Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2017, 65, 9980-9986
Bioconcentration and Metabolism of Pyriproxyfen in Tadpoles of African Clawed Frogs, Xenopus laevis Keiko Ose,*,† Mitsugu Miyamoto,† Takuo Fujisawa,† and Toshiyuki Katagi‡ †
Environmental Health Science Laboratory, Sumitomo Chemical Company, Limited, 4-2-1 Takatsukasa, Takarazuka, Hyogo 665-8555, Japan ‡ Environmental Health Science Laboratory, Sumitomo Chemical Company, Limited, 3-1-98 Kasugade-naka, Konohana-ku, Osaka-city, Osaka 554-8558, Japan ABSTRACT: Bioconcentration and metabolism of pyriproxyfen uniformly labeled with 14C at the phenoxyphenyl ring were studied using tadpoles of African clawed frog, Xenopus laevis, exposed to water at the nominal concentrations of 3 and 300 ppb for 22 days under the flow-through conditions, with a following 3 day depuration phase. Neither meaningful mortality nor abnormal behavior was observed in control and exposure groups throughout the study. After the rapid uptake to tadpoles, pyriproxyfen was extensively metabolized and excreted, and as a result, steady-state bioconcentration factors and depuration halflives ranged from 550 to 610 and from 0.34 to 0.54 days, respectively. The metabolites were mostly distributed in the liver or gastrointestinal tract. The major metabolic reactions were hydroxylation at the 4′ position of the phenoxyphenyl group and cleavage of the ether linkage, followed by sulfate conjugation. KEYWORDS: pyriproxyfen, metabolism, bioconcentration, amphibian
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INTRODUCTION Pyriproxyfen (1) [4-phenoxyphenyl (R,S)-2-(2-pyridyloxy)propyl ether] is an insect growth regulator by interfering with metamorphic changes in insects, and it exhibits insecticidal activities against various pest insects, such as houseflies, mosquitoes, cockroaches, as well as thrips and whiteflies.1−3 Surface water may be contaminated with compound 1 by spray drift or runoff as well as erosion of farm soil, but the aquatic exposure should be very limited as a result of its rapid degradation in soil and water sediment systems.4−6 However, the ecotoxicological profiles of compound 1 in various species (aquatic and terrestrial organisms) have been extensively studied,4,7 taking into account its mode of action, and they are useful for exhaustive ecological risk assessment of compound 1. Amphibians change their habitats from aquatic to terrestrial environments through metamorphosis and, therefore, are possibly exposed to contaminants in each environment. They can be prey as well as predator at each life stage and involved in bioaccumulation of a contaminant via a food web. Tadpoles are the first candidate for risk assessment by taking into account the mode of action and potential exposure route of compound 1, but the ecological effects on amphibians are usually evaluated using fish data as a surrogate. Not only the various differences in physiology, including possible dermal uptake of pesticides and life cycle/morphology between amphibian and fish, but also typical metamorphosis in amphibian may raise some concerns on the validity of such an approach. In both the European Union (EU) and the U.S., requests of risk assessment on amphibians are growing under no specific test guidelines available. Moreover, research on amphibians is in the forefront of assessing the endocrine-disrupting potential of chemicals in the world,8,9 and the United States Environmental Protection Agency (U.S. EPA) has started to use amphibians as one of the © 2017 American Chemical Society
Tier 1 and 2 studies in the endocrine disruptor screening program (EDSP) to evaluate functions of the hypothalamic− pituitary−thyroid (HPT) axis.10−12 In fact, as the Tier 1 study, an amphibian metamorphosis assay (AMA) was conducted to investigate effects of compound 1 on the HPT axis.13 The toxic effects of a chemical are generally controlled by its uptake, distribution in tissues, metabolism, and excretion, and such information is indispensable to understand the relationship between exposure and toxic effects. In vivo and/or in vitro metabolism of compound 1 have been reported in relation to its ecological effects for not only invertebrates but also vertebrates, such as fish and mammals.4,14−17 Although many acute toxicities of pesticides for amphibians have recently been reviewed,18 there appears to be less knowledge on bioconcentration of pesticides in relation to distribution, metabolism, and excretion.19 For more robust hazard characterization and risk assessment in amphibians, we examined bioconcentration and metabolism behavior of 14C-labeled compound 1 with tadpoles of Xenopus laevis.
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MATERIALS AND METHODS
Chemicals. Compound 1 uniformly labeled with 14C at the phenoxyphenyl ring (specific activity, 13.1 MBq mg−1; radiochemical purity, 99.1%) as well as non-radiolabeled compound 1 (purity, 99.1%) were prepared in our laboratory.4,5,14,15 The following unlabeled standards of potential metabolites were also synthesized in our laboratory, with each chemical purity being >97%:4,5,14,15 4-(4hydroxyphenoxy)phenyl (R,S)-2-(2-pyridyloxy)propyl ether (2), 4phenoxyphenyl (R,S)-2-hydroxypropyl ether (3), 4-phenoxyphenol (4), and 4-hydroxyphenyl (R,S)-2-(2-pyridyloxy)propyl ether (5). The Received: Revised: Accepted: Published: 9980
September 14, 2017 October 25, 2017 October 30, 2017 October 30, 2017 DOI: 10.1021/acs.jafc.7b04184 J. Agric. Food Chem. 2017, 65, 9980−9986
Article
Journal of Agricultural and Food Chemistry structures of these compounds are illustrated in Figure 1. βGlucuronidase from bovine liver and sulfatase from Helix pomatia
typical retention times and Rf values of compound 1 and each metabolite are listed in Table 1.
Table 1. Typical Chromatographic Properties of Compound 1 and Its Metabolites TLC Rf value compound 1 2 sulfate conjugate of 2 3 sulfate conjugate of 3 4 5 sulfate conjugate of 5
Figure 1. Proposed metabolic pathways of compound 1 in tadpoles. (∗) 14C-labeled position. Common paths at 3 and 300 ppb in blue, and specific paths at 300 ppb in red.
HPLC retention time (min)
system Aa system Bb system Cc
35.8 29.2 23.5
0.63 0.37
26.9 22.5
0.37
25.6 22.6 18.0
0.46 0.38
0.61 0.49 0.61 0.60 0.47 0.65 0.32 0.40
a n-Hexane/ethyl acetate/acetic acid (10:4:1). bToluene/ethyl formate/formic acid (5:7:1). cn-Butanol/acetic acid/water (8:1:1).
were purchased from Sigma-Aldrich Japan (Tokyo, Japan). All other chemicals were of a reagent grade and obtained from commercial suppliers. Test Organisms. Tadpoles of African clawed frog, X. laevis, at the Nieuwkoop and Faber (NF) stage20 of approximately 45 were purchased from Watanabe-Breeding Center (Hyogo, Japan). They were acclimated and grew up until the appropriate NF stage in the dilution water (tap water dechlorinated with activated charcoal, typical total hardness of 65−75 mg L−1 and total alkalinity of 60 mg L−1) for at least 7 days under 12 h light and dark cycle and regularly fed with a commercial food (sera Micron, Sera Japan, Co., Ltd., Tokyo, Japan). Radioanalysis. Radioactivity in the exposure water and extracts of tadpoles, mixed with Packard Emulsifier Scintillator 299 or PerkinElmer Emulsifier Scintillator Plus, was determined by liquid scintillation counting (LSC) with Packard model 2000CA or 2900TR liquid scintillation analyzers (Packard Instrument Co., Inc., Meriden, CT, U.S.A.). Unextractable residues in tadpoles were quantified by LSC after combustion, using a Packard Tri-Carb model 307 sample oxidizer and Packard Carb-CO2 absorber with a Packard Permafluor scintillator (14C recovery, greater than 95%). Chromatography. High-performance liquid chromatography (HPLC) analysis was conducted using a Shimadzu LC-20AT pump linked in series with a L-7405 ultraviolet (UV) detector set (Shimadzu Co., Kyoto, Japan) at 254 nm and a Packard Radiomatic 505TR radio detector equipped with a 500 μL liquid cell. Packard Ultima-Flo AP was used as a scintillator. A Sumipax ODS A-212 column (5 μm, 6 mm inner diameter × 150 mm, Sumika Chemical Analysis Service, Ltd. Osaka, Japan) was employed for analyses at a flow rate of 1 mL min−1. The following linear gradient system was used for typical analysis of metabolites with 0.03% acetic acid in acetonitrile (solvent A) and 0.03% acetic acid in water (solvent B): 0 min, 10% A−90% B; 30 min, 90% A−10% B; and 40 min, 90% A−10% B. Each 14C peak was identified by HPLC co-chromatography by comparing its retention time to those of non-radiolabeled authentic standards. Thin-layer chromatography (TLC) was conducted using silica gel 60F254 thin-layer chromatoplates (20 × 20 cm, 0.25 mm thickness, E. Merck, Kenilworth, NJ, U.S.A.). The following solvent systems were used for co-chromatographic analysis: n-hexane/ethyl acetate/acetic acid (10:4:1, v/v/v) and toluene/ethyl formate/formic acid (5:7:1, v/ v/v) in two-dimensional (2D) TLC for identification of metabolites and n-butanol/acetic acid/water (8:1:1, v/v/v) in one-dimensional (1D) TLC for isolation of conjugates. The non-radiolabeled reference standards were detected by exposing TLC plates to UV light. Autoradiograms were prepared by exposing TLC plates to a BAS-IIIS Fuji Imaging Plate (Fujifilm Co., Tokyo, Japan), and the radioactivity on an imaging plate was detected using an Amersham Variable Mode Imager Typhoon 9200 (Amersham plc, Buckinghamshire, U.K.). The
Bioassay. The flow-through exposure of tadpoles was conducted with five volume replacements per day (70 mL min−1) under the same conditions of their acclimation. The water quality during the exposure was kept within an acceptable range for the test organisms: pH, 6.9− 7.6; dissolved oxygen (DO) concentration, 5.3−8.2 mg L−1; and temperature, 22.0−23.6 °C. The tadpoles were regularly fed with the commercial food (sera Micron) according to the feeding schedule outlined in the AMA guidelines.8,10 Two test concentrations of 3 and 300 ppb was conveniently selected on the basis of an acute toxicity of pyriproxyfen to tadpoles of X. laevis, whose 96 h LC50 value was greater than 300 ppb, taking into account the OECD 305 guideline.22 Two stock solutions were prepared at the concentrations of 60 and 6000 ppm by dissolving 14C-1 and its nonradiolabeled one in N,N-dimethylformamide (DMF), whose specific activity was 1.3 and 0.013 MBq mg−1, respectively. Each stock solution and dechlorinated water was continuously flown into a mixture chamber at 3.5 μL mL−1 and 70 mL min−1, respectively, and wellmixed to obtain a nominal concentration of 3 or 300 ppb, and this exposure solution was introduced into each 20 L aquarium (30 × 30 × 30 cm). A solvent control group was similarly prepared using only DMF at 50 ppb. Exposure was initiated by introducing 100 tadpoles at the NF stage 51 into each test vessel (exposure day 0). Mortality and toxic symptoms of the tadpoles were monitored daily, and dead organisms were removed to maintain good water quality. After 22 days, all surviving tadpoles exposed to compound 1 at each concentration were separately transferred to 20 L of clean water to initiate the 3 day depuration phase. Approximately 5 g of surviving tadpoles was randomly sampled at 1, 3, 7, 15, and 22 days of exposure and 1, 2, and 3 days of depuration. Each tadpole was euthanized, blotted dry with a paper towel, and weighed individually on an electronic balance (model AE200, Mettler Toledo, Tokyo, Japan). The tadpoles were subjected to a NF stage observation and stored under frozen conditions from −10 to −20 °C until further radioanalyses. To examine the metabolic profile of compound 1, five tadpoles at the NF stage 51 were separately exposed for 3 days to 14C-1 at 300 ppb in 1 L glass beakers under static conditions. A similar 3 day exposure at 3 and 300 ppb was additionally conducted using five tadpoles at the NF stage approximately 57 for a whole-body autoradiography to clarify the 14C distribution in the tadpole. Water and Tadpole Analyses. A 5 mL aliquot of the exposure water was taken in triplicate for radioassay on a daily basis, except for 8 and 12 days of exposure. In addition, 1 L exposure water was taken at the same time of sampling tadpoles and extracted twice with 240 mL of ethyl acetate/ethanol (5:1, v/v) after adding 100 g of ammonium sulfate and 1 mL of 1 N HCl. The concentrated residue of the 9981
DOI: 10.1021/acs.jafc.7b04184 J. Agric. Food Chem. 2017, 65, 9980−9986
Article
Journal of Agricultural and Food Chemistry Table 2. Concentration of Total 14C and Compound 1 in the Test System concentration, ppm in water and tadpole uptake phase 3 ppb
a
day 1
day 3
day 7
day 15
day 22
day 1
day 2
day 3
2.40 1.83
2.40 1.81
3.00 1.92
2.90 1.83
2.80 1.07
a a
a a
a a
0.640 0.298
0.797 0.309
1.50 0.619
1.58 0.844
1.36 0.823
0.305 0.201
0.133 0.103
0.237 0.177
a a
a a
5.89 4.90
4.18 4.18
water 14
300 ppb
depuration phase
C 1 whole tadpole 14 C 1 water 14 C 1 whole tadpole 14 C 1
199 162
219 161
49.1 32.0
86.7 55.0
240 195
307 245
281 131
a a
131 65.8
162 104
153 101
18.4 14.4
Not applicable.
Table 3. Metabolite Distribution of Compound 1 at 3 ppb Exposure % TRR in water and tadpole uptake phase
depuration phase
day 1
day 3
day 7
day 15
day 22
day 1
day 2
day 3
76.5 4.0 15.4 NDc 4.2 ND 100.0
75.6 7.8 11.7 ND 4.9 ND 100.0
64.1 5.3 16.9 4.3 9.3 0.1 100.0
63.0 6.1 17.9 1.8 11.3 0.1 100.0
38.2 10.0 30.2 10.0 11.7 0.1 100.0
a a a a a a a
a a a a a a a
a a a a a a a
46.5 3.7 46.9 2.9 ND ND 100.0
38.8 5.7 51.7 3.9 ND ND 100.0
41.3 6.0 48.7 4.0 ND ND 100.0
53.5 3.9 39.7 2.8 ND ND 100.0
60.7 4.1 35.3 ND ND ND 100.0
65.7 3.2 31.0 ND ND ND 100.0
77.7 ND 22.3 ND ND ND 100.0
74.8 ND 25.2 ND ND ND 100.0
water organic layer 1 2 sul. conjugateb of 2 5 others aqueous layer total whole tadpole extractable 1 2 sul. conjugate of 2 sul. conjugate of 5 others unextractable total a
Not applicable. bSul. conjugate = sulfate conjugate cND = not detected.
combined organic fraction was redissolved in 1 mL of methanol and subjected to HPLC and 2D TLC analyses. The unextracted 14C residues in the aqueous layer were also assayed by LSC. The pooled tadpoles at each sampling were homogenized with 11 mL of methanol using an Ace homogenizer (Nippon Seiki Co., Ltd., Niigata, Japan) at 10000 rpm for 15 min on ice. The 14C extract was separated by centrifugation at 2000 rpm for 15 min at 4 °C with a HIMAC CR5B centrifuge (Hitachi Koki Co., Ltd., Tokyo, Japan). The same extraction procedure was further repeated 2 times. The combined supernatant was evaporated and dissolved in 1 mL of methanol for the LSC, HPLC, and 2D TLC assays. The bound 14C residues were dried, and their portion was subjected to the combustion analysis. To identify the aglycone of a conjugated metabolite, the corresponding fraction isolated by 1D TLC was subjected to enzymatic hydrolyses at 37 °C overnight in 3 mL of 10 mM acetic acid buffer at pH 5.0 in the presence of 8 mg of β-glucuronidase or sulfatase. The reaction was quenched by adding 0.3 mL of 1 N HCl and 1.8 g of ammonium sulfate, followed by extracting 3 times with 3 mL of ethyl acetate/ ethanol (5:1, v/v). The combined organic fraction was concentrated and dissolved in 1 mL of ethyl acetate/ethanol (5:1, v/v) for the
HPLC and 2D TLC co-chromatographies with the reference standards. Whole-Body 14C Distribution. Freeze-dried tadpoles embedded in paraffin were sectioned in a horizontal plane using a microtome (RUB-2100, Yamato Kohki Industrial Co., Ltd., Saitama, Japan) until all internal organs appeared on the cutting surface. Then, the cutting surface of the paraffin block was placed on the imaging plate for several days in darkness, and the whole-body autoradiogram was prepared by an Amersham Variable Mode Imager Typhoon 9200. Furthermore, the liver, gastrointestinal tract (gut), heart, kidneys, fore limbs, hind limbs, head, and tail were separately taken to quantify and characterize 14C in each organ obtained from 300 ppb exposed tadpoles. A representative lipid content in each organ was measured with acclimated tadpoles at the NF stage 58 using the modified sulfo-phospho-vanillin (SPV) method21 after extraction with chloroform/methanol. Statistical Analysis. Mean values of body weights and the developmental stage throughout exposure to compound 1 at each concentration were subjected to Student’s t test to evaluate any significant modification from the control group (p < 0.05) using Stat Light (version 2.10, Yukms Co., Ltd., Kanagawa, Japan). 9982
DOI: 10.1021/acs.jafc.7b04184 J. Agric. Food Chem. 2017, 65, 9980−9986
Article
Journal of Agricultural and Food Chemistry Table 4. Metabolite Distribution of Compound 1 at 300 ppb Exposure % TRR in water and tadpole uptake phase
depuration phase
day 1
day 3
day 7
day 15
day 22
day 1
day 2
day 3
81.5 NDb 8.9 ND 9.4 0.2 100.0
73.7 5.4 9.6 3.2 8.2 0.1 100.0
81.3 4.1 5.3 4.2 4.8 0.2 100.0
79.8 3.9 6.0 6.0 4.3 0.1 100.0
46.7 9.5 14.7 16.3 12.9 0.1 100.0
a a a a a a a
a a a a a a a
a a a a a a a
65.2 5.1 14.8 ND 9.1 ND 5.9 ND 100.0
63.4 8.4 12.5 ND 12.1 ND 3.5 ND 100.0
50.3 7.7 12.2 4.7 13.4 4.4 7.2 ND 100.0
64.0 10.2 8.3 6.1 8.3 ND 3.1 ND 100.0
66.0 8.4 11.8 2.7 5.8 ND 5.2 0.1 100.0
78.2 ND 21.8 ND ND ND ND ND 100.0
83.1 ND 16.9 ND ND ND ND 0.1 100.0
99.9 ND ND ND ND ND ND 0.1 100.0
water organic layer 1 2 sul. conjugatec of 2 4 others aqueous layer total whole tadpole extractable 1 2 sul. conjugate of 2 3 sul. conjugate of 3 sul. conjugate of 5 others unextractable total a
Not applicable. bND = not detected. cSul. conjugate = sulfate conjugate.
Calculation of Bioconcentration Factor and Depuration Half-Life. The bioconcentration factor in a steady-state (BCFss) was conveniently calculated by dividing the apparent mean concentration of compound 1 in the tadpoles by that in the exposure water. The tissue concentration (Ct in μg kg−1) of compound 1 at time t in the uptake and depuration phases can be described by the following two equations: uptake phase, Ct/Cw = (ku/kd)[1 − exp(−kdt)]; depuration phase, Ct = Ct,0 exp(−kdt).22 Cw and Ct,0 are the mean water concentrations (μg L−1) during the uptake phase and tissue concentration at the start of the depuration period, respectively. The rate constants in the uptake (ku in L kg−1 day−1) and depuration (kd in day−1) were obtained by the nonlinear fitting of the equations to the experimental data using the software SigmaPlot (version 10.0, SPSS, Inc., Chicago, IL, U.S.A.). The depuration half-life (t1/2 in days) was calculated assuming the first-order kinetics, t1/2 = 0.693/kd, and the kinetic bioconcentration factor (BCFk) was estimated by the ratio of the ku and kd values, BCFk = ku/kd.
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RESULTS Development of Tadpoles. During the uptake phase at the nominal concentrations of 3 and 300 ppb, the exposure concentrations on the 14C basis were kept almost constant, with the mean values of 2.80 and 278 ppb, but the content of compound 1 decreased especially at the end of exposure (day22), as listed in Tables 2−4. Neither >4% of an incidental mortality nor abnormal behavior was observed in both the control and exposure groups throughout the study. The mean developmental NF stage was 57, 57, and 56 for the control and 3 and 300 ppb, respectively, at the end of exposure (Figure 2A). No statistical differences in the mean body weight were observed during the study, except for the 7 and 15 day exposure at 300 ppb (Figure 2B), with more food leftover on the bottom of the aquarium. Therefore, the slightly retarded NF stage with the sporadically decreased body weight was likely to originate from systemic toxic effects with reduction of food consumption. The minimal effects by exposure sufficiently met the criteria
Figure 2. (A) NF stage of tadpoles (n = 6) at termination of the uptake phase and (B) mean body weights (n = 6−20) at each sampling. The error bars represent standard deviation (∗, p < 0.05; ∗∗, p < 0.01).
prescribed in OECD 30522 for the evaluation of metabolism and bioconcentration in aquatic organisms. Bioconcentration of Compound 1 in Tadpoles. The 14 C concentrations in tadpoles gradually increased and almost reached the plateau of 1.36−1.58 ppm after 7 days and 153− 162 ppm after 15 days at the treatment levels of 3 and 300 ppb, respectively (Tables 2−4 and Figure 3). The corresponding 9983
DOI: 10.1021/acs.jafc.7b04184 J. Agric. Food Chem. 2017, 65, 9980−9986
Article
Journal of Agricultural and Food Chemistry
Figure 5. Distribution of compound 1 and its metabolites in each organ at 300 ppb exposure.
gut and liver was 38 and 3.2% of total lipid in the tadpole, respectively, while those in the other organs ranged from 11 to 12%. Metabolite Identification and Distribution. The amounts of compound 1 and its metabolites in tadpoles are summarized in Tables 2−4. The chemical identity of the metabolites was confirmed directly or after enzymatic hydrolysis by HPLC and 2D TLC co-chromatographies with the corresponding authentic standards. All of the conjugates detected in the tadpoles were considered to be the sulfate form, because the release of aglycone was only observed with sulfatase treatment but not with β-glucuronidase treatment. Irrespective of the exposure concentration, major metabolite 2, produced via hydroxylation at the 4′ position of the phenoxyphenyl ring, was mainly detected as the sulfate conjugate at the maximum amount of 51.7% TRR (day 3) and 14.8% TRR (day 1) for 3 and 300 ppb exposure, respectively. The corresponding amounts of its free form were 6.0% TRR (day 7) and 10.2% TRR (day 15). Another major metabolite 3, formed via ether cleavage at the pyridyloxyl moiety, was only detected at 300 ppb exposure, amounting to 13.4% TRR (day 7) as the sulfate conjugate and 6.1% TRR (day 15) as the free form at a maximum. As a minor metabolite, the sulfate conjugate of compound 5 amounted to
