“Biomonitoring of Organophosphate

Pesticide Metabolites in Urban Children”

THESIS SUBMITTED TO OSMANIA UNIVERSITY

FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY
IN
ENVIRONMENTAL SCIENCE

BY
VENKAT REDDY .B

NATIONAL INSTITUTE OF NUTRITION

(INDIAN COUNCIL OF MEDICAL RESEARCH)
HYDERABAD-500007, INDIA
2018

CERTIFICATE

This is to certify that Mr. VENKAT REDDY.Bhas carried out the research work presented in

this thesis titled “Biomonitoring of Organophosphate Pesticide Metabolites in Urban

Children”under my supervision at the National Institute of Nutrition, Hyderabad. The work is

original and has not been submitted either in part or in full for the award of any other degree or

diploma. I hereby recommend the submission of the thesis for the award of the degree of Doctor

of Philosophy in Environmental Science.

Date: 25May 2018

(Dr.Sukesh Narayan Sinha)
Scientist-E
National Institute of Nutrition
Hyderabad – 500007.

3
 DECLARATION

I hereby declare that the research work described in this thesis entitled “Biomonitoring of

Organophosphate Pesticide Metabolites in Urban Children” has been carried out by me at

the National Institute of Nutrition, Hyderabad, under the guidance of Dr.Sukesh Narayanan

Sinha, Scientist-E. The work is original and has not been submitted either in part or in full for

the award of any other degree or diploma.

Date: 25 May 2018 (VENKAT REDDY.B)

4
 ACKNOWLEDGEMENTS

I would like to extend my sincere thanks and gratitude to many people who so willingly and
whole heartedly helped me while I was doing my research work which is being presented in this
thesis.

Special mention goes to my energetic, active and dynamic supervisor, Dr.S.N.Sinha who has
been always a guiding force during my PhD work. My PhD has been an amazing experience,
and I thank him, not only for his uncompromised desire which was resolute to help me
complete my PhD, and also giving me space to think independently and carry out my work. I
strongly feel this has been a privilege bestowed upon me by the almighty. My sincere thanks go
to the Director, NIN, and HOD, Dr.Arjun Khandare, who stood by me during thick and thin of
my tenure without their support it wouldn’t have been easy for me to carry out my research
work.

I convey my special thanks with profound sincerity to Professor Dr. Nirmala Babu Rao who has
always been a helping hand and spared her precious time in spite of her busy schedule, and I am
running short of words to express my love and affection as she had always treated me child-
likely and helped me in all possible ways to carry out my research work. My special thanks go
to HOD and Professor Dr. Hari Rama Krishna who as a keen observer always expected me to
be meticulous in planning the work, even though I was little scared to approach him initially but
his perfectionism helped me carry out my research work smoothly, and I will always have a
special place for him in my heart throughout my life.

I express my special thanks to Dr. Shasikala and Dr. Azim Unnisa Begum and all the other staff
members of the Department of Environmental Science, who supported me whenever I needed
support and visited their department.

I am grateful to my colleagues, who have maintained a harmonious atmosphere in the lab

throughout my tenure. I am also grateful to my friends who have supported me along the way. I
am also hugely appreciative of Mr. K. Vasudev (S.T.O), who acted like a secondary supervisor
and being like a mentor to me, especially for sharing his lab expertise so willingly and without
his language editing and corrections for my manuscripts; I wouldn't have published any paper.
It was so nice of Mr.Paul, Mr. Noor Ahmed, Mr. Asif Shaik, Mr.Suresh, Mr.Mahender,

5
Mr.Rama Krishna, Mr.Rajesh Kumar, Mr. Balaji, Mrs Joshna, Ms. Hema, Ms Veena,
Ms.Divya, and Ms.Mamatha for lending their helping hands while I was doing my field work,
lab work, and data analysis.

Finally, but by no means least, my thanks goes to my inner soul being the most important
person, friend, mentor and guide in my world, and whenever I was mentally and morally weak
it was always with me and made me do my best and guided me on the right path, even though
sometimes I did not listen to it, due to becoming selfish and getting into my comfort
zoneinstead of treading the thorn filled right path of truth.

I dedicate this thesis to myself and if there is anything positive in this, it belongs to the good
people who made me realize this and if there is anything negative which has not been discussed
about, it is me and I always hold myself responsible for all the mistakes done knowingly or
unknowingly.

VENKAT REDDY .B

6
 TABLE OF CONTENTS

Content Title Page No.

Chapter – I Introduction 010-021

Chapter – II Methods and Materials 022-048

Chapter – III Development of a new method for exposure 049-057

assessmentof organophosphate pesticide

metabolites

Chapter – IV Biomonitoring of organophosphate 058-95

pesticide metabolitesin Hyderabad urban

children

Chapter – V In-silico studies on Human Reproductive 96-105

Hormonesand Interactions with

Organophosphate.

Chapter – VI Final conclusions of studies 106-112

Bibliography 113-137

Publications 138-139

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 ABBREVIATIONS

AChE Acetylcholinesterase

CDC Centers for Disease Control and Prevention

CRFD Chronic oral reference dose (U.S. EPA)

DAP Dialkyl phosphate

DEDTP Diethyl dithiophosphate

DEP Diethyl phosphate

DEs Molar concentrations of Diethyl Metabolites

DETP Diethyl thiophosphate

DMDTP Dimethyl dithiophosphate

DMP Dimethyl phosphate

DMs Molar concentrations of Dimethyl Metabolites

DMTP Dimethyl thiophosphate

EPA Environmental Protection Agency

GM Geometric mean

F Female

FIA Federal Insecticide, Fungicide, and Rodenticide Act

FQA Food Quality Protection Act

LC-MS Liquid Chromatography – Mass Spectrometry

LC-MS/MS Liquid Chromatography – Mass Spectrometry/ Mass Spectrometry

LOD Limit of detection

LOQ Limit of quantification

M Male

Max Maximum

8
MUA Morning unadjusted urine samples.

MA Morning adjusted urine samples.

24-hr 24hour urine samples.

FMV First morning voids

Min Minimum

HIG High Income Group

LIG Low Income Group

MIG Medium Income Group

GC Gas Chromatography

OP Organophosphate/Organophosphorus

RSD Relative standard deviation

MRM Multiple reaction monitoring

N Number of samples

OP Organophosphate / Organophosphorus

Q Quadrupole

CV Coefficient of variation

Y/yrs Years

SD Standard deviation

FNTN Fenitrothion

CLPS Chlorpyrifos

US EPA (United States Environmental Protection Agency)

9
10
More than 10,000 years ago humans started the practice of agriculture in the Mesopotamia
[Kislevet al. 2004]. Farmed crops observed to suffer from pests and diseases causing a large loss
in yield. However,even today with advanced agricultural technology losses range from 10-90%
due tothe pests[Singh et al. 2002].Many pesticides are being used for different pests.In general,
pesticide is a chemical or biological substance intended to prevent or repel or destroy pests
causing damage or disturb the growth or health of living organisms which may be plants or
animals [Baldwin et al. 1962].Pesticides are generally classified into:Bio-pesticides and
Chemical pesticides,out of the two, chemical pesticides are largely used worldwide for
controlling pests. For example, using Boric acid for cockroach control; DDT
(Dichlorodiphenyltrichloroethane) for mosquitoes control and large-scale usage of chemicals for
crop protection [Baldwin et al. 1962].

Mainly pesticides are designed to kill or inhibit organisms that cause damage to the crops or
animals, but unfortunately they also have different harmful effects. Their high levels in the
environment may even be lethal sometimes. Chemical pesticides, in general, act on the vital
metabolic pathways of pests, either by interfering or by inhibiting the biological mechanism.
Exposure to pesticides causes mild to lethal health effects of organisms such as instant reactions,
allergies, asthma,and hypersensitivity, and long term effects such as to cancer, hormone
disruption, and problems with reproduction, fetal development, fetal death[EPA 2007, Sanborn
et al. 2007], and birth defects. It can cause neurodevelopmental disorders, neurological problems
such as memory loss, loss of coordination, reduced speed of response to stimuli, reduced visual
ability, altered or uncontrollable mood and general behavior, and reduced motor skills [Jurewicz
et al. 2008].

Among all the other pesticides, organophosphate or organophosphate (OP) compounds are one
of the most important classes of present-day pesticides. They constitute a heterogeneous category
of chemicals precisely designed for the control of pests, weeds or plant diseases. Constituents
with a great variety of insecticidal properties are found among the organophosphorus
compounds.Organophosphates form the basis of many insecticides, herbicides, and nerve agents
and are responsible for a number of poisonings and have many domestic and industrial uses.They
are also widely used inside homes in home gardens, for flea control on petsto control pests like

11
termites and ants, but these uses have been discontinued, as several of them are highly toxic.
They play a vital role in the commercial agriculture to control pests on fruit and vegetable crops.

1.1. Chemistry of Organophosphate Compounds:

Organophosphate pesticides have a synthetic origin; amides, esters, or thiol derivatives of

phosphonic, phosphoric, phosphorothioic, or phosphonothioic acids. General chemical structure
of an organophosphate is illustrated in figure 1.1, which comprises a central phosphorus atom (P)
and the characteristic phosphoric (P=O) or thiophosphoric (P=S) bond and the symbol X is the
leaving group. In less toxic OPs the leaving group usually contains alkyl or aryl groups. Side
groups R1 and R2 are usually alkoxy groups [kazemi et al. 2012].

Those bearing the P=S functionality in thiophosphoryl compounds are much less toxic than other
related phosphoryl derivatives. Thiophosphoryl compounds are not vigorous inhibitors of
acetylcholinesterase in either mammals or insects. In mammals, the metabolism involves the
removal of lipophilic side groups from the phosphorus atom, whereas in insects it is to oxidize
the compound, thus removing the terminal sulfur and replacing it with a terminal oxygen, which
allows the compound to more efficiently act as an acetylcholinesterase inhibitor.

Figure 1.1 General structure of OP compound, X represents the leaving group, R1 and R2 are the
side chains usually alkoxy groups.

On the basis of structural characteristics, organophosphorus compounds are divided into at least
10 types, including phosphates, phosphonates, phosphinates, phosphorothioates,
phosphonothioates,phosphorothioates,phosphonothioates, phosphorodithioates,
phosphorotrithioates, and phosphoramidothioates (Gupta 2011).

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Among all the OP pesticides the ten most commonly used are:1. Malathion, 2.Parathion,
3.Denitrothion, 4.Diazinon, 5.Dichlorvos, 6.Phosmet, 7.Methyl parathion, 8.Chlorpyrifos,
9.Azinphos-methyl, 10.Azamethiphos.

Almost all the OP pesticides have toxic effectson living organisms. Toxicity of OP compounds is
determined by many factors, few of them are structure and chemistry of OP compounds,
metabolism of the organism, levels of toxins in the exposed organism, and degree of
decomposition. Among all the toxicities of OP compounds the most studied is the
neurotoxicity.[Slotkin et al. (2006)].

1.2. Organophosphate Toxicity:

The OP pesticides mostly inhibit the carboxylester hydrolyzes in humans. OP pesticides and their
derivatives function as inhibitors of the enzyme acetylcholinesterase (AChE), hence called
anticholinesterases. The primary machinery of OP toxicity is well contemplated which involves
inhibition of acetylcholinesterase (AChE) by phosphorylating and forming a covalent bond
between OP and an amino acid residue serine in the active site of AChE, in which a proton is
transferred from the same amino acid to the leaving group of OP compound from its active site.
The result is inhibition of catalysis of acetylcholine to choline and acetic acid. This leads to
accumulation of acetylcholine at cholinergic receptor sites and produce effects equivalent to
excessive stimulation of cholinergic receptors throughout the central and peripheral nervous
systems, resulting into neuromuscular paralysis (i.e. incessant muscle contraction) all over the
body. Spontaneous hydrolysis of OP from the active serine amino acid site is very slow,
sometimes irreversible, resulting in long-term toxic effects. [Senanayake et al.1987, Pope et
al.2006, Lionetto et al. 2013].

Regarding the effects of OP pesticides on health, few reports were published and some studies
still being carried out every year especially among agricultural workers and children. Although
exposures of OP pesticides leads us to get many abnormalities in our physiological conditions,
but major toxicological effects are neurotoxic effects which have been most frequently
described as consequences of OP exposure[Alavanja et al. 2004, Engel et al. 2007, Eskenazi et
al. 2008, Rosas et al. 2008, Handal et al. 2007, Jurewicz et al. 2008].

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1.3. Organophosphate Metabolites:

The assessment of OP exposure is usually done by estimating blood and urine biomarkers. OP
pesticides have a tendency to be metabolized relatively quickly and get excreted primarily in the
urine. Most of the OP pesticides are metabolized up to three of six common dialkyl phosphate
(DAP) metabolites [Table 1.1]. These six metabolites are dimethyl phosphate (DMP), diethyl
phosphate (DEP), dimethyl thiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethyl
thiophosphate (DETP), and diethyl dithiophosphate (DEDTP) [Bravo et al. 2002][Figure 1.2,
Table 1]. Quantifying those six urinary dialkyl phosphate (DAP) metabolites is one of the best
methods of assessment of exposure to OP pesticides [CDC 2009, Cynthia et al. 2003]. However,
these six metabolites are very nonspecific because there is no direct sign to know which DAP
metabolite originates from which OP pesticide, so it is not possible to recognize individual
pesticides by these metabolites. However, most of the OP pesticides give one to three of the
given six DAP metabolites which provide precious data regarding cumulative exposure to OP
pesticides.

Figure 1.2.The structures of different urinary metabolites of organophosphate pesticides.

14
Table 1.1.The 28 EPA-registered organophosphorus pesticides and their potential dialkyl
phosphate metabolites. [Lesliam et al. 2012]

S.NO Pesticide Metabolites Produced
1 Azinphos methyl DMP DMTP DMDTP
2 Chlorethoxyphos DEP DETP
3 Chlorpyrifos DEP DETP
4 Chlorpyrifosmethyl DMP DMTP
5 Coumaphos DEP DETP
6 Dichlorvos(DDVP) DMP
7 Diazinon DEP DETP
8 Dicrotophos DMP
9 Dimethoate DMP DMTP DMDTP
10 Disulfoton DEP DETP
11 Ethion DEP DETP DEDTP
12 Fenitrothion DMP DMTP
13 Fenthion DMP DMTP
14 Isazaphos-methyl DMP DMTP
15 Malathion DMP DMTP DMDTP
16 Methidathion DMP DMTP DMDTP
17 Methylparathion DMP DMTP
18 Naled DMP
19 Oxydemeton-methyl DMP DMTP
20 Parathion DEP DETP
21 Phorate DEP DETP DEDTP
22 Phosmet DMP DMTP DMDTP
23 Pirimiphos-methyl DMP DMTP
24 Sulfotepp DEP DETP
25 Temephos DMP DMTP
26 Terbufos DEP DETP DEDTP
27 Tetrachlorviphos DMP
28 Trichlorfon DMP
DMP:dimethylphosphate; DEP:diethylphosphate; DMTP:dimethylthiophosphate; DMDTP:
dimethyldithiophosphate; DETP:diethylthiophosphate; DEDTP: diethyldithiophosphate.

Numerous studies have been conducted on urinary dialkyl phosphate (DAP) metabolites as
exposure biomarkers for assessment of OP exposure. Some of those studies have used urinary
DAP metabolites to derive OP pesticide dose estimates by estimating metabolite levels in urine.
[Fenske et al. 2000, Lambert et al. 2005, Huen et al. 2012]. While others were depending on

15
blood OP levels for the same exposure assessment. However, there are chances of degradation of
OP pesticides in the environment, which cause the presence of preformed DAPs in food and
environmental media. The reports of the presence of DAPs in fruit juices and other produce have
strengthened this [Weerasekera et al. 2009, Lu et al. 2005, Zhang et al. 2008] and the previously
published studies show that DAPs in dust and documented the existence of DAPs in house dust
samples from urban and agricultural homes. Thus, urinary DAP levels may not only represent
exposure to parent OP pesticides but also to the preformed degradation products (i.e., DAPs)
present in food and environmental media.

1.4. Methods for analyzing organophosphatepesticide metabolites:

Number of methods has been developed for quantifying the six DAP urinary metabolites. Reid
and Watts developed one of the earliest methods for quantification of OP metabolites, which
include azeotropic distillation of urine with acetonitrile, followed by derivatization with
pentafluorobenzyl bromide (PFBBr) to prepare urine samples for analysis of OP metabolites in
given samples [Reid and Watts 1981]. But in analytical estimations improvement of methods is
necessary in order to get more and more accurate and reproducible quantifications. Later in 1982
Sultatos, Costa and Murphy made some changes in the earlier method and used high-pressure
liquid chromatography (HPLC) for determination of organophosphorus insecticides, their oxygen
analogs and metabolites [Sultatos et al. 1982]. Continuing with this, Moate and Lu et al (1999)
have developed an improved cleanup of urine using solid-phase extraction, followed by
derivatization and GC analysis for determination of dialkyl phosphates in the urine of children
who were exposed to organophosphorus insecticides [Moate et al 1999]. According to them,
additional cleanup of urine samples allows for increasing sample size and improving sensitivity
while minimizing interferences and variability associated with derivatization. By using their
improved cleanup of urine using solid-phase extraction there could have increased the sensitivity
and recorded limits of quantitation as 10 ng/mL for DMP and DEP and 2 ng/mL for DMTP,
DETP, and DMDTP. By using this smooth method researcher could increase sample throughput,
get clean extract for analysis, and without any need of custom glassware.

Various studies have reported the measurements of urinary dialkyl phosphate (DAP) metabolites
of OP pesticides [Aprea et al. 1996; Moate et al. 1999; Fenske et al. 2000; Hardt and Angerer
2000; Lu et al. 2001; Oglobine et al. 2001a, b; Bravo et al. 2004]. There are many approaches for

16
OP metabolites analysis, but one approach that has been widely accepted in quantitating
concentrations of OP biomarkers is the one developed by Barr and Needham in 2002 [Barr and
Needham 2002]. Researchers started using more sensitive LC-MS/MS technique and producing
more accurate and precise results. The limits of detection would be in the µg/L (parts per billion)
to pg/ml range (parts per trillion).

1.5. Exposure in children:

With regard touptake and adverse effects of pesticides, children are exclusively vulnerable as
compared to adults due to their physiological conditions, developmental issues, dietary habits
[Goldman 1993; Jacqueline et al. 2004; Landrigan et al. 1999; Tanner 1971]. Exposure to
pesticides may happen through ingestion, inhalation, or dermal contact. Unintentional ingestion
by children may be in considerably high doses due to greater intake of food or fluids per kg of
body weight as compared to adults.In particular, the potential effects of pesticide exposures to
the developing fetus and child are of great interest to society and regulatory agencies (Rauh et al.
2011). Neurotoxic associations of high-level prenatal and early childhood exposure to certain
pesticides are well established, the implications of potential effects observed at low exposures
are less straightforward, particularly in the absence of a clinically defined adverse outcome.
Studies evaluating potential neurodevelopment effects associated with pesticide exposure are
challenging to interpret due to the diversity of types and classes of chemicals, differences in
exposure measures, and the wide range of instruments used to assess outcomes [Slotkin and
Seidler 2012; Rauh et al. 2011; Rauh et al. 2006; Eskenazi, et al. 2007]. Nevertheless, it is
important to critically evaluate the evidence to date, as well as to identify important research
gaps and methodological issues that require further attention to advance our understanding of
observed effects.

The affected groups to those pesticides may vary from a large population of the country or state
or city to a small group of farmworkers and their children [Kapka-Skrzypczak et al. 2011]. As
children are more vulnerable to OP pesticides most studies were focused on levels of OP
metabolites and their health effects in children of various age groups. Number of studies has
been conducted regarding the ill effects on health due to OP pesticides.Very few studies are
available in the Indian subcontinent on the subject, but are very regular in the USA, Canada, and
in many European countries. Recently, public health concerns about pesticide exposures to

17
young children have received higher attention [Hayes 1980, Saito 1884, Timoroğlu 2014and
Dhanushka 2017]. Meanwhile, bio-monitoring studies on different populations have also
demonstrated that children are widely exposed to a number of pesticides, including
organophosphorus (OPs), pyrethroid, fungicide, and organochlorine(OC)pesticides through
multiple routes[Bradman et al., 1997; Aprea et al., 2000; Adgate et al., 2001, Barr et al., 2005].In
the report “Pesticides in the Diets of Infants and Children” (NRC 1993) the National Research
Council (NRC) has reported that the major source of pesticide exposure in children is dietary
intake and it may lead to the increased pesticide-related health risks in children. This has also
been reported through inhalation in the paper by [Damalas 2011].

Apreaand other researchers in the study of OP exposure to 195 children who lived in the
municipality of Siena, Tuscany, Italy found detectable concentrations of two methyl metabolites
DMP and DMTP in the greatest number of samples (96 % and 94%, respectively). The
geometric means they reported of DMP (96% of detection), DMTP(94%), DMDTP (34%), DEP
(75 %), DETP (48%) and DEDTP (12 %) were 116.7 nmol/g, 104.3 nmol/g, 14.1 nmol/g, 33.2
nmol/g, 16.0 nmol/g, and 7.7 nmol/g creatinine respectively[Aprea 2000]. In the bio-monitoring
survey of preschool children residing in Seattle metropolitan in 99 % urine samples of children,
at least one DAP metabolite was detected.

Thereare many possible ways of exposure of OP pesticides especially in children. Inhalation,

ingestion, and dermal contact are some of them. Even children’s handling of food
couldcontribute 20-80% of the total dietary intake of pesticides[Akland et al. 2000]. When
compared to children from non -agricultural communities there is a more possibility of pesticide
exposure to pesticides of agricultural communities. Some studies could reveal the factors which
determine the exposure of OP pesticides in children belonging to agricultural communities such
as the closeness to farmland [Fenske et al. 2000], types and amounts of OP pesticides used, the
timing of applications and degradation of pesticide, modus operandi used, the proximity of
housing to agricultural area and frequency of to and fro movement of workers [Lambertet al.
2005], consumption of agricultural produce [Bradman et al].

Other factors such as house dust, cloth, through which children get exposed [Bradman 2007] but
Quirós-Alcalá et al. studieshave shown ingestion of dust is not a significant source of DAPs in
urine [Quirós-Alcalá 2012]. Holme et al. 2016 study indicates the levels of dimethyl metabolite

18
correlates with vegetable consumption, the same group noted there is no significant trends
between fruit and vegetable consumption and diethyl (DEAP) metabolites[Holme et al. 2016].

1.6. Health Effects in children:

Organophosphate pesticides may cause the following health effects in children like
neurodevelopment and growth, mental development and developmental problems [Eskenazi et
al. 1999].High exposure of OPs to pregnant women in Shenyang and China was the predominant
risk factor for neonatal neurobehavioral development [Zang et al. 2014]. It was reported that the
early-life exposure to OP pesticides was associated with respiratory symptoms consistent with
possible asthma in childhood [Raanan 2015]. The study group Raanan et al. found that early-life
organophosphate exposure was adversely associated with lung function in 7-year-old children
[Raanan et al. 2016].

But in their study Oulhote and Bouchard did not observe any significant association between OP
metabolite levels and behavioral scores in children and is in contrast with the earlier studies
[Oulhote and Bouchard 2013]. About 20 articles were published regarding the neurological
effects of OP exposures in children there.González-Alzaga and his team took a systematic review
for analyzing the scientific evidence published on potential neurodevelopmental and behavioral
effects of prenatal and postnatal exposure to OP. They observed a large variability in
epidemiological designs and methodologies across the studies which made them difficult to
compare. They comprehended there is a need for more standardized methodologies to compare
the results in the best way [González-Alzaga et al. 2014].

1.7. Indian context:

In India, the green revolution initiatives, occurring between the 1930s and the late 1960s,
resulted in the adaption of chemical fertilizers and agro-chemicals [Pingali 2012]. After 1961,
India was adopting the same and slowly started the use of chemical fertilizers and pesticides
[Posgate 1974]. From then onwards, use of pesticides became a necessary evil. Although organic
foods are a healthier alternative, its production at a sufficient level is difficult. In order to manage
crop growth and increase production, the use of pesticides has become essential. During the
1960s, however, it became evident that persistent use of pesticides was having an adverse
impact on the ecological communities [Ramade, F., Ecotoxicology, John Wiley, New York,
1987.]. Currently, India is the largest producer of pesticides in Asia and ranks in top 20 in the

19
world for the use of pesticides [Abhilash and Singh 2009]. Around 230 pesticides are registered
for use in agriculture in India against various pests and diseases(Singh and Battu 2008). Several
organochlorine pesticides (OCPs) like hexachloro-cyclohexane (HCH), dichloro-
diethyltrichloroethane (DDT), aldrin, and dieldrinhave have shown persistent occurrence in
various studies [Battu et al. 1989; Dikshith et al. 1990; Gupta et al. 1982; Kang et al. 2000;
Kannan et al. 1992; Kaphalia et al. 1990; Kole et al. 2002; Kumari et al. 2002; Noronha et al.
1980; Singh, 2002; Shukla et al. 2006].

Among all the pesticides being used organophasphate pesticidesrepresent the largest group of
chemical insecticides that are used in plant protection throughout the world. In the United States,
organophosphorus pesticides represent about half of the total insecticides used. At the same time,
these are also major pesticides being extensively used in India at par with the global usage.
Andhra Pradesh and Telangana are no exceptions and account for a hefty 24 percent share of
pesticide consumption in the country and are grappling with increased pesticide residues in food
commodities.

The present study area Andhra Pradesh (302 g/hectare) is one of the largest states for pesticides
consumption, followed by Punjab (889 g/hectare), Haryana (3827 g/hectare). Consumption of
Pesticides in erstwhile Andhra Pradesh during 1999-2000 (technical grade, MT) was 4054 MT
whilst it was 2200 MT during 2004-05 and at Rangareddy 228391 MT/2004-05 Year one of the
largest vegetable-cultivating districts in the state so; there is a higher probability of using
pesticides around Hyderabad.

Andhra Pradesh (including Telangana & Seemandhra), Maharashtra and Punjab arethe top three
states consuming 45% of pesticide in India. Andhra Pradesh is the leading consumer with 24%
share. The top seven states together account for more than 70% of crop protection chemicals
usage in India.

The central sector scheme “Monitoring of Pesticide Residues at National Level” conducted by
the government of India for 2014-15 shows that, out of all samples of commodities, pesticide
residues were detected in 18.7 % of samples and out of that 2.6 % of samples contained pesticide
residues above the Maximum Residues Limits (MRLs). The major pesticides found in
vegetables, fruits, and cereals were organophosphate pesticides e.g. Chlorpyriphos, Acephate. In

20
part of the above scheme, the Hyderabad centers National Institute of Plant Health Management
(NIPHM), and Professor Jayashanker Telangana State Agricultural University (PJTSAU) during
their analysis, found that Organophosphorus pesticides like Endosulfan, Acephate,
Monocrotophos and Acetamiprid etc., which are also OP pesticides were among the major
pesticides being used around the Hyderabad.

Objectives:

1. To develop a new method for extraction and analysis of OP metabolites.

2. To estimate organophosphate (OP) pesticide metabolite levels in children of different

socioeconomic group of Hyderabad.

3. To assess OP exposure in children of Hyderabad urban area.

4. To elucidate interactions of organophosphate on human reproductive hormones.

21
22
The bio-monitoring of OP pesticide metabolites in urban children is a longitudinal cohort study
investigating OP pesticide exposures of children living in the Hyderabad city, Telangana, India.
The selected area is urban and densely populated. This cross-sectional study included urine
sample collection and aims to assess children’s exposure to OP pesticides. We conducted this
study between July 2012 and July 2017. The target population of the study was the children
residing in Hyderabad city urban area from different socioeconomic status and two age groups.
In each group, an equal number of children of the same age group were selected. During the
study for the sake of convenience the total Hyderabad urban area has been divided into five areas
namely 1.West zone, 2.East zone, 3.North Zone, 4.South Zone, and 5.Central Zone [Figure 2.1].
The socioeconomic status of the participating families in the study differed distinctly. We
divided families and the recruiting subjects into the following three income groups depending
upon their economic status [Figure 2.2]:

1. Low-income family background children (LIG)

2. Medium income family background children (MIG)
3. High-income family background children (HIG)

Figure 2.1.Demographic divisions of (West, South, North, East and Central) Hyderabad urban
area.

23
Figure 2.2.Flow chart representing different categories based on socioeconomic status, age and
sex of children for sample collection.

2.1. Subjects Recruitment:

2.1.1. Participants
Children between ≥6 years and <15 years old, who took conventional food, and were living
under the Hyderabad urban area were eligible for the study. They were in good health condition
with no history of diabetes or renal disease, toilet-trained, free of involuntary urination,
especially at night, and had local language (Telugu or Hindi or English) speaking mothers, who
were at least 24 years old. We limited our analysis to these children because information of other
studies showed that they are more vulnerable towards pesticides [Oulhote 2013].

2.1.2. Enrollment:
Participants for this study were recruited in three stages a) selection of areas b) enumeration and
random selection of households with children and c) Interviewing parents. During the first visit,
we explained the purpose and protocol of the study, eligibility of children to be part of this study
and requested them to participate in it. To confirm the willingness of families for the
participation, we contacted them directly over telephone. Approximately 95% of families with
eligible children agreed to participate. Once subjects were enrolled, an in-house appointment was
made for the second visit to go over the study protocol and to obtain written consent from
parents and older children, or oral consent from younger children. In the present study we

24
enrolled just one child of either sex from a household. In few instances, the child wasnot
available during the visit, so the parent obtained the child’s assent at a later time. The study and
procedures used in the present study were reviewed and approved by the Scientific Advisory
Committee and Ethical Committee of National Institute of Nutrition, Hyderabad, Telangana,
India.

2.1.2.1. Questionnaire:
Interviews were conducted in Telugu, or Hindi or English by Trilingual interviewers.
Information was collected on the child’s age, weight, time spent at home or in child care or in
school. Information collected also included demographics, parents' age, household enumeration,
occupational status, annual family income, home ownership, the length of time at the current
residence, and housekeeping practices, home pesticide use, the presence of pets. We have
inquired the parents about the use of pesticides in and around the home, on the home structure, in
the garden, on the lawn, and on the pets. And, they also asked, how long it had been since the
most recent application of the pesticides in each of these areas, whether the applicator had been
someone from the home or not. They were requested, to see any applied pesticide products, if
available, recorded the product name, Indian Pesticide Registration number, and date, and
location of the application.

Further, all the information regarding the dietary consumption pattern of children was obtained
from their mothers. The mothers were, asked about their children’s diets and the children, whose
parents stated that their children’s food consumption was conventional, were selected for the
study while those consuming organic foods were excluded.

The interview also included daily servings of child, fruit and vegetable consumption based on a
modified food frequency questionnaire, a Child Food Habit Checklist (CFHCL) which uses a
standardized format to assess parent-reported food habits of children. Based on the CFHCL, we
selected child taste of food indicators that we hypothesized could be associated with food habits
that affect pesticide exposure of child through the type of food intake: “Vegetarian”, “Non-
vegetarian”, “Eggetarian” or “Flexitarian.”

25
Shortly after each interview, we conducted a home inspection. Recorded information included
distance between the home and School, types of floor surfaces, carpeting, housekeeping quality,
and detailed record of home pesticides. Home visits were conducted for 100% of the enrolled
children of both at 6-10, and 11-15 years old age.

Parents were provided with a detailed study protocol, a food diary form, Food measuring cups,
an overview of the urine sampling procedures, a urine collection form, and supplies for urine
sample collection. Urine samples, details of urine collection recording forms, and completed
food diaries were collected during second visit.

2.1.2.2. Food diaries:

In our study, we have used the "three cups method" to measure the quantities of intake foods of
the children. The three cups were C1, C2, and C3. The first cup, C1 (volume was 200 ml) was
used to measure the quantities of cooked foods such as plain rice, lemon rice, fried rice, and
Upma. The second cup, C2 (volume was 150 ml) was used to measure cooked vegetables such as
eggplant, cabbage, cauliflower, tomato and okra and fruits such as grapes. And, the third cup, C3
(volume was 100-mL) was used to measure the liquid food items such as fruit juices, milk, and
beverages. We provided the parents, the three cups, to measure the quantity of food before
feeding their children and a food diary to note down the quantity.

Parents were instructed to note down the food consumed by the children on two days before and
on the day of urine sampling. They were also directed to include in the consumption list, the
foods which were eaten outside the home (e.g., food consumed during lunch at the school). They
recorded the types and the quantities of food, by using given measuring cups, all conventional
foods consumed by their children for breakfast, lunch, and dinner and the time of consumption.
All the consumed foods, it may include water, juices, and fresh produce and or any other, by the
child was recorded by the parents in the food diaries and then it was converted into “units of
servings” for each child. Juice and fresh produce and other food items consumption as recorded
by the parents in the food diaries were later converted into units of “servings,” and number of
servings were calculated for each child. Parents were asked to record all food items consumed by
the child during the 24-h period after the consent was obtained. Information recorded included
type of food, portion size, and time of consumption and we reviewed the food diary.

26
Calculation procedure for servings was followed as from the U.S. Department of Agriculture’s
“5 A Day” method for tallying fruit and vegetable intake (USDA 1995). According to this
method, fruits usually eaten whole, such as apples, bananas, and peaches, are counted as single
servings. For most other produce, raw or cooked, full of C1 or C2 or C3 cups were counted as
one serving of respective cup [USDA. 1995].

2.3. Sampling:

2.3a.Urine Sampling and Extraction

2.3a.1. SamplesCollection:

On the first visit, the study staff provided a urine collection bag which contained the sampling
supplies include sampling record forms, commode specimen collection pan for children who
were unable to urinate directly into the bottle (usually female and younger), five units of each
250-mL volume sized polypropylene bottles, gloves, and large container into which the ten
bottles could fit, with blank labels and instructed to let their children urinate into the commode
inserts, or directly into the bottles. If the insert was to be used for sample collection, the parent
was asked to transfer the urine from the insert to the provided sample collecting bottle.

Along with all the instructions they were also instructed to collect all urine produced by their
children for a 24-hour period it includes first urination early in the morning, and all the
remaining voids including the last void before their bedtime. Sample collection was not
scheduled on the day children spent at daycare centers and schools; however, parents were asked
to collect the sample the preceding day. If the child could not provide the sample during the visit,
a sample was collected on the next day at the child’s home. The collected urine bottles were kept
in the plastic container and stored in the refrigerator or maintained overnight on dry ice provided
to the parents who didn’t have a refrigerator until retrieval on the following day.

Parents identified, separated and labeled each sample bottle as an FMV (First-morning void) or a
non-FMV spot sample, and even noted down the time of the urination event. Sampling record
form given to the familieswas reviewed to assure its accuracy and completeness. All samples
were picked up within 24 hour after collection, mostly in less than24 hour. All urine samples in
sample carrying boxes wereput in a container with dry ice and transportedto the laboratory of the

27
National Institute of Nutrition and processed immediately. The transport time did not exceed 45
minutes. Some parents found it difficult to collect all urine produced by their child over the 24-
hour study period. Reasons for missed voids were out-of-wash room, toileting accidents, and
participant’s errors. Collection efficiencydiffered with age group. Twenty-four-hour samples
included 6 - 10 collected voids (the mean was 7.8). Overall, 63% of the parents collected a full
24-hr sample. Another 9% of parents missed just one void. Thirteen percentages (13%) of
parents missed two voids, and 23% of parents missed three voids. On average, children in this
study urinated 7.8 times per day. In instances where parents reported missing samples, we
calculated an adjusted total volume by estimating the average volume per void for that child.
After adjustment for missed voids, the average volume, collected per child, was 817 ml and
ranged from 150 ml to 1,584 ml.

2.3a.2. Sample processing and analysis:

Samples were processed at National Institute of Nutrition, Hyderabad, India. During processing
we measured the volume in milliliters. Individual voids from 24-hour sampling sessions were not
selected for individual analysis, except the first morning void, but these samples were pooled.
After that, aliquotting, samples were stored at –20°C until being analyzed at our institute. Urine
samples were analyzed for six DAP metabolites: dimethylphosphate (DMP),
dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP),
diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP). These metabolites represent the
urinary breakdown products of the agricultural OP pesticides used in the Telangana.Samples
were lyophilized to remove water, re-dissolved in a 1:1 solution of acetonitrile, and analyzed
using liquid chromatography - mass spectrometry (LC-MS/MS) using a validated method. We
estimated Creatinine concentrations by using a commercially available Jaffe kit method.
However, because the samples represented full or near 24-hour voids, creatinine adjustment to
normalize for hydration was not necessary. OP metabolite levels in 24-hour voids were
estimated as an average of concentrations in all samples collected on sampling days including the
FMV samples from the same day. We expressed metabolite concentration in three ways: a)
unadjusted metabolite concentration (micro grams metabolite per liter of urine); b) creatinine-
adjusted metabolite concentration (micro grams metabolite per gram of creatinine); and c) 24
hours metabolite concentrations (micro grams metabolite per liter of urine) of each metabolite.

28
2.3a.3. Extraction: Liquid-liquid extraction (LLE)
Liquid-liquid extraction(LLE) is a conventional technique involving the partitioning of solutes
between two immiscible liquids. The selection of appropriate solvents is important for this
purpose, the solvent should match the analytes polarity while still being immiscible with water
and it should preferably be compatible with the following detection method. A volume of the
extraction solvent should be higher compared to the sample. However, the sample extract can
easily be evaporated if a volatile solvent is used to increase the analyte concentration. Other
factors, such as pH and ionic additives may affect the extraction efficiency.

All urine samples to be analyzed, reagents and standards were brought to room temperature. A
volume of 10 mL urine from each urine sample was placed into 20 mL respective test tubes then
samples were initially frozen in the ethanol bath for 1 hour at –110oC temperature and
atmospheric pressure. After the samples were sufficiently frozen, the vacuum was set to 25.5 mT
and the samples remained at –84oC for an additional 4 hours and placed in a commercial bench-
top lyophilizer (ScanVac Cool Safe 110-4 PRO 4litre) for freeze drying. The lyophilizer was
operated overnight.

On the following day, after completion of the lyophilization process, to each lyophilized sample
test tube, 4 mL of acetonitrile was added and procedure as per flow chart given below followedto
prepare dried sample for analysis of for six non-specificDAP metabolites of OP, vortex for 10
min and then transferred into 15 mL centrifuge tubes. The samples were further extracted using 5
mL of acetonitrile for 2 min and then transferred to the acetonitrile-extracted sample made in the
preceding step. In the last step, added a mixture of 2 mL Acetonitrile (ACN) and 2 mL ether to
the same lyophilized urine sample tubes. The extract obtained was pooled with first extract in the
centrifuge tube. Extract was transferred into a 15 mL centrifuge tube. When the extraction was
complete, all the tubes pooled extracts were centrifuged for 8 min at 3000 rpm. Next, the
supernatant solution was transferred into a new set of 15 mL tubes for drying and then placed in

a TurboVap at 20◦C under 15 psi of nitrogen gas to ensure that it was completely dried. The dry
residues were reconstituted in 1 mL acetonitrile and run on LC-MS/MS for the quantification of
six DAP metabolites (Sinha et al. 2014). The extraction procedure is diagrammatically shown in
below flow chart.

29
 From each sample, 10 ml urine was taken in 20 ml vol. test tube.

The test tubes were kept for Lyophilisation to remove the water content.

4 mL Acetonitrile (ACN) was added to each lyophilized sample tube.

Vortex for10 minutes.

Extract was transferred into a 15 mL centrifuge tube.

For second round the samples were extracted with 5 mL Ether.

Vortex for 10 minutes.

The extract obtained was pooled with first extract in the centrifuge tube.

Again, added a mixture of 2 mL Acetonitrile (ACN) and 2 mL ether to the same Lyophilised
urine sample tubes.

The final extract was pooled with the previous extracts in the centrifuge tube

30
 The tubes were centrifuged at 3000 rpm for 8 min.

Next, the supernatant transferred into test tubes.

The tubes were Placed in a TurboVap at 20◦C under 15 psi of for drying.

The dried samples were reconstituted with 1 mL ACN for analysis.

Reconstituted urine samples were analyzed for six non-specificDAP metabolites:

dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate
(DMDTP), diethylphosphate (DEP), and diethylthiophosphate (DETP), diethyldithiophosphate
(DEDTP as reported by Moate et al. (1999).

2.3b. Food Samples’Collection and Extraction:

Five zones were selected from Hyderabad urban city namely; Erragodda, Kukatpally,
Mahdipatnam, Falakanuma and L.B.Nagar. From each zone and each selected area vegetables
and fruits were collected from vegetable and fruit outlets and food items such as Rice, Pulses,
and Wheat flour collected from grocery stores, cool drinks and fruit juices collected from
respective outlet. After collection of samples the extraction of sample were carried out by Quick
Easy Cheap Effective Rugged and Safe (QuEChERS) and shown as below.

The extraction procedure has been shown diagrammatically as:

Food item

Homogenized

Extraction by QuEChERS

Analysis on LC-MMS/MS

31
2.4. LC-MS/MS analysis

2.4.1. Materials:

All solvents (LC-MS grade) and reagents of analytical grade (AR) were used during this
study. Water, methanol, acetonitrile (LC-MS grade) were purchased from Sigma Aldrich
GmbH., while formic acid (analytical grade) and acetone were also obtained from Merck,
Germany. The standards of DAP metabolites dimethyl phosphate (DMP),
dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethyl phosphate
(DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) were purchased
from Organic Toxicology Branch (pesticide toxicology), Centre for Disease Control and
Prevention,Division of Laboratory Sciences,Mail Stop F-20,4770 Buford Highway, NE
Atlanta, GA 30341-3724, USA. The primary secondary amine, PSA tubes were purchased
from Agilent (USA). The LC-MS column was purchased from Agilent (USA). All reagents
and standard were made fresh in LC-MS grade water or solvent before use.

2.4.2. Standards:
2.4.2.1. Stock solutions-1 (10 mg L-1)
Individual stock standards at a concentration of 10 ppm for DMP, DMTP, DMDTP, DEP, DETP,
and DEDTP were prepared from the standards received from CDC, Atlanta USA of strength 318
pg µL-1.

2.4.2.2. Stock solutions - 2 (l mg L-1)

Stock solutions of l mg L-1 for the standard mixtures of DMP, DMTP, DMDTP, DEP, DETP,
and DEDTP were prepared by diluting 1 mL of each standard from stock solutions-1 into 10 mL
of acetonitrile using a 10-mL volumetric flask. The stock solutions -2 were stored at -20°C.

2.4.2.3. Working solutions

Six working standard sets were prepared using the serial dilution method for DMP, DMTP,
DMDTP, DEP, DETP, and DEDTP at concentrations of 1, 5, 10, 50, 150, and 250ng mL-1 (CDC
2005). The standard sets were divided into aliquots, sealed in ampoules and stored at -40°C.
These standards were then used for the determination of the limits of detection (LOD), limits of
quantification (LOQ), the recovery experiment and the linearity experiment.

32
2.4.2.4. Preparation of blanks
Urine samples from 30 donors were collected and pooled. The pooled sample was diluted (1:1
v/v) with water to reduce the endogenous levels of the analytes of interest and then kept on a
shaker with magnetic beads for overnight mixing. After that, the pooled sample was pressure
filtered with a 0.2 µ filter capsule and divided into aliquots of 10 mL into 20 mL glass test tubes
and stored at 20°C [Driskell 1999, Odetokun 2010]. After bringing them to room temperature the
stored aliquots were then screened for metabolites using the method described earlier. Only,
after finding no traces of the metabolites, the pooled samples were considered as blanks.

2.4.2.5. Spiking concentration of standards

Urine samples were spiked with a standard of each compound DMP, DMTP, DMDTP, DEP,
DETP, and DEDTP at different concentration levels of 0.2, 2.5, 1, 10, 30 and 50ngmL-1.

2.4.2.6. Working standard solutions

Six solutions, spiked with different concentrations of DMP, DMTP, DMDTP, DEP, DETP, and
DEDTP (ranging from 1 to 250 ngmL-1), were prepared in acetonitrile using the serial dilution
technique for the construction of a linear curve. These standard curves were used throughout the
study to maintain the accuracy and precision of the sample analysis.

2.5. Percent recovery of different OP metabolites:

The urine samples free from pesticides were spiked with standards of DMP, DMTP, DMDTP,
DEP, DETP and DEDTP at different levels. The percent recoveries and coefficients of variation
(% CV) from these experiments are shown in [Table 2.1]. Excellent extraction efficiency was
obtained for all of the tested pesticides.

2.6. Instrumentation:
The extracted samples for six DAP metabolites were analyzed using a 4000-QTRAP triple-
quadrupole hybrid mass spectrometer (Applied Biosystems MDS Sciex, USA) equipped with a
liquid chromatograph (LC, Shimadzu, LC 20 AD, and binary pump) fitted with a reverse phase
column and operated in the negative turbo ion spray (ESI) mode.

33
2.6.1. Mass spectrometer configuration
The analysis of the analytes was carried out in the multiple reaction monitoring (MRM) negative
ESI modes with high resolution. The interface heater was held at temperature of 550◦C, and the
ion spray (IS) voltage used was 5500 eV. A full auto tune of the mass spectrometer was
performed before the analysis of every set of samples. A full scan of the mass spectra of all the
metabolites was recorded to select the most abundant mass to charge ratio (m/z) ion (Q1) using a
continuous infusion of each metabolite in the negative ionization mode of ESI. The product mass
spectra were obtained with continuous infusion of each analyte, so the Q1, corresponding to the
protonated precursor ion, remained constant. The most abundant product ion for each compound
was then selected for MRM analysis. At least two of the most intense product ions were isolated;
one ion was used for quantification, whereas the other was used for confirmation, as per the three
principle criteria given for mass spectrometry studies of OP pesticides.Optimization of the
source dependent parameters, such as nebulizing gas (GS1), heating gas (GS2) and curtain gas,
were carried out in the flow injection analysis (FIA) mode. The GS1, GS2 and curtain gas
pressures were then maintained at 35, 40 and 30 psi respectively, during the entire study.
Furthermore, declustering potential (DP), collision energy (CE), entrance potential (EP) and
collision exit potential (CXP) were used as per the required sensitivity of the method (Table 2.2).

2.6.2. Mass spectrometer instrument control using the analyst program

The Analyst 4.1.2 software controlled the MRM operational mode of the 4000 Q-Trap mass
spectrometer was used. The operational procedure of the instrument was also controlled by using
the same software. This program set the instrument to acquire data using the ESI in the negative
mode for the analysis of the various analytes. The operational details for the precursor ions and
the product ions are shown in Table 2.2. Different DP (Declustering Potential), CE, EP and CXP
parameters were applied for the selection of confirmation and quantification ions of each analyte
depending on their physical and chemical properties. However, the maximum sensitivity for the
quantification ions was achieved at different energy sets shown in Table 2.2. Precursor ions were
therefore isolated using the maximum sensitivity, specificity and linear dynamic range. The
product ions of the precursor ions were then used for confirmation, adding to the selectivity of
the analysis (Table 2.1).

34
2.6.3. Tuning of mass spectra. Continuous infusion of each pesticide was carried out in positive
ionization mode by an ESI negative source. Full-scan mass spectra were recorded in order to
select the most abundant mass to charge ratio (m/z). Full scan product mass spectra were
obtained with continuous infusion of each analyte in product ion scan mode, keeping Q1
constant on m/z value corresponding to deprotonated ions. The most abundant product ion for
each compound was chosen for LC-MS/MS analysis in Multiple Reaction Monitoring (MRM)
mode. The optimization of source dependent parameters like nebulizing gas and auxiliary gas
was carried out in Flow Injection Analysis (FIA) mode. The MRM transitions and the energy
profile at which the maximum sensitivity were achieved have been taken into consideration for
analysis (Table 2.2).

2.6.5. Liquid Chromatography Conditions (LC):

An ultrafast liquid chromatography (UFLC) system equipped with a Triple-Quadra pole ion trap
hybrid mass detector operating in the MRM mode was used for this study. Chromatographic
separation was achieved for the six DAP metabolites using a liquid chromatograph equipped
with a SB-C18 600 bar reversed phase column with a particle size of 1.8 µm and a dimension of
4.6 mm. Samples (10 µl) were injected into the instrument using a Shimadzu auto-sampler fitted
with a Hamilton 100 µl syringe. Different gradient compositions of 10 mM ammonium acetate in
water (gradient-A) and acetonitrile (gradient-B) with a flow rate of 0.2 mL min-1 were used, but
the best sensitivity and separation for allof the compounds of interest were achieved using the
gradient compositions shown in Figure 2.3, and the column oven temperature was held constant
at 20°C.

2.7. Quantifications:

2.7.1. Linearity: Correlation Coefficient (R2):

Calibration curves were prepared with 6 sets of different concentrations (1, 5, 10, 50, 100, 150,
and 250ngL-1) for each DAP metabolite (DMP, DEP, DMTP, DETP, DMDTP and DEDTP)
during each analytical run. The concentrations of each metabolite were plotted against the known
concentrations to show the correlation coefficient and percentage accuracy of this method at low
concentration levels. The calibrated standard concentration curves were used during the entire
study to ensure the linearity range of analysis for each run. The lowest standard concentrations
used for construction of linearity were at lower level of the spike for each metabolite. The

35
unknown sample’s concentrations in each of the urine samples were determined by linear
regression analysis of calibration plots using the slopes and the intercepts. The obtained
correlation coefficient for all metabolites was ≥ 0.9999.

2.8. Method Validation:

2.8.1 Sample analysis protocol as per quality assurance:

A total of 30 samples were included in one typical batch for confirmation and quantification. Six
standard samples and one blank reagent were used for analysis. The first sample was a blank
solution while samples 2 to 7 were standards of different concentrations, and the 8th position was
a blank solution in the first batch. In another batch, the 30 studying samples were placed in
positions 9-39.

2.8.2. Limit of Detection (LOD):

The LOD was defined as three times the standard deviation of the noise at zero concentration
(3S0), where S0 was estimated as the y-intercept of a linear regression analysis of a plot of the
standard deviation of three lowest standards versus the expected concentration from 10 runs
[Oglobline et al. 2001]. Furthermore, the LOD was compared with results of the calibration
standard samples and low-level spiked samples to ensure that calculated values agreed with the
peak observed and that a minimum signal-to-noise ratio of 3 was present at these low levels.

2.8.3. Limit of quantification (LQ):

The lower level where measurements become quantitatively meaningful was called the limit of
quantification, and it was calculated as described by the previously reported method.

2.8.4. Percent recovery:

Recoveries of the method were determined by spiking blank urine samples (free from OP
metabolites) with different known concentrations of the reference standards. Recovery of each
metabolite was calculated at each of the known concentrations levels by comparing the measured
concentrations with the spiked concentrations as per the reported method. A ratio of 1.00
indicated 100 percent recovery.

36
2.8.5. Accuracy:
To determine accuracy, 6 blank urine samples were fortified with different concentrations of
metabolites, mixed and allowed to stand for 1hr so that all the metabolites were absorbed
thoroughly by samples before performing the extraction, while the blank urine sample served as
the negative control. The accuracy was calculated by spiking urine samples with a known
concentration of the metabolite reference standard, preparing and analyzing the samples, and
then comparing the calculated and expected concentrations. The linear regression analysis was
carried out on linearity curves of the calculated concentrations versus the expected
concentrations. After performing the analysis, if, obtained slope was 1.0, it was indicative of 100
percent accuracy.

2.8.6. Selectivity:
The degree of selectivity was determined by using LC–MS/MS in the MRM transition mode.
The chromatographic sensitivity increased when the LC was interfaced with a MS/MS detector,
which was utilized for this study as the most selective analytical technique (Sinha 2011). This
technique was capable of separating the OP metabolite on the basis of the selected ion.
Additionally, background noise was easily removed with high selectivity, which increased signal
intensity.

2.8.7. Quality control assurance:

Urine samples previously determined to be free of DAP metabolites were taken and were
analyzed along with actual samples. For laboratory quality control and assurance, one blank
sample and two spiked samples were analyzed along with six actual samples in each analytic
batch. Laboratory duplicates were analyzed for 8% of the samples, and quality control criteria
required that there be no more than 12% variation between laboratory duplicates.

2.8.8. Method Validation:

The best sensitivity and separation for all of the compounds of interest were achieved using the
gradient compositions shown in Figure 2.3. The column oven temperature was held constant at
20○C.

37
Figure.2.3 Different gradient profiles of the percent acetonitrile (B) in 10 mM ammonium acetate
in water (A) at different time intervals.

First, the full scan spectra were recorded. In thespectra, the characteristic stable ions
were isolatedforMRMtransition for the confirmation and quantification of thesixDAP
metabolites in the urine samples. The extracted ion chromatograms of the six DAPs of

interest (10 mg L -1 spiked level)are shown in Figure. 2.4and the structure schemes of
the six OP pesticidemetabolites(DMP,DEP,DMTP,DETP,DMDTPand
DEDTP)areshowninFigure 1.2.

38
Figure-2.4: Extracted ion chromatogram of OP urinary metabolites a spiked at 10µgL-1
(1)DMDTP (RT;4.79), (2)DEDTP (RT;4.78), (3)DMTP(RT;7.48), (4)DETP(RT;7.48)
(5)DEP(RT;17.68), (6)DMP(RT;17.38).

The detailed precursor and product ions for each analyte used for quantification are shown in
Table 2.2. In addition to these two ions, a third ion was isolated at m/z 79, 125, 95,142, 95 and
157 for the confirmation of DMP, DEP, DMTP, DMDTP, DETP, and DEDTP, respectively, by
using different energy parameters. Certain DAP metabolites proved to be amenable to separation
by LC attached with MS/MS (Table 2.2).

39
Table. 2. 1. Inter-day percent recoveries (mean) and CV (coefficient of variation) of pesticides at
different spiked concentrations of OP metabolites in urine samples.
Spiked
concentration 0.2 1 2 30 50
(ng/mL)
DMP CV 1.175 1.055 0.549 0.248 0.66
%Recovery 99 99 100 98 99
N 5 5 5 5 5
DMTP CV 0.4 1.55 0.652 1.381 0.205
%Recovery 98 103 98 100 99
N 5 5 5 5 5
DEP CV 0.4 1.107 2.119 0.25 0.316
%Recovery 99 101 100 99 99
N 5 5 5 5 5
DMDTP CV 0.4 1.568 3.56 0.822 0.479
%Recovery 107 93 100 99 100
N 5 5 5 5 5
DETP CV 7.98 6.137 10.85 10.758 6.306
%Recovery 102 98 100 99 100
N 5 5 5 5 5
DEDTP CV 6.321 1.236 1.904 0.668 0.482
%Recovery 104 99 102 99 100
N 5 5 5 5 5

N= Number of samples.

Table 2.2.The isolated precursor and product ions in multiple reactions monitoring mode (MRM)
using different energy profiles.
PI(Q- DT
OP Metabolite PDI (Q-2) DP CE EP CXP RT(min)
1) (min)
DMP 125 62.8 68.24 28.9 4.86 1.56 200 17.38
DMTP 140.8 125.8 61.72 19.89 14.67 0.9 200 7.48
DEP 152.9 78.9 47.78 26.65 5.34 1.58 200 4.79
DMDTP 156.7 112 56.99 23.52 4.94 10.76 200 17.68
DETP 168.8 140.8 54.47 18.03 5.65 9.71 200 7.48
DEDTP 184.7 110.8 54.37 29.06 13.57 3.55 200 4.78

PI Precursor ion; PDI Product ion; DP Declustering potential; CE Collision energy;

EP Entrance potential; CXP Collision exit potential; RT Retention time.

40
2.8.8.1. Percentage recovery:
The blank urine samples were spiked with the different concentrations of standards, i.e., DMP,
DEP, DMTP, DETP, DMDTP and DEDTP. The inter day percent recovery (Table 2.2) and
coefficient of variation (CV) were determined based on the reported methodology [Taylor et al.
1987]. Excellent extraction efficiency was obtained for these compounds. The obtained percent
recovery for all of these pesticides is in the range of 93–107% as per the standard value [Tayloret
al. 1987].The CV value obtained was below 10%, which is an acceptable value. The lowest spike
level was 0.2 pg mL-1 of urine for DMP, DEP, DMTP, DETP, DMDTP and DEDTP. The overall
recoveries for the solvent were greater than 93% for all of the metabolites, indicating the
selectivity and precision of this method. The CV obtained was below 10% for all of metabolites
themselves, which explains the importance, sensitivity, precision and selectivity of this method.

Six replicas of six point calibration curves were preparedusing ten different spiked
concentrations (1 ng mL-1, 5 ng mL-1, 10 ng mL-1, 50 ng mL-1, 150 ng mL-1 and250 ng mL-1) and
plotted against area, and they were evaluated by linear square regression analysis. The observed
r2 values were DMP, 1; DEP, 1; DMTP, 1; DETP, 0.99; DMDTP, 1 and DEDTP, 1 (Table 2.3).
The precision was below 2%. The correlation coefficient, which was below 0.9999 (r > 0.999),
was obtained for all of these metabolites throughout the study, maintaining an acceptable range
[Bravo et al. 2004]. The 100% accuracy achieved shows the high degree of effectiveness of this
method. The accuracy of the method was also assessed by determination of the correlation
coefficient and the r2 value by spiking different concentrations in blank urine samples and
plotting area of standard concentration against area of spiked concentration (Table 2.3). The
observed accuracy for all metabolites was 100%.

41
Table 2.3. Determination of recovery accuracy of metabolites using r2 linear regression equation
with various spiked concentrations.

Concentration SD in
Metabolite r2 Intercept Slope
(ngmL-1) slope
DMP 1,5,10,50,150,250 1 -0.053 1.001 0.003

DMTP 1,5,10,50,150,250 1 -0.065 1.001 0.004

DEP 1,5,10,50,150,250 1 0.093 0.999 0.003

DMDTP 1,5,10,50,150,250 1 0.111 0.999 0.003

DETP 1,5,10,50,150,250 0.99 -1.395 1.018 0.012

DEDTP 1,5,10,50,150,250 1 -0.364 1.005 0.004

r2=Coefficient of Determination, SD = standard deviation.

The limit of detection (LOD) and limit of quantification (LOQ) were determined as per the
reported method. The limits of detection (ng mL-1) obtained for DMP, DEP, DMTP, DETP,
DMDTP and DEDTP were 0.058, 0.009, 0.004, 0.0148, 0.008 and 0.019, respectively (Table
2.4). The LOQ (ng mL-1) obtained for DMP, DEP, DMTP, DETP, DMDTP and DEDTP were
0.074, 0.0299, 0.040, 0.49, 0.0287 and 0.066, respectively (Table 2.4). The observed LOD of our
method is very low compared to other reported methods [Bravo 2002, Bravo2004] No other
method reported a LOQ as good as that obtained in our method. The LODs of this method
ranged from 0.004 ng mL-1 to 0.058 ng mL-1 with CVs less than 5%. Both the LODs and CVs
were much lower than those in other reported methods (Table 2.4), and it was achieved by
routine analysis using azeotropic distillation (from 0.25 mg L-1 for DEDTP to 1.6 mg L-1 for
DMTP) resulting in a CV value less than 20%. In addition, our LODs were lower than those
reported in the literature for most methods, which typically ranged from approximately 1 to 20
mg L-1.

42
Table 2.4. LOD, accuracy, and coefficient of determination of six OP metabolites
Linear
LOD LOQ Accuracy
Metabolite Regression R2 S/N C.V
ngmL-1 (ngmL-1) (%)
Equation
DMP y=0.004x+0.007 1 34 0.058 0.074 0.793 100
DMTP y=0.003x+0.001 1 32 0.009 0.029 0.42 99
DETP y=0.005x+0.004 1 135 0.004 0.04 0.771 99
DEP y=0.005x+0.003 1 95 0.014 0.49 0.53 100
DEDTP y=0.007x + 0.06 1 53 0.008 0.028 1.059 98
DMDTP y=0.007x+0.002 1 35 0.019 0.066 0.867 99

R2=Coefficient of Determination; S/N=Signal to noise ratio; LOD = Limit of detection;

LOQ=Limit of quantification; CV= co-variation.

There are some reported methods in the literature for the quantifications of DAP metabolites
with higher LODs and lower CV values [Hardt et al. 2000,Bravo et al. 2002,Ogloblin et al.
2001,Hernández et al. 2002].We used the LC-MS/MS technique for analysis of these metabolites
in selective mode, which is capable of removing the matrix and also minimizing interference
peaks leading to a maximum sensitivity.In 2004,Bravo et al. 2004 attempted high-performance
liquid chromatography-tandem mass spectrometry (LC/MS/MS) to minimize the matrix effect on
sample preparation time using 3-hour derivatization time and the 12-hour lyophilization steps.
They observed good results with neat standards; however, the sensitivity was inadequate when
they spiked them into a urine matrix. The reason for less sensitivity might be due to the structural
reactivity of DAP metabolites. They reported LODs of 1 mg L 1 for DEP, DETP and DEDTP
and 2 mg L 1 for DMTP, and the CVs were lower than 12%. They used the LC-MS/MS
technique for analysis and analyzed direct spiked extracted urine samples.

2.8.9. Significance:
The extraction process is simple and accurate. In the case of urine samples, several methods have
been used to extract OP from urine, but they could not optimize the extraction of the metabolites
due to complex matrix of urine. We used a simple method for the extraction of the OP analyte
from urine and achieved good recovery. The achieved percent recoveries and coefficientof

43
variation (CV) obtained were well within acceptable analytical limit (<10%) [Bravo et al.
2004]. The use of an acetonitrile solvent resulted in a 93–107% extraction efficiency for DMP,
DEP, DMTP, DETP, DMDTP and DEDTP in the urine samples (Table 2.2).

Actually, ‘polar likes polar’ and ‘non-polar likes non-polar’ mechanisms prevent us from using
polar solvents, such as methanol, for the extraction of these metabolites directly from urine
samples. Therefore, lyophilization was used to completely dry the samples, and acetonitrile was
used to extract the DAP metabolites.This method has been modified as reported in ref.[Odetokun
et al. 2010]to reduce the amount of solvent required and the time for quantification of the DAP
metabolites in urine samples. DAP metabolites are the most important urinary biomarkers for
calculation of the cumulative OP pesticide exposure in humans. The purpose of this method is to
assess the presence of the OP metabolites and correlate them with the OP pesticide exposure in
the general population. The general population is exposed to OP pesticides through food almost
daily, and to date, no studies on DAP metabolites have been reported from India for health risk
assessment. Therefore, we modified a more sensitive and cost effective method for urinary
metabolite analysis for calculation of OP exposure using urine samples. One of the most
important steps in analyzing DAP metabolites is their extraction from the polar urinematrix. A
variety of methods reported in the literature have been shown to use azeotropic distillation
liquid–liquid extraction or solid phase extraction for extracting DAPs from the urine matrix.
However, the sample preparation proposed here is simple, efficient and reproducible. In 2010,
Martin et al. employed solid phase extraction using weak anion exchange sorbent on a 96-well
plate followed by isotope dilution. In this method [Odetokun et al. 2010], DAP metabolites were
extracted in a procedure using anion exchange solid phase extraction (SPE) in an automated
system. This reported method is very costly and cannot be used by many laboratories in
developing countries because of its high costs. The extraction recoveries we obtained are
superior than those previously reported, which used reverse phase cartridges and solid phase
extraction for most of the analytes, the recoveries were greater than 93% for all DAP
metabolites[Odetokun et al. 2010].The liquid–liquid extraction, solid phase extraction,
azeotropic distillation and lyophilization methods were reported[Bravo et al. 2002]for the
isolation of different OP metabolites at the Centers for Disease Control and Prevention in
Atlanta, USA. However, the reported LOD was much higher than those from our described

44
method. The reported method is complex, and the weak anion exchange cartridges followed by
solid phase extraction cause a loss of analyte during extraction, which directly affects the
extraction efficiencyand LOD. While in our method, an appropriate quantity of solvent was
preferred for the extraction of metabolites to avoid matrix effects, as this may be required to
achieve better LOD and LOQ. The solvent effect plays an important role in liquid–liquid and
solid–liquid extraction depending upon the medium, pH, and nature of the samples. However,
duringlyophilization, the polar and non-polar matrix causes a minimal effect on the analyte in
solid urine samples. The quantity of solvent used also plays a vital role for proper extraction.
Therefore, the optimal solvent volume was used for extraction of analytes from the urine
samples. Lyophilization was also a very good technique for removing water from the
samples[Driskell et al. 1999, Oglobline et al. 2001, and Whyatt et al. 2001]and ensuring that the
sample is completely dry for extraction. Therefore, in our method, the lyophilization technique
was used for the removal of water from the urine samples.

Different compositions of solvent were used, but the best separations of all of the metabolites
were achieved in the solvent gradient of the LC method as described above [Fig. 2.1]. The
extracted ions from the DAP standards are shown inTable 2.2. First, the full scan spectra were
recorded. In the full scan spectra, the characteristic stable ions were isolated forMRM transition
for the confirmation and quantification of the six DAP metabolites in the urine samples. The
extracted ion chromatograms of the six DAPs of interest (10 mg L-1 spiked level) are shown
infigure. 2.2; and the structure schemes of the six OP pesticide metabolites (DMP, DEP, DMTP,
DETP, DMDTP and DEDTP) are shown infigure 2.3. The combination of LC and mass
spectrometry (MS) is capable of eliminating interfering components and matrices in the urine
sample extracts, which in turn provided the low detection limits shown with this method.
Analysis in the simple scan mode resulted in recurring interferences for many of the analytes.
These specificity requirements precluded the use of single quadrupole and ion trap mass
spectrometry. To increase sensitivity for quantification at the pg level, this method was a shield
to Multiple Reaction Monitoring (MRM). The obtained isolated ion in MRM for different
metabolites is shown inTable 2.2. The isolation of ions was carried out as per the reported
method. To increase the sensitivity, at the time of tuning of compounds on MRM, the energy
levels giving highest sensitivity were taken into consideration for the analysis of compounds as
shown in Table 2.2.

45
The comparison of LOD is shown inTable 2.5. The detection limits for the metabolites were 0.5
mg L-1, DMP; 0.1 mg L-1, DEP; 0.1 mg L-1, DMTP; 0.04 mg L-1, DMDTP; 0.04 mg L-1, DETP
and 0.02 mg L-1, DEDTP, and the CV for the analytical method was between 4 and 14% for the
six metabolites as reported earlier [Whyatt et al. 2001, Oglobline et al. 2001,Odetokun et al.
2010]. Our CVs are better than those reported in the literature for methods with higher LODs.
The reported LODs ranged from 0.044 to 1.549 ng mL-1 for all six DAP metabolites using solid
phase extraction with a weak anion exchange sorbent on a 96-well plate with isotope dilution and
HPLC with electrospray ionization-tandem mass spectroscopy (HPLC/ESI- MS/MS). The
accuracy of the method was also assessed by calculating the coefficient of determination (r2) by
spiking urine samples with different concentrations of pesticides and plotting the values obtained
against the area of the concentration. The observed accuracy for all compounds was >99.99%.
The uncertainty parameters, such as intercept (>0.053), slope (>0.999) and standard error in the
slope (>0.003) were also calculated, and these values were well within the prescribed limits,
which proved the accuracy and reproducibility of the described method (Table 2.3). These data
suggest that manual and laboratory errors were not significant.

Table: 2.5. Comparison of different reported LODs with our LODs of different metabolites using
different methods.
Bravo et al. Martin et al.
Analyte Bravo et al. 2002 CDC 2005 Our results
2004 2010
LODa LOQb LOD LOQb LODa LOQb LODa LOQ LODa LOQ
(ngmL (ngmL (ngmL (ngmL-
- - -
(ngmL- (ngmL- (ngmL- (ngmL- (ngmL- (ngmL-
1 1 1 1 1 1 1 1 1 1
) ) ) ) ) ) ) ) ) )
DMP 0.6 NRC 1000 NR 0.6 NR 0.468 NR 0.058 0.074
DEP 0.2 NR 720 NR 0.2 NR 0.044 NR 0.009 0.029
DMTP 0.2 NR 1600 NR 0.2 NR 0.066 NR 0.004 0.04
DETP 0.1 NR 380 NR 0.1 NR 0.11 NR 0.014 0.49
DMDTP 0.1 NR 780 NR 0.1 NR 0.073 NR 0.008 0.028
DEDTP 0.1 NR 250 NR 0.1 NR 10549 NR 0.019 0.066

Note: LOD = Limit of detection in ngmL-1; LOQ=Limit of quantification in ngmL-1; NR= Not
reported.

The reported method by different researchers has a high detection limit with lower percent
recovery in human urine samples. However, in some methods (Table 2.5), the percent recoveries,
CV and LOD in the urine matrix were higher than our method. Similarly, a new analytical

46
method was reported by Van den Eedea and Neelsa,[Van den Eede 2013]for the determination of
dialkyl and diaryl phosphates (DAPs), which are metabolites of organophosphate triesters
(PFRs), in human urine. In this method, sample preparation was performed by solid phase
extraction using a weak anion exchange sorbent (Oasis WAX). A reversed phase liquid
chromatography-negative electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS)
was used. The method accuracy was determined by running recoveries at 3 ng mL-1 in pooled
urine samples, which ranged between 69 and 119%, while inter-day imprecision (as relative
standard deviation) was <31%. The limits of quantification (LOQ) were 0.3 and 0.15 ng mL-1.

Moreover, in the method reported by Davis[Davis 2013]and others, urine was extracted by a
semi-automated solid phase extraction technique and an isotope quantification technique
followed by reversed-phase high performance liquid chromatography analysis.

In comparison, our method is much more cost-effective than the method adopted by Davis et al.,
where SPE cartridges and isotopes were used, which are very costly and some of them are not
readily available in all parts of the world. As we used only 10 mL of acetonitrile, we could
achieve better LODs and LOQs than the above reported methods (Table 2.5).

Our method employs a lyophilization followed by simple solvent (economic and cheaply
available) extraction analysis using selective LC-MS/MS. This method is selective and has
good precision and sensitivity. The limit of detection (LOD) is in the parts per billion (ppb)
ranges with CVs of typically <10%. The vital points of our method are its cost-effectiveness,
time saving and sensitive factors which are a matter of significance in days of recession and
unemployment. We used lyophilization followed by extraction without compromising on
sensitivity, selectivity and precision. This method may be used for analysis of DAP metabolites
in human urine samples for cumulative exposure due to OP pesticides especially in the
developing countries. Some polar compounds may also be extracted by OP metabolites during
extraction process by using acetonitrile. But due to the use of MRM, LC-MS/MS the other
organic polar compounds, except metabolites are removed. This method was used for analysis of
the OP, DAP metabolites in exposed population for health risk assessment, especially in children
of different age groups.

47
2.9. Dose estimation
Our earlier study [Sinha et al 2012] showed that the chlorpyrifos, fenitrithion and,
Imidachloropid were present in vegetables and fruits samples, where the urine samples were
collected for bio-monitoring and exposure assessment. On the basis of this early report we
selected chloripyrifos and fenitriothion pesticides for dose estimations. We calculated dose
estimates from urinary dialkyl metabolites and from agricultural pesticide use data, assuming
that all exposure came from a two pesticides (Fenitrothion and Chlorphyrifos) mainly used in
Hyderabad City.All urine samples containing concentrations below the LOD, assumed to have
concentrations equal to one-half of the LOD. Total dimethyl and diethyl molar quantities
havebeen calculated according to the reported formula [Cynthia et.al 2003].

[Dimethyl DAP] = [DMP]/125

+ [DMTP]/141

+ [DMDTP]/157, --------- [1]

[Diethyl DAP] = [DEP]/153

+ [DETP]/169

+ [DEDTP]/184, --------- [2]

Total molar metabolite quantities were multiplied by the volume of the 24-hr urine sample and
the molecular weight of the parent pesticide, and were divided by the child’s body weight
according to the formula reported as

Fenitrothion (Dose)* = [Dimethyl DAP] ×Volume × MW pesticide ×1/body weight. ----- [3]
Chlorpyifos (Dose) = [Diethyl DAP] ×Volume × MW pesticide ×1/body weight. ----- [4]

*Where Dose units are micrograms per kilograms per day (µg/Kg/Day), the metabolite
concentrations are in units of micromoles per liter (µmol/L), volume is in liters, units for
molecular weight are grams per mole (gm), and body weight measured in kilograms (Kg).

2.10.Statistical analysis: Statistical analysis was performed using SPSS 19.0 version.
Descriptive statistics were performed for different age groups and correlation matrix (Pearson
correlation) was computed for metabolites. The data skewed in nature therefore, non-parametric
Mann –Whitney U test, & spearman’s correlation applied for statistical analysis.

48
 Chapter 3
Development of a new method for
exposure assessment of
organophosphate pesticide
metabolites

49
3.1. Introduction:
Although both blood and urine have OP biomarkers, researchers prefer to use urine samples for
the OP exposure assessment studies due to its ease of collection and analysis. Urine is one of the
excretory liquid products of the body which is produced by filtration of blood by the kidneys.
Along with the main components urine also has often metabolic waste compounds. It is also one
of the body fluids frequently used for exposure assessment studies. It has excreted products of
the rapid metabolism of some environmental contaminants as different biomarkers like
organophosphate pesticide metabolites. However temporal variation of exposure, physiological
processes, and timing of sampling for biomarkers estimation may affect analytical results of
exposure estimations (Hinwood et al., 2002). Generally, 24-hour output urine is collected for
exposure studies to ensure utmost precision, however, in many cases; the collection of 24-hour
urine is a cumbersome procedure. Scientists used random spot urine samples collection as more
practical and convenient alternative for the above limitation (James and Roberts 2012,
Berenbaum and Snyder 1995). WHO reports of 1986 showed that biological half-lives of most
OP pesticide metabolites are relatively short generally on the order of 2 days or less when
compared to the other urinary biomarkers used for bio-monitoring studies (WHO, 1986). Whilst
Kissel et al. in their percent deviation analysis of spot sample time points they found that first-
morning void samples were consistently the best predictors of weighted-average daily metabolite
concentration. And, first-morning urine void is relatively more incorporated, provides an
integrated and relatively long exposure and is less influenced by factors such as hydration and
physical activity; hence it is to be preferred over a spot urine sample. To reinforce it, here we
developed a method for exposure assessment of OP pesticides.

3.2. Material and Methods

All materials and methods are explained in the chapter 2

3.3. Subjects:
The Scientific Advisory Committee and Ethical Committee of National Institute of Nutrition
approved the study. Total urine samples used for this method development were collected from
196 children and all of them are residing in the Hyderabad city. Out of them, 123 were 6-10
year-old and, 73 were 11-15-year-old age groups.

50
3.4. Results

3.4.1. Model construction:

Here we tried to formulate a model using the first morning, and 24-hour urine samples and found
that the first-morning urine sample is the bestpredictor of 24-hour exposure to pesticides. We
formulated generalized estimating equation (GEE) models using DAP concentrations in a 24-hr
sample as the outcome variable and concentration in the same-day first-morning void (FMV)
sample as the best predictor variable. Table 3.1 presents results of GEE models examining how
well same-day early morning samples predict 24-hr metabolite concentrations. For models
examining the concentrations of DAP in a single unadjusted morning sample (3.05 µgL-1) and
its respective 24-hr sample (1.7 µg L-1), the predictive power of the model, defined by the
coefficient of determination (R2), was highest for FMVs [r = 0.997, R2= 0.999 for DAP, P<0.00
and 99.6 % detection (Table 3.3, Figure 3.1)]. The predictive power of the model was lower for
creatinine-adjusted metabolites compared with 24-hr urine samples (DAP;r = 0.001, R2 =
0.0.031, p < 0.230 with 3.10 %detection). The best-fitting model was obtained when the
arithmetic mean of the FMV sample was used to predict the 24-hr values (Table 3.1). The model
fit was strongest for analyses of total dimethyl metabolites concentrations (DMs) =
DMP+DMTP+DMDTP) [r = 0.999, R2≈ 0.999, p <0.000, 99% detection, (Table3.1, Figure 3.1)],
and also stronger for diethyl metabolites concentrations (DEs) = DEP+DETP+DEDTP) (r =
0.915, R2 = 0.915, p<0.00, accuracy 91.5 % detection.) (Table3.2, Figure 3.3).

Table 3.1.Correlation between the concentrations of OP metabolites of 24-hr and early morning urine samples.

N Model
Type Metabolites r* R2 % Det. Intercept Slope p- value
DMs (µmolL-1) 197 0.999 0.999 99.9 0.07 1.482 <0.00
DEs (µmol L-1) 197 0.915 0.838 83.8 0.7 1.324 <0.00
M vs 24 hr
DAPs (µmol L-1) 197 0.996 0.996 99.6 0.344 1.11 <0.00

DMs (µmolL-1) 197 0.02 0.141 14.1 -0.003 -0.01 0.047
CA vs. 24 hr DEs (µmol L-1) 197 0.37 0.192 19.2 0 -0.017 0.551
197 0.001 0.031 3.1 -0.002 -0.015 0.23
DAPs (µmol L-1)

*Non parametric Spearmen’s correlation (significant at the 0.01 level; 2-tailed)

FMU = First Morning unadjusted samples

51
24hour = 24hour urine

N= Number of samples.

% Det. = % determination

DMs (µmol L-1) = Dimethyl phosphate metabolites(DMP+DMTP+DMDTP),

DEs (µmol L-1) = Diethyl phosphate metabolites (DEP + DETP+ DEDTP),

DAP (µmol L-1) = dialkyl phosphate metabolites (DMs +DEs).

Generally, compliance with 24-hr urine samples can be low in population-based studies and first-
morning void urine samples are often collected for convenience. Interpretation of pesticides
concentrations in urine is influenced by a range of factors unrelated to exposure. To reduce the
influence of such factors, creatinine adjustment is routinely used. This study is aimed to
determine whether first-morning void urinary metabolite concentrations approximate 24-hr
urinary metabolite concentrations and whether creatinine adjustment improved the correlation
between first-morning urinary metabolite concentrations and 24-hr urinary metabolite
concentrations. For this purpose, one hundred ninety-six first morning urine samples and
corresponding 24-hr urine samples were collected from the children living in urban areas and
extracted and analyzed for metabolites using LC-MS/MS. Correlation showed that there is no
significant difference between the first-morning urinary metabolite concentrations for the
different sample types. Therefore we suggest that concentrations of metabolites found in first-
morning urine sample may be a good predictor of 24-hr-exposure instead of concentration found
in 24-hr urine samples and there is no need to collect the 24-hr urine samples for assessment of
exposures. DAP metabolites in single or multiple spot samples are strongly correlated with levels
in same-day 24-hr samples, children’s urinary DAP concentrations vary greatly from day to day,
and use of spot samples to characterize a child’s cumulative weekly exposure results in a
moderate degree of misclassification. Exposure misclassification resulting from urinary
metabolite variability has the potential to bias measures of association between early childhood
OP exposures and developmental outcomes in epidemiologic research toward the null
hypothesis; such misclassification may account for the weak or null findings often reported to
date between pediatric urinary DAP concentrations and child development (Bouchard et al. .
2011; Marks et al. .2010). Additional research on variability in measures of no persistent
compounds in children is needed to assure that exposure biomarkers are valid and that

52
epidemiological studies have adequate power to detect health outcomes that may result from
these exposures.

3.4.2. Concentrations of metabolites with creatinine and without creatinine.

Central tendency measures (e.g., means) for unadjusted concentration values were lower for
FMV (3.05 µmol L-1) samples and 24-hr samples (1.7 µmol L-1), which were generally lower
than levels in non-FMV spot samples. In contrast, central tendency measures for creatinine-
adjusted concentration values were higher for FMVs (5.17 µmol L-1, Table 3.2).

Table 3.2 Comparison of mean and median concentrations of OP metabolites in the creatinine
adjusted and unadjusted of first morning and 24-hr urine samples.

DETP DAP
Type of DMP DMTP DMDTP DEP DEDTP
Age group N (µg L- (µmol L-
samples (µg L-1) (µg L-1) (µg L-1) (µg L-1) 1 (µg L-1)
) 1)

6-15 yrs 123 0.06 0.21 0.01 1.13 0.32 0.02 2.92
MUA 11-15 yrs 73 0.06 0.25 0.01 0.83 0.63 0.02 3.22
Total 196 0.06 0.22 0.01 0.89 0.45 0.02 3.05
DETP
DMP DMTP DMDTP DEP DEDTP
N (µg g- DAP
(µg g-1) (µg g-1) (µg g-1) (µg g-1) 1
)
(µg g-1)

6-15 yrs 113 0.13 0.39 0.02 1.62 0.48 0.05 5.06
MA 11-15 yrs 71 0.13 0.46 0.02 1.31 0.95 0.05 5.27
Total 184 0.13 0.41 0.02 1.45 0.64 0.05 5.17
DETP DAP
DMP DMTP DMDTP DEP DEDTP
N (µg L- (µmol L-
(µg L-1)* (µg L-1) (µg L-1) (µg L-1) 1
) (µg L-1) 1)

6-15 yrs 123 0.04 0.12 0.01 0.55 0.17 0.02 1.4
24-hr 11-15 yrs 73 0.05 0.2 0.01 0.51 0.38 0.02 2.39

Total 196 0.04 0.15 0.01 0.53 0.25 0.02 1.7

MUA = Morning unadjusted urine samples.

MA = Morning adjusted urine samples.

24-hr = 24hour urine samples.

N= number of samples.

*µg g-1 = µg per g creatinine

yrs = years.

53
3.5. Discussion:

The correlation between FMVs and 24-hr samples indicate that they are the best predictor of 24-
hr exposure rather than any spot samples. On the basis of this observation, the estimated doses of
the total pesticide exposure to children of different age groups were calculated. This study of
variability in DAP metabolites of OP pesticides in urine samples from children aged 6–15 years
old indicates high variability within a time frame. We observed strong correlations between full-
day 24-hr samples and same-day early morning samples (Table 3.1, Figure 3.4), with strong
correlation for DMs and DEs. There was weak correlation between DAP metabolites levels in
24-hrsamples collected with creatinine-adjusted early morning samples on the same day, and
associations were weaker for DEs compared with DMs and DAP metabolites. In our study the
early morning urine samples without creatinine adjustment had strong correlation with 24-hr
concentrations of pesticides. Recently, Bradman et al. (2013) collected spot urine as well as 24-
hr urine samples from 25 children aged 3-6 yrs. age group and to assess the reproducibility of
urinary DAP metabolites concentrations. In this report authors observed that the DAP
concentration in single spot urine samples were moderately associated with concentrations in
same-day 24-hr samples (r =0.6, p<0.01), but in our study we found that the DAP concentrations
in morning urine samples were strongly associated with concentrations in same-day 24-hr
samples (r =0.996, p < 0.00). When compared to the routine procedure of OP exposure
assessment, by using spot urine samples, our approach is better due to greater sample size, no
need for creatinine adjustment and a simple calculation procedure (Figure 3.4).

54


[*Coefficient of determination, Model R2 for FMV, R2 = (0.99, P<0.000, accuracy 99%) of

DM’s (DMP+DMTP+DMDTP)]

Figure 3.1: Model R2 of DM’s between FMUS (first morning urine samples) and 24- hour
samples

55


[*Coefficient of determination, Model R2= (0.95, P<0.000, accuracy 83.8%) of DE’s

(DEP+DETP+DEDTP)]

Figure 3.2: Model R2 of DEs between FMUS (first morning urine samples) and 24- hour
samples.

56


[*Coefficient of determination, R2 for FMV, R2 = 0.996 for DAP, P<0.000 and 99.6% accuracy]

Figure3.3. Model R2 of DAPs between FMUS (first morning urine samples) and 24-hour
samples.

Correlation btn. 24-hr and first morning

urine samples.
2
FMV vs 24 hr

1.5
1 DMs (µmolL-1)
0.5 DEs (µmol L-1)
0
DAPs (µmol L-1)
2

pt
*

lue
pe
R
r

ce

Slo

va
el

r
te
od

p-
In
M

Figure3.4. Correlation between the concentrations of DAP metabolites of 24-hour and early
morning urine samples.

57
 Chapter 4
Biomonitoring of organophosphate
pesticide metabolites in Hyderabad
urban children

58
4.1. Introduction:
Bio-monitoring is an efficient and cost-effective way to measure environmental chemicals, or
their breakdown products, in the human body. Biological specimens such as blood and urine are
tested to check whether certain chemicals are present and to what levels. Bio-monitoring can be
used to reinforce regulatory actions, to improve exposure assessment, to draw baselines or
reference values, to tackle environmental health-related problems, to identify health disparities in
communities.

The committee on Human Bio-monitoring for Environmental Toxicants has defined “bio-
monitoring is one method for assessing human exposure to chemicals by measuring the
chemicals or their metabolites in human specimens, such as blood or urine” (CDC 2005).

Bio-monitoring of organophosphate pesticide metabolites in different populations, groups and

communities is the best method researchers are adapting now to assess the OP exposure in the
respective group of the study population.

Applications:

• To analyze the actual internal levels of bodily substances from all potential routes of
exposure at one time.
• Whether a chemical measured in an individual or a population may cause a health risk
and determine its sources.
• To understand the presence of chemicals in the human body and their effects on
humanhealth
• To improve exposure assessment, to draw baselines or reference values, to tackle
environmental health-related problems, to identify health disparities in communities.

As far as we know usage of OP pesticides around the world started very decades back and
enough research has been conducted which is at par with the usage and studies are still going on
to check toxicity of OP pesticides. When compared to other countries especially the US, India is
lagging behind in this area of study. The studies on OP pesticides which include, occupational
exposure of organophosphates [Hayes 1980, Saito 1884 ] and more recent studies on mutagenic
and cytotoxic effects of organophosphorus insecticides [Eslava 2013] and Cytotoxic and
Genotoxic Effects of OP pesticides[Timoroğlu 2014, Dhanushka 2017 ]and Chromosomal

59
abnormalities due to OP pesticides[Figueroa 2015]werealso among those studies. These studies
even focused sufficiently on children of parents who were engaged in Agricultural activities
[Loewenherz 1997, Stuart 2003, Chensheng 2004] Preschool Children [Lu 2001, Fenske, Curl
2003,] and in Italian children [Aprea 2000], and also how diet effects the exposure in children
[Lu 2006, Kissel 2005,Morgan 2005] and effects on health due to OP pesticides [Eskenazi 1999].

In India studies on OP pesticides and their exposure are very few, some of them are, the
evaluation of pesticide residues in farmgate and market samples of Vegetables [Singh2002,
Srivastava 2011,AnandaGowda 2012], Organophosphorus pesticide residues in fodder and milk
samples[Kotinagu 2015] and in breast milk [Sanghi 2003], Contamination of vegetables with
these pesticides and related health risk assessment [Bhanti 2007], Chronic pesticide exposure
and Health effects [Mathew 2015]. Except thesefew studies, research on OP exposure especially
in children is very rare in India.

So here we made a small attempt to fill this gap by bio-monitoring OP metabolites in children of
Hyderabad urban area.

4.2. Methods and materials: Mentioned in Chapter 3

4.3. Results
Bio-monitoring study of OP metabolites in Hyderabad urban children was initiated in 2012 to
investigate exposure levels of OP metabolites in children from five urban areas: West, South,
North, East, and Central zones.Figure 2.1 [in chapter 2]presents the demographic characteristics
of the subjects recruited and samples collected. Participants were recruited using random home
visits, and were 6–15 years of age at enrollment with an approximately equal gender ratio.We
recruited a convenience sample size of 456 boys, 449 girls from the children residing in
Hyderabad city (6-10 years and 11-15 years), who consume conventional food. Equal number of
children of the same age from both the groups were (6-10 and 11-15 years)selected. Parents of
those children in the 6–15-year-old age range were asked to fill forms about their children’s
diets. The Scientific Advisory Committee and Ethical Committee of National Institute of
Nutrition approved the study, and parents provided written informed consent.

60
Total subjects initially enrolled for of this study were 1050, but 64 dropped from the study and
986 (93.9%) participated. Out of 986, only 905 (91.7%) families could provide urine sample of
their children. Hence we collected urine samples from 91.7% (905 of 986) out of total
participating children. Out of the 905 subjects, 52.7 % (477 of 905) children were at 6-10 years
old, and 42.7 % (428 of 905) of children were at 11-15 years old. Participating children turned 6-
10 years of age between 2006 and 2002 in 6-10 years group; 11-15 years of age between 2003
and 1999 in 11-15 years age group.

4.3.1. Demographic Characteristics:

The number of participants from each zone were; 163(18%) from west zone, 181(20%) from
south zone, 200(22.1%) from north zone, 180(19.8%) from east zone and 181 (20%) from
Central zone [Fig.2.1 & Fig. 2.2].

The mean ages of the participating children were 8.32 years for male and 8.16 years for female
children of age group 6-10 and the mean ages were 12.81 years for male and 12.74 years for
female children of age group 11 – 15 years old. While the mean weights of the subjects from the
6 - to 10 -year-old children was 27.44 kg for males and 28.05 kg respectively for females.
Likewise, the children aged 11- to 15-year-old, did not vary by mean weightasmean weights
were 30.29 kg and 30.32 kg for males and females respectively [Table 4.1].

61
Table 4.1.Demographic characteristics of participating children (n = 905).

Category N % of samples
6-10 Yrs. 477 52.71
11-15 Yrs. 428 47.29
Male 456 50.39
Female 449 49.61
LIG 304 33.59
MIG 325 35.91
HIG 276 30.5
West zone 163 18.01
South zone 181 20
North zone 200 22.1
East zone 180 19.89
Central zone 181 20
Total 905 100

4.3.2.OP Metabolite Levels Across Agegroups:

Eighty percentof the study group (905 children) had at least one measurable DAP metabolite. Of
the five DAP metabolites targeted for analysis; DMTP was the dominant metabolite, as it was
found at detectable levels (>LOD) in 88.1% of all urine samples. The DEAP metabolites were
dominated byDEP and DETPwhich were found with frequencies of 87.2% and 86.6%
respectively. DMP, DMDTP and DEDTP have shown similar detection frequencies of 75.3%,
73.4%, and 74.3% respectively. DMTP, DEP and DETP concentrations were substantially higher
than concentrations of the other three compounds.DMTP concentrations were substantially
higher than concentrations of the other five compounds ( Non parametric Spearmen’s significant
test, p ≤ 0.0002). However there was no significant difference found between dimethyl DAP
levels and total diethyl DAP levels (medians, 0.07 and 0.07 µmol/L, respectively; Non
parametric Spearmen’s significant test, p > 0.0012). [Table 4.2 and Table 4.3].

62
Table 4.2.Mean and median concentrations of OP metabolites in Hyderabad urban children.

OP Mean Median
Category N DF SD
Metabolite (µg/L) (µg/L)
DMP Total 905 75.30% 6.82 1.9 20.48
DMTP Total 905 88.10% 9.4 2.61* 29.46
DMDTP Total 905 73.40% 4.48 1.85 9.26
DEP Total 905 87.20% 8.75 2.33 23.45
DETP Total 905 86.60% 14.29 2.88 40.68
DEDTP Total 905 74.30% 2.09 0.12 5.7
#Total DMTP > other four metabolites;p ≤ 0.0001 ( Non parametric Spearmen’s significant test).

Table 4.3.Mean and median molar concentrations of dimethyl and diethyl OP metabolites in
Hyderabad urban children.

Total Molar Mean Median

Category N SD
Concentrations (µmol/L) (µmol/L)
DMAP# Total 905 0.151 0.07* 0.29
DEAP# Total 905 0.154 0.07* 0.3
DAP$ Total 905 0.3042 0.14 0.48

*Total dimethyl = total diethyl; p > 0.0001 (Non parametric Spearmen’s significant test).

Most participants found to have been detected with at least one DAP were within 6-10 and11-15
years, DMAP metabolite levels were similar to the DEAP metabolite levels. DMAP metabolite
levels were almost equal in 6-10 years compared to the 11-15 year old children. There is no clear
pattern in the change in DMAP and DEAP levels with age.Methyl (DMAP), ethyl (DEAP) and
alkyl (DAP) metabolite concentrations were compared across two age groups (6-10 years. and
11-15 years.). We found no significant differences for either dimethyl or diethyl concentrations
(Non parametric Spearmen’s significant test, p > 0.001). We then averaged the two samples from
each child to represent the DAP concentrations during the study period (2012-2017). We found
no significant differences even in the median concentrations of either dimethyl or diethyl
metabolites.

Table 4.4 presents descriptive statistics for the concentrations of dialkyl phosphate metabolites
and molar concentrations of DEAP, DMAP and DAPin urine samples of children of age groups
6-10 years and 11-15 years. Among 6-10 years DMAP metabolites were dominated by DMTP

63
(mean7.82 µg/L; median; 2.59 µg/L) while DMP and DMDTP detected with median
concentrations of 1.57 and 1.85 µg/L (mean 6.55 µg/L and 4.42 µg/L) respectively whereas the
DEAP metabolites were dominated by DETP and DEP with concentrations with medians; 2.6
µg/L and 2.09 µg/L (mean 13.8 µg/L and 8.43 µg/L)

However, both dimethyl and diethyl DAP concentrations in both age groups and in total
samples are similar and as the median DMAP levels were 0.07 μ mol/L and DEAP levels
were 0.07 μmol/L in 6-10 years old age group. While median DMAP and DEAP
concentrations were 0.8μmol/L and 0.8 μmol/L (mean; 0.163 μ mol/L and 0.159 μmol/L)
in the age group 11-15 old children. Pooling data from the two groups shows that the mean
concentrations of dimethyl and diethyl DAP median concentrations were 0.07 μmol/L and
0.07 μmol/L (mean 0.151 μmol/L and 0.154 μmol/L) respectively. And Total DAP median
concentrations were 0.14 μmol/L(mean 0.304 μmol/L).

In the two age groups 6-10 years and 11-15 years, both median DAP values (0.07μmol/L
and 0.07μmol/L respectively), and mean DAP values(0.151μmol/L and 0.154 μmol/L),did
not differ by any significant factor.

64
Table 4.4.Comparision of Children’s urinary DAP metabolite levels between 6-10 year and 11-
15 year age groups.

Compound Mean Median SD

6-10 yrs. (N=477)
DMP (µg/L) 6.55 1.57 22.24
DMTP (µg/L) 7.82 2.59 22.58
DMDTP (µg/L) 4.42 1.85 9.78
DEP (µg/L) 8.43 2.09 24.79
DETP (µg/L) 13.8 2.6 41.88
DEDTP (µg/L) 2.2 0.11 7.07
@DMAP (µmol/L) 0.14 0.07* 0.27
#DEAP (µmol/L) 0.15 0.31
0.07**
$DAP (µmol/L) 0.29 0.14 0.48
11-15 yrs. (N=428) Mean Median SD
DMP (µg/L) 6.74 2.33 15.83
DMTP (µg/L) 11.2 2.92 35.75
DMDTP (µg/L) 4.6 1.85 8.79
DEP (µg/L) 9.21 2.91 22.05
DETP (µg/L) 14.88 3.18 39.53
DEDTP (µg/L) 2 0.18 3.65
DMAP (µmol/L) 0.163 0.8* 0.305
DEAP (µmol/L) 0.159 0.8** 0.286
DAP (µmol/L) 0.32 0.183 0.497
Total (905)
DMAP (µmol/L) 0.151 0.07* 0.29
DEAP (µmol/L) 0.154 0.07* 0.3
DAP (µmol/L) 0.304 0.14** 0.48

Abbreviations: yrs., years; SD, Standard deviation;

@DMAP is sum of DMP, DMTP, and DMDTP concentrations.

#DEAP is sum of DEP, DETP, and DETP concentrations.

$DAP is sum of DMAP and DEAP concentrations

*p < 0.001(Non parametric Spearmen’s significant test).

*Dimethyl concentration in 6-10 years old ~ dimethyl concentration in 11-15 years; p > 0.0003
(Non parametric Spearmen’s significant test).

65
*Diethyl concentration in 6-10 years old ~ dimethyl concentration in 11-15 years; (Non
parametric Spearmen’s significant test, p = 0.13)

**Total dimethyl total diethyl; p > 0.0001 ( Non parametric Spearmen’s significant test).

4.3.3. OP Metabolite Levels Across Gender Groups:

Methyl (DMAP), ethyl (DEAP) and alkyl (DAP) metabolite concentrations were compared
across two genders (male and female).Table 4.5 presents descriptive statistics for the
concentrations for DAP metabolites and molar concentrations of DEAP, DMAP and DAP in age
male and female children of Hyderabad city. Among 6-10 years The DMAP metabolites were
dominated by DMTP (median 3.05 µg/L) while DMP and DMDTP detected with median
concentrations 2.29 and 1.96 µg/L respectively; whereas the DEAP metabolites were dominated
by DETP and DEP with median concentrations 3.08 µg/L and 2.09 µg/L. Among male and
female children, DMAP metabolite median levels were similar to the DEAP metabolite levels.
The DMAP concentrations were 0.08µmol/L and 0.06µmol/L in male and female subjects
respectively. Likewise the diethyl concentration in boys was 0.07µmol/L while it was
0.08µmol/L in girls were. And total DAP median molar concentrations were in both the genders
0.14 µmol/L. DMAP metabolite levels were almost equal in male children as compared to the
female children. There is no clear pattern in the change in DMAP and DEAP levels with
sex.Neither, median dimethyl nor diethyl DAP concentrations were significantly different
between male and female children (Table 4.5; Non parametric Spearmen’s significant test, p
>0.005).

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Table 4.5.Comparision of Dialkylphosphate metabolite concentrationsin urine samples collected
from both male and female children living in the Hyderabad urban area.

Metabolite Mean Median SD

Male (N=456)
DMP (µg/L) 7.51 2.29 20.55
DMTP (µg/L) 10.2 3.05 32.06
DMDTP (µg/L) 4.7 1.96 8.45
DEP (µg/L) 7.58 2.09 19.95
DETP (µg/L) 13.6 3.08 39.04
DEDTP (µg/L) 2.38 0.2 5.36
DMAP (µmol/L) 0.163 0.08* 0.306
DEAP (µmol/L) 0.143 0.07* 0.272
DAP (µmol/L) 0.306 0.14 0.5

Female (N=443)
DMP (µg/L) 6.12 1.62 20.39
DMTP (µg/L) 8.6 2.41 26.57
DMDTP (µg/L) 4.26 1.8 10.08
DEP (µg/L) 9.95 2.77 26.51
DETP (µg/L) 15 2.72 42.32
DEDTP (µg/L) 1.81 0.1 6.03
DMAP (µmol/L) 0.137 0.06* 0.276
DEAP (µmol/L) 0.164 0.08* 0.325
DAP (µmol/L) 0.301 0.14 0.475

Abbreviations: yrs., years; SD, Standard deviation; Max, maximum; Min, minimum.

@DMAP is sum of DMP, DMTP, and DMDTP concentrations.

#DEAP is sum of DEP, DETP, and DETP concentrations.

$DAP is sum of DMAP and DEAP concentrations.

*p > 0.005 (Non parametric Spearmen’s significant test).

67
4.3.4. Male (6-10 yrs.):
In the male children with 6-10 years old highest level was of DETP; 13.2µg/L and least was of
DEDTP with 3.09 µg/L. And mean concentration of DMAP was 0.17 µmol/L, DEAPs was 0.128
µmol/L. and DAP was 0.3 µmol/L [Table 4.6].

4.3.3.1. Male (11-15 years.): In the male children with 11-15 years old the highest metabolite
level was of DETP; 13.82µg/L and least was of DEDTP with concentration of 2.03 µg/L. And
mean concentration of DMAP was 0.156 µmol/L, DEAPs was 0.153 µmol/L. and DAP was 0.3
µmol/L [Table 4.6].

Table 4.6.Comparison of Dialkylphosphate metabolite concentrations in urine samples collected

from male children of both 6-10 and 11-15 years old living in the Hyderabad urban area.

Male (6-10 yrs.) N=158 Male (11-15 yrs.) N=291

Metabolite Mean Median SD Mean Median SD
DMP (µg/L) 9.44 2.84 26.66 5.93 2.17 11.93
DMTP (µg/L) 9.08 3.37 25.21 10.83 2.5 35.47
DMDTP (µg/L) 4.67 2.38 5.47 4.79 1.8 9.78
DEP (µg/L) 5.01 1.64 9.99 9.07 2.69 23.74
DETP (µg/L) 13.2 2.59 47.7 13.82 3.11 33.81
DEDTP (µg/L) 3.09 0.33 7.42 2.03 0.13 3.84
DMAP(µmol/L) 0.17 NC 0.297 0.156 NC 0.295

DEAP(µmol/L) 0.128 NC 0.297 0.153 NC 0.259

DAP (µmol/L) 0.3 0.15 0.53 0.31 0.16 0.48

4.3.3.2. Female (6-10 yrs.):

In the female children with 6-10 years old highest level was of DETP; 14.09µg/L and least was
of DEDTP with 1.76 µg/L. And mean concentration of DMAP was 0.12 µmol/L, DEAPs was
0.16 µmol/L. and DAP was 0.28 µmol/L [Table 4.7].

68
4.3.3.2. Female(11-15 years):
In the female children with 11-15 years old, the highest metabolite level was of DETP;
17.24µg/L and least was of DEDTP with concentration of 1.93 µg/L. And mean concentration of
DMAP was 0.181 µmol/L, DEAPs was 0.175 µmol/L. and DAP was 0.36 µmol/L [Table 4.7].

Table4.7. Comparison of dialkylphosphate metabolite concentrations in urine samples collected

from female children of both 6-10 and 11-15 years old who were living in the Hyderabad urban
area.
Female (6-10 yrs.) N=391 Female (11-15 yrs.) N=137
Metabolite Mean Median SD Mean Median SD
DMP (µg/L) 5.12 1.33 19.58 8.55 2.73 22.16
DMTP (µg/L) 7.2 2.02 21.16 12.04 3.24 36.48
DMDTP (µg/L) 4.29 1.66 11.33 4.2 2.04 6.02
DEP (µg/L) 10.12 2.63 29.35 9.53 2.99 17.76
DETP (µg/L) 14.09 2.66 38.75 17.24 3.33 50.11
DEDTP (µg/L) 1.76 0.09 6.85 1.93 0.25 3.21

DMAP(µmol/L) 0.12 NC 0.251 0.181 NC 0.34

DEAP(µmol/L) 0.16 NC 0.32 0.175 NC 0.286

DAP (µmol/L) 0.28 0.12 0.45 0.36 0.21 0.54

4.5.8. High Income Group (HIG)(N=304):

In the high income group children highest metabolite level found was of DETP; 22.07µg/L and
least was of DEDTP with2.73µg/L. And mean concentration of DMAP was 0.18
µmol/L,DEAPs was 0.19 µmol/L. and DAP was 0.37 µmol/L [Table 4.8].

69
Table 4.8.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
HIG group children.

Mean Median
Metabolite SD
(µg/L) (µg/L)
HIG (N=304)

DMP (µg/L) 7.08 2.38 17.11

DMTP (µg/L) 13.68 3.21 44.09

DMDTP (µg/L) 4.22 2.08 5.42

DEP (µg/L) 6.17 2.32 12.46
DETP (µg/L) 22.07 5.14 58.18
DEDTP (µg/L) 2.73 0.53 5.18
DMAP(µmol/L) 0.18 NC 0.38
DEAP(µmol/L) 0.19 NC 0.38
DAP (µmol/L) 0.37 0.17 0.66

4.5.9. Medium Income Group (MIG) (N=325):

In the medium income group children highest metabolite levelfound was of DETP; 9.82µg/L
and least was of DEDTP with 1.91µg/L. And mean concentration of DMAP was 0.14 µmol/L,
DEAPs was 0.13 µmol/L. and DAP was 0.27 µmol/L [Table 4.9].

70
Table 4.9.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
MIG group children.

Mean Median
Metabolite SD
(µg/L) (µg/L)
MIG (N=325)

DMP (µg/L) 7.4 1.86 26
DMTP (µg/L) 7.38 2.21 16.95

DMDTP (µg/L) 4.56 1.6 12.38

DEP (µg/L) 9.77 1.96 28.6

DETP (µg/L) 9.82 2.15 22.22

DEDTP (µg/L) 1.91 0.09 7.19

DMAP(µmol/L) 0.14 NC 0.26

DEAP(µmol/L) 0.13 NC 0.24
DAP (µmol/L) 0.27 0.13 0.37

4.5.10. Low Income Group (LIG) (N=276):

In the low income group children highest metabolite level found was of DETP; 11.01µg/L and
least was of DEDTP with1.61µg/L. And mean concentration of DMAP was 0.13 µmol/L
DEAPs was 0.14 µmol/L. and DAP was 0.27 µmol/L [Table 4.10].

Table 4.10.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
LIG group children.

Mean Median
Mtabolite SD
(µg/L) (µg/L)
LIG (N=276)
DMP (µg/L) 5.85 1.59 16.06
DMTP (µg/L) 7.1 2.43 18.5
DMDTP (µg/L) 4.69 2.08 8.42
DEP (µg/L) 10.41 3.61 25.72
DETP (µg/L) 11.01 2.57 32.05
DEDTP (µg/L) 1.61 0.08 3.97
DMAP(µmol/L) 0.13 NC 0.2
DEAP(µmol/L) 0.14 NC 0.26
DAP (µmol/L) 0.27 NC 0.27

71
4.5.11. Across the zones:
4.5.11.1. West Zone (N=163):
In the west zone children highest metabolite level found was of DETP; 16.86 µg/L and least was
of DMDTP with 4.37 µg/L. And mean concentration of DMAP was 0.18 µmol/L, DEAPs was
0.16 µmol/L. and DAP was 0.34 µmol/L[Table 4.11].

Table 4.11.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
west zone children.
Metabolite Category N Mean Median SD
DMP (µg/L) West Zone 163 9.12 3.51 28.23
DMTP (µg/L) West Zone 163 11.74 4.53 27.81
DMDTP (µg/L) West Zone 163 4.37 2.39 4.75
DEP (µg/L) West Zone 163 4.71 2.71 5.72
DETP (µg/L) West Zone 163 16.86 5.26 47.07
DEDTP (µg/L) West Zone 163 4.4 2.04 9.36
DMAP(µmol/L) West Zone 163 0.18 NC 0.33
DEAP(µmol/L) West Zone 163 0.16 NC 0.29
DAP (µmol/L) West Zone 163 0.34 0.21 0.51

4.5.11.2. South Zone (N=161): In the west zone children highest metabolite level found was of
DEP; 13.35 µg/L and least was of DEDTP with 0.57 µg/L. And mean concentration of DMAP
was 0.16 µmol/L, DEAPs was 0.26 µmol/L. and DAP was 0.26 µmol/L [Table 4.12].

Table 4.12.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
south zone children.

Metabolite Category N Mean Median SD

DMP (µg/L) South Zone 181 4.11 0.77 24.02
DMTP (µg/L) South Zone 181 5.82 1.38 14.76
DMDTP (µg/L) South Zone 181 4.59 1.72 7.75
DEP (µg/L) South Zone 181 13.35 1.78 35.58
DETP (µg/L) South Zone 181 11.17 1.29 29.93
DEDTP (µg/L) South Zone 181 0.57 0.02 1.59
DMAP(µmol/L) South Zone 181 0.16 NC 0.31
DEAP(µmol/L) South Zone 181 0.26 NC 0.39
DAP (µmol/L) South Zone 181 0.26 0.12 0.39

72
4.5.11.3. East Zone (N=180):
In the west zone children highest metabolite levelfound was of DEP; 21.24 µg/L and least was of
DEDTP with 2.37 µg/L. And mean concentration of DMAP was 0.18 µmol/L, DEAPs was
0.17µmol/L. and DAP was 0.35 µmol/L [Table 4.13].

Table 4.13.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
east zone children.
Metabolite Category N Mean Median SD
DMP (µg/L) East Zone 180 7.15 2.58 13.11
DMTP (µg/L) East Zone 180 13.53 3.17 39.61
DMDTP (µg/L) East Zone 180 3.67 1.22 6.65
DEP (µg/L) East Zone 180 5.46 1.46 13.78
DETP (µg/L) East Zone 180 21.24 3.32 49.13
DEDTP (µg/L) East Zone 180 2.37 0.25 6.09
DMAP(µmol/L) East Zone 180 0.18 NC 0.36
DEAP(µmol/L) East Zone 180 0.17 NC 0.34
DAP (µmol/L) East Zone 180 0.35 0.13 0.6

4.5.11.4. North Zone (N=200):

In the north zone children highest metabolite level found was of DMP; 9.11µg/L and least was of
DEDTP with 1.45 µg/L. And mean concentration of DMAP was 0.15 µmol/L, DEAPs was
0.11µmol/L. and DAP was 0.26 µmol/L [Table 4.14].

Table 4.14.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
north zone children.
Metabolite Category N Mean Median SD
DMP (µg/L) North Zone 200 9.11 2.31 22.45
DMTP (µg/L) North Zone 200 6.46 2.56 18.67
DMDTP (µg/L) North Zone 200 5.41 3.12 8.7
DEP (µg/L) North Zone 200 8.66 3.4 23.56
DETP (µg/L) North Zone 200 6.93 1.79 28.25
DEDTP (µg/L) North Zone 200 1.45 0.09 2.59
DMAP(µmol/L) North Zone 200 0.15 NC 0.23
DEAP(µmol/L) North Zone 200 0.11 NC 0.23
DAP (µmol/L) North Zone 200 0.26 0.13 0.39

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4.5.11.5. Central Zone (N=200):
In the west zone children highest metabolite level found was of DETP; 16.35µg/L and least was
of DEDTP with 1.97µg/L. And mean concentration of DMAP was 0.14 µmol/L, DEAPs was
0.18 µmol/L. and DAP was 0.32 µmol/L.

Table 4.15.Mean and median molar concentrations of diemethyl and diethyl OP metabolites in
central zone children.
Metabolite Category N Mean Median SD
DMP (µg/L) Central 181 4.6 2.31 7.73
DMTP (µg/L) Central 181 10.06 2.6 38.12
DMDTP (µg/L) Central 181 4.26 1.11 14.97
DEP (µg/L) Central 181 11.18 5.02 24.73
DETP (µg/L) Central 181 16.35 4.13 44.83
DEDTP (µg/L) Central 181 1.97 0.11 5.48
DMAP(µmol/L) Central 181 0.14 NC 0.29
DEAP(µmol/L) Central 181 0.18 NC 0.32
DAP (µmol/L) Central 182 0.32 0.16 0.52

Among total zones west zone and central zone children had high levels of DAP followed by east,
south and north [Table 4.16]

Table 4.16. Mean molar concentrations of diemethyl and diethyl and dialkyl OP metabolites
infive zones ofchildren.
TM AM DMAP DEAP DAP
Zone N (µg/L) (µg/L) (µmol/L) (µmol/L) (µmol/L)
West 163 51.2 8.53 0.18 0.16 0.34
South 181 39.63 6.6 0.1 0.16 0.26
North 200 38.03 6.34 0.15 0.11 0.26
East 180 53.41 8.9 0.18 0.17 0.35
Central 181 48.42 8.07 0.14 0.18 0.32

4.6. Distribution of pesticides fenitrothion (FNTN) and chlorpyrifos (CLPS):

4.6.1. Age Group wise:In 6-10 years age group the exposure of fenitrothion (FNTN) was
0.82 µg/Kg/day (<<CRFD) and exposure of chlorpyrifos (CLPS) was 1.24 µg/Kg/day(>CRFD).
While in 11-15 years age group the exposure of fenitrothion (FNTN) was 0.72

74
µg/Kg/day(<<CRFD) and exposure of chlorpyrifos (CLPS) was 1.02 µg/Kg/day (~
CRFD)[Table 4.17 & 4.18].

Table 4.17.Distribution of mean dose levels of pesticides fenitrothion (FNTN) and chlorpyrifos
(CLPS) children.
FNTN CLPS
Category Subcategory N (µg/Kg/day) (µg/Kg/day)
Mean SD Mean SD
6-10 yrs. 477 0.82 2.17 1.24 3.01
Age Group
11-15 yrs. 421 0.72 1.73 1.02 2.09

Male 456 0.804 1.81 1.037 2.45

Sex
Female 449 0.74 2.129 1.228 2.767

Male 158 1.06 2.07 1.1 2.99

6-10 yrs.
Female 319 0.7 2.21 1.31 3.03
Male 291 0.66 1.63 1.02 2.14
11-15 yrs.
Female 130 0.86 1.92 1.03 1.98

HIG 276 0.94 1.13 1.34 3.3

Income
MIG 325 0.77 1.84 0.97 1.98
Group
LIG 304 0.6 2.61 1.1 2.39

West 163 0.93 1.9 1.01 2.53

South 181 0.50 1.71 1.19 2.49
Zones North 200 0.79 1.57 0.86 2.24
East 180 0.97 2.67 1.27 2.84
Central 181 0.68 1.84 1.35 2.93
Total All 905 0.77 1.97 1.13 2.61

The US EPA (Environmental Protection Agency) released Chronic Reference Dose (CRFD) of
Fenitrothion as 1.3µg/Kg/day and of Chlorpyrifos as 1.0 µg/Kg/day.
Chronic oral reference dose in units of µg/kg/day (U.S. EPA 2002)

4.6.2. Sex wise:On observation it was found that dose exposure of OP pesticides gender wise
in children of Hyderabad city was of fenitrothion (FNTN) 0.804 µg/Kg/day (<<CRFD) and of
chlorpyrifos (CLPS) was 1.037(~CRFD) µg/Kg/day in boys and incase of girls the exposure of

75
fenitrothion (FNTN) was 0.74µg/Kg/day (~ CRFD) and exposure of chlorpyrifos (CLPS) was
1.22 µg/Kg/day (>CRFD)[Table 4.17 & 4.18].

4.6.3. Among Male children of 6-10 years: It was observed that the dose exposure of
OP pesticides as per age and gender wise, in the male children of 6-10 years old, found to be of
fenitrothion (FNTN) was 1.06µg/Kg/day (<CRFD) and exposure of chlorpyrifos (CLPS) was
1.1µg/Kg/day (>CRFD)[Table 4.17 & 4.18].

4.6.4. Among Female Children of 6-10years: In the girls of 6-10 years old age group
the exposure of fenitrothion (FNTN) was 0.7 µg/Kg/day (<CRFD) and exposure of chlorpyrifos
(CLPS) was 1.31 µg/Kg/day (>CRFD)[Table 4.17 & 4.18].

4.6.5. Among Male children of 11-15 years:However, in the male children of 11-15
years old, it was found that the exposure of fenitrothion (FNTN) was 0.66 µg/Kg/day (<CRFD)
and exposure of chlorpyrifos (CLPS) was 1.02 µg/Kg/day (=CRFD)[Table 4.17 & 4.18].

4.6.6. Among Female children of 11-15 years:In girls of 11-15 years old age group the
exposure of fenitrothion (FNTN) was 0.86 µg/Kg/day (<CRFD) and exposure of chlorpyrifos
(CLPS) was 1.03 µg/Kg/day (=CRFD)[Table 4.17 & 4.18].

in HIG group the children got exposed to fenitrotion with 0.94µg/Kg/day (<CRFD) and to
chlorphyrifos with 1.34 µg/Kg/day (>CRFD) doses. In MIG groupchildren it was fenitrothion
dose 0.77µg/Kg/day (<CRFD) and to chlorphyrifos dose 0.97µg/Kg/day (<CRFD). In the low
income group children the exposure of fenitrotion and chlorphyrifos were 0.6µg/Kg/day
(<CRFD) and 1.1µg/Kg/day (>CRFD)respectively. There was no significant pesticide exposure
difference between children of MIG and LIG however, therewas found to be a significant (p <
0.00125) difference between HIG group children and the children of other two income groups;
MIG and LIG [Table 4.17 & 4.18].

4.6.8. Zone Wise:

It was observed that the pesticide dose exposures demographically in the Hyderabad city, in
west zone the children’s exposure towards fenitrothion and chlopyrifos were 0.93 µg/Kg/day

76
(<CRFD) and 1.01µg/Kg/day (>CRFD) respectively. In south zone area, the children were
exposed to 0.50µg/Kg/day (<CRFD)secondhighest,of fenitrothion and 1.19 µg/Kg/day
(>CRFD)doses of chlopyrifos. The children of north zone area were exposed to 0.79 µg/Kg/day
(<CRFD)of fenitrothion and 0.86µg/Kg/day (<CRFD), least exposure, of chlorpyrifos.East zone
children had highest exposure 0.97 µg/Kg/day (<CRFD)offenitrothion and second highest
exposure of chlopyrifos 1.27 µg/Kg/day (>CRFD). The children of central zone were being
exposed to 0.68 µg/Kg/day (<CRFD) of fenitrothion and to 1.35 µg/Kg/day (>CRFD) highest of
chlorpyrifos [Table 4.17 & 4.18].

Among all zones, the children of east zone showed highest exposureof 0.97 µg/Kg/day (<CRFD)
of fenitrothion and children of south zone showed the least exposure of 0.50
µg/Kg/day(<CRFD) It was observed that the exposure of chlorpyrifos, among all zone children,
the children of central zone showed the highest exposure 1.35µg/Kg/day (>CRFD) while north
zone children showed the least exposure 0.86 µg/Kg/day (<CRFD) to the same pesticide[Table
4.17 & 4.18].

4.6.9. Among Total Children:Among all children who participated in the study e were being
exposed to 0.77µg/Kg/day (<<CRFD of 1.3 µg/Kg/day) of fenitrothion and 1.13 of µg/Kg/day
(>CRFD of 1.0 µg/Kg/day)chlorpyrifos[Table 4.17 & 4.18].

77
Table 4.18.Comparision of mean concentrations of pesticides, FNTN and CLPS with the US
FDA (FOOD and Drug Administration) released Chronic Reference Doses (CRFD) of those
FNTN and CLPS pesticides.

FNTN CLPS
(µg/Kg/day) (µg/Kg/day)
Category Subcategory N
Relative to CRFD Relative to CRFD
Mean Mean
(1.3 µg/Kg/day) (1 µg/Kg/day)
Age 6-10 yrs. 477 0.82 < 1.24 >>
Group 11-15 yrs. 421 0.72 < 1.02 >

Male 456 0.804 < 1.037 >

Sex
Female 449 0.74 < 1.228 >>

Male 158 1.06 < 1.1 >

6-10 yrs.
Female 319 0.7 < 1.31 >>

11-15 Male 291 0.66 < 1.02 >

yrs. Female 130 0.86 < 1.03 >

HIG 276 0.94 < 1.34 >

Income
MIG 325 0.77 < 0.97 <
Group
LIG 304 0.6 < 1.1 >

West 163 0.93 < 1.01 >

South 181 0.5 < 1.19 >>
ZONES North 200 0.79 < 0.86 <
East 180 0.97 < 1.27 >>
Central 181 0.68 < 1.35 >>
Total All 905 0.77 < 1.13 >

4.7. Diet Consumption pattern in children: We assessed dietary OP exposure in 905 children
of Hyderabad urban area by typical dietary intake with average residue levels in those items. We
further assessed the relationship between these estimates and urinary DAP concentrations in a
subset of participants with conventional diets (N = 905). This analysis of inter method
comparability was intended as a check for the face validity of our estimates. After identifying the
specific pesticides and food items to be included in this analysis, we calculated the average
concentration of each OP measured in each food item. When we calculated average consumption
of each food item in total children, we found that the consumption of cool drinks is 62.5 ml/day,

78
fruit juices is 15.9 ml/day, fruits are 154 g/day, vegetables are 100g/day, plain food is
779.09g/day and 1234.1 g/day in 6-10 years and 11-15 years age groups respectivelyor it should
be 1006.6g /day[Table 4.19].

Table 4.19.Diet consumption pattern in children of Hyderabad urban area.

Count Mean SD

Total Cool Drinks (ml/day) 905 62.545 15.28

Total Fruit Juices(ml/day) 905 15.965 3.9

Total Fruits (gm/day) 905 154 134.1

Total Vegetables (gm/day) 905 100.2 120.7

Plain Food (gm/day) 905 1006.595 245.86

4.7.1. Age Group wise:When we see the food consumption pattern in age group wise it is found
that the food irrespective of food item consumption levels are higher in 11-15 years when
compared with 6-10 years age group [Table 4.20].

Table: 4.20. Diet consumption pattern in children of two age groups.

Age Group

6-10 years 11-15 years

Count Mean SD Count Mean SD

Total Cool Drinks (ml/day) 477 48.41 7 421 76.68 8.28

Total Juices(ml/day) 477 12.36 1.79 421 19.57 2.11

Total Fruits (gm/day) 477 139.9 61.43 421 168.1 72.67

Total Vegetables (gm/day) 477 74.1 55.29 421 126.3 65.41

Plain Food (gm/day) 477 779.09 112.63 421 1234.1 1233.23

79
4.7.2. Sex-wise: When we see the food consumption pattern in age and sex wise we find that
there is not much difference in the consumption of cool drinks, fruit juices and plain food
between male and female children of both age groups of 6-10 years and 11-15 years. But, there is
a considerable difference in the consumption of fruits and vegetables between male and female
children of both 6-10 -years –old-and 11-15 years- old-age groups [Table 4.21].

Table4.21. Comparison of pattern of food consumption in children with age and sex groups.

Gender

Male Female

Age Group 6-10 yrs. 10-15 yrs. 6-10 yrs. 10-15 yrs.

N 158 291 319 137

Food item Mean SD Mean SD Mean SD Mean SD

Total Cool Drinks

(ml/day) 48.4 6.9 77.6 8 47.4 7 74.6 8.4
Total Juices (ml/day) 12.4 1.8 19.8 2.1 13.4 1.8 19 2.2

Total Fruits (gm/day) 136.7 61 151.3 70.6 125.1 61.8 184.8 74.1

Total Vegetables
(gm/day) 102.2 54.9 113.2 63.6 92.6 55.6 139.3 66.7

Plain Food (gm/day) 778.6 111.7 1249.1 129.5 779.4 113.2 1200.4 135.9

4.7.3. Income Group wise: Our results indicate that the socio economic status of children
affects the food consumption pattern. The high income group children consumption levels are
higher compared to MIG and LIG children. But interestingly both MIG and LIG group children’s
consumption is relatively similar and consumption levels in these two groups are lower than HIG
group. Among all food items the significant difference is found with the consumption levels of
fruits and vegetables among three income groups when compared to the other food items [4.22].

80
Table.14.22. Food consumption levels in children with different socioeconomic status.

Income Groups

HIG MIG LIG

N 304 325 276

Food item Mean SD Mean SD Mean SD

Total Cool Drinks

63.52 16.18 65.87 15.76 41.38 16.79
(ml/day)

Total Juices
25.22 4.13 16.54 4.02 15.67 4.29
(ml/day)
Total Fruits
197.64 42.07 154.4 78.34 138.87 47.36
(gm/day)

Total Vegetables
145.87 27.86 130.95 64.5 124.97 32.62
(gm/day)

Plain Food (gm/day) 1022.3 260.45 979.71 253.62 987.91 270.15

4.7.4.Zone Wise:On checking of food consumption levels in children of 5 zones of Hyderabad

city it was found that the consumption levels are higher in west zone followed by central, east,
south and north zones. When compared to other food items the differences are relatively higher
in consumption of fruits and vegetables among children of five given zones [Table 4.23].

81
Table 4.23. Variation in food consumption levels in children of different zones of Hyderabad
urban area.

Zones

West South North East Central

N 163 181 200 180 181

Mean SD Mean SD Mean SD Mean SD Mean SD

Total Cool
Drinks 63.1 16.3 59.8 15.4 60.3 15.6 61.1 17.2 65.8 16.3
(ml/day)

Total Juices
16.6 4.2 15.5 3.9 15.1 4 15.6 4.4 16.3 4.2
(ml/day)

Total Fruits
196.6 42.7 134.1 55.5 120.7 36.8 146.7 51.2 188.9 42.9
(gm/day)

Total Vegetables
149.5 68.4 98.7 41.5 128.6 23.2 133 36.1 139.9 28.6
(gm/day)
Plain Food
1048 261.6 979.2 247.5 954.6 250.9 983.8 277.3 1026.5 262
(gm/day)

4.8.Dietary exposure assessment:

Mean OP pesticide levels in Food items: After identifying the specific pesticides and food items
to be included in this analysis, we calculated the average concentration of each OP measured in
each food item. We calculated mean concentrations of most frequently used nine OP pesticides
(Acephate, Monocrotofos, Fenitrothione, Diazinon, Malathion, Chlorpyrifos, Phoslone, Ethion,
Chlorfenvinfo) levels in commonly consumed food items such as cool drinks, fruit juices, fruits,
vegetables, and plain food in and around Hyderabad. The average OP pesticide level found in
fruits is 10.15µg/kg,in cool drinks is 0.12µg/L, in fruit juices is 10.12µg/L, in vegetables is
7.9µg/kg, and in plain food is 0.93µg/kg[Table 4.24].

82
Table 4.24. Average OP pesticide levels in selected food items collected from different outlets in
Hyderabad.

µg/Kg µg/L

Plain Cool Fruit

Food item Fruits Veg.
Food drinks juices

N 34 65 56 12 30

Detection Frequency 79.4 % 81.5% 58.9% 40.0% 76.7%

Average 10.148 7.9 0.93 0.12 10.12

SD 0.72 0.97 0.69 0.72 0.46

Veg = vegetables

4.8.1. Mean levels of OP pesticide consumption in the given diet of children: To calculate
dietary OP exposure, we combined individual diet intake data from our questionnaire and
average residue. Based on the average levels of OP pesticides in each food item and quantity of
consumption of each food item by children daily, we calculated the ingestion of OP pesticides
through the diet. These estimates were calculated exclusively based on food intake information.
Individual-level exposures were calculated in the following way. We multiplied each
individual’s typical intake of each food item by the average residue of each OP measured of that
food.

Here, we converted average OP residue levels in each food item to their microgram equivalents,
and multiplied that quantity by each individual’s reported typical intake of each food item:

Average Pesticide ingestion = [average daily intake (gm food/day) × average concentration OP
pesticides in given food item (µg OP/Kg food).

We estimated average OP pesticide ingestion in units of microgram OP equivalents per kilogram

food item per day (mg/kg-day) for all of these participants (Table 2).

Exposure (µg OP/day) = [average daily intake (gm food/day) × concentration (µgm OP/gm food)

83
On checking the OP pesticide consumption in each group of children, in all the groups OP
pesticides were ingested majorly through fruits and vegetables followed by plain food. In the
following tables we have given OP pesticide consumption through each food item in each group
of children [Table 4.25, Figure 4.1].

Table 4.25: Mean consumption levels of OP pesticides through the given diet in each group of
children.

Sex Male Female

Age group 6-10 yrs. 10-15 yrs. 6-10 yrs. 10-15 yrs.

N 158 291 319 137

Through Cool
0.01 0.01 0.01 0.01
Drinks(µg/day)

Through Juices (µg/day) 0.13 0.20 0.13 0.19

Through Fruits (µg/day) 1.39 1.54 1.27 1.88

Through Vegetables
0.81 0.89 0.73 1.10
(µg/day)
Through Plain Food
0.72 1.16 0.72 1.12
(µg/day)

Total (µg/day) 3.05 3.80 2.86 4.29

2
1.8 1.54
1.6
1.4 1.16
1.2 0.89
1
0.8 Male 6-10 yrs.
0.6
0.4 0.2
0.2 0.01 Male 10-15 yrs.
0
Female 6-10 yrs.
l…

…
s…
y)

od
y)
oo

ble

Female 10-15 yrs.

da

da

Fo
h C

g/

ta
g/

n
( µ

eg
ug

(µ

lai
ro

h V
es

its

P
Th

gh
uic

ru

ug

ou
h F
h J

ro

hr
Th
ug

ug
ro

ro
Th

Th

Figure4.1 Mean consumption levels of OP pesticides through the diet.

84
Among the three income groups HIG children were consuming more OP through the diet when
compared to MIG and LIG [Table 4.26]. Whilst among children of five zones children of west
zone children are consuming high OP followed by central zone, East, South and North zone
[Table 4.27].

Table 4.26: Mean consumption levels of OP pesticides through the given diet in respective
income group of children

Income group HIG MIG LIG

N 304 325 276

Through Cool Drinks(µg/day) 0.01 0.01 0.01

Through Juices (µg/day) 0.16 0.16 0.16

Through Fruits (µg/day) 2.00 1.57 1.41

Through Vegetables (µg/day) 1.15 1.03 0.99

Through Plain Food (µg/day) 0.95 0.91 0.92

Total (µg/day) 4.28 3.68 3.48

Table 4.27: Mean consumption levels of OP pesticides through the given diet
in respective zone of children

ZONE West South North East Central

N 163 181 200 180 181

Through Cool Drinks(µg/day) 0.01 0.01 0.01 0.01 0.01

Through Juices (µg/day) 0.17 0.16 0.15 0.16 0.16

Through Fruits (µg/day) 2.20 1.39 1.19 1.35 1.91

Through Vegetables (µg/day) 1.44 0.76 0.91 1.00 1.06

Through Plain Food (µg/day) 0.97 0.91 0.89 0.91 0.95

Total (µg/day) 4.79 3.23 3.15 3.43 4.10

85
On average the total OP pesticide consumed through all the given food items among total
children is 3.74µg/day. While the highest OP intake is through the fruits and vegetables which
itself indicates that fruits and vegetables are major source of OP pesticide ingestion in children
[Table 4.28]

Table 4.28: Mean consumption levels of OP pesticides through the given diet in total
group of children (6-15 years)

Age Group 6-15 years (N = 905)

Through Cool Drinks(µg/day) 0.01

Through Juices (µg/day) 0.16

Through Fruits (µg/day) 1.35

Through Vegetables (µg/day) 1.29

Through Plain Food (µg/day) 0.93

Total (µg/day) 3.74

4.8.2.Correlation between dietary exposure of OP pesticide levels and estimated OP

metabolites levels in urine samples: When we compare ingested OP levels and OP metabolites
found in each group of children it is found that there is a strong correlation between consumed
OP levels andestimated OP metabolites, among age and sex groups, females of 11-15 years are
consuming 4.29 µg/day of OP and the same group has shown high levels of DAP levels i.e. 0.36
µmol/L (r = 0.983).Among income groups also we find a strong correlation as HIG group
children consumed highest OP (µg/day) (r = 0.893) through the diet and the same HIG group has
shown highest levels of DAP levels of 0.37 (µmol/L). The same pattern of correlation isalso
found among children of five zones of Hyderabad city i.e. among all the five zones the west zone
children have shown OP consumption as 4.78 µg/day and DAP levels as 0.35 µmol/L ( r =
0.888) followed by central zone which has 4.10 µg/day and 0.35 µmol/day (0.999) [Table 4.29].

86
Table 4.29. Correlation between OP pesticide exposure through diet and DAP metabolite levels
in respective groups of children

Category Group (N) r* p-Value

M(6-10 Y) (158) 0.853 <0.0011

M(11-15 Y) (291) 0.952 <0.0012

Age & Sex
F(6-10 Y) (319) 0.889 0.001

F(11-15 Y) (137) 0.983 0.001

HIG (304) 0.893 <0.001

Income
MIG (325) 0.886 0.001
Groups
LIG (276) 0.875 0.001

West (163) 0.888 <0.0013

Central (181) 0.999 0.001

Zones East (180) 0.993 0.001

South (181) 0.953 <0.001

North (200) 0.932 <0.001

All Groups Total (905) 0.796 0.0011

*Non parametric Spearmen’s significant test

Y = years

4.9. Discussion
In the present-day situation, the most common and wide spread environmental health problem
facing mankind, especially young children is getting exposed to pollution. Millions of Indian
children have been exposed to polluted food and water and now they suffer from elevated levels
of toxic compounds in their bodies. Once inside the body, the toxic compounds can cause a wide
variety of physical and mental problems. The problems associated with this toxicity are
constituted by the fact that many people do not know what it is and how their children get it, as
users rarely are aware of its presence. It can very well be said that toxicity is one of the most
severe health threats of today.

87
After the green revolution initiative India started adapting chemical fertilizers and agro-
chemicals along with the induction of modern agricultural technologies. In this study we
analyzed urine samples of children living in Hyderabad urban area, for six dialkylphosphate
(DAP) compounds, the common metabolites of the OP pesticides i.e. dimethylphosphate (DMP),
dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP),
diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP).The concentrations are
classified by age, sex, and income groups, of dialkyl phosphate (DAP) metabolites of
organophosphorus pesticides. Only one child was selected from each family who participated in
this study with families having more than one childgot enrolled in the study, to remove
anyhousehold dependence.

Analysis was performed using a liquid chromatography equipped with a Triple-Quadra pole ion
trap hybrid mass detector (4000-QTRAP, Applied BiosystemsMDS Sciex, USA) operating in
MRM positive electrospray ion (ESI) mode has been used for this study. Sample preparation
procedures included liquid- liquid extraction.

The dimethyl (DMP, DMTP, and DMDTP) and diethyl (DEP and DETP) metabolite
concentrations were converted to their molar concentrations (µmol/L) and summed to produce a
single methyl or ethyl dialkylphosphate concentration for each sample [Lu C 2004]. Asmost of
the urine samples contained all the six metabolites in detectable concentration, hence all the six
were included in the data analysis and none was excluded. Statistical analyses were performed
with the help of nonparametric tests using SPSS 19.0.

The DMDTP metabolite was least dominated by detection frequency of 73.4%.However there
was no significant difference found between dimethyl DAP levels and total diethyl DAP levels
(medians, 0.07 and 0.07 µmol/L, respectively; Non parametric Spearmen’s significant test, p >
0.00012). Among 6-10 years the DMAP metabolites were dominated by DMTP (median3.05
µg/L)whereas the DEAP metabolites were dominated by DETP and DEP with median
concentrations 3.08 µg/L and 2.09 µg/L.DMAP metabolite levels were almost equal in 6-10
years as compared to the 11-15 year-old children. There is no clear pattern in the change in
DMAP and DEAP levels with age. Methyl (DMAP), ethyl (DEAP) and alkyl (DAP) metabolite
concentrations were compared across two age groups (6-10 yrs. and 11-15 yrs.). We found no

88
significant differences for either dimethyl or diethyl concentrations (Non parametric Spearmen’s
significant test, p > 0.005). We then averaged the two samples from each child to represent the
DAP concentrations during the study period (2012-2017). We found no significant differences
even in the median concentrations of either dimethyl or diethyl metabolites.

Among male and female children, DMAP metabolite median levels were similar to the DEAP
metabolite levels. Total DAP median molar concentrations in both the genders were 0.14
µmol/L. DMAP metabolite levels were almost equal in male children compared to the female
children. There is no clear pattern in the change in DMAP and DEAP levels with sex.Neither
median dimethyl nor diethyl DAP concentrations significantly differed between male and female
children.

In the male children of 6-10 years old, highest level was of DETP and least was of DEDTP. In
the male children of 11-15 years old the highest metabolite level was of DETP; and least was of
DEDTP. In the female children of 6-10 years age group, highest level found was of DETP; and
least was of DEDTP. In the female children of 11-15 years old, the highest metabolite level was
of DETP; and least was of DEDTP. Among the three income groups, HIG children had
comparatively high levels of molar DAP 0.37 µmol/L compared to levels of 0.27 µmol/L in
MIGand LIG group children withlevel of 0.27 µmol/L. Reciprocally HIG children had more
intakes of food while MIG and LIH children were consuming relatively less quantity of
food.[Tables 4.8-410, Table 4.22].

On checkingDAP metabolite levels of children in each zone it was found that west (0.36
µmol/L) east (0.35 µmol/L) and central zones (0.32 µmol/L) have shown relatively high and
similar DAP levels among those three zones( while remaining two zones, south and north
shows less levels of DAP levels (0.26 µmol/L) [Table 4.16].

The participating children of Hyderabad city were being exposed to 0.77µg/Kg/day (<<CRFD of
1.3 µg/Kg/day) of fenitrothion and 1.13 of µg/Kg/day (>CRFD of 1.0 µg/Kg/day) of
chlorpyrifos. Among all zones, the children of east zone showed highest exposure 0.97
µg/Kg/day (<CRFD) of fenitrothion and children of south zone showed the least exposure 0.50
µg/Kg/day(<CRFD) of fenitrothion. It was observed that exposure of chlorpyrifos, among all
zone children, the children of central zone werefacing the highest exposure of 1.35µg/Kg/day

89
(>CRFD) while north zone children shown the least exposure 0.86 µg/Kg/day (<CRFD) of the
same pesticide[Table 4.17 & 4.18].

It can be concluded thatthe pesticide dose exposure levels relative to CRFD of respective
pesticidesof participatingchildren, level of chlorpyrifos was higher than its CRFD value of 1
µg/Kg/day and the level of fenitrothion was lower than its CRFD value of 1.3 µg/Kg/day
meaning thereby that children of Hyderabad city are highly exposed to Chlorpyrifos andwithin
limitsof Fenitrothion.

4.9.1. Comparison with similar studies conducted in other populations:

The Centers for Disease Control and Prevention (CDC) have periodically analyzed biologic
samples from the National Health and Nutrition Examination Survey IV [NHANES-IV]. In
2007-08, the NHANES-IV was conducted across the country [CDC 2007]. During this, six DAP
metabolites were also analyzed in urine samples collected from over 600 subjects 6–59 years old.
Here we compared the OP metabolite concentrations of Hyderabad children of age groups 6-10
years and 11-15 years with their US counter parts of s 6-11 years old and 12-29 years old
population. In the top 50% of these values (50th percentile and higher), DMP levels in the 50th
percentile and higher of the U.S. population are found nearer corresponding levels for Hyderabad
urban children. DMTP levels in the urine of Hyderabad children with 6-11years were less than
the their counter age group of 6-11 years in US population, but 11-15 years old children of
Hyderabad have greater levels of the same metabolite than the 12-29 years old group of US
population. The concentrations of all the methyl metabolites found greater in Hyderabad children
group than the US corresponding population. However, the magnitude of difference in urinary
levels between the study children and the NHANES adult population was much greater for
DETP and DEDTP than for DEP. This suggests that overall children of Hyderabad city
havehigher dimethyl and diethyl OP metabolites than the counter parts of US population covered
underNHANES-IV [Table 4.30].

90
Table. 4. 30. Comparison of urinary DAP concentrations (µg/L) of Hyderabad urban children
with NHANES-IV data for DAP concentrations in the general U.S. population.

Percentile
Metabolite Group N Mean
50 75 90 95
DMP (µg/L)
6-11 yrs. 477 6.55 1.57 6.05 12.5 20.32
Hyderabad children
11-15 yrs. 428 14.88 2.33 6.86 13.28 26.84
6-11 yrs. 385 < LOD 4.31 12.9 20.2
NHANESb
12-29 yrs 391 < LOD 3.81 10.7 23.3

DMTP (µg/L)
6-11 yrs. 477 7.82 2.59 6.03 15.66 29.72
Hyderabad children
11-15 yrs. 428 6.74 2.92 7.84 20.59 41.91
6-11 yrs. 385 3.43 3.5 9.9 32 52.5
NHANESb
12-29 yrs 391 2.1 1.82 5.18 13.6 31.5

DMDTP (µg/L)
6-11 yrs. 477 4.42 1.85 5.56 11.49 16
Hyderabad children
11-15 yrs. 428 11.2 2.91 9.42 23.03 32.19
6-11 yrs. 379 < LOD 0.95 3.8 6.66
NHANESb
12-29 yrs 384 < LOD < LOD 1.45 2.45

DEP (µg/L)
6-11 yrs. 477 8.43 2.09 6.93 15.18 33.74
Hyderabad children
11-15 yrs. 428 4.6 2.91 9.42 23.03 32.19
6-11 yrs. 385 < LOD 4.67 17.2 28.2
NHANESb
12-29 yrs 389 < LOD 2.86 8.56 17

DETP (µg/L)
6-11 yrs. 477 13.8 2.6 9.5 25.26 59.09
Hyderabad children
11-15 yrs. 428 9.21 3.18 9 36.88 60.84
6-11 yrs. 385 < LOD 1.18 2.93 6.44
NHANESb
12-29 yrs 390 < LOD 1.04 2.2 4.02

DEDTP (µg/L)
6-11 yrs. 477 2.2 0.11 1.79 5.64 8.2
Hyderabad children
11-15 yrs. 428 2 0.18 2.35 6.59 9.69
6-11 yrs. 383 < LOD < LOD < LOD < LOD
NHANESb
12-29 yrs 390 < LOD < LOD < LOD < LOD

91
a<LOD equals “less than the limit of detection.” For the NHANES study, the LOD for DMP was
0.37 µg/L, forDMTP the LOD was 0.47 µg/L, and for DMDTP, the LODwas 0.56 µg/L, for DEP
0.55, for DETP 0.39, for DEDTP 0.51 (CDC, 2017).b,Data from updated NHANES-IV as
reported by CDC (CDC 2017).

On comparing the concentrations of DMPs and DEPs of this study with the earlier studies, it was
found that the levels of DEPs in our study are higher than the reported levels of DEPs, of 0.0105
µmol L-1 in US population and 0.05 µmol L-1 in the preschool children of Seattle metropolitan
area [Barr et al. 2004; Lu et al. 2001]. A study conducted in Canada showed that the level of
DEPs of young children was 0.025 µmol L-1 and DMP’s was 0.0625 µmol L-1 in children aged
6-11 yr. [Oulhote and Maryse 2013]. The levels of DEPs in case of Italian (6-7 yr.) and
American young children (3-6 yr.) were 0.0663 and 0.065 µmol L-1 respectively and the levels
of DMPs, in the same groups, were 0.277 µmol L-1and 0.23 µmol L-1 respectively (Aprea et al.
2000; Bradman et al. 2013) (Table- 4.31). Previous research has largely dealt with OP exposure
in children, without reporting the sex wise differences in the exposure of OP pesticides in
children in general (Bradman et al 2007; Bradman et al 2013; Curl 2003; Lambert et.al2005).

Table4.31. Comparison of our results with the concentrations of DAP metabolites observed in
different populations in different studies.

DMs DEs DAP

GM GM GM
Country Sex Age N. (µmol (µmol Reference
(µmol L-1)
L-1) L-1)

M/F 6-59 yrs 1949 0.049 0.010 0.076

M/F 6-11 yrs 471 0.07 0.013 0.101
USA M/F 12-19 yrs 664 0.063 0.011 0.096 Barr DB (2004)
M 952 0.053 0.011 0.082
F 997 0.046 0.009 0.07

M 2-5 yrs 49 0.19 0.05
USA Lu C (2001)
F 2-5 yrs 47 0.18 0.04

M 6-7 yrs 92 0.277 0.066 0.356
Italy,Tuscany Aprea C (2000)
F 6-7 yrs 103 0.258 0.063 0.334

California, Both 3-6 yrs 50 0.23 0.065 0.296 Bradman A(2013)

92
 USA

M/F ≥20 yrs 876 0.083 0.009 0.114

USA, New
M ≥20 yrs 345 0.11 0.005 0.122 McKelvey W(2013)
York
F ≥20 yrs 531 0.076 0.013 0.108

6-11 yrs 1035 0.062 0.025 0.099
M/F
M/F 6–8 yrs 458 0.1039 Oulhote & Maryse
Canada M/F 9–11 yrs 572 0.0872 (2013)
M 6-11 yrs 529 0.094
F 6-11 yrs 501 0.0956

DMs DEs DAP
Mean Mean Mean
(µmol L- (µmol
M 6-10 yrs 158 (µmol L-1)
1) L-1)
F 6-10 yrs 391 0.12 0.16 0.28
Hyderabad, Total 477 0.14 0.15 0.29
M 11-15 yrs 291 0.156 0.153 0.31 Our Results
India
F 11-15 yrs 137 0.181 0.175 0.36
Total 428 0.163 0.159 0.324
6-15 yrs 905 0.151 0.154 0.304

GM = geometric mean, n = number of samples, M = Male, F = female,

4.8.2. Dietary Exposure:

After identifying the specific pesticides and food items to be included in this analysis, we
calculated the average concentration of each OP measured in each food item. For comparison
with measurements of urinary DAPs, we calculated individual-level exposure in units of µg per
day.Exposure estimates were calculated in units of µg/day for all study participants; the
distribution of these estimates is presented in Figure 4.24.

This study provides estimates of long-term dietary OP exposure in a population. The estimates
are consistent with the results of urinary DAP bio-monitoring, increasing our confidence in this
methodology. DAP biomarkers are imperfect measures of long-term exposure, due to their short
half-lives, lack of specificity to parent compounds, and potential to represent exposure to
preformed metabolites (Sudakin and Stone 2011).In the present study, we used these DAP
biomarkers in a novel way: to assess the face validity of our proposed exposure assessment

93
method, which suffers from none of the aforementioned limitations of the DAPs. We found that
low DAP levels were measured when exposure estimates were low and higher DAP levels were
measured when exposure estimates were higher. We estimated chronic dietary OP exposure for
the Hyderabad urban children in µg of OP equivalents per day. Unlike urinary biomarkers, these
estimates can be used to inform risk.

4.8.3. Outcome: Data obtained from the parental interview and questionnaire helped identify
factors that may influence food habits of a child. In general, the survey achieved its purpose of
obtaining information relevant to the scope of this study. Parents were able to answer most of the
questions with certainty, but some households provided much more detailed information in their
responses than did others. When parents were asked about residential children’s food
consumption, they were not normally able to provide information on the type of food consumed
and the frequency of different foods being consumed.

Although the concentrations between sexes, age groups, among income groups and zones varied,
but not much significant differences were observed.The most interesting observation of this
study is that the concentration of pesticide metabolites, in male children, is not significantly
different, from that in females, in the 6- to 10-year-old age group but, in the 11- to 15-year-old
age group, female children exhibit higher pesticide metabolite levels than of males.

In conclusion, it is found that children living in an urban area are likely to be exposed to OP
pesticides from limited pathways, and total urinary DAP, in particular DMAP, metabolite levels
did not show big co-variations with age and sex. Diet may be the main possible exposure source.
Given the health benefits of fresh fruit and vegetable consumption, we do not suggest that
children limit intake of these foods but encourage washing of all the produce before eating.
While the OP pesticide metabolite levels in this population do appear higher than populations
other than India, there is very limited reference data available in Indian context to make valid
comparisons.

Given the significance of these health studies, additional research is needed to better explain the
trend of increasing OP urinary metabolites with age and the dietary, behavioral, and other factors
to determine exposure [Marks 2010, Bouchard 2010,and Ruckart 2004].This study would also
help in the generation of proxy indicators for large-scale health studies on OP pesticides. This

94
data will be important in evaluating the impact of organophosphorus pesticide exposure in the
Hyderabad urban children and the effectiveness of regulatory actions. Furthermore, this study
also suggests that the spray of pesticides should be discouraged as this will increase the burden
of diseases in developing countries. This report highlights the exposure of children’s with
different types of OP pesticides on determinants such as DM’s and DE’s of children’s exposure
due to conventional diets.

95
96
5.1. Introduction
Pesticides and its related chemicals destroy the delicate balance between species that characterize
a functioning ecosystem. Pesticides produce many physiological and biochemical changes in
freshwater organisms by influencing the activities of several enzymes. Alterations in the
chemical composition of the natural environment usually affect behavioral and physiological
systems of the inhabitants; particularly humans [Radhaiah et al 1987] Hormone-disrupting
effects in biota as a result of chemicals are caused by a wide variety of mechanisms. Pesticides
and related chemicals are substances that can cause adverse effects by interfering in some way
with the body’s hormones or chemical messengers. These substances are, therefore, called
hormone disruptors or endocrine disruptors, as the endocrine glands secrete the hormones
directly into the blood. Hormones play a crucial role in guiding normal cell differentiation in
early life forms, and so exposure to endocrine disrupting substances in the egg or in the womb
(mammals) can alter the normal process of development. Lately, most attention has been focused
on estrogens; natural or synthetic compounds that elicit a feminizing effect by binding to the
cellular estrogen receptor in organisms [Zaheer Khan 2005].

There are many studies stating neurotoxicity of OP pesticides. Recent studies confirmed OPs
could cause other toxic effects like immunotoxicity, genotoxicity, carcinogencity, endocrine
alterations and adverse effects on reproductive health (Sultatos, 1994; Tamura et al.,
2001, 2003; Kang et al., 2004).

Main toxic action of organophosphates (OP) is phosphorylation of proteins. Chemical changes in

sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis,
is part of the cause of male reproductive toxicity [Esmail et al 2009] Toxicity of OP mainly
results in neurotoxicity due to its oxygen analogues (oxons), formed during the OP oxidative
activation.

Some of the studies have also experimentally shown OP pesticides as endocrine disruptors, for
example decreasing serum levels of LH in quails acutely exposed to OP pesticide [Rattner et al.
(1986)]. While Sarkar et al., 2000 reported that chronic exposure to sublethal doses of OP
pesticides increased LH (Lieutinizing harmone), prolactin and testosterone serum levels in rats
[Sarkar et al., 2000]. In contrasting to those, Rawlings et al., 1998 found decreased LH
(Lieutinizing harmone) serum levels in sheep exposed to OP [Rawlings et al., 1998]. Very few

97
reports are available to conclude the effects of OP pesticides on dysfunction of human
reproductive health like altered basal levels of prolactin, FSH (follicle stimulating Harmone),
adrenocorticotropic hormone (ACTH) and cortisol in patients suffering acute OP pesticides
intoxication [Güven et al. (1999)]. Whilst, Recio et al. (2005) found a negative association
between urinary levels of dialkyl phosphates (DAP), which are non-specific OP pesticides
metabolites, and serum levels of FSH and LH without significant associations with estradiol,
prolactin or testosterone levels.
OP alters semen quality and sperm chromatin and DNA at different stages of spermatogenesis.
Oxons are more toxic than the parent compounds. These compounds can influence the testes, for
example decreasing the testis weight, sperm motility, grading and sperm viability;and they
alsoincrease the sperm abnormal forms [Kolstad 1999].

But, still there is lack of knowledgeof OP moieties on mechanism of action on reproductive

hormones hence; by using simulation studies it was tried to decipher possible mechanism of
interaction of OP compounds with reproductive hormones. Molecular structural descriptor
generation and conformations of the structures, it was hypothesized that the best orientation of
OP moieties was dependent on the following two processes: (1) the penetration of OP moieties
through bio-membrane and reaching the target site of action, and (2) the interactions between the
OP moieties and progesterone, testosterone, and estradiol. Computational studies of alternate
orientation structures show that better orientations of the structures have the better binding to the
hormones [Dixit 2011]. Insilico studies with alternate orientations of the organophosphates on
our hormones have shown high binding affinity [Ubuka 2013]. This study was undertaken to
investigate the structural analysis of the three alternate plasticizers using in silico approaches,the
computational systems approach-was derived with molecular mechanics and dynamic action by
root mean square energy (RMSE) , the distinctive energy values and energy minimizations
scores.

5.2. METHODS AND MATERIALS:

Biologically active molecules namely; estradiol, progesterone and testosterone (Ligands) were
retrieved from PUBCHEM database [Pubchem 2006]. The molecules were drawn by using
Chemsketch tool (http://www.acdlabs.com/home/) [ACD 2016] and designed ligands are shown
in (Figure 5.1). Estrogen molecule was designed and added to the Phosposulphate group to all

98
possible positions of the structure and minimized by using Accelrys Discovery Studio v2.1
(ADS) [Accelrys USA] by adding the hydrogen atoms to the structure and applying the CHARM
forcefield [Guvench 2010], later the molecules were minimized using steepest descent algorithm
with 1000 steps followed by conjugate gradient up to minimization gradient tolerance satisfied
i.e. at 2000 steps and noted the energy variation values in terms of RMS. The best possible
conformation was selected based on least RMS value; same methodology was followed for
progesterone and testosterone.

5.3. RESULT AND DISCUSSIONS

Bioinformatics study was also carried out to find out the interaction of phosphate moiety of OP
pesticides with different sex hormone of male and female. In this study Estradiol [Butcher et al
1974, Fishman 1975] was considered as scaffold for the study, and the (PO3S) was added to all
the possible positions of the scaffold. Out of the PO3S attached at Position-8 (Carbon 8 of cyclo
hexane) as shown in Table-5.2, responded with least RMS value out of 12 possible positions for
the molecule which are provided in the (Table-5.1Suppl. Fig.5.2, Table 5.2). Based on
stereochemistry and valance of the estradiol, which has the potency to attach PO3S group the
same observation was found in the study which has the initial RMS (root mean square energy) of
816.32630 and showed final RMS of 0.08094 at 2000 steps of conjugate gradient. Testosterone
was considered as scaffold for the study, and the PO3S was added to all the possible positions of
the scaffold. Out of the PO3S attached at Position-17 (Carbon17-alpha of cyclo pentane) as
shown in Table-5.2, responded with least RMS value, of 11 possible positions for the molecule
which was provided in the (Table-5.1). In this study it was found that 17th Position has shown
initial RMS of 78133.35251 and the best final RMS of 0.08961 at 2000 steps of conjugate
gradient. Progesterone was considered as scaffold for the study, and the PO3S was added to all
the possible positions of the scaffold. Out of the PO3S attached at position-1 (Carbon 1 of A
ring) (Table 5.2) responded with the least RMS value, which is named as molecule-9, out of 11
possible positions for the molecule as provided in the (Table-2). In this study it was found that
the 1st Position has initial RMS of 45.86071 and the best final RMS of 0.07532 at 2000 steps of
conjugate gradient. The binding affinities of 10OP flame retardant (OPFRs) to the recombinant
zebrafish p53 protein were determined by surface plasma resonance(SPR). OPFRs could induce
the expression of p53 mRNA and protein. Molecular docking and dynamics simulation

99
simulations suggested that H-bonds and electrostatic interactions are of great importance on the
binding interactions between OPFRs and p53. The Quantitative structure activity relationship
(QSAR) model was developed to describe the binding affinities and find the mechanism of
action. The OPFRs had greater ability to form H-bonding with the p53, exhibiting high binding
affinity. The developed QSAR model has good robustness, predictability and mechanism
interpretability.

5.4. Exposure pathways in girls (11-15 years)

In this age period the hormonal changes in a girl are much more pronounced as compared to
male children of same age group. This can be understood by examining the bioinformatics
predication of male sex hormones sensitivity towards phosphate moiety of OP pesticides (Table-
5.2). The root mean square energy (RMS) is much lower (0.07532) in case of female hormone
(progesterone) as compared to male hormone (0.08961) (testosterone) which suggests that the
female hormone is more reactive to phosphate moiety of OP pesticides group leading to higher
exposure in girls as compared to boys in same age group.

When puberty begins, the set points for androgens and estrogens rise in the hypothalamus, and
the set point for androgens rises higher in males than in females. Similarly, the set point for
estrogens rises higher in females than in males. Hence, generally in the age of 11-15 production
of female hormone is much more as compared to 6-10 years age girl which leads to menarche
and sexual organ development [Rose and Rudolph 2006] (Fig.5.2). However, once puberty
begins the balance changes dramatically, with females producing more estradiol [Butcher et al
1974, Fishman 1975] than males and males producing more testosterone than females (Fig.5.2).
By the mid-teens, estradiol [Fishman 1975] production is about eight times as high in females as
it was before puberty, but only about twice as high in males [Ira et al 1941]. Our bioinformatics
study also suggested that the estradiol hormone (0.08094) is more reactive towards phosphate
moiety as compared to male hormone(0.08961) (Table-5.2) Puberty usually occurs in girls
between the ages of 11 and 14 and between the ages of 12 and 16 in boys. Girls typically reach
the beginning of their sexual growth [Ira et al. 1941, Esmail et al. 2009] spurt as well as their
peak height velocity about 2 years earlier than boys. Similarly, the other factors like deposition
of fats (Fig.5.3), environmental condition [Hines 1982] and genetic changes also might play a
vital role for absorption of pesticides in female as compared to male in 11-15 years of age group.

100
But in our study the dietary habit, environment condition, weight, volume of urine samples,
sample size, and mean age is almost same among the boys and girls. The fat deposition may be
one of the factors for absorption of pesticides in females as compared to males. Fat cells produce
a protein, leptin that provides the signal to the hypothalamus. Levels of body fat also surge
during puberty, but body fat increases more in girls than in boys (Figure. 5.3). As a consequence
of these sex differences in muscle and fat growth[Esmail et al 2009], by the end of puberty boys
have a muscle-to-fat ratio of about 3:1, whereas the muscle-to-fat ratio for girls is 5:4.[16]
Therefore, the pesticide groups deposited in the fat cells are major contributors to the exposure.
As females are prone to excess fat deposits, more exposure can be seen in females rather than in
males. Reported literature also showed that OP pesticides accumulate rapidly in fat, liver,
kidneys, and salivary glands. OP pesticides has phosphorothioate (P=S) group and are therefore
stored extensively in fat which may account for the prolonged intoxication and clinical relapse
after apparent recovery which has been observed in poisoning from these OP insecticides. OP
compounds are generally lipophilic and therefore cross the blood / brain barrier in most cases.
On the basis of this study it can be concluded that by minimizing the fat deposition in body we
can minimize the pesticides exposures. To the best of our knowledge this type of observation has
been reported for the first time.

CH3

H3C

O Testosterone

OH
CH3

HO Estradiol

101
 O
CH3
CH3

CH3

O Progesterone

Figure.5.1. Structures of the Progesterone, Testosterone, and Estradiol.

Table-5.1; Bioinformatics Comparative analysis of male and female sex hormones

Progesterone 9 45.86071 Steepest 0.155
Descent 21
(1st Position )
Conjugate 0.075
Gradient (1000) 32

Conjugate 0.075
Gradient (2000) 32

Testosterone 78133.352 Steepest 0.097

11 51 Descent 85

(17th Position ) Conjugate 0.089

Gradient (1000) 61

Conjugate 0.089
Gradient (2000) 61

Estradiol 5 Steepest
Descent 0.566
(8th position)
816.32630 22
Conjugate 0.080
Gradient (1000) 94

Conjugate 0.080
Gradient (2000) 94

102
 Table 5.2, Comparative study of sex hormones by Bio-Informatics study

Molecule Molecule Structure RMS (kcal/(mol x Angstrom)

name and
Initial Final
Position
Steepest 0.56622
Descent
OH
CH3
816.32630 Conjugate 0.08094
Gradient (1000)

O
Estradiol 5 OH Conjugate 0.08094
HO P
HO S Gradient (2000)

502.20193 Steepest 0.09806

Descent
S OH
P OH Conjugate 0.08531
HO CH3
O
Estradiol 10 Gradient (1000)

Conjugate 0.08531
Gradient (2000)
HO

45.86071 Steepest 0.15521

H3C
Descent
S
HO
P Co Conjugate 0.07532
O OH CH3
Progesterone 9 Gradient (1000)
CH3

Conjugate 0.07532
O Gradient (2000)

40.24169 Steepest 0.20746

Descent
Testosterone 1 OH
CH3 OH
O S Conjugate 0.09349
P
CH3
OH
Gradient (1000)

Conjugate 0.09349
O
Gradient (2000)

103
Testosterone 2 121.0179 Steepest 0.09836
Descent
OH
CH3
Conjugate 0.08833
CH3
Gradient (1000)
O

P OH Conjugate 0.08833
O HO
S Gradient (2000)

Testosterone 8 1634.0808 Steepest 0.58601

Descent
OH OH
CH3
P Conjugate 0.09933
S O
OH CH3 Gradient (1000)

O
Conjugate 0.09933
Gradient (2000)

104
Figure 5.2: Sex differences in hormonal changes during puberty. (Source: Nottelmann et al., 1987)

Figure 5.3: Sex differences in muscle mass and body fat during puberty. Source: Adapted from Grumbach
et al. (1974)

105


106
6.1. Method development for extraction and analysis of OP metabolites:
During the study a sensitive method for the quantification of six dialkyl phosphate (DAP)
urinary metabolites (DMP, DEP, DMTP, DETP, DMDTP, and DEDTP) in urine samples at ng
mL-1 concentration was developed. A solvent extractionwas followed by selective analysis by
using a liquid chromatography-mass spectrometric method. This method was found to be very
accurate and exhibited the limits of detection as follows: DMP, 0.058 ng mL-1; DEP, 0.009 ng
mL-1; DMTP, 0.004 ng mL-1; DETP, 0.0148 ng mL-1; DMDTP, 0.00861 ng mL-1; and DEDTP,
0.0198 ng mL1. The limits of quantification were in the range of 0.0287 ng mL-1 to 0.0743ng
mL-1. The value of the coefficient of variation was found to be > 0.999 for all of the
metabolites. The percent recovery of the pesticides, at the lowest levels of detection, ranged
from 93–107% and the coefficient of variation was found in the range of 0.400 –7.98 ng mL-1,
which indicates high degree of accuracy and precision. This method is found to be more cost-
effective as the quantity of chemicals involved is much less compared to other methods. This
method is also found to be simpleras the number of steps for e sample spillage are reduced,
and it can also be used for quantification of pesticide concentrations in urine samples collected
from the general population through various parts of the world. Initially, 30 samples were
analyzed, and the mean concentration of the metabolites ranged from 0.058 to 118.27 ng mL-1,
which suggests that the children of Hyderabad city were exposed to OP pesticides.

It is highly sensitive and selective method for the low level quantification of DAP metabolites in
urine samples. Our method employs lyophilization followed by simple solvent extraction and
analysis using selective LC-MS/ MS. This method is selective and has good precision and
sensitivity. The limit of detection (LOD) is in the parts per billion (ppb) range with CVs of
typically <10%. The advantages of this method are its cost-effectiveness and its time-saving
and sensitivity factors, which are importantfactors during times of recession and
unemployment. We used lyophilization followed by extraction without compromising on the
sensitivity, selectivity and precision of the method. This method may be used for the analysis of
DAP metabolites in human urine samples for the cumulative exposure determination of OP
pesticides.

107
6.1.2 Limitations:
• This method can’t be used to estimate specific metabolites of an OP pesticide from which
they were produced.
• Due to its reliance on freeze drying, makes this method consume more time.
• It uses same number of equipment thatwere used in earlier methods. It reduces number
of steps but not number of equipment.
• This method still depends on same principle of old solvent extraction.

6.1.3. Future directions:It would serve as a reference for future scientists to work on
developing more sensitive extraction methods for OP metabolites.

6.2. Construction of new model for exposure assessment of OP metabolites:

Dialkyl phosphate metabolites (DAP) of urine samples are generally used to characterize
children’s exposure to organophosphate pesticides. Several authors have reported that any spot
urine sample is a good predictor of cumulative exposure in humans (James and Roberts 2012;
Berenbaum and Snyder 1995) and other authors suggested that the early morning samples are
more suitable for assessment (Brenda et al. 2007; Barr et al.2004; Eskenazi et al.2007; Witte et
al. 2009); recently assessment of OP pesticide exposure in the selected population was done by
estimating OP pesticide metabolites in spot urine samples (Bouchard et al. 2011; Bradman et al.
2013). Therefore, the question arose which urine samples are better predictors of 24-hr exposure.
Also, concerns were raised whether metabolites measured in a single spot sample reflect
estimation of 24-hr exposure assessment due to pesticides.This study was designed to test
whether spotor 24hr urine samples are good for exposure assessment of
OPmetabolites.Collecting 24-hr urine samples from children is challenging, and missed voids
due to occasional toileting accidents or other circumstances is beyond the control of any study
researcher. So generally researcher prefers to collect spot urine sample for exposure assessment
after adjustment with creatinine. Therefore, we tried to formulate a model using, first morning,
and 24-hr urine samples. We enrolled a sample of 196 children, 123, aged 6-10 and 73, aged 11-
15 year old residing in Hyderabad, India and collected their urine samples at different time points
during the day. The study suggested that organophosphate pesticide exposure, based on first-
morning urine samples, is a good predictor of exposure for 24-hour(24-hr) exposure assessment.
The mean concentrations of dialkyl phosphate metabolites (DAP) in first-morning urine samples

108
(10.201 µmol L-1) were strongly correlated with concentrations of the same-day 24-hr samples
(4.866 µmol L-1) (model R 2 ≈ 0.748, p < 0.0001) with 74.8% accuracy.

This method reveals that the first morning urine samples are the most ideal and best predictors
for assessing cumulative exposure and these early morning urine samples can be used for
epidemiological studies. This study can be helpful in adopting new methods for OP metabolite
concentration estimations and OP exposure studies.Collecting 24-hr urine samples from children
is challenging and more time consuming process and voids may be missed due to occasional
toileting accidents or other circumstances beyond the control of any study researcher. The data
generated in the present study will add to the existing knowledge of exposure assessment of
children due to the pesticide metabolites concentration.

6.2.1. Limitations:
• Early morning urine samples may give 24 hr exposure but they don’t give cent percent
accuracy in concentration levels in urine samples of 24 hour.
• If we cannot get the first morning void we can’t adjust the void as this is the only sample
we collect from the child.
• Some children urinate less in the early morning.

6.2.2. Future directions: It will help in the development of new methods for exposure
assessments.

6.3. Biomonitoring of Organo Phosphate pesticide metabolites in Hyderabad urban

children:

This report is a biological monitoring survey for a total organophosphorus pesticide exposure
study, conducted among children living in Hyderabad urban area of Telangana, during 2012 to
2015 in a sample of 905 children 6-15 years of age. It documents exposures to OP pesticides
among children living in urban communities. Detection was made for each DAP metabolite in
more than 80% of the samples, with DEP being detected most frequently (87%) with a limit of
detection of 0.2 µg/L. The geometric means for the metabolites detected in more than 60% of the
samples were 1.85µg/L for DMTP and 2.33µg/L for DEP. The 95th percentiles for each
metabolite were DMP, 23 µg/L; DMTP, 33 µg/L; DMDTP, 16 µg/L; DEP, 32 µg/L; DETP, 59
µg/L; and DEDTP, 9.36 µg/L. The molar sums of the dimethyl-containing and diethyl-containing

109
metabolites were determined; their mean concentrations were 0.151 and 0.154 nmol/L,
respectively.

Most participants had at least one DAP detected within 6-10 and11-15 years, DMAP metabolite
levels were similar to the DEAP metabolite levels. DMAP metabolite levels were almost equal in
6-10 years compared to the 11-15 year old children. There is no clear pattern in the change of
DMAP and DEAP levels with age.Methyl (DMAP), ethyl (DEAP) and alkyl (DAP) metabolite
concentrations were compared across two age groups (6-10 yrs. and 11-15 yrs.). No significant
differences were found for either dimethyl or diethyl concentrations (Non parametric
Spearmen’s significant test, p > 0.005). It was then averaged of the two samples from each child
to represent the DAP concentrations during the study period (2012-2017). No significant
differences even in the median concentrations of either dimethyl or diethyl metabolites were
found.

No clear pattern of DMAP and DEAP levels with sex of the children could be found. Neither
median dimethyl nor diethyl DAP concentrations were significantly different between male and
female children (Non parametric Spearmen’s significant test, p >0.05). Among the three income
groups HIG children were having comparatively high levels of DAP levels.When metabolite
levels of children in each zone were checked, it was found that west and central zones showed
relatively high and similar DAP levels among those three zones, while remaining two zones,
south and north less levels of DAP were seen.In two age groups 6-10 years and 11-15 years,
both median DAP values (0.07μmol/L and 0.07μmol/L respectively), and mean DAP
values(0.151μmol/L and 0.154 μmol/L) did not differ by any significant factor.

Total median molar concentrations of DAP in both the genders were0.14 µmol/L. In male
children, DMAP metabolite levels were almost equal to that of the levels in the female children.
There is no clear pattern in the change of DMAP and DEAP levels with sex. Neither median
dimethyl nor diethyl DAP concentrations differed significantly between male and female
children (Non parametric Spearmen’s significant test, p >0.005).

The exposures among all children who participated in the study showed exposure of
0.77µg/Kg/day (<<CRFD of 1.3 µg/Kg/day) of fenitrothion and 1.13 of µg/Kg/day (>CRFD of
1.0 µg/Kg/day) chlorpyrifos.The dose levels of fenitrothion were less than its FDAs reported

110
CRFD level, but the dose levels of chlorpyrifos were greater than corresponding CRFD
values in children, it indicates that children of Hyderabad are being highly exposed to
chlorpyrifos when compared to fenitrothion.

The estimated consumption levels of OP pesticides through the diet are strongly correlated
with the OP metabolite levels found in the urine samples of urban children under the study.
The average OP pesticide exposure through the diet is 3.74 µg/day and the mean levels of
OP metabolites in children is 0.30 µmol/day. When compared to the other food items both
fruits and vegetables are major sources of the exposure of OP pesticides in children and the
least source is cool drinks. It strengthens the statement that the primary route of exposure in
the general population is through diet (U.S. EPA 2006).

The study showed that children, who are residing in Hyderabad, Telangana, India; eating
conventional foods are exposed to OP pesticides. The yield of these two (DEP and DMP)
metabolites significantly depends mainly on the concentration of OP pesticides.

6.3.1. Limitations: There are few limitations of this work.

• The 905 children who participated in this study are not necessarily representative
of children in the Hyderabad urban area. When compared to the children population
of Hyderabad city the samples collectedare very little.
• No residential pesticide usage was recorded based on interview data but there may
be chances of pesticides being used in households in indirect ways.
• Only 63% parents could collect all of the samples represented full 24-hr voids. Some
parents (23 %) missed as many as three voids and 13% parents missed two voids
during the collection period, and it is difficult to determine the effect of the missing
voids on the overall results.
• We hypothesized this model by assuming that 100% of the absorbed dose of OP
pesticides is expressed as dimethyl and diethyl metabolites, which islikely to lead to
an underestimate of the true dose [Fenske et al. 2000].For example, an intravenous
dosing study of Feldmann andMaibach in 1974, using human volunteers
demonstrated that only approximately 70% of original dose of pesticide was
actually excreted in urine [Feldmann andMaibach 1974].

111
 • This limitation involves dose attribution; lack of definitive calculation of dose from
biological monitoring results. The specific pesticides responsible for exposure
cannot be identified. It could give only possible exposure but not complete and real
exposure of a child.
• It relies on only one day urine samples of a subject we can’t tell the long term exposure
of a child by estimating metabolite concentrations in just one day urine sample. And there
are more chances of food he/she took in the preceding day to influence the concentration
levels.

6.3.2 Future directions: It may be useful for future researchers to get an idea for OP exposure
studies in Hyderabad children.Policy makers may use this data to draft health concerning
programs in near future.

6.4.In-silico studies on Human Reproductive Hormones and Interactions with

Organophosphate:
Exposure of humans and wildlife to pollutants released in the environment is a center of attention
nowadays. Lots of these chemicals (generally referred to as environmental pollutants) have been
shown to hamper with normal hormonal signaling and natural biological functions, leading to
reproductive disorders or infertility, which has been a matter of concern within the recent
decades. From the literature reviewed it is seen that pollutants appear as environmental toxicants,
especially heavy metals and organic chemicals of synthetic and microbiological origins, disrupt
hormone production and action in the mammalian testes. The endocrine effects of long-term,
low-level exposure to organophosphate (OP) and Phosphosulphate compounds in our current
investigations has proven that changing the organophosphate position on the hormones such as
progesterone, testosterone, estradiol and molecular simulation studies evidenced for the stability
of the structures, when changing the R-groups on scaffolds with energy minimization and RMS
scoreand found t structural alterations. This study was undertaken to evaluate and predict the
potential endocrine disrupting activity of the three most commonly sexual hormones in male and
female due to exposure of organophosphate compounds.Our Molecular docking and dynamics
simulation simulations suggested that progesterone; 1st position, testosterone; 17th position,
estradiol; 8th position have best and stable orientations to inhibit the Organophosphate activity.

112


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 Publications

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Publications from thesis:

1. Venkat Reddy Banda, Shyam Perugu, Rajesh Kumar K, Sukesh Narayan Sinha, In-Silico
Studies on Human Reproductive Hormones and Interactions With Organophosphate, EJBPS,
2018, Volume 5, Issue 4: 592-597.

2. Sukesh Narayan Sinha, B. Venkat Reddy, Kasturi Vasudev,M. Vishnu Vardhana Rao, M.
Noor Ahmed, Shaik Ashu, Alka Kumaria and Vijay Bhatnagar (2014) Analysis of dialkyl urine
metabolites of organophosphate pesticides by a liquid chromatography mass spectrometry
technique. Anal. Methods. 6:1825.

Publications out of thesis:

1. Banda Venkat Reddy, Bommena Rajendra Prasad, Sukesh Narayan Sinha and Noor
Ahmed (2014) New mathematical derivations for calculation of ATP yield due to the complete
oxidation of different types of fatty acids. Indian Journal of Biochemistry & Biophysics
(IJBB). 51:52-57.

Conferences/symposium/workshops attended:

2012 “Laboratory Animal Science Adhoc Training Course” Conducted at National Centre for
Laboratory Animal Sciences. NIN, Hyderabad, India.

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