Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil

Article abstract of DOI:10.1016/j.envint.2017.03.012

Occupational medical surveillance is highly desirable in manufacturing facilities where exposure to chemicals is significant. The insecticide fipronil is generally considered safe for humans but with increasing use, exposure to fipronil is of concern. Ide

Full text of DOI:10.1016/j.envint.2017.03.012

EI-03602; No of Pages 8
Environment International xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Environment International
journal homepage: www.elsevier.com/locate/envint
Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil
Natalia Vasylieva a, Bogdan Barnych a, Debin Wan a, El-Sayed A. El-Sheikh b, Hai M. Nguyen c, Heike Wulff c,
Rebecca McMahen d, Mark Strynar d, Shirley J. Gee a, Bruce D. Hammock a,
⁎
a
Department of Entomology and Nematology, and UCD Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, United States
b
c
Faculty of Agriculture, Plant Protection Department, Zagazig University, Egypt
Department of Pharmacology, University of California Davis, Davis, CA 95616, United States
d
United States Environmental Protection Agency, National Exposure Research Laboratory, Research Triangle Park, NC, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Occupational medical surveillance is highly desirable in manufacturing facilities where exposure to chemicals is
significant. The insecticide fipronil is generally considered safe for humans but with increasing use, exposure to
fipronil is of concern. Identification of urinary metabolites of fipronil may allow development of affordable, cheap
and rapid procedures for human exposure evaluation. In this study we developed a fast and easy approach for
synthesis of hydroxy-fipronil, a potential urinary metabolite of fipronil. This standard was used to develop a sen-
sitive analytical LC-MS/MS method with a limit of quantification (LOQ) of 0.4 ng/mL. Fipronil sulfone, a known
metabolite, and hydroxy-fipronil were quantified in urine samples from rats treated with a fipronil containing
diet. Fipronil sulfone concentration centered around 20 ng/mL, while the concentration of hydroxy-fipronil
was dose-dependent ranging in 10–10,000 ng/mL and thus being a more sensitive marker of fipronil exposure.
A fipronil immunoassay with cross-reactivity to hydroxy-fipronil showed a good correlation in signal intensity
with LC-MS data. It was also used to demonstrate the applicability of the method for sample screening in the eval-
uation of exposure levels.
Received 5 January 2017
Received in revised form 15 March 2017
Accepted 16 March 2017
Available online xxxx
Keywords:
Insecticide
GABA receptor bioassay
Metabolite
LC-MS/MS
Antibody
Immunoassay
Screening
© 2017 Elsevier Ltd. All rights reserved.
Sensor
1. Introduction
it in the fields and other corresponding sites. In addition to occupational
exposure, the general population is also exposed to fipronil through
Fipronil is a broad spectrum insecticide from the phenylpyrazole
family. It is often used in four major domains: pest control in a wide va-
riety of field crops, urban pest management, veterinary applications (es-
pecially in topical pet care products), and to control rice water weevil
infestation in rice-crawfish rotation (EPA, 2011; Jackson et al., 2009;
Simon-Delso et al., 2015; Watts, 2012). Fipronil is a neurotoxic com-
pound acting on invertebrate GABA-gated chloride channels in the cen-
tral nervous system and thus has found a wide application mainly
because the target insects have not developed resistance to the pesti-
cide. Fipronil is also relatively persistent in the environment providing
long-term protection, with half-life (t1/2) over 100 days in certain con-
ditions (Gunasekara et al., 2007). A recent report by the U.S. Environ-
mental Protection Agency (EPA) indicated a significant increase in the
amount of fipronil used between 1998 and 2008 (Simon-Delso et al.,
2015).
household uses of fipronil. In particular, human exposure may occur
through application of fipronil to animals and interaction with pets. An-
imals may spread fipronil residues by rubbing against carpet and furni-
ture, suggesting sub-chronic or chronic exposure in the general
population (Cochran et al., 2015). Although, fipronil is accepted to be
relatively safe for humans because of its lower binding affinity to verte-
brate GABA-gated chloride channels (Hainzl et al., 1998), it has been re-
cently demonstrated that exposure to micromolar concentrations of
fipronil induced cell death (Vidau et al., 2009; Vidau et al., 2011). Inter-
estingly, cytotoxic effects of fipronil were shown to be stronger with
lower micromolar concentrations and less pronounced with higher con-
centrations (Das et al., 2006), indicating that continuous exposure to
low levels of fipronil as in the case of occupational exposure may have
a pronounced impact on human health. A number of studies with ani-
mals showed that continuous exposure to fipronil leads to significant
hepatic effects (e.g. periacinar hypertrophy) in mice and rats even at
low doses. Mice showed reduced bodyweight gains at the high doses
tested (4.5 mg/kg body weight (bw)/day for 13 weeks) (Hamernik,
1998). Rats, in particular females, showed a significant increase in
liver weight following treatment with fipronil even with low doses
(3.4 mg/kg bw) (Hamernik, 1998). Dogs treated with a diet containing
With increasing amounts of insecticide use, human exposure to
fipronil is of greater concern, in particular in the case of people directly
working with the chemical at manufacturing plants and those applying
⁎
Corresponding author.
E-mail address: bdhammock@ucdavis.edu (B.D. Hammock).
http://dx.doi.org/10.1016/j.envint.2017.03.012
0160-4120/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

2
N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
low fipronil doses (up to 3 mg/kg bw/day) did not experience abnormal
weight gain or severe neurological effects (usually 6–30 mg/kg bw for
flea and tick control). However, other signs of toxicity were observed in-
cluding convulsions, head nodding and muscles twitching (Hamernik,
1998; Woodward, 2012). Herin et al. (2011) assessed serum concentra-
tions of thyroid-stimulating hormone (TSH), fipronil and fipronil sul-
fone in 159 workers in a factory manufacturing fipronil-containing
veterinary drugs. They found a significant positive correlation between
exposure and fipronil/fipronil sulfone concentration in the blood. In ad-
dition, fipronil sulfone concentration correlated negatively with serum
TSH. Authors suggested the possibility that fipronil has a central inhibi-
tory effect on TSH secretion in humans that potentially leads to adverse
health consequences, the major one being loss of bone density and oste-
oporosis (Clark et al., 2010; Ozkaya et al., 2015). Thereby they empha-
sized that close occupational medical surveillance is highly desired to
monitor the exposure of factory workers to fipronil in order to maintain
health and well-being.
A number of studies were conducted to determine fipronil distribu-
tion and metabolism in mammals, as well as to identify its metabolites
as potential biomarkers of exposure to fipronil (Cravedi et al., 2013;
FAO, 2001; McMahen et al., 2015). Fipronil sulfone is a commonly ac-
cepted and proven major metabolite in all tissues, excrement, and
blood (Cravedi et al., 2013; FAO, 2001; McMahen et al., 2015). Patients
with signs of distress indicating potential poisoning with fipronil are
usually subjected to blood analysis to identify the parent compound or
fipronil sulfone (Mohamed et al., 2004). However, blood screening is
an invasive method causing discomfort, pain and carries a risk of devel-
2.2. Enzymatic hydrolysis of urine
Urine aliquots of 250 μL were mixed with 150 μL of methanol and
hydrolyzed using a 100 μL solution containing 33 μL (85,000/
7500 U/mL) of β-glucuronidase/sulfatase and 1.1 mL of 1 M ammonium
acetate buffer at pH 5.5. The reaction was left overnight at 37 °C with
gentle mixing.
2.3. Hydroxy-fipronil identification
Analysis was carried out with a method reported by McMahen et al.
(2015). Briefly, an Agilent 1100 HPLC interfaced with an Agilent 6210
(TOF) mass spectrometer fitted with an electrospray ionization (ESI)
source was used. The HPLC was performed with a Zorbax Eclipse Plus
C18 column (2.1 × 50 mm, 3.5 μm, Agilent Technologies) fitted with a
Phenomenex guard column (Torrance, CA). The method consisted of
the following: 0.2 mL/min flow rate; at 30 °C; mobile phases: A: ammo-
nium formate buffer (0.4 mM) and DI water:methanol (95:5 v/v), and
B: ammonium formate (0.4 mM) and methanol:DI water (95:5 v/v);
gradient: 0–5 min a linear gradient from 50:50 A:B to 100% B; 5–
15 min, 100% B; 15–18 min re-equilibration to 50% A and 50% B.
2.4. Method 1 (the Hammock group). For fipronil sulfone and hydroxy-
fipronil
Rat urine samples (40 μL) were mixed with 10 μL of 1 μM 12-(3-
cyclohexyl-ureido)-dodecanoic acid (CUDA) methanol solution. We
used CUDA as an external standard since it showed a retention time
close to fipronil sulfone, as well as spike-recovery studies with blank
urine matrix showed CUDA to be an appropriate standard to account
for ion suppression. The samples were cleared with a centrifugal filter
device under 20,000g for 5 min. The resulting solutions were transferred
to vials with 100 μL volume inserts, and stored at −20 °C prior to anal-
ysis. Separation of the target compounds was performed on an UPLC
system (Waters Corp., Milford, MA). Samples were stored in an
autosampler at 4 °C, and 10 μL were injected by a partial loop with nee-
dle overfill. The UPLC column Kinetex C18 (1.7 μm, 2.1 × 100 mm, 1.7
μm particle size, Kinetex, Phenomenex) was kept at 40 °C. Mobile
phases were composed of water with 0.1% acetic acid (phase A) and ace-
tonitrile with 0.1% acetic acid (phase B). The following gradient was ap-
plied: 0–3 min 20% B, 3.1–6 min, a linear gradient from 20 to 80% B, 6.1–
8 min, a linear gradient from 80 to 100% B, 8.1–9.1 min, 100% B, 9.1–
10 min re-equilibration to 20%. The flow rate remained constant at
0.4 mL/min. The run event was designed to pre-purify samples online,
0–3 min to waste; 3–8 min to MS; 8–10 min to waste. The UPLC system
was interfaced with the Quattro Premier MS equipped with an ESI
source operated in positive ionization mode. MS operating parameters
and compound specific information are provided in the SI, Tables S1
and S2. The data were acquired and processed using Masslynx 4.1 soft-
ware with instrument and Masslynx 4.1 with TargetLynx.
oping infection. Therefore, urine is
a much more convenient
biospecimen for screening tests. Two recent studies performed a de-
tailed analysis of urine samples obtained from rats treated with a
fipronil-containing diet (Cravedi et al., 2013; McMahen et al., 2015).
Based on mass fragmentation of the compound both teams assigned a
structure of hydroxy-fipronil but it has not been proven by analytical
methods. Nevertheless, based on signal ratios in chromatograms
McMahen et al. (2015) provided an estimate of concentration of metab-
olites present in the urine. From these data, hydroxy-fipronil appears to
be a dominant metabolite among the other identified metabolites of
fipronil.
The specific objectives of this study were to develop an approach for
the synthesis of hydroxy-fipronil and use it as a standard to verify the
identity of the discovered metabolite. A synthetic standard of hy-
droxy-fipronil was tested on mammalian GABAA receptors to character-
ize the biological activity of the new compound. It was also used to
assess its toxicity to insects. Finally, hydroxy-fipronil was used to devel-
op a high performance liquid chromatography method (HPLC) coupled
with tandem mass spectrometry detection (MS/MS) to provide a quan-
titative estimate of the biomarker in the urine of treated animals.
2. Materials and methods
Information concerning chemicals, instruments, buffers, reagents
and synthesis is detailed in the Supporting Information (SI) or in the
sections below.
2.5. LC/MS/MS analysis. Method 2 (the EPA group). For fipronil sulfone only
Method 2 is an alternative method for fipronil sulfone quantification
and for independent evaluation of the accuracy of method 1. Analysis
was carried out with a method reported by McMahen et al. (2015).
Briefly, rat urine (100 μL) was precipitated with 900 μL of cold acetoni-
trile and centrifuged for 8 min at 12,500 ×g. An aliquot of the superna-
tant was extracted and mixed 50:50 with 10 mM ammonium acetate
buffer before LC/MS analysis. Quantification analysis (LC/triple-quad)
was carried out using an Agilent 1100 HPLC interfaced with a Sciex
3000 triple quadrupole mass spectrometer (Applied Biosystems/MDS
Sciex) fitted with an ESI operated in the negative ionization mode.
Fipronil sulfone specific transitions used for quantification were 451.1/
415, 451.1/281.9, 451.1/243.9. The HPLC system consisted of a
Phenomenex Luna C18 column (50 × 3 mm, 5 μm; Torrance, CA, USA)
2.1. Urine samples
Urine samples were generated as part of a study reported by
Freeborn et al. (2015). Briefly, rats were treated by oral gavage with a
fipronil-containing corn oil using an 18 Ga feeding needle with a blunt
tip. Animals were treated daily at 5 or 10 mg/kg either for two weeks
(repeated) or on a single day only. Urine was collected in a syringe ei-
ther from voids on a clean table or via bladder puncture and transferred
to a micro-centrifuge tube, immediately frozen on dry ice, and stored at
−80 °C.
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
3
with a Security-guard guard column (Phenomenex). The method
consisted of the following: 0.4 mL/min flow rate which increased to
0.75 mL/min at time = 2 min; temperature: 30 °C; mobile phases —
A: ammonium acetate buffer (0.2 mM) and DI water:methanol (95:5,
v/v), and B: ammonium acetate buffer (0.2 mM) and acetonitrile:DI
water (95:5, v/v); gradient: 0–2 min 50% A and 50% B; 2.1–4 min, a lin-
ear gradient from 50:50 A:B to 10:90 A:B; 4–6 min 10% A and 90% B;
6.1–10 min re-equilibration to 50% A and 50% B.
were voltage clamped at −80 mV and control currents were recorded
under the application of 25 μM GABA, using a gravity-fed fast perfusion
system, for 5–6 s followed by a 30–40 s wash with external solution. In-
hibition of currents were determined by the reduction in current level
elicited by the same amount of GABA after the pre-application of fipronil
and its derivatives for at least 3 min. Test solutions of fipronil and its de-
rivatives were freshly prepared immediately before each application
onto cells. Data analysis was performed using Excel (Microsoft) and Or-
igin 7.0 (OriginLab Corp.) software. Data fitting to the Hill equation to
obtain IC50 values was performed with Origin 7.0. Data are presented
as the mean S.D.
2.6. Insect bioassay
2.6.1. Rearing of Spodoptera frugiperda (fall armyworm)
The eggs of S. frugiperda were obtained from Benzon Research (Car-
lisle, PA) and maintained at 26 2 °C until hatching. The larvae were
reared on a premixed artificial diet (Bioserv#F9781B, Frenchtown, NJ)
and maintained at 26 2 °C, with a photoperiod of 14:10 h (L:D).
2.8. Indirect competitive Fipronil ELISA
For ELISA all buffers and water solutions were prepared with DI
water; phosphate-buffered saline (PBS, 10 mM, pH 7.5); wash buffer
PBST (PBS containing 0.05% Tween 20); coating buffer (14 mM
Na2CO3, 35 mM NaHCO3, pH 9.8); blocking buffer (1% BSA in PBST); sub-
strate buffer (0.1 M sodium citrate/acetate buffer, pH 5.5). Substrate so-
lution contained 0.2 mL of 0.6% TMB (in dimethyl sulfoxide, DMSO w/v),
0.05 mL of 1% H2O2 in 12.5 mL of substrate buffer. Stop solution was 2 M
H2SO4.·The immunoassay was performed as previously described
(Vasylieva et al., 2015). Briefly, plates were coated with 1 μg/mL antigen
BDH-297-7-CON diluted in coating buffer (100 μL/well). After incuba-
tion for 1 h at RT, the solution was replaced with blocking buffer (200
μL/well) and plates were incubated over night at 4 °C or for 1–4 h at
RT. Plates were washed with 300 μL/well of PBST 3 times prior to sample
loading. Standards in assay buffer were prepared in glass vials and load-
ed onto the coated plate in triplicate (50 μL/well). Urine samples were
directly loaded onto the plate at 5 μL in 45 μL of the assay buffer. An
equal volume (50 μL/well) of anti-fipronil serum #2268 diluted in PBS
was added at 1:4000 dilution, giving final 1:8000 dilution in the plate.
The plate was incubated for 1 h at RT and then washed 5 times with
wash buffer. Goat anti-rabbit IgG-HRP secondary antibody was added
at 100 μL/well in a 1:10,000 dilution as instructed by manufacturer.
The plate was incubated for 1 h at RT and washed 5 times. Substrate so-
lution was added (100 μL/well). The color development was stopped by
addition of 2 M H2SO4 (50 μL/well) and absorbance was read at 450 nm.
SigmaPlot 11.0 software was used for curve fitting and data analysis.
2.6.2. Bioassays procedure
Bioassays were conducted on second instar (~3.5 days old) larvae of
S. frugiperda using a diet surface contamination method. Five concentra-
tions ranging from 1 to 100 μg/mL from each agent were prepared in DI
water and used with 3 replicates for each concentration. The diet was
prepared according to the manufacturer instructions by adding
139.7 g dry mix (BioServ, NJ; Product #F9781B) and 4.8 g potassium hy-
droxide solution into 24 g boiled agar in 831 mL DI H2O to form 1 L of
diet. An aliquot of 15 mL was poured in 120 mL cup (Frontier Agricultur-
al Services, Newark, DE) (with base and top diameters equal to 6 and
6.5 cm, respectively). One mL of each concentration or DI H2O (control)
was separately spread on the diet surface, and then cups moved several
times to ensure covering the whole surface of the diet with solution. Im-
mediately after covering the whole diet surface, the remainder of the so-
lution was completely removed using a pipette. Cups were left for
15 min at RT to dry. Ten 2nd instar larvae of S. frugiperda were trans-
ferred into each cup with a total number of 30 larvae/concentration or
control. A total of 180 larvae were used for each bioassay and control,
and repeated in triplicate. Data were recorded at 12 h intervals for
3 days. The larvae were considered dead when they did not respond
to a gentle touch with a brush to provoke movement. Median lethal
concentrations (LC50) and median lethal times (LT50) were calculated
using the Probit analysis 3.1 program and ViStat 2.1 program
respectively.
3. Results
2.7. GABA receptor bioassay
3.1. Synthesis
2.7.1. Preparation of cells expressing the α1β3γ2L GABAA receptors
The human GABAA receptors α1, β3 and γ2L cloned into pcDNA3.1
expression vectors were a gift from Dr. Robert L. Macdonald, Vanderbilt
University, TN. Fibroblast L929 cells were cultured in Dulbecco's modi-
fied Eagle's medium (Lonza) supplemented with 10% fetal bovine
serum, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen)
and maintained in humidified 95% air and 5% CO2 air at 37 °C. Cells
were transfected using FuGENE 6 (Roche) transfection reagent with
an equal amount of each of the subunits in combination with pEGFP-
C1. The transfection ratio of total cDNA to transfection reagent was
3:1. 36 h post-transfection, cells were plated on glass coverslips and
transfected cells were identified using an epifluorescence microscope
for electrophysiological whole-cell voltage-clamp studies.
5-Amino-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-hydroxy-
1H-pyrazole-3-carbonitrile (hydroxy-fipronil, (5)) and methyl 5-
amino-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-1H-pyrazole-3-
carbimidate (imidate derivative of fipronil desulfinyl, (6)) were synthe-
sized as described in Scheme 1. The synthesis of hydroxy-fipronil (5)
began with preparation of the aceto-intermediate (3) according to the
previously described procedure (Banks, 2000). Briefly, iodination of
the fipronil detrifluoromethylsulfinyl with N-iodosuccinimide followed
by the Sonogashira coupling of the resulting iodo derivative with
trimethylsilyl acetylene gave the acetylenic derivative (2) which was
further subjected to potassium carbonate catalyzed cleavage of the
trimethylsilyl group and acid catalyzed hydration of the acetylene moi-
ety to give the desired intermediate (3). Baeyer-Villiger oxidation of (3)
with mCPBA in the presence of para-toluensulfonic acid provided the
acetoxy intermediate (4) in 33% yield (68% based on the recovered
starting material). Hydrolysis of the acetoxy function completed the
synthesis of the hydroxy-fipronil 5.
2.7.2. Electrophysiological recordings
Whole-cell voltage-clamp recordings were performed at RT with an
EPC-10 HEKA amplifier. Cells were bathed in an external Ringer solution
consisting of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2,
10 mM HEPES, pH 7.4, 311 mOsm. Recording electrodes were pulled
and fire-polished to resistances of 1.4–1.8 MΩ and filled with an inter-
nal solution consisting of 154 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
10 mM HEPES and 10 mM EGTA with pH 7.3 and 308 mOsm. Cells
The imidate (6) was synthesized in 54% yield (quantitative based on
the recovered starting material) by stirring the solution of fipronil
detrifluoromethylsulfinyl (1) and sodium hydroxide in methanol
under an atmosphere of ambient air or oxygen. NMR and HRMS spectra
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

4
N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
Scheme 1. Synthetic approach toward hydroxy-fipronil (5) and the imidate derivative of fipronil desulfinyl (6).
obtained for the synthesized compound were in a good agreement with
expected structure. The detailed analysis is provided in the SI.
McMahen et al., 2015). For this purpose, we used an enzymatic solution
of β-glucuronidase/sulfatase since it provides mild conditions for hy-
drolysis, decreasing the possibility of destroying the hydroxy-fipronil
by harsh acidic hydrolysis conditions. The hydrolyzed urine samples
were loaded on the chromatography column directly without a prelim-
inary extraction step. To remove any urinary components that could
suppress ionization, after loading and prior to elution, the column was
washed excessively with aqueous phase followed by elution and MS/
MS detection. Table 1 shows a good relationship between the concen-
tration of hydroxy-fipronil detected in the samples and the amount of
fipronil in the diet received by the corresponding rats. Thus, when rats
received a single meal with a fipronil dose of 5 or 10 mg/kg, the hy-
droxy-fipronil concentration in the urine did not differ much and was
about 20 ng/mL. However, when animals received continual treatment
over 14 days, the concentration of hydroxy-fipronil was 2025 and
5340 ng/mL for animals treated with 5 mg/kg (2 rats tested) and
10,200 ng/mL for the animal treated with 10 mg/kg. The developed
method for hydroxy-fipronil quantification was also applied for the
quantification of fipronil sulfone. Fipronil sulfone was detected only in
samples obtained from animals treated with fipronil repeatedly (sam-
ples 42, 81 and 82). All samples were tested blindly and results agreed
between two laboratories that tested the samples independently with
different sample preparation protocols and instrumental set-up (Table
1).
3.2. Identification and quantification analysis
High resolution TOF MS in negative ionization mode was used to
evaluate compound (5) (Scheme 1). Hydroxy-fipronil was observed as
a formate adduct with a major ion at m/z 380.9786 while lower intensi-
ty ions at m/z 382.9751 and 384.9716 appeared due to the presence of
chlorine isotopes (Fig. S1). The mass-to-charge values obtained experi-
mentally correspond perfectly to calculated values compared at three
decimal places. This isotopic pattern is very characteristic of compounds
containing two chlorine atoms. There were also additional chlorinated
peaks in the spectrum, most probably related to hydroxy-fipronil (reac-
tion side products or intermediate materials) suggesting that the stan-
dard contained some impurities. However, the compound was
purified by flash column chromatography and as judged by TLC and
NMR spectroscopy it was pure at N95%. Therefore, chlorinated peaks ob-
served in the spectra may indicate presence of impurities at no N5%.
The spectrum looked similar when hydroxy-fipronil was spiked in
the urine matrix (Fig. S2). Prior to quantitative analytical method devel-
opment, we ran a standard solution of hydroxy-fipronil and a sample of
hydrolyzed but non-spiked urine sample from a treated rat on LC/TOF to
compare retention times of the standard and the metabolite. We
showed clearly a match in retention times of the peaks with the same
exact molecular mass of 380.9786 corresponding to the formic adduct
of hydroxy-fipronil thus further supporting the identity of the metabo-
lite (Fig. S3).
The LC-MS/MS chromatograms of a standard mixture containing hy-
droxy-fipronil, fipronil sulfone and CUDA as an internal standard are
provided in the SI (Fig. S4). The limit of quantification (LOQ) was deter-
mined as 10 times the signal-to-noise ratio. In methanol the LOQ was
0.4 ng/mL and 1.5 ng/mL for hydroxy-fipronil and fipronil sulfone, re-
spectively. The LOQ for hydroxy-fipronil in the urine matrix was deter-
mined to be 1.0 ng/mL. The sensitivity achieved is comparable to other
methods reported in the literature (Bichon et al., 2008; Cazorla-Reyes et
al., 2011; Lacroix et al., 2010). All replicates for all experiments had a rel-
ative standard deviation (RSD) of b18%. Recoveries were determined
from spiked urine samples with CUDA used as an external standard. Re-
covery values for hydroxy-fipronil spiked at 1.0 ng/mL were 110 6%.
Prior to quantitative analysis all urine samples were hydrolyzed to
cleave possible glucuronide and sulfate conjugates of hydroxy-fipronil
which occur according to literature reports (Cravedi et al., 2013;
3.3. Insect toxicity
Table S3 indicates that fipronil is toxic to S. frugiperda larvae with an
LC50 of 4.1 μg/mL. The level of mortality increased with increasing con-
centrations and reached 100% after 48 h of treatment using 50 μg/mL,
and after 12 h of exposure using 100 μg/mL. On the other hand, the
fipronil analogues, both hydroxy and imidate, did not cause any mortal-
ity on 2nd instar larvae of S. frugiperda even at doses as high as 100 μg/
mL which was sufficient to kill 100% of the treated S. frugiperda larvae
with fipronil in b12 h.
3.4. Human GABA receptor bioassay
To test the activity of fipronil and its derivatives in inhibiting GABA-
induced Cl− currents, their effect on currents produced by cells tran-
siently expressing the α1β3γ2L GABAA receptor were measured using
whole-cell voltage-clamp. Fipronil showed the highest potency in re-
ducing currents and had an inhibitory effective concentration (EC50
)
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
5
Table 1
Quantification of fipronil sulfone and hydroxy-fipronil in urine samples.
Conc sulfone, ng/mL
LOQ*
Conc sulfone, ng/mL
LOQ**
Conc sulfone, ng/mL
LOQa
Conc FipOH, ng/mL
LOQa
Sample ID
Dose info
1 ng/mL
10 ng/mL
1.5 ng/mL
0.4 ng/mL
17
35
90
3
60
42
81
52
82
0 mg/kg
0 mg/kg
0 mg/kg
5 mg/kg single
5 mg/kg single
5 mg/kg repeated
5 mg/kg repeated
10 mg/kg single
10 mg/kg repeated
2
2
8
8
13
12
18
7
n/a
b10
b10
n/a
b10
26
24
b10
23
N/D
N/D
N/D
N/D
N/D
17
14
N/D
19
N/D
N/D
N/D
23
24
5340
2025
14
29
10,200
n/a, not tested, N/D-non-detectable.
Determined with different LC-MS/MS methods by the EPA group in: *the study of McMahen et al. (2015); **this study.
a
Determined in this work with a LC-MS/MS method different from the EPA group.
of 1.15 0.25 nM (n = 6, Fig. 1), in agreement with its previously pub-
lished nanomolar activity (Hainzl and Casida, 1996). Fipronil desulfinyl
imidate blocked GABA-induced currents with a similar EC50 of 2.78
1.42 nM (n = 23). In contrast, the metabolite hydroxy-fipronil was
not as potent and required up to a 1000 fold higher concentrations to
elicit similar current inhibition (EC50 = ~1 μM).
fipronil derivatives and the sera cross-reactivity is not known for all de-
rivatives, the signal from the immunoassay was given a (+) when the
concentration of analyte was lower than 5 ng/mL but still significantly
detectable; (++) for the concentration range between 10 and
1000 ng/mL and (+++) for N1000 ng/mL. Table 2 shows a relatively
good correlation between data obtained with LC-MS/MS and immunoas-
say screening of hydrolyzed urine samples.
3.5. Immunoassay screening for independent evaluation
4. Discussion
We previously developed 2 immunoassays using different sera for
the detection of fipronil (Vasylieva et al., 2015). Cross-reactivity (CR)
to hydroxy-fipronil was tested with these polyclonal antibodies 2265
and 2268 (pAbs). In both assays sensitivity to hydroxy-fipronil was sig-
nificantly lower with CR being only 3.2 (n = 5) and 0.7% (n = 4) for
sera 2265 and 2268 respectively (Fig. S5). The assay 2268 is more robust
to changing sample conditions like pH or ionic strength (Vasylieva et al.,
2015). We therefore chose this assay to assess the applicability of the im-
munoassay for screening of urine samples for the presence of biomarkers
of exposure to fipronil. Although minimal CR is observed, based on our
previously determined concentrations for hydroxy-fipronil, it should
be high enough to produce a pronounced result in the immunoassay.
Since the literature data indicates that urine may contain a number of
Serum and urine are two common body fluids used for general
health analysis but also for monitoring of human exposure to chemicals.
Even though both specimens bring complementary information, serum
samples are more often used to monitor global health parameters. Nev-
ertheless, urine is much more suitable for large monitoring campaigns
since the sample collection is fast, cheap, does not require special train-
ing, could be accomplished by the patient and provides large amounts if
needed. It is also noninvasive and thus does not have any side effects
such as pain or inflammation. However, its use is limited only to the tar-
get analytes that are cleared from the body into the urine in the form of
the initial parent compound or its corresponding metabolites. To date,
fipronil has mainly been detected in the serum of patients in its parent
Fig. 1. Inhibition of GABAA current by fipronil and its derivatives. A) Whole-cell patch-clamp recordings showing the inhibition of GABA-induced Cl− currents by fipronil (top), fipronil
desulfinyl imidate (middle) and hydroxy-fipronil (bottom) at concentrations of 100 nM and 1000 nM. Scale bars indicate 1 nA and 5 s, respectively. B) Concentration-response curves
showing the percentage of α1β3γ2L GABAA currents blocked by increasing concentrations of fipronil, fipronil desulfinyl imidate and hydroxy-fipronil. The inhibitory EC50 values are
1.2 0.2 nM (n = 6) for fipronil, 2.8 1.4 nM (n = 23) for fipronil desulfinyl imidate, and 1000 341 nM (n = 7) for hydroxy-fipronil.
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

6
N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
Table 2
(UM10, Scheme 2). Despite the actual match in exact mass and frag-
ments analysis, the identity of the compound has not been proven by
comparison with a synthetic standard.
Screening urine samples for hydroxy-fipronil using immunoassay.
Animal
Dose
Sulfone, μg/L
Fip-OH, μg/L
ELISA screening
A novel and easy approach to the synthesis of hydroxy-fipronil was
developed here to extend previous studies and provide data supported
with a synthetic standard for detection of hydroxy-fipronil. Since
fipronil detrifluoromethylsulfinyl (1), a late-stage intermediate for the
synthesis of fipronil, is commercially available and differs from the
structure of hydroxy-fipronil by only the absence of the oxygen atom
at the 4th position of the pyrazole moiety that could be potentially in-
troduced by an oxidation reaction, it was considered as an attractive
starting material for the synthesis. The pyrazole moiety of fipronil
detrifluoromethylsulfinyl has a high degree of similarity with enols of
β-ketocarbonyl compounds (Fig. 2). Keeping in mind that enols and
enolates are very reactive toward molecular oxygen and undergo addi-
tion reactions with it at ambient temperature (Cubbon and Hewlett,
1968; Schottner et al., 2010; Wang et al., 2011) yielding α-hydroperoxy
carbonyl compounds, a natural question arose: is a similar reaction pos-
sible for the appropriately substituted heterocycles and in particular for
17
35
90
3
60
42
81
52
82
0 mg/kg
0 mg/kg
0 mg/kg
5 mg/kg single
5 mg/kg single
5 mg/kg repeated
5 mg/kg repeated
10 mg/kg single
10 mg/kg repeated
N/D
N/D
N/D
N/D
N/D
17
14
N/D
19
N/D
N/D
N/D
23
−
+
+
+
24
+
5340
2025
14
+++
+++
++
+++
10,200
− non-detectable, + b5 ng/mL, ++ N10 ng/mL, +++ N1000 ng/mL.
N/D – non-detectable.
form or as fipronil sulfone (Mohamed et al., 2004). Two recent studies
performed a detailed analysis of urine samples obtained from rats treat-
ed with fipronil-containing diet (Cravedi et al., 2013; McMahen et al.,
2015). Both research teams identified a novel potential metabolite in
urine, hydroxy-fipronil (UM6, Scheme 2), in its conjugated form
Scheme 2. Urinary fipronil metabolites identified by high resolution MS and fragment analysis. Summary from 3 independent studies (Cravedi et al., 2013; McMahen et al., 2015; Powles et
al., 1992).
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
7
undiluted urine samples using HPLC. Deconjugation with a β-glucuron-
idase/sulfatase did not change the elution profile. Fourteen compounds
were resolved of which seven were identified, while 4 remained un-
known. These authors concluded that fipronil sulfone was a minor con-
stituent in deconjugated and original urine. Our results strongly suggest
that at least one of those unidentified metabolites might be hydroxy-
fipronil because in addition to other evidence shown above, it also pos-
sesses polar properties after enzymatic deconjugation as described by
Powles et al. (1992). As an interesting addition, we also observed that
concentration of hydroxy-fipronil detected in the samples before and
after enzymatic hydrolysis did not change significantly (Table S4).
4.1. Bioactivity studies
Fig. 2. Similarity between aminopyrazol moiety (A) and enols of β-ketocarbonyl
compounds (B).
Since the data reported here demonstrate strong evidence that hy-
droxy-fipronil is a urinary metabolite in humans, further characteriza-
tion of its bioactivity properties was conducted. Our goal was to
identify if the metabolite had any remaining activity and if it presented
any potential concern after being released from the body. Its toxicity
was studied in an insect bioassay, as well as its activity on a major
human GABAA receptor to which fipronil has a high affinity. In addition,
in the bioassay studies we included the imidate derivative of fipronil.
This compound has never been characterized before and since it has a
chemical structure similar to fipronil, we tested it to extend our knowl-
edge about this compound class. In our hands, fipronil showed an LC50
(4.1 μg/mL) (Table S3) on 2nd larval instar of S. frugiperda very close
to what has been previously detected in other lepidopterans (Li et al.,
2006) even though the method of treatment was different (diet surface
contamination method vs incorporation in the food). In the current
study hydroxy-fipronil and fipronil desulfinyl imidate did not show
any mortality against 2nd instar larvae of S. frugiperda even when
used in high concentrations (i.e. 24-fold higher than fipronil LC50). Lar-
vae treated with both fipronil derivatives did not show any differences
compared with control and survivors completed the life cycle normally
at least until the pupae stage. We did not monitor possible abnormali-
ties with adult emergence. Our results reflect the apparent safety of
these compounds in the environment.
The activity of fipronil and derivatives on GABAA α1β3γ2L receptor
currents was then evaluated. As expected, hydroxy-fipronil did not have
a significant inhibitory effect on GABAA receptor currents and thus this
metabolite might be considered safe in terms of neurotoxicity to
humans. Surprisingly, the imidate derivative that did not show any in-
secticidal activity turned out to be approximately as potent as fipronil
on human α1β3γ2L receptors. This discrepancy is even more surprising
in light of previous research suggesting that the fipronil binding site lies
within the second trans-membrane region of the β3 subunit, a region
that shares high amino-acid sequence homology to the analogous re-
gion of the insect GABAR, the target of the fipronil (Ratra and Casida,
2001). One possible explanation for this observation could be the met-
abolic instability of fipronil desulfinyl imidate.
the pyrazole moiety of fipronil detrifluoromethylsulfinyl? Unfortunate-
ly, treatment of (1) with sodium hydroxide in methanol in the presence
of ambient air only resulted in formation of the imidate (6) but not the
desired peroxide. The precise structural assignment for compound (6)
was proven by spectroscopic analyses. Since the peroxidation-reduction
approach failed to give the desired hydroxy-fipronil product, a new suc-
cessful approach was designed that involved Baeyer-Villiger oxidation
of known compound (3) as the key step (Scheme 1A).
The newly developed synthetic standard of hydroxy-fipronil and
commercial fipronil sulfone were used to develop an analytical method
for their separation and quantification with LC-MS/MS. Prior to the
quantification of hydroxy-fipronil the developed method was used to
quantify fipronil sulfone in urine samples. These results were compared
to the data obtained using a different analytical set-up (McMahen et al.,
2015), tested independently by the EPA group in a blind fashion (Table
1). The concentrations of fipronil sulfone determined in urine samples
using the LC-MS/MS method developed in this work (17, 14 and
19 ng/mL for rat samples 42, 81 and 82 respectively) were very close
to the values provided by the EPA group (26, 24 and 23 ng/mL) that
were obtained with two separate methods having different LOQs: 26,
24 and 23 ng/mL at LOQ 10 ng/mL; and 12, 19 and 29 ng/mL at LOQ
1 ng/mL (the EPA data provided for this study). These urine samples
were previously tested for free fipronil and no detectable amount was
reported (McMahen et al., 2015), which also agrees with literature re-
ports (Cravedi et al., 2013; Powles et al., 1992).
Using the analytical method developed here to quantify hydroxy-
fipronil in the urine of rats treated with fipronil containing diet, we ob-
tained a very good positive trend between the concentration of metab-
olite detected and fipronil dose in the diet (Table 1). Control animals did
not have detectable hydroxy-fipronil (LOQ 0.4 ng/mL). Those rats that
received a single treatment dose of 5 or 10 mg/kg of fipronil had similar
hydroxy-fipronil concentrations in the urine at about 20 ng/mL. Howev-
er, when the animals were treated with the same doses for a period of
two weeks, about 80 to 700 times difference in metabolite concentra-
tion was observed with the highest values detected in animals treated
with dose of 10 mg/kg. Interestingly, Powles et al. (1992) in metabolism
studies with 14C-fipronil and a similar dosing regimen showed that after
a single dose at 4 mg/kg, recovery of radioactivity in the urine of treated
rats was 5.6%, while after repeated dosing over 14 days, the recovery of
radioactivity in the urine was 14–16%. They also showed that a single
high dose (150 mg/kg) resulted in an even higher recovery of 22–30%.
Thus, our results correlate with data on fipronil metabolism reported
in the literature (Powles et al., 1992). Theoretically, a single exposure
to a low amount of fipronil may lead to a distribution of the compound
over the body and mostly to the adipose tissue. However, when repeat-
ed exposure occurs and the compound is no longer accumulating, a
higher amount is excreted, leading to higher detected amounts in the
urine.
4.2. Urine samples screening by immunoassay
A sensitive fipronil immunoassay was employed to assess the pres-
ence of fipronil-like compounds in the urine of treated animals. It was
used to evaluate the presence of fipronil metabolites in the samples as
an independent method that has a different way of sensing the target
analyte as an alternative to LC-MS/MS. Two false positive read outs for
samples 35 and 90 were detected. It could have been any other metab-
olite that the pAb may recognize, but since these samples were from
negative control animals, it was more likely due to contamination of
the sample or presence of a small matrix effect. Aside from this, there
is a strong trend in signal intensity with (++) read out for samples con-
taining hydroxy-fipronil and no fipronil sulfone and (+++) for sam-
ples with a significant amount of fipronil sulfone and hydroxy-fipronil
detected with LC-MS/MS. These data further support the hypothesis
In the same study with 14C-fipronil Powles et al. (1992) reported
high levels of very polar radiolabeled material detected in unextracted
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012

8
N. Vasylieva et al. / Environment International xxx (2017) xxx–xxx
Das, P.C., Cao, Y., Cherrington, N., Hodgson, E., Rose, R.L., 2006. Fipronil induces CYP iso-
forms and cytotoxicity in human hepatocytes. Chem. Biol. Interact. 164, 200–214.
EPA, 2011. Fipronil Summary Document Registration Review: Initial Docket June 2011.
Docket Number: EPA-HQ-OPP-2011-0448. Case No.7423.
FAO, 2001. JMPR evaluations: fipronil. Accessible at: http://www.fao.org/fileadmin/
templates/agphome/documents/Pests_Pesticides/JMPR/Evaluation01/08_Fipronil.
pdf.
Freeborn, D.L., McDaniel, K.L., Moser, V.C., Herr, D.W., 2015. Use of electroencephalogra-
phy (EEG) to assess CNS changes produced by pesticides with different modes of ac-
tion: effects of permethrin, deltamethrin, fipronil, imidacloprid, carbaryl, and
triadimefon. Toxicol. Appl. Pharmacol. 282, 184–194.
that hydroxy-fipronil is a urinary metabolite of fipronil. In addition, this
demonstrates that an immunoassay for analysis of urine samples could
be a useful and convenient method for exposure assessment and evalu-
ation. Immunoassays are proven to be the tools of choice for high
throughput large screening studies because they provide an inexpen-
sive and fast alternative to instrumental methods. Immunoassays can
also be converted into a field portable lateral flow sensor format. To-
gether with non-invasive urine sample collection the availability of
field portable sensors would simplify the screening campaigns at
manufacturing plants and other related sites of exposure. This would
also make occupational medical surveillance affordable and allow time-
ly treatment of affected persons in order to maintain health and well-
being.
Gunasekara, A.S., Truong, T., Goh, K.S., Spurlock, F., Tjeerdema, R.S., 2007. Environmental
fate and toxicology of fipronil. J. Pest. Sci. 32, 189–199.
Hainzl, D., Casida, J.E., 1996. Fipronil insecticide: novel photochemical desulfinylation
with retention of neurotoxicity. PNAS 93, 12764–12767.
Hainzl, D., Cole, L.M., Casida, J.E., 1998. Mechanisms for selective toxicity of fipronil insec-
ticide and its sulfone metabolite and desulfinyl photoproduct. Chem. Res. Toxicol. 11,
1529–1535.
In conclusion, we have developed an easy and fast method for syn-
thesis of hydroxy-fipronil, a urinary metabolite of the fipronil. The
newly developed synthetic standard of hydroxy-fipronil was used to
develop an analytical LC-MS/MS method for its quantification in the
urine of treated animals. Immunoassay with cross-reactivity to fipronil
metabolites and to hydroxy-fipronil was used as alternative method to
confirm the presence of the metabolite in the urine and to evaluate its
level. Our results obtained in a blind fashion, correlated well with the
data obtained in the second laboratory that used a different analytical
set-up. New compounds synthesized in this work were evaluated in
bioassays for neurotoxicity, and results suggested that they did not
show GABAergic activity.
Hamernik, K.L., 1998. Pesticide Residues in Food-1997, Evaluations 2005, Part II - Toxico-
logical Joint FAO/WHO Meeting on Pesticide Residues. World Organization, Geneva.
Herin, F., Boutet-Robinet, E., Levant, A., Dulaurent, S., Manika, M., Galatry-Bouju, F., Caron,
P., Soulat, J.M., 2011. Thyroid function tests in persons with occupational exposure to
fipronil. Thyroid 21, 701–706.
Jackson, D., Cornell, C.B., Luukinen, B., Buhl, K., Stone, D., 2009. Fipronil Technical Fact
Sheet. National Pesticide Information Center. Oregon State University Extension Ser-
vices. http://npic.orst.edu/factsheets/archive/fiptech.html.
Lacroix, M.Z., Puel, S., Toutain, P.L., Viguie, C., 2010. Quantification of fipronil and its me-
tabolite fipronil sulfone in rat plasma over a wide range of concentrations by LC/UV/
MS. J. Chromatogr. B 878, 1934–1938.
Li, A.G., Yang, Y.H., Wu, S.W., Li, C., Wu, Y.D., 2006. Investigation of resistance mechanisms
to fipronil in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 99,
914–919.
McMahen, R.L., Strynar, M.J., Dagnino, S., Herr, D.W., Moser, V.C., Garantziotis, S.,
Andersen, E.M., Freeborn, D.L., McMillan, L., Lindstrom, A.B., 2015. Identification of
fipronil metabolites by time-of-flight mass spectrometry for application in a human
exposure study. Environ. Int. 78, 16–23.
Acknowledgment
Mohamed, F., Senarathna, L., Percy, A., Abeyewardene, M., Eaglesham, G., Cheng, R., Azher,
S., Hittarage, A., Dissanayake, W., Sheriff, M.H., Davies, W., Buckley, N.A., Eddleston,
M., 2004. Acute human self-poisoning with the N-phenylpyrazole insecticide fipronil
- a GABA(A)-gated chloride channel blocker. J. Toxicol. Clin. Toxicol. 42, 955–963.
Ozkaya, H.M., Keskin, F.E., Haliloglu, O.A., Senel, T.E., Kadioglu, P., 2015. Life-threatening
hypercalcemia due to Graves' disease and concomitant adrenal failure: a case report
and review of the literature. Case Rep Endocrinol.
This work was supported by National Institute of Environmental
Health Sciences, Superfund Research Program, P42 ES04699 and the Na-
tional Institute for Occupational Safety and Health Western Regional
Center for Agricultural Health Science U50 OH07550. The research
was also supported by the CounterACT Program, National Institutes of
Health Office of the Director, and the National Institute of Neurological
Disorders and Stroke, grant number U54 NS079202.
Powles, P., Biol, C., Biol, M.I., 1992. In: EPA (Ed.), 14C Fipronil (14C-M&B 46 030): Absorp-
tion, Distribution, Metabolism, and Excretion in the Rat.
Ratra, G.S., Casida, J.E., 2001. GABA receptor subunit composition relative to insecticide
potency and selectivity. Toxicol. Lett. 122, 215–222.
Schottner, E., Wiechoczek, M., Jones, P.G., Lindel, T., 2010. Enantiospecific synthesis of the
cubitane skeleton. Org. Lett. 12, 784–787.
Appendix A. Supplementary data
Simon-Delso, N., Amaral-Rogers, V., Belzunces, L.P., Bonmatin, J.M., Chagnon, M., Downs,
C., Furlan, L., Gibbons, D.W., Giorio, C., Girolami, V., Goulson, D., Kreutzweiser, D.P.,
Krupke, C.H., Liess, M., Long, E., McField, M., Mineau, P., Mitchell, E.A.D., Morrissey,
C.A., Noome, D.A., Pisa, L., Settele, J., Stark, J.D., Tapparo, A., Van Dyck, H., Van
Praagh, J., Van der Sluijs, J.P., Whitehorn, P.R., Wiemers, M., 2015. Systemic insecti-
cides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites. En-
viron. Sci. Pollut. R. 22, 5–34.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.envint.2017.03.012.
References
Vasylieva, N., Ahn, K.C., Barnych, B., Gee, S.J., Hammock, B.D., 2015. Development of an im-
munoassay for the detection of the phenylpyrazole insecticide fipronil. Environ. Sci.
Technol. 49, 10038–10047.
Vidau, C., Brunet, J.L., Badiou, A., Belzunces, L.P., 2009. Phenylpyrazole insecticides induce
cytotoxicity by altering mechanisms involved in cellular energy supply in the human
epithelial cell model Caco-2. Toxicol. in Vitro 23, 589–597.
Vidau, C., Gonzalez-Polo, R.A., Niso-Santano, M., Gomez-Sanchez, R., Bravo-San Pedro,
J.M., Pizarro-Estrella, E., Blasco, R., Brunet, J.L., Belzunces, L.P., Fuentes, J.M., 2011.
Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis
in human neuronal cell line SHSY5Y. Neurotoxicology 32, 935–943.
Wang, G.W., Lu, Q.Q., Xia, J.J., 2011. Three types of products obtained unexpectedly from
the reaction of dimedone with chalcones. Eur. J. Org. Chem. 4429–4438.
Watts, M., 2012. Pesticide Action Network Asia and the Pacific, Highly Hazardous Pesti-
cides: Fipronil. A PAN AP Factsheet Series. http://archive.panap.net/en/p/post/
pesticides-info-database/1209.
Banks, B.J., 2000. Parasiticidal Compounds. Pfizer Inc., USA.
Bichon, E., Richard, C.A., Le Bizec, B., 2008. Development and validation of a method for
fipronil residue determination in ovine plasma using 96-well plate solid-phase ex-
traction and gas chromatography-tandem mass spectrometry. J. Chromatogr. A
1201, 91–99.
Cazorla-Reyes, R., Fernandez-Moreno, J.L., Romero-Gonzalez, R., Frenich, A.G., Vidal, J.L.,
2011. Single solid phase extraction method for the simultaneous analysis of polar
and non-polar pesticides in urine samples by gas chromatography and ultra high
pressure liquid chromatography coupled to tandem mass spectrometry. Talanta 85,
183–196.
Clark, L., Mohammed, A., Pillai, A., Klotsas, A., Masters, P., Lawson, N., 2010. Hyperthyroid-
ism, an overlooked cause of severe hypercalcaemia. Grand Rounds,Specialities: Endo-
crinology 10, 110–112.
Cochran, R.C., Yu, L., Krieger, R.I., Ross, J.H., 2015. Postapplication fipronil exposure follow-
ing use on pets. J. Toxicol. Environ. Health A 78, 1217–1226.
Cravedi, J.P., Delous, G., Zalko, D., Viguie, C., Debrauwer, L., 2013. Disposition of fipronil in
rats. Chemosphere 93, 2276–2283.
Woodward, K.N., 2012. In: Marrs, T.C. (Ed.), Chapter 12.2.6 Fipronil. Mammalian Toxicol-
ogy of Insecticides. The Royal Society of Chemistry.
Cubbon, R.C.P., Hewlett, C., 1968. Organic peroxides containing functional groups.1. Prep-
aration and properties of some alpha-oxo-hydroperoxides. J. Chem. Soc. C (2978-&).
Please cite this article as: Vasylieva, N., et al., Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil, Environ Int (2017), http://
dx.doi.org/10.1016/j.envint.2017.03.012