ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
21,207-2 14 (199 1)
Studies on the Environmental Persistence of S-31 183 (Pyriproxyfen): Adsorption onto Organic Matter and Potential for Leaching through Soil C. H. SCHAEFER, E. F. DUPRAS, JR., AND F. S. MULLIGAN Mosquito Control Research Laboratory, 9240 S. Riverbend Avenue, Parker. Received
University California
III
of Calr;fornia, 93648
July 21, 1990
Analytical methods were developed to allow the extraction and analysis of S-3 1183 (pyriproxyfen), 2-[ 1-methyl-2-(4-phenoxyphenoxy)ethoxyJ pyridine, in organic matter from animal wastewater lagoons. Analysis of water and organic debris from a treated lagoon showed that the active ingredient readily adsorbed onto organic matter. S-3 1183 persisted on organic matter for over a 2-month period, during which time the concentration decayed at an exponential rate. In leaching trials with four different soil types, over 50% of the active ingredient applied remained in the upper 6 cm of a 30-cm soil column; there was no indication of a rapid potential for downward migration. The use of S-31 183 for treating wastewater lagoons at doses which are effective for mosquito control did not result in any apparent problems of environmental incompatibility. 0 I99 I Academic
Press. Inc.
INTRODUCTION Mosquito larvae are an ideal target for insect juvenile hormone-type compounds because they are very susceptible and delayed mortality does not result in increased damage problems as in the case of plant feeding larvae. When mosquito larvae are exposed to juvenile hormone-type compounds, they do not show direct toxicity, except at very high treatment levels, but die in the pupal stage. At doses just below that required to cause pupal mortality, emergence of abnormal adults occurs; their legs remain attached to the pupal case, they are unable to fly and they die on the water surface. Three natural insect juvenile hormones have been identified (Judy et al., 1973; Meyer et al., 1968; Roller et al., 1967) and they have isoprenoid structures. One commercial mosquito larvicide, methoprene or Altosid, has a similar structure and has been used by mosquito abatement agencies since 1975. During the past 5 years another series of compounds have been evaluated against mosquito larvae that exhibit juvenile hormone-type activity but are not isoprene-type structures. These compounds include fenoxycarb, 2-(4-phenoxyphenoxy)thyl ethylcarbamate (Schaefer et al., 1987); Rol6- 1294,2-[p(m-chlorophenoxy)phenoxy]ethyl ethylcarbamate; and Ro 16- 1295,2-[pm-fluorophenoxy)phenoxy]ethyl ethylcarbamate (Schaefer et al., 1985). These carbamates are not inhibitors of acetylcholine esterase and exhibit only the type of biological activity typical of juvenile hormone-type compounds. Another compound which lacks the carbamate moiety and yet has high juvenile hormone-type activity is S-3 1183 (also referred to as pyriproxyfen and Nylar), 2-[ 1-methyl-2-{ 4-phenoxyphenoxy}ethoxy] pyridine. S-3 1183 was effective against Aedes spp. larvae on pasture plots at doses as low as 0.005 kg (AI)/ha and chemical 207
0147-6513/91 $3.00 Copyright 0 1991 by Academic Press, Inc. AI1 rigM.s of reproduction in any form reserved.
208
SCHAEFER, DUPRAS, AND MULLIGAN
residues in the water were not detectable 48 hr after treatment. In small rice ponds Culex quinquefusciatuslarvae were controlled up to 14 days after treatment and again residues in water were not detected after 48 hr. It was speculated that S-3 1183 adsorbed onto organic matter and that extended control was due to larvae acquiring the active ingredient through feeding (Schaefer et al., 1988). Following single treatments of animal wastewater lagoons with 0.11 kg (AI)/ha, S-3 1183, control of C. quinquefasciutuscontinued for periods of up to 2 months even though waters had been periodically removed by pumping. (Schaefer et al., 1988; Mulligan and Schaefer, 1990). Attempts to measure the levels of S-3 1183 on organic matter in these studies were not successful due to the lack of suitable methodology. Also, the degree to which S-3 1183 might be lost from water by leaching through the soil column was not investigated. This paper describes methods for analyzing residues of S-3 1183 in water and organic matter from a treated animal wastewater lagoon and the vertical movement of S3 1183 through a series of different soil types in leaching trials. MATERIALS
AND METHODS
Nunes Dairy wustewuter lagoon. The Nunes Dairy is located in Fresno County, California, and milks ca. 200 cows. The daily wastes, including wash water, urine, feces, and wasted food, are washed into a holding lagoon (Fig. 1); The water from the lagoon is periodically pumped onto adjacent fields. The lagoon is a known breeding source of C. quinquefusciutus mosquitoes and has been used in previous studies on the efficacy of S-31 183 against the latter (Mulligan and Schaefer, 1990). The lagoon
FIG. 1. Nunes dairy drain lagoon.
ENVIRONMENTAL
PERSISTENCE
OF PYRIPROXYFEN
209
is ca. 0.32 ha in area and ca. 2 m in depth. It was sprayed on 10/2/89 with 0.11 kg (AI)/ha S-3 1183 using an emulsifiable concentrate (EC) formulation diluted with water. Samples of floating organic debris were collected around the perimeter before treatment and at 1,4, and 8 days and at weekly intervals thereafter for 8 additional weeks. Water samples were collected before and at 1 hr after treatment as well as at all of the above sampling times for organic matter. All samples were transported to the laboratory and then frozen until analysis. Eficacy of treatment. Before treatment and at weekly intervals throughout the study, samples of pupae were collected by dipping around the lagoon’s perimeter and transported to the laboratory for determination of biological activity. Two subsamples of 50 pupae each were held until mortality or adult emergence occurred. In addition weekly field observations were made to detect the emergence of abnormal adults. Analytical methods.Organic matter: (1) Extraction. Organic matter was compressed by hand, to remove excess water, and a 100-g sample was placed in a Sorval Omnimixer with 50 g anhydrous NaS04 and 150 ml hexane. After blending 3-4 min at 400-500 rpm, the contents were filtered through Whatman No. 1 paper and then through a Millipore, Millex-RS OS-grn unit. One hundred milliliters of the filtrate was concentrated to l-2 ml using a rotating evaporator. (2) Liquid/liquid partition. The sample from ( 1) was transferred into a separatory funnel in a total of 25 ml hexane and then partitioned 3X against 25-ml aliquots of acetonitrile. The combined acetonitrile phases were reduced to dryness and the residues dissolved in l-2 ml hexane. (3) Cleanup on NH2 column. A chromatographic column (1 X 30 cm) was packed with 2 g Bond Elut-NH2 (Analytichem International, Harbor City, CA) and eluted with 10 ml hexane. The sample in hexane from (2) was added and the column eluted with 15 ml hexane, which was discarded. The active ingredient was then eluted with 40 ml hexane and the latter concentrated to l-2 ml. (4) Aluminum oxide column cleanup. A chromatographic column (1.5 X 30 cm) was packed with 20 g neutral aluminum oxide (Aldrich Chemical Co. No. 19,997-4, 150 mesh) and eluted with 25 ml hexane/ethyl acetate (100:4 v/v). The sample in hexane from (3) was added and the column was eluted with 90 ml hexane/ethyl acetate (100:4 v/v) and the latter was discarded. The active ingredient was then eluted with 75 ml hexane/ethyl acetate (100: 4 v/v). The latter was reduced to dryness and then dissolved in 1 ml hexane. (5) Florisil column cleanup. A chromatographic column (1.5 X 30 cm) was packed with 15 g Florisil (J. T. Baker No. 3372-7, 60-100 mesh) and preeluted with 50 ml of hexane/ ethyl acetate (100:2 v/v). The sample from (4) was then added and the column was eluted with an additional 50 ml hexane/ethyl acetate (100:2 v/v), which was then discarded. The active ingredient was then eluted with 75 ml hexane/ethyl acetate ( 100: 4 v/v) and reduced to dryness. The residue was dissolved in 1 ml acetonitrile for analysis by high-performance liquid chromatography (HPLC). The moisture content of organic matter was determined by weighing 30-50 g of material, drying overnight at 105 “C, and then reweighing. Triplicate samples of untreated organic matter from the Nunes Dairy lagoon were fortified at 1.O, 0.1, and 0.01 ppm with S-3 1183 and analyzed as above to determine recoveries. Dairy lagoon water: Water samples from the Nunes Dairy lagoon were filtered through glass filter paper (Whatman 934AH) to remove the bulk of suspended solids. Then a l-liter sample was partitioned 2X against 200 ml methylene chloride. The combined methylene chloride phase was reduced to dryness and the residue was dis-
210
SCHAEFER, DUPRAS, AND MULLIGAN
solved in l-2 ml hexane. The latter was then subjected to cleanup on aluminum oxide and then on Florisil, as described above, and then analyzed by HPLC. Triplicate l-liter dairy lagoon water samples were fortified at 0.001, 0.0005, and 0.0001 ppm with S-3 1183 and analyzed to determine recoveries. Soil leaching. Design of soil leaching experiments was based on EPA guidelines (USEPA, 1988). Soils representing four different soil types (loamy sand, sandy loam, loam, and clay) were used. Specific soil series representing these soil types were selected from soil maps, collected, and subjected to mechanical analysis (Black et al., 1965) to verify composition. Prior to leaching trials, all soils were adjusted to a 5% moisture content. A metal column (1.5 X 90 cm) was filled with soil to a 30-cm depth above a glass wool plug and 2.5 g of soil was removed. A 10-g sample of the same soil was treated with an acetone solution of S-3 1183 to give ca. 2.0 pg (AI) per 2.5 gm soil; the solvent was removed and the soil mixed in a rotating evaporator. Two 2.5-g soil aliquots were extracted and analyzed, as described below, and a third 2.5-g aliquot was placed on top of the soil column. This resulted in a target dosage of 0.112 kg (AI)/ha, which is anticipated to be the highest use rate for S-3 1183 in mosquito control. A 90-ml aliquot of distilled water (a water volume equal to 20 times the cross-sectional area of the column) was added and leaching was achieved using a 40-p& head pressure (helium). Each metal column was graduated in 6-cm segments and after leaching these were separated using a tubing cutter, and the soil in each segment was analyzed. The total aqueous eluate was also analyzed for S-3 1183. Each soil was leached on two different dates. Extraction and cleanup of soil samples. The soil removed from each 6-cm segment of the metal column was placed in a blender cup with 20 g anhydrous NaSo4 and blended twice with 150~ml aliquots of hexane. The combined hexane phase was filtered, concentrated to ca. 25 ml, and partitioned 3X against 25 ml of acetonitrile. The combined acetonitrile phase was concentrated to dryness and the residues were dissolved in 5 ml hexane. The latter was then subjected to cleanup by column chromatography on Florisil, as described for the dairy drain organic matter samples, and then analyzed by HPLC. The water used to elute soil columns was partitioned 2X against 100 ml methylene chloride and the combined methylene chloride phase was dried over anhydrous NaS04 and then concentrated to dryness. The residues were dissolved in 5 ml hexane, cleaned up on Florisil, as above, and then analyzed by HPLC. High-pe@rmance liquid chromatography. A Perkin-Elmer Model 400 instrument was equipped with a uv detector (270 nm). The C 18 column (octadecylsilane bonded to 3-pm silica spheres) was 4.6 X 83-mm and was held at 40°C; the mobile phase was acetonitrile-HZ0 (70:30 v/v at 1 ml/min.). The detector was coupled to a PerkinElmer LCI- 100 computing integrator. S-3 1183 had a retention time of 2.25 min (1.45 min adjusted for void volume). Confirmation of peak assignment by mass spectrometry. The peak assigned to be S-3 1183 by HPLC was based on retention time and the lack of emergence of peaks from untreated samples at the same time. However, since the dairy lagoon habitat contained an abundance of potentially interfering components, it was considered important to verify the peak assigned as the active ingredient. A Hewlett-Packard Model 5988 mass spectrometer (MS) was coupled to Hewlett-Packard Model 5890 gas chromatograph (GLC) and a Hewlett-Packard Model 98 16 data system. GLC conditions
ENVIRONMENTAL
PERSISTENCE
OF
PYRIPROXYFEN
211
were as follows: cross-linked 50% phenylmethyl silicone capillary column (0.2 X 25 mm), carrier gas, helium at a 21 cm/set linear velocity, temperatures, column 280, injection port 300, transfer line 280, and MS source 200°C. The MS was operated in the electron ionization mode at 70 eV. Five ions were selected and monitored: m/z 4 I, 77, 78,96, and 136 (base peak). For confirmation a peak was required to have the same GLC retention time as an S-3 1183 standard (8.4 min), show the same approximate mass as by HPLC, and have all 5 ions present and in the same ratios as a S-31 183 technical standard. RESULTS Validation of analytical methods. Samples of untreated organic matter from the Nunes Dairy lagoon fortified (in triplicate) with 1.O, 0.1, and 0.01 ppm S-3 1183 and then analyzed by the methods described yielded recoveries of 94.7 * 2.4, 96.1 + 1.3, and 92.2 + 6.2%, respectively. The lowest detectable limit (minimum peak of twice background noise) was estimated as 0.0005 ppm. Water samples from the Nunes Dairy lagoon fortified (in triplicate) with 0.001,0.0005, and 0.000 1 ppm S-3 1183 and analyzed as described yielded recoveries of 97.3 + 0.4, 97.0 & 1.1 and 85.5 +- 9.6%, respectively; the lowest detectable limit was estimated to be 0.00004 ppm. The minimum detectable limits for S-3 1183 in nonpolluted water (soil eluant) and in soil were 0.00005 ppm and 0.001 pg, respectively (Schaefer and Miura, 1990). Bioassay of mosquito pupae from Nunes Dairy lagoon. Pupae collected on three occasions prior to treatment (6 days, 5 days, and 1 hr) emerged into adults normally. After treatment, all pupae collected up to 29 days died; however, after 29 days time some pupae emerged into abnormal adults in the laboratory. No adults emerged normally throughout the 64-day sampling period. In the field, abnormal adults were observed on the water surface commencing on Day 29 and thereafter. Thus, complete control of mosquitoes resulted from the treatment throughout the duration of the test. Residues of S-31183 on organic matter and water from the Nunes Dairy lagoon. The average concentrations of S-31 183 on organic matter samples collected from Nunes Dairy lagoon are shown in Table 1. The concentrations slowly declined during the 64&y study. The data were subjected to analysis using the SAS nonlinear regression model (SAS Institute 1985) and fit the standard chemical decay curve Y = Y&“. This yielded values for the initial concentraiton ( YO)of 0.126 +- 0.0 154 ppm (wet wt) and for the decay constant (X) of 0.0927 + 0.0215. Using this equation, the half life (t$) of S-3 1183 on organic matter in the Nunes lagoon was calculated to be 7.47 days. The concentration of S-3 I 183 in triplicate water samples collected 1 hr after treatment showed 0.0013 + 0.00041 ppm; The active ingredient was not detected (
AND
ENVIRONMENTAL
SAFETY
21,207-2 14 (199 1)
Studies on the Environmental Persistence of S-31 183 (Pyriproxyfen): Adsorption onto Organic Matter and Potential for Leaching through Soil C. H. SCHAEFER, E. F. DUPRAS, JR., AND F. S. MULLIGAN Mosquito Control Research Laboratory, 9240 S. Riverbend Avenue, Parker. Received
University California
III
of Calr;fornia, 93648
July 21, 1990
Analytical methods were developed to allow the extraction and analysis of S-3 1183 (pyriproxyfen), 2-[ 1-methyl-2-(4-phenoxyphenoxy)ethoxyJ pyridine, in organic matter from animal wastewater lagoons. Analysis of water and organic debris from a treated lagoon showed that the active ingredient readily adsorbed onto organic matter. S-3 1183 persisted on organic matter for over a 2-month period, during which time the concentration decayed at an exponential rate. In leaching trials with four different soil types, over 50% of the active ingredient applied remained in the upper 6 cm of a 30-cm soil column; there was no indication of a rapid potential for downward migration. The use of S-31 183 for treating wastewater lagoons at doses which are effective for mosquito control did not result in any apparent problems of environmental incompatibility. 0 I99 I Academic
Press. Inc.
INTRODUCTION Mosquito larvae are an ideal target for insect juvenile hormone-type compounds because they are very susceptible and delayed mortality does not result in increased damage problems as in the case of plant feeding larvae. When mosquito larvae are exposed to juvenile hormone-type compounds, they do not show direct toxicity, except at very high treatment levels, but die in the pupal stage. At doses just below that required to cause pupal mortality, emergence of abnormal adults occurs; their legs remain attached to the pupal case, they are unable to fly and they die on the water surface. Three natural insect juvenile hormones have been identified (Judy et al., 1973; Meyer et al., 1968; Roller et al., 1967) and they have isoprenoid structures. One commercial mosquito larvicide, methoprene or Altosid, has a similar structure and has been used by mosquito abatement agencies since 1975. During the past 5 years another series of compounds have been evaluated against mosquito larvae that exhibit juvenile hormone-type activity but are not isoprene-type structures. These compounds include fenoxycarb, 2-(4-phenoxyphenoxy)thyl ethylcarbamate (Schaefer et al., 1987); Rol6- 1294,2-[p(m-chlorophenoxy)phenoxy]ethyl ethylcarbamate; and Ro 16- 1295,2-[pm-fluorophenoxy)phenoxy]ethyl ethylcarbamate (Schaefer et al., 1985). These carbamates are not inhibitors of acetylcholine esterase and exhibit only the type of biological activity typical of juvenile hormone-type compounds. Another compound which lacks the carbamate moiety and yet has high juvenile hormone-type activity is S-3 1183 (also referred to as pyriproxyfen and Nylar), 2-[ 1-methyl-2-{ 4-phenoxyphenoxy}ethoxy] pyridine. S-3 1183 was effective against Aedes spp. larvae on pasture plots at doses as low as 0.005 kg (AI)/ha and chemical 207
0147-6513/91 $3.00 Copyright 0 1991 by Academic Press, Inc. AI1 rigM.s of reproduction in any form reserved.
208
SCHAEFER, DUPRAS, AND MULLIGAN
residues in the water were not detectable 48 hr after treatment. In small rice ponds Culex quinquefusciatuslarvae were controlled up to 14 days after treatment and again residues in water were not detected after 48 hr. It was speculated that S-3 1183 adsorbed onto organic matter and that extended control was due to larvae acquiring the active ingredient through feeding (Schaefer et al., 1988). Following single treatments of animal wastewater lagoons with 0.11 kg (AI)/ha, S-3 1183, control of C. quinquefasciutuscontinued for periods of up to 2 months even though waters had been periodically removed by pumping. (Schaefer et al., 1988; Mulligan and Schaefer, 1990). Attempts to measure the levels of S-3 1183 on organic matter in these studies were not successful due to the lack of suitable methodology. Also, the degree to which S-3 1183 might be lost from water by leaching through the soil column was not investigated. This paper describes methods for analyzing residues of S-3 1183 in water and organic matter from a treated animal wastewater lagoon and the vertical movement of S3 1183 through a series of different soil types in leaching trials. MATERIALS
AND METHODS
Nunes Dairy wustewuter lagoon. The Nunes Dairy is located in Fresno County, California, and milks ca. 200 cows. The daily wastes, including wash water, urine, feces, and wasted food, are washed into a holding lagoon (Fig. 1); The water from the lagoon is periodically pumped onto adjacent fields. The lagoon is a known breeding source of C. quinquefusciutus mosquitoes and has been used in previous studies on the efficacy of S-31 183 against the latter (Mulligan and Schaefer, 1990). The lagoon
FIG. 1. Nunes dairy drain lagoon.
ENVIRONMENTAL
PERSISTENCE
OF PYRIPROXYFEN
209
is ca. 0.32 ha in area and ca. 2 m in depth. It was sprayed on 10/2/89 with 0.11 kg (AI)/ha S-3 1183 using an emulsifiable concentrate (EC) formulation diluted with water. Samples of floating organic debris were collected around the perimeter before treatment and at 1,4, and 8 days and at weekly intervals thereafter for 8 additional weeks. Water samples were collected before and at 1 hr after treatment as well as at all of the above sampling times for organic matter. All samples were transported to the laboratory and then frozen until analysis. Eficacy of treatment. Before treatment and at weekly intervals throughout the study, samples of pupae were collected by dipping around the lagoon’s perimeter and transported to the laboratory for determination of biological activity. Two subsamples of 50 pupae each were held until mortality or adult emergence occurred. In addition weekly field observations were made to detect the emergence of abnormal adults. Analytical methods.Organic matter: (1) Extraction. Organic matter was compressed by hand, to remove excess water, and a 100-g sample was placed in a Sorval Omnimixer with 50 g anhydrous NaS04 and 150 ml hexane. After blending 3-4 min at 400-500 rpm, the contents were filtered through Whatman No. 1 paper and then through a Millipore, Millex-RS OS-grn unit. One hundred milliliters of the filtrate was concentrated to l-2 ml using a rotating evaporator. (2) Liquid/liquid partition. The sample from ( 1) was transferred into a separatory funnel in a total of 25 ml hexane and then partitioned 3X against 25-ml aliquots of acetonitrile. The combined acetonitrile phases were reduced to dryness and the residues dissolved in l-2 ml hexane. (3) Cleanup on NH2 column. A chromatographic column (1 X 30 cm) was packed with 2 g Bond Elut-NH2 (Analytichem International, Harbor City, CA) and eluted with 10 ml hexane. The sample in hexane from (2) was added and the column eluted with 15 ml hexane, which was discarded. The active ingredient was then eluted with 40 ml hexane and the latter concentrated to l-2 ml. (4) Aluminum oxide column cleanup. A chromatographic column (1.5 X 30 cm) was packed with 20 g neutral aluminum oxide (Aldrich Chemical Co. No. 19,997-4, 150 mesh) and eluted with 25 ml hexane/ethyl acetate (100:4 v/v). The sample in hexane from (3) was added and the column was eluted with 90 ml hexane/ethyl acetate (100:4 v/v) and the latter was discarded. The active ingredient was then eluted with 75 ml hexane/ethyl acetate (100: 4 v/v). The latter was reduced to dryness and then dissolved in 1 ml hexane. (5) Florisil column cleanup. A chromatographic column (1.5 X 30 cm) was packed with 15 g Florisil (J. T. Baker No. 3372-7, 60-100 mesh) and preeluted with 50 ml of hexane/ ethyl acetate (100:2 v/v). The sample from (4) was then added and the column was eluted with an additional 50 ml hexane/ethyl acetate (100:2 v/v), which was then discarded. The active ingredient was then eluted with 75 ml hexane/ethyl acetate ( 100: 4 v/v) and reduced to dryness. The residue was dissolved in 1 ml acetonitrile for analysis by high-performance liquid chromatography (HPLC). The moisture content of organic matter was determined by weighing 30-50 g of material, drying overnight at 105 “C, and then reweighing. Triplicate samples of untreated organic matter from the Nunes Dairy lagoon were fortified at 1.O, 0.1, and 0.01 ppm with S-3 1183 and analyzed as above to determine recoveries. Dairy lagoon water: Water samples from the Nunes Dairy lagoon were filtered through glass filter paper (Whatman 934AH) to remove the bulk of suspended solids. Then a l-liter sample was partitioned 2X against 200 ml methylene chloride. The combined methylene chloride phase was reduced to dryness and the residue was dis-
210
SCHAEFER, DUPRAS, AND MULLIGAN
solved in l-2 ml hexane. The latter was then subjected to cleanup on aluminum oxide and then on Florisil, as described above, and then analyzed by HPLC. Triplicate l-liter dairy lagoon water samples were fortified at 0.001, 0.0005, and 0.0001 ppm with S-3 1183 and analyzed to determine recoveries. Soil leaching. Design of soil leaching experiments was based on EPA guidelines (USEPA, 1988). Soils representing four different soil types (loamy sand, sandy loam, loam, and clay) were used. Specific soil series representing these soil types were selected from soil maps, collected, and subjected to mechanical analysis (Black et al., 1965) to verify composition. Prior to leaching trials, all soils were adjusted to a 5% moisture content. A metal column (1.5 X 90 cm) was filled with soil to a 30-cm depth above a glass wool plug and 2.5 g of soil was removed. A 10-g sample of the same soil was treated with an acetone solution of S-3 1183 to give ca. 2.0 pg (AI) per 2.5 gm soil; the solvent was removed and the soil mixed in a rotating evaporator. Two 2.5-g soil aliquots were extracted and analyzed, as described below, and a third 2.5-g aliquot was placed on top of the soil column. This resulted in a target dosage of 0.112 kg (AI)/ha, which is anticipated to be the highest use rate for S-3 1183 in mosquito control. A 90-ml aliquot of distilled water (a water volume equal to 20 times the cross-sectional area of the column) was added and leaching was achieved using a 40-p& head pressure (helium). Each metal column was graduated in 6-cm segments and after leaching these were separated using a tubing cutter, and the soil in each segment was analyzed. The total aqueous eluate was also analyzed for S-3 1183. Each soil was leached on two different dates. Extraction and cleanup of soil samples. The soil removed from each 6-cm segment of the metal column was placed in a blender cup with 20 g anhydrous NaSo4 and blended twice with 150~ml aliquots of hexane. The combined hexane phase was filtered, concentrated to ca. 25 ml, and partitioned 3X against 25 ml of acetonitrile. The combined acetonitrile phase was concentrated to dryness and the residues were dissolved in 5 ml hexane. The latter was then subjected to cleanup by column chromatography on Florisil, as described for the dairy drain organic matter samples, and then analyzed by HPLC. The water used to elute soil columns was partitioned 2X against 100 ml methylene chloride and the combined methylene chloride phase was dried over anhydrous NaS04 and then concentrated to dryness. The residues were dissolved in 5 ml hexane, cleaned up on Florisil, as above, and then analyzed by HPLC. High-pe@rmance liquid chromatography. A Perkin-Elmer Model 400 instrument was equipped with a uv detector (270 nm). The C 18 column (octadecylsilane bonded to 3-pm silica spheres) was 4.6 X 83-mm and was held at 40°C; the mobile phase was acetonitrile-HZ0 (70:30 v/v at 1 ml/min.). The detector was coupled to a PerkinElmer LCI- 100 computing integrator. S-3 1183 had a retention time of 2.25 min (1.45 min adjusted for void volume). Confirmation of peak assignment by mass spectrometry. The peak assigned to be S-3 1183 by HPLC was based on retention time and the lack of emergence of peaks from untreated samples at the same time. However, since the dairy lagoon habitat contained an abundance of potentially interfering components, it was considered important to verify the peak assigned as the active ingredient. A Hewlett-Packard Model 5988 mass spectrometer (MS) was coupled to Hewlett-Packard Model 5890 gas chromatograph (GLC) and a Hewlett-Packard Model 98 16 data system. GLC conditions
ENVIRONMENTAL
PERSISTENCE
OF
PYRIPROXYFEN
211
were as follows: cross-linked 50% phenylmethyl silicone capillary column (0.2 X 25 mm), carrier gas, helium at a 21 cm/set linear velocity, temperatures, column 280, injection port 300, transfer line 280, and MS source 200°C. The MS was operated in the electron ionization mode at 70 eV. Five ions were selected and monitored: m/z 4 I, 77, 78,96, and 136 (base peak). For confirmation a peak was required to have the same GLC retention time as an S-3 1183 standard (8.4 min), show the same approximate mass as by HPLC, and have all 5 ions present and in the same ratios as a S-31 183 technical standard. RESULTS Validation of analytical methods. Samples of untreated organic matter from the Nunes Dairy lagoon fortified (in triplicate) with 1.O, 0.1, and 0.01 ppm S-3 1183 and then analyzed by the methods described yielded recoveries of 94.7 * 2.4, 96.1 + 1.3, and 92.2 + 6.2%, respectively. The lowest detectable limit (minimum peak of twice background noise) was estimated as 0.0005 ppm. Water samples from the Nunes Dairy lagoon fortified (in triplicate) with 0.001,0.0005, and 0.000 1 ppm S-3 1183 and analyzed as described yielded recoveries of 97.3 + 0.4, 97.0 & 1.1 and 85.5 +- 9.6%, respectively; the lowest detectable limit was estimated to be 0.00004 ppm. The minimum detectable limits for S-3 1183 in nonpolluted water (soil eluant) and in soil were 0.00005 ppm and 0.001 pg, respectively (Schaefer and Miura, 1990). Bioassay of mosquito pupae from Nunes Dairy lagoon. Pupae collected on three occasions prior to treatment (6 days, 5 days, and 1 hr) emerged into adults normally. After treatment, all pupae collected up to 29 days died; however, after 29 days time some pupae emerged into abnormal adults in the laboratory. No adults emerged normally throughout the 64-day sampling period. In the field, abnormal adults were observed on the water surface commencing on Day 29 and thereafter. Thus, complete control of mosquitoes resulted from the treatment throughout the duration of the test. Residues of S-31183 on organic matter and water from the Nunes Dairy lagoon. The average concentrations of S-31 183 on organic matter samples collected from Nunes Dairy lagoon are shown in Table 1. The concentrations slowly declined during the 64&y study. The data were subjected to analysis using the SAS nonlinear regression model (SAS Institute 1985) and fit the standard chemical decay curve Y = Y&“. This yielded values for the initial concentraiton ( YO)of 0.126 +- 0.0 154 ppm (wet wt) and for the decay constant (X) of 0.0927 + 0.0215. Using this equation, the half life (t$) of S-3 1183 on organic matter in the Nunes lagoon was calculated to be 7.47 days. The concentration of S-3 I 183 in triplicate water samples collected 1 hr after treatment showed 0.0013 + 0.00041 ppm; The active ingredient was not detected (
