Blood Pharmacokinetic of 17 Common Pesticides in Mixture Following a Single Oral Exposure in Rats: Implications for Human Biomonitoring and Exposure Assessment

Archives of Toxicology (2019) 93:2849–2862 https://doi.org/10.1007/s00204-019-02546-y

TOXICOGENOMICS

Blood pharmacokinetic of 17 common pesticides in mixture following a single oral exposure in rats: implications for human biomonitoring and exposure assessment

Caroline Chata1,2 · Paul Palazzi1 · Nathalie Grova1 · Serge Haan3 · Claude Emond1,4 · Michel Vaillant5 · Brice M. R. Appenzeller1

Received: 3 May 2019 / Accepted: 14 August 2019 / Published online: 19 August 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Human biomonitoring provides information about chemicals measured in biological matrices, but their interpretation remains uncertain because of pharmacokinetic (PK) interactions. This study examined the PKs in blood from Long–Evans rats after a single oral dose of 0.4 mg/kg bw of each pesticide via a mixture of the 17 pesticides most frequently measured in humans. These pesticides are β-endosulfan; β-hexachlorocyclohexane [β-HCH]; γ-hexachlorocyclohexane [γ-HCH]; carbofuran; chlorpyrifos; cyhalothrin; cypermethrin; diazinon; dieldrin; difufenican; fpronil; oxadiazon; pentachlorophenol [PCP]; permethrin; 1,1-dichloro-2,2bis(4-chlorophenyl)ethylene [p,p′-DDE]; 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane [p,p′- DDT]; and trifuralin. We collected blood at 10 min to 48-h timepoints in addition to one sample before gavage (for a control). We used GS–MS/MS to measure the pesticide (parents and major metabolites) concentrations in plasma, determined the PK parameters from 20 sampling timepoints, and analyzed the food, litter, and cardboard in the rats’ environment for pesticides. We detected many parents and metabolites pesticides in plasma control (e.g., diethyl phosphate [DEP]; PCP; 3-phenoxybenzoic acid [3-PBA]; 3,5,6-trichloro-2-pyridinol [TCPy], suggesting pre-exposure contamination. The PK values post-exposure showed that the AUC0−∞ and Cmax were highest for TCPy and PCP; β-endosulfan, permethrin, and trifuralin presented the lowest values. Terminal T1/2 and MRT for γ-HCH and β-HCH ranged from 74.5 h to 117.1 h; carbofuran phenol presented the shortest values with 4.3 h and 4.8 h. These results present the frst PK values obtained through a realistic pattern applied to a mixture of 17 pesticides to assess exposure. This study also highlights the issues of background exposure and the need to work with a relevant mixture found in human matrices.

Keywords Background exposure · Mixture · Pesticides · Pharmacokinetic · Rat

Abbreviations ATSDR Agency for Toxic Substances and Disease 3-PBA 3-Phenoxybenzoic acid Registry ADME Absorption, distribution, metabolism, and AUC0−∞ Area under the curve excretion Cmax Maximum concentration in plasma Cl2CA Cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid Electronic supplementary material The online version of this ClCF3CA 3-(2-Chloro-3,3,3-trifuoro-1-propenyl)- article (https://doi.org/10.1007/s00204-019-02546-y) contains 2,2-dimethylcyclopropanecarboxylic acid supplementary material, which is available to authorized users.

* Claude Emond 3 Life Sciences Research Unit, University of Luxembourg, 6 [email protected] Avenue du Swing, Belvaux, Luxembourg 4 PhysioKinetic Simulations to Human Inc. (PKSH Inc), 1 Human Biomonitoring Research Unit, Luxembourg Institute Mascouche, QC J7K 0M6, Canada of Health (LIH), Rue Henri Koch 29, 4354 Esch‑sur‑Alzette, Luxembourg 5 Competence Center for Methodology and Statistics, Luxembourg Institute of Health (LIH), Rue Thomas Edison 2 Faculty of Science, Technology and Communication, 1A‑B, 1445 Luxembourg, Luxembourg University of Luxembourg, 2 Avenue de l’Université, 4365 Esch‑sur‑Alzette, Luxembourg

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DEP Diethyl phosphate in Europe from 2010 through 2014 (EFSA 2017). The DETP Diethyl thiophosphate European Food Safety Authority (EFSA) analyzed more ED30 Efective dose than 78,000 samples from 750 diferent food products and EDTA Ethylenediaminetetraacetic acid detected 800 diferent pesticides in them (EFSA 2017). EFSA European Food Safety Authority Human exposure to pesticides can occur through dif- FNR Luxembourg National Research Fund ferent pathways (e.g., ingestion of food, water, or dust; (Fonds National de la Recherche) inhalation of particles, vapors, or gases; through dermal GC Gas chromatography contact). Nevertheless, ingestion is the major route of GC–MS/MS Gas chromatography with tandem mass exposure for the general population (Curl et al. 2015; Shel- spectrometry don and Berry 1999). Researchers have discussed the pos- HCH Hexachlorocyclohexane (gamma [γ], beta sible association between exposures to pesticides and other [β]) environmental contaminants and the development of meta- LD50 Lethal dose 50% bolic disorders, diferent types of cancer, or several human MRT Mean residence time diseases, including Parkinson’s disease (Bastias-Candia PAH Polycyclic aromatic hydrocarbon et al. 2019; Hernandez et al. 2013; Jaga and Dharmani PCP Pentachlorophenol 2005; Lebov et al. 2015; Saeed et al. 2017; Storm et al. PK Pharmacokinetics 2000; van der Mark et al. 2014). For exogenous chemicals p,p′-DDD 1,1-Dichloro-2,2-bis(p-chlorophenyl) such as pesticides, the physico-chemical properties and ethane the tissue interactions drive the pharmacokinetics (PKs), p,p′-DDE 1,1-Dichloro-2,2bis(4-chlorophenyl) resulting in short or long half-lives in humans, which ethylene could subsequently cause health efects. p,p′-DDT 1,1,1-Trichloro-2,2-bis(p-chlorophenyl) Biomonitoring is the measurement of contaminants ethane (parents and metabolites) in conventional biological SPME Solid-phase microextraction matrices such as blood or urine (NRC/NAS 2006) or in T1/2 Terminal elimination half-life non-conventional matrices such as hair (Beranger et al. TCPy 3,5,6-Trichloro-2-pyridinol 2018; Palazzi et al. 2018). Biomonitoring integrates all Tmax Time to reach the Cmax exposure routes, thereby providing important and useful WHO World Health Organization information; it is frequently used during studies involving environmental epidemiology; and it provides aggregate information about multi-residues. Many published stud- Introduction ies have discussed the use of diferent human matrices (e.g., urine, blood, hair) for evaluating chemical concen- Pesticides are widely used during crop production to con- trations. For example, results from a Canadian survey, trol insects, weeds, or other organisms. According to the which evaluated urine and blood samples collected from World Health Organization (WHO), there are more than 2007 to 2011 by Health Canada, showed that parents and 1000 pesticide formulations used around the world to ensure metabolites of organochlorines, organophosphates, pyre- that pests do not damage or destroy food (WHO 2018). The throids, and carbamates were detected and ranged from widespread use of pesticides for agricultural activities rep- 56% to 99% (Haines et al. 2017). During a European bio- resents thousands of molecules with a large range of phys- monitoring study, researchers detected urinary metabolites ico-chemical properties, and each pesticide or family has of organophosphates ranging from 61 to 100% and pyre- diferent properties and toxicological efects (WHO 2018). throids ranging from 60 to 98% in 600 German children Even though the European Union developed an extensive (Schulz et al. 2009). Another European biomonitoring methodology of assessment that covers the chemical, bio- study, researchers detected urinary metabolites of organo- logical, toxicological, and environmental behaviors of the phosphates ranging from 0 to 28% and pyrethroids ranging active ingredients before a pesticide is approved for use on from 5 to 100% in 1077 French women who were preg- the market (Tsaboula et al. 2016), we are still exposed to a nant (Dereumeaux et al. 2016). A study was conducted in mixture of pesticides through our diet and our environment. Luxembourg on 14 volunteers who provided hair samples, According to the literature, 912 million of pounds of which, when analyzed, revealed exposure to 11 diferent common popular pesticides are used annually in the United pesticides (Salquebre et al. 2012). More recently, a study States (Barr 2008). During agricultural activities, manage- of 101 volunteers living in Burkina Faso, West Africa, ment and crop production are rely on the use of herbicides, detected organochlorines (64%), pyrethroids (52%), and fungicides, and insecticides, amounting to approximately organophosphate (18%) pesticides in their hair samples 3.3 × 106 t/year worldwide, or specifcally 4.2 × 105 t/year (Lehmann et al. 2018).

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As previously mentioned, human biomonitoring provides pesticides for the mixture mainly based on their prevalence information about many contaminants (multi-residues) in several human biomonitoring studies reviewed (Appen- measured in biological matrices, but their interpretation zeller and Tsatsakis 2012; Salquebre et al. 2012). remains uncertain regarding the PKs. In addition, only a Sixty healthy Long–Evans rats (females aged 12 weeks few experimental studies have described the PK parame- and weighing between 180 and 200 g each) were purchased ters for a mixture exposure scenario to low or background from Janvier Labs (Saint-Berthevin, France). Before the human exposure doses, and then calculated the PK param- study, the rats were acclimated for least 1 week to the facil- eters individually. To quantitatively measure the impacts of ity, which was maintained on a 12-h light and 12-h dark these interactions, a study focusing on rodent exposure is cycle, at an ambient temperature of 22 ± 2 °C, and a relative an excellent approach because the exposure doses and time humidity of 40 ± 5%. The rats were housed three per cage points of measurement are controlled. as set forth in the 2010/63/EU directive regarding the pro- There were two objectives of this current study. The frst tection of animals used for scientifc purposes. Each cage objective was to study the PKs of 17 pesticides in a mixture contained litter, as well as cardboard for nesting (both prod- and their major metabolites following a single oral dose of ucts from Tecnilab-BMI, Someren, the Netherlands). Feed the exposure via gavage in female Long–Evans rats. The (Tecnilab-BMI, Someren, The Netherlands) and water were second objective was to assess the concentration changes available to the rats ad libitum. in blood to provide a biomonitoring perspective. We measured the parents and major metabolites that corresponded Preparation of solutions in gel pellets to the most frequent chemicals investigated during human biomonitoring studies and at the same order of magnitude. The exposure dose was prepared in a solution of 0.4 mg/kg This study helped improve our knowledge of PKs of some bw of each chemical in the mixture. This dose was estab- specifc chemicals in a mixture. For this purpose, we col- lished by considering the 1/20 of the LD (rat, 5 mg/kg, lected sequential blood samples over 48-h post-exposure 50 oral) of carbofuran, which is considered to be the most toxic period. We used gas chromatography with tandem mass compound in the mixture (Gupta 1994). The dose that was spectrometry (GC–MS/MS) for analysis, and then deter- selected allowed detectable concentrations in the plasma mined the PK parameters for parents and major metabolites to be obtained and is representative of human background measured in blood. exposure level, as demonstrated during previous research to compare γ-HCH (lindane) and pyrethroid metabolite Materials and methods concentrations in hair and urine between rats and humans (Appenzeller et al. 2017a). Chemicals Each chemical was prepared in ethanol at 2 g of chemical per liter. Gels were prepared by following the procedure discussed by Appenzeller et al. (2017). Briefy, hot HydroGel The mixture of 17 pesticides was purchased from Sigma- and MediGel sucralose (Bio Services BV, Uden, Nether- Aldrich (Diegem, Belgium). The 17 pesticides were lands) were mixed at a 1:1 ratio (v/v), poured into aluminum β-endosulfan, β-hexachlorocyclohexane (HCH), γ-HCH, car- molds, allowed to cool at room temperature (approximately bofuran, chlorpyrifos, cyhalothrin, cypermethrin, diazinon, 25 °C), and supplemented with the appropriate volume of dieldrin, difufenican, fpronil, oxadiazon, pentachlorophenol stock solutions of pesticides (Appenzeller et al. 2017b). Eth- (PCP), permethrin, 1,1-dichloro-2,2bis(4-chlorophenyl) ethanol was evaporated at room temperature (approximately ylene (p,p’-DDE), 1,1,1-trichloro-2,2-bis(p-chlorophenyl) 25 °C) for at least 4 h, and a second layer of gel was depos- ethane (p,p’-DDT), and trifuralin. ited to trap the pesticides inside the gel. Animals and treatment Treatment of animals during exposure This current study was approved by the Ministry of Agri- culture, Viticulture, and Consumer Protection of Luxem- Before the current experiment, the rats were acclimated for bourg and conformed to the national and European directives 1 week. On the day of the experiment, each rat was weighed, regarding the care and treatment of animals in a laboratory. and the weights were logged. The rats were exposed by gav- During the current study, we used the Long–Evans strain age (via a cannula) with a single dose of 0.4 mg/kg bw of of rat, because previous studies employed this same strain, each pesticide in a mixture dissolved in 1 mL of a low-cal- thereby facilitating comparison (Appenzeller et al. 2017a; orie gel. The assumption was that metabolism interactions Chata et al. 2016). The gender (i.e., females) and strain were negligible at that level of dose; therefore, the PKs of were selected because of their sociability. We selected the chemicals in plasma are not infuenced.

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The protocol was optimized to reduce the number of rats Analysis of pesticide concentrations in plasma and to obtain enough time points and replicates for each pesticide in the mixture (Fig. 1). The analysis of plasma was performed by following the pro- To allow for fve blood samplings, each rat was admin- cedures described previously by Chata et al. (2016). Briefy, istered isoflurane anesthesia, and then, a catheter was for the parents’ pesticides, the plasma was supplemented inserted into each rat’s lateral tail vein and flled with a with the internal standard (Dr. Ehrenstorfer, Cambridge Iso- heparin:sodium chloride 0.9% solution (1:10 ratio) to inhibit tope Laboratories, Inc., Tewksburry, MA) and cold methanol coagulation. The frst blood sampling (henceforth referred to (− 20 °C). After adding 1 M of a phosphate bufer at pH, as the internal control blood sample) was conducted during two successive liquid–liquid extractions were achieved with the catheterization and was used to determine the presence a 1:1:1 ratio (v/v/v) of an acetonitrile:cyclohexane:ethyl ace- and concentrations of pesticides before exposure, which tate mixture. The organic phases were evaporated to dryness, constituted for each rat their own control. To allow the rats the residue was reconstituted in acetonitrile, and then, 1 M time to recover and to limit the efects from the isofurane of a phosphate bufer at pH 7 was added. The sample was anesthesia, the rats were orally administered the pesticide analyzed by using direct-immersion solid-phase microex- doses 1 h after the catheters were implemented. traction (SPME; fber exposure at 60 °C for 80 min) followed Each rat was randomly enrolled in a time sampling sched- by desorption in the gas chromatography (GC) injector at ule, with the intent to assess 20 samples over time points 260 °C for 10 min before GC–MS/MS analysis. spanning 48 h, including 12 rats for each time point (Fig. 1). For the metabolites, plasma was supplemented with the For each rat, a total of fve blood samples (i.e., 4 × 300 µL internal standard, hydrolyzed with concentrated hydrochlo- with the catheter plus one at sacrifce) were collected in ric acid (32%) in water, and then incubated at 90 °C for 1 h. tubes spray rinsed with ethylenediaminetetraacetic acid Sodium chloride was added to the sample, and then, two suc- (EDTA). Each tube was centrifuged to retain the plasma, cessive liquid–liquid extractions were achieved with a 1:1:1 and then was stored at − 80 °C until analysis. ratio (v/v/v) of acetonitrile:cyclohexane:ethyl acetate. After The assumption was that the measured concentration in centrifugation, the two organic phases were evaporated to plasma before the exposure was due to the background expo- dryness. The extract was derivatized with a 1:3 ratio (v/v) of sure of the chemicals before treatment. The entire physical pentafuorobenzyl bromide:acetonitrile, and then incubated environments for the rats (i.e., feed, litter, and cardboard for at 80 °C for 30 min. The extract was evaporated to dryness. nesting) were also controlled for the presence of the pesti- Finally, the residue was reconstituted with ethyl acetate and cides selected in the mixture. injected into GC injector (Chata et al. 2016).

Fig. 1 Experimental design to collect the samples after administering the single oral exposure via gavage of 17 pesticides in a mixture for rats

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Chromatography was performed using an Agilent 7890 We plotted PK profle curves for 22 biomarkers measured GC system equipped with an HP-5MS capillary column in rats’ plasma (i.e., 14 out of the 17 parent pesticides were (30 m, 0.25-mm inner diameter, 0.25-μm flm thickness) used in the exposure mixture and 8 of their metabolites). We coupled to an Agilent CTC PAL autosampler and to an Agi- included chlorpyrifos and diazinon as part of the pesticide lent 7000 A triple quadrupole mass spectrometer operating mixture, but did not detect either in the blood samples. We in negative chemical ionization mode with methane as the did not analyze carbofuran for technical reasons. The break- chemical reagent gas. The mass spectrometry parameters down of the 22 biomarkers is as follows: seven organochlo- used for the current study were detailed by Chata et al. rines, three organophosphates, six pyrethroids (i.e., three (2016). parents and three metabolites), and six from other pesticides (i.e., fve parents, including fpronil and its metabolite, and Calculation of pharmacokinetic parameters fpronil sulfone). Twenty out of the 22 biomarkers presented a PK profle describing phases such as Absorption, Distri- PK Solutions™ software package (version 2.0.6), a non- bution, Metabolism, and Excretion (ADME) (Fig. 2a–v). compartmental model (Summit, Ashland, OH, USA), was Because the PK of the early time points of each biomarker used to calculate the PK parameters. The PK parameters were relatively similar, a small graph presenting the frst were area under the curve (AUC0−∞ ), mean residence time 10 h of the time point sequence was inserted into a graph for (MRT), terminal elimination half-life (T1/2), maximum con- each kinetics profle, for a better visibility of the PK profle. centration (Cmax), and time to reach the Cmax (Tmax). The PK The analysis of PK profles of biomarkers showed that parameters were calculated or estimated from the plasma carbofuran phenol presented the fastest decrease in the concentration with time curves. A total of 12 replicates (cor- plasma concentration. However, the absorption phase was responding to 12 rats) were measured for each time point, not visible, even after the post-exposure time point of and the mean concentration was used to establish the PK 10 min, which suggests a very fast absorption rate (Fig. 2v). time curve (Fig. 1). In contrast, fpronil sulfone (Fig. 2r), which is a metabolite of fpronil, showed only an increased concentration over the 48-h time point, making it impossible to determine the PK Results elimination constant. Based on the standard deviation, carbofuran phenol also had the highest inter-animal variability, Animals’ treatment which can be explained by its short MRT and half-life (T1/2). This short half-life suggests that carbofuran phenol was Each Long–Evans female rat was weighed the day of the already in the elimination phase at the time point of 10-min study. The mean ± standard deviation was 206 ± 6 g. During post-exposure. In contrast, PCP had the lowest inter-animal the exposure and the sequential sampling, we monitored the variability over the 48-h time point sequence. rats for distress due to the exposure dose, but no problems The highest AUC0−∞ values calculated were for the were observed. We conducted plasma sampling of the rats metabolite of chlorpyrifos the TCPy (103,655.7 pmol h/ for total of 20 time points per pesticide, corresponding to 5 ml), and for the parent PCP (83,188.8 pmol h/ml). In con- time points per rat [which includes the pre-exposure sample trast, the lowest AUC0−∞ values calculated were for parents (i.e., the internal control blood sample)]. Although 60 rats β-endosulfan (26.7 pmol h/ml), permethrin (38.2 pmol.h/ were purchased for study, we quantifed the internal control ml), and trifuralin (29.0 pmol h/ml) (Table 1). blood sample for 59 rats, because one sample had insuf- Regarding organochlorine, we observed three types fcient plasma volume. Then, we subtracted the concentra- of chemicals based on their Cmax values. The frst type of tion of each pesticide for each time point from the control chemical had a low absorption rate with the Cmax less than concentration (sample before gavage); therefore, each rat had 50 pmol/ml. The second type had a fast absorption rate with its own control for the 17 pesticides of the mixture orally a Cmax greater than 100 pmol/ml. The last type of chemical administered via gavage. had a high Cmax greater than 1000 pmol\ml. The PK profle shows that most chemicals under study reached the Cmax at Pharmacokinetic analysis or before the 2-h time point. The pyrethroids parent compounds demonstrated rela- During the current study, we investigated eight families of tively low absorption rates but fast metabolization rates pesticides because they are routinely found in biomonitor- based on the formations of 3-phenoxybenzoic acid (3-PBA), ing study as parents or metabolites. The eight families of 3-(2-chloro-3,3,3-trifuoro-1-propenyl)-2,2-dimethylcyclo- pesticides are as follows: anilines, carbamates, carboxam- propanecarboxylic acid (ClCF 3CA), and cis-/trans-3-(2,2- ides, organochlorines, organophosphates, oxadiazines, phe- dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid nylpyrazoles, and pyrethroids. (Cl2CA) concentrations in plasma, which ranged from 100

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Fig. 2 Pharmacokinetic profles of the diferent pesticides (mean ± standard deviation of the 12 replicated concentrations per time point). For each compound, the inset graphs present the concentration in semi-logarithmic scale over the frst 10 h, only for better visibility to 1000 pmol/ml (Fig. 2n through p). The higher concentra- metabolites (i.e., one from the fpronil parent and the other tion in 3-PBA can be explained by the fact that this metabo- from carbofuran parent) were measured in plasma (Fig. 2q lite comes from three parent pesticides: cyhalothrin, cyper- through v). The plasma concentration of carbofuran phenol methrin, and permethrin. In the group called Others, four was very high, suggesting a rapid conversion of carbofuran parent compound pesticides from diferent families and two into carbofuran phenol, a hydrolyzed form of carbofuran not

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Fig. 2 (continued)

detected in this study. In addition, carbofuran phenol was b; Table 1). Carbofuran phenol presented the shortest T1/2 quickly removed from plasma (Fig. 2v). (with 4.3 h) and MRT (with 4.8 h) (Fig. 2v; Table 1). The We observed the longest T1/2 and MRT for two chemi- PK parameters obtained using the PK Solutions software cals: γ-HCH and β-HCH. The longest T1/2 for γ-HCH was package were also confrmed by employing the PK Solver, 74.5 h and for β-HCH was 79.7 h. The longest MRT for which is another type of PK software (Zhang et al. 2010). γ-HCH was 109.6 h and for β-HCH was 117.1 h (Fig. 2a,

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Fig. 2 (continued)

We compared four pairs of parents and metabolites that ClCF3CA increased progressively with the increase (Fig. 3). The pair of fpronil parent from the phenylpyra- of cyhalothrin after oral absorption until the 7-h post- zoles family and the metabolite, fpronil sulfone, showed a exposure time point, and then followed the elimination variation in time regarding the concentrations between the profle of the parent. At the 13-h post-exposure time point, parent and the metabolite (Fig. 3a). Specifcally, fpronil the concentration of cyhalothrin became lower than the sulfone increased with time until the 32-h post-exposure ClCF 3CA. We also observed two other instances in which time point, and then, the concentration decreased. In several parent pesticides metabolized the same metabolite. contrast, fpronil decreased with the increase of fpronil The frst instance involved cypermethrin and permethrin, sulfone, with an interlace at approximately the 17-h post- with both metabolized into Cl2CA (Fig. 3c). The second exposure time point. This observation suggests that the instance involved cyhalothrin, cypermethrin, and perme- metabolite formation rate for the conversion of fpronil thrin, with all three metabolized into 3-BPA (Fig. 3d). into fpronil sulfone is lower than the absorption rate of Because Cl 2CA and 3-PBA metabolites come from several fpronil after oral exposure of the mixture. Three other parent pesticides, the metabolite concentration observed comparisons showed an association between parents and was much higher than the individual parents, which tends metabolites in the pyrethroids family. Cyhalothrin has to make the analysis and the interpretation more difcult one major metabolite: ClCF 3CA (Fig. 3b). We observed (Fig. 3c, d).

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Table 1 Pharmacokinetic Chemicals AUC (pmol h/ml) C (pmol/ml) T (h) T (h) MRT (h) parameters calculated during 0−∞ max max 1/2 the current study Organochlorines β-Endosulfan 26.7 4.2 0.8 10.0 18.0 β-HCH 1861.2 23.7 0.8 80.0 117.0 γ-HCH 1388.5 21.1 0.8 75.0 110.0 Dieldrin 2084.8 47.2 2.0 13.0 66.0 PCP 8388.8 3395.1 4.0 12.7 19.0 p,p′-DDE 1972.6 84.9 2.0 20.0 31.0 p,p′-DDT 1221.8 72.9 2.0 15.0 23.0 Organophosphates DEP 1466.5 91.0 2.0 11.0 17.0 DETP 335.3 68.3 0.8 4.0 8.0 TCPy 103655.7 10986.3 5.0 6.0 9.0 Pyrethroids λ-Cyhalothrin 1750.1 137.0 2.0 7.0 10.0

Cl2CA 4407.1 238.0 7.0 8.0 14.0

ClCF3CA 2344.0 103.2 7.0 11.0 19.0 Cypermethrin 103.2 10.4 1.5 7.0 10.0 3-PBA 9870.7 727.4 4.0 7.0 10.0 Permethrin 38.2 2.4 1.5 9.0 16.0 Others Carbofuran phenol 348.7 380.6 0.2 4.3 4.8 Difufenican 190.1 15.6 0.8 12.4 17.0 Fipronil 1264.0 52.7 0.8 14.4 21.8 Fipronil sulfone – 41.9 28.0 – – Oxadiazon 772.0 95.0 0.8 9.0 11.1 Trifuralin 29.0 6.3 1.5 16.1 18.1

All values were calculated by the software except when it was not possible due to a profle issue

Environmental exposure to contaminants measured in plasma were less than 5% of the Cmax for all the compounds measured post-exposure. The additional, We also observed an unanticipated source of exposure other unexpected exposure dose is considered to be negligible than the single oral exposure of the mixture via gavage. The when compared with the administered dose (Table S1, Sup- analysis showed that 18 out of the 22 target compounds plemental Materials). were detected; 12 in the feed, 6 in the litter, and 12 in the Regarding the last time point of the PK (at 48 h), several cardboard (Table 2). The pesticides detected were difer- plasma concentrations with pesticides returned to the back- ent, depending of the environmental matrices. Difufeni- ground concentration level (e.g., carbofuran phenol and die- can and p,p’-DDT were the major concentrations detected thyl thiophosphate [DETP]). They represent the fastest PK in the feed, and TCPy, a metabolite of chlorpyrifos, was profle of the pesticides measured in this current study. How- found in high concentration in the litter and in the cardboard ever, some pesticides or metabolites (e.g., fpronil sulfone, (Table 2). γ-HCH [lindane], β-HCH) had concentrations higher than We compared the pesticide concentrations found in lit- the background concentration levels, 48-h post-exposure. ter, feed, or cardboard to the control plasma concentrations observed. The highest plasma concentrations in declining order were as follows: TCPy, PCP, diethyl phosphate (DEP), Discussion and 3-PBA. The plasma sample corresponding to the internal control blood sample group (i.e., the frst sample of each Biomonitoring is an analytical measurement of biomark- rat) showed a large range of variability between rats for most ers, mostly blood and urine, but a non-conventional matrix of the compounds (e.g., the plasma concentration for TCPy such as hair can also be used. Biomarkers are any substances ranged from 14.2 to 278 pmol/ml). Nevertheless, the results such as exogenous chemicals that are measured to indicate showed that the background concentration of pesticides the level of exposure. Biomonitoring is referred generally,

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of interest are not always the only one present; they are frequently infuenced by other chemicals. The experimental in vivo studies can provide essential information to better understand the interaction between the PKs and the pesticides. In fact, this current study of rats is very helpful, because it examines the PK of 17 pesticides in mixture that are frequently found in human matrices of the general population. There were two objectives of this current study. The frst objective was to study the PKs of 17 pesticides in a mixture and their major metabolites following a single oral dose of the exposure via gavage in female Long–Evans rats. The second objective was to assess the concentration changes in blood to provide a biomonitoring perspective. The results from this study clearly point out the importance of the exposure design (time of diet versus time of sampling), but also highlight the challenge of indirect and unintentional exposure during experimental studies (i.e., environmental background exposure). Compared with previously published PK studies, the design applied during the current study was sophisticated regarding the number of samplings, the sequential sampling over 48 h, and the number of duplicates at each time point (Table S2, Supplemental Materials). In the context of this current study, it is important to ensure that this experimental mixture exposure design is in follow with human biomonitoring observation. Both cases require attention to handle multi-residues measured in a matrix, low-exposure doses (environmental background exposure), and potential interactions with other pesticides and subsequent implications. A PK profle is diferent depending on whether the chemical is persistent or non-persistent in a biological matrix and whether it is based on the capacity of enzyme to metabolize the parent compounds or to eliminate the parent quickly or slowly. Literature stated that in diferent biological matrices, persistent chemicals are relatively stable (in terms of months to years) (Luo et al. 2016). In contrast, for the non- persistent chemicals, the concentration can change relatively quickly (in terms of a few hours to a few days) after exposure (LaKind et al. 2014). In fact, in both cases (i.e., persistent and non-persistent), the body burden increases during the exposure and for some time in the post-exposure condi- tion. Indeed, for non-persistent chemicals, the body burden decreases quickly during and after the exposure (Tornero- Velez et al. 2012). Rather than, the concentration persistent Fig. 3 Pharmacokinetics of parent pesticides with their metabolites chemicals also increase rapidly and might decrease rapidly (mean ± standard deviation of the 12 replicated concentrations per in blood, but the body burden will not decrease that much time point). a, b Are on linear scales, whereas c, d are on semi-logarithmic scales and then may even increase (Mühlebach et al. 1991). This information is very important for biomonitoring and PKs, because depending on the exposure time, the concentration but not exclusively, to evaluating one chemical at a time detected might not refect the reality of current exposure. or multi-residues (He et al. 2017; Rodriguez-Gomez et al. For the current study, we selected the pesticides from 2017). It is important to note that PK in humans for environ- different chemical families to assess their behaviors in mental contaminants can be challenging, because chemicals mixtures. Literature reported that some pesticides were

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Table 2 Semi-quantitative Chemicals Feed (pg/mg) Litter (pg/mg) Cardboard Plasma concen- analysis of feed, litter, and (pg/mg) tration (pmol/ml) cardboard, and the value of the plasma background Organochlorines concentration for 59 rats −2 −1 β-Endosulfan > 10 > 0.1 – 5.34E −5.07E β-HCH – – – 1.07E−4−7.14 γ-HCH > 1 > 0.1 – 9.63E−2−6.94 Dieldrin > 1 – – 2.14E−3−8.16 PCP – > 0.1 > 10 1.33−3.01E2 p,p′-DDE – – – 5.95E−2−1.19E1 p,p′-DDT > 100 – > 0.1 1.15E−1−9.51 Organophosphates Chlorpyrifos > 10 > 0.1 > 1 – DEP – > 1 – 1.03−1.18E1 DETP > 1 – > 1 5.85E−2−2.62 Diazinon – – – – TCPy – > 100 > 100 1.42E1−2.78E2 Pyrethroids λ-Cyhalothrin – – – 7.84E−3−1.33 −1 Cl2CA > 0.1 – > 1 3.45E −4.66 −2 −1 ClCF3CA – – – 3.68E −9.25E Cypermethrin > 10 – > 1 1.92E−3−9.98E−1 3-PBA > 1 – > 1 1.35−1.05E1 Permethrin – – > 1 4.88E−2−8.32E−1 Others Carbofuran phenol – – – 9.94E−3−9.63E−1 Difufenican > 100 – > 1 7.32E−3−1.25 Fipronil – – > 0.1 2.14E−3−1.27E1 Fipronil sulfone – – > 0.1 1.60E−2−2.94E1 Oxadiazon > 1 – – 1.39E−2−2.17 Trifuralin > 1 – – 3.86E−2−8.64E−1 Total detected compounds 12 6 12

investigated individually (Mucke et al. 1970; Reigner et al. study. In another publication, the researchers used two difer- 1992; Srinivasan and Radhakrishnamurty 1983). Other ent doses for pyrethroids that corresponded to 10–20 times researchers presented the PKs of parents and metabolites of higher when compared to the current study (Starr et al., the pyrethroids family after rats were administered a mixture 2014). Therefore, it is reasonable to mention that the dose exposure (Starr et al. 2014). Some exposed rats via gavage used in the current manuscript represents one of the lowest to parents or metabolites of the organophosphates family doses published so far. individually and concluded that the PKs of the metabolite We compared the PK parameters that we calculated for were longer with the parent administration (Timchalk et al. the current study with those published in the literature. In 2007). These observations confrm the importance of using one study, Timchalk et al. (2007) calculated the T1/2 for DEP a mixture of pesticides that are considered to be more rep- (118 h), DETP (31 h), and TCPy (8.5 h), which were much resentative of human exposure. higher than in the current work with 11.0 h for DEP, 4.1 h Out of all of the experimental designs reviewed in the lit- for DETP, and 6.4 h for TCPy—especially for the frst two erature, the dose applied during the current study was lower metabolites. This fnding may suggest a possible competition when compared with previous PK studies published. For in the formation of metabolism in their study in compari- instance, in one publication, the researchers used a concen- son with the current investigation due to the concentration tration 100 times higher for chlorpyrifos, which is in the applied. Another groups exposed rats to cyhalothrin at a organophosphate family (Timchalk et al. 2007). As another dose 50 times higher than was used in the current study example, Kumar et al. (2014) exposed rats to cyhalothrin and observed an AUC0−∞ and Cmax 50 times higher than at doses 50 times higher than was used during the current in the current investigation; however, the T1/2 (5.1 h) and

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MRT (7.3 h) were quite similar to the values observed dur- in which the pesticides are solubilized, may infuence the ing the current study (6.8 h and 9.9 h) (Kumar et al. 2009). PK. A group compared diferent routes of exposure (i.e., In another study, the authors used two diferent doses of oral gavage, and subcutaneous and intravenous tail veins cypermethrin (i.e., 1.5 times and 5 times the ED 30) and cal- injections of parent compounds diluted in saline or in corn culated T1/2 as 3.3 h for both doses, whereas for 3-PBA, the oil), observed diferences in PKs, and concluded that the metabolite, the T1/2 was 4.0 h and 5.2 h, respectively (Starr exposure route and dosing vehicle can substantially impact et al. 2014). In addition, from the same study, the T1/2 for target tissue dosimetry (Smith et al. 2009). permethrin (low dose) and Cl2CA (high dose) enantiomers The blood concentration time point measured before the were 2.7 h and 3.3 h, respectively (Starr et al. 2014). The mixture was administered via gavage confrms that the rats Starr et al. (2014)’s values are similar to the current study, had environmental background levels of exposure before we although the exposure dose was at least ten times higher. administered the doses during the current study. The envi- This observation also confrms that using the 0.4 mg/kg con- ronmental background levels of exposure that we observed centration for the current study, we were not saturating the are potentially due to the rats’ contact with the feed (dif- metabolism. Another observation in this comparison is the lufenican and p,p’-DDT) and with the litter and cardboard similarity between the half-life discussed in literature and (TCPy). This exposure infuences the values of PK param- the results previously detailed in this manuscript. eters. For instance, during the current study, we detected Organochlorines are persistent in the environment (Man- several pesticides in blood before administering the dose via souri et al. 2017). However, in this current study, PCP, gavage. Nevertheless, the concentrations were below 5% of p,p′-DDT, p,p′-DDD, and β-endosulfan presented T1/2 in the Cmax post-gavage, suggesting that this exposure was not rats between 12.7 and 19.6 h, and their quantifcation in signifcant enough to afect the PKs. Thus, 18 out of the 22 blood can yield a high variability, because the blood con- target compounds were identifed in feed, litter, or cardboard centration will be halved after a half day. The Agency for used in the rat cages. Our group previously discussed in the Toxic Substances and Disease Registry (ATSDR) reports literature the presence of contaminants in control animals that the complete body burden half-life elimination of DDE due to background exposure; specifcally, 19 out of 27 pes- is approximately 120 days for adult rats (ATSDR 2002). ticides that we analyzed in plasma were also detected in This discrepancy is partially explained by the accumula- control rats (Appenzeller et al. 2017a). Another study from tion of p,p′-DDE or p,p′-DDT in adipose tissue; the release the same group showed the presence of polycyclic aromatic takes much more time in adipose tissue than in blood based hydrocarbon (PAH) metabolites in the control animals: how- on partition coefcient. In the current study, we detected ever, the contaminants were not detected in their surround- fpronil sulfone at a high, but stable concentration after 24 h, ings and feed during the experiment. The authors, therefore, whereas the parent, fpronil, presented a T1/2 of 14.4 h; there- suggested that animals were probably contaminated before fore, fpronil sulfone appears to be a more stable biomarker their arrival at the animal facility (Grova et al. 2011). than fpronil. Finally, during the current study, we noted that the behavior of ClCF3CA as a specifc metabolite of cyhalothrin when compared with 3-PBA demonstrated that ClCF3CA was a better biomarker of exposure than 3-PBA. Conclusion The human PK studies published in literature for environmental chemicals are infrequent. For example, literature The current study provided important information about the reported a PK study of six volunteers who were exposed to PKs of mixture pesticides in plasma that have similar pro- cypermethrin (Ratelle et al. 2015). The T1/2 reported were fles as those observed in the literature for biomonitoring. 5.1 ± 2.5 h for trans-Cl2CA, 6.9 ± 8.1 h for cis-Cl2CA, and The current study illuminates the importance of generating 9.2 ± 6.9 h for 3-PBA; these fndings are in line with the val- experimental data using a mixture of the most common pes- ues we observed during our current study of rats. The same ticides identifed in biomonitoring studies to understand the group published a similar observation with λ-cyhalothrin, implications of their interactions. The information acquired observing that the T1/2 was 5.3 h for ClCF 3CA and 6.4 h for during the current study regarding PKs is comparable to 3-PBA (Khemiri et al. 2017). These results are in the same human biomonitoring exposure in the literature. This experi- range as the current study with 11.7 h and 6.6 h, respectively. mental study points out the importance of measuring the The variabilities of the half-life are also observed in matrix control group, because this step is crucial for accurate human PK studies. These variabilities can be explained by data, but it is frequently neglected. ethnicity, age, sex, and physiological and metabolic dif- ferences, including enzymatic polymorphism (Ma and Lu Acknowledgements Caroline Chata benefted from a Ph.D. grant from the Luxembourg National Research Fund (Fonds National de la 2003; Song et al. 2012; Tang et al. 2005). In the current rat Recherche [FNR]) (AFR 7009593), Luxembourg. study, other factors, such as the route of exposure or vectors

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