Toxicology Letters 221 (2013) 1522

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Toxicology Letters
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Variability of urinary excretion of pyrethroid metabolites in seven persons over
seven consecutive daysImplications for observational studies
Bartosz Wielgomas

Department of Toxicology, Medical University of Gda nsk, Al. Gen. J. Hallera 107, 80-416 Gda nsk, Poland
h i g h l i g h t s

Urinary 3PBA concentrations show fairly stable levels in seven adults over one week.

Spot or multiple urine spot samples should be considered as the most reliable for exposure assessment.

Creatinine adjustment was shown to improve overall 3PBA measurement reliability in spot samples.
a r t i c l e i n f o
Article history:
Received 22 January 2013
Received in revised form15 May 2013
Accepted 18 May 2013
Available online xxx
Keywords:
Biomarker
Exposure
Human biomonitoring
Synthetic pyrethroids
Temporal changes
a b s t r a c t
Concentration of urinary metabolites is frequently used for biomonitoring of exposure to synthetic
pyrethroids, the class of non-persistent insecticides. These chemicals are currently widely used in agricul-
ture, households and public health all over the world. Most of them are easily metabolized in mammals
and in the form of metabolites excreted in urine. The concentration in urine is thus susceptible to
signicant variations, even within a short period of time. In this study, temporal changes in urinary
metabolites concentrations in seven subjects (four females and three males aged: 2471) were moni-
tored over seven consecutive days. All urine voids (281 in total) were collected and analyzed for cis- and
trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylic acid (cis-Cl
2
CA and trans-Cl
2
CA), cis-
3-(2,2-dibromo-vinyl)-2,2-dimethylcyclo-propanecarboxylic acid (Br
2
CA) and 3-phenoxybenzoic acid
(3PBA) using a validated gas chromatography ion-trap mass spectrometry method. Only 3PBA was
detectable in more than 60% of the collected samples enabling a reliable statistical analysis. Statisti-
cal analysis was performed to evaluate temporal variability in urinary excretion of 3PBA over the studied
period. Both volume and creatinine (Cre) adjusted concentrations were evaluated with the latter one
being the most reliable. Among all samples, rst morning voids (FMV) were the least reproducible (inter-
class correlation coefcient ICC, 0.551 and 0.350 for volume and creatinine adjusted concentrations,
respectively). Spot and reconstructed 24-h samples were more reproducible in this study. ICC values for
ng/mL concentrations were 0.599 and 0.681 (in spot and 24-h samples) and 0.846 and 0.796 for g/g
creatinine concentrations.
Results of this study suggest fairly constant short-term exposure to pyrethroids metabolized to 3PBA
among the urban population in Poland. Creatinine adjustment should be performed in epidemiological
studies and spot or multiple spot samples should be preferentially collected for the highest reliability of
the measurement.
2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Insecticides play a very important role in the protection of
human health and signicantly improve agricultural productivity.
This is a group of pesticides of particular interest in toxico-
logy because they exhibit the same toxicity mechanism in target
organisms as in humans. One of the most widely used classes

Tel.: +48 58 349 16 72; fax: +48 58 349 16 75.

E-mail addresses: bartek@gumed.edu.pl, bartosz.wielgomas@gumed.edu.pl
of insecticides are actually synthetic pyrethroids (PYRs) which
partly replaced more toxic organophosphates and carbamates.
PYRs as active ingredients are components of many commercial
formulations employed in agriculture, gardening, households and
are of special importance in public health to prevent spreading
of the vector transmitted infections like malaria, tropical fevers,
etc. This group of insecticides is the only one recommended by
WHO for impregnation of anti-mosquito bednets (WHO, 2009).
Numerous biomonitoring surveys demonstrated wide exposure
amongthe populations of several countries: Germany(Heudorf and
Angerer, 2001), Japan (Kimata et al., 2009), Canada (Fortin et al.,
0378-4274/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxlet.2013.05.009
16 B. Wielgomas / Toxicology Letters 221 (2013) 1522
2008a) and USA (Barr et al., 2010). Recently, quantiable concen-
trations of 3PBA in the urine of a convenience sample of the Polish
population have been also reported (Wielgomas et al., 2013). Esti-
mation of systemic exposure is usually performed using urinary
metabolite concentrationmeasurement, because once ingestedthe
human body, PYRs undergo effective biotransformation and are
quite rapidly excreted in urine partly in conjugated forms as glu-
curonides and sulphates (Eadsforth et al., 1988; Sams and Jones,
2012; Eadsforth and Baldwin, 1983). This approach based on the
urinary metabolite excretion combines all routes and possible
sources of exposure. In the general population, non occupationally
exposedtopesticides, dietaryexposure seems tobe the most signif-
icant source of pyrethroids, however episodic exposure can occurs
also fromthe other sources (periodic or systematic indoor pesticide
application, dust, clothes and wool carpet impregnation). Besides
the diet, also pet care products and other household products con-
taining PYRs may play a signicant source of exposure (Keenan
et al., 2009). The dose recovered in urine is a result of combined
exposure to both parent compounds (native pyrethroids) and their
degradationproducts/metabolites that couldbeingestedwithfood,
air, dust or water (Morgan, 2012). It is still unidentied to what
extent exposure to degradation products plays a role in systemic
exposure, measured as urinary excretion. Based on the animal and
human experiments, toxicokinetics for most pyrethroids is well
known. It is ascertained that the half-lives for most PYRs are short.
In a few hours, the majority of the adsorbed dose is eliminated
through the kidneys, thus, urine analysis can estimate very recent
exposure (Eadsforth and Baldwin, 1983; Eadsforth et al., 1988).
Since the excretion is efcient and rapid, single measurement of
urinary concentration would be inappropriate and not reliable to
evaluate averaged individual exposure. Biomonitoring studies are
designed frequently to measure relationships or predict health
outcomes and exposure to hazardous factors. Many variables can
affect temporal variability of urinary excretion of specic biomark-
ers: gender, time of the year, diet, episodic pesticide application,
indoor/outdoor activity, etc. (Meeker et al., 2005; Lambert et al.,
2005). It is of the highest importance to precisely design a samp-
ling strategy in large scale observational studies. Depending on the
type of collected urine samples (spot sample, rst morning void or
24h collections), a study outcome could be affected due to the dif-
ferences in concentrations in those samples. Thus, studying short-
and long-term reliability of sampling is very important. If a single
spot sample is to be collected per day, rst morning voids (FMV)
are often preferred on the basis of the assumption that the FMV
provides an integrated sample over a relatively long part of the day
and is therefore more likely to be representative. However, this
assumption has not been validated, and no clear standards exist for
the correction for urine volume, contaminant excretion, dilution,
or time between exposure and void (Hwang et al., 1997).
Due tothe short half-lives andmixedexposure sources, PYRs are
suspected to show high variability over time that could aggravate
interpretation of the biomonitoring results, over- or underestimat-
ingof real exposure basedona single measurement approach. Since
levels of biomarkers of exposureinurinereect temporal variations
inbothexposure andphysiological processes, sampling designmay
signicantly affect analytical results (Hinwood et al., 2002).
Until now, short-term variability was studied for urinary con-
centrations of polycyclic aromatic hydrocarbon metabolites (Li
et al., 2010), bisphenol A (Ye et al., 2011), phthalate metabolites
(Peck et al., 2010; Preau et al., 2010), and organophosphate pes-
ticide metabolites (Kissel et al., 2005). Usually the concentrations
exhibited moderate to high variability described by an interclass
correlation coefcient (ICC).
In this work, we attempted to evaluate intra- and inter-day
personal variations in urinary 3-phenoxybenzoic acid over seven
consecutive days in seven people. The study was designed to
provide detailed information on exposure variability by observing
urinary biomarker concentrations. In regard to the epidemiolog-
ical studies, this knowledge can be used for designing exposure
assessment strategies and for adjusting for measurement errors in
pyrethroid exposure.
2. Materials and methods
2.1. Chemicals
The following chemicals were obtained from SigmaAldrich (Germany):
1,1,1,3,3,3-hexauoroisopropanol (HFIP), diisopropylocarbodiimide (DIIC), 3-
phenoxybenzoic acid (3PBA) and 2-phenoxybenzoic acid (2PBA) which was
used as an internal standard. Other metabolites: cis-3-(2,2-dibromo-vinyl)-2,2-
dimethylcyclo-propanecarboxylic acid(Br
2
CA), cis- andtrans-3-(2,2-dichlorovinyl)-
2,2-dimethylcyclopropane-carboxylic acid (cis-Cl
2
CA and trans-Cl
2
CA) were from
Dr. Ehrenstorfer GmbH (Germany). All other reagents were of analytical grade.
Standard stock solutions of 1mg/mL were prepared in acetonitrile and stored at
20

C, protected fromlight. Working standard solutions were prepared in acetoni-

trile and were stored at +4

C.
2.2. Subjects and sample collection
Inthis study(JuneOctober 2011), we recruitedfour womenandthree men aged
2471, residents of Gda nsk (Poland), who agreed to collect urine samples according
to a study protocol. All procedures were approved by the Ethics Committee of the
Medical University of Gda nsk, Poland (NKEBN/301/2011). Prior to enrolment in the
study, each subject signed the informed consent form. The study participants were
asked to collect all urine voids in precleaned, calibrated glass beakers (to measure
total voidvolume) andsaveabout 30mL fromeachvoidinthepolypropylenestorage
tubes. No dietary restrictions were forced, so the volunteers were allowed to follow
their own, regular dietary habits. All seven participants declared no occupational
exposure to pesticides or recent application at place of residence. Two male partic-
ipants (P2 and P3) were active cigarette smokers. Study participants characteristics
are shown in Table 1.
Collected urine samples were stored at the participant location in a portable
freezer (18/20

C) until theendof thecollectionperiodandwerethentransported

to the laboratory and stored further at 20

C until analysis.
All subjects collected in total 281 spot samples including 49 rst morning voids.
None of the subjects declared to miss any sample.
2.3. Chemical analysis
The concentrations of 3PBA were measured by gas chromatography ion-trap
mass spectrometry (GCMS) using electro-ionization operated in selected ion stor-
age detection mode. The extraction step was adopted froma previously established
method (Schettgen et al., 2002). Briey, 3mL of thawed urine were transferred into
10mL screw-top glass tube and 25L of IS solution (2PBA, 1g/mL of acetoni-
trile) along with 0.6mL concentrated hydrochloric acid were added. Samples were
incubated at 95

C in a laboratory oven for 90min. After bringing samples to room

temperature, 4mL of hexane were added and tubes were shaken for 15min. Fol-
lowing centrifugation, the hexane layer was transferred to the next screw-top glass
tube, and extraction with the same volume of hexane was repeated. Extracts were
combined and then reextracted with 0.5mL of 0.1MNaOH. Hexane was discarded,
while 0.1mL of concentrated HCl and 2mL of hexane were added to the remaining
aqueous phase. Samples were shaken again, centrifuged and the resulting super-
natant was evaporated to dryness under the streamof nitrogen at 45

C. The residue
was treated with 10L of HFIP, 15L of DIIC and 250L of hexane. Samples were
mixed for 10min at roomtemperature and then 1mL of 5% K
2
CO
3
was added. After
vigorous shaking, tubes were centrifuged and hexane layer was transferred to an
autosampler vial. Two microliters of the nal extract were analyzed by GCMS.
Analyses were performed using a Varian GC-450 gas chromatograph equipped
with 220-MS ion-trap mass spectrometer working in the selected ion storage (SIS)
mode. Derivatives were separated on VF-5ms (Varian, Palo Alto, USA) low bleed
column (30m0.25mmI.D., 0.25mlmthickness) using the following oven pro-
gram: 60

C 1min, 60150

C (8

C/min), 150280

C (30

C/min), 280

C 11min.
Selectedion(364m/z) was monitoredfor quantitationof 2PBAand3PBA. Two micro-
liters of the nal extract were injected (injector temperature 280

C) in splitless
mode with 25psi pulse pressure lasting 1.0min. Chromatograph was equipped with
1079 programmable temperature vaporizing injector and SGE focusliner glass inlet.
Urinary creatinine was measured with spectrophotometric Jaffe method.
2.4. Internal and external quality control
Quality of the assay was systematically monitored throughout the study. It was
accomplished by the inclusion in each analytical batch a series of quality control
samples. Physiological human urine spiked with analytes at three levels (0.5ng/mL,
1 ng/mL and 5ng/mL) was prepared in our lab, frozen and used when needed. In
addition, ClinCal

Urine Calibrator (Cat. No. 9969), lyophilized, for Toxic Organic

B. Wielgomas / Toxicology Letters 221 (2013) 1522 17
Table 1
Study participants characteristics.
Subject
number
Age Gender Body weight
(kg)
Number of
samples provided
Current smoking
status (Yes/No)
Occupation
1 34 Female 60 32 No Pharmacist
2 26 Male 92 55 Yes Pharmacist
3 27 Male 89 28 Yes Pharmacist
4 24 Female 49 56 No Student
5 34 Female 72 34 No Academic teacher
6 30 Male 69 38 No Student
7 71 Female 62 38 No Retired
Compounds acquired from Recipe Chemicals and Instruments GmbH (Munich,
Germany) was used to assure reliability of the analytical method. The calibrator
with declared concentrations of pyrethroid metabolites served as a quality control
material (3.6ng/mL, 3.3ng/mL, 2.5ng/mL and 6.6ng/mL, respectively for cis-Cl
2
CA,
trans-Cl
2
CA, Br
2
CA and 3PBA). In each analytical batch we analyzed 30 samples
including: 25 unknown, 4 spiked at different levels and one ClinCal

Urine Cali-
brator. For all metabolites a limit of detection (LOD) of 0.1ng/mL was established.
Coefcient of variation was in the range 4.68.3%, representing between day vari-
ability of the assay.
External quality control. The laboratory successfully participated in The German
External Quality Assessment Scheme For Analyses in Biological Materials (Round
No. 50) for pyrethroid metabolites.
2.5. Statistical analysis
The statistical analyses were performed with Statistica software from Statsoft
(Tulsa, USA) and Excel from Microsoft (USA). Concentrations below the detection
limit (LOD) were replaced with LOD/square root of two to reduce a bias (Baccarelli
et al., 2005). None of the outliers were excluded before statistical operations.
Descriptive statistics was calculated and is presented in Table 2. Simulated 24h
metabolite excretion was calculated as the volume-weighted average of all samples
collected by a single participant during this period. Due to the log-normal distribu-
tion the results were log transformed before statistical operations. Results where
p <0.05 were classied as statistically signicant. Graphs were constructed to visu-
ally evaluate temporal changes in urinary 3PBA (Fig. 1) as well as cis-, trans-Cl
2
CA
and Br
2
CAconcentrations (Fig. S1). Reproducibilityof thesinglemeasurement of uri-
nary 3PBA was assessed by a calculation of interclass correlation coefcient (ICC).
The interclass correlation coefcient is a measure of reproducibility of replicate
measures fromthe same subject. Calculation of ICC using a one-way random-effects
ANOVA model is described as a ratio of the between-person variance divided by the
sum of the between-person and the within-person variance. ICC is expressed by
a value in the range 01.0 (from non-reproducible to perfectly reproducible), ICC
lower than 0.4 indicates poor reproducibility, in the range of 0.40.75 fair to good
and over 0.75 excellent reproducibility (Rosner, 2011).
3. Results and discussion
Study participants characteristics are presented in Table 1.
A total of 281 spot samples including 49 FMVs were collected
during a 7 day study from seven participants and analyzed for
pyrethroid metabolites by a validated GCMS method. Each sub-
ject contributed on average 5.7 urine samples per day which
corresponds to about 40 urine samples per personweek. The con-
centrations of 3PBA ranged from less than LOD (0.1ng/mL) to
5.494ng/mL and 3.095ng/mL in spot urine samples and FMVs,
respectively. The remaining pyrethroid metabolites (cis-, trans-
Cl
2
CA and Br
2
CA) were detected with various but low frequency
(below 50%) and thus only descriptive statistics is presented
in this paper. According to a questionnaire none, of the voids
were missed by any subject. Concentrations of 3PBA, higher than
LOD were noticed in 81.9% and 93.9% of all spot and FMV sam-
ples, respectively. Only in one out of the seven persons, 3PBA
was at a quantiable level in all spot samples (participant P2).
Remaining metabolites: cis-, trans-Cl
2
CA and Br
2
CA were detected
in 40.9%, 47.3% and 3.2% of all spot urine samples collected in
this project. However, in individual participants detection ratio
for those metabolites ranged from 0.0% to 91.2% in all spot sam-
ples. When considering the FMVs and reconstructed 24-h samples
slightly higher detectivity was observed for cis- and trans-Cl
2
CA
(Table 2). The specic biomarker of deltamethrin (Br
2
CA) was the
least detectable: 2.0% in both FMVs and 24-h samples.
Detectivity and its range, arithmetic mean, median and geo-
metric mean concentrations in spot, FMVs and reconstructed 24-h
samples are given in Table 2. Mean creatinine adjusted concen-
trations of 3PBA, concentrations in FMV and reconstructed 24-h
samples are summarized in Table 3. Daily excretion of 3PBAranged
from 31ng to 1247ng (Table 4). Mean amount of 3PBA excreted
daily was from283.4 (P4) to 1055.0ng (P5) with the coefcient of
variation (CV%) ranged 15.572.4% among seven participants of the
study. Excretion of the other metabolites was not calculated.
Excretion of creatinine as expected was gender dependent.
Males excreted signicantly more creatinine daily than women
(1744 vs. 1120mg; test U MannWhitney, p<0.0001).
In general, creatinine adjusted concentrations in FMVs were
higher than the mean concentrations of spot samples for person-
day in 32 of 49 cases. In 16 cases FMV concentrations were lower
thantheaveragespot andinonecasetheconcentrations wereequal
in FMV and spot samples. Mean concentrations of 3PBA in FMVs
were about 10% higher than mean calculated for spot urine sam-
ples (0.300 vs. 0.271g/g Cre) but similar to 24-h reconstructed
samples.
High Spearman correlations (r >0.8) were observed between
FMVs, spot and 24-h collections. The highest association was
observed between 24-h and spot samples (r =0.9468), medium
(r =0.8803) between FMVs and 24-h and the lowest for FMVs and
mean spot (r =0.8158).
In this work, we calculated daily excretion of creatinine for each
subject during the study periodandfurthermore ICCwas calculated
for 24-h excretion of creatinine and its value 0.792 suggests good
repeatability.
Reproducibility of daily 3PBAexcretionamong subjects was also
evaluated by calculation of ICC=0.715 (Table 5). Weekly excre-
tion of 3PBA was calculated as a sum of seven consecutive 24-h
excretions expressed in nanograms and total amount of excreted
metabolite ranged from826ng (P4) to 7154ng (P5).
Reliability of each sampling category (FMV, spot and simu-
lated 24-h) was evaluated by calculating interclass correlation
coefcientICC, the parameter describing the reliability of repeated
samplings. In Table 5, ICC values are summarized for both: volume
and creatinine adjusted 3PBA concentrations. In general, moder-
ate to low variability was observed for urinary 3PBA over a 7 day
periodinsevenstudyparticipants (ICCintherange0.3500.846) for
creatinine adjusted concentrations. Higher overall variability (ICC:
0.5510.681) was noticed in the volume expressed concentrations
(ng/mL). The only exception is 3PBA measured in the rst morning
voids, ICC 0.551 vs. 0.350 (ng/mL vs. ng/g Cre).
4. Discussion
Since the biomonitoring of exposure to pyrethroids among gen-
eral populations of different countries started over decade ago,
therearestill somegaps inknowledgetobelledfor morecomplete
understanding of human exposure. This is particularly important
18 B. Wielgomas / Toxicology Letters 221 (2013) 1522
Table 2
Descriptive statistics of pyrethroid metabolites concentrations in urine samples (n=281) obtained fromseven study participants.
Sample type Metabolite %>LOD (range)
a
Concentrations
Range GM Median Mean (SD)
Spot (n=281) 3PBA 81.9 (60.6100.0) ng/mL <LOD5.494 0.278 0.263 0.489 (0.675)
g/g Cre <LOD3.095 0.271 0.253 0.385 (0.397)
cis-Cl
2
CA 40.9 (1.891.2) ng/mL <LOD5.602 nc
b
nc nc
g/g Cre <LOD3.079 nc nc nc
trans-Cl
2
CA 47.3 (3.691.2) ng/mL <LOD9.362 nc nc nc
g/g Cre <LOD3.048 nc nc nc
Br
2
CA 3.2 (0.07.9) ng/mL <LOD1.070 nc nc nc
g/g Cre <LOD0.503 nc nc nc
FMV (n=49) 3PBA 87.8 (71.4100.0) ng/mL <LOD2.781 0.317 0.315 0.512 (0.535)
g/g Cre <LOD3.096 0.300 0.270 0.479 (0.549)
cis-Cl
2
CA 45.2 (0.0100.0) ng/mL <LOD1.575 nc nc nc
g/g Cre <LOD0.632 nc nc nc
trans-Cl
2
CA 59.5 (14.3100.0) ng/mL <LOD2.346 nc nc nc
g/g Cre <LOD0.941 nc nc nc
Br
2
CA 2.0 (0.014.3) ng/mL <LOD0.680 nc nc nc
g/g Cre <LOD0.343 nc nc nc
24-h (n=49) 3PBA 93.9 (71.4100.0) ng/mL <LOD2.150 0.305 0.270 0.441 (0.435)
g/g Cre <LOD1.506 0.299 0.247 0.402 (0.350)
cisCl
2
CA 54.8 (28.6100.0) ng/mL <LOD1.897 nc nc nc
g/g Cre <LOD1.329 nc nc nc
trans-Cl
2
CA 59.5 (0.0100.0) ng/mL <LOD3.170 nc nc nc
g/g Cre <LOD2.220 nc nc nc
Br
2
CA 2.0 (0.014.3) ng/mL <LOD0.159 nc nc nc
g/g Cre <LOD0.213 nc nc nc
LOD: limit of detection, 0.1ng/mL; GM: geometric mean; SD: standard deviation; and 24-h: simulated 24h samples.
a
Minimumand maximumvalues found among seven participants.
b
Not calculated due to the lowdetectivity (<60%).
because of the expected increase in the use of pyrethroids in the
next years.
Despite the wide use of pyrethroid insecticides, evaluation
of exposure among Polish population is limited. In this paper,
some additional data on pyrethroid exposure in non-occupational
settings are presented. Results of this study expand our knowl-
edge on exposure characteristics and allow for a more accurate
designing of observational studies to be performed on general pop-
ulations.
As expectedfromour previous ndings (Wielgomas et al., 2013),
widespread exposure to pyrethroids among the adult populationof
Poland is conrmed. The overall frequency of detection and deter-
mined mean concentrations of 3PBA are similar to earlier results,
where only FMVs were collected due to the ease of collection. The
Table 3
Mean concentrations of 3PBA (g/g Crea) for each subject. For spot samples standard deviation of the mean is presented in parenthesis.
Day Sample Participant
P1 P2 P3 P4 P5 P6 P7
Mon Spot 0.154 (0.083) 0.358 (0.136) 0.195 (0.023) 0.299 (0.112) 1.031 (0.516) 0.260 (0.169) 0.604 (0.245)
FMV 0.146 0.641 0.168 0.329 1.404 0.578 0.997
24-h 0.124 0.362 0.192 0.489 0.912 0.419 0.779
Tue Spot 0.590 (1.107) 0.531 (0.391) 0.093 (0.044) 0.340 (0.064) 1.246 (0.218) 0.131 (0.044) 0.244 (0.098)
FMV 3.095 0.424 0.046 0.430 1.332 0.202 0.280
24-h 0.573 0.509 0.096 0.347 1.271 0.142 0.224
Wed Spot 0.214 (0.037) 0.622 (0.278) 0.150 (0.036) 0.241 (0.071) 1.210 (0.185) 0.440 (0.790) 0.263 (0.114)
FMV 0.224 0.644 0.164 0.299 1.143 0.122 0.375
24-h 0.212 0.624 0.150 0.238 1.215 0.561 0.257
Thu Spot 0.134 (0.054) 0.266 (0.128) 0.087 (0.034) 0.222 (0.071) 1.529 (0.205) 0.324 (0.215) 0.224 (0.045)
FMV 0.134 0.573 0.048 0.248 1.531 0.111 0.190
24-h 0.125 0.318 0.089 0.211 1.505 0.174 0.221
Fri Spot 0.327 (0.051) 0.216 (0.031) 0.236 (0.160) 0.193 (0.068) 1.202 (0.342) 0.400 (0.257) 0.235 (0.155)
FMV 0.375 0.253 0.109 0.266 1.267 0.683 0.165
24-h 0.335 0.225 0.161 0.195 1.155 0.384 0.169
Sat Spot 0.466 (0.092) 0.236 (0.044) 0.184 (0.086) 0.299 (0.111) 0.954 (0.186) 0.276 (0.174) 0.268 (0.100)
FMV 0.380 0.271 0.104 0.124 0.949 0.456 0.369
24-h 0.481 0.225 0.177 0.268 1.012 0.315 0.247
Sun Spot 0.197 (0.074) 0.198 (0.122) 0.270 (0.106) 0.146 (0.026) 0.972 (0.148) 0.156 (0.056) 0.110 (0.083)
FMV 0.270 0.271 0.395 0.161 1.115 0.079 0.110
24-h 0.208 0.199 0.271 0.152 0.981 0.128 0.080
Week Spot 0.297 0.347 0.174 0.249 1.163 0.284 0.278
FMV 0.661 0.440 0.148 0.265 1.249 0.319 0.355
24-h 0.294 0.352 0.162 0.271 1.150 0.303 0.282
B. Wielgomas / Toxicology Letters 221 (2013) 1522 19
Fig. 1. The urinary 3PBA concentrations in seven persons over one week observation. One vertical segment of each graph represent one day. A dotted lineunadjusted
concentrations (ng/mL) and solid linecreatinine adjusted concentrations (g/g Cre).
primary route of exposure to pyrethroids in Poland among non-
occupationally exposed population is considered to be the diet,
however incidental high exposure to locally applied pyrethroids
cannot be excluded. Thus the results of current study reect
only short-term variability in the urbanized regions of Poland
where insecticides are used incidentally mainly during the sum-
mer season. The results of this study should be considered as a
representative to the adult population, not exposed occupationally
to pesticides, not using themat home and having a regular lifestyle
throughout the week (diet, physical activity, outdoorindoor activ-
ity ratio). Relatively constant levels of metabolites in each subject
over seven consecutive days could suggest a stable, background
average exposure from all available sources in an urban microen-
vironment. However, in different regions of the world temporal
variability in the urinary concentrations of pyrethroid metabolites
can differ due to the many reasons: lifestyle, place of residence
20 B. Wielgomas / Toxicology Letters 221 (2013) 1522
Table 4
Summary of daily excretion of 3PBA, creatinine and total 24-h urine volume.
Subject Mon Tue Wed Thu Fri Sat Sun Mon-Sun
Mean SD CV%
P1 3PBA, ng 117.8 706.7 233.2 177.9 391.1 592.6 244.5 352.0 222.1 63.1
Creatinine, mg 952.0 1232.6 1100.4 1422.3 1167.3 1232.3 1177.0 1183.4 143.0 12.1
Urine volume, mL 1350.0 2620.0 1570.0 1975.0 1010.0 1360.0 1620.0 1643.6 522.4 31.8
P2 3PBA, ng 701.3 1243.0 1177.8 763.4 458.1 615.0 437.9 770.9 323.1 41.9
Creatinine, mg 1936.9 2443.1 1888.8 2402.1 2039.5 2733.3 2204.7 2235.5 307.5 13.8
Urine volume, mL 1230.0 1295.0 1105.0 1435.0 1660.0 1440.0 1430.0 1370.7 179.0 13.1
P3 3PBA, ng 339.9 155.5 304.9 169.4 384.6 303.6 537.2 313.6 130.2 41.5
Creatinine, mg 1768.6 1611.0 2039.3 1906.3 2386.4 1711.4 1980.1 1914.7 257.3 13.4
Urine volume, mL 1095.0 885.0 1240.0 1070.0 3720.0 1400.0 1580.0 1570.0 975.1 62.1
P4 3PBA, ng 344.8 411.7 278.1 266.6 222.4 321.1 139.1 283.4 88.1 31.1
Creatinine, mg 705.2 1186.4 1166.2 1264.2 1143.3 1199.0 915.9 1082.9 199.3 18.4
Urine volume, mL 1610.0 1585.0 1570.0 2070.0 1920.0 2050.0 1400.0 1743.6 265.4 15.2
P5 3PBA, ng 1213.2 1078.7 1100.7 1246.9 996.7 992.4 756.5 1055.0 163.8 15.5
Creatinine, mg 1329.9 848.9 906.1 828.2 862.8 980.3 771.3 932.5 186.9 20.0
Urine volume, mL 1100.0 1260.0 750.0 580.0 750.0 1580.0 580.0 942.9 380.6 40.4
P6 3PBA, ng 408.7 210.2 573.1 91.2 516.8 362.2 182.9 335.0 179.8 53.7
Creatinine, mg 975.5 1475.8 1021.9 521.2 1346.7 1148.8 1428.9 1131.2 332.3 29.4
Urine volume, mL 1680.0 1230.0 1450.0 1230.0 610.0 800.0 1240.0 1177.1 365.3 31.0
P7 3PBA, ng 864.7 206.6 281.4 343.0 200.4 349.7 127.7 339.0 245.3 72.4
Creatinine, mg 1234.5 921.6 1095.8 1552.6 1184.4 1417.5 1680.3 1298.1 266.1 20.5
Urine volume, mL 1490.0 930.0 1420.0 970.0 925.0 910.0 1315.0 1137.1 259.4 22.8
(rural or urban location), vicinity of orchards and cultivating crops,
frequency of application or different application rate of pesticides.
The aim of this work was to quantitatively describe the vari-
ability in excretion of 3PBA in humans over seven consecutive
days. Innumerous studies onexposure to non-persistent chemicals
(phthalates, phenols, bisphenol A, etc.) repeated measurements of
urinary metabolites in the same person were not well correlated
over the relatively short period of time. Moreover, the within-
person variability was in some cases shown to be higher than the
between-person variability (Grifth et al., 2011; Braun et al., 2012).
For optimal exposure classication it seems reasonable to perform
several repeatedmeasurements withinone subject collecting urine
voids at different times of the day. The relationship between a sin-
gle urinary metabolite measurement and the mean of a series of
measurements from the same participant was evaluated among
samples collected over seven days. The reliability of a particular
measurement in representing a true longer-termaverage is a func-
tion of the variability of a series of measurements. It is a function
of the magnitude of the intra-person component of variance with
respect tothe total variance, or the interclass correlationcoefcient
of reliability (ICC). An ICC of at least 0.80 is generally considered to
indicate good reliability (Egeghy et al., 2011; Rosner, 2011).
A 24-h urine collection is recognized as a gold standard in expo-
sure assessment inbothoccupational and experimental studies but
it is not practical for the large cross sectional studies for many rea-
sons (cost, subjects burden, etc.). From a practical point of view,
Table 5
Interclass correlationcoefcients for volumeandcreatinineadjustedconcentrations
of 3PBAinurinesamples fromsevensubjects intheperiodof sevenconsecutivedays.
ICC (95% CI)
ng/mL g/g Cre
FMV 0.551 (0.2600.871) 0.350 (0.0970.766)
Spot 0.599 (0.3080.890) 0.846 (0.6580.966)
24 h 0.681 (0.4040.919) 0.796 (0.5740.953)
Creatinine excretion
(g/24h)
0.792 (0.5660.951)
3PBA (ng/24h) 0.715 (0.4540.929)
single urine voids (spot samples referred to a single urine void col-
lected at any time of the day) as well as rst morning voids (FMV)
are collected mostly due to simplicity. Non-persistent chemicals
excreted as their metabolites with urine can exhibit high intraindi-
vidual variability due to the episodic exposure and toxicokinetics
(short half lives). Among spot urine samples, the rst morning void
is sometimes used as a compromise between ease of collection and
reliability. FMV samples are usually more concentratedbecause the
collection (production) period is longer than the average spot sam-
ple. According to studies of Kissel et al. (2005) and Grifth et al.
(2011) concentrations of organophosphate metabolites found in
FMVs are the best predictors of estimated total daily excretion
in pre-school children. Contrary for bisphenol A, the other non-
persistent chemical, its concentrations in the rst morning voids
were may not be good surrogates for 24-h collections (Ye et al.,
2011).
Urine dilution may affect the evaluation of temporal variability
andcreatinine correctionprovides a more representative approach.
Creatinine adjustment is often used for compensation in urine
dilution, however its excretion rate is also dependent on many
variables other than gender: physical activity, age, pregnancy, diet
etc. (Lorber et al., 2011; Barr et al., 2005). Fortin et al. (2008b)
reported creatinine excretion and urine volume production in 15
adult Canadians with coefcients of variation being respectively in
the range of 35.644.6% and 38.748.2%. In the current study CVs
were respectively 13.162.1% and 9.029.4% for urine volume and
creatinine excretion. The same authors (Fortin et al., 2008b) con-
cluded that for a more accurate assessment of individual adsorbed
dose, it is better to determine total amounts of metabolites in urine
collectedover thelongest feasibleandpractical periodof time. Rela-
tively constant exposure levels to pyrethroids or their degradation
products documented in this work suggests that dietary sources
remain the major route of exposure in Poland among the non-
occupationally or accidentally exposed individuals. It is worth to
stress that urine collection for this study was started during the
summer and nished in the early autumn. In Poland it is the period,
when insecticides are used in households in higher quantities than
during the rest of the year. None of the study participants lived in
a house, but all in apartments in the city where either mosquitoes
B. Wielgomas / Toxicology Letters 221 (2013) 1522 21
or ies are rare and in general insecticide application rate is low.
The insecticide usage prole could be quite different for rural and
suburban areas and needs further attention.
Daily excretion of 3PBA in study participants are in the range
comparable with those observed in other studies. Saieva et al.
(2004) reported daily (based on the 24-h urine samples) excre-
tion of 3PBA in inhabitants of two Italian cities at a mean level of
6nmol/day which corresponds to 1285ng (3PBA m.w. 214.22).
Our results conrmprevious observations of temporal changes
in 3PBA urinary levels in US children (Egeghy et al., 2011). Authors
noticed fairly constant (ICC 0.69) creatinine adjusted concentra-
tions in children, participants of CTEPP study (The Childrens Total
Exposure to Persistent Pesticides and Other Persistent Organic Pol-
lutants study), reporting a recent application of any pesticide. In
current work none of the subjects declared insecticide application
in their home, however, the use of pesticides in the past cannot
be ruled out. It is known that the half-life of certain pesticides in
house dust can reach up to several months or years. Pyrethroids
have a very lowvapor pressure and showhigh absorption to mate-
rials, such as textiles, and to dust. They are persistent indoors, with
the half-life of long-lasting pyrethroids (e.g. deltamethrin) being
estimated at about 10 years (Kolaczinski and Curtis, 2004).
Individual exposure level from sources other than diet could
be stable for a long period when the given person is in the same
local environment and with the assumption that levels of con-
taminants in this environment show little variability over time.
Non-persistent pesticides like organophosphates or pyrethroids
are usually foundindoor inhigher concentrations thanoutdoor due
to the lack of, or lower intensity of environmental factors respon-
sible for their degradation: light, microorganisms, humidity, etc.
(Simcox et al., 1995).
The limitations of this study included: only adult volunteers
were involved while children may demonstrate dissimilar pat-
tern of exposure due to e.g. high hand to mouth activity, higher
house dust ingestion, indoor/outdoor activity ratio, etc. (Butte and
Heinzow, 2002). Moreover, usually during the 7 day observational
period, food products consumed by each participant were of the
same origin and repeatable day-to-day lifestyle activities partly
explain the low variability in the exposure. Thus it is advisable to
monitor long-term changes in exposure to pyrethroids as well by
analyzing urine samples fromthe same subjects over the period of
several months.
The volunteers that participated in this study lived in an urban
environment, while in rural and agricultural areas the exposure
may differ signicantly.
It is alsostill unansweredtowhat extent unchangedpyrethroids
are present in food and in the environment in comparison to their
metabolites or degradation products. Further studies are needed
to identify the fate of pyrethroids in food and in the environment
including behavior of indoor applied formulations. Most of the
biomonitoring studies concluded diet as a major source of expo-
sure to pyrethroids for non-occupationally exposed humans. High
pesticide residues in food products, thanks to a reliable systemfor
food and feed monitoring are now uncommon, and thus pose lit-
tle threat to public health. A more signicant problemseems to be
the use of insecticides in dwellings, particularly when children, the
elderly or pregnant womenmight be exposed. Therefore, it appears
advisable to focus further research on the estimation of exposure
in such cases and the development of appropriate guidelines to
protect the most vulnerable subpopulations.
5. Conclusions
Inthis study we monitoredurinary concentrations of pyrethroid
metabolites in seven healthy persons to describe short-term
intra- and inter-person variability. A knowledge on temporal
changes in urinary excretion is a prerequisite for the correct
exposure classication in observational studies. It is particularly
important for pollutants with short half-lives. Our ndings sug-
gest a relatively stable exposure level to synthetic pyrethroids
based on the urinary concentrations of 3PBA in the Polish urban,
non-occupationally exposed population. In our study we examined
all urine samples from seven individuals over the period of one
week and revealed fairly stable biomarker concentration in urine,
which means that the determination of the concentration in a sin-
gle randomurine sample with high reliability reects the average
exposure.
Conict of interest
None declared.
Acknowledgments
This study was funded by grant ST-5 fromMedical University of
Gda nsk. The excellent technical assistance of Ms. Teresa Pawowska
is greatly acknowledged. Author would like to thank all study par-
ticipants for their valuable contribution.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.toxlet.2013.
05.009.
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