This study measured levels of the chlorpyrifos metabolite TCPy in urine samples from adolescent Egyptian cotton field workers over multiple time points. The workers were divided into applicators and non-applicators from two villages with different chlorpyrifos application schedules. TCPy levels increased in applicators during application periods and remained elevated for at least 74 days after. Non-applicators also showed increased TCPy levels during application, indicating environmental exposure. The study demonstrated urinary TCPy is a sensitive biomarker for chlorpyrifos exposure that can show both occupational and environmental exposures over time in this population.

1. Table 2: Descriptive statistics of urinary TCPy (ug/g
creatinine) of Al-Shohada on June 20, 2010 (top) and Sept.
29, 2010 (bottom) from region population sizes(N) of 31 and
30, respectively.
Table 1: Descriptive statistics of urinary TCPy (ug/g
creatinine) of Berket El-Sabe on June 20, 2010 (top) and
Sept. 29, 2010 (bottom) from region population sizes(N) of
25 and 29, respectively.
Using Biomarkers to Assess Chlorpyrifos Exposure in
Adolescent Egyptian Cotton Field Workers: A Longitudinal Study
Ashley Peppriell, SUNY Geneseo
Dr. James R. Olson & Lai Har Chi, Pharmacology andToxicology, University at Buffalo
Introduction
Methods
Results
• All workers, regardless of job category or region, showed detectable levels of TCPy at
baseline.
• TCPy levels in applicators increased compared to their baseline values. These remained
elevated from baseline for at least 74 days.
• TCPy levels in applicators increase during the CPF application period. The average levels
approach baseline, but don’t’ return over the course of this study.
Acknowledgements
Study Population
• Adolescent male field workers (12-21 years of age) from the Menoufia Governate, Egypt;
a region in the Nile Delta north of Cairo
• Two job categories: applicators and non-applicators
• Two villages: Berket El-Sabe and El Shohada
• Two different spray schedules (Figure 3)
On a global scale, Chlorpyrifos(CPF) is one of the most
commonly applied organophosphate pesticides (OP). However,
the EPA in the United States has gradually tightened restrictions
on CPF since 2000, and has recently issued a proposal to revoke
all tolerances by Dec. 2016. There is concern because of the
neurotoxic potential of this OP insecticide.
In Egypt, seasonal agricultural workers report some of the highest
occupational exposures of CPF in the world (Crane, et al. 2013).
To assess chlorpyrifos exposure in an adolescent cohort of
Egyptian agriculture workers, urinary TCPy levels were measured
over the course of a longitudinal study. Adolescents were studied
because their still-developing bodies may be more susceptible to
the neurological effects of pesticide exposure. Neurological
deficits result from eventual inhibition of acetylcholinesterase
(AChE) by the oxon metabolite of chlorpyrifos. As shown in Figure
1, the CPF-oxon inhibits cholinesterases such as
butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE)
(Farahat, et al., 2011). In addition, chlorpyrifos is detoxified into
trichloro-2-pyridinol(TCPy), a non-toxic metabolite. In this study,
TCPy has been used as a biomarker for exposure to Chlorpyrifos.
Aims
1. Demonstrate that urinary levels of TCPy are a sensitive & specific
biomarker of occupational and environmental exposure to
Chlorpyrifos.
2. Investigate the temporal decline in urine TCPy levels following the
end of daily CPF application to cotton fields.
.
Figure 4 : Average urinary TCPy (ug/g creatinine) levels of adolescent Egyptian cotton field workers dates Baseline, 6/20/10,
and 9/29/10. Baseline indicates the first sample collection date on 4/12/10. All groups report TCPy at baseline, which is over two
months prior to the start of scheduled CPF spray for either group. TCPy levels were compared using Wilcoxon Rank-Sum test
for significant differences between dates, job-category(applicator v. non-applicator), and region(Berket El-Sabe v. Al-Shohada).
* Indicates significant difference.
Figure 1 (left): Metabolism of
chlorpyrifos(CPF) pesticide.
CPF first undergoes oxidation
into an intermediate , followed
by either bioactivation to the
chlorpyrifos-oxon (light blue) or
detoxification into trichloro-2-
pyridinol (TCPy- green). The
CPF-oxon is potent inhibitor of
cholinesterases such as AChE,
while TCPy is a non-toxic
metabolite and is excreted in
the urine.
Figures 2a-2b: log mean urinary TCPy (ug/g creatinine) of applicators (figure 2a) and non-applicators (figure 2b) across
32 time points of a longitudinal study of adolescent Egyptian cotton field workers. Sample collection dates are indicated at
each solid black dot to represent the study span from 4/12/10-7/17/11. The colored, vertically spaced dots indicate the
individual samples collected on 6/20(green) and 9/29(blue) for both regions. The pink and blue shading represents the
different CPF sprays in Berket El Sabe and Al-Shohada, respectively, while purple indicates overlap of the two schedules.
Conclusions
1.Urinary TCPy is a sensitive and specific biomarker of chlorpyrifos exposure,
which directly reflects elevated exposures during the period of CPF application
and a prolonged recovery period necessary to reach baseline (pre-application)
levels.
2.In non-applicators, TCPy levels also increased during the application season,
which suggests environmental exposure during the application season.
3.Marked variability in urinary TCPy levels at a given time point indicates the wide
range of individual exposures and the importance of utilizing this biomarker to
more accurately reflect the magnitude of pesticide exposure for each individual.
Urinary TCPy levels
TCPy was extracted from urine using the
method outlined in Farahat et al., 2011.
Each urine was spiked with the internal
standard, 13
𝐶−15 𝑁 -3,5,6-TCPy. Samples
were hydrolyzed and extracted with
toluene. The extracts were derivatized and
analyzed by GC/MS. TCPy levels in urine
were normalized to creatinine, and
expressed as ug/g creatinine. The Jaffe
reaction was used to measure the urinary
creatinine concentrations (Fabiny, et al.,
1971) for all samples. TCPy levels were
compared via Wilcoxon Rank-Sum test for
non-parametric, paired samples.
1
10
100
1000
10000
logMeanUrinaryTCPy
(µg/creatinine)
June 20th Sept. 29thapplicator
1
10
100
1000
10000
logMeanUrinaryTCPy
(µg/creatinine)
Sample Collection Dates (M/D/Y)
controls June 20th Sept. 29th
Figure 3: Timeline for CPF application in Berket El- Sabe, which lasted from 6/17/10 - 7/17/10, and Al-Shohada, which lasted
from 6/24/10 - 7/17/10 and again on 8/4/10-8/8/10. The different spray schedules of the two regions allowed comparison of
CPF exposure pre-spray(Al-Shohada) and during spray(Berket El-Sabe) on 6/20/10, as well as the TCPy levels in groups
having temporal differences from the last spray to 9/29/10 (74 and 53 days, respectively). Baseline indicates the first sample
collection date on 4/12/10)
Shohada Non-Applicator Applicator
N 14 17
Mean 6.11 9.79
Median 4.99 5.37
SD 4.89 13.26
Max 20.17 55.12
Min 1.46 1.03
Berket Non-Applicator Applicator
N 10 15
Mean 5.83 53.33
Median 6.34 16.86
SD 2.55 84.83
Max 8.84 259.30
Min 2.60 1.94
Shohada Non-Applicator Applicator
N 13 17
Mean 6.17 24.52
Median 5.37 16.94
SD 3.26 23.04
Max 12.84 89.44
Min 2.36 4.71
Berket Non-Applicator Applicator
N 9 20
Mean 13.51 18.97
Median 7.10 6.71
SD 16.05 25.15
Max 48.81 102.39
Min 3.31 2.47
6/20/109/29/10
Berket El-Sabe Al-Shohada
• The Olson lab; specifically Dr. James R. Olson , Lai Har Chi, Barbara McGarrigle,
Mallorey Himmel & Sam VanEtten for mentoring, experience, and support .
• CLIMB UP program at UB
• SUNY Geneseo
• Research supported by the Zannoni SURF Award, The American Society for
Pharmacology and Experimental Therapeutics
Figure
2b
Figure
2a
Urine sample
& standards
+ Alkaline
picrate
reagent
Incubate ~30
minutes
Read at 490nm
Determine
creatinine
Hydrolysis
Extraction
Derivatization
GC/MS
analysis
Determine
TCPy
References
-Crane et.al.(2013). Longitudinal Assessment of Chlorpyrifos Exposure and Effect Biomarkers in Adolescent
Egyptian Agricultural Workers. 2013. Journal of Exposure Science and Environmental Epidemiology, 23: 356-362.
-Farahat et al. (2011) Biomarkers of chlorpyrifos exposure and effect in
Egyptian cotton field workers. Environmental Health Perspectives, 119: 801-806.
-Rohlman (2016) 10-month prospective study of organophosphorus pesticide exposure and neurobehavioral
performance among adolescents in Egypt. Cortex 74:383-395.