INDOXACARB (216) First draft prepared by Denis Hamilton, Department of Primary Industries and Fisheries, Brisbane, Australia.

Transcription

1 Indoxacarb IDXACARB (2) First draft prepared by Denis Hamilton, Department of Primary Industries and Fisheries, Brisbane, Australia. EXPLAATI Indoxacarb is an indeno-oxadiazine insecticide that is used for control of lepidoptera and other pests. Indoxacarb is a new pesticide in the Codex System. The CCPR, in 22 (4 th Session), requested an evaluation by the present meeting of JMPR. The manufacturer submitted studies on metabolism, supervised field trials, processing, farm animal feeding, analytical methods and freezer storage stability. IDETITY IS common name Synonyms: IUPAC name Chemical Abstracts name CAS umber indoxacarb: DPX-MP2: 4--9 CIPAC umber 2 Indoxacarb DPX-K S-enantiomer, i.e. indoxacarb I-K2 is R-enantiomer of indoxacarb DPX-MP2 is parts indoxacarb + part I-K2 (S)--chloro--[methoxycarbonyl-(4-trifluoromethoxy-phenyl)- carbamoyl]-2,-dihydro-indeno[,2-e][,,4]oxadiazine-4a(h)- carboxylic acid methyl ester (S)-methyl -chloro-2,-dihydro-2-[[(methoxycarbonyl)[4- (trifluoromethoxy)phenyl]amino]carbonyl]indeno[,2- e][,,4]oxadiazine-4a(h)-carboxylate Molecular formula C 22 H ClF Molecular mass 2.84 g/mol Structural formula CCH CCH Cl CF Physical and Chemical Properties Pure active ingredient. Ref Appearance white powder AMR 4- dour mild innocuous odour AMR 4- Melting point 88. ±.4 C AMR 4- Relative density.44 ±. AMR 4- Vapour pressure Pa at 2 C AMR Pa at 2 C Henry s Law constant - Pa.m mol - at 2 C AMR 49- Solubility in 2 ± 2 µg/l at 2 C AMR 4-

2 2 Indoxacarb Ref Solubility in organic solvents at acetonitrile > 2 g/l DuPont C: dimethylformamide > 2 g/l ethyl acetate > 2 g/l n-hexane. g/l methanol 9.9 g/l methylene chloride > 2 g/l n-octanol. g/l o-xylene > 2 g/l Dissociation constant in no pk a observed in ph range AMR 4- ctanol/ partition coefficient: log K ow = 4. at 2 C AMR 4- Technical material DPX-MP2 ( parts indoxacarb + part I-K2, R-enantiomer of indoxacarb) ph buffer stable Hydrolysis (sterile soln) at 2 C ph buffer DT See below. 22 days ph 9 buffer DT. days Photolysis in at 2 C Half-life days at ph (experiment days, i.e. full-time exposure) DuPont-98 DuPont-98 Hydrolysis of indoxacarb technical (Lentz, 22a) Two hydrolysis products were identified at more than % of starting material. CH C H H H CCH ph CCH ph and 9 Cl Cl Ca CCH CF CF CF Indoxacarb hydrolysis Chiral analysis showed that indoxacarb and its R enantiomer hydrolysed in sterile at the same rate. Photolysis of indoxacarb technical (Lentz, 22b) A number of photolysis products, including C 2 were identified in amounts exceeding % (on at least one sampling occasion) of the starting material when technical indoxacarb at. mg/l in ph sterile aqueous solution at 2 C was subjected to photolysis for days, equivalent to days' midday natural sunlight at latitude.

3 Indoxacarb Cl CCH CCH CF Cl CCH CH C H H H CF Cl CH CH CH C H CF Cl CH C 2 CH Indoxacarb photolysis Chiral analysis showed that indoxacarb and its R enantiomer photolytically degraded in sterile at the same rate. Formulations Racemic indoxacarb was developed originally, followed by technical material (TC) containing parts of indoxacarb and part of inactive enantiomer. A current formulation described as a contains g/kg indoxacarb and g/kg inactive enantiomer. The following table shows the manufacturer's codes for the various components. Code Indoxacarb (DPX-K) Inactive enantiomer (R enantiomer) (I-K2) Description (TC = technical material) DPX-JW2 % % TC DPX-MP2 % 2% TC For the purposes of this evaluation DPX-JW2 will be referred to as " indoxacarb." DPX-MP2 will be referred to as "indoxacarb ". Residues, where the two enantiomers are not in a defined ratio, will be described as "indoxacarb + R enantiomer". Indoxacarb is available in the following formulations: WP WP WP g/l, g/l, g/l g/kg g/kg g/kg indoxacarb + 2 g/kg teflubenzuron g/kg indoxacarb + g/kg etofenprox

4 4 Indoxacarb METABLISM AD EVIRMETAL FATE Animal and plant metabolism and environmental fate studies used indoxacarb and indoxacarb labelled at the -indanone carbon or uniformly labelled in the aromatic ring of the trifluoromethoxyphenyl moiety. Indanone - C label CCH CCH Cl CF Trifluoromethoxyphenyl(U)- C label Structures, names and codes for metabolites are summarised below. -H-DPX-JW2 methyl -chloro-2,-dihydro--hydroxy-2-[[(methoxycarbonyl)[4- (trifluoromethoxy)phenyl]amino]carbonyl]indeno[,2-e][,,4]oxadiazine- 4a(H)carboxylate -H-I-JT methyl -chloro-2,-dihydro--hydroxy-2-[[[4- (trifluoromethoxy)phenyl]amino]carbonyl]indeno[,2-e][,,4]oxadiazine- 4a(H)carboxylate I-JT [CAS o. 4-9-] methyl -chloro-2,-dihydro-2-[[[4- (trifluoromethoxy)phenyl]amino]carbonyl]indeno[,2-e][,,4]oxadiazine-4a(h)- carboxylate I-JU8 [CAS o ] methyl -chloro-2,-dihydro-2-hydroxy--[[[[4- (trifluoromethoxy)phenyl]amino]carbonyl]hydrazono]-h-indene-2- carboxylate I-KB8 [CAS o. 9--] methyl [4-(trifluoromethoxy)phenyl] carbamate I-KG4 [S-enantiomer CAS o ] (E)-methyl -chloro-2,-dihydro-2-hydroxy--[[[(methoxycarbonyl)[4- (trifluoromethoxy)phenyl]amino]carbonyl]hydrazono]-h-indene-2-carboxylate I-KT9 (Z)-methyl -chloro-2,-dihydro-2-hydroxy--[[[(methoxycarbonyl)[4- (trifluoromethoxy)phenyl]amino]carbonyl]hydrazono]-h-indene-2-carboxylate I-KT4 indeno[,2-e][,,4]oxadiazine-4a(h)-carboxylic acid, -chloro-2,-dihydro-2- [[(methoxycarbonyl)[4-(trifluoromethoxy) phenyl]amino]carbonyl]-, sodium salt I-MF methyl 2-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]hydrazine carboxylate Cl Cl Cl Cl Cl Cl Cl H H CCH CCH CCH CCH H H CCH H H H CF CF CF H CF CH C CCH H H CCH H H Ca CH H H C I-MK8 [CAS o ] H 2 C H -[4-(trifluoromethoxy)phenyl]urea I-MK4 H,2-dihydro--(trifluoromethoxy)-2H-benzimidazol-2-one H CF H CCH CCH CCH CF CF CF CF CF CF

5 Indoxacarb I-ML48 -chloro-2,4-dihydro-4-[4-(trifluoromethoxy)phenyl]-h-indeno[2,-e]-,2,4-triazin--one I-M4 urea, -(2-hydroxyethyl)-'-[4-(trifluoromethoxy)phenyl]urea I-MP89 indeno[,2-e][,,4]oxadiazine-(2h)-carboxylic acid, -chloro-,-dihydro-2-[[[4- (trifluoromethoxy)-phenyl]amino]carbonyl]-, methyl ester I-MU -chloro-2,-dihydro--oxoindeno[,2-e][,,4]oxadiazine-4a(h)- carboxylic acid I-MX829 [CAS o ] (as sulphate) -chloro-,-dihydro-2h-indene-2-one I-P [CAS o ] 4-(trifluoromethoxy)aniline Metabolite F, hen metabolism, proposed structure [tentative name] -(-(-chloro--hydroxy-2-methoxycarbonylindene)-4-(4- trifluoromethoxyphenyl)-,2,4-triazole-2,,4,-tetrahydro-,-dione CF Cl H H H H CF Cl H CHC CF CH Cl H Cl S 2 H H 2 CF H CCH Cl CF H proposed Animal metabolism The Meeting received animal metabolism studies with indoxacarb in rats, lactating dairy cows and laying hens. Rats Himmelstein (99, HL-99-49) dosed rats orally with [ C] indoxacarb labelled in the indanone ring and showed that it was readily absorbed followed by extensive metabolism and excretion. Major metabolites identified were I-JT, I-MU and I-MX829 sulphate. Himmelstein (99, HLR -) dosed rats orally with [ C] indoxacarb labelled in the indanone ring or in the trifluoromethoxyphenyl ring. The major urinary metabolite was I- MU while I-JT was the major metabolite in the fat. Dairy cows Two lactating Friesian dairy cows weighing 2 and 4 kg on the initial day were dosed orally once daily for consecutive days by gelatin capsule with 2 mg/animal/day of [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring, equivalent to ppm in the feed (Scott, 99, AMR ) for a 2 kg/day feed consumption. Milk was collected twice daily; a day's sample began in the afternoon after dosing and ended with the morning milking preceding the next dose. The animals were slaughtered 2. hours after the final dose for tissue collection. Recovery of administered C was 4% for the indanone label and 82% for the trifluoromethoxyphenyl ring label. The majority of the administered C (indanone label quoted first each time) was excreted in the faeces (% and %) and urine (9.% and 9.8%). Milk accounted for.8% and.% of the administered C while tissues accounted for.9% and.84%. The distribution of the radiolabel and identified metabolites in milk and tissues are summarised in Table and Table 2. Parent compound was the major identified component of the residue in milk and each of the tissues.

6 Indoxacarb Chiral HPLC analysis of parent in milk (day and pooled) and kidneys showed S:R enantiomer ratios of 2: and 2-2.: respectively, a change from the starting ratio of :. Table. Distribution of C residue and identified metabolites in milk and tissues of dairy cows dosed orally for days with 2 mg/animal/day of [ C] indoxacarb, labelled in the indanone ring, equivalent to ppm in the feed (Scott, 99, AMR ). Concentration, mg/kg, expressed as parent Compound Composite Foreleg muscle Liver Kidney Perirenal fat milk Total C residue Extracted residue Indoxacarb + R enantiomer CCH I-JT nd nd <. nd. Cl H I-MP89 Cl ote -H-I-JT CHC H CCH H CF CF.2 <. nd nd nd <. nd Cl H -H-DPX- H CCH CF JW2 Cl CCH nd <...2 CF -H-DPX-JW2 glucuronide nd nd.24 nd nd nd: not detected, limit of detection ~. mg/kg. ote: I-MP89 may be an artefact of the degradation of I-KT4. Table 2. Distribution of C residue and identified metabolites in milk and tissues of dairy cows dosed orally for days with 2 mg/animal/day of [ C] indoxacarb, labelled in the trifluoromethoxyphenyl ring, equivalent to ppm in the feed (Scott, 99, AMR ). Compound Composite milk Concentration, mg/kg, expressed as parent Foreleg muscle Liver Kidney Perirenal fat Total C residue Extracted residue Indoxacarb + R enantiomer CCH I-JT nd nd <. nd.8 Cl H I-MP89 Cl ote -H-I-JT CHC H CCH H CF CF. <. nd nd nd <. nd Cl H -H-DPX- H CCH CF JW2 Cl CCH nd <... CF -H-DPX-JW2 glucuronide nd nd.9 nd nd I-M4 + I-MF nd nd. nd nd I-KB8 nd nd.22 nd nd H CF CHC nd: not detected, limit of detection ~. mg/kg. ote: I-MP89 may be an artefact of the degradation of I-KT4.

7 Indoxacarb CCH CCH Cl indoxacarb CF CH C H H H CCH H CCH I-MF CCH CF Cl H Cl I-JT CF -H-DPX-JW2 CF Glucuronide of -H-DPX-JW2 Cl H CCH Cl H CH C I-MP88 CF H CH C I-KB8 CF -H-JT CF H H H I-M4 CF Figure. Proposed metabolic pathway for indoxacarb in cows. Hens Two groups of white leghorn laying hens ( birds in each group) mean body weight.44 kg at study initiation were dosed orally once daily for consecutive days with.2 mg/bird/day of [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring, equivalent to ppm in the feed (Li, 99, AMR 8-94) for a 2 g/day feed consumption. Eggs were collected daily. The birds were slaughtered hours after the final dose for tissue collection. Recovery of administered C was 89.% for the indanone label and 89.% for the trifluoromethoxyphenyl ring label. The majority of the administered C (indanone label quoted first each time) was excreted in the faeces (88% and 8%). Eggs accounted for.4% and.% of the administered C while tissues, blood and skin accounted for.4% and.%. The distribution of the radiolabel and identified metabolites in eggs and tissues are summarised in Table and Table 4. Total C concentrations in egg yolks at day were approximately times the concentrations at day, suggesting that indoxacarb residues had not reached a plateau at day. More residue appeared in the egg yolk than in the egg white, suggesting a tendency for fat solubility in the residue components. Parent compound constituted 4% of the total C in egg yolk. Major metabolites in egg yolk were I-KG4 + I-KT9 at 8-2% of total C and Metabolite F at - % of total C.

8 8 Indoxacarb Metabolites I-KG4 and I-KT9 are enantiomers. In egg yolks, the ratio of I-KG4 (the S enantiomer) concentration to I-KT9 concentration was approximately 2.4:, suggesting different rates of formation or depletion. Fat contained the highest concentration of residue, where the main component, Metabolite F constituting 4 and 8% of the total C in the fat, was tentatively identified as a product of I- KG4, initially produced by fracture of the oxadiazine ring. Parent compound constituted and % of the total C in the fat. Metabolite I-JT constituted and 8% of the total C in the fat. Residues in breast muscle and thigh muscle were generally too low for metabolite identification. Parent compound accounted for approximately 4% of the total C in liver. Table. Distribution of C residue and identified metabolites in tissues of laying hens dosed orally for days with.2 mg/bird/day of [ C] indoxacarb, labelled in the indanone ring, equivalent to ppm in the feed (Li, 99, AMR 8-94). Concentration, mg/kg, expressed as parent Compound Fat Skin + Liver Gizzard Thigh Breast Egg white, Egg yolk, fat muscle muscle day day Total C residue Extracted residue Indoxacarb + R enantiomer <. <.. I-KG4 CCH H CCH. H. <. <. <..4 Cl I-KT9 I-JU8 -H-JT Cl Cl Cl H CCH H H CCH H CCH H H CCH H CF CF CF.2.. <. <..2. < <. <..4 Metab F H Cl proposed CCH H CF CF <. <.. I-JT Cl CCH H CF.8.4. <. <..2

9 Indoxacarb 9 Table 4. Distribution of C residue and identified metabolites in tissues of laying hens dosed orally for days with.2 mg/bird/day of [ C] indoxacarb, labelled in the trifluoromethoxyphenyl ring, equivalent to ppm in the feed (Li, 99, AMR 8-94). Concentration, mg/kg, expressed as parent Compound Fat Skin + Liver Gizzard Thigh Breast Egg white, Egg yolk, fat muscle muscle day day Total C residue Extracted residue Indoxacarb + R enantiomer <. <.. CCH H CCH I-KG4 Cl H I-KT9 CCH H CCH Cl H CF CF..2 <. < <. <..2 CCH H Cl I-JU8 H CCH -H-JT Cl H H H CF CF < H Metab F Cl proposed CCH H CF <. <..4 CCH I-JT Cl H CF.9.. <. <..2 I-KB8 CHC H CF.... <. <... I-MK8 H 2 C H CF... <...

10 Indoxacarb CCH CCH Cl indoxacarb CF CCH CCH H CCH Cl H Cl H I-JT CF I-KG4 + I-KT9 CF H CCH H CH C I-KB8 CF CCH H Cl H Cl H H -H-JT CF I-JU8 CF H H 2 C H I-MK8 CF Cl Metab F CCH H CF Figure 2. Proposed metabolic pathway for indoxacarb in poultry. Plant metabolism The Meeting received plant metabolism studies with indoxacarb on cotton, lettuce, grapes and tomatoes. In each crop tested, parent compound mostly represented more than 9% of the total C residue and was essentially the only compound detected. In grapes and tomatoes the residue was found to be mostly a surface residue. Chiral HPLC analysis of the residues in tomatoes showed that the enantiomers remained in a : ratio. Cotton In a plant metabolism study in Mississippi, cotton plants were treated with a single application of formulated [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring at the equivalent of g indoxacarb/ ha (Scott and Guseman, 99, AMR 29-9). Plants

11 Indoxacarb were sampled at,, and 9 days post-treatment and at maturity, 9 days after treatment. In plant samples, parent compound mostly represented more than 9% of the total C residue. Chiral analysis of parent compound demonstrated that it remained. An isolated cotton leaf was treated with labelled compound and housed in a closed vessel while exposed to UV light. After 2 days, approximately 4.% of the C had been converted to C 2. Table. C residues on cotton plants treated with formulated [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring at the equivalent of g indoxacarb/ ha (Scott and Guseman, 99, AMR 29-9). Concentration, mg/kg of C expressed as parent Day Indanone label trifluoromethoxyphenyl label TRR TRR Racemic TRR TRR TRR TRR Racemic TRR TRR extracted indoxacarb hydrolysed unextracted extracted indoxacarb hydrolysed unextracted plant seed. <.. <. Lettuce In a plant metabolism study, lettuce plants at the 4- leaf stage were treated with a single application of formulated [C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring at the equivalent of g indoxacarb/ ha (Gaddamidi and Hashinger, 99, AMR 2-9). Plants were sampled at,,, 2, and days (mature) post-treatment. Even on day less than half of the residue was on the surface and the % on the surface decreased further with time after treatment. Parent compound was the only component found in the residue. Also in an experiment with an exaggerated application rate ( ) of indoxacarb, only parent compound was detected in the residue. Table. Residues in and on lettuce plants treated with formulated [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring at the equivalent of g indoxacarb/ ha (Gaddamidi and Hashinger, 99, AMR 2-9). Concentration, mg/kg of C expressed as parent Day Indanone label trifluoromethoxyphenyl label TRR TRR surface TRR extracted Racemic indoxacarb TRR TRR surface TRR extracted Racemic indoxacarb Grapes Grape vines at the early fruit development stage were treated with a single foliar application of formulated [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring at the equivalent of g indoxacarb/ ha (Gaddamidi and Hashinger, 99, AMR 229-9).

12 2 Indoxacarb Grape leaves and grapes were sampled on days,, 4 and (mature) post-treatment. In addition, grape leaves were sampled on days and. The majority of the residue associated with the fruit was surface residue, with 2% and % still surface residues days after treatment. Parent compound was essentially the only component of the residue at all times. The majority of the residue on leaves was also a surface residue. Table. Residues in and on grapes from vines treated with formulated [ C] indoxacarb, labelled in the indanone ring or the trifluoromethoxyphenyl ring at the equivalent of g indoxacarb/ ha (Gaddamidi and Hashinger, 99, AMR 229-9). Concentration, mg/kg of C expressed as parent Day Indanone label trifluoromethoxyphenyl label TRR TRR surface TRR extracted Racemic indoxacarb TRR TRR surface TRR extracted Racemic indoxacarb Tomatoes Tomato vines, variety Heinz 8892, were treated with 4 foliar applications, approximately - days apart, of formulated [ C] indoxacarb, labelled in the trifluoromethoxyphenyl ring at the equivalent of 4 g indoxacarb/ ha (Brown and Young, 99, AMR -9). Tomato leaf and fruit samples were taken before and after each treatment (except the first where no fruit were available) and, and days (final harvest) after the final application, or 8 days after the first application. The majority of the residue associated with the fruit was surface residue, mostly around 9% of the residue. Parent compound was essentially the only component of the residue at all times. The majority of the residue on leaves was also a surface residue. Parent compound isolated from the leaf extracts from samples collected before the second application and at harvest, days after final application, were subjected to chiral HPLC analysis, which demonstrated that the two enantiomers remained in a : ratio. Table 8. C residues in and on tomatoes from plants treated with formulated [ C] indoxacarb, labelled in the trifluoromethoxyphenyl ring at the equivalent of 4 g indoxacarb /ha (Brown and Young, 99, AMR -9). Concentration, mg/kg of C expressed as parent Timing TRR TRR surface TRR extractable / TRR unextractable Racemic indoxacarb Before 2 nd treatment.4.4 <. After 2 nd treatment.. <..4 Before rd treatment.2.. <.. After rd treatment Before 4 th treatment.... After 4 th treatment days after final treatment.... days after final treatment days after final treatment.8... / TRR extractable only conducted on sample from before rd treatment.

13 Indoxacarb Environmental fate in soil The Meeting received information on the environmental fate of indoxacarb in soil, including studies on aerobic soil metabolism, field dissipation and crop rotational studies. Because indoxacarb is used on peanuts and potatoes (edible portion in soil), studies on aerobic soil metabolism are needed and studies on field dissipation are helpful. Soil metabolism A study on the aerobic soil metabolism of [ C] indoxacarb in a silt loam soil showed that indoxacarb + R enantiomer degraded quickly and identifiable metabolites were a minor part of the residue and mostly also degraded relatively quickly (Singles, 22, AMR -9). The indanone ring was mineralized more quickly than the trifluoromethoxyphenyl ring. The non-extractable C had begun to decline within 9 days. Aerobic soil metabolism Ref: Singles, 22, AMR -9 Test material: [ C] indoxacarb, indanone ring label Dose rate: mg/kg dry soil Silt loam ph:.2,. rganic matter: 2.4, 2.%. Duration: 4 days Temp: 2 C Moisture: % of. bar moisture Half-life of indoxacarb: approx 2- days (methods would not have detected epimerization) % indoxacarb + R enantiomer remaining, day 4 =. % % mineralization, day 4 = % Metabolites Max (% of dose) Day I-JT % I-KG4 9.9% 2 I-ML48. % on-extractable C 2 % 9 2. Duration: 2 days Temp: 2 C Moisture: % of. bar moisture Half-life of indoxacarb: approx 2- days (methods would not have detected epimerization) % indoxacarb + R enantiomer remaining, day 2 = 9.9 % % mineralization, day 2 = 2 % Metabolites Max (% of dose) Day I-JT 2 % I-KG4. % I-ML48. % 2 on-extractable C 42 % 2 Aerobic soil metabolism Ref: Singles, 22, AMR -9 Test material: [ C] indoxacarb, TFP ring label Dose rate: mg/kg dry soil Silt loam ph:.2,. rganic matter: 2.4, 2.%. Duration: 4 days Temp: 2 C Moisture: % of. bar moisture Half-life of indoxacarb: approx 2- days (methods would not have detected epimerization) % indoxacarb + R enantiomer remaining, day 4 = 2.2 % % mineralization, day 4 =. % Metabolites Max (% of dose) Day I-JT % 2 I-KG4 % I-ML48.8 % I-MK on-extractable C % 9 2. Duration: 2 days Temp: 2 C Moisture: % of. bar moisture Half-life of indoxacarb: approx 2- days (methods would not have detected epimerization) % indoxacarb + R enantiomer remaining, day 2 =.2 % % mineralization, day 2 =.4 % Metabolites Max (% of dose) Day I-JT % I-KG4.2 % I-ML48.2 % 2 I-MK4. % - on-extractable C % 9

14 Indoxacarb Singles (24, DuPont-) reviewed the environmental fate studies on indoxacarb mixtures in soil and aquatic systems, also explaining some of the analytical difficulties in identifying metabolites in the soil metabolism studies and the progress with methods since the early studies on indoxacarb. Acidic mobile phases in HPLC degrade some of the soil metabolites, so mobile phases have been modified. ew stationary phases provide better separation of degradation products than previously achieved. Metabolite I-KT4 rearranges to I-MP89 in pure acetonitrile, but can be stabilised in extractants containing at least 2%. Indoxacarb and its R enantiomer were shown, by chiral chromatography analysis, to degrade at the same rate in soil and systems. CCH CCH Cl CF Cl Ca CCH Cl CCH H H CCH I-KT4 CF I-KG4 CF CCH Cl I-JT H CF CH C H I-KB8 CF Cl CH C H CCH H I-MP89 CF Cl H H Cl I-ML48 H H 2 CF C H I-MK8 CF I-JU8 H H I-MK4 Figure. Proposed metabolic pathway for indoxacarb in aerobic soil (Singles, 24, DuPont- ). CF CF Field dissipation Rühl (99, AMR 4-9) tested the persistence and mobility of [ C] labelled indoxacarb when applied to the bare soil in 4 different soils in the USA in 99. Very little of the applied indoxacarb moved below the top cm of the soil during the trials of duration up to 8 months. Indoxacarb + R enantiomer concentrations declined to half of their initial values in days to months (Figure 4).

15 Indoxacarb. Soil dissipation, indoxacarb, 4 sites in USA, AMR mg/kg.4. DE FL TX CA Figure 4. Disappearance of indoxacarb + R enantiomer from the top cm of soil at 4 sites in USA (Rühl, 99, AMR 4-9) after application of.8- kg/ha on to bare soil. days At two of the sites after 2 months the indoxacarb + R enantiomer levels declined to approximately 8 % of their initial values and at the other two sites they had declined to approximately % of the initial values (Table 9). nly low concentrations of indoxacarb + R enantiomer were detected in the cm soil (2..% of initial soil concentration in the cm soil). Metabolite I-JT was detected in the cm soil in the 4 trials, reaching..4% of the initial indoxacarb + R enantiomer concentrations. Metabolite I-KG4 was detected in 2 of the 4 soils reaching.4.% of the initial indoxacarb + R enantiomer concentrations. Table 9. Field dissipation of [ C] labelled indoxacarb in 4 different soils in the USA in 99 (Rühl, 99, AMR 4-9). Trial Applic rate, kg/ha Initial conc, mg/kg / Indoxacarb + R enantiomer as % of original concentration in - cm soil. Residues are reported as mean of 2 treatments, one with indoxacarb labelled in the indanone ring and the other in the trifluoromethoxyphenyl ring. month months months 2 months longest time measured USA (DE) 99 Plot area.4 sq m. Silt loam: ph, 2% sand, % silt, % clay, 2.9% organic matter. treat in July..48 (.) 29%.4%.%.%.% (8 months) USA (FL) 99 Plot area.4 sq m. Loamy sand: ph., 8% sand, % silt, 4% clay,.2% organic matter. treat in January.8. (.44) % 48% % 8.8%.% ( months) USA (TX) 99 Plot area.4 sq m. Sandy clay loam: ph 8, 44% sand, 24% silt, % clay,.2% organic matter. treat in August..29 (.) %.%.%.% ( months) USA (CA) 99 Plot area.4 sq m. Sandy loam: ph 8, % sand, % silt, % clay,.% organic matter. treat in ctober.8. (.44) 9% 9% % 9.% 4.% (8 months) / Theoretical initial concentration is shown in parentheses. Vincent et al. (99, AMR 42-9) studied the field persistence and mobility of indoxacarb at two sites in the USA. Soil characteristics in the top cm at the Florida site were: ph

16 Indoxacarb., sand 92%, silt 4.%, clay 4.%, organic matter., classified as a sand; and at the California site were: ph., sand %, silt %, clay %, organic matter.%, classified as a loam or sandy loam. Racemic indoxacarb was applied to bare soil 4 times at. kg/ha on days,, and 9 of the trials. Soil cores of,, 4, 4 and 9 cm were periodically taken from the treated plots at each site for residue analysis. Soil moistures were determined by oven drying separate subsamples and residues were expressed on soil dry weight. Soil samples were analysed for indoxacarb + R enantiomer and metabolites I-JT and I-KG4. Residues of indoxacarb + R enantiomer disappeared from the top cm of soil with half-lives of days for the Florida site and days for the California site, calculated on residue data from 9 days (final application) to year (Figure ). Indoxacarb +R enantiomer occurred at low levels around. mg/kg occasionally and intermittently in soils from the cm profile at both sites. Metabolite I-JT reached its peak concentration in the Florida soil on day (.2 mg/kg) and on day in the California soil (. mg/kg). Metabolite I-KG4 did not appear in the Florida soil, but was detected, up to. mg/kg, in the California soil. either I-JT nor I-KG4 appeared in the cm profile soil. Soil dissipation, indoxacarb, 2 sites in USA, AMR mg/kg.2 FL CA days Figure. Disappearance of indoxacarb + R enantiomer from the top cm of soil at 2 sites in USA (Vincent et al., 99, AMR 42-9) after 4 applications of. kg/ha on to bare ground on days,, and 9. Crop rotation studies Information on the fate of radiolabelled indoxacarb in a confined crop rotational study was made available to the meeting. In a confined rotational crop study in USA (Freeman and Terranova, 99, AMR 429-) soil (Delaware sandy loam,.% organic matter, ph.8) was treated directly with C labelled indoxacarb at. kg/ha equivalent to. kg ai/ha. Crops of carrots, lettuce, wheat and soybeans were sown into the treated soil at intervals of, 9 and 2 days after treatment. Crops were grown to maturity, subsequently harvested and analysed for C content (Table ). Samples were further examined by extraction and HPLC analysis but no parent compound or potential

17 Indoxacarb metabolite (I-JT) was observed. Low levels (. mg/kg) of unidentifiable components were observed, with different patterns for the two different label positions suggesting that the parent compound was fragmented. Table. Confined rotational crop studies with labelled indoxacarb. Soil was treated with C labelled indoxacarb at. kg/ha, equiv to. ai/ha. Application country, year, ref. Bare soil, USA, 9 AMR 429- [ C]indanone label Bare soil, USA, 9 AMR 429- [ C]TFP label Rotational crop TSI / days carrot (Fontana) spring wheat (Katepawa) soybean (A2242) carrot (Fontana) 9 9 THI 2/ days 9 2 soil soil soil soil tops roots Sample C as indoxacarb + R enantiomer mg/kg Residues, mg/kg lettuce (Prizehead) leaves. / 4 forage. / straw.49 / grain.24 / forage hay seed tops roots lettuce (Prizehead) 9 leaves. na spring wheat (Katepawa) forage straw grain / / / soybean (A2242) carrot (Fontana) forage hay seed tops roots lettuce (Prizehead) 2 99 leaves. na spring wheat (Katepawa) forage straw grain.2.2. / / / soybean (A2242) forage hay seed soil soil soil soil tops roots carrot (Fontana) / / lettuce (Prizehead) leaves. / spring wheat (Katepawa) soybean (A2242) carrot (Fontana) forage straw grain forage hay seed tops roots lettuce (Prizehead) 9 leaves. na / / / / / / na / / / na na / / / / / / / / / / na

18 8 Indoxacarb Application country, year, ref. Rotational crop TSI / days spring wheat (Katepawa) soybean (A2242) carrot (Fontana) 2 2 spring wheat (Katepawa) soybean (A2242) THI 2/ days Sample forage straw grain forage hay seed tops roots C as indoxacarb + R enantiomer mg/kg Residues, mg/kg lettuce (Prizehead) 2 99 leaves. na forage. / straw. / grain. / 2 2 forage hay seed na: not analysed. / TSI: interval between treatment on soil and sowing of rotation crop, days. 2/ THI: interval between treatment on soil and harvest of rotation crop (or sampling of soil), days. / HPLC analysis did not detect (LQ. mg/kg) indoxacarb or metabolite I-JT in any raw agricultural commodity / / / / / / na na / / / METHDS F RESIDUE AALYSIS Analytical methods The Meeting received descriptions and validation data for analytical methods for residues of indoxacarb in raw agricultural commodities, processed commodities, feed commodities, animal tissues, milk and eggs. Methods rely on HPLC-UV, GC-ECD and GC-MSD for analysis of indoxacarb in the various matrices. Indoxacarb and its R enantiomer are determined and reported together in all these methods. Signal enhancement by extracts of some matrices may require the preparation of standards in matrix extracts for measurement at low concentrations. Extraction efficiency has been tested with various solvent mixtures on [ C] indoxacarb incorporated into crop and animal commodities. Crops and processed fractions (Gagnon and Guinivan, 9, AMR 49-9) Analyte: Indoxacarb + R enantiomer GC-MSD Method AMR 49-9 LQ: Typically.2 mg/kg Description Analytes are extracted from crop samples into ethyl acetate after the addition of. Cotton seed is an exception, requiring extraction into acetonitrile after the addition of hexane to the sample matrix. An aliquot from the extraction solution is concentrated by evaporation under nitrogen and cleaned up by solid phase extraction with silica and carbon. The cleaned-up extract is then analysed by GC-MSD. Signal enhancement by matrix may require standards to be prepared in control matrix solutions, especially for low analyte concentrations. Crops and processed fractions (Gagnon et al., 99, AMR 49-9 s2 r) Analyte: Indoxacarb + R enantiomer GC-MSD - enforcement Method AMR 49-9 LQ: Typically.2-. mg/kg for most commodities, mg/kg for tomato paste Description See Gagnon and Guinivan, 9, AMR Standards may be prepared in organic solvent, rather than in control matrix solutions, for residue levels of. mg/kg and higher if the final analysis solution is more dilute than in the original method. The revised method is suitable as an enforcement method

19 Indoxacarb 9 Crops (Klemens et al., 99, AMR 22-9) Analyte: Indoxacarb + R enantiomer HPLC-UV Method AMR 22-9 LQ: Typically. mg/kg. Cotton gin trash and maize fodder. mg/kg. Description Residues are extracted from crop matrix with hexane-acetonitrile and the acetonitrile extract is concentrated and cleaned up by solid-phase extraction with a combination of silica and strong anion exchanger. The analytes are measured by reversed-phase HPLC (2-column system with switching) with UV detection at nm. Plant materials (Schmidt, 99, AMR 42-) Analyte: Indoxacarb + R enantiomer GC-ECD and GC-MS Method AMR 42- LQ:. mg/kg for apple, cabbage, grape, tomato..2 mg/kg for cotton seed. Description AMR 42- is based on multi-residue method DFG S9 with a modified extraction solvent. Samples are extracted with /acetone/ethyl acetate/cyclohexane. Cotton seed is extracted with acetone/acetonitrile. Extracts are cleaned up by gel permeation chromatography (GPC) and by adsorption chromatography on silica gel. Analytes are determined by capillary GC- ECD on a non-polar stationary phase. Full scan GC-MS is used for identification. Animal commodities (Amoo and Beaver-Stetser, 99, AMR -9) Analyte: Indoxacarb + R isomer and I-JT HPLC-UV Method AMR LQ:. mg/kg for whole milk, skim milk, cream, fat, muscle, kidney, liver Description Residues are extracted from aliquots of milk, cream or tissues with acetonitrile (milk, cream, liver and kidney) or ethyl acetate (fat and muscle). Milk and cream extracts are further cleaned up with C 8 cartridges. Tissue extracts are cleaned up with additional hexane washes, with liver and kidney extracts further cleaned up by silica solid phase extraction. Extracts are evaporated and taken up in HPLC mobile phase (acetonitrile + ph. phosphate buffer) for analysis, with UV detection at nm. Further cleanup is affected on a 2-column switching system with the first column (cyano) acting as the cleanup system and the second column (DS) as the analytical column. nce the analytes are transferred to the analytical column, the cyano column is flushed with a solvent prior to injection of the next sample. Animal commodities (Linkerhagner and Guinivan, 2, Du-Pont-) Analyte: Indoxacarb + R isomer and I-JT GC-ECD Method DFSG S9 LQ:. mg/kg for milk, eggs, bovine muscle, poultry meat.. mg/kg for fat Description Samples are homogenized with a mixture of, acetone and sodium chloride. Ethyl acetate and cyclohexane are added and residues are partitioned into the organic phase, which is separated and dried with sodium sulphate. The extract is concentrated to near dryness and the residue is taken up in ethyl acetate and cyclohexane ready for cleanup through a gel permeation column. The selected eluate is collected, concentrated and made to volume with ethyl acetate ready for cleanup through a silica column. Selected fractions are evaporated and the residue is taken up in acetone for GC-ECD analysis on a medium-polarity capillary column. A non-polar column or GC-MSD may be used for confirmation of residue identity. Different conditions and extraction solvent mixtures are chosen to suit different sample types. Matrix effects from some substrates tended to produce high recoveries at low concentrations. HPLC method AMR 22-9 was subjected to independent laboratory validation (ILV) where successful recoveries were obtained for the cotton and maize commodities tested (Lochhaas, 99, AMR 42-9). Successful ILV recoveries were obtained in GC-MSD method AMR 49-9 for spinach leaves and tomatoes at concentrations relevant for enforcement purposes (Lyle and James, 99, AMR 42-9). Independent laboratory validation of GC-ECD method AMR 42-, based on multiresidue method DFG S9, was carried out with numerous recoveries on apples, cabbage, cotton seed, grapes and tomatoes (Class, 2, DuPont-29). GC-ECD method DuPont-, a modification of multiresidue method DFG S9, was subjected to independent laboratory validation (ILV) where acceptable recoveries were obtained for indoxacarb and metabolite I-JT in the animal commodities tested - liver and kidney (Class, 2, Du-Pont-224) and milk, bovine muscle, bovine fat and whole egg (Class, 2, DuPont-2).

20 2 Indoxacarb Recoveries were more variable for analyses on a second column used for confirmation of residue identity. Column-switching HPLC method AMR -9 for residues of indoxacarb and metabolite I-JT in animal commodities was subjected to independent laboratory validation (Miller and James, 99, AMR 424-9). Satisfactory recoveries at. and.2 mg/kg in whole milk and ground beef were achieved after slight modifications to the method. Improved recoveries were achieved when extra care was taken to ensure the residue was all redissolved after an evaporation step. Recovery data from the independent laboratory validation (ILV) testing are summarised in Table. Table. Analytical recoveries for spiked indoxacarb in various substrates. Commodity Spiked analyte Spike conc, mg/kg n Mean recov% Range recov% Method Apple indoxacarb AMR 49-9 AMR 49-9 Apple indoxacarb AMR 49-9 AMR 49-9 Apple indoxacarb., AMR 49-9 AMR 49-9 Apple indoxacarb.2,.2, DFG S9 modified AMR 42- Apple indoxacarb AMR 42- DuPont-29 Apple indoxacarb AMR 42- DuPont-29 Apple indoxacarb.,. 2, 94 AMR 42--GCMS DuPont-29 Apple juice indoxacarb AMR 49-9 AMR 49-9 Apple juice indoxacarb AMR 49-9 AMR 49-9 Apple juice indoxacarb., AMR 49-9 AMR 49-9 Apple pomace, indoxacarb AMR 49-9 AMR 49-9 wet Apple pomace, indoxacarb AMR 49-9 AMR 49-9 wet Apple pomace, indoxacarb., AMR 49-9 AMR 49-9 wet Apple sauce indoxacarb AMR 49-9 AMR 49-9 Apple sauce indoxacarb AMR 49-9 AMR 49-9 Apple sauce indoxacarb., AMR 49-9 AMR 49-9 Beef indoxacarb.,. -8 DuPont- / DuPont- Beef I-JT., DuPont- / DuPont- Beef, ground indoxacarb., AMR -9 AMR Beef, ground I-JT., AMR -9 AMR Broccoli indoxacarb AMR 49-9 AMR 49-9 Broccoli indoxacarb AMR 49-9 AMR 49-9 Broccoli indoxacarb., AMR 49-9 AMR 49-9 Cabbage indoxacarb AMR 49-9 AMR 49-9 Cabbage indoxacarb AMR 49-9 AMR 49-9 Cabbage indoxacarb., AMR 49-9 AMR 49-9 Cabbage indoxacarb AMR 42- DuPont-29 Cabbage indoxacarb AMR 42- DuPont-29 Cabbage indoxacarb.,. 2 8, 2 AMR 42--GCMS DuPont-29 Cabbage, white indoxacarb.2,.2, DFG S9 modified AMR 42- Cauliflower indoxacarb AMR 49-9 AMR 49-9 Cauliflower indoxacarb AMR 49-9 AMR 49-9 Cauliflower indoxacarb., AMR 49-9 AMR 49-9 Cauliflower indoxacarb.2,.2, DFG S9 modified AMR 42- Cotton forage indoxacarb + R. 2, 9 AMR 22-9 AMR 22-9 Cotton forage isomer indoxacarb + R.2,. 8-8 AMR 22-9 AMR 22-9 Cotton gin trash isomer indoxacarb + R AMR 22-9 AMR 22-9 Cotton gin trash isomer indoxacarb + R. 9-2 AMR 22-9 AMR 22-9 Cotton gin trash indoxacarb isomer., AMR 22-9 AMR 42-9 Cotton hulls indoxacarb + R. 9-8 AMR 22-9 AMR 22-9 Cotton hulls isomer indoxacarb + R.2, AMR 22-9 AMR 22-9 Cotton meal indoxacarb isomer + R isomer AMR 22-9 AMR 22-9 Ref

21 Indoxacarb 2 Commodity Spiked analyte Spike conc, mg/kg Cotton meal indoxacarb + R Cotton oil, ref isomer indoxacarb + R Cotton oil, ref isomer indoxacarb + R Cotton seed isomer indoxacarb + R Cotton seed isomer indoxacarb + R n Mean recov% Range recov% Method.2, AMR 22-9 AMR AMR 22-9 AMR , AMR 22-9 AMR AMR 22-9 AMR ,. 2-2 AMR 22-9 AMR 22-9 Cotton seed isomer indoxacarb AMR 49-9 AMR 49-9 Cotton seed indoxacarb., AMR 49-9 AMR 49-9 Cotton seed indoxacarb., AMR 22-9 AMR 42-9 Cotton seed indoxacarb.2-4 AMR 42- DuPont-29 Cotton seed indoxacarb.2 8- AMR 42- DuPont-29 Cotton seed indoxacarb.2,.2 2 8, 8 AMR 42--GCMS DuPont-29 Cream indoxacarb + R AMR -9 AMR -9 Cream I-JT isomer AMR -9 AMR -9 Egg indoxacarb., DuPont- / DuPont- Egg I-JT., DuPont- / DuPont- Egg indoxacarb., DuPont- / DuPont-2 Egg I-JT., DuPont- / DuPont-2 Fat indoxacarb + R AMR -9 AMR -9 Fat I-JT isomer AMR -9 AMR -9 Fat indoxacarb., DuPont- / DuPont- Fat I-JT.,. -8 DuPont- / DuPont- Fat, bovine indoxacarb.,. - DuPont- / DuPont-2 Fat, bovine I-JT.,. 44- DuPont- / DuPont-2 Grape juice indoxacarb AMR 49-9 AMR 49-9 Grape juice indoxacarb AMR 49-9 AMR 49-9 Grape juice indoxacarb., AMR 49-9 AMR 49-9 Grape pomace, indoxacarb AMR 49-9 AMR 49-9 dry Grape pomace, indoxacarb., AMR 49-9 AMR 49-9 dry Grape pomace, indoxacarb AMR 49-9 AMR 49-9 wet Grape pomace, indoxacarb AMR 49-9 AMR 49-9 wet Grape pomace, indoxacarb., AMR 49-9 AMR 49-9 wet Grapes indoxacarb AMR 49-9 AMR 49-9 Grapes indoxacarb AMR 49-9 AMR 49-9 Grapes indoxacarb., AMR 49-9 AMR 49-9 Grapes indoxacarb.2,.2, DFG S9 modified AMR 42- Grapes indoxacarb. - AMR 42- DuPont-29 Grapes indoxacarb AMR 42- DuPont-29 Grapes indoxacarb.,. 2, 88 AMR 42--GCMS DuPont-29 Kidney indoxacarb + R AMR -9 AMR -9 Kidney I-JT isomer AMR -9 AMR -9 Kidney indoxacarb.,. 8-8 DuPont- / DuPont-224 Kidney I-JT.,. 8-9 DuPont- / DuPont-224 Lettuce indoxacarb AMR 49-9 AMR 49-9 Lettuce indoxacarb AMR 49-9 AMR 49-9 Lettuce indoxacarb., AMR 49-9 AMR 49-9 Liver indoxacarb + R AMR -9 AMR -9 Liver I-JT isomer AMR -9 AMR -9 Liver indoxacarb., DuPont- / DuPont-224 Liver I-JT., DuPont- / DuPont-224 Maize fodder indoxacarb + R AMR 22-9 AMR 22-9 Maize fodder isomer indoxacarb + R AMR 22-9 AMR 22-9 Maize forage indoxacarb isomer + R AMR 22-9 AMR 22-9 Maize forage indoxacarb isomer + R.2, AMR 22-9 AMR 22-9 Maize forage indoxacarb isomer., AMR 22-9 AMR 42-9 Milk indoxacarb.,. 9- DuPont- / DuPont- Milk I-JT.,. -9 DuPont- / DuPont- Milk indoxacarb., DuPont- / DuPont-2 Milk I-JT.,. 9 - DuPont- / DuPont-2 Ref

22 22 Indoxacarb Commodity Spiked analyte Spike conc, mg/kg Milk, skim indoxacarb + R isomer Milk, whole indoxacarb + R isomer n Mean recov% Range recov% Method AMR -9 AMR -9 Milk, skim I-JT AMR -9 AMR AMR -9 AMR -9 Milk, whole I-JT AMR -9 AMR -9 Milk, whole indoxacarb., AMR -9 AMR Milk, whole I-JT., AMR -9 AMR Muscle indoxacarb + R AMR -9 AMR -9 Muscle I-JT isomer AMR -9 AMR -9 Muscle, bovine indoxacarb.,. 8-9 DuPont- / DuPont-2 Muscle, bovine I-JT., DuPont- / DuPont-2 Peach indoxacarb.2,.2, DFG S9 modified AMR 42- Pear indoxacarb AMR 49-9 AMR 49-9 Pear indoxacarb AMR 49-9 AMR 49-9 Pear indoxacarb., AMR 49-9 AMR 49-9 Peppers indoxacarb AMR 49-9 AMR 49-9 Peppers indoxacarb., AMR 49-9 AMR 49-9 Peppers indoxacarb.,.2, AMR 49-9 AMR 49-9 Poultry meat indoxacarb.,. -8 DuPont- / DuPont- Poultry meat I-JT.,. -88 DuPont- / DuPont- Raisins indoxacarb AMR 49-9 AMR 49-9 Raisins indoxacarb AMR 49-9 AMR 49-9 Raisins indoxacarb., AMR 49-9 AMR 49-9 Spinach leaves indoxacarb.,., AMR 49-9 AMR 42-9 Sweet corn k+c indoxacarb + R AMR 22-9 AMR 22-9 Sweet corn k+c isomer indoxacarb + R.2, AMR 22-9 AMR 22-9 Sweet corn k+c indoxacarb isomer., AMR 22-9 AMR 42-9 Tomato indoxacarb AMR 49-9 AMR 49-9 Tomato indoxacarb AMR 49-9 AMR 49-9 Tomato indoxacarb., AMR 49-9 AMR 49-9 Tomato indoxacarb.2,.2, DFG S9 modified AMR 42- Tomato indoxacarb., AMR 49-9 AMR 42-9 Tomato indoxacarb AMR 42- DuPont-29 Tomato indoxacarb AMR 42- DuPont-29 Tomato indoxacarb.,. 2 8, 98 AMR 42--GCMS DuPont-29 Tomato juice indoxacarb AMR 49-9 AMR 49-9 Tomato juice indoxacarb.2 8- AMR 49-9 AMR 49-9 Tomato juice indoxacarb., AMR 49-9 AMR 49-9 Tomato ketchup indoxacarb AMR 49-9 AMR 49-9 Tomato ketchup indoxacarb AMR 49-9 AMR 49-9 Tomato ketchup indoxacarb., AMR 49-9 AMR 49-9 Tomato paste indoxacarb AMR 49-9 AMR 49-9 Tomato paste indoxacarb., AMR 49-9 AMR 49-9 Tomato paste indoxacarb.2,., AMR 49-9 AMR 49-9 Tomato puree indoxacarb AMR 49-9 AMR 49-9 Tomato puree indoxacarb AMR 49-9 AMR 49-9 Tomato puree indoxacarb., AMR 49-9 AMR 49-9 Wine indoxacarb AMR 49-9 AMR 49-9 Wine indoxacarb AMR 49-9 AMR 49-9 Wine indoxacarb., AMR 49-9 AMR 49-9 / Based on multi-residue method DFG S9 Ref Extraction efficiency of analytical methods Plant metabolism studies have shown that parent indoxacarb isomers are the only isomers of interest in plant tissues. Studies were designed to test the extraction efficiencies of the solvent systems used in the analytical methods. In a series of studies in 99, confined crops were treated with radiolabelled indoxacarb and commodity was harvested at suitable intervals to produce aged residues. Extraction

23 Indoxacarb 2 efficiency of incurred residue was tested by measuring the % of the TRR extracted from the crop matrix (Table 2). In a second series of studies in 99, radiolabelled indoxacarb was topically applied to samples of agricultural commodities, which were aged uncapped at room temperature for 2- days. Extraction efficiency of residue was then tested by measuring the % of applied C that was extracted from the sample (Table ). Two extraction procedures were used:. Ethyl acetate -. Homogenized sample was further homogenized with a mixture of distilled and ethyl acetate ( g sample, 2 ml, ml ethyl acetate), then the mixture was centrifuged. The ethyl acetate solution was removed and evaporated to dryness, and the residue taken up in acetonitrile for C measurement. 2. Acetonitrile - hexane. Homogenized sample was soaked in a mixture of acetonitrile and hexane ( g sample, ml saturated acetonitrile, ml saturated hexane) then further homogenized and centrifuged. The organic layer was separated and the hexane discarded. The pellet was reextracted and extracts were combined. The acetonitrile was evaporated to a small volume for C measurement. Table 2. Extraction efficiency of aged incurred C residues from raw agricultural commodities resulting from the treatment of confined crops with [trifluoromethoxyphenyl (U) C] indoxacarb. Crop Applic rate PHI, Commodity Solvent % extracted TRR Ref kg/ha days range mean n Corn. maize fodder / acetonitrile - hexane AMR 2-9 Corn. maize fodder / ethyl acetate AMR 2-9 Corn. maize forage acetonitrile - hexane -8 8 AMR 2-9 Corn. maize forage ethyl acetate AMR 2-9 Corn. sweet corn 2/ acetonitrile - hexane -8 8 AMR 2-9 Corn. sweet corn 2/ ethyl acetate AMR 2-9 Lettuce.2 / lettuce acetonitrile - hexane -9. AMR -9 Lettuce.2 / lettuce ethyl acetate AMR -9 Potato. tuber acetonitrile - hexane 84-9 AMR 4-9 Potato. tuber ethyl acetate AMR 4-9 / Maize fodder. Samples were cut at day after treatment and allowed to dry in the field until day 2. 2/ Sweet corn. Kernels + cobs with husks removed. / Lettuce. Four applications at weekly intervals for a total of 2. kg/ha of indoxacarb. Table. Extraction efficiency of C residues from samples of raw agricultural commodities following topical application of [trifluoromethoxyphenyl (U) C] indoxacarb and storage at room temperature. Commodity Storage Solvent % recovery of applied C Ref at room temp range mean n Cotton gin trash days acetonitrile - hexane -9 8 AMR Cotton gin trash days ethyl acetate AMR Cotton seed, with lint days acetonitrile - hexane AMR Cotton seed, with lint days ethyl acetate AMR 494-9

24 24 Indoxacarb Commodity Storage Solvent % recovery of applied C Ref at room temp range mean n Grapes 48 hours acetonitrile - hexane AMR 4-9 Grapes 48 hours ethyl acetate AMR 4-9 Tomato 48 hours acetonitrile - hexane -8 8 AMR 4-9 Tomato 48 hours ethyl acetate AMR 4-9 The extraction efficiency of ethyl acetate and acetonitrile were tested on samples of milk, cream and tissues from the dairy cow metabolism study (Amoo and Beaver-Stetser, 99, AMR - 9). Extraction efficiencies of 99,, 2 and % C were achieved with acetonitrile on whole milk. Extraction efficiencies of 88 and % C were achieved with ethyl acetate on muscle and 2 and 2% on fat. Extraction efficiencies of 49 and 2% C were achieved with acetonitrile on liver. Stability of residues in stored analytical samples The Meeting received information on the stability of residues of indoxacarb and indoxacarb in alfalfa, apple juice, apple pomace, apples, fat, grape pomace, grapes, lettuce, liver, milk, muscle, peanut hay, peanut kernels, peanut meal, peanut oil, sweet corn, sweet corn forage, sweet corn stover, tomatoes and wine. Storage stability data are recorded in the tables unadjusted for concurrent procedural recoveries. If the concurrent procedural recoveries were outside of the 2% range the data from that sampling occasion was not taken into account. Klemens (99, AMR 29-9) fortified aliquots (approximately g) of sweet corn commodities with indoxacarb for freezer storage stability testing at approximately -8 C. After each storage interval, two aged aliquots and two freshly fortified aliquots acting as procedural recoveries were analysed (Table ). Table. Freezer storage stability data for indoxacarb () spiked into sweet corn (kernels + cob with husk removed, K+CWHR) and sweet corn forage and stover (Klemens, 99, AMR 29-9). Months stored Procedural recov % Conc., mg/kg Months stored Procedural recov % Conc., mg/kg Months stored Procedural recov % Conc., mg/kg K+CWHR Sweet corn forage Sweet corn stover / repeat / Mean procedural recovery <%. Guinivan and McVicker (2, AMR 449-9) measured the stability of indoxacarb spiked in peanut kernels and peanut hay and stored at - ± C. Desmond and Guinivan (2, AMR 4-9) measured the stability of indoxacarb spiked in peanut oil and meal and stored at -2 ± C.