaves Spirotetramat

150 OD 44

72
112
184
288 Corrected

mortality 5.3

7.9
2.6
0
5.3 no effect on reproduction Roehlig 2005

Coccinella septempunctata
larvae contact with

dried residues
on treated bean leaves, feeding with treated aphids Spirotetramat

150 OD 33

57
97
168
288 Corrected

mortality -14.8

-22.2
0
22.2
25.9 no effect on reproduction Waltersdorfer

2006b

n.d. not determined, n.p. no assessment performed negative figures indicate a positive effect compare d to the control

B.9.5.4 Risk assessment for non-target arthropods

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".– 124 –

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

B.9.6 Effects on earthworms (OECD IIA 8.9, IIIA 10. 6.3)

B.9.6.1 Acute toxicity (OECD IIA 8.9.1, IIIA 10.6.1 .1)

Active substance

Reference: BYI 08830 (techn.): Acute toxicity to earthworms ( Eisenia fetida ) tested in artificial soil with 5 % peat Author(s), year: Lechelt-Kunze, C., 2004a Report/Doc. number: LKC/RG-A-18/03, Edition Number: M-104105-01-2, 07.01.2004 Guideline(s): OECD 207 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.5 %, batch: 08045/0014 Material and methods: Test species: Earthworm ( Eisenia fetida ) Number of organisms: 4 replicates each with 10 ind ividuals per treatment and control Weight, age: Average weight: 360 mg/worm, clitella te adults > 2 months Type of test, duration: Laboratory acute test, 14 d ays Applied concentrations: Nominal: 0 (control, quartz sand), 32, 100, 178, 316, 562, 1000 mg/kg dw soil Solvent/vehicle: Quartz sand Toxic standard: 2-Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mg/kg dw soil Test conditions: Test substrate: 5 % sphagnum peat, 20 % kaolinite clay, 74 % quartz sand, about 0.2 – 1 % CaCO 3 to adjust pH to 6.0 ± 0.5, max. WHC between 40 - 60 % , pH and the water content were measured at the beginning and the end of the study Substrate/test vessel: 625 g wet weight (500 g dw) in 1.5 L test containers Temperature: Measured temperatures were not stated Light regime: Continuous illumination (400 – 800 L ux) Test parameters: Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 14 days) and mean body weight (measured at 0 and 14 da ys) Statistics: NOEC: Dunnett’s’ Test (mortality, mean body weight) LC 50 (toxic standard): Probit Analysis Findings: Water content: About 21 % (0 h), 19.6 – 20.1 % (14 d) pH: 6.4 (0 h), 6.4 (14 d) Mortality: Spirotetramat: No mortality was noticed in the control and all treatments up to 562 mg/kg dw soil. At 1000 mg/kg dw soil the mortality was 3 % (do not differ statistically significantly in Dunnett test). Toxic standard: LC 50 : 23 mg/kg dw soil (95% C.I.: 21 – 24 mg/kg dw soil ) Behavioural effects: No effects were observed in t he control and all treatments Body weight: See table B.9.6.1-1– 125 –

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

Table B.9.6.1-1: Effects of BYI 08330 (spirotetrama t) on the mean body weight of Eisenia fetida Mean body weight (SD) Spirotetramat [mg/kg dw soil] Day 0 [g] Day 14 [g] Mean change in body weights at day 14, compared to control 0 (control) 0.35 (0.02) 0.32 (0.02) -- 32 0.36 (0.03) 0.33 (0.02) +2.3 % 100 0.37 (0.03) 0.34 (0.03) +5.5 % 178 0.37 (0.02) 0.33 (0.02) +3.9 % 316 0.36 (0.02) 0.33 (0.02) +2.3 % 562 0.36 (0.02) 0.33 (0.03) +3.1 % 1000 0.35 (0.03) 0.32 (0.02) ± 0.0 % NOEC (body weight): 1000 mg/kg soil dw EC 50 > 1000 mg/kg soil dw

Conclusions: LC 50 > 1000 mg/kg soil dw, NOEC (body weight, mortality ): 1000 mg/kg soil dw Comments: While no deviations to the OECD guideline 207 are notable, deviations to the OPPTS guideline are remarkeable: The recommended test duration is 28 days, with obse rvations and measured concentrations taken on Days 0, 7, 14, 21, and 28; this study only lasted 14 days and measured concentrations are not reported

Metabolites

Reference: BYI 08830 cis-ketohydroxy: Acute toxicity to earthw orms ( Eisenia fetida ) tested in artificial soil Author(s), year: Lechelt-Kunze, C., 2005 Report/Doc. number: LKC/RG-A-47/05, Edition Number: M-258925-01-2, 17.10.2005 Guideline(s): OECD 207 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08830 cis-ketohydroxy (metabolite of spirotetr amat), purity: 98.6 %, batch: NLL7549-7 Material and methods: Test species: Earthworm ( Eisenia fetida ) Number of organisms: 4 replicates each with 10 ind ividuals per treatment and control Weight, age: Average weight: 0.34 g/worm, clitella te adults > 2 months Type of test, duration: Laboratory acute test, 14 d ays Applied concentrations: Nominal: 0 (control, quartz sand), 32, 100, 316, 1000 mg/kg dw soil Solvent/vehicle: Quartz sand Toxic standard: 2-Chloroacetamide (purity > 97 %): 5.6, 10, 18, 24, 32 mg/kg dw soil Test conditions: Test substrate: 10 % sphagnum peat, 20 % kaolinite clay, 70 % quartz sand, about 0 % CaCO 3 to adjust pH to 6.0 ± 0.5, max. WHC between 40 - 60 % , pH and the water content were measured at the beginning and the end of the s tudy Substrate/test vessel: 625 g wet weight (500 g dw) in 1.5 L test containers Temperature: Measured temperatures were not stated Light regime: Continuous illumination (400 – 800 L ux)– 126 –

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

Test parameters: Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 7 and 14 days) and mean body weight (measured at 0 an d 14 days) Statistics: NOEC: Dunnett’s’ Test (mortality, mean body weight) LC 50 (toxic standard): Probit Analysis Findings: Water content: 23.3 % (0 h, mean), 24.7 % (14 d, m ean) pH (mean): 6.4 (0 h), 6.2 (14 d) Mortality: No mortality was noticed in the control and all treatments. Behavioural effects: No effects were observed in t he control and all treatments Body weight: See table B.9.6.1-2

Table B.9.6.1-2: Effects of BYI 08330-ketohydroxy o n the mean body weight of Eisenia fetida Mean body weight (SD) Spirotetramat- ketohydroxy [mg/kg dw soil] Day 0 [g] Day 14 [g] Mean change in body weights at day 14, compared to control 0 (control) 0.35 (0.01) 0.36 (0.01) -- 10 0.33 (0.01) 0.34 (0.01) -5.9 % 32 0.35 (0.01) 0.36 (0.02) -0.7 % 100 0.35 (0.01) 0.35 (0.01) -2.8 % 316 0.35 (0.01) 0.35 (0.01) -4.2 % 1000 0.35 (0.01) 0.36 (0.01) ± 0.0 % NOEC (body weight): 1000 mg/kg soil dw EC 50 > 1000 mg/kg soil dw

Toxic standard: LC 50 : 23 mg/kg dw soil (95% C.I.: 21 – 24 mg/kg dw soil ) Conclusions: LC 50 > 1000 mg/kg soil dw, NOEC (body weight, mortality ): 1000 mg/kg soil dw Comments: While no deviations to the OECD guideline 207 are notable, deviations to the OPPTS guideline are remarkeable: The recommended test duration is 28 days, with obse rvations and measured concentrations taken on Days 0, 7, 14, 21, and 28; this study only lasted 14 days and measured concentrations are not reported

Reference: Spirotetramat – 4-methoxycyclohexanone: Acute toxic ity to earthworms ( Eisenia fetida

) tested in artificial soil with 5 % peat Author(s), year: Leicher, T., 2008 Report/Doc. number: LRT-Rg-A-98/07, Edition Number: E310 3411-3, 09.01.2008 Guideline(s): OECD 207 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: 4-methoxycyclohexanon (metabolite of spirotetramat ), purity: 97.9 %, batch: SVA 5506-001 Material and methods: Test species: Earthworm ( Eisenia fetida ) Number of organisms: 4 replicates each with 10 ind ividuals per treatment and control Weight, age: Average weight: 0.29 g/worm, clitella te adults > 2 months Type of test, duration: Laboratory acute test, 14 d ays Applied concentrations: Nominal: 0 (control, quartz sand), 62.5, 125, 250, 500, 1000 mg/kg dw soil Solvent/vehicle: Quartz sand– 127 –

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

Toxic standard: Chloroacetamide (purity > 97 %): 5. 6, 10, 18, 24, 32 mg/kg dw soil Test conditions: Test substrate: 5 % sphagnum peat, 20 % kaolinite clay, 74.8 % quartz sand, about 0.2 % CaCO 3 to adjust pH to 6.0 ± 0.5, max. WHC between 40 - 60 % , pH and the water content were measured at the beginning and the end of the study Substrate/test vessel: 580 g wet weight (500 g dw) in 1.5 L test containers Temperature: Measured temperatures were not report ed Light regime: Continuous illumination (400 – 800 L ux) Test parameters: Mortality (assessed after 7 and 14 days), condition and behaviour (observed at 7 and 14 days) and mean body weight (measured at 0 an d 14 days) Statistics: NOEC: Dunnett’s’ Test (mortality, mean body weight) LC 50 (toxic standard): Probit Analysis Findings: Water content: 20.7 % (0 h, mean), 20.4 % (14 d, m ean) pH (mean): 5.76 (0 h), 5.53 (14 d) Mortality: No mortality was noticed in the control and all treatments. Behavioural effects: No effects were observed in t he control and all treatments Body weight: See table B.9.6.1-3

Table B.9.6.1-3: Effects of 4-methoxycyclohexanone on the mean body weight of Eisenia fetida

4-methoxycyclohexanone [mg/kg dw soil] Weight alteration of the surviviors Mean ± standard devision 0 (control) + 8,93 ± 6,07 62.5 + 3,19 ± 2,32 125 + 2,51 ± 3,14 250 - 4,24 ± 2,32*
500 - 13,66 ± 5,36*
1000 - 20,88 ± 7,45*

NOEC (body weight): 125 mg/kg soil dw EC 50 > 1000 mg/kg soil dw * significant difference when compared to control ( Dunnett test, alpha = 0.05)

Toxic standard: LC 50 : 11.1 mg/kg dw soil (95% C.I.: 10.1 – 12.2 mg/kg d w soil) Conclusions: LC 50 > 1000 mg/kg soil dw, NOEC (body weight, mortality ): 125 mg/kg soil dw Comments: While no deviations to the OECD guideline 207 are notable, deviations to the OPPTS guideline are remarkeable: The recommended test duration is 28 days, with obse rvations and measured concentrations taken on Days 0, 7, 14, 21, and 28; this study only lasted 14 days and measured concentrations are not reported

B.9.6.2 Sublethal effects (OECD IIA 8.9.2, IIIA 10. 6.4)

Active substance Not submitted. Not required because the maximum app lication number is two times per year and spirotetramat is not persistent in soil.– 128 –

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

Metabolites

Reference: BYI 08330-Enol: Reproduction toxicity to the earthw orm Eisenida fetida in artificial soil Author(s), year: Moser, T.; Scheffczyk, A., 2005 Report/Doc. number: P18RR, Edition Number: M-243305 -01-2, 19.01.2005 Guideline(s): ISO Guideline 11268-2 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08830-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005 Material and methods: Test species: Earthworm ( Eisenia fetida ) Number of organisms: 4 replicates each with 10 ind ividuals per treatment and control Weight, age: Mean: 404 mg/worm, clitellate adults Type of test, duration: Laboratory sublethal test, 8 weeks Applied concentrations: Nominal: 0 (control, quartz sand), 10, 32, 100, 316, 1000 mg/kg dw soil Solvent/vehicle: Quartz sand Toxic standard: Carbendazim (applied as SC formulat ion Derosal): 1, 3, 5 mg a.s./kg dw soil Test conditions: Test substrate: 10 % sphagnum peat, 20 % kaolinite clay, 69.55 % quartz sand, 0.45 % CaCO 3 to adjust pH to 6.0 ± 0.5, WHC max : 50 ± 10 %, pH and the water content were measured at the beginning and the end of the study Substrate/test vessel: 500 g dry weight/test conta iner Temperature: 17 – 22 °C The temperature was only for few and short time per iod below the recommended 18 °C, thus this did not affected the results of th e test. Light regime: 16 hours light (485 – 558 Lux)/8hour s dark Feeding: At 24 hours and then weekly (until 4 week s after application): 5 g manure per test vessel on the soil surface, sprinkled with 10 ml de ionised water Test parameters: Mortality of adults (assessed afte r 28 days), mean body weight of adults (measured at day 0 and after 28 days), morphologica l and behavioural changes of adults (observed at day 28), number of juvenile ear thworms (counted after 8 weeks) and condition and behaviour of juveniles (ob served after 8 weeks) Statistics: NOEC: Dunnett’s multiple test, EC 50 (reproduction): Probit analysis using max. likelihood regression Findings: Water content: Start: 32.1 – 35.4 % (53 – 58.6 % o f WHC max ) End: 28.9 – 33.8 % (49 – 60.7 % of WHC max ) pH: 6.1 – 6.2 (start), 6.8 – 7.0 (end) Effects test item: See table B.9.6.2-1– 129 –

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

Table B.9.6.2-1: Effect of BYI 08330-enol (metaboli te of spirotetramat) on mortality, growth and reproduction of Eisenia fetida
BYI 08330-enol [mg/kg dw soil] Mean mortality [%] Mean biomass increase

after 4 weeks [%] Mean number of juveniles (SD)

Control 0 34.7 148 (43.3) 10 0 33.7 133 (30.3) 32 0 40.0 133 (9.6) 100 0 34.5 130 (25.2)* 316 2.5 42.6 113 (18.8)* 1000 0 41.5 76 (20.9)* NOEC [mg/kg dw] 1000 32 LOEC [mg/kg dw] > 1000 100 LC 50 [mg/kg dw] > 1000 1269 (95% C.I. 1007 – 1896) * significantly different to control (alpha = 0.05)

Effects toxic standard: Reproduction: LOEC = 1 mg a.s./kg dw soil Validity criteria: Adult mortality ( ≤ 10 %): 0 % after 4 weeks Weight loss of adult worms ( ≤ 20 %): + 37.7 % after 4 weeks Number of juveniles/vessel ( ≥ 30 %): 148 ± 43.3 % Coefficient of variation ( ≤ 30 %): 29.3 % Conclusions: EC 50 (reproduction): 1269 mg/kg soil dw NOEC (reproduction) 32 mg/kg soil dw

B.9.6.3 Field studies

No study submitted. Not required, because the risk assessment for earthworms (see B.9.6.5) indicated a n acceptable risk for the intended uses and no refine ment is necessary.

B.9.6.4 Risk assessment for earthworms (OECD IIIA 1 0.6.1)

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".– 130 –

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

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

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

Metabolites

Reference: BYI 08330-Enol: Effects on survival and reproduction of the predaceous mite Hypoaspis aculeifer Canestrini (Acari: Laelapidae) in standard soil (LUFA 2.1) Author(s), year: Moser, T.; Scheffczyk, A., 2005 Report/Doc. number: P11HR, Edition Number: M-243307 -01-1, 19.01.2005 Guideline(s): SECOFASE, final report (Lokke & von G estel 1996), Guidance document on regulatory testing procedures for pesticides with nontarget arthropods (Barret et al. 1994) GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: BYI 08830-enol (metabolite of spirotetramat), puri ty: 99.1 %, batch: 692-101-09- 0005 Material and methods: Test species: Gamasid mite ( Hypoaspis aculeifer ) Number of organisms: 4 replicates with 20 mites pe r treatment, 5 replicates with 20 mites per control and 3 replicates with 20 mites per toxic reference Age: Protonymphes 2 day old Type of test, duration: Laboratory reproduction tes t, 34 days Applied concentrations: Nominal: 0 (control, deioni sed water), 10, 32, 100, 316, 1000 mg /kg dw soil Solvent: Deionised water Toxic standard: Dimethoat (applied as EC formulatio n “Perfekthion”): 5 mg/kg soil dw Test conditions: Test substrate: LUFA 2.1 standard soil (type: sand ): WHC max : 34.6 %, pH 6.2 ± 0.7, C org : 1.23 ± 0.3 % Substrate/test vessel: 5.1 – 5.3 g wet weight (app rox. 3 mm layer) / 30 mL glass container Temperature: 25 ± 2 °C Light regime: Permanent dark Feeding: At day 0 mites were fed with prey mites ( Tyrophagus putrescentiae ). Twice a week the food was checked and was added on demand. After 21 days enchytraeids ( Enchytraeus crypticus ) were fed. Test procedure: 0-day: Introduction of 20 individu als on the soil surface of each test unit randomly. 14-day: Surviving adult mites were transferred to u ntreated mating units (replicate groups together with sex ratio of 1:5 male to femal e). 21-day: Females from the control and the three high est concentration levels which caused < 50 % mortality (i.e. 100, 316, 1000 mg/kg) were transferred individually to the first series of reproduction units (one fema le per unit). 24-day: Females from the first series of reproducti on units were transferred individually to a second reproduction unit (one fem ale per unit). Day 28 – 30: Counting of juveniles from the first s eries. Day 32 – 34: Counting of juveniles from the second series.. Test parameters: Mortality of adults after 14 days, reproduction after a 7-day period Statistics: NOEC (mortality adults): ANOVA followed by Dunnetts test, NOEC (reproduction):– 131 –

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

Welch-t-test with Bonferroni Adjustment Findings: Water content: Start: 18.8 – 20.9 % (= 54.2 – 60.2 % WHC) End: Not reported pH: Start: 5.4 – 5.5 End: not reported Effects test item: See table B.9.7.1-1

Table B.9.7.1-1: Effects of BYI 08830-enol (metabol ite of spirotetramat) on mortality and reproduction of gamasid mites Mortality of parental mites Number of juveniles per female after 7 d BYI 08330-enol [mg a.s./kg dw] Number of dead mites at 14 days Corrected mortality a) [%] Mean ± SD Reduction [%]

Control 3 0.0 25.6 ± 7.3 0.0 10 8 7.2 n.d. n.d. 32 12 12.4 n.d. n.d.
100 4 2.1 30.1 ± 3.2 -14.8 316 8 7.2 26.4 ± 8.6 -3.4 1000 34 40.7* 23.7 ± 5.9 7.4 NOEC 316 mg/kg soil dw 1000 mg/kg soil dw LOEC 1000 mg/kg soil dw > 1000 mg/kg soil dw LC/EC50 > 1000 mg/kg soil dw > 1000 mg/kg soil dw a) corrected according to Abbott (1925) *Significant different by Dunnetts test (alpha = 0.0 5)

Effects toxic standard: 88 % corrected mortality a t 5 mg/kg soil dw Validity criteria: Adult mortality in control afte r 14 days ( ≤ 25 %): 3 % Mean reproduction rate in control ( ≥ 10 fertile eggs per female): 25.6 ± 7.3 Adult mortality in toxic standard after 14 days (50 – 99.5 %): 88 % Conclusions: NOEC (mortality): 316 mg/kg soil dw LC/EC 50 > 1000 mg/kg soil dw

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

Not submitted. Study is not required because spirot etramat is not persistent and the effects on soil m icroand macro-organisms are acceptable.

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

Substance Species Endpoint Time EC/LC 50 [mg/kg dw] NOEC

[mg/kg dw] Reference/study

acceptable

BYI 08330-enol Gamasid mite

( Hypoaspis aculeifer) Mortality Reproduction 28 d > 1000 316 1000 Moser & Scheffczyk

2005/yes– 132 –

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

B.9.7.4 Risk assessment for other soil non-target m acro-organisms

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".

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

B.9.8.1 Nitrogen mineralization

Active Substance

Reference: BYI 08330 tech.: Determination of effect s on nitrogen transformation in soil

Author(s), year: Lechelt-Kunze, C., 2004 b Report/Doc. number: LKC-N-32/04, Edition Number: M- 087831-01-1, 01.09.2004 Guideline(s): OECD 216 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 Material and methods: Test species: Soil microflora Type of test, duration: Nitrogen transformation tes t, 28 days Applied concentrations: 0 (control, purified water) , 0.13 and 1.32 mg/kg dry soil Solvent/vehicle: Quartz sand Toxic standard: Sodium chloride: 16 g/kg soil dw Test conditions: Test substrate: Natural Soil, collected on April 1 3 th , 2004 at a depth of 0-20 cm, from experimental field on Lacherhof Germany: C org : 0.7, pH: 5.6, texture (DIN): 6.3 % clay, 15.6 % silt, 78.1 % sand

, microbial biomass: 393 mg microbial C/kg soil dw, additionally 5 g/kg soil dw Lucerne-grass-green mea l was added after the application. Substrate/test vessel: 300 g soil dw/500 mL brown glass bottles Incubation: 20 ± 2 °C in the dark, about 40 % MHC Test parameters: Samples (10 g dry soil) were extra cted with 50 mL 1M KCl and soil particles were removed by filtration, NH 4 + and NO 3 - were determined using a “Bran & Lübbe Autoanalyzer 3”. Samples were taken at 0, 7, 14 and 28 days after incubation. Statistics: F-test (homogeneity of variance), T-tes t (significant differences)– 133 –

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

Findings: pH: 5.6 (start), 5.9 – 6.0 (end) Effects test item: See table B.9.8.1-1

Table B.9.8.1-1: Effects of spirotetramat on nitrog en transformation (mean values)

Treatment Time

(days) NH 4 -N

(mg N/kg dw) NO 3 -N

(mg N/kg dw) Nitrification rate

(mg NO 3 -N/kg soil dw/d) % Deviation from control

0 3.27 ± 0.00 28.34 ± 0.41 - - 7 1.93 ± 0.08 13.35 ± 1.18 -2.14 ± 0.22 - 14 1.56 ± 0.07 13.46 ± 1.47 0.02 ± 0.37 - Control

28 1.48 ± 0.14 26.02 ± 1.46 0.90 ± 0.07 -

0 3.42 ± 0.17 30.07 ± 0.55 - - 7 1.95 ± 0.07 14.76 ± 0.65 -2.19 ± 0.02 2 14 1.58 ± 0.06 13.77 ± 1.24 -0.14 ± 0.22 980 0.13 mg a.s./kg (0.099 kg a.s./ha)
28 1.57 ± 0.02 25.94 ± 0.76 0.87 ± 0.08 3 0 3.37 ± 0.21 30.00 ± 0.22 -2.38 ± 0.12 11
7 1.93 ± 0.13 13.32 ± 1.02 - - 14 1.76 ± 0.33 13.60 ± 0.54 0.04 ± 0.09 143 1.32 mg a.s./kg (0.99 kg a.s./ha)
28 1.71 ± 0.18 28.19 ± 1.22 1.04 ± 0.05 16

Validity criteria: Coefficient of variation in the control ( ≤ 15 %): max. 11 % Conclusions: After 28 days of incubation the deviations between control soil and soil treated with spirotetramat (applied with dose rates of 0.13 mg/kg soil and 1.32 mg/kg soil, corresponding to an application rate of 0.099 kg a. s./ha and 0.99 kg a.s./ha) was < 25 %, thus spirotetramat had no adverse effects o n N-transformation of soil microorganisms.

B.9.8.2 Carbon mineralization

Active substance

Reference: BYI 08330 tech.: Determination of effect s on carbon transformation in soil

Author(s), year: Lechelt-Kunze, C., 2004 c Report/Doc. number: LKC-C-30/04, Edition Number: M- 087840-01-1, 01.09.2004 Guideline(s): OECD 217 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.4 %, batch: 08045/0014 Material and methods: Test species: Soil microflora Type of test, duration: Carbon transformation test, 28 days Applied concentrations: 0 (control, purified water) , 0.13 and 1.32 mg/kg dry soil Solvent/vehicle: Quartz sand Toxic standard: Sodium chloride: 16 g/kg soil dw Test conditions: Test substrate: Natural Soil, collected on April 1 3 th , 2004 at a depth of 0-20 cm, from experimental field on Lacherhof Germany:– 134 –

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

C org : 0.7, pH: 5.6, texture (DIN): 6.3 % clay, 15.6 % silt, 78.1 % sand

, microbial biomass: 393 mg microbial C/kg soil dw, in addition glucose was added to the soil samples ( 2 g/kg soil dw) to induce maximum respiration rate. Substrate/test vessel: 350 g soil dw/500 mL brown glass bottles, three replicates per treatment Incubation: 20 ± 2 °C in the dark, about 40 % MHC Test parameters: Evolved CO 2 was measured with a gas analyser for at least 12 ho urs. Samples were taken at 0, 7, 14 and 28 days after in cubation. Statistics: F-test (homogeneity of variance), T-tes t (significant differences) Findings: pH: 5.6 (start), 5.5 – 5.6 (end) Effects test item: See table B.9.8.2-1

Table B.9.8.2-1: Effects of NOA446510 on glucose-in duced short-term respiration Day 0 Day 7 Day 14 Day 28

Treatment Mean

CO 2 (mg/h/kg) %

variation from control Mean CO 2

(mg/h/kg)) %

deviation from control Mean CO 2

(mg/h/kg)) %

deviation from control Mean

CO 2 (mg/h/kg) %

deviation from control
Control 268.3±4.2 - 284.6±2.3 - 290.56.4 - 264.5±1. 7 - 0.13 mg a.s./kg 251.7±7.7 6 256.9±3.1 10 267.85.9 8 247.3±5.2 6 1.3 mg a.s./kg 248.7±0.6 7 253.8±5.1 11 269.54.6 7 245.9±2.6 7

Validity criteria: Coefficient of variation in the control ( ≤ 15 %): max. 2.3 % Conclusions: After 28 days of incubation the deviations between control soil and soil treated with spirotetramat (applied with dose rates of 0.13 mg/kg soil and 1.33 mg/kg soil, corresponding to an application rate of 0.099 kg a. s./ha and 0.99 kg a.s./ha) was < 25 %, thus spirotetramat had no adverse effects o n C-transformation of soil micro-organisms.

B.9.8.3 Summary of effects on non-target micro-orga nisms

Test substance Test parameter Test concentration Ti me Effects (deviation from control) 0.13 mg a.s./kg (0.099 kg a.s./ha) 3 % Spirotetramat Nitrogen mineralization 1.3 mg a.s./kg (0.99 kg a.s./ha) 28 days 16 %
0.13 mg a.s./kg (0.099 kg a.s./ha) 6 % Spirotetramat Carbon mineralization 1.3 mg a.s./kg (0.99 kg a.s./ha) 28 days 7 %

B.9.8.4 Risk assessment for non-target micro-organi sms

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.– 135 –

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

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".

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

IIA 8.12, IIIA 10.8)

B.9.9.1 Effects on terrestrial non-target plants (O ECD IIIA 10.8.1)

B.9.9.1.1 Formulation

Reference: Non-target terrestrial plants: an evaluation of the effects of BYI 08330 OD 150 in the vegetative vigour test (Tier 1) Author(s), year: Gosch, H., Nguyen, D.H. (2006a) Report/Doc. number: VV06/03-Tier 1; M-273381-01-1 Guideline(s): OECD 208 B (July 2000, draft) GLP: No Deviations: None Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD, Batch no.: 08030 /0233(0152), Tox no.: TOX07087-00, content: 148.28 g a.s./L Material and Methods: Test species: Corn Zea mays , oats Avena sativa (Monocotyledonae: Poaceae), cucumber Cucumis sativus

(Dicotyledonae: Cucurbitaceae), oilseed rape Brassica napus (Dicotyledonae: Brassicaceae), soybean Glycine max (Dicotyledonae: Leguminosae), sunflower Helianthus annuus (Dicotyledonae: Asteraceae) Type of test: Vegetative vigour test Number of organisms: 4 to 5 replicates with 4 to 5 plants per pot for each species (19 - 20 plants per treatment group) Treatments: Control: deionised water; test item: 28 8 g a.s./ha in 200 L/ha. The test item was sprayed on the leaves and above-ground portions of the plants. Duration: 21 days after treatment application Soil characteristic: Standard soil: 20.7 % clay, 65 .1 % silt, 14.2 % sand, organic carbon: 1.19 %, pH: 7.4 Test design: At the 2 - 4 leaf stage the test item was sprayed on the leaves and above-ground portions of the plants. Test conditions: Glasshouse conditions: temperature : 18°C at night, 23°C at day; natural daylight supplemented by artificial lighting, 16 h photoperi od; Plants were watered from the bottom. Test parameter: Visual phytotoxicity ratings were d one at days 7, 14 and 21 after application. Plant biomass was determined per replicate (pot). Statistics: Pairwise Mann-Whitney-U-test, one sided smaller Findings: There were no adverse effects on survival.– 136 –

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

There were visible signs of phytotoxicity such as c hlorosis, necrosis, leaf deformation, bleaching, and/or stunting in all crop s tested. There were differences in growth stages in corn and oats. Biomass was statistically significantly reduced in all species tested. The differences exceeded the 50 % trigger in three of t he species tested. A summary of all the assessments for the day 21 veg etative vigour test (Tier 1) for the effects of BYI 08330 OD 150 is shown in the tab le below.
Table B.9.9.1.1-1: Phytotoxicity assessments for th e day 21 of the vegetative vigour test (Tier 1) for the effects of BYI 08330 OD 150 Cucumber Oilseed rape Soybean Sunflower Corn Oats Survival in number (out of 20) and (%) 19 (95) 20 (100) 20 (100) 20 (100) 20 (100) 20 (100 )

Inhibition of survival * (%) 5 0 0 0 0 0
Phytotoxicity (see below) B-C D A B-C C B
Dry Weight ** (% inhibition) 41.3 83.3 40.2 43.1 56.9 56.6

* compared to the control, where survival was 100 % (20 plants) in all cases ** inhibition expressed on a per plant basis (figur es in parentheses indicate that there was an increa se in dry weight when compared to the control) phytotoxicity ratings: 0 = no injury or effect A = slight symptom(s) throughout the whole plant or more moderate symptom(s) on a small area, i.e. one leaf B = moderate symptom(s) throughout the whole plant o r severe symptom(s) on a limited area, i.e. one-two leaves C = severe symptom(s) throughout the whole plant wi th younger or newly developed leaves growing normal ly D = total plant symptom(s) with the plant showing p oor vigour.
E = moribund Conclusion: An application rate of 288 g a.s./ha BYI 08330 OD 150 showed several significant adverse effects (i.e. greater than 50 %) to represe ntative non-target plants in the vegetative vigour test. Oilseed rape was the most s ensitive species tested. These results trigger further testing. Comments: Following deviations from US guideline requirement s are notable: - The EPA recommends using at least 4 monocot speci es including corn, and at least six dicot species in four families including soybean and a root crop. - US EPA recommends that shoot length/plant height effects be measured and reported.

Reference: Tier I and Tier II Vegetative Vigor Non-Target Plan t Study using BYI 08330 150 OD Author(s), year: Christ, M.T., Lam, C.V. (2006a) Report/Doc. number: EBFNX016; M-276131-01-1 Guideline(s): OECD 208 B (July 2000, draft), US EPA Guideline 122-1 & 123-1 GLP: Yes Deviations: None. Validity: Acceptable

Test substance: BYI 08330 150 OD, Batch no.: 08030/0189(0152), Tox no.: TOX07087-00, purity: 15.1 % w/w Material and Methods:– 137 –

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

Test species: 4 monocotyledonous plants: corn ( Zea mays, Poaceae), oat ( Avena sativa, Gramineae), ryegrass ( Lolium perenne , Poaceae), onion ( Allium cepa , Alliaceae) 6 dicotyledonous plants: cucumber ( Cucumis sativus, Cucurbitaceae), canola ( Brassica napus, Brassicaceae), soybean ( Glycine max. ), sunflower ( Helianthus

annuus L., Asteraceae), sugarbeet ( Beta vulgaris , Chenopodiaceae), tomato ( Lycopersicon esculentum , Solanaceae) Type of test: Vegetative vigour test Number of organisms: 8 replicates with 5 plants per pot for each species (40 seeds per treatment) Treatments: Control: deionised water; test item (no minal): monocotyledones: 11, 22, 44, 88, 176 g a.s./ha; Dicotyledones: 176 g a.s./ha. Nomina l test concentrations (mg/L): 0, 118, 235, 471, 941, 1882; measured test concentrati ons (mg/L): 0. 123, 252, 527, 1072, 2113; Calibration had been performed before a pplication. Duration: 21 days after treatment application Soil characteristic: Silt loam: 22 % clay, 50 % sil t, 28 % sand, organic matter: 2.4 % (organic carbon 1.2 %), pH: 7.8 Test design: At the 2 - 4 leaf stage the test item was sprayed using an Allen Track Sprayer from above the plant canopy. The test units were th en transferred to the greenhouse. Test conditions: Glasshouse conditions: temperature : 18.6 - 22.5°C; 37.1 - 61.2 % relative humidity; 16 h photoperiod; bottom watering. Test parameter: Visual phytotoxicity ratings were d one at days 7, 14 and 21 after application using the following rating system: 0 (no injury/effect), 10 to 20 % (indicates slight effect), 30 to 40 % (indicates moderate effect), 50 to 60 % (indicates severe effect), 70 to 80 % (indicates total plant effect), 90 % and above (plant death). Shoot height measurements were performed on day 21. Plant biomas s was determined per replicate (pot). Statistics: Descriptive statistics were performed u sing EXCEL. One-way-ANOVA and Dunett`s test were applied. Statistical analyses we re performed using SAS procedure. In Dec 2007 the applicant was asked to calculate, i f possible, EC 05 values. Due to the nature of the non-linear regression program, th e dose-response slope and standard error values could not be calculated. Most of the EC 05 values were not calculable because of the nature of the data. Findings: Measured test concentrations ranged from 104 to 11 4 % of nominal. Survival in all control groups was 100 %. Canola: Plant survival was not significantly affected at t he 176 g a.s./ha treatment level in comparison to the controls. Phytotoxicity was observed in one replicate on day 21 with minor observations of stunting. Plants were not significantly different for plant dry weight or length at the 176 g a.s./ha treatment by Dunnett’s Test as compared to the controls. Corn: Plant survival was not significantly affected at a ny treatment level in comparison to the controls. Phytotoxicity was obser ved throughout the study with observations of stunting, bleaching, leaf curling, and necrosis with severity of effects increasing with treatment concentration. Pl ants were significantly different for plant length and dry weight at the 88 and 176 g a.s./ha treatments by Dunnett’s Test as compared to the controls. The inhibition co mpared to the control for these two dosages were 18 % and 51 %, respectively for pl ant length, and 28 % and 62 % for dry weight. The EC 25 and EC 50 dry weight values were determined as 76 g a.s./ha and 134 g ai/ha (95 % cl: 117 to 155) by non-linear regression, respectively. The EC 50 for corn length was determined as 172 g ai/ha (95 % cl:– 138 –

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157 to 189). Oat: Plant survival was not significantly affected at a ny treatment level in comparison to the controls. Random phytotoxicity wa s observed throughout the study with observations of stunting, bleaching, lea f curling, and necrosis with severity of effects lessening by Day 21 in the high er treatment rates. Plants were not significantly different for plant length and dr y weight for any treatment by Dunnett’s Test as compared to the controls. Onion: Plant survival was not significantly affected at a ny treatment level in comparison to the controls. Minor phytotoxicity was observed throughout the study with observations of stunting, leaf curling, and ne crosis. Plants were not significantly different for plant length and dry we ight for any treatment by Dunnett’s Test as compared to the controls. Ryegrass: Plant survival was not significantly affected at a ny treatment level in comparison to the controls. Phytotoxicity was obser ved throughout the study with observations of stunting, bleaching, leaf curling, and necrosis with severity of effects increasing with treatment concentration. Pl ants were significantly different for plant length and dry weight at the 176 g a.s./h a treatment by Dunnett’s Test as compared to the controls. The inhibition compared t o the control was 14 % and 35 %, respectively, for these two parameters. The E C 25 dry weight values were determined as 168 g a.s./ha. Cucumber, soybean, sugarbeet, sunflower, tomato: Plant survival was not significantly affected at the 176 g a.s./ha treatme nt level in comparison to the controls. No phytotoxicity was observed in any trea tment. Plants were not significantly different for plant dry weight or len gth at the 176 g a.s./ha treatment by Dunnett’s Test as compared to the controls. The EC 25 plant length and dry weight values were determined as > 176 g a.s./ha. Endpoint results are summarised in table B.9.9.1.1- 2.

Table B.9.9.1.1-2a: Phytotoxic effects on ten plant species in a vegetative vigour test [11 – 176 g a.s./ha]. EC figures ± 95 % CI

Survival Plant length NOEC LOEC EC25 NOEC LOEC EC05 EC25 EC50
Canola 176 >176 >176 176 >176 >176 >176 >176

Corn 176 >176 >176 44 88 53 (38-72) 106

(92-122) 172

(157-189) Cucumber 176 >176 >176 176 >176 >176 >176 >176 Oat 176 >176 >176 176 >176 >176 >176 >176 Onion 176 >176 >176 176 >176 >176 >176 >176 Ryegrass 176 >176 >176 88 176 >176 >176 >176 Soybean 176 >176 >176 176 >176 >176 >176 >176 Sugarbeet 176 >176 >176 176 >176 >176 >176 >176 Sunflower 176 >176 >176 176 >176 >176 >176 >176 Tomato 176 >176 >176 176 >176 >176 >176 >176– 139 –

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

Plant dry weight NOEC LOEC EC05 EC25 EC50
Canola 176 >176 >176 >176 >176 Corn 44 88 34 (22-54) 76 (60-96) 134 (117-155) Cucumber 176 >176 >176 >176 >176 Oat 176 >176 >176 >176 >176 Onion 176 >176 >176 >176 >176 Ryegrass 88 176 176 168 (153-184) >176 Soybean 176 >176 >176 >176 >176 Sugarbeet 176 >176 >176 >176 >176 Sunflower 176 >176 >176 >176 >176 Tomato 176 >176 >176 >176 >176

Table B.9.9.1.1-2b: Mean plant length and dry weigh t data of control plants

Control Mean plant length (cm) Mean plant dry weigh t (g) Canola 35.7 8.652 Corn 78.8 6.424 Cucumber 34.4 9.132 Oat 58.2 3.533 Onion 31.7 0.628 Ryegrass 51.9 3.433 Soybean 27.6 3.857 Sugarbeet 22.8 5.568 Sunflower 70.6 14.429 Tomato 33.5 4.908

Conclusion: In a vegetative vigour study with 10 non-target te rrestrial plant species, following a 21 day post-emergent foliar application of BYI 0833 0 150 OD, the most sensitive endpoint was corn dry weight with an EC 25 value of 76 g a.s./ha (95 % CI: 60-96) and an EC 50 value of 134 g a.s./ha (95 % cl: 117 to 155). The NOEC for corn was 44 g a.s./ha.

Reference: Non-target terrestrial plants: an evaluation of the effects of BYI 08330 OD 150 in the seedling emergence and growth test (Tier 1) Author(s), year: Gosch, H., Nguyen, D.H. (2006b) Report/Doc. number: SE06/03-Tier 1; M-273373-01-1 Guideline(s): OECD 208 A (July 2000, draft) GLP: No Deviations: None Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030 /0233(0152), Tox no.: TOX07087-00; analytical content: 148.28 g a.s./L Material and Methods: Test species: 2 monocotyledonous plants: corn ( Zea mays, Poaceae), oat ( Avena sativa, Gramineae), 4 dicotyledonous plants: cucumber ( Cucumis sativus, Cucurbitaceae), oilseed rape ( Brassica napus, Brassicaceae), soybean ( Glycine

max. ), sunflower ( Helianthus annuus L., Asteraceae) Type of test: Seedling emergence and growth test Number of organisms: 20 plants per treatment group (4 pots with 5 seeds each)– 140 –

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Treatments: Control: deionised water; test item: 28 8 g a.s./ha in a spray volume of 200 L/ha. Calibration performed before application. Duration: 14 days following the emergence of 70 % o f control seedlings Soil characteristic: Silty loam: 20.7 % clay, 65.1 % silt, 14.2 % sand, organic content: 1.19 %, pH: 7 .4 Test design: The day before application seeds were introduced manually in the soil. They were covered by about 2 to 5 mm with soil. Plant pots we re sprayed. Test conditions: Temperature: 18°C at night, 23°C a t day (short deviations); humidity: 28 - 98 %, 16 h photoperiod of natural daylight supplemented b y artificial lighting; Plants were initially top watered and then bottom watered. Commercial plastic flower pots of 10-cm diameter containing 420 g soil were used. Test parameters: Germination: Daily checks were made to identify the date when 7 0 % of the seedlings emerge in the control for each species. T he number of plants was counted after 7 and 14 days. Mortality/Survival: Dead plants were removed after each assessment. Th e number of plants that survived after application was recor ded at the final assessment. Phytotoxicity records: Visual phytotoxicity ratings were recorded (e.g. c hlorosis, necrosis, stunting, abnormal growth) 7 and 14 days after the emergence of 70 % of seeds in the controls. Phytotoxicity was assesse d according to EPPO Standard 135. Growth stages: Growth stages were recorded at the final assessmen t according to BBCH-Monograph - Growth stages. Plant biomass: The dry weights were determined at the final asses sment (day 14 after 70 % emergence in the untreated control). The plants of one pot represent one replicate. Statistics: Statistical analysis was carried out us ing the Pairwise Mann-Whitney-U-test (one sided smaller). Findings: The germination of the control plants was at least 95 % and all controls exhibited normal growth. There was no significant adverse effect of BYI 0833 0 OD 150 on the six species tested. Biomass was less compared to control in soybean, co rn and oats by 0.2 %, 10.6 % and 8.1 %, respectively. Biomass was greater compared to control in cucumber, oilseed rape and sunflower by 0.9 %, 8.1 %, and 1.1 % respectively. None of these differences were significant at the 9 5 % confidence limits and no adverse effect reached or exceeded the 50 % trigger for further testing. No phytotoxicity was observed in any of the tested species, except a marginal stunting in oats. There were no differences in grow th stages, except a marginal difference in corn.– 141 –

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Table B.9.9.1.1-3: Effects of Spirotetramat 150OD o n six plant species in a seedling emergence and survival test [288 g a.s./ha] 14 d seedling emergence 14 d seedling survival

Treatment Control 288 g a.s./ha Control 288 g a.s. /ha

Species % Emergence % Emergence % Survival % Survival

Cucumber 90 100 100 100 Oilseed rape 100 100 100 100 Soybean 100 100 100 100 Sunflower 95 100 100 100 Corn 100 100 100 95 Oat 95 95 100 100 % Emergence = (No. plants emerged / No. seeds plante d) x 100 % Survival = (No. plants surviving / No. seeds emerg ed) x 100

Conclusion: An application of 288 g a.s./ha BYI 08330 OD 150 s howed no significant adverse effect (i.e. greater than 50 %) to representative n on-target plants in the seedling emergence and growth test. The EC 50 figures for all tested species were therefore > 28 8 g a.s./ha. Comments: Following deviations from US guideline requirement s are notable: - The EPA recommends using at least 4 monocot speci es including corn, and at least six dicot species in four families including soybean and a root crop. - US EPA recommends that shoot length/plant height effects be measured and reported. - The EPA recommends 10 seeds/replicate and 4 repli cates/dose; only 5 seeds/replicate and 4 replicates/dose were used in this study.

Reference: Tier I Seedling Emergence Non-Target Plant Study us ing BYI 08330 150OD Author(s), year: Christ, M.T., Lam, C.V. (2006b) Report/Doc. number: EBFNX017; M-276244-01-1 Guideline(s): US-EPA 122-1 GLP: Yes Deviations: Short deviation in the photoperiod from 13 to 15 h. The EPA recommends 10 seeds/replicate and 4 replicates/dose; however, 5 s eeds/replicate and 8 replicates/dose were used in this study. Organic ca rbon content of the soil was slightly higher (2.4 %) than what EPA recommends (2 %). These deviations do not affect the validity of the study. Validity: Acceptable

Test substance: Spirotetramat (BYI 08330) 150 OD; Batch no.: 08030 /0189(0152); analytical content: 15.1 % w/w; Material and Methods: Test species: 4 monocotyledonous plants: corn ( Zea mays, Poaceae), oat ( Avena sativa, Gramineae), ryegrass ( Lolium perenne , Poaceae), onion ( Allium cepa , Alliaceae) 6 dicotyledonous plants: cucumber ( Cucumis sativus, Cucurbitaceae), canola ( Brassica napus, Brassicaceae), soybean ( Glycine max. ), sunflower ( Helianthus

annuus L., Asteraceae), sugarbeet ( Beta vulgaris , Chenopodiaceae), tomato ( Lycopersicon esculentum , Solanaceae) Type of test: Seedling emergence and growth test Number of organisms: 40 plants per treatment group (8 pots with 5 seeds each)– 142 –

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Treatments: Control: deionised water; test item: 17 6 g a.s./ha in a spray volume of 93.5 L/ha. Calibration performed before application. The nomin al a.s. concentration in the spray solution was 1882 g a.s./L, the measured conc entration was 1954 g a.s./L, representing 104 % of nominal. Duration: 21 days Soil characteristic: 22 % clay, 50 % silt, 28 % san d, organic content: 2.4 %, pH: 7.8 Test design: The day before application seeds were introduced in the soil. They were covered by about 1.2 or 2.5 cm with soil. The spray solutio n was applied to the soil surface using an Allen Track Sprayer. Test conditions: Temperature: 18.6 - 24.4°C; humidi ty: 28 - 71 %, 16 h photoperiod of natural daylight supplemented by artificial lighting; Plant s were bottom watered; commercial plastic flower pots were used. Test parameters: Samples of the spray stock solutio ns were collected at study initiation on day 0 and analysed for the active substance. The endpoints evaluated on Day 7, Day 14, and Day 2 1 following spray application were the number of emerged seedlings, n umber of seedlings surviving, and phytotoxicity of each replicate and were evaluated by visual enumeration using the following rating system: 0 (n o injury/effect), 10 to 20 % (indicates slight effect), 30 to 40 % (indicates mo derate effect), 50 to 60 % (indicates severe effect), 70 to 80 % (indicates to tal plant effect), 90 % and above (plant death). Plants were excised at the soil surface on Day 21 f or plant height and dry weight determination. Plant shoot height was measured by e xtending cut seedlings to their maximum length and recording. Plant dry weigh t was performed by placing all replicate plants within labelled paper bags. Th e plants were placed in a drying oven and allowed to dry for at least 48 hours. Statistics: All Day 21 data were subjected to a Sha piro-Wilk’s Test to assess departures from a normal distribution and a Levene’s Test to d etermine homogeneity of variance. To assess treatment effects between the c ontrol and treatment group, a Student’s t-test was performed. All statistical ana lyses were performed using the computer program SAS, descriptive statistics were p erformed using EXCEL. Findings: The measured a.s. concentration was 104 % of nomin al. Percent seedling emergence for the 10 plant species ranged from 88 to 100 % in the controls and from 80 to 100 % in the 176 g a.s. /ha treatment. Percent seedling survival for the 10 plant species ranged from 91 to 100 % in the controls, and 84 to 100 % in the 176 g a.s./ha treatment. Phytotoxicity ratings for the seedling emergence te st ranged from 1 to 13 % for the 176 g a.s./ha treatment for all species. Simila r phytotoxicity of minor necrosis and stunting was observed in the controls and treat ed levels of canola, the species most sensitive in this respect. The observe d phytotoxicity was considered to be not treatment related as the level of inciden ce was similar in both the control and treated levels. Percent inhibition for plant length for the 10 plan t species ranged from -4 to 8 % in comparison to the controls and the 176 g a.s./ha tr eatment. Percent inhibition for plant dry weight ranged from -13 to 19 % in compari son to the controls and the 176 g a.s./ha treatment. The percent effect for each endpoint (i.e. emergenc e, survival, plant length, dry weight) was 19 % or less for each plant species. No significant effects were observed to any of the 10 species tested. Summarised results are given in table B.9.9.1.1-4.– 143 –

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Table B.9.9.1.1-4: Phytotoxic effects of Spirotetra mat 150OD on ten plant species in a seedling emergence and survival test [176 g a.s./ha] 21 d seedling emergence 21 d seedling survival

Treatment Control 176 g a.s./ha Control 176 g a.s. /ha

Species % Emergence % Emergence % Survival % Survival

Canola 88 80 91 84 Corn 93 98 100 100 Cucumber 95 98 97 100 Oat 98 98 100 100 Onion 100 98 100 100 Ryegrass 100 95 100 100 Soybean 98 93 100 100 Sugarbeet 93 95 100 97 Sunflower 95 100 100 100 Tomato 90 90 100 100 % Emergence = (No. plants emerged / No. seeds plante d) x 100 % Survival = (No. plants surviving / No. seeds emerg ed) x 100 Conclusion: There were no significant adverse effects for any measured parameter at or above 25 % as compared to the controls in a Tier I seedli ng emergence study at the single application rate of 176 g a.s./ha. Because t here were no effects at or above 25 %, a Tier 2 study is not required. The EC 50 figures for all tested species were therefore > 17 6 g a.s./ha.

Reference: Spirotetramat OD 150: Vegetative vigour test on non -target terrestrial plants Author(s), year: Friedrich, S. (2006) Report/Doc. number: M-279698-01-1 Guideline(s): OECD 227 (Draft 2005) GLP: Yes (certified laboratory) Deviations: None Validity: Acceptable

Test substance: Spirotetramat OD 150; Batch no.: 08030/0189(0152), purity: 15.1 % w/w analysed Material and Methods: Test species: 2 monocotyledonous plants: corn ( Zea mays, Poaceae), oat ( Avena sativa, Gramineae), 1 dicotyledonous plant: canola ( Brassica napus, Brassicaceae) Type of test: Vegetative vigour test Number of organisms: 20 / 10 / 8 replicates with 2 / 4 / 5 plants per pot for corn / rape / oats (40 p lants per treatment) Treatments: Control: deionised water; test item (no minal): 0.17, 0.48, 0.96, 1.92 L product/ha in 400 L water; nominally equivalent to 26, 72, 144 , 288 g a.s./ha; corresponding to actually applied 0.17, 0.47, 0.94, 1.86 L produc t/ha; Calibration had been performed before application. Duration: 21 days after treatment application Soil characteristic: Agricultural soil, sandy loam: 12.4 % clay, 48.8 % silt, 38.9 % sand, organic carbon: 0.9 %, pH: 6 Test design: At the 2 leaf stage the test item was sprayed onto the leaves and the soil substrate using an automatic application cabin. Test conditions: Glasshouse conditions: temperature : 11 - 41°C; 17 - 99 % relative humidity; 16 h photoperiod; bottom watering.– 144 –

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Test parameter: Mortality and visual phytotoxicity were assessed at days 7, 14 and 21 after application. The severity of phytotoxic symptoms wa s described, e.g. chlorosis, necrosis, wilting (%/plant). Shoot fresh weight mea surements were performed on day 21. Plant biomass was determined per replicate (pot). Statistics: Computation of EC 50 (e.g. probit analysis). Software ToxRat Profession al used. In Dec 2007 probit analysis using max. likelihood r egression was conducted to derive EC 05 and EC 25 values with regard to shoot fresh weight. Findings: Measured test concentrations ranged from to 82.1 t o 90.9 % of nominal. Survival was 100 % in all treatment and the control group. B. napus: no phytotoxic symptoms. Growth inhibition effects i n the two highest rates (11 and 35 %, respectively). A. sativa: no phytotoxic symptoms. Growth inhibition effects i n the two highest rates (9 and 44 %, respectively). Z. mays : Necrotic, chlorotic and growth inhibitory effects in the two highest rates (with regard to the latter 7 and 41 %, respectively ). Endpoint results are summarised in table B.9.9.1.1- 5. Details of probit analysis of the shoot fresh weigh t: B. napus : Slope: 2.53929, Intercept: 4.04718, Variance of slope: 27.86593, Ch i 2 : 0.00871, degrees of freedom: 2, p(Chi 2 ): 0.99565, r 2 : 0.964 A. sativa : Slope: 2.94907, Intercept: 3.88868, Variance of s lope: 44.36333, Chi 2 : 0.01456, degrees of freedom: 2, p(Chi 2 ): 0.99275, r 2 : 0.931 Z. mays : Slope: 4.62209, Intercept: 3.49296, Variance of s lope: 148.26663, Chi 2 : 0.00001, degrees of freedom: 2, p(Chi 2 ): 1.00000, r 2 : 1.000

Table B.9.9.1.1-5: Phytotoxic effects on three plan t species in a vegetative vigour test after treatme nt with Spirotetramat OD 150 [L product/ha] 21 DAA Survival Shoot fresh weight species LC50 NOEC EC50 BBCH

B. napus > 1.92 0.48 > 1.92 15-17 A. sativa > 1.92 0.96 > 1.92 16-21 Z. mays > 1.92 0.96 > 1.92 16-18

Table B.9.9.1.1-6: Effects on shoot fresh weight (g ) of three plant species after 21 d exposure to Spirotetramat OD 150 [L product/ha, nominal] Treatment group Control 0.17 0.48 0.96 1.92 B. napus

Mean ± SD 20.76 ± 2.19 20.41 ± 2.21 19.39 ± 2.97 17.87 ± 1.80 12.16 ± 1.95 % reduction - 2 7 14* 41* A. sativa

Mean ± SD 15.22 ± 1.24 15.45 ± 1.52 14.25 ± 2.08 13.67 ± 1.42 9.18 ± 1.75 % reduction - -2 6 10 40* Z. mays

Mean ± SD 34.76 ± 3.01 36.58 ± 3.57 35.13 ± 2.47 32.81 ± 2.76 20.10 ± 3.35 % reduction - -5 -1 6 42* * statistically significant (Dunnett test, p ≤ 0.05), SD standard deviation– 145 –

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

Tab. B.9.9.1.1-7: Results of the probit analysis of shoot fresh weight data: Selected effective concentrations (ECx, L product/ha) of the test item and their 95 %- and 99 %-confidence limits Parameter EC05 EC25 EC50 EC75 EC90 B. napus Value [L product/ha] 0.534 1.290 2.373 4.363 7.575

lower 95 %-cl 0.114 0.866 1.849 2.838 4.067 upper 95 %-cl 0.818 1.614 4.907 20.750 78.794 lower 99 %-cl 0.031 0.577 0.977 0.774 0.587 upper 99 %-cl 2.970 2.423 9.284 76.109 546.253 A. sativa Value [L product/ha] 0.659 1.409 2.381 4.024 6.470

lower 95 %-cl 0.008 0.564 1.807 2.550 3.358 upper 95 %-cl 1.039 1.880 16.834 342.214 5434.059 lower 99 %-cl 0.000 0.257 0.421 0.104 0.027 upper 99 %-cl 25.149 4.129 72.336 8399.393 678412.3 75 Z. mays Value [L product/ha] 0.934 1.516 2.119 2.961 4.009

lower 95 %-cl 0.920 1.508 2.108 2.929 3.942 upper 95 %-cl 0.947 1.524 2.130 2.994 4.080 lower 99 %-cl 0.902 1.497 2.094 2.887 3.854 upper 99 %-cl 0.966 1.535 2.144 3.038 4.173

Conclusion: In a vegetative vigour study with three non-target terrestrial plant species, following a 21 day post-emergent foliar application of Spirotetramat OD 150, the most sensitive endpoint was shoot fresh weight of o ilseed rape with a NOEC value of 72 g a.s./ha. However, the EC50`s for all species were > 288 g a.s./ha. Comments: The study does not comply with EPA guidelines (e.g . EPA commented the variation in temperature and relative humidity to b e too high).

B.9.9.2 Risk assessment for terrestrial non-target plants

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".– 146 –

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

B.9.10 Effects on biological methods of sewage trea tment (OECD IIA 8.15)

B.9.10.1 Activated sludge, respiration inhibition

Active substance

Reference: BYI 08330 - Toxicity to bacteria Author(s), year: Bruns; Caspers, N. 2004 Report/Doc. number: 1312 N/04 B, Edition Number: M- 275364-01-1, 05.10.2004 Guideline(s): OECD 209 GLP: Yes Deviations: None of relevance Validity: Acceptable

Test substance: Spirotetramat (BYI 08330), purity: 97.2 %, batch: 08045/0014 Material and methods: Test species: Activated sludge Source: Aeration tank of a domestic sewage treatmen t plant Type of test, duration: Laboratory aerobic activate d sludge inhibition test, 3 hours Applied concentrations: Nominal: 1000, 1800, 3200, 5600, 10000 mg/L Toxic standard: 3,5-dichlorophenol (97 %): 2.5, 5. 0, 10, 20, 40 mg/L Test conditions: Substrate/test vessel: Activated sludge (solid con centration: 480 mg/L) treated with active substance

, reference substance or water (control) Incubation: Aerated for 3 hours at 20 ± 2°C Test parameters: Inhibition of respiration rate (ra te of oxygen uptake) Statistics: None, because the inhibition at highest tested concentration was 22.2 mg/L Findings/conclusions: See table B.9.10.1-1

Table B.9.10.1-1: Effect of spirotetramat on the re spiration rate of activated sludge

Test Substance Nominal concentration [mg/L] pH Respiration rate [mg O 2 /L/h] Inhibition [%]

3,5-DCP a) 2.5 7.8 26.4 9.9 3,5-DCP 5.0 7.7 22.8 22.2 3,5-DCP 10 7.6 10.5 64.2 3,5-DCP 20 7.6 4.0 86.3 3,5-DCP 40 7.7 2.0 93.2 Control 0 7.7 (mean) 29.3 (mean) -- BYI 08330 1000 7.6 30.0 0.0 BYI 08330 1800 7.6 27.6 5.8 BYI 08330 3200 7.6 27.0 7.9 BYI 08330 5600 7.6 26.0 11.3 BYI 08330 10000 7.6 22.8 22.2
3 hours EC 50 > 10000 mg/L a) 3,5-dichlorophenol

Conclusions: No inhibitory effects on aerobic waste water micro -organisms are expected if the test substance is correctly applied at the intended application rate.– 147 –

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

B.9.10.2 Risk assessment for biological methods of sewage treatment

The risk assessment for the EU can be found in the Appendix at the end of section B.9. The risk assessment for the PMRA and the US-EPA is not included in this monograph.

The Proposed Registration Decision – Spirotetramat (PRD2008-07), containing the detailed Canadian review and risk assessment, has been published by t he PMRA and can be found at the PMRA’s web site www.pmra-arla.gc.ca

.

The registration decision made by the U.S. EPA's Of fice of Pesticide Program is supported by the risk assessments found at our Federal Docket website loc ated at: http://www.regulations.gov/fdmspublic/component/mai n?main=AdvancedDocket The Docket ID is "EPA-HQ-OPP-2007-0475".– 148 –

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B.9.11 References relied on

Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

Annex II Data and Information IIA 8.1.1 Barfknecht, R. 2003 Acute oral toxicity for bobwhite quail ( Colinus virginianus ) for the test item BYI 08330 (tech.) Bayer CropScience AG, Report No.: M-115911-02-2, Edition Number: M-115911-02-2 Date: 29.10.2003, Amended: 13.01.2006 GLP unpublished Yes BCS

IIA 8.1.2 Barfknecht, R. 2004 BYI 08330 techn. a.s.: 5 -day-dietary LC50 bobwhite quail ( Colinus virginianus ) Bayer CropScience AG, Report No.: BAR/LC017, Edition Number: M-084796-01-2 Date: 18.08.2004 GLP unpublished Yes BCS

IIA 8.1.3 Stoughton, T. L. 2006 Technical BYI 08330: A subacute dietary LC50 with mallards Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX012, Edition Number: M-274385-01-1 Date: 03.05.2006 GLP unpublished Yes BCS

IIA 8.1.4 Sabbert, T. J.;

Nuessle, C. 2006

a Effect of technical BYI 08330 on northern bobwhite reproduction Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNO220, Edition Number: M-277757-01-1 Date: 21.09.2006 GLP unpublished Yes BCS

IIA 8.1.4 Sabbert, T. J.;

Nuessle, C. 2006

b Effect of technical BYI 08330 on mallard reproduction Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX013, Edition Number: M-277794-01-2 Date: 22.09.2006 GLP unpublished Yes BCS

IIA 8.1.4 Stafford, J. M. 2007

a Long-Term Mallard Duck Dermal Exposure to BYI 08330 Bayer CropScience AG, Report No.: EBFNP033 Edition Number: M-290165-01-1 Date: 05.07.2007 GLP unpublished Yes BCS– 149 –

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Y/N-R/NR Owner

IIA 8.1.4 Stafford, J. M. 2007 b Semi-Field Study of the Effects of Dermal and Dietary Exposure to BYI 08330 in Mallard Ducks ( Anas platyrhynchos ) Bayer CropScience AG, Report No.: EBFNP034 Edition Number: M-290000-01-1 Date: 26.06.2007 GLP unpublished Yes BCS

IIA 8.1.4 /03 Temple, D.L;

Martin, K.H.; Beavers, J.B.; Jaber, M. 2006 BYI 08330 technical: A reproduction study with the mallard Wildlife International Ltd., Easton, MD, USA Bayer CropScience AG, Report No.: EBFNP024, Edition Number: M-278058-01-1 Date: 29.09.2006 GLP unpublished Yes BCS

IIA 8.2.1.1 Dorgerloh, M. 2004

a Acute toxicity of BYI 08330 (tech.) to fish (Oncorhynchus mykiss ) Bayer CropScience AG, Report No.: DOM 24025, Edition Number: M-182649-01-2 Date: 16.12.2004 GLP unpublished Yes BCS

IIA 8.2.1.2 Dorgerloh, M. 2004 b Acute toxicity of BYI 08330 (tech.) to fish ( Cyprinus carpio ) Bayer CropScience AG, Report No.: DOM24022, Edition Number: M-128667-01-2 Date: 25.11.2004 GLP unpublished Yes BCS

IIA 8.2.1.2 Dorgerloh, M. 2005

a Acute toxicity of BYI 08330 (tech.) to fish ( Lepomis macrochirus ) Bayer CropScience AG, Report No.: DOM 24056, Edition Number: M-242689-01-2 Date: 12.01.2005 GLP unpublished Yes BCS

IIA 8.2.1.3 Nieden, D. 2005

a Acute toxicity of BYI 08330-enol to fish ( Oncorhynchus mykiss ) Bayer CropScience AG, Report No.: EBFNM013, Edition Number: M-251850-01-2 Date: 18.05.2005 GLP unpublished Yes BCS– 150 –

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Y/N-R/NR Owner

IIA 8.2.1.3 Neuhahn, A. 2006

a 4-Methoxycyclohexanone - Acute fish toxicity Bayer Industry Services, Leverkusen Bayer CropScience AG, Report No.: 2006/0032/02, Edition Number: M-274305-01-1 Date: 10.07.2006 GLP unpublished Yes BCS

IIA 8.2.4 Dorgerloh, M. 2005 b Early-life stage toxicity of BYI 08330 tech. to fish ( Pimephales promelas ) Bayer CropScience AG, Report No.: EBFN0305, Edition Number: M-260676-01-2 Date: 16.11.2005 GLP unpublished Yes BCS

IIA 8.3.1.1 Dorgerloh, M. 2005

c Acute toxicity of BYI 08330 (tech.) to the waterflea Daphnia magna under static conditions Bayer CropScience AG, Report No.: DOM 24004, Edition Number: M-242683-01-2 Date: 14.01.2005 GLP unpublished Yes BCS

IIA 8.3.1.1 Dorgeloh, M. 2005 d Acute toxicity of BYI 08330-enol to the waterflea Daphnia magna in a static laboratory test system -limit test Bayer CropScience AG, Report No.: EBFNM012, Edition Number: M-254487-01-2 Date: 14.07.2005 GLP unpublished Yes BCS

IIA 8.3.1.1 Neuhahn, A. 2006 b 4-Methoxycyclohexanone - Acute daphnia toxicity Bayer Industry Services, Leverkusen, Germany Bayer CropScience AG, Report No.: 2006/0032/01, Edition Number: M-274275-01-1 Date: 10.07.2006 GLP unpublished Yes BCS

IIA 8.3.2.1 Dorgerloh, M. 2005

e Influence of BYI 08330 (tech.) on development and reproductive output of the waterflea Daphnia magna in a static renewal laboratory test system Bayer CropScience AG, Report No.: EBFN0245, Edition Number: M-251843-01-2 Date: 13.05.2005 GLP unpublished Yes BCS– 151 –

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Y/N-R/NR Owner

IIA 8.4 Dorgerloh, M. 2004

c Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330 (tech.) Bayer CropScience AG, Report No.: DOM 23092, Edition Number: M-128874-01-2 Date: 29.11.2004 GLP unpublished Yes BCS

IIA 8.4 Kern, M.E.;

Lam, C.V. 2005 Toxicity of BYI 08330 to the freshwater diatom Navicula pelliculosa Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX008, Edition Number: M-252794-01-1 Date: 15.06.2005 Non GLP unpublished Yes BCS

IIA 8.4 Kern, M. E.;

Lam, C. V. 2006 Toxicity of BYI 08330 to the blue-green alga Anabaena flos-aquae Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX007, Edition Number: M-264055-01-1 Date: 12.01.2006 GLP unpublished Yes BCS

IIA 8.4 Dorgerloh, M. 2005f Pseudokirchneriella subcapitata - Growth inhibition test with BYI 08330-enol Bayer CropScience AG, Report No.: EBFNM010, Edition Number: M-252531-01-2 Date: 06.06.2005 GLP unpublished Yes BCS

IIA 8.4 Weyers, A. 2006 4-Methoxycyclohexanone - Alga growth inhibition test Bayer Industry Services, Leverkusen, Germany Bayer CropScience AG, Report No.: 2006/0032/03, Edition Number: M-275954-01-1 Date: 28.07.2006 GLP unpublished Yes BCS

IIA 8.5.1 Dorgerloh, M. 2005 g Acute toxicity of BYI 08330 (tech.) to larvae of Chironomus riparius in a 48 h water-only study Bayer CropScience AG, Report No.: EBFNX072, Edition Number: M-262632-01-2 Date: 08.12.2005 GLP unpublished Yes BCS– 152 –

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Y/N-R/NR Owner

IIA 8.5.1 Dorgerloh, M. 2005 h Acute toxicity of BYI 08330-enol to larvae of Chironomus riparius in a 48 h water-only study Bayer CropScience AG, Report No.: EBFNX073, Edition Number: M-262638-01-2 Date: 08.12.2005 GLP unpublished Yes BCS

IIA 8.5.1 Dorgerloh, M. 2006

a Acute toxicity of BYI 08330-ketohydroxy to larvae of Chironomus riparius in a 48 h static laboratory test system Bayer CropScience AG, Report No.: EBFNO347, Edition Number: M-263956-01-2 Date: 16.01.2006 GLP unpublished Yes BCS

IIA 8.5.1 Dorgerloh, M. 2007

a Acute toxicity of 4-Methoxycyclohexanon to Larvae of Chironomus riparius in a 48 h static laboratory test system (Limit-Test) Bayer CropScience AG, Report No.: EBFX104, Edition Number: M-287218-01-1 Date: 27.04.2007 GLP unpublished Yes BCS

IIA 8.5.1 Dorgerloh, M. 2007 b Acute toxicity of BYI 08330-cis-methoxycyclohexylamino carboxylic acid to Larvae of Chironomus riparius in a 48 h static laboratory test system (Limit-Test) Bayer CropScience AG, Report No.: EBFX105, Edition Number: M-287220-01-1 Date: 27.04.2007 GLP unpublished Yes BCS

IIA 8.5.2 Dorgerloh, M. 2005i Chironomus riparius 28-day chronic toxicitytest with BYI 08330 (tech.) in a watersediment system using spiked water Bayer CropScience AG, Report No.: EBFN0050, Edition Number: M-248099-02-2 Date: 16.03.2005, Amended: 29.06.2006 GLP unpublished Yes BCS

IIA 8.6 Dorgerloh, M. 2005j Lemna gibba G3 - Growth inhibition test with BYI 08330 (tech.) under static-renewal test conditions Bayer CropScience AG, Report No.: DOM 24019, Edition Number: M-255296-01-2 Date: 29.07.2005 GLP unpublished Yes BCS– 153 –

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Y/N-R/NR Owner

IIA 8.6 Dorgerloh, M. 2005 k Lemna gibba

G3: Growth inhibition test with BYI 08330-enol under static conditions Bayer CropScience AG, Report No.: EBFNX065, Edition Number: M-252412-01-2 Date: 07.06.2005 GLP unpublished Yes BCS

IIA 8.7.1 Schmitzer, S. 2004 Effects of BYI 08330 (acute contact and oral) on honey bees ( Apis mellifera L.) in the laboratory Ibcaon GmbH, Rossdorf, Germany Bayer CropScience AG, Report No.: 20091035, Edition Number: M-081227-01-2 Date: 12.07.2004 GLP unpublished Yes BCS

IIA 8.7.4 Schur, A. 2004

a Assessment of the side-effects of BYI 08330 OD 100 on the honey bee ( Apis mellifera L.) in a feeding test GAB Biotechnologie GmbH & IFUUmweltanalytik GmbH, NiefernOeschelbronn, Germany Bayer CropScience AG, Report No.: 20031168/01-BFEU, Edition Number: M-000345-01-2 Date: 23.02.2004 GLP unpublished also filed: IIIA2 10.4.2.1 also filed: IIIA2 10.4.2.2 Yes BCS

IIA 8.7.4 Schur, A. 2004 b Assessment of the side-effects of BYI 08330 SC 240 on the honey bee ( Apis mellifera L.) in a feeding test GAB GmbH, Niefern-Oeschelbronn, Germany Bayer CropScience AG, Report No.: 20031166/01-BFEU, Edition Number: M-121877-01-2 Date: 05.02.2004 GLP unpublished also filed: IIIA3 10.4.2.1 Yes BCS

IIA 8.8.1.1 Waltersdorfer,

A. 2005

a Toxicity to the parasitoid wasp Aphidius rhopalosiphi

(DeStephani-Perez) (Hymenoptera: Bracconidae) in the laboratory; BYI 08330 150 OD; Code: AE 1302943 00 OD15 A101 Bayer CropScience AG, Report No.: CW05/023, Edition Number: M-254442-01-1 Date: 15.07.2005 GLP unpublished Yes BCS– 154 –

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Y/N-R/NR Owner

IIA 8.8.1.2 Waltersdorfer,

A. 2005

b Toxicity to the predatory mite Typhlodromus pyri

Scheuten (Acari, Phytoseiidae) in the laboratory, BYI 08330 150 OD; Code AE 1302943 00 OD15 A101 Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: CW05/024, Edition Number: M-254269-01-1 Date: 12.07.2005 GLP unpublished Yes BCS

IIA 8.8.2.1 Waltersdorfer,

A. 2006

a Toxicity to the parasitoid wasp Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera: Braconidae) using an extended laboratory test with BYI 08330 150 OD Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: CW05/071, Edition Number: M-264179-01-1 Date: 18.01.2006 GLP unpublished Yes BCS

IIA 8.8.2.2 Waltersdorfer,

A. 2005

c Toxicity to the predatory mite Typhlodromus pyri

SCHEUTEN (Acari, Phytoseiidae) using an extended laboratory test BYI 08330 150 OD Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: CW05/030, Edition Number: M-256306-01-1 Date: 25.08.2005 GLP unpublished Yes BCS

IIA 8.8.2.4 Roehlig, U. 2005 Dose-response toxicity (LR50) of BYI 08330 150 OD to the green lacewing Chrysoperla carnea

(STEPH.) under extended laboratory conditions (including food-application) BioChem agrar, Gerichshain, Germany Bayer CropScience AG, Report No.: 051048082, Edition Number: M-262650-01-1 Date: 14.12.2005 GLP unpublished Yes BCS– 155 –

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Y/N-R/NR Owner

IIA 8.8.2.4 Waltersdorfer,

A. 2006

b Toxicity to the ladybird Coccinella septempunctata

L. (Coleoptera, Coccinellidae) using an extended laboratory test including exposure to and oral uptake of BYI 08330 150 OD Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: CW05/053, Edition Number: M-264175-01-1 Date: 18.01.2006 GLP unpublished Yes BCS

IIA 8.9.1 Lechelt-Kunze,

C. 2004

a BYI 08830 (techn.): Acute toxicity to earthworms ( Eisenia fetida ) tested in artifical soil with 5 % peat Bayer CropScience AG, Report No.: LKC/RG-A-18/03, Edition Number: M-104105-01-2 Date: 07.01.2004 GLP unpublished Yes BCS

IIA 8.9.1 Lechelt-Kunze,

C. 2005 BYI 08330-cis-ketohydroxy: Acute toxicity to earthworms ( Eisenia fetida ) tested in artificial soil Bayer CropScience AG, Report No.: LKC/RG-A-47/05, Report includes Trial Nos.: Rg08/05 Edition Number: M-258925-01-2 Date: 17.10.2005 GLP, unpublished Yes BCS

IIA 8.9.1 Moser, T.;

Scheffczyk, A. 2005 BYI 08330-Enol: Reproduction toxicity to the earthworm Eisenida fetida in artificial soil ECT Oekotoxikologie GmbH, Floersheim, Germany Bayer CropScience AG, Report No.: P18RR, Edition Number: M-243305-01-2 Date: 19.01.2005 GLP, unpublished Yes BCS

IIA 8.9.1 Leicher, T. 2008 Spirotetramat - 4-methoxyc yclohexanon: Acute toxicity to earthworms (Eisenia fetida) tested in artificial soil with 5 percent peat Bayer CropScience AG, Report No.: LRT-RG-A-98/07, Edition Number: M-296072-01-1 Date: 09.01.2008 GLP, unpublished Yes BCS

IIA 8.10.1 Lechelt-Kunze,

C. 2004

b BYI 08330 tech.: Determination of effects on nitrogen transformation in soil Bayer CropScience AG, Report No.: LKC-N-32/04, Edition Number: M-087831-01-1 Date: 01.09.2004 GLP, unpublished Yes BCS– 156 –

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Y/N-R/NR Owner

IIA 8.10.1 Lechelt-Kunze,

C. 2004

c BYI 08330 tech.: Determination of effects on carbon transformation in soil Bayer CropScience AG, Report No.: LKC-C-30/04, Edition Number: M-087840-01-1 Date: 01.09.2004 GLP, unpublished Yes BCS

IIA 8.11.1 Banman, C. S.;

Lam, C. V. 2005 Acute toxicity of BYI 08330 technical to the sheepshead minnow ( Cyprinodon variegatus ) under flow-trough conditions Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX006, Edition Number: M-255363-01-1 Date: 27.07.2005 GLP unpublished Yes BCS

IIA 8.11.1 Cafarelle, M. A. 2005

a BYI 08330 - Acute toxicity to mysids ( Americamysis bahia ) under flow-through conditions Springborn Smithers Laboratories, Wareham, MA, USA Bayer CropScience AG, Report No.: EBFNX010, Edition Number: M-270200-01-1 Date: 06.01.2005 GLP unpublished Yes BCS

IIA 8.11.1 Cafarella, M. A. 2005 b BYI 08330 - Acute toxicity to eastern oysters ( Crassostrea virginica ) under flowthrough conditions Springborn Smithers Laboratories, Inc., Wareham, MA, USA Bayer CropScience AG, Report No.: EBFNX011, Edition Number: M-257677-01-1 Date: 15.06.2005 GLP unpublished Yes Springborn

IIA 8.11.1 Banman, C. S.;

Lam, C. V. 2006 Toxicity of BYI 08330 technical to the saltwater diatom Skeletonema costatum Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX009-1, Edition Number: M-271037-02-1 Date: 23.02.2006, Amended: 16.06.2006 GLP unpublished Yes BCS– 157 –

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Y/N-R/NR Owner

IIA 8.12 Gosch, H.;

Nguyen, D.H. 2006

a Non-target terrestrial plants: an evaluation of the effects of BYI 08330 OD 150 in the vegetative vigour test (Tier 1) Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: VV06/03, Edition Number: M-273381-01-1 Date: 20.06.2006 Non GLP unpublished Yes BCS

IIA 8.12 Christ, M. T.;

Lam, C. V. 2006

a Tier I and Tier II - Vegetative vigor nontarget plant study using BYI 08330 150 OD Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX016, Edition Number: M-276131-01-1 Date: 09.08.2006 GLP unpublished Yes BCS

IIA 8.12 Friedrich, S. 2006 Spirotetramat OD 150: Veget ative vigour test on non-target terrestrial plants BioChem agrar, Gerichshain, Germany Bayer CropScience AG, Report No.: 061048126, Edition Number: M-279698-01-1 Date: 30.10.2006 GLP unpublished Yes BCS

IIA 8.12 Gosch, H.;

Nguyen, D.H. 2006

b Non-target terrestrial plants: an evaluation of the effects of BYI 08330 OD 150 in the seedling emergence and growth test (Tier 1). Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: SE06/03, Edition Number: M-273373-01-1 Date: 20.06.2006 Non GLP unpublished Yes BCS

IIA 8.12 Christ, M. T.;

Lam, C. V. 2006

b Tier I seedling emergence - Non-target plant study using BYI 08330 150 OD Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: EBFNX017, Edition Number: M-276244-01-1 Date: 09.08.2006 GLP unpublished Yes BCS– 158 –

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Y/N-R/NR Owner

IIA 8.14 Moser, T.;

Scheffczyk, A. 2005 BYI 08330-Enol: Effects on survival and reproduction of the predaceous mite Hypoaspis aculeifer

Canestrini (Acari: Laelapidae) in standard soil (LUFA 2.1) ECT Oekotoxikologie GmbH, Floersheim, Germany Bayer CropScience AG, Report No.: P11HR, Edition Number: M-243307-01-1 Date: 19.01.2005 GLP unpublished Yes BCS

IIA 8.15 Bruns; Caspers,

N. 2004 BYi 08330 - Toxicity to bacteria Bayer Industry Services GmbH, Leverkusen, Germany Bayer CropScience AG, Report No.: 1312 N/04 B, Edition Number: M-275364-01-1 Date: 05.10.2004 GLP unpublished Yes BCS

IIA 8.16 Schneider, K. 2008 Residues of Spirotetramat i n arthropods after spray application in citrus - magnitude and time course of residue decline RIFCON GmbH, Heidelberg, Germany Bayer CropScience AG, Report No.: RA07-031, Edition Number: M-296043-01-1 Date: 09.01.2008 GLP, unpublished Yes BCS

Annex III Data and Information IIIA1 10.1.2 Wolf, C.;

Gerlach, J. 2006 Insectivorous birds in southern European citrus plantations focal species and their diet composition RIFCON GmbH, Hirschberg, Germany Bayer CropScience AG, Report No.: RC06-067, Edition Number: M-277503-01-1 Date: 15.09.2006 Non GLP unpublished Yes BCS

IIIA1 10.1.2 Ebeling, M.

Kyriazi, K. 2008 Refinement for the risk assessment for the Great Tit in citrus orchards following treatment with Spirotetramat Bayer CropScience AG, Edition Number: not yet finalised Non GLP, unpublished Yes BCS

IIIA1 10.1.2 Wikens, S.,

Schneider, K. 2008 Residues of Spirotetramat in arthropods after spray application in citrus - magnitude and time course of residue decline RIFCON GmbH, Heidelberg, Germany Bayer CropScience AG, Report No.: RA07-031, Edition Number: M-296043-01-2 Date: 09.01.2008 GLP, unpublished Yes BCS– 159 –

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Y/N-R/NR Owner

IIIA1 10.1.7 Fischer, D. R.,

Ramanarayana n, T. & Ortego, L. 2006 BYI08330 150 OD - Magnitude of the residue in/on soybeans/wheat, potential wildlife feed items Bayer CropScience, Stilwell, KS, USA Bayer CropScience AG, Report No.: RAFNP002, Edition Number: M-277315-01-1 Date: 06.09.2006 GLP unpublished Yes BCS

IIIA1 10.1.7 Wolf, C. 2004 Generic field monitoring of birds in potato cultivation in northern Germany Bayer CropScience AG, Report No.: WFC/FS 019, Edition Number: M-090336-02-1 Date: 29.09.2004, Amended: 04.01.2005 GLP unpublished Yes BCS

IIIA 10.2.2.1 Nieden, D. 2005 d Acute toxicity of BYI 08330 OD 150 to fish ( Oncorhynchus mykiss ) under static conditions Bayer CropScience GmbH, Monheim, Germany Bayer CropScience AG, Report No.: EBFNM008, Edition Number: M-253916-01-2 Date: 29.06.2005 GLP, unpublished Yes BCS

IIIA 10.2.2.2 Dorgerloh, M. 2006 d Acute toxicity of BYI 08330 OD 150 to larvae of Chironomus riparius in a 48 h static laboratory test system Bayer CropScience AG, Report No.: EBFNM007, Edition Number: M-264018-01-2 Date: 19.01.2006 GLP, unpublished Yes BCS

IIIA 10.2.2.3 Dorgerloh, M. 2006

c Pseudokirchneriella subcapitata growth inhibition test with BYI 08330 OD 150 Bayer CropScience AG, Report No.: EBFNM006, Edition Number: M-264263-01-2 Date: 16.01.2006 GLP unpublished Yes BCS

IIIA1 10.3.1.3 G.D. Cappon,

T.L. Fleeman, R.E. Chapin, and M.E. Hurtt 2005 Effects of feed restriction during organogenesis on embryo-fetal development in rabbit Publisher:2005 Wiley-Liss, Inc., Journal:Birth Defects Research (Part B), Volume:74, Pages:424-430, Year:2005, Report No.: Lit. 8639, Edition Number: M-274544-01-1 Non GLP published No– 160 –

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Y/N-R/NR Owner

IIIA1 10.4.2 Barth, M. 2005

a Acute toxicity of BYI 08330 150 OD to the honeybee Apis mellifera L. under laboratory conditions BioChem agrar, Gerichshain, Germany Bayer CropScience AG, Report No.: 051048032, Edition Number: M-259122-01-2 Date: 18.10.2005 GLP unpublished Yes BioChem

IIIA1 10.4.2.1 Barth, M. 2005 b Acute toxicity of BYI 08330 150 OD to the honeybee Apis mellifera L. under laboratory conditions BioChem agrar, Gerichshain, Germany Bayer CropScience AG, Report No.: 051048032, Edition Number: M-259122-01-2 Date: 18.10.2005 GLP unpublished Yes BioChem

IIIA1 10.4.2.2 Barth, M. 2005 Acute toxicity of BYI 083 30 150 OD to the honeybee Apis mellifera L. under laboratory conditions BioChem agrar, Gerichshain, Germany Bayer CropScience AG, Report No.: 051048032, Edition Number: M-259122-01-2 Date: 18.10.2005 GLP unpublished Yes BioChem

IIIA1 10.4.5 Schur, A. 2006 Assessment of side effects of Spirotetramat OD 150 on the honey bee ( Apis mellifera L.) in the field eurofins-GAB GmbH, Niefern-Öschelbronn, Germany Bayer CropScience AG, Report No.: 20061133/S1-BFEU, Edition Number: M-277194-02-2 Date: 11.09.2006 GLP unpublished Yes BCS

IIIA1 10.4.7 Schur, A. 2005 Assessment of side effects of BYI 08330 OD 100 on the honey bee ( Apis mellifera L.) in the semi-field GAB GmbH, Niefern-Oeschelbronn, Germany Bayer CropScience AG, Report No.: 20041144/01-BZEU, Edition Number: M-244490-01-2 Date: 02.02.2005 GLP unpublished Yes BCS– 161 –

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Annex point/ reference number Author(s) Year Title Source (where different from company) Company, Report No GLP or GEP status (where relevant) Published or not Data Protection Claimed

Y/N-R/NR Owner

IIIA1 10.4.7 Koelzer, U. 2007 Summary of orientating r esearch tunnel trials on effects of spirotetramat to honey bees according to EPPO 170 RIFCon GmbH, Leichlingen, Germany Bayer CropScience AG, Report No.: PA07-174, Edition Number: M-294216-01-1 Date: 17.10.2007 Non GLP, unpublished Yes BCS

IIIA1 10.5.2 Waltersdorfer,

A. 2006

c BYI 08330 150 OD; Toxicity to the predatory mite Typhlodromus pyri SCHEUTEN (Acari, Phytoseiidae) using an extended laboratory test (under semi-field conditions aged residues on apple trees); AE 1302943 00 OD15 A101 Bayer CropScience GmbH, Frankfurt am Main, Germany Bayer CropScience AG, Report No.: CW05/028, Edition Number: M-264153-01-1 Date: 18.01.2006 GLP unpublished Yes BCS

IIIA1 10.5.4 Aldershof, S.A. 2006 Evaluating effects of BYI 08330 OD 150 applications on mite fauna (Acari) in the field (grape vines, France) MITOX Consultants, Amsterdam, The Netherlands Bayer CropScience AG, Report No.: B127AFG, Edition Number: M-269107-01-1 Date: 14.03.2006 GLP unpublished Yes Mitox– 162 –

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# Appendix



# Spirotetramat

Environmental Risk Assessment (Ecotoxicology)

for the EU (Annex III)– 163 –

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B.9.1.8 Risk assessment for birds

Birds may be exposed to spirotetramat by eating con taminated vegetation, seeds and fruits, invertebrat e prey like arthropods (i.e. insects) or earthworms o r vertebrate prey. Toxicity Exposure Ratios (TER) and Estimated Theore tical Exposure (ETE) were calculated based on the methods, i.e. equations and tabulated values descri bed in the “Guidance Document on Risk Assessment fo r Birds and Mammals Under Council Directive 91/414/EE C” (Sanco/4145/2000 of 25. September 2002, http://ec.europa.eu/food/plant/protection/evaluatio n/guidance/wrkdoc19_en.pdf). The risk assessment is based on toxicity figures of the active substance. Data for the formulation are considered not required, as results from mammalian testing with the formulation do not give an indicat ion that the formulated active ingredient is significan tly more toxic than the technical active ingredient . In addition, birds would typically be exposed to dry r esidues of spirotetramat on their food items follow ing the dilution and spraying of the formulated product.

The principles of ETE calculation according to the guidance document are presented below. The TER is calculated as toxicity endpoint/ETE. Maximum ETE values were estimated for use in acute and short-term risk assessments while time-weighted average ETE values were calculated for use in asses sing long-term risks. ETE values were calculated us ing the following equation:

ETE (mg as/kg bw/day) = FIR/bw x RUD x Application rate x MAF x f TWA x PT x PD x AV

where: FIR Food Intake Rate (g fresh weight per d ay) bw Body weight (g) RUD Residue per unit dose (mg/kg fresh weight) MAF Multiple Application Factor f TWA Time-weighted average factor (only used for calcula ting long-term ETE) PT Proportion of diet obtained in treated area PD Proportion of food-type in diet AV Avoidance factor (no avoidance =1) As recommended by the EU Expert Group on Higher Tie r Risk Assessment for Birds and Mammals, the 90 th percentile residues on food items were used for the acute risk assessment, and the 50 th percentiles for the short and long-term risk assessments. The f TWA was calculated from the following equation: f twa = (1-e -kt )/kt where: k = ln2/DT 50 t = averaging time

The relevant toxicity figures were taken from labor atory studies with spirotetramat. These endpoints a re listed in table B.9.1.7-1 (above) for the different time scales. The reproductive NOAEL of 4.0 mg/kg b w is derived from a 2-generation reproduction study in t he mallard. It is also the no effect level with reg ard to duck foot lesions. The risk assessment is based on the lowest toxicity endpoints (see table above), which were considered ecotoxicologically relevant with regard to the risk assessment.– 164 –

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B.9.1.8.1 First tier risk assessment (OECD IIIA 10. 1.1; OECD IIIA 10.21.2)

The calculations are based on the following represe ntative application scenarios in the EU:

• Lettuce: 2 x 0.072 kg a.s./ha (interval 14 d), Nor thern + Southern Europe

• Citrus: 2 x 0.096 kg a.s./m canopy height/ha (inte rval 21 d), max. 0.288 kg a.s./ha, Southern Europe

Assessment of exposure / Selection of scenarios:
The following relevant exposure scenarios were cons idered: Lettuce : - dietary exposure of a medium herbivorous bird fe eding on treated herbage (category “leafy crops”) - dietary exposure of a small insectivorous bird f eeding on contaminated arthropods Citrus : - dietary exposure of a small insectivorous bird f eeding on contaminated arthropods

In order to calculate the estimated theoretical exp osure the respective figures (e.g. RUD, FIR/bw) wer e chosen from the guidance document to estimate food intake rates and concentrations in the food of conc ern (see Table 9.1.8.1-1). For both crops two applicati ons are intended. However, as for citrus the focus is on insectivorous species and no residue accumulation i s considered on insects, a MAF (multiple applicatio n factor) is included in the ETE estimations for lett uce only. The MAF is set at 1.2 for the acute and 1 .4 for the short- and long-term scenario. A default half-life of 10 d is assumed for estimating residue decline o n the crop. For acute exposure, the 90 th percentile and for long-term exposure mean initial residue levels in the respective food items are applied. In the first tie r worst case assessment it is assumed that the bird s feed on a single food type, which is not avoided and that t hey satisfy their entire food demand in the treated area (PD and PT = 1). Possible food items like earthworms, for which no r espective risk assessment was performed, are covere d by the calculations presented because residues in t hese items are expected to be much lower.

Discussion of foot lesions observed in chronic mall ard studies: During avian reproduction studies with the mallard duck dietarily exposed to spirotetramat the occurre nce of dried and deeply cracked skin on the underside of t he feet was observed. These dermal effects to the f eet may have been exacerbated by an interaction between the wire mesh of the caging (possible physical damage) and food contact with excreta (possible che mical exposure) on the cage floors. As the causal mechanism was not fully understood in the standard reprotox studies, the notifier performed two furthe r studies to gain more information. However, in these new, specially designed studies, the following res ults were obtained: 1. Ducks only dermally exposed to spirotetramat exp erience much less severe and transient symptoms compared to dietarily exposed birds. 2. Ducks kept in a semi-natural outdoor environment (turf ground) that were exposed to spirotetramat through contaminated diet and through contact with oversprayed turf, also exhibited severe foot symptoms. Thus, the hypothesis, that the foot lesions were pa rtly caused by the contact with artificial pen floo r, could not be verified. No foot effects were seen in bobwhite quails in the chronic reproduction study and in acute and subchr onic studies with the bobwhite or the mallard. It can be assumed, that the specific pathology observed is somehow specific to duck feet, but as only two spec ies were investigated chronically, this can not be concluded for sure. In conclusion, the effects on feet skin are conside red as ecotoxicologically relevant, as they lead to a severe impairment of the bird`s general condition and beha viour. The NOAEL of 4 mg a.s./kg bw used in the chronic/reproductive risk assessment is also protec tive with regard to foot effects.– 165 –

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Effective application rate with regard to insectivo rous birds in citrus = 0.096 kg/ha The diet of canopy-dwelling insectivorous birds obt ained from within the citrus orchard may contain re sidues from spray applications. Realistically, residues on foliage dwelling arthropods in the canopy should b e calculated as product of the RUD (residue per unit dose) and that fraction of the application rate tha t can be expected to reach the respective compartment (“effe ctive rate”) of the canopy, and not as product of t he RUD and the maximum application rate for trees of 3 m height. The application rate is defined per m ca nopy height. Likewise, the maximum spray concentration a nd the predicted residues in the sprayed canopy (e. g. foliage, arthropods) are comparable regardless of w hether a tree of 1, 2 or 3 m height has been spraye d. Thus, an application rate corresponding to 0.096 kg a.s./ha (= amount per 1 m tree height) is consider ed in the respective ETE calculations for citrus.

Table B.9.1.8.1-1: Acute, short-term and long-term exposure of birds to spirotetramat and calculated toxicity exposure ratios, Tier 1 Crop / Time horizon Indicator

species Endpoint

(mg/kg bw) FIR/bw RUD MAF ftwa ETE TER
Lettuce, 72 g a.s./ha

Medium herbivorous bird 0.76 87 1.2 / 5.7 > 350 Acute

Insectivorous

bird > 2000 1.04 52 / / 3.9 > 514

Medium herbivorous bird 0.76 40 1.4 / 3.1 > 155 Short term

Insectivorous

bird > 475 1.04 29 / / 2.2 > 219

Medium herbivorous bird 0.76 40 1.4 0.53 1.6 2.46 Long term

Insectivorous

bird 4 1.04 29 / / 2.2 1.84
Citrus, 96 g a.s./ha Acute Insectivorous

bird > 2000 1.04 52 / / 5.2 > 385

Short term Insectivorous

bird > 475 1.04 29 / / 2.9 > 164
Long term Insectivorous

bird 4 1.04 29 / / 2.9 1.38

FIR/bw...food intake rate in relation to body weight; RUD...residue per unit dose; MAF...multiple applicatio n factor; ftwa...time weighted average factor; ETE...estimated theoretical exposure; TER...toxicity exposure ratio

The resulting acute and short-term TER`s are above the respective triggers of Annex VI of Directive 91 /414 and indicate a low risk to wild bird populations af ter oral intake of feed items contaminated with spi rotetramat. The long-term TER`s, however, indicate potential ri sk at tier 1 level. Further refinement of the expos ure is needed.

B.9.1.8.2 Higher tier risk assessment

The long-term risk is refined based on actual resid ue decline data of spirotetramat on plant foliage a nd by using information on focal species.

Refinement of long-term risk to herbivorous birds i n lettuce:

Focal species: Wood Pigeon ( Columba palumbus ), FIR/bw = 0.76 The Wood Pigeon can be considered relevant as a mod el species for herbivorous birds in leafy crops. However, direct and quantitative reports about the extent of Wood Pigeons feeding on lettuce are not– 166 –

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available. The notifier further argues: “This is no t surprising, given the very limited nutritive valu e of lettuce on one hand, and the protective action vegetable farme rs are prepared to take against herbivorous birds ravaging their crops. There are well known examples for Wood Pigeons feeding on vegetable seedlings, particularly on rape and cabbage. In northern Franc e, Wood Pigeons are typically considered even as a pest on cabbage.” The FIR/bw figure is derived from Sanco/4145/2000.
Refinement of PD for the Wood Pigeon: PD = 0.15 Overall, Wood Pigeons are herbivorous and feed main ly on grains, fruits, seeds, buds on trees, leaves, root crops and may also take invertebrate food, especial ly to feed their young (English Nature: Woodpigeon – Columba palumbus: http://www.plantpress.com/wildlife/o9- woodpigeon.p hp ). They are known to be particularly fond of the leaves of cabbages and oth er brassica vegetables. In a study on the feeding biology of Wood Pigeons i n the agricultural landscape, Murton et al. (1963) examined the crop contents of Wood pigeons over the year. During April and May, legume or brassica leaves made up less than about 15 % and 10 % of the diet of 56 and 90 Wood Pigeons, respectively. In t he winter period (December - February), Wood Pigeons a re forced to forage on leafy crops (particularly wi nter oil seed rape), since alternative food sources are at the minimum at this time of year (Inglis et al. 1989). However, no applications of spirotetramat are made on lettuce in the winter season. Very similar to the results of Murton et al., Colqu houn (1951) reported the share of cabbage to the cr op content of shot Wood Pigeons at its maximum of arou nd 25 % in January, but with only 13 % (% weight) i n March and no more incidence until next winter. Thus , Wood Pigeons are unlikely to forage preferentiall y foliage treated with spirotetramat, particularly du ring the breeding season when plenty and more attra ctive alternative food is available. Despite considerable searching efforts, no single r eport was detected about wood pigeons - or another bird species - feeding on lettuce foliage. This lack of reports suggests that the occurrence of birds forag ing on lettuce is probably not wide-spread, and certainly less important than that of birds foraging on cabba ge. The PD-factor of 0.15 as identified for the Wood Pi geon and its wide-spread and well reported foraging preference on cabbages can therefore be considered as appropriate surrogate PD-factor for the use in t he exposure assessment of herbivorous birds on lettuce during spring/summer. It is thus considered conservative to use a PD-fact or of 0.15 as worst-case estimation for the exposur e of herbivorous birds to residues in young leafy crops treated with Spirotetramat.
Refinement of the residue decline of spirotetramat on lettuce (MAF, f TWA ): In the study of Fischer et al. (2006), the half liv es of spirotetramat on leaves of soybean, short gra ss (wheat), and tall grass (wheat) were determined. The mean ha lf-life for soybean was 3.42 days and this figure h as been proposed for the risk assessment by the notifi er. However, when the half-life of one trial that r eceived rain one day after application is excluded, the hal f-life is 4.45 d (see B.9.1.4.). This figure is use d in the RMS risk assessment as representative worst-case. Soybe an is considered as a model plant for the type of d iet relevant for herbivorous birds in vegetable crops ( non-grass herbs). A MAF of 1.1 and a 14-d twa factor of 0.41 can be d erived according to the formulas given in SANCO/4145/2000-Guidance Document. To summarize, for the refined long-term risk assess ment for herbivores the following assumptions are applied in the TER-calculation: Wood Pigeon: FIR/bw 0.76; DT 50 4.45 d, MAF 1.1, 14d f TWA 0.41, PD 0.15

Table B.9.1.8.2-1: Long-term exposure of herbivorou s birds to spirotetramat and calculated toxicity exposure ratio, refined Crop / indicator species Endpoint

(mg/kg bw) FIR/bw RUD MAF ftwa PD PT ETE TERlt

Lettuce Wood Pigeon 4 0.76 40 1.1 0.41 0.15 1 0.15 27– 167 –

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The resulting TER-value indicates a low risk to her bivorous birds after the use of spirotetramat in le ttuce. If there is doubt on the use of the PD factor of 0. 15 or on the relevance of the Wood Pigeon, it shoul d be considered that under the unrealistic assumption th at a medium sized bird would feed on 80 % treated lettuce, the TER would be equal to 5 and thus accep table.

Refinement of the long-term risk to insectivorous b irds in lettuce:

Focal Species - Yellow wagtail ( Motacilla flava ) The default Tier 1 model species for insectivorous birds according to SANCO/4145/2000, the Wren ( Troglodytes troglodytes ) or the Blue Tit ( Parus caeruleus ) cannot be considered particularly relevant for th e use of Spirotetramat in lettuce (“leafy crops”). Cramp (1998) described the habitat preference of th e Wren to be within a predominantly moist mild clim atic range, offering a wide variety of low cover and for aging opportunities, within or outside woodland, cr ops and aquatic vegetation, fallen trees and branches or he aps of brash, hedgerows, gardens, parks, and shrubberies. In a multi-annual study conducted in t he years 1982-84 on the habitat requirements of bir ds on farmland in south-western Germany (Seitz 1989), Wre ns were recorded 66 times in total per year from wh ich 21 records were made in spring and summer. From the se 21 observations recorded during spring and summer, no Wrens were observed foraging in arable f ields (average 0 %), but most of them foraging in forests (average 63.7 %), hedgerows (average 9.7 %) , trees (average 9.7 %) and small forbs (average 5.6 %). Likewise, the Blue Tit, the other insectivorous lig ht weight bird proposed as theoretical worst case m odel is also of low relevance to vegetable/leafy crops. Rat her, the Blue Tit is associated with the presence o f trees (Cramp 1998). In the multi-annual study by Seitz (1 989) referred to above, only 0.4 % of all Blue Tits were observed in agricultural fields. Based on its ecology, and the occurrence on arable land, the Yellow Wagtail ( Motacilla flava ) is considered more relevant for the refined risk assessment of sp irotetramat in lettuce at the crop stage BBCH 42-43 . This species is a summer visitor in Europe from April to September. Its nesting habitat is generally associ ated with areas of low dense herbage either natural or m anaged particularly along the fringes of wetlands a nd where low perches are available (Buxton et al., 199 8).
The preference of the Yellow Wagtail for fields cro pped with spring sown vegetables/leafy crops has be en reported by various authors: a) In a 3-year field study in agricultural land in UK, Mason and Macdonald (2000) monitored the territories of eight bird species in eastern Englan d and reported a preference index. The land use in the study area was characterised by winter wheat (3 2 %), winter barley (22 %), potatoes (6.4 %), oilseed rape (5.2 %), salad crops (1.2 %), beans (0 .8 %), grass (8.6 %), and set-aside (4.3 %). Highest densities of Yellow Wagtails were found in spring crops (potatoes, peas, beans and salad crops). Only a small fraction of the nests monitore d (4 %) were found in grassland, with a similar proportion in set-aside. Potatoes held the greatest proportion of territories, although nests were found also in spring sown cereals, oil-seed rape, s ugar beet, linseed and maize. b) The typical association of Yellow wagtails with spring-sown leafy row crops was also underlined by Anthes et al. (2002). The authors monitored territo ries and nests of Yellow wagtails during their breeding season from May to June 2002 on arable lan d in Southern Germany. Clearly most territories were counted in spring-sown row crops, (mainly sugar beet and potato but also strawberries) which were significantly preferred ov er their proportional share. c) The importance of spring sown row crops for the Yellow Wagtail was finally confirmed by a radiotracking study in potatoes (Wolf 2005, see above un der B.9.1.4).
This information suggests that the Yellow Wagtail i s an appropriate focal species for the refined risk assessment for insectivorous birds in lettuce. Alth ough only limited information is directly available for lettuce fields, there are consistent reports of this specie s in other spring sown leafy row crops. As ecologic al habitat for insectivorous birds, spring crops like potatoes , sugar beet, and salad crops bear many similaritie s– 168 –

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particularly in late spring / early summer, which i s the season relevant for this refined risk assessm ent addressing the reproductive phase: the leafy crop i s grown in rows between which birds can walk and fo rage, with only very limited growth of weeds. In consequence, the refined risk assessment for ins ectivorous birds in lettuce is based on the Yellow Wagtail as focal species.
Yellow Wagtail: FIR/bw = 0.88 The food intake rate to body weight ratio for the Y ellow Wagtail is calculated to be 0.88 according to SANCO/4145/2000 (2002) based on an energy content f or arthropods of 21.9 kJ/g dry weight, a water content of the food of 70.5 %, an assimilation effi ciency of 76 % and a body weight of 17 g (Dittberne r and Dittberner, 1984). The relationship between body we ight and daily energy expenditure is described by t he following equation according to Crocker et al. (200 2): Log(DEE) = 1.0017 + 0.7034 x log bw.

Refinement of diet composition: PD: 80 % large + 20 % small invertebrates. The Yellow Wagtail is primarily an insectivorous bi rd but also feeds on other epigaeic invertebrates ( e.g. spiders). Its foraging behaviour is well known and comprises mainly three techniques: picking (picks i tems from the ground while walking), run-picking (quick darting run at prey, picking it up either from the ground or as it takes off), and fly-catching (makes short fli ght from the ground or perch, catching the prey in mid-air). Based on these foraging techniques the Yellow Wagta il can be expected to feed on a mixed arthropod die t mainly comprising of insects that at least partly d well on or visit the ground. A targeted study into the prey spectrum of Yellow W agtails on arable land has been conducted by Wolf (2005). According to his results the Yellow Wagtail s foraged on Diptera, Coleoptera, Aphidoidea, Hymenoptera and Araneae, and targeted specimen larg er than the respective average size. Based on these results the notifier considered it t o be conservative to assume that the typical diet o f Yellow Wagtails from lettuce fields consists of 50 % large and 50 % small insects, taken equally from the fol iage and from the ground. The diet composition Wolf (2005) found in his study is in accordance with the data of Davies (1977). I n this study the feeding behaviour of Yellow Wagtails in f locks or solitary was examined on non-flooded areas of a meadow, where the birds fed predominantly on flies around dung pats. Five main prey items were identif ied. The smallest (Staphilinidae and small Spheroceridae ) were rejected. The largest (Scatophagidae) were patchily distributed and difficult to capture when in swarms and so the next largest prey (large Spheroceridae) were also incorporated into the diet . The size of Scatophagidae eaten corresponds to th e optimum in terms of energy intake per unit handling time. The birds’ behaviour in leaving patches coul d also be predicted by optimal foraging theory. According to these data Yellow Wagtails prefer insects of 3 - 4 mm and greater. The size of the insects and the defini tion of small and large feed items are essential si nce the estimation of the residues of plant protection prod ucts on insects as proposed by Kenaga (1973) is bas ed on the residues on weed seeds and wheat seeds which ar e considered to be comparable to small or large insects. As wheat seeds are typically 4 - 5 mm in l ength, insects of ≥ 3 mm body length are defined as large. In consequence, the diet of yellow wagtails consist s of 76.4 % large insects (Scatophagidae and large Spheroceridae) and 23.6 % small insects. With regar d to the mass of insects it is reasonable to consid er that these values are rather conservative as a given num ber of large insects would contribute more to the d aily energy intake as the same number of small insects. As described above, the selection of the food items is probably determined by the rate of energy conte nt to handling time and therefore not stringently a quest ion of species composition. This can be supposed to be similar in different habitats/crops. In the table below, the diet composition of the Yel low Wagtail as found in Wolf (2005) is shown in det ail.– 169 –

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Table B.9.1.8.2-2: Composition of Yellow Wagtail fo od Based on 25 samples of faeces; size of individual a rthropods estimated after the size of remains in fa eces.

* values of stones estimated

From these figures it can be derived that minimum 8 0 % of arthropods eaten have a size of 3 mm or more

. These can be defined as large, as described above. For large, predominantly ground dwelling insects a mean RUD = 5.1 is recommended in SANCO/4145/2000. Based on these data the diet is considered to consi st of 80 % large and 20 % small arthropods (corresponding to PD figures of 0.8 / 0.2). This is also in accordance with figures used for the refin ement of other EU peer-reviewed substances.
Refinement of the time spent in the treated area: P T = 0.4 The specific preference of the Yellow Wagtail for s pring sown vegetable row crops has been confirmed b y various authors. Mason & McDonald (2002) reported t he highest preference of the Yellow Wagtail for potatoes, beans and salad crops, with very similar preference indices of 0.702, 0.693 and 0.752, respectively. Anthes et al (2002) confirmed the cle ar preference of Yellow Wagtails particularly for p otatoes. No data on the specific use of lettuce fields are a vailable but it is reasonable to assume that the us e of lettuce fields is unlikely to be higher than that o f the consistently most attractive crops, i.e. pota toes. As mentioned before, a radio-tracking study has been c onducted on the foraging pattern of Yellow Wagtails (Wolf 2005) in and around potato fields. On average , Yellow Wagtails spent 38.4 % of their time in pot ato fields, 39.8 % in cereal fields, 6.8 % in oil seed rape, 3.7 % on streets and field paths, and 11.3 % in other habitats. No data from lettuce fields are available from this study since this crop was not cultivated to a significant extent in the study area, but the resul ts allow nevertheless to expect that Yellow Wagtail s will not be exclusively foraging in any specific type of ara ble fields, including potato fields. Thus, for the refined risk assessment with the Yell ow Wagtail as insectivorous species of concern, the mean portion of diet taken from treated lettuce fields ( PT) is considered to be similar to the maximum PT r eported from the most preferred crops (potatoes: PT = 0.38) . Therefore a PT-value of 0.4 is considered a conservative estimation for the average portion of diet Yellow Wagtails may obtain from lettuce fields treated with Spirotetramat. The TER calculation is presented in table B.1.8.2-2

. (Without refinement of PT the resulting TER is 6.3)

.– 170 –

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Refinement of long-term risk to insectivorous birds in citrus orchards:

The tier 1 model species for insectivorous birds in orchards, the Wren ( Troglodytes troglodytes ) or the Blue Tit ( Parus caeruleus ), are considered not relevant as typical birds inh abiting citrus orchards. Published literature (Barba & Gil-Degado 1990, Beld a et al. 1998, Monros et al. 1999) provides evidenc e for the relevance of the Great Tit ( Parus major ) as typical insectivorous bird species in citrus p lantations. Several ecological investigations show that Great T its find favourable breeding habitats in Spanish ci trus groves, that they occur there with relatively high population densities, and that, at least in certain areas of Spain, and at certain times of the year, citrus orc hards are even more favourable habitats for Great T its than the surrounding natural habitats (literature source s are summarised and evaluated in Wolf & Gerlach 20 06, see RMS summary under B.9.1.4). In the first risk assessment provided by the notifi er, the refinement of the exposure scenario was mai nly based on the percentual diet composition of large a nd small arthropods and respective RUD figures as g iven in SANCO 4145/2000 (The derived TER was 6.6). Howev er, in the meantime an arthropod residue study after spirotetramat application is available and it has also become scientifically more accepted that for the residue concentration in prey items, the location o f the arthropod (on foliage or on the ground) is mo re decisive than its size.

Refinement of spirotetramat residues on arthropods using values measured in the field: The results of the insect residue study (Wilkens & Schneider 2008) were reported separately for foliag e dwelling arthropods (21 d twa residue 1.9 mg/kg) an d for ground dwelling arthropods (21 d twa residue 0.35 mg/kg, see B.9.1.4). The 21-d time-weighted-average residue values for spirotetramat + -enol were used for the ETE refinement. The total measured residue, inc luding the spirotetramat-ketohxdroxy-metabolite is not the ecotoxicollogically relevant figure, as the ket ohydroxy metabolite can be considered as (eco)toxicologically inactive (see also B.9.1.8.3). The application rates in the field study were abou t 15 % higher than the GAP, however, the residues as deriv ed in the study are included in the calculation.

Refinement of PD-portion of arthropods taken from t he canopy or from the ground: Based on feeding observations reported in the liter ature an adapted exposure scenario for the great ti t feeding in citrus groves on foliage and ground dwel ling arthropods is developed in a second risk asses sment provided by the notifier. Great tits mainly feed in the canopy. However, prey is also taken from the ground. In mixed woodlands in central Spain, the portion of ground foraging in Ap ril and May was reported to be 32 % (Atienza & Ille ra, 1997). In this field work activities of the observe d great tits in the microhabitat were recorded at 3 0-s intervals. From all foraging activities of the grea t tit (n=48), 32 % were performed on the ground and the remainder in tree- and bush canopies. For the long-term scenario an average portion of 32 % of ground dwelling arthropods is therefore consi dered as a realistic estimation of this part of the diet.

Proportion of caterpillars / other arthropods in th e diet: Barba & Gil-Delgado (1990) investigated the seasona l variation of the nestling diet of the great tit i n orange groves in eastern Spain. The proportion of caterpil lars peaked in early May and declined to about 10 % in the period from May 26 - June 05, which fits very well the expected application timing of spirotetramat, a nd it declines even more in the following weeks (see figu re below). This result confirms that caterpillars are not expe cted to be a significant part of the prey of great tits at the time when spirotetramat will be applied. However, b ecause of the different energy content of caterpill ars compared to other arthropods, and consequently FIR, the portion should be considered. This is also consistent with the observation that f ew caterpillars were sampled in the arthropod resid ue study conducted end of May / early June in a Spanish citr us orchard (Wilkens & Schneider 2008, see B.9.1.4). A PD value of 0.1 will therefore be included for ca terpillars in the foliage dwelling arthropod part o f the ETE calculation.– 171 –

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Great Tit: FIR/bw = 0.85 for arthropods, 1.23 for c aterpillars Based on bodyweights, energetic requirement and ene rgy values of respective feed items, the food intak e per day in terms of fresh weight can be estimated a ccording to SANCO/4145/2002. For the Great Tit (approximate weight 19 g, based on Buxton et al. 19 98) this has been calculated to be 16.2 g of arthro pods, giving an FIR/bw of 0.85 for arthropods, or 23.4 g and 1.23 for caterpillars.

Refinement of PT - Proportion of diet obtained in t he treated area The results of the investigation of Crocker et al. (1998): “Contract PN0903: Improving the assessment of pesticide risk to birds in orchards” are reported i n the EU Guidance Document SANCO/4145/2000. The dat a indicate that for blue tits - a common example of a small insectivore and closely related to the great tit – 95 % of the local population spent less than 61 % of pot ential foraging time in orchard trees. As this is a conservative refinement approach (95 th percentile, the median figure would be 20 %), and because of additional information presented below, it is consi dered acceptable to extrapolate this proportion fro m another tit species in orchards to great tits in ci trus. This corresponds with a factor of 0.61 for refineme nt of PT. In a study conducted by Mols (2003) in the Netherla nds on the foraging behaviour of great tits, the bi rds were attracted to the study orchards by providing nest b oxes to compensate for missing nesting sites in mod ern orchards. This is similar to the situation in citru s groves studied in Spain (Barba & Gil-Delgado, see above). The birds were monitored with binoculars. An averag e proportion of foraging trips inside a one-ha orch ard of 48 % was observed for nine breeding pairs. This wou ld correspond to a PT of 0.48. However, as that proportion is not verified by means of time-budget, it is considered as evidence only, that great tits strongly tend to spend foraging time outside orchards. The notifier also mentioned another new study with data from 25 radio-tracking sessions of 22 great ti ts in German pome fruit orchards, delivering a PT of 0.25 . However, this study was not yet available to the RMS. Overall, considering all reported data on active fo raging time of tits in- and outside orchards, the i nclusion of a PT-value of 0.61 seems to represent a conservativ e estimation with regard to populations of great ti ts inhabiting monocultural orchards. Because of the statement that “Great Tits find favo urable breeding habitats in Spanish citrus groves, that they occur there with relatively high population de nsities, and that, at least in certain areas of Spa in, and at certain times of the year, citrus orchards are even more favourable habitats for Great Tits than the surrounding natural habitats” (reported in Wolf & G erlach 2006, based on Belda et al. 1998), some more justification on the refinement of the PT to < 1 wi th regard to the intended use of spirotetramat shou ld be added. Taken on it`s own, this may appear to sugges t a high habitat attractiveness. However, taking a closer look into the underlying study it becomes clear tha t there are several additional factors to be consid ered (e.g. altitude of the locations and time of observations) . Belda et al. (1998) compared the laying date and the size of the first clutch of great tits in a Spanish oran ge grove with those in other, more natural habitats . Nest boxes were installed in the orange grove and two ho lm oak forests at different altitudes, a pine fores t and a (semi-evergreen) zeen oak forest. Whilst the orange grove was close to the sea level, the forest sites were located at 500, 900-950, 1000-1050 and 900-1100 m a ltitude. The mean laying date of the first clutch i n the orange plantation (21. April ± 6.2 d) was about 10- 15 days earlier than in the forest habitats. Howeve r, this time point is significantly earlier than the first applications of spirotetramat (end of May / early J une). The laying dates were also earlier at low compared to h igh altitudes. Overall, the earlier laying time and the greater size of the first clutch in the orange grov e were linked to the earlier food availability in t he evergreen habitats and particularly in the habitats with lowe r altitude. An earlier availability of food in the (evergreen) citrus grove cannot be translated into better food availability in May and June. In contrary, the typically preferr ed food of great tits (caterpillars) becomes rather scarce after the initial peak (Barba & Gil-Delgado 1990). Thus, an earlier peak of caterpillars is rather lin ked to an earlier disappearance than indicative of a good cat erpillar supply later on. In conclusion, the results of Belda et al. (1998) o nly concerned the first clutch in April, whilst spi rotetramat will typically be applied not before end of May / e arly June. Any early season effects which may favou r evergreen sea-side level habitats like citrus grove s in Spain in the early spring (e.g. early availabi lity of– 173 –

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

caterpillars) may even have reversed at the spirote tramat application time, with metamorphosis of the caterpillars, increasing temperature and dryness. T herefore the results of Belda et al. (1998) do not put into question any proposal for a PT refinement for great tits nesting in citrus. The comparison of the bree ding habitat quality of the citrus grove to not-nearby f orests at about 1000 m altitude in early spring can not be seen as indicative for the breeding habitat quality of the citrus grove compared to its surroundings i n early

summer.

In conclusion, the following refinement factors are included in the revised ETE calculation for citrus : FIR/bw for the great tit: 0.85 for arthropods, 1.23 for ca terpillars; PD 0.58 for foliage dwelling arthropods , 0.1 for foliar dwelling caterpillars, 0.32 for ground dwelling art hropods; PT 0.61; measured 21-d twa residues on fol iage and ground dwelling arthropods: 1.9 and 0.35 mg/kg.

All refined TER lt calculations are presented in the tables below:

Table B.9.1.8.2-3: Long-term exposure of birds to s pirotetramat and calculated toxicity exposure ratios, higher-tier refinement for lettuce

Indicator species AR (kg

a.s./ha) Food type FIR/bw RUD F twa MAF PT PD ETE Endpoint (mg/kg bw) TER trigger

Wood Pigeon 0.072 plants 0.76 40 0.41 1.1 1 0.15 0.15 4 27 5

large arthropods 0.88 5.1 - - 0.4 0.8 0.10

small arthropods 0.88 29 - - 0.4 0.2 0.15 Yellow Wagtail 0.072

sum: 0.25 4 16 5

Table B.9.1.8.2-4: Long-term exposure of birds to s pirotetramat and calculated toxicity exposure ratios, higher-tier refinement for citrus

Indicator species Food type FIR/bw C 1 PT PD ETE Endpoint (mg/kg bw) TER trigger

foliage-dwelling arthropods 0.85 1.9 0.61 0.58 0.571

caterpillars 1.23 1.9 0.61 0.10 0.143
ground dwelling arthropods 0.85 0.35 0.61 0.32 0.058 Great Tit

sum: 0.772 4 5.2 5

1 21-d twa concentration of spirotetramat + -enol me asured in arthropods in the field [mg/kg f.w.]

Because the resulting TER for great tits in citrus is only marginally above the trigger, the following arguments could be additionally considered: - the residues in arthropods were derived after app lication rates 15 % higher than the GAP, a 15 % overestimation can therefore be assumed - The NOAEL of 4 mg/kg bw is the lowest figure from two mallard duck reproduction studies and one study with the bobwhite quail (NOAEL repro for the quail: 74 mg/kg bw, highest tested dose). The use of a geometric mean of the NOAELs for the two speci es might be a refinement option.

Southern EU MS during their national risk evaluatio ns should consider that the risk refinement for cit rus as presented above does not cover a potential spirotet ramat use during the period from March to mid of Ma y (because a higher portion of caterpillars in the di et and potentially a higher PT fraction could be ex pected during this period).– 174 –

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

Overall conclusion: All resulting TER`s indicate a low risk to populati ons of birds after the use of spirotetramat accordi ng to GAP.

References : Anthes, N.; Gastel, R.; Quetz, P.-Ch. (2002): Bestand und Habitatwahl einer Ackerpopulation der Schafstelz e ( Motacilla f. flava ) im Landkreis Ludwigsburg, Nordwuerttemberg. Orn.J h.Bad.-Württ, Vol. 18, p. 347-361. Atienza J.C. & J.C. Illera (1997): Tree species selec tion to perform singing and foraging behaviour by G reat and Blue Tits: a trade-off between food gathering and territ orial behaviour ? Bird study 44, p. 117-119. Barba & Gil-Delgado (1990): Seasonal variation in nes tling diet of the Great Tit Parus major in orange groves in eastern Spain. Ornis scandinavica 21, p. 296-298. Belda et al. (1998): Laying date and clutch size of great tits Parus major in the Mediterranean region: a comparison of four habitat types. J. Ornithologie 139: 269-276. Colquhoun, M. K. (1951): The woodpigeon in Britain. Ag ricultural Research Council, London, The woodpigeon in Britain, Vol. 10, p. 1-74. Crocker et al. (1998): Contract PN0903: Improving th e assessment of pesticide risk to birds in orchards . CSL report EH 18/02, 38 pp. Dittberner, H. & Dittberner, W (1984): Die Schafstel ze - Motacilla flava . Publisher:Ziemsen Verlag, Wittenberg, Die neue Brehm Bücherei, p. 72-79, 90-97, 128-131. Inglis, i. R.; Thearle, R. J. P. & Isaacson, A. J. (1 989): Woodpigeon ( Columba palumbus ) damage to oilseed rape. Crop Protection, Vol. 8, p. 299-309. Mason, C.F. & Macdonald, S.M. (1999): Influence of la ndscape and land-use on the distribution of breedin g birds in farmland in eastern England. The Zoological Society o f London, J. Zool. Lond., Vol. 251, p. 339 - 348. Mols, Ch.M.H. (2003): Great Tits ( Parus major ) foraging for caterpillars contribute to biologica l control in apple orchards. Ph.D. Thesis, University of Utrecht, 104 pp. Murton, R.K.; Westwood, N.J. and Isaacson, A.J. (1963 ): The feeding habits of the woodpigeon Columba palumbus

, stock dove C. oenas

and turtel dove Streptopelia turtur . Ibis, Vol. 106, p. 174-188. Seitz, B.-J. (1989): Beziehungen zwischen Vogelwelt und Vegetation im Kul turland, Untersuchungen im suedwestdeutschen Huegelland - Beihefte zu den Veroef fentlichungen fuer Naturschutz und Landschaftspfleg e in Baden-Wuerttemberg. Landesanstalt für Umweltschutz Ba den - Wuerttemberg, Beihefte zu den Veroeffentlichung en fuer Naturschutz und Landschaftspflege in Baden-Wuerttem berg, Volume 54, p.1-236. Wolf, C. & Gerlach, J. (2006): Insectivorous birds in southern European citrus plantations - focal spec ies and their diet composition. Unpublished report.

B.9.1.8.3 Risk to birds from plant metabolites

The following metabolites of spirotetramat were fou nd in lettuce metabolism at levels above 10 % of th e total radioactive residue (TRR): BYI 08330-enol (17.8 %) and BYI 08330-enol glycoside (11.4 %). In smaller amounts the following metabolites were observed in plants metabolism studies at levels above 0.01 mg/k g: BYI 08330-ketohydroxy, BYI 08330- monohydroxy, BYI 08330-di-hydroxy, BYI 08330-desmethyl-enol, and BYI 08330-desmethyl-ketohydroxy. According to curre nt guidance, only metabolites > 10 % have to be considered, however, as the notifier presented info rmation also to minor metabolites, this is reported here. The enol metabolite is also the primary and main me tabolite in the rat and hen metabolism study (see B .7 part of this monograph). In so far, it can be consi dered to be toxicologically well characterised and to be covered by the respective avian toxicological studi es which were conducted with spirotetramat. The enol glycoside metabolite is immediately and qu antitatively metabolised to BYI 08330-enol after di etary uptake. Thus, this metabolite can be considered to be ecotoxicologically sufficiently covered. BYI 08330-desmethyl-enol was also observed in the r at and hen metabolism study and thus is covered by respective toxicological studies conducted with par ent compound. Likewise, the ketohydroxy metabolite was detected i n the rat, and furthermore, an acute toxicity study on rat is available for this metabolite (Schüngel, 2005) w hich reveals that this metabolite shows no toxicity to mammals (LD50 > 5000 mg a.s./kg bw). This is suppor ted by the observation that the ketohydroxy– 175 –

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metabolite proved less toxic than the parent compou nd and the enol metabolite in all ecotoxicological studies conducted. There is no reason to assume that these metabolites would be more toxic to birds. The metabolites BYI 08330-monohydroxy, BYI 08330-di -hydroxy and BYI 08330-desmethyl-ketohydroxy showed no toxicity in acute rat studies (LD50 > 500 0 mg a.s./kg bw, Schüngel, 2005, Schüngel, M., 2006 a and Schüngel, 2006b, respectively); it can thus be considered highly unlikely that exposure might resu lt in unacceptable effects to terrestrial vertebrates. Hence, it is concluded that the plant metabolites o f spirotetramat are covered by existing ecotoxicolo gy or toxicology studies, or they were shown not to be to xic to terrestrial vertebrates. In conclusion, the risk to birds from plant metabol ites appears low.

B.9.1.8.4 Risk to birds from secondary poisoning

The log P OW of spirotetramat is 2.51 at pH 7 and hence it`s po tential to bio-accumulate is considered low. An assessment of the risk through bioaccumulation i s not triggered according to SANCO/4145/2002. This applies also to the main metabolites of spirot etramat as the log P OW of BYI 08330-cis-ketohydroxy is 1.3 and of BYI 08330-enol is 0.3 (at pH 7).

B.9.1.8.5 Risk to birds from the consumption of con taminated drinking water

The risk to birds from uptake of contaminated drink ing water is calculated according to SANCO/4145/200 0. As the exposure via drinking from surface water bod ies is considered to be negligible, the exposure fr om puddles of spray liquid is assessed as a worst case . The calculations are based on the intended use in lettuce (72 g a.s./ha), which covers also the citru s rate, because the concentration in the spray liqu id is higher. Furthermore the formation of spray puddles in citrus orchards in Southern EU is highly unreali stic.

TER = LD 50 /Dailydose

Dailydose = (PEC drinking water x Totalwater ingestion rate)/W

Total water ingestion rate = 0.059 x W 0.67

W = bw in kg

Table B.9.1.8.5-1: Parameters and calculations for the assessment of the acute and short-term risk to birds from the uptake of contaminated drinking wate r from puddles and leaf axils
Parameter Value Application rate [g ai/ha] 72 Application volume [L/ha] 500 (- 1000) PEC drinking water [mg ai/L], assuming a dilution factor of 5 28.8 W [kg], Body weight of a relevant indicator bird 0.015 Total water ingestion rate [L/d] 0.0035 Daily dose [mg ai/kg bw/d] 6.8 LD 50 acute [mg ai/kg bw] > 2000 LD 50 short-term [ mg ai/kg bw/d] > 475 TER acute > 294 TER short-term > 70

The TER-values for acute and short-term exposure vi a contaminated drinking water indicate that the ris k to birds via this route is low.– 176 –

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Overall conclusion

After the use of spirotetramat according to the rel evant GAP no unacceptable effects on populations of wild birds are expected.

B.9.2.4 Risk assessment for aquatic organisms

The risk assessment for aquatic organisms is based on the recommendations of the Guidance Document on Aquatic Ecotoxicology ( http://ec.europa.eu/food/plant/protection/evaluatio n/guidance/wrkdoc10_en.pdf ) and follows a tiered approach. The standard risk assessment (first tier) is based on the calculation of the toxicity/exposure ratios (TER) taking into consideration the most sensitive organi sm of each group. TER values will be estimated as t he ratio of EC/LC 50 or NOEC (toxicity, see table B.9.2.4-1) to PEC sw (exposure, see table B.9.2.4-2 and B.9.2.4- 3) calculated with the FOCUS surface water model. T his model based on scenarios considering a tiered sequence of exposure assessment steps (step 1, 2, 3 and 4). If trigger values for the acute risk (TER A ) or the chronic risk (TER LT ) stated in directive 91/414/EEC are failed and hen ce a potential risk is indicated according to the standard risk assessment, appropri ate refinement steps can be considered (higher tier risk assessment). Usually, in the higher tier risk asses sment higher tier data, e.g. additional single spec ies tests and micro- and mesocosms can be considered.

The aquatic risk assessment includes spirotetramat (as active substance and formulated in the product BYI 08330 OD 150) and the environmentally relevant meta bolites:

• BYI 08330-enol (> 10 % in water and sediment, max 97.5 % in total water/sediment system at day 7)

• BYI 08330-ketohydroxy (> 10 % in water and sedimen t, max 40.5 % in total water/sediment system at day 120)

• 4-Methoxycyclohexanone (> 10 % in photolysis study )

• 4-Methoxycyclohexylamin carboxylic acid (> 10 % in photolysis study).

One metabolite, spirotetramat-MA-amide, is relevant for the groundwater risk assessment, but was not predicted to exceed 0.1 μg/L. Therefore aquatic tes ts are not required and an aquatic risk assessment is not triggered.

Table B.9.2.4-1: Summary of acute and chronic toxic ity data for aquatic organisms which are relevant for risk assessment (most sensitive species of each group)

Group Test substance Time-scale (Test type) Endpoint Toxicity 1 (mg/L)
Fish Cyprinodon variegatus Spirotetramat 96 h (flowthrough) Mortality, LC50 1.96 (mm)

Pimephales promelas Spirotetramat 33 d (flowthrough) Fry survival, NOEC 0.534 (mm)

Oncorhynchus mykiss BYI 08330 OD 150 (formulation) 96 h (flowthrough) Mortality, LC50 9.48 (nom) (1.41 mg as/L)

Oncorhynchus mykiss BYI 08330-enol 96 h (static, limit) Mortality, LC50 > 100 (nom)

Danio rerio 4-Methoxycyclohexanone 96 h (static,

limit) Mortality, LC50 > 100 (nom)

Aquatic invertebrate Daphnia magna Spirotetramat 48 h (static) Mortality, EC50 > 42.7 (mm) Crassostrea virginica Spirotetramat 96 h (static) Shell deposition, EC50 0.8 5 (mm)– 177 –

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Group Test substance Time-scale (Test type) Endpoint Toxicity 1 (mg/L) Daphnia magna Spirotetramat 21 d (static) Mortality adults, NOEC 2. 0 (nom) Daphnia magna BYI 08330-enol 48 h (static, limit) Mortality, EC50 > 100 (nom)

Daphnia magna 4-Methoxycyclohexanone 48 h (static,

limit) Mortality, EC50 > 100 (nom)
Sediment dwelling organisms Chironomus riparius Spirotetramat 48 h (static, water only) Mortality, EC50 1.3 (nom)

Chironomus riparius Spirotetramat 21 d (static, spiked water) Emergence, NOEC 0.1 (nom)

Chironomus riparius BYI 08330 OD 150 (formulation) 48 h (static,

water only) Mortality, EC50 4.41 (mm) (0.66 mg as/L)

Chironomus riparius BYI 08330-enol 48 h (static, water only) Mortality, EC50 74.9 (nom)

Chironomus riparius BYI 08330- ketohydroxy 48 h (static,

water only) Mortality, EC50 > 100 (nom)

Chironomus riparius 4-Methoxycyclohexanone 48 h (static,

water only) Mortality, EC50 > 100 (nom)

Chironomus riparius 4-Methoxycyclohexylamin carboxylic acid 48 h (static,

water only) Mortality, EC50 > 100 (nom)

Algae Skeletonema costatum Spirotetramat 72 h (static) Biomass: E b C50 Growth rate: E r C50 0.36 (mm) 0.96 (mm) Pseudokirch subcap. BYI 08330 OD 150 (formulation) 72 h (static) Biomass: E b C50

Growth rate: E r C50 43.4 (mm) (6.56 mg as/L) > 54.3 (mm) (> 8.2 mg as/L) Pseudokirch subcap. BYI 08330-enol 72 h (static) Biomass: E b C50 Growth rate: E r C50 > 100 (mm) > 100 (mm) Desmodesmus subcap. 4-Methoxycyclohexanone 72 h (static,

limit) Biomass: E b C50

Growth rate: E r C50 > 100 (nom) > 100 (nom) Higher plant Lemna gibba Spirotetramat 7 d (static) Yield: E y C50 Growth rate: E r C50 4.49 (mm) 6.21 (mm) Lemna gibba BYI 08330-enol 7 d (static) Yield: E y C50 Growth rate: E r C50 5.4 (mm) 19.3 (mm)

1 Toxicity based on nominal ( nom ) or mean measured concentrations ( mm )

The risk to aquatic organisms from spirotetramat, i ts metabolites and the formulation was assessed for applications in citrus (worst case) and lettuce. Gl obal maximum PEC SW values for the intended application scenarios were calculated with the FOCUS surface wa ter model. For the active substance and the environmentally relevant metabolites, BYI 08330-eno l and BYI 08330-ketohydroxy (both major in water an d sediment), 4-methoxycyclohexylamino carboxylic acid and 4-methoxycyclohexanone (major metabolites in photolysis studies) the maximum PEC SW values were calculated with FOCUS step 1 and step 2. In addition, FOCUS step 3 and step 4 calculations w ere performed for the application of spirotetramat in citrus (worst case). Details of PEC SW calculations with the FOCUS surface water modellin g are presented in the appendix to the fate section B.8 and the maximum observed PEC SW values are summarised in table B.9.2.4-2 (citrus) and B.9.2.4-3 (lettuce).– 178 –

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Table B.9.2.4-2: Citrus: Summary of maximum observe d PEC SW values of spirotetramat and metabolites using FOCUS step 1 – 4.
max. PEC SW [μg L -1 ] FOCUS STEP 1 FOCUS

STEP 2 FOCUS STEP 3 FOCUS STEP 4 Substance

South EU D6 – ditch* R4 – stream* D6 – ditch* R4 – s tream* Spirotetramat 84.9 11.6 8.43 6.51 5.86 nc BYI 08330-enol 168 15.6 nc nc nc nc BYI-ketohydroxy 46.5 8.59 nc nc nc nc4-Methoxycyclohexanone 8.38 1.39 nc nc nc nc
4-Methoxycyclohexylaminocarboxylic acid 1.58 1.21 nc nc nc nc

* worst case scenario nc...not calculated

Table B.9.2.4-3: Lettuce: Summary of maximum observ ed PEC SW values of spirotetramat and metabolites using FOCUS step 1 and 2.
max. PEC SW [μg L -1 ] FOCUS STEP 1 FOCUS STEP 2 Substance

South EU (worst case) Spirotetramat 18.1 0.585 BYI 08330-enol 37.1 0.950 BYI-ketohydroxy 9.48 0.662 4-Methoxycyclohexanone 1.72 0.071 4-Methoxycyclohexylaminocarboxylic acid 0.069 0.061

Acute and chronic risk

First a conservative risk assessment was performed and TER A and TER LT -values were estimated based on the maximum observed PEC SW from FOCUS Step 1 and 2. If a TER value fails the r elevant trigger value at the step 2 a refinement is required on the basis of FOCUS step 3 and step 4 calculations.

Active substance, metabolites and formulation The TER-values were calculated for the most sensiti ve species of fish, invertebrates, algae and aquati c plants for the active substance, its metabolites an d the formulation. For spirotetramat additionally endpoints from studi es with marine test organisms were available and th e results were considered for the acute risk assessme nt (see argumentation in the main section B.9 under point B.9.2.1.5 Effects on marine or estuarine spec ies). Thus the acute risk is based on the endpoint of marine fish species Cyprinodon variegates and the risk to aquatic invertebrates was assessed with the results of the eastern oyster. This endpoint (EC50: 0.85 mg a.s./L) is comparable with the endpoints f rom the most sensitive sediment dwelling species Chironomus riparius (EC50: 0.66 mg a.s./L, endpoint from test with the formulation). Furthermore the risk for algae wa s assessed by considering the endpoint from marine diatom Skeletonema costatum, the most sensitive algae species.

A summary of toxicity (EC 50 /LC 50 and NOEC), relevant global maximum PEC SW and calculated TER values for spirotetramat, metabolites and the formulation are presented in tables B.9.2.4-4 until B.9.2.4-8:– 179 –

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

Table B.9.2.4-4: Citrus: Acute and chronic toxicity exposure ratios (TER A and TER LT ) based on worst case PEC SW from FOCUS STEP 1

Organism Test substance Toxicity endpoint (mg a.i./L) Time

scale PEC

(mg/L) TER Annex VI Trigger
Fish Cyprinodon variegatus Spirotetramat 1.96 96 h 0.0849 23 100 Pimephales promelas Spirotetramat 0.534 33 d 0.0849 6 10 Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.0849 17 100 Oncorhynchus mykiss BYI 08330-enol > 100 96 h 0.1680 > 595 100 Danio rerio 4-Methoxycyclohexanone > 100 96 h 0.0084 > 11933 10 0 Aquatic invertebrates Daphnia magna Spirotetramat > 42.7 48 h 0.0849 > 503 100 Crassostrea virginica Spirotetramat 0.85 48 h 0.0849 10 100 Daphnia magna Spirotetramat 2.0 21 d 0.0849 24 10 Daphnia magna BYI 08330-enol > 100 48 h 0.1680 > 595 100 Daphnia magna 4-Methoxycyclohexanone > 100 48 h 0.0084 > 11933 10 0 Sediment dwelling organisms Chironomus riparius Spirotetramat 1.3 48 h 0.0849 15 100 Chironomus riparius Spirotetramat 0.1 21 d 0.0849 1 10 Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0849 8 100 Chironomus riparius BYI 08330-enol 74.9 48 h 0.1680 446 100 Chironomus riparius BYI-ketohydroxy > 100 48 h 0.0465 > 2151 100 Chironomus riparius 4-Methoxycyclohexanone > 100 48 h 0.0084 > 11933 10 0 Chironomus riparius 4-Methoxycyclohexylamino-carboxylic acid > 100 48 h 0.0016 > 63291 100
Algae Skeletonema costatum Spirotetramat 0.36/0.96 72 h 0.0849 4/ 11 10 P. subcapitata BYI 08330 OD 150 6.56/>8.2 72 h 0.0849 77/>97 10 P. subcapitata BYI 08330-enol > 100 72 h 0.1680 > 595 10 Desmodesmus subcapitata 4-Methoxycyclohexanone > 100 72 h 0.0084 > 11933 10 Higher plant Lemna gibba Spirotetramat 4.49/6.21 7 d 0.0849 53/73 10 Lemna gibba BYI 08330-enol 5.4/19.3 7 d 0.1680 32/115 10

Table B.9.2.4-5: Lettuce: Acute and chronic toxicit y exposure ratios (TER A and TER LT ) based on worst case PEC SW from FOCUS STEP 1

Organism Test substance Toxicity endpoint (mg a.i./L) Time

scale PEC

(mg/L) TER Annex VI Trigger
Fish Cyprinodon variegatus Spirotetramat 1.96 96 h 0.0181 108 100 Pimephales promelas Spirotetramat 0.534 33 d 0.0181 30 10 Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.0181 78 100 Oncorhynchus mykiss BYI 08330-enol > 100 96 h 0.0371 > 2695 100 Danio rerio 4-Methoxycyclohexanone > 100 96 h 0.0017 > 58140 100 Aquatic invertebrates Daphnia magna Spirotetramat > 42.7 48 h 0.0181 > 2359 100 Crassostrea virginica Spirotetramat 0.85 48 h 0.0181 47 100 Daphnia magna Spirotetramat 2.0 21 d 0.0181 110 10 Daphnia magna BYI 08330-enol > 100 48 h 0.0371 > 2695 100 Daphnia magna 4-Methoxycyclohexanone > 100 48 h 0.0017 > 58140 100 66 Chironomus riparius Spirotetramat 1.3 48 h 0.0181 72 100 Chironomus riparius Spirotetramat 0.1 21 d 0.0181 6 10– 180 –

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Organism Test substance Toxicity endpoint (mg a.i./L) Time

scale PEC

(mg/L) TER Annex VI Trigger Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0181 36 100 Chironomus riparius BYI 08330-enol 74.9 48 h 0.0371 2019 100 Chironomus riparius BYI-ketohydroxy > 100 48 h 0.0095 10549 100 Chironomus riparius 4-Methoxycyclohexanone > 100 48 h 0.0017 > 58140 100 Chironomus riparius 4-Methoxycyclohexylamino-carboxylic acid > 100 48 h 0.00007 > 14.4 x 10 5 100
Algae Skeletonema costatum Spirotetramat 0.36/0.96 72 h 0.0181 20/53 10 P. subcapitata BYI 08330 OD 150 6.56/>8.2 72 h 0.0181 362/>453 10 P. subcapitata BYI 08330-enol > 100 72 h 0.0371 > 2695 10 Desmodesmus subcapitata 4-Methoxycyclohexanone > 100 72 h 0.0017 > 58140 10 Higher plant Lemna gibba Spirotetramat 4.49/6.21 7 d 0.0181 248/343 10 Lemna gibba BYI 08330-enol 5.4/19.3 7 d 0.0371 146/520 10

Table B.9.2.4-6: Citrus: Acute and chronic toxicity exposure ratios (TER A and TER LT ) based on worst case PEC SW from FOCUS STEP 2 – mentioned are only these organ isms which failed the trigger at Step 1

Organism Test substance Toxicity endpoint (mg a.s./L) Time

scale PEC

(mg/L) TER Annex VI Trigger
Fish Oncorhynchus mykiss Spirotetramat 1.96 96 h 0.0116 169 100 Oncorhynchus mykiss Spirotetramat 0.534 33 d 0.0116 46 10 Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.0116 122 100 Aquatic invertebrates Crassostrea virginica Spirotetramat 0.85 48 h 0.0116 73 100 Sediment dwelling organisms Chironomus riparius Spirotetramat 1.3 48 h 0.0116 112 100 Chironomus riparius Spirotetramat 0.1 21 d 0.0116 9 10 Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0116 57 100 Algae Skeletonema costatum Spirotetramat 0.36/0.96 72 h 0.0116 31/83 10

Table B.9.2.4-7: Lettuce: Acute and chronic toxicit y exposure ratios (TER A and TER LT ) based on worst case PEC SW from FOCUS STEP 2 – mentioned are only these organ isms which failed the trigger at Step 1

Organism Test substance Toxicity endpoint (mg a.s./L) Time

scale PEC

(mg/L) TER Annex VI Trigger
Fish Oncorhynchus mykiss BYI 08330 OD 150 1.41 96 h 0.00059 2410 100 Aquatic invertebrates Crassostrea virginica Spirotetramat 0.85 48 h 0.00059 1453 100 Sediment dwelling organisms Chironomus riparius Spirotetramat 1.3 48 h 0.00059 2222 100 Chironomus riparius Spirotetramat 0.1 21 d 0.00059 171 10 Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.00059 1128 100– 181 –

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

Table B.9.2.4-8: Citrus: Acute and chronic toxicity exposure ratios (TER A and TER LT ) based on worst case PEC SW from FOCUS STEP 3 – mentioned are only these organ isms which failed the trigger at Step 2

Organism Test substance Toxicity endpoint (mg a.s./L) Time

scale PEC*

(mg/L) TER Annex VI Trigger Aquatic invertebrates Crassostrea virginica Spirotetramat 0.85 48 h 0.0084 101 100 Sediment dwelling organisms Chironomus riparius Spirotetramat 0.1 21 d 0.0084 12 10 Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0084 78 100 *highest PEC sw

from the worst case scenario D6 (Thiva) ditch

Table B.9.2.4-9: Citrus: Acute toxicity exposure ra tios (TER A ) based on worst case PEC SW from FOCUS STEP 4 (5 m buffer zone) – mentioned are only these organisms which failed the trigger at Step 3

Organism Test substance Toxicity endpoint (mg a.s./L) Time

scale PEC*

(mg/L) TER Annex VI Trigger Sediment dwelling organisms Chironomus riparius BYI 08330 OD 150 0.66 48 h 0.0059 113 100 *highest PEC sw

from the worst case scenario D6 (Thiva) ditch

Acute risk from active substance and formulation: Spirotetramat and the formulation BYI 08330 OD 150 are toxic to standard test species of fish, aquatic invertebrates, algae and higher plants. The most se nsitive species is the sediment dwelling organism Chironomus riparius

with an LC50 of 0.66 mg a.s./L, based on a study p erformed with the formulation. In general the toxicity of the formulation is slightly higher when compared to the toxicity of the active substance alone.

Citrus: All TER A values for fish, daphnids, algae and higher plants calculated with global maximum PEC SW from FOCUS step 2 (first tier risk assessment) meet the relevant triggers established in directive 91/414/EEC. However, the calculated TER value for the most sens itive species Chironomus riparius (based on test with the formulation) is 8, 57 and 78 calculated with FO CUS STEP 1, 2 and 3 (worst case scenario D6 ditch), respectively. Since all TER values are below the re levant trigger of 100 the TER is also calculated wi th PECsw from FOCUS STEP 4 considering a 5 m buffer zo ne to surface water. This calculation based on the PECsw of 0.00586 mg/L from the worst case scenario D6 (Thiva) ditch and resulted in a TER of 113. Lettuce: All TER A values for fish, aquatic invertebrates (daphnids a nd sediment dwelling organisms), algae and higher plants based on calculations with the gl obal maximum PEC SW from FOCUS STEP 2 are clearly above the relevant trigger values established in di rective 91/414/EEC, thus the risk is considered acc eptable.

In conclusion, the acute risk can be considered as acceptable for applications in citrus provided that risk mitigation measures (at least a buffer zone of 5 m to surface water) are implemented. For applications in lettuce an acceptable risk is indicated without ris k mitigation measures.

Acute risk from metabolites: Aquatic toxicity studies with fish, aquatic inverte brate and algae have been conducted with the metabo lites BYI 08330-enol and 4-methoxycyclohexanone. Tests wi th the metabolites BYI 08330-ketohydroxy and 4- methoxycyclohexylamino carboxylic acid were perform ed with the most sensitive organism, Chironomus riparius, only.– 182 –

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

In conclusion all environmental relevant metabolite s are of low acute toxicity to aquatic organisms an d all calculated TER values for all intended uses are wel l above the relevant trigger values. Thus the risk from metabolites of spirotetramat to aquatic organisms i s expected to be low.

Chronic risk from active substance and formulation: The assessment of the chronic toxicity is based on long term studies with fish ( P. promelas ) and aquatic invertebrates ( C. riparius ) with the active substance.

Citrus: For fish the safety factor (TER LT ) was calculated to be 46 based on PEC sw from FOCUS step 2. For aquatic invertebrates a TER LT of 12 was estimated based on PEC sw from FOCUS step 3. Both TER LT values are above the relevant trigger of 10. Lettuce: The safety factors for fish and aquatic invertebra tes were calculated to be 30 (based on FOCUS step 1 calculation) and 171 (based on FOCUS step 2 calculation). Thus both TER LT values are clearly above the relevant trigger of 10.

In conclusion for the intended uses in citrus and l ettuce the chronic risk of spirotetramat for aquati c organisms is expected to be low.

Bioaccumulation

Spirotetramat has a logP OW of 2.51 and studies on bioconcentration are not tr iggered. Therefore the potential of bioconcentration of spirotetramat in aquatic org anisms is expected to be low and the risk of bioaccumulation in aquatic ecosystems is considered acceptable. The bioaccumulation potential of all major metaboli tes in water and sediment (spirotetramat-enol, spirotetramat-ketohydroxy, 4-methoxycyclohexanone a nd 4-methoxycyclohexylamino carboxyl acid) is also assumed to be low, due to logP OW values clearly lower than 3. Thus, it can be concl uded that the risk of bioaccumulation of the major metabolites in the aqu atic ecosystem is acceptable.

B.9.3.1 Risk assessment for mammals

Wild mammals may be exposed to spirotetramat by eat ing contaminated vegetation, seeds and fruits, invertebrate prey like arthropods (i.e. insects) or earthworms or vertebrate prey. Toxicity Exposure Ratios (TER) and Estimated Theore tical Exposures (ETE) were calculated based on the methods, i.e. equations and tabulated values descri bed in the “Guidance Document on Risk Assessment fo r Birds and Mammals Under Council Directive 91/414/EE C” (Sanco/4145/2000 of 25. September 2002). The risk assessment is based on toxicity values of the active substance and the acute oral LD 50 for the EU lead formulation (Spirotetramat OD150). The relevant toxicity figures were taken from labor atory studies with spirotetramat. All of them were considered acceptable. A detailed evaluation of the respective mammalian toxicology studies can be fou nd in the B.6 part of this monograph. These endpoints are listed in table B.9.3.3-1 (below) for the diffe rent time scales.

Maximum ETE values were estimated for use in the ac ute risk assessment while time-weighted average ETE values were calculated for use in assessing long-te rm risks. ETE values were calculated using the foll owing equation:

ETE (mg as/kg bw/day) = FIR/bw x RUD x Application rate x MAF x f TWA x PT x PD x AV

where: FIR Food Intake Rate (g fresh weight per d ay)– 183 –

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

bw Body weight (g) RUD Residue per unit dose (mg/kg fresh weight) MAF Multiple Application Factor f TWA Time-weighted average factor (only used for calcula ting long-term ETE) PT Proportion of diet obtained in treated area PD Proportion of food-type in diet AV Avoidance factor (no avoidance =1) As recommended by the EU Expert Group on Higher Tie r Risk Assessment for Birds and Mammals, the 90 th percentile residues on food items were used for the acute risk assessment, and the 50 th percentiles for the short and long-term risk assessments. The f TWA was calculated from the following equation: f twa = (1-e -kt )/kt where: k = ln2/DT 50 t = averaging time

The risk assessment is based on the following toxic ity endpoints, which were considered ecotoxicologic ally relevant with regard to risk assessment:

Table B.9.3.1-1: Ecotoxicologically relevant mammal ian toxicity endpoints for spirotetramat

Species mg/kg bw/day Reference
Acute oral LD 50 Rat, female > 2000 Eigenberg (2004) Acute oral LD 50 formulation Rat, female > 2000* Schüngel (2005) Reproductive NOAEL Rat 70.7 / 82.5 (m / f) Young ( 2006) * equivalent to 306 g a.s.

Choice of relevant NOAEL for long-term risk assessm ent: The two-generation reproduction study in the rat, a nd the developmental toxicity studies in the rat an d the rabbit have been evaluated in order to derive an ap propriate endpoint for use in the long-term risk assessment. A clear NOAEL of 70.7 / 82.5 mg/kg bw/d ay in male / female rats is derived from the twogeneration study which is based on effects in the n ext higher dosage group on male reproductive performance, sperm cells and offspring body weight development. The value for males is used in risk assessment as this figure is considered as protecti ve with regard to wild mammal populations. The NOAE Ls derived from developmental studies were higher.

B.9.3.1.1 First tier risk assessment to mammals

The calculations are based on the following represe ntative application scenarios in the EU:

• Lettuce: 2 x 0.072 kg a.s./ha (interval 14 d), Nor thern + Southern Europe

• Citrus: 2 x 0.096 kg a.s./m canopy height/ha (inte rval 21 d), max. 0.288 kg a.s./ha used in risk assessment, Southern Europe

Assessment of exposure / Selection of scenarios: The default values for the acute and long-term expo sure in the tier 1 risk assessment are selected acc ording to recommendations of the “Guidance Document on Ris k Assessment for Birds and Mammals Under Council Directive 91/414/EEC”. According to this guidance d ocument, crop specific generic indicator species wi th specific daily food intake rates (FIR) related to t he body weight (bw) should be used for the tier 1 r isk assessment. Spirotetramat OD 150 is an insecticide that is applied by spray application in lettuce and citrus. For spray applications in leafy crops the risk asse ssment should be based on generic data for medium herbivorous mammals (e.g. hare). For spray applicat ion in orchards small herbivorous mammals are used as model organisms. To summarise, the following relevant exposure scena rios were considered:– 184 –

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

- dietary exposure of a medium herbivorous mammal f eeding on treated herbage in lettuce (category “leafy crops”) - dietary exposure of a small herbivorous mammal fe eding on short grass in citrus (category “orchard”)

In order to calculate the estimated theoretical exp osure the respective figures were chosen to estimat e food intake rates and concentrations in the food of conc ern (see Table B.9.3.3.1.-1). For both intended uses two applications are intende d. Accordingly the MAF (multiple application factor ) is set according to the recommendations of the guidance do cument at 1.2 for the acute scenarios and to 1.4 or 1.2 for long-term in lettuce and citrus, respectively. A default half-life of 10 d is assumed for estimati ng residue decline on the crop. For acute exposure, the 90 th percentile and for long-term exposure mean initial residue levels in the respective food items were applied. I n the first tier worst case assessment it is assume d that the mammals feed on a single food type, which is not av oided and that they satisfy their entire food deman d in the treated area. Possible food items like soil-dwelling arthropods o r earthworms, for which no respective risk assessme nt was performed, are covered by the calculations presente d because residues in these items are expected to b e much lower.

Table B.9.3.1.1-1: Acute and long-term exposure of mammals to spirotetramat and calculated toxicity exposure ratios, tier 1 Crop / Time horizon Indicator species Endpoint (mg a.s./kg bw) FIR/bw RUD MAF ftwa ETE TER

lettuce Acute Medium herbivorous

mammal > 2000 0.28 87 1.2 / 2.1 > 950

Acuteformulation Medium herbivorous

mammal > 306 0.28 87 1.2 / 2.1 > 145

Long term Medium herbivorous

mammal 70.7 0.28 40 1.4 0.53 0.6 118

citrus Acute Small herbivorous

mammal > 2000 1.39 85 1.2 / 41 > 49

Acuteformulation Small herbivorous

mammal > 306 1.39 85 1.2 / 41 > 7.5

Long term Small herbivorous

mammal 70.7 1.39 46 1.2 0.53 12 5.9

FIR/bw...food intake rate in relation to body weight; RUD...residue per unit dose; MAF...multiple applicatio n factor; ftwa...time weighted average factor; ETE...estimated theoretical exposure; TER...toxicity exposure ratio

The TERs except the TERacute for the formulation an d the citrus application are above the respective t rigger values of 10 (acute) or 5 (reproductive) and indica te a low risk to mammals after consumption of contaminated vegetation. As the TERacute is a “bigger than” value based on t he usually highest tested rate of 2000 mg/kg bw in acute oral toxicity studies, and as no mortalities were o bserved at this dosage, this figure can be consider ed to exceed the trigger of 10. The risk to wild mammals after consumption of conta minated diet after the use of spirotetramat accordi ng to GAP is low.

B.9.3.1.2 Higher tier risk assessment to mammals

Not required.– 185 –

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

B.9.3.1.3 Risk to mammals from plant metabolites

The following metabolites of spirotetramat were fou nd in lettuce metabolism at levels above 10 % of th e total radioactive residue (TRR): BYI 08330-enol (17.8 %) and BYI 08330-enol glycoside (11.4 %). In smaller amounts the following metabolites were observed in plants metabolism studies at levels above 0.01 mg/k g: BYI 08330-ketohydroxy, BYI 08330- monohydroxy, BYI 08330-di-hydroxy, BYI 08330-desmethyl-enol, and BYI 08330-desmethyl-ketohydroxy. According to curre nt guidance, only metabolites > 10 % have to be considered, however, as the notifier presented info rmation also to minor metabolites, this is reported here. The enol metabolite is also the primary and main me tabolite in the rat and hen metabolism study (see B .7 part of this monograph). In so far, it can be consi dered to be toxicologically well characterised and to be covered by the respective mammalian toxicological s tudies available which were conducted with the pare nt compound. The enol glycoside metabolite is immediately and qu antitatively metabolised to BYI 08330-enol after di etary uptake. Thus, this metabolite can be considered to be ecotoxicologically sufficiently covered. BYI 08330-desmethyl-enol was also observed in the r at and hen metabolism study and thus is covered by respective toxicological studies conducted with the parent compound. Likewise, the ketohydroxy metabolite was detected i n the rat, and furthermore, an acute toxicity study on rat is available for this metabolite (Schüngel, 2005) w hich reveals that this metabolite shows no toxicity to mammals (LD50 > 5000 mg a.s./kg bw). This is suppor ted by the observation that the ketohydroxy metabolite proved less toxic than the parent compou nd and than the enol metabolite in all ecotoxicolog ical studies conducted. The metabolites BYI 08330-monohydroxy, BYI 08330-di -hydroxy and BYI 08330-desmethyl-ketohydroxy showed no toxicity in acute rat studies (LD50 > 500 0 mg a.s./kg bw, Schüngel, 2005, Schüngel, M., 2006 and Schüngel, 2006, respectively); it can thus be c onsidered highly unlikely that exposure might resul t in unacceptable effects to terrestrial vertebrates. Hence, it is concluded that the plant metabolites o f spirotetramat are covered by existing ecotoxicolo gy or toxicology studies, or they were shown not to be to xic to terrestrial vertebrates. The risk to wild ma mmals from plant metabolites appears low.

B.9.3.1.4 Risk to mammals from secondary poisoning

The log P OW of spirotetramat is 2.51 at pH 7 and hence it`s po tential to bio-accumulate is considered low. An assessment of the risk through bioaccumulation i s not triggered according to SANCO/4145/2002. This applies also to the main metabolites of spirot etramat as the log P OW of BYI 08330-cis-ketohydroxy is 1.3 and of BYI 08330-enol is 0.3 (at pH 7).

B.9.3.1.5 Risk to mammals from the consumption of c ontaminated drinking water

The risk to mammals from uptake of contaminated dri nking water is calculated according to SANCO/4145/2000. As the exposure via drinking from surface water bodies is considered to be negligible

, the exposure from puddles of spray liquid is assess ed as a worst case. The calculations are based on t he intended use in lettuce (72 g a.s./ha), which cover s also the citrus rate, because the concentration i n the spray liquid is higher. Furthermore the formation o f spray puddles in citrus orchards in Southern EU i s highly unrealistic.

TER = LD 50 /Dailydose

Dailydose = (PEC drinking water x Totalwater ingestion rate)/W

Total water ingestion rate = 0.099 x W 0.90– 186 –

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

Table B.9.3.3.5-1: Parameters and calculations for the assessment of the acute and short-term risk to mammals from the uptake of contaminated drinking wa ter from puddles and leaf axils
Parameter Value Application rate [g ai/ha] 72 Application volume [L/ha] 500 (- 1000) PEC drinking water [mg ai/L], assuming a dilution factor of 5 28.8 W [kg], Body weight of a relevant indicator mammal ( pygmy shrew) 0.006

Total water ingestion rate [L/d] 0.001 Daily dose [mg ai/kg bw/d] 4.8 LD 50 acute [mg ai/kg bw] technical substance / formulated s. > 2000 / > 306 TER acute technical substance / formulation 421 / 64

The TER-value for acute exposure via contaminated d rinking water indicates that the risk to wild mamma ls via this route is negligible.

Overall conclusions

After the use of spirotetramat according to GAP no unacceptable effects on populations of wild mammals are expected.

B.9.4.8 Risk assessment for honey bees

Honey-bees may be exposed to formulated spirotetram at by direct spraying of the plant protection produ ct while bees are foraging on flowers and weeds presen t in or adjacent to the crop treated. They may also be exposed through contact with fresh or dry residues or by oral uptake of contaminated pollen, nectar an d honey dew. The LD 50 -values (48 h) for both oral and contact toxicity o f the active substance are above 100 μg/bee, indicating that spirotetramat is of low toxicity to adult bees. The acute toxicity of the lead formulation Spirotetramat OD150 is slightly higher by the oral route. The intended rates for spirotetramat are 2 x 72 g a .s./ha in lettuce and 2 x 96 g a.s./ m canopy heigh t in citrus. With regard to citrus the rate per m canopy height is considered as effective application rate for bee exposure (see also B.9.5.4). In accordance with the Guidance Document on Terrestrial Ecotoxicology und er Council Directive 91/414/EEC (SANCO/10329/2002) the acute risk for bees was expressed as a Hazard Quotient calculated by the following formula (singl e application rate in g/ha, LD 50 in μg/bee): Hazard quotient (Q H ) = application rate/LD 50– 187 –

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

Table B.9.4.8-1: Risk to honey-bees from oral and c ontact exposure to technical and formulated Spirotetramat, expressed as Hazard Quotient Q H Test substance Exposure route Application rate [g a.s./ha] Endpoint Value [μg a.s./bee] Q H

lettuce Spirotetramat techn. oral contact 72 72 LD 50

LD 50 > 107.3

> 100 < 0.67

< 0.72 Spirotetramat OD150 oral contact 72 72 LD 50

LD 50 91.7

162 0.79

0.44 citrus Spirotetramat techn. oral contact 96 96 LD 50

LD 50 > 107.3

> 100 < 0.89

< 0.96 Spirotetramat OD150 oral contact 96 96 LD 50

LD 50 91.7

162 1.04

0.59

All resulting Hazard Quotients are significantly be low the trigger of 50 and indicate a low risk to be es with respect to adult bee mortality after the use of spi rotetramat according to GAP.

In a brood feeding test, where bee colonies were fe d a sugar solution containing 0.0144 % of Spirotetr amat OD 100, brood effects, seen as a high termination r ate, were detected (Schur 2004 a and b). However, according to its design, this test can be seen as a screening test with unrealistically sever e exposure conditions, which is appropriate to evalua te an intrinsic hazard potential of a compound but is not necessarily indicative for a risk under realistic c onditions (EPPO risk assessment scheme). In order to test for potential brood effects under more realistic exposure conditions, a semi-field br ood test (Schur, 2005) was conducted. A worst-case applicati on scenario was applied with three pre-flowering applications at 72 g a.s./ha followed by two applic ations at 96 g a.s./ha into the blooming crop durin g bee flight. In this study, an irritation of brood development in the earlier a ssessments in one of 3 colonies of the 5x treatment was observed. However, there was no cl ear treatment relation and recovery took place. No effects to other endpoints, as adult mortality, for aging activity, or bee behavior were detected. An additional semi-field study with special focus o n brood effects was conducted (Schur, 2006). Two application scenarios with applications into the bl ooming crop were tested, covering the lettuce scena rio (even with additional pre-flowering applications) a nd the citrus scenario. The study was conducted in a model crop ( Phacelia ) which can be considered to represent worst-case c onditions in terms of flower density and bee attractiveness. No adverse effects of the compo und could be seen, neither in brood development or colony condition, nor on any other parameter assess ed. A series of additional, non GLP-tunnel trials, whic h have not been conducted for regulatory purposes, was also reported by the notifier, because bee brood tu rned out to be negatively affected in these studies . After different application scenarios, the proportion of the larval cell comb area appeared to be reduced fr om about 3 up to 14 days after application. In all cases the effects were transient; i.e. recovery was seen and the overall survival of the affected colonies was not e ndangered. At the time of writing this DAR (spring 2008), the notifier is conducting a field study to enable a re asonable conclusion with regard to the bee safety of Spirote tramat containing products under realistic exposure conditions. As soon as these results are available, the RMS will report them in an addendum.

Overall conclusion

The hazard quotients for oral and contact toxicity of spirotetramat are below the trigger of 50, as sp ecified in 91/414/EEC, thus indicating a low risk for adult be es after application according to the intended use conditions.– 188 –

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

Spirotetramat applications into blooming, bee-attra ctive crops may cause transient effects on honey-be e brood. However, many semi-field trials covering dif ferent application scenarios and worst case situati ons were conducted, all of them of good quality, and in no case a lasting effect on brood development occu rred, and the overall condition and survival of colonies was not disturbed. As regards the potential risk to bees for the repre sentative uses, the RMS recommends precautionary labelling as “Spe8: Dangerous to bees. To protect b ees and other pollinating insects do not apply to c rop plants when in flower or when flowering weeds are p resent. Do not use when bees are actively foraging. ”. Especially for the citrus use, which possibly takes place also during blooming, EU Southern MS should apply their regional risk mitigation measures.

B.9.5.4 Risk assessment for non-target arthropods

Non-target arthropods may be exposed to formulated spirotetramat by direct spraying, contact with fres h or dry residues or by oral uptake of contaminated prey , nectar and pollen or via host organisms. A risk assessment for non-target arthropods was per formed according to the recommendations of ESCORT II. In the first tier hazard quotients were calculated for exposure in in-field and off-field a reas according to the following formulas:

50 field in LR MAF

rate n applicatio HQ ×

=

−

50 field

- off LR factor on distributi vegetation factor drift MAF rate n applicatio
factor correction HQ        
× ×

× =

drift factor = % drift/100 (90 %ile drift according to Ganzelmeier et al. 1995)

The correction factor and the vegetation distributi on factor were set to 10. Drift figures were chosen according to crop type in 1 m distance (lettuce) or 3 m distance (citrus).

Effective application rate with regard to foliage d welling arthropods in citrus = 0.096 kg/ha Foliage dwelling arthropods in the canopy are expos ed to that fraction of the application rate that ca n be expected to reach the respective compartment (“effe ctive rate”) of the canopy, and not to the maximum application rate for trees of 3 m height. The appli cation rate is defined per m canopy height. Likewis e, the maximum spray concentration and the predicted resid ues in the sprayed canopy (e.g. foliage, arthropods ) are comparable regardless of whether a tree of 1, 2 or 3 m height has been sprayed. Thus, an applicati on rate corresponding to 0.096 kg a.s./ha (= amount pe r 1 m tree height) is considered in the non-target arthropods risk assessment for citrus.

Multiple application factors (MAF) were calculated taking into account the measured half-life on plant surface of 4.45 days (for more details see B.9.1.8.2). For citrus as a fruit crop the respective spray drift f or “late” use of 15.73 % was chosen, because the figure for “earl y” use is related to the early stage of orchard tre es (i.e. apples) where only buds and no or very small leaves are present and the drift would be accordingly hig her. In citrus leaves are fully developed during all sta ges, however.– 189 –

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

Table B.9.5.4-1: HQ calculations for Aphidius rhopalosiphi and Typhlodromus pyri

Crop Application rate [g a.s./ha] MAF Drift [%] LR 50 [g a.s./ha] HQ in-field HQ off-field

Aphidius rhopalosiphi

lettuce 72 1.2 2.77 114.7 0.75 0.02

citrus 96 1.1 15.73 114.7 0.92 0.14 Typhlodromus pyri

lettuce 72 1.2 2.77 0.333 259 7

citrus 96 1.1 15.73 0.333 317 50

Both HQ-values for Aphidius rhopalosiphi are below the trigger of 2 and indicate an accepta ble risk. The triggers are definitely breached for Typhlodromus pyri for the in- and off-field area and indicate the ne ed of further higher tiered data on this indicator specie s and additional species. In the following the in-c rop- and off-crop risk to the four relevant functional group s of arthropods are assessed seperately.

Larvicidal mode of action of spirotetramat: In bees spirotetramat caused transient effects on b ee larvae. With regard to non-target-arthropods few guidance on the evaluation of larvicidal / “insect growth regulator” - effects is currently available. However, “worst-case” toxicity studies have been conducted w ith larvae of two different insect taxa: Coccinella septempunctata

and Chrysoperla carnea , and the resulting effects at the intended rates w ere below 10 %. Hence it is considered that potential effects of sp irotetramat on insect larvae / development is cover ed by the risk assessment presented below.

In-crop risk assessment:
Parasitoid wasps ( Aphidius rhopalosiphi ): At the in-field rates of both intended uses spirote tramat effectuates only low mortality rates in this species in the laboratory. Under extended lab conditions effec ts are even lower. Hence the risk to parasitoids ap pears low. This is also indicated by the Hazard Quotients for this species (see table B.9.5.4-1). Predatory mites ( Typhlodromus pyri ): Very low doses of spirotetramat lead to complete mo rtality of T. pyri in the laboratory. If the mites are exposed to fresh residues on treat ed plants (extended lab), this still results in 100 % mortality at rates lower than intended. Even residues aged fo r up to 49 days yield in mortality > 50 %. However, after 56 days of aging mortality was low and no effects o n reproduction were seen in the extended lab. Based on the results from laboratory and extended l aboratory testing, it is therefore considered that the incrop populations of predatory mites will have the p ossibility to recover after an approximate period o f 8 weeks or rather that mites from the off-crop area m ay immigrate. However, according to the results of the field study, it can be assumed that the initial tox icity under “real world” conditions will be lower. Southern EU member states during their national authorisations for citrus should evaluate the field study with res pect to it`s representativeness for their purposes. Foliage-dwelling arthropods: In an extended laboratory study with Chrysoperla carnea , mortality of < 10 % was observed at rates up to 288 g a.s./ha. In Coccinella septempunctata , no mortality occurred at the intended rates and a t higher rates it stayed < 30 %. Hence the risk to foliage-dwelling a rthropods can be considered low. Ground-dwelling arthropods: No studies were performed with ground-dwelling spec ies, however, their risk is covered by two species of foliage-dwellers, which are well representing the a rthropod fauna, especially with regard to the citru s use.– 190 –

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

Off-crop risk assessment:
Parasitoid wasps (Aphidius rhopalosiphi) : The risk to this species/group is low, as at drift rates no mortality or sublethal effects are expecte d. Predatory mites (Typhlodromus pyri) : According to the laboratory and extended laboratory data high mortality may happen also at drift rates

. However, in a field study in vineyards at rates app roximately comparable to GAP no significant effects were detected on the acarine fauna. Hence under natural outdoor conditions off-crop mortality apparently is low and the risk to predatory mites after the use of sp irotetramat according to GAP appears low. The poten tial for recolonisation of mites in the in-crop area is give n. Foliage-dwelling arthropods: The risk to this species/group is low, as at drift rates no mortality or sublethal effects are expecte d according to the results of extended laboratory testing. Ground-dwelling arthropods: The risk to this group is covered by the data on fo liage-dwelling species.

Overall conclusion

Both in- and off-field exposure to formulated spiro tetramat according to the intended use conditions d o not cause unacceptable effects to populations of terres trial non-target arthropods.

B.9.6.4 Risk assessment for earthworms

The TER values for earthworms were calculated as th e ratio between the acute effect concentrations (LC 50 ) and the sublethal no observed effect concentration (NOEC), respectively, and the maximum PEC s . The relevant endpoints (EC50 and NOEC) and overall wors t case PEC s values for the active substance and its metabolites are summarised in table B.9.6.4-1 and t able B.9.6.4-2, respectively. Details for PEC S calculation by FOCUS modelling are presented in the appendix to the fate section B.8.

In the risk assessment for earthworms the active su bstance spirotetramat and its major metabolites in soil are considered:

• BYI 08330-enol (100 % AR after 1 day, DT50 of 2.6 days in laboratory test)

• BYI 08330-ketohydroxy (max. 24 % after 1 day, DT50 of 12.1 days in laboratory test)

• 4-methoxycyclohexanone (max. 10 % after 2 days, DT 50 of 0.6 days in soil photolysis study).

Spirotetramat has a logP OW of > 2 and usually a correction of the relevant en dpoints is necessary. However, in this case the study with the active substance wa s performed using an artificial soil containing 5 % sphagnum peat and therefore a correction of the end points is not needed.– 191 –

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

Table B.9.6.4-1: Summary of effects of spirotetrama t and its metabolites on earthworms

Substance Species Endpoint Time EC/LC 50 [mg/kg dw] NOEC

[mg/kg dw] Reference Studyacceptable Spirotetramat (BYI 08330)) Eisenia fetida Mortality Body weight 14 d > 1000 > 1000 1000

1000 LecheltKunze 2004 yes

BYI 08330- ketohydroxy (metabolite) Eisenia fetida Mortality Body weight 14 d > 1000 > 1000 1000

1000 LecheltKunze 2005 yes

4-Methoxycyclohexanone (metabolite) Eisenia fetida Mortality Body weight 14 d > 1000 > 1000 1000

125 Leicher

2008 yes

BYI 08330-enol (metabolite) Eisenia fetida Mortality Body weight Reproduction 4 w

4 w 8 w > 1000

> 1000 1269 1000

1000 32 Moser &

Scheffczyk 2005 yes

Table B.9.6.4-2: Summary of maximum observed PEC Soil values of spirotetramat and its metabolites max. PEC S [mg kg -1 ] Substance Citrus Lettuce Spirotetramat 0.115 0.029 BYI 08330-enol 0.093 0.024 BYI-ketohydroxy 0.031 0.009 4-Methoxycyclohexanone 0.004 0.001

Acute effects

Table B.9.6.4-3: Citrus: TER acute for earthworms

Test substance 14 days LC 50 [mg/kg soil dw] max PEC s [mg/kg] TER A Trigger value Refined risk

assessment Spirotetramat (BYI 08330)) > 1000 0.115 > 8696 10 no refinement required BYI 08330-ketohydroxy (metabolite) > 1000 0.031 > 32258 10 no refinement required
4- Methoxycyclohexanone > 1000 0.004 > 250000 10 no refinement required

Table B.9.6.4-4: Lettuce: TER acute for earthworms

Test substance 14 days LC 50 [mg/kg soil dw] max PEC s [mg/kg] TER A Trigger value Refined risk

assessment Spirotetramat (BYI 08330)) > 1000 0.029 > 34483 10 no refinement required BYI 08330-ketohydroxy (metabolite) > 1000 0.009 > 111111 10 no refinement required
4- Methoxycyclohexanone > 1000 0.001 > 1000000 10 no refinement required– 192 –

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

Long term effects

Table B.9.6.4-5: Citrus: TER long term for earthwor ms

Test substance NOEC [mg/kg soil dw] max PEC s [mg/kg] TER LT Trigger value Refined risk

assessment
BYI 08330-enol (metabolite) 32 0.093 344 5 no refinement required

Table B.9.6.4-6: Lettuce: TER long term for earthwo rms

Test substance NOEC [mg/kg soil dw] max PEC s [mg/kg] TER LT Trigger value Refined risk

assessment
BYI 08330-enol (metabolite) 32 0.024 1333 5 no refinement required

Active substance The calculated TER A value for spirotetramat is well above the trigger value of 10, thus the acute risk to earthworms after the application according to GAP c an be considered as low. Since the representative use of spirotetramat inclu ded only a low number of applications in citrus and lettuce (maximum of 2 applications in both crops) and spiro tetramat is not persistent a reproduction test was not required. Therefore the risk for earthworms after the applica tion of spirotetramat is considered acceptable.

Metabolites Three major soil metabolites were identified: BYI 0 8330-enol, BYI 08330-ketohydroxy and 4- methoxycyclohexanone. Acute tests with BYI 08330-ke tohydroxy and 4-methoxycyclohexanone are available and the toxicity of the metabolite BYI 08330-enol w as assessed in a reproductive test.

For the worst case application in citrus the TER A values for BYI 08330-ketohydroxy and 4- methoxycyclohexanone were estimated to be > 32258 a nd > 250000, respectively. The acute risk of BYI 08330-enol is covered with the study on the active substance due to the rapid and complete formation o f this metabolite (100 % after 1 day). In addition an endp oint from reproductive study is available and the T ER LT value for this long term exposure of BYI 08330-enol was calculated to be 344. Thus, all calculated val ues meet the relevant trigger. Therefore risk for earthworms from the metabolites BYI 08330-enol, BYI 08330-ketohydroxy and 4- methoxycyclohexanone is expected to be low.

In conclusion, all calculated TER values for spirot etramat and its relevant metabolites (BYI 08330-eno l, BYI 08330-ketohydroxy and 4-methoxycyclohexynone) are w ell above the relevant trigger values, thus the ris k to earthworms after the application according to GAP c an be considered as low.– 193 –

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

B.9.7.4 Risk assessment for other soil non-target m acro-organisms

One study to assess the risk to gamasid mites was p erformed with the major metabolite BYI 08330-enol i n soil. This major metabolite was used because the ac tive substance spirotetramat will be rapidly conver ted to BYI 08330-enol (max. 100 % after 1 day) in soil.

Table 9.7.4-1: Summary of effects on soil non-targe t macro-organisms
Substance Species Endpoint Time EC/LC 50 [mg/kg dw] NOEC

[mg/kg dw] Reference/study

acceptable

BYI 08330-enol Gamasid mite

( Hypoaspis aculeifer) Mortality Reproduction 28 d > 1000 316 1000 Moser & Scheffczyk

2005/yes

The TER value was calculated as the ratio between t he NOEC and the maximum PEC s . The worst case PEC s value of BYI 08330-enol after applications in citr us and in lettuce was calculated to be 0.093 mg a.s ./kg and 0.024 mg a.s./kg, respectively. For details of calculations see the appendix to the fate section B .8. A correction factor is not necessary because a natu ral soil with a low C org content was used in the test.

Table 9.7.4-2: Citrus: TER long term for gamasid mi tes

Test substance NOEC [mg a.s./kg soil] maximum PECs [mg/kg] TER lt Trigger value

BYI 08330-enol 316 0.093 3399 5

Table 9.7.4-3: Lettuce: TER long term for gamasid m ites

Test substance NOEC [mg a.s./kg soil] maximum PECs [mg/kg] TER lt Trigger value

BYI 08330-enol 316 0.024 13167 5

The calculated TER values of 3399 (citrus) and 1316 7 (lettuce) for spirotetramat indicate an acceptabl e risk for soil non-target macro-organisms. The calculated TER values indicate an acceptable risk for soil no ntarget macro-organisms. Furthermore it is indicated that spirotetramat, which will be converted in soi l immediately to the BYI 08330-enol, has no adverse e ffects on juvenile life stages of soil dwelling art hropods. Thus the risk from spirotetramat for other non-targ et soil macro-organisms is expected to be low when applied according to GAP.– 194 –

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

B.9.8.4 Risk assessment for non-target micro-organi sms

Table 9.8.4-1: Summary of effects on non-target mic ro-organisms
Test substance Test parameter Test concentration Ti me Effects (deviation from control) 0.13 mg a.s./kg (0.099 kg a.s./ha) 3 % Spirotetramat Nitrogen mineralization 1.3 mg a.s./kg (0.99 kg a.s./ha) 28 days 16 %
0.13 mg a.s./kg (0.099 kg a.s./ha) 6 % Spirotetramat Carbon mineralization 1.3 mg a.s./kg (0.99 kg a.s./ha) 28 days 7 %

Active substance The effects of spirotetramat (tested at two doses: 0.13 mg/kg and 1.3 mg/kg) on the activity of the so il microflora were studied in standard laboratory tests. At both applied concentrations the impact on soil nit rogen transformation and soil respiration was < 25 %. The highest exposure concentration in the test was approximately 10 times higher than the expected PEC soil, which was calculated to be 0.115 mg/kg soil. Thus, it can be concluded that spirotetramat, when applied according to GAP, will not significantly af fect the soil micro-flora.

Metabolites In the fate section for the compartment soil three major metabolites were identified: BYI 08330-enol BYI 08330-keto-hydroxyd and 4-methoxycyclohexanone. In soil BYI 08330-enol immediately occurred after application with a maximum of 100 % after 1 day, BY I 08330-keto-hydroxy reached the maximum on day 1 with 16.3 %. Therefore, it can be concluded that th e study performed with the active substance covered the possible effects on soil micro-organisms for these both metabolites. However, 4-methoxycyclohexanone (metabolite of photolysis) will not be formed under test condition without illumination. But toxicity test with aquatic organisms and earthworm indicate that 4- me thoxycyclohexanone is not toxic to non-target organisms. In addition, under natural exposure cond ition in the field an exposure of non-target soil-o rganisms to relevant environmental concentrations of 4- meth oxycyclohexanone is extremely unlikely. Because, th e initial PECs is very low with 0.004 mg/kg at day 0 and from day 2 it can be expected that no measurabl e concentration of the metabolite will occur (PECs < 0.001 mg/kg). Thus, also a long term exposure can b e excluded. In conclusion, the risk from soil degradation produ cts for the activity of the soil micro-flora is con sidered to be low.– 195 –

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

B.9.9.2 Risk assessment for terrestrial non-target plants

Terrestrial non-target plants may be exposed to for mulated spirotetramat by spray drift in the vicinit y of the treated area. The effects of Spirotetramat 150 OD on non-target p lants were assessed in one vegetative vigour screen ing test, two dose-response vegetative vigour tests and two seedling emergence screening tests. The result s are summarised in table 0.1-1 above. A quantitative risk assessment is performed which f ollows a deterministic TER approach. As specified i n the “Guidance Document on Terrestrial Ecotoxicology und er Council Directive 91/414/EEC” the risk for nontarget plants is considered acceptable if the TER f or the most sensitive species is greater than 5.

Spray drift is considered to be the relevant exposu re route for terrestrial non-target plants. Consequ ently the PECs are calculated based on the drift models produ ced by the BBA (Ganzelmeier et al., 1995; updated b y Rautmann et al., 2001). TERs were determined based on drift deposition at 1 respectively 3 m from the edge of the field considering a multiple application fac tor (MAF) (as for non-target arthropods, measured h alf-life on plants considered; guidance see the respective s ection B.9.5) and the application scenarios for let tuce and citrus. The most sensitive endpoint is the EC 50 for corn dry weight of 134 g a.s./ha derived from a tier 2 vegetative vigour test; this figure is considered as relevant for the risk assessment. For citrus as a fruit crop the respective spray dri ft / ground deposition for “late” use of 15.73 % wa s chosen, because the figure for “early” use is related to th e early stage of orchard trees (i.e. apples) where only buds and no or very small leaves are present and the dri ft would be accordingly higher. In citrus leaves ar e fully developed during all stages, however. The respective TER calculations are presented in ta ble B.9.9.2-1 below.

Table B.9.9.2-1: Risk assessment for non-target pla nts for Spirotetramat OD150
TER values Application rate [g a.s./ha] x MAF Distance [m] Spray drift

[%] Exposure 1

[g a.s./ha] EC 50

[g a.s./ha] No drift

reduction 50 % drift

reduction

lettuce

72 x 1.2 1 2.77 2.39 134 56

citrus

3 15.73 50 2.7 5.4

5 8.41 27 5 288 x 1.1

10 3.60 11.4 134

12 MAF...Multiple application factor

1 based on Ganzelmeier drift data

The submitted studies on terrestrial plants and the calculated TER-values indicate that the risk for n on-target plants is low at a distance of 1 m to the treated a rea for lettuce and at a distance of 5 m for citrus (3 m high trees as worst case) without drift reducing measure s. When drift reducing nozzles are used in citrus, buffer zones can be reduced according to the table above ( to 3 m with 50 % drift reduction).

Overall conclusions

The risk to non-target plants due to application of Spirotetramat OD150 at the intended representative uses is considered acceptable at 1 m (lettuce) and 5 m ( citrus) distance to the field edge. The buffer zone for citrus may be reduced when drift reducing measures are applied.– 196 –

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

B.9.10.2 Risk assessment for biological methods of sewage treatment

For the effects of spirotetramat on the inhibition of the respiration rate of aerobic wastewater micro

organisms an EC50 > 10000 mg/L was determined. Ther efore it can be assumed that adverse effects on methods of sewage treatment are unlikely, when spir otetramat is applied according to GAP.