Toxicokinetics of Imidacloprid-Coated Wheat Seeds in Japanese Quail

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Ecotoxicology and Human Environmental Health

Toxicokinetics of imidacloprid-coated wheat seeds in Japanese quail (Coturnix japonica) and an evaluation of hazard Thomas G. Bean, Michael S. Gross, Natalie K. Karouna-Renier, Paula F.P. Henry, Sandra L Schultz, Michelle L Hladik, Kathryn M. Kuivila, and Barnett A. Rattner Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07062 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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1 low dose 10 low doses 1 high dose 10 high doses

Imidacloprid (ng/g liver)

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Environmental Science & Technology

75 50 25 0 0

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Time post-exposure (h)

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Bean, Gross et al. 2018

Toxicokinetics of imidacloprid-coated wheat seeds in Japanese quail (Coturnix japonica) and an evaluation of hazard

1 2 3 4 5

Thomas G. Bean†, Michael S. Gross‡, Natalie K. Karouna-Renier§, Paula F.P. Henry§,

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Sandra L. Schultz§, Michelle L. Hladik‡, Kathryn M. Kuivila║ and Barnett A. Rattner§*

7 8

†Department

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Park, Maryland 20742, United States

of Environmental Science and Technology, University of Maryland, College

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‡U.S.

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United States

12

§U.S.

13

United States

14

║U.S.

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States

Geological Survey California Water Science Center, Sacramento, California 95819,

Geological Survey Patuxent, Wildlife Research Center, Beltsville, Maryland 20705,

Geological Survey Oregon Water Science Center, Portland, Oregon 97201, United

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1 ACS Paragon Plus Environment

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Bean, Gross et al. 2018 17

ABSTRACT: Birds are potentially exposed to neonicotinoid insecticides by ingestion of

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coated seeds during crop planting. Adult male Japanese quail were orally dosed with wheat

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seeds coated with an imidacloprid (IMI) formulation at either 0.9 mg/kg body weight (BW)

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or 2.7 mg/kg BW (~3 and 9% of IMI LD50 for Japanese quail, respectively) for 1 or 10 days.

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Quail were euthanized between 1 and 24 h post-exposure to assess toxicokinetics. Analysis

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revealed rapid absorption (1 h) into blood, and distribution to brain, muscle, kidney and liver.

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Clearance to below detection limits occurred at both dose levels and exposure durations in all

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tissues within 24 h. Metabolism was extensive, with 5-OH-IMI and IMI-olefin detected at

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greater concentrations than IMI in tissues and fecal samples. There was no lethality or overt

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signs of toxicity at either dose level. Furthermore, no evidence of enhanced expression of

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mRNA genes associated with hepatic xenobiotic metabolism, oxidative DNA damage or

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alterations in concentrations of corticosterone and thyroid hormones was observed.

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Application of the toxicokinetic data was used to predict IMI residue levels in liver with

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reasonable results for some field exposure and avian mortality events. It would appear that

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some affected species are either consuming larger quantities of seeds or exhibit differences in

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ADME or sensitivity than predicted by read-across from these data.

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INTRODUCTION

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Neonicotinoids are the most widely applied class of insecticides worldwide.1 Between 2010

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and 2012, 72-100% of neonicotinoids applied to corn, soybean, and wheat were in the form

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of seed treatments.1 Imidacloprid (IMI), clothianidin and thiamethoxam are the most widely

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applied neonicotinoids, constituting nearly all usage in the U.S. in 2011.1 Some reports

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suggest that the concentration of IMI present on a few treated canola seeds or a 10th of an

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IMI-treated corn seed can cause sublethal effects and even lethality in passerines.2-4 Seed

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treatments can be a single neonicotinoid [e.g., IMI (Gaucho®), clothianidin (Poncho®)] or a

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combination of neonicotinoids along with other insecticides and/or fungicides (e.g.,

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Poncho®Plus, Gaucho® XT, Latitude®, Sativa® IMF Max). However, the fungicide

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components appear to pose less of an acute risk to granivorous birds than neonicotinoids

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[acute toxicities exceed limit dose test; >2000 mg/kg body weight (BW) and >5000 mg/kg

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feed].5 Nonetheless, it is possible that the presence of fungicides and inert ingredients in a

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formulation could enhance neonicotinoid toxicity through synergistic interactions or produce

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sublethal effects (i.e., reproductive toxicity).



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Neonicotinoids are highly toxic to many invertebrates, acting as agonists of nicotinic

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acetylcholine receptors (nAChRs).6,7 They bind reversibly (acting as false

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neurotransmitters),6-8 and the over activation of nAChRs can result in sedation, reduced

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locomotion, tremor, seizure, paralysis and eventual death.6-10 Neonicotinoids are more toxic

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to invertebrates than vertebrates due to their greater affinity for insect nAChRs; for example,

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IMI has a 565 times greater affinity for insect nAChRs than for vertebrate nAChRs.7 Despite

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lower affinity for vertebrate nAChRs, controlled exposure studies have demonstrated that

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neonicotinoids adversely affect genomic, cellular, endocrine, immunological, growth,

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reproductive, and neurobehavioral endpoints and can cause lethality in birds.3,10-13 Based on

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its median lethal dose, technical grade IMI is the most potent neonicotinoid seed treatment in

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birds, being classed as highly toxic to grey partridge (Perdix perdix; LD50 = 13.9 mg/kg

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BW), Japanese quail (Coturnix japonica; LD50 = 31 mg/kg BW), feral pigeon (Columba

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livia domestica; LD50 = 25-50 mg/kg BW), house sparrow (Passer domesticus; LD50 = 41

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mg/kg BW) and canary (Serinus canaria domestica; LD50 = 25-50 mg/kg BW).14

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Imidacloprid and other neonicotinoids have occasionally been detected in free-ranging

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birds,15-18 and implicated with varying degrees of certainty in a few bird mortality

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events.15,16,18,19

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Despite evidence that exposure of wild birds to IMI following seed ingestion could

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result in toxicity, toxicokinetic data (absorption, distribution, metabolism, excretion; ADME)

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for IMI (or any neonicotinoid) are lacking for avian species. In laboratory rats (Rattus

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norvegicus), orally administered IMI was found to be extensively (>92%) and rapidly

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absorbed from the gastrointestinal (GI) tract.20 Peak plasma levels occurred after 1.1-2.5 h.

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The principal sites for IMI distribution were liver, kidney, lung, skin and plasma, with very

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low levels found in the brain and testes. Elimination was rapid with a half-life of 3 h.20 Over

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90% of the dose was excreted in fecal samples within 48 h; only 10-16% of the dose was

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excreted as parent compound, with nine metabolites detected (some more potent than parent

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compound).7 Metabolism in mammals involves oxidation to the 5-hydroxy and olefin

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metabolites, and reduction to nitrosoguanidine, aminoguanidine, desnitro-IMI and IMI-urea,

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and phase II glucuronidation.21-23 Some of the IMI metabolites are more potent than the

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parent compound.7 After 48 h, only 0.5% of the dose remained in rats meaning that

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bioaccumulation is unlikely.20 There is interest in drawing upon toxicokinetic data from

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laboratory mammals to use in risk assessments in “read-across” approaches to predict effects

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in free-ranging wildlife.24 However, the absence of toxicokinetic data for birds makes

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interspecific extrapolation (mammal to bird) uncertain. Without at least an estimate of 3 ACS Paragon Plus Environment

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toxicokinetics in birds, it is difficult to i) understand the time course of toxicity, and ii) bridge

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the gap between residues detected in wild birds and threshold values for toxicity.

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To investigate the ADME of IMI in birds, we used Japanese quail as a model species

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for upland game birds that are commonly used in registration studies. Wheat was selected

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over corn and soybean as we found quail could ingest and digest such seeds more easily, and

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there is evidence that birds ranging from small passerines to geese consume wheat seeds.25,26

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The objectives of this study were to describe ADME and some toxicological endpoints (e.g.,

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DNA damage, hepatic gene expression, endocrine responses and overt signs of intoxication)

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in quail orally dosed with neonicotinoid-coated seeds.

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MATERIALS AND METHODS

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Animal Husbandry. Procedures involving Japanese quail were approved by the Institutional

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Animal Care and Use Committee of the U.S. Geological Survey Patuxent Wildlife Research

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Center (USGS-PWRC). Quail were raised from a colony at USGS-PWRC. Adult male quail

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were housed indoors and kept on a 16 h light: 8 h dark photoperiod, with ambient

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temperature ranging from 20-25°C. They were housed individually (22.8 cm wide×33.0 cm

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high×38.1 cm deep cages, Wahmann Manufacturing Co., Baltimore, MD), fed Purina Layena

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complete ration game bird diet (20% protein; St. Louis, MO) ad libitum, and were given free

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access to water during a 2 week acclimation period. Feed was removed 2 h prior to dosing

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and euthanasia.

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Soybeans, Wheat Seeds and Pesticide Coating. Untreated soybeans were obtained from the

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Central Maryland Research and Education Center (Laurel, MD) and wheat seeds (untreated

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and treated) were obtained from Kansas Foundation Seed Project (Department of Agronomy,

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Kansas State University). Wheat seeds (32.8±5.2 mg/seed) coated with Sativa® IMF Max

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(formulation containing 120 g IMI/L, 6.4 g metalaxyl/L, 3.84 g/L and 4.8 g tebuconazole/L;

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Nufarm Americas Inc.; EPA Reg. No. 55146-119) were obtained commercially. Sativa®

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IMF Max (hereafter SIMF) wheat was analytically verified (see SI for details) and found to

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contain (mean±SD) IMI at 9.04±3.72 µg/seed (~0.276 mg/g wheat seed). This IMI

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concentration detected on the wheat is within the range that would result from the SIMF

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application described on the product label (i.e., 100.5-147.9 mL SIMF per 45.36 kg wheat;

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8.7-12.8 µg IMI/seed). Inter-seed variability was high, but variability in dose received by

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quail would be offset by numerous seeds in each administered dose. The fungicide residues



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were quantified, and are the subject of a subsequent publication; the kinetics and toxicity of

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IMI are the focus of this paper.

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Design of Experiments. The study consisted of three phases. A preliminary radiographic

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trial sought to determine how many seeds would fill a quail’s crop and gizzard and how long

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it took to digest and pass through the gastrointestinal (GI) tract. This exposure scenario was

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designed to mimic a bird gorging itself on spilled seeds. A range finding trial aimed to

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determine appropriate post-exposure sampling times by examining elimination of IMI from

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plasma and liver. Finally, in a definitive oral toxicity trial quail received gelatin capsules

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containing untreated wheat seeds (sham-dosed), 10 pesticide-coated seeds or 30 pesticide-

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coated seeds for 1 or 10 d. Gelatin capsules containing wheat seeds were administered to the

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level of the crop using a modified pet pilling device (Jorgensen Laboratories, Loveland, CO).

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This method of toxicant administration ensures accurate dose and overcomes variable food

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intake rate, food aversion and spillage that accompany ad libitum dietary exposure.

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Radiographic Trial. Soybeans were prepared by pipetting barium sulfate (E-Z-Paque,

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Bracco Diagnostics, Inc., Monroe Twp., NJ) into a 0.3175 mm hole drilled into the bean that

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was then sealed using dental filament (Flowable Composite, Henry Schein, Melville, NY).

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Barium sulfate was also pipetted onto wheat seeds and allowed to air dry. Birds were

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provided food and grit only during daylight hours. Three adult male quail were orally dosed

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(1-4 soybeans) and re-dosed a day later (4-8 seeds). Soybean passage and digestion within the

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quail GI tract were observed by taking radiographic images (VetTek, Universal Imaging

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Radiograph, Bedford Hills, NY; settings 3.3 mA, 80 kVp) at 5-30 min intervals (initially

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short intervals extending to 30 min after 3 h) for a total of 6.75 h. After 48 h, birds were then

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re-dosed with barium sulfate-coated wheat seeds. Quail received 1, 2 or 3 capsules (10

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seeds/size 2 gelatin capsule; Eli Lilly, IN) and a series of radiographic images were taken

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initially at 5 min intervals for 30 min, and then at 15 min intervals for 3 h.

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Range Finding Trial. To determine the post-exposure time points for euthanizing birds in

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the principal study, we conducted a range finding study using 9 sham-dosed quail (controls)

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and 9 quail dosed with SIMF. The controls received 30 untreated wheat seeds per dose (3

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capsules each containing 10 untreated seeds) and the SIMF-treated birds received 30 treated

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wheat seeds per dose (~2.7 mg IMI/kg BW). A single bird from each group was euthanized 1,

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2, 4, 8, 24 and 48 h. The remaining 6 birds were re-dosed on days 2 and 3, and an individual

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bird from each group was euthanized 1, 4 and 24 h after the final dose. Samples of plasma 5 ACS Paragon Plus Environment

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and liver were collected and frozen (-20°C), and residues of parent compound active

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ingredient were quantified by liquid chromatography tandem mass spectrometry

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(LC/MS/MS).

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Oral Toxicity Trial. Based upon findings of the radiographic and range finding trials, quail

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were orally gavaged with 3 gelatin capsules each containing 10 wheat seeds for 1 d or daily

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for 10 d (Table 1). Sham-dosed controls (N=15) received 30 untreated wheat seeds per dose,

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the low dose group (N=30) received 20 untreated seeds and 10 SIMF-treated seeds per dose

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(0.904 mg IMI/kg BW; i.e., [10 IMI-coated seeds x (0.00904 mg IMI/seed)]/0.1 kg quail),

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and the high dose group (N=31) received 30 SIMF-treated wheat seeds per dose (2.712 mg

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IMI/kg BW). The birds that were dosed once (hereafter “1 dose birds”) were euthanized 1, 2,

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4 and 24 h post-exposure. The birds that were dosed for 10 consecutive days (hereafter “10

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dose birds”) were euthanized at 1, 4 and 24 h after the final dose. These dose levels and their

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frequencies reflect a range of potential environmental exposures (e.g., one instance of

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ingesting a few seeds to ingestion of many seeds on consecutive days). In addition, 3

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“unmanipulated” controls (not gavaged) were sacrificed at the 10 d time point. Toxicological

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endpoints assessed included gene expression in liver, oxidative DNA damage in liver, brain

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and whole blood, corticosterone in plasma, and thyroid hormones in plasma and gland.

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The daily doses administered to the low and high dose groups were approximately 3

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and 9% of the estimated IMI LD50 in Japanese quail.14 The toxicity of the three fungicides

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contained in the SIMF formulation is very low for birds and would take hundreds of

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thousands to millions of treated wheat seeds to cause lethality (metalaxyl) or even reach the

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avian limit dose (fluodioxinil, tebuconazole).



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Bean, Gross et al. 2018

Table 1: Number of birds euthanized at each time point

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Day

Time euthanized after final dose

Unmanipulated Control

Sham-dosed Control

Low dose

High dose

1h

5

5

6

2h

1

4

4

4h

1

4

4

24 h

1

4

4

1h

5

5

5

4h

1

4

4

24 h

1

4

4

15

30

31

1

10

Total

0h

3

3

170 171

Collection of Tissues and Fecal Samples. For the range finding and oral toxicity trials, birds

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were euthanized by decapitation, and whole blood was collected into lithium heparin

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Monovette tubes (Sarstedt, Newton, NC). For the subsets of birds euthanized 1 h after dosing

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and those repetitively dosed for 10 d, samples of whole blood (~50 µL), liver (>30 mg of

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right lobe) and brain (portion of mid-brain) were placed in cryovials, snap frozen in dry ice-

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ethanol, and stored at -80°C for assessment of oxidative DNA damage. For all birds,

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remaining whole blood was centrifuged (10 min 1500 x g), and aliquots of plasma were

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transferred into cryovials and stored frozen at -80°C for residue analyses (IMI; metabolites

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measured in only definitive oral toxicity trial), thyroid hormones and corticosterone.

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For evaluation of hepatic gene expression associated with metabolism of

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neonicotinoids, ~ 100 mg portions of the right lobe of the liver were placed in tubes

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containing 1 mL of RNAlater (Qiagen, Valencia, CA), stored at 4°C for 24-96 h, and then

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kept at -20°C until analysis. The remaining liver, kidney and a 2-3 g portion of pectoral

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muscle were placed in cryovials, and the remainder of the brain was placed in a chemically

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clean 20 mL amber glass jar, and frozen for neonicotinoid residue analyses.

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Cloacal droppings (fecal matter including urates, hereafter “fecal samples”) produced

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in the 24 h period after dosing from 9 single-dosed birds were collected for residue analysis.

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Fecal samples were separated from spilled feed using cotton swab, placed in 1.5 mL tubes 7 ACS Paragon Plus Environment

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and stored at -80°C. Samples collected for residue determination were shipped frozen to

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USGS California Water Science Center, where they were stored at -20°C prior to analysis.

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Analytical Methods for IMI and Metabolite Residues in Seeds, Tissues and Fecal

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Samples. A detailed description of the analytical methods can be found in the SI. In short,

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treated and untreated wheat seeds were extracted via sonication with methanol. An aliquot

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was removed for analysis of metabolites (imidacloprid-olefin, imidacloprid urea, and 5-OH-

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imidacloprid) and a second aliquot was diluted tenfold for IMI analysis. Tissue samples were

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homogenized with sodium sulfate. Tissue masses varied from approximately 0.15 g for brain

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and kidney to 0.25 g for liver and muscle. Samples were fortified with d4-IMI as a recovery

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surrogate and extracted with 50/50 (v/v) acetone/dichloromethane via pressurized liquid

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extraction at 1500 psi and 100 °C. Following extraction, samples were protein precipitated in

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acetonitrile and cleaned-up via pass-through solid phase extraction (SPE) using Oasis®

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PRiME HLB cartridges (Waters, Milford, MA). Plasma samples (100 µL) were spiked with

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d4-IMI prior to extraction and clean-up. Samples were protein precipitated with acetonitrile

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and pass-through SPE was performed for clean-up. Fecal samples were homogenized and

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approximately 0.25 g was weighed for extraction. Samples were spiked with d4-IMI as a

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surrogate and extracted by sonication with acetonitrile. Fecal samples were cleaned-up via

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pass-through SPE. All samples were fortified with d3-thiamethoxam as an internal standard

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and analyzed via LC/MS/MS. All values are expressed on a wet weight (ww) basis.

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Gene Expression. There are very limited data on IMI metabolism in birds,27 with

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predominant metabolites seemingly similar to those identified in rodents.20 We targeted genes

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based on metabolism of nicotine and neonicotinoids in mammals, and examined expression

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of 5 cytochrome P450 (CYP450) genes (1A4, 1A5, 2H1, 3A7, 3A12) and one aldehyde

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oxidase gene in liver (AOX1).21

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RNA extraction and reverse transcription. RNA was extracted from liver using RNAzol RT

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(Molecular Research Center, Cincinnati, OH) following the manufacturer’s protocol with

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minor modifications. Tissue was homogenized using stainless steel beads with a Bullet

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Blender (Next Advance, Troy, NY). Residual DNA, protein and polysaccharides were

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removed and RNA precipitated as a pellet using isopropanol. The concentration and purity of

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RNA were quantified using UV absorbance (260 nm; NanoDrop 8000, Thermo Fisher

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Scientific, Wilmington, DE). Samples with low yield (

Toxicokinetics of Imidacloprid-Coated Wheat Seeds in Japanese Quail

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