Exposures of Children to Organophosphate Pesticides and Their Potential

Adverse Health Effects

Brenda Eskenazi, Asa Bradman, and Rosemary Castorina

Center for Children's Environmental Health Research, School of Public Health, University of California, Berkeley, California USA

Recent studies show that young children can be exposed to pesticides during normal oral

exploration of their environment and their level of dermal contact with floors and other surfaces.

Children living in agricultural areas may be exposed to higher pesticide levels than other children

because of pesticides tracked into their homes by household members, by pesticide drift, by

breast milk from their farmworker mother, or by playing in nearby fields. Nevertheless, few studies

have assessed the extent of children's pesticide exposure, and no studies have examined whether

there are adverse health effects of chronic exposure. There is substantial toxicologic evidence that

repeated low-level exposure to organophosphate (OP) pesticides may affect neurodevelopment and

growth in developing animals. For example, animal studies have reported neurobehavorial effects

such as impairment on maze performance, locomotion, and balance in neonates exposed in utero

and during early postnatal life. Possible mechanisms for these effects include inhibition of brain

acetylcholinesterase, downregulation of muscarinic receptors, decreased brain DNA synthesis,

and reduced brain weight in offspring. Research findings also suggest that it is biologically

plausible that OP exposure may be related to respiratory disease in children through dysregulation

of the autonomic nervous system. The University of California Berkeley Center for Children's

Environmental Health Research is working to build a community-university partnership to study

the environmental health of rural children. This Center for the Health Assessment of Mothers and

Children of Salinas, or CHAMACOS in Monterey County, California, will assess in utero and

postnatal OP pesticide exposure and the relationship of exposure to neurodevelopment, growth,

and symptoms of respiratory illness in children. The ultimate goal of the center is to translate

research findings into a reduction of children's exposure to pesticides and other environmental

agents, and thereby reduce the incidence of environmentally related disease. Key words: asthma,

children, environment, exposure, growth, neurodevelopment, organophosphate, pesticide.

- Environ Health Perspect 1 07(suppl 3):409-419 (1999).

http.//ehpnetl.niehs.nih.gov/docs/1999/suppl-3/409-419eskenazi/abstract. html

Recent studies have demonstrated that

home environments throughout the

United States are commonly contaminated

with pesticides, including organophosphate (OP), carbamate, organochlorine,

pyrethroid, and herbicide compounds

(1-12). Young children may be highly

exposed to these pesticides because of their

normal tendency to explore their environment orally, combined with their proximity to potentially contaminated floors,

surfaces, and air. Physiologic characteristics

of young children, such as high intake of

food, water, and air per unit of body

weight, may also increase their exposures

(13). Because children are developmentally

immature, they may also be at higher risk

for adverse health effects (13). This paper

reviews information on children's exposure

to OP pesticides and potential adverse

health effects. We also outline our planned

research in Monterey County, California,

to investigate exposures to children living

in an agricultural area and possible effects

on growth, neurobehavioral development,

and respiratory disease.

Nationally, approximately 750-800

million pounds of conventional pesticides

are used annually in agriculture, excluding

sulfur, oils and repellants (14). Total conventional pesticide use, including home,

structural, and other applications, averages about 1 billion pounds in the United

States. During the mid-1990s, national

pesticide use levels have been stable

(15), although trends vary by region. In

California, which has the largest agricultural output of all 50 states, approximately 200 million pounds of pesticidal

active ingredient are used annually in agriculture (16). Pesticide use data for

California suggest a trend of increasing

use between 1991 and 1995 for production agriculture, postharvest treatment,

structural fumigation, and landscape

maintenance (16). These changes may be

due, in part, to unique meteorologic and

economic factors, including heavy rains,

shifts to lower toxicity compounds that

require higher volumes, and changes in

planted acreage (16-18). Agricultural use

of neurotoxic pesticides, including the

OPs chlorpyrifos and diazinon, was also

higher in 1995 than in 1991, most likely

due to increased use on cotton, and to a

lesser extent on oranges, alfalfa, apples, and

broccoli (16,17). Overall, pesticide use in

California appears to be stable or increasing, with annual fluctuations making it

difficult to identify long-term trends.

Pesticide residue in food may also

contribute to children's exposures. In

response to concern about low-level exposure, the Food Quality Protection Act of

1996 (P.L. 104-170) (19) was unanimously passed by the U.S. Congress to

address pesticide food safety issues raised

by the seminal 1993 National Academy of

Sciences report Pesticides in the Diets of

Infants and Children (13). This report

drew the public's attention to the specific

vulnerability of children to many pesticides. The National Academy of Sciences

committee found that current tolerances

for pesticide levels in food are not health

based and may not adequately protect

children. Congress specifically directed the

U.S. Environmental Protection Agency

(U.S. EPA) to reevaluate food tolerances

and establish health-based standards that

account for children's unique sensitivity to

environmental toxicants. The law requires

the U.S. EPA to consider all nonoccupational sources of pesticide exposure, especially exposure to compounds with similar

mechanisms of toxicity. The National

Academy of Sciences recommended in

1993 that the U.S. EPA modify its

This article is part of the monograph on Children's

Environmental Health and Disease Prevention

Research.

Address correspondence to B. Eskenazi, School of

Public Health, University of California, Berkeley, 140

Warren Hall, Berkeley, CA 94720-7360. Telephone:

(510) 642-3496. Fax: (510) 642-5815. E-mail:

eskenazi@uclink4.berkeley.edu

The authors thank M. O'Rourke for preliminary information on pesticide levels in Arizona children. We also

thank G. Solomon for review of early drafts. Finally, we

thank our collaborators in the Center who have contributed to parts of this review, including 1. Tager, J.

Leckie, V. Zartarian, T. Boyce, and M. Lipsett.

This research is jointly funded by the U.S.

Environmental Protection Agency and the National

Institute of Environmental Health Sciences award

R826709-01-0.

Received 2 February 1999; accepted 7 April 1999.

Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999 409
ESKENAZI ET AL.

decision-making process for setting

pesticide tolerances to reflect the unique

characteristics of the diets of infants and

children and account also for all nondietary intake of pesticides (13). Findings

from several small, cross-sectional studies

(3-6,8,12) indicate that nondietary

exposures to young children from residential contamination may be an important

component of total pesticide exposure.

Unfortunately, our knowledge about the

actual levels of pesticide exposures of children from food and environmental exposures and their potential health effects is

extremely limited.

Children's Exposure to

Pesticides

National population-based surveys of

pesticide urinary metabolites in adults indicate widespread exposure to pesticides,

including organophosphates, carbamates,

wood preservatives, and fungicides (20,21).

For example, Hill et al. (20) detected chlorpyrifos, an OP pesticide, in 82% of 993

adults tested through the National Health

and Nutrition Examination Survey, and

found a 5-fold increase in the proportion of

adults with levels over 5 pg/L compared to

earlier surveys, suggesting increasing exposure in the general population. OPs are

eliminated from the body after 3-6 days

(22), so the widespread detection of these

compounds indicates continuing exposure.

Biologic information on children's

pesticide exposure is very limited. Hill et al.

(23) reported detections of dichlorobenzene

and wood preservatives in 96% and 100%,

respectively, of 197 Arkansas children,

whereas phenoxy herbicide metabolites were

found in 20% of all samples. Preliminary

results from the federal Agricultural Health

Study indicate detectable pesticide residues

in children's urine (8). Loewenherz et al.

(24), working in Washington state, found

that 44% of children of pesticide applicators

and 27% of nonfarm, rural children had

detectable OP residues. In preliminary data

from Arizona, chlorpyrifos was detected in

100% of about 40 children >6 years of age

sampled in a population-based survey, and

approximately 25% of 150 children <6

years of age sampled in an agricultural area.

Detection limits in the second survey were

higher (25). Comparison of these studies to

each other and to data reported by Hill et al.

(20) is difficult because of differences in

detection limits, sample type (spot samples

vs first morning void), and ages of participants. Overall, these studies suggest the

potential for widespread low-level pesticide

exposure in children and the need for

population-based studies to establish norms.

Pesticides enter children's bodies via

dermal absorption, ingestion, and inhalation. Exposure in the home depends on the

frequency, duration, and nature (i.e., dermal contact, hand-to-mouth behavior) of

the child's interaction with contaminated

media such as house dust. Children may

have higher exposure to pesticides than

other residents living in the same contaminated environment, in part because

young children spend more of their time

indoors at home (26,27). Thus, they are

likely to spend more time in proximity to

any pesticides present in their immediate

environment. The importance of specific

exposure-related behaviors, such as handto-mouth activity, will be age dependent,

suggesting that consequent exposure levels

and pathways will vary with age, as has

been observed for lead exposure (28). For

example, children younger than 6 months

of age may receive their greatest exposures

through breast milk or inhalation, but dermal absorption and ingestion may be the

major pathway of exposure when children

begin crawling and placing their hands on

dusty surfaces and increasing their hand-tomouth behavior. The level of exposure may

continue to increase, given that the normal

tendency of young children to explore their

environment orally increases through 2

years of age. The actual dose to the child

will depend on environmental concentrations and the efficiency of pesticide uptake

for the different types of exposure routes,

i.e., dermal contact versus ingestion. To

date, direct observation and quantification

of children's exposure-related activity

patterns and their interaction with their

environment is very limited. Time-activity

analysis thus could provide information

about age-specific exposure pathways.

To assess time-activity patterns, most

researchers have preferred self-administered

time diaries and interviews (29,30). However, these diaries are subject to inaccurate

recall and thus have limited validity

(31-33). Moreover, they fail to document

microactivities such as dermal and hand-tomouth contacts, which are important pathways of exposure in young children.

Observational techniques are more detailed

and accurate than conventional methods of

questionnaires and diaries for recording

such microscopic data (34-37). Leckie and

Zartarian and co-workers (36-39) have

successfully developed videotaping methodologies and video translation software to

quantify children's activity patterns for

dermal and nondietary contacts, and have

piloted these techniques on 2- to 4-year-old

children of Mexican American farmworkers

in California (36). More extensive data collection is needed on children of various ages

to assess the changing pathways and routes

of exposure as children develop.

Exposures of Farmworkers

and Their Families

Nationally, an estimated 5 million farmworkers work on America's farms, including

approximately 1 million California residents

(40). A growing body of literature indicates

that resident farm families, hired farmworkers, and their children are among those most

highly exposed to pesticides (5,6,8, 10,

12,24,41-49). These studies suggest that

farm children can be exposed by the same

pathways as other children, namely through

consumption of contaminated food, by

household use of pesticides, as a result of

drift from nearby agricultural applications,

by contaminated breast milk from their

farmworker mothers, by playing in the

fields, and through pesticides tracked into

their homes by their parents or other household members working in fields (5,6,8,10,

12,24,41-43,50,51). For example, preliminary data from pilot studies conducted for

the Agricultural Health Study in North

Carolina and Iowa indicate elevated levels of

recently applied pesticides in the food,

homes, and bodies of farm families and their

children (8,10,12,43,44). In a study of 88

children in the Yakima Valley, Washington,

Loewenherz et al. (24) reported more

frequent detection of OP metabolites in

children of pesticide applicators compared

to nonapplicators, particularly those living

less than 200 ft from orchards. Trends of

increasing exposure with decreasing age also

suggested that child activity is an important

exposure variable.

Simcox et al. (5) studied 59 families in

the Yakima Valley, and compared levels of

four organophosphate (OP) pesticides in

the homes of hired farmworkers, families

residing on farms, and nonagricultural families. Chlorpyrifos was detected in 95% of

the homes. House dust concentrations were

consistently higher for agricultural families

than for nonagricultural families, and pesticide applicators tended to have higher

house dust concentrations compared to

nonapplicators. There was a 3-fold difference in median chlorpyrifos house dust

concentration between farmworkers who

did not directly handle pesticides and reference families of nonfarmworkers living

in agricultural communities (median

410 Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999
CHILDREN AND PESTICIDES

=172 ng/g for farmworker vs 53 ng/g for

nonagricultural families).

Bradman et al. (6) conducted a pilot

study of pesticide exposures to children of

migrant farmworkers and nonfarmworkers

living in a largely Latino community in

California's Central Valley. Floor dust samples and child hand wipes were collected

from the homes of 10 families, 5 of which

had at least one resident farmworker.

Higher levels of the OPs diazinon (maximum = 160 ppm), chlorpyrifos (maximum

= 33 ppm), and malathion (maximum = 1.6

ppm) were found in house dust in farmworker homes (6). Residues of diazinon and

chlorpyrifos were detected on the hands of

two and three farmworker toddlers, respectively, who also lived in the homes with the

highest dust concentrations. A preliminary

risk assessment suggested that diazinon

exposures in children could exceed the U.S.

EPA Office of Pesticide Program's oral

reference dose of 9E-5 mglkg/day.

Overall, the findings from these studies

suggest that inadvertent carry-homes of

occupational pesticides are occurring and

that contamination in the homes of farm

families are likely to be higher than in other

homes. Further, a significant source of

exposure to farmworker families may derive

from their residential proximity to fields.

Potential Health Effects of

Exposure to OP Pesticides

Studies of the effects of pesticide exposure

on children's health have been limited to

those of birth defects, childhood cancer,

and acute poisonings following ingestion.

Several case-control studies have associated

parental exposure to pesticides or pesticide

use in the home with childhood brain

tumors, leukemias and lymphomas, testicular cancers, and other cancers (52-55).

Other studies have reported that parental

exposure to pesticides or application of

pesticides in the home is associated with

certain birth defects including neural tube

and other birth defects (56,57).

To date, only one small ecologic study

has examined whether low-level chronic

exposure of children to pesticides can lead

to adverse health consequences (58). This

study of Yaqui children in Mexico found

that children 4-5 years of age (n = 33) living in an agricultural valley with presumably higher pesticide exposure had deficits

in tests of stamina, coordination, recall,

and ability to draw a person, compared to

children (n = 17) living in the foothills

where there was mostly ranching. This

study, although suggestive of an effect of

pesticides, is limited by small sample size,

utilization of a convenience sample, the

lack of individual exposure data, and no

statistical control of potential confounders.

At present, the only prospective study

investigating pesticides and adverse health

effects is the National Cancer Institute/

U.S. EPA Agricultural Health Study,

which is a large cohort study of cancer in

midwestern and eastern farmers and their

families (49). In spite of the paucity of

information concerning the potential

health effects in children of chronic lowlevel exposure to organophosphates, there

is substantial evidence in developing

rodents and limited evidence in adult

humans who have been chronically

exposed to OPs that low-level chronic

exposure to organophosphates may affect

neurologic functioning, neurodevelopment, and growth. Because OP exposure

may cause dysregulation of the autonomic

control of airways, it is biologically plausible that exposure may be related to the

occurrence of asthma in children.

Effects of Acute Exposure to

OP and Carbamate Pesticides

in Children

The primary effects of OP and carbamate

acute exposure are on the parasympathetic,

sympathetic, and central nervous system.

These pesticides interfere with the metabolism of acetylcholine (ACh) by inhibiting

the enzyme that hydrolyzes it, acetylcholinesterase (AChE) (59). ACh accumulates at the neuronal junctions, resulting in

the continued stimulation and then suppression of neurotransmission to organs.

ACh is the chemical transmitter of somatic

motor neurons to skeletal muscle, postganglionic parasympathetic nerve fibers, preganglionic fibers of both sympathetic and

parasympathetic nerves, and some fibers in

the central nervous system. The accumulation of ACh at the motor nerves results in

weakness, fatigue, muscle cramps, fasciculations, and muscular weakness of respiratory

muscles. Accumulation at the autonomic

ganglia results in increased heartbeat and

blood pressure, pallor, and hypoglycemia.

Accumulation of ACh at muscarinic receptors results in visual disturbances, tightness

in the chest and wheezing due to bronchoconstriction and increased bronchial

secretions, and increased salivation, lacrimation, sweating, peristalsis (resulting in

nausea, vomiting, cramps, diarrhea), and

urination. Central nervous system effects

from ACh accumulation include anxiety,

headache, confusion, convulsions, ataxia,

depression of respiration and circulation,

slurred speech, tremor, and generalized

weakness (59,60). Carbamates, unlike OPs,

do not irreversibly inhibit ACHE. Thus,

their activity is quickly reversed after excretion of the pesticide (61). Pregnancy may

pose a time of increased risk because plasma

AChE activity is already reduced, at least

during the first two trimesters (62,63).

The most frequent acute symptoms of

OP poisoning in children include miosis,

excessive salivation, nausea and vomiting,

lethargy, muscle weakness, tachycardia,

hyporeflexia, and hypertonia, and respiratory distress (60,64). Duration of symptoms depends on the dose, with the highest

doses resulting in death. In one study,

pneumonitis developed in about one-third

of poisoned children (64). OP-induced

delayed onset peripheral neuropathy

(OPIDN), reported for adults, has not

been reported in children.

Long-Term Sequelae of

Acute Exposure to OP

and Carbamate Pesticides

in Adults

Although no studies have examined the

long-term sequelae of acute pesticide poisoning in children, some studies in adults

suggest that there are residual effects.

Neuropsychologic investigations of poisoned farmworkers, pest control workers,

and industrial workers tested a number of

months to years after acute exposure to

various OP pesticides have revealed deficits

in overall abstraction, verbal and visual

attention, visual memory, visuomotor

speed, sequencing, visuomotor problem

solving, motor steadiness, motor dexterity,

and fine motor speed (65-68). These

workers report anxiety, depression, irritability, confusion, and impaired concentration and memory. On neurologic exam,

lower vibrotactile sensitivity has been

reported (68,69). High acute or subchronic exposures to OPs may also result

in delayed neurotoxicity or OPIDN

(59,70,71). OPIDN usually manifests 1 to

6 weeks after exposure and may result in

moderate to severe peripheral neuropathies

lasting months, years, or indefinitely (59).

Effects of Chronic Exposure

to OP and Carbamate

Pesticides in Adults

Although there are no studies in children

on the neuropsychologic effects of chronic

pesticide exposure, small studies of chronic

low-level exposures of farmers or pest

Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999 411
ESKENAZI ET AL.

control workers who had levels of AChE

within normal limits found no differences

in tests of their neurobehavioral functioning compared to those of unexposed

controls (72) or in a pre/post exposure

comparison (73). Results of other studies in

adults indicate that there may be mild

peripheral effects of chronic lower level

exposure as indicated by slower reaction

times (74), impaired proprioception (postural sway) (75), decreased conduction

velocities in motor (median and peroneal)

and sensory (median and sural) nerves

(76), wider two-point discrimination (77),

as well as some neurobehavioral effects such

as increased anxiety (78), decreased visuomotor speed (65,79,80), and short-term

verbal memory (79). Daily exposure to

OPs that are insufficient to cause signs and

symptoms of acute poisoning may also produce an influenza-type illness characterized

by weakness, anorexia, and malaise (81). In

chronic lower level exposures, depression of

cholinesterase activity may be cumulative,

and there is no predictable correlation

between the severity of symptoms and the

degree of cholinesterase inhibition.

Animal Evidence for

Neurodevelopmental Effects

of Exposure to OP Pesticides

There is a strong and growing body of

evidence linking exposure to OP pesticides

during gestation or the early postnatal

period and neurodevelopmental effects in

animals. These effects may be due to the

direct impact of OPs on the cholinergic

system of the fetus, although effects on cellular intermediates such as adenylyl cyclase

(82) and altered DNA synthesis in the

brain through noncholinergic mechanisms

(83-85) have been hypothesized. Table 1

summarizes animal studies investigating

different organophosphate pesticides, dosing regimes, and exposure routes and their

impacts on the developing nervous system.

Chlorpyrfos

Neurobehavioral tests given postnatally

found that animals exposed in utero

demonstrated decreased balance (86),

increased righting reflex time, and poorer

cliff avoidance (87,88). When exposure

occurred in the early postnatal period, there

was a lowered threshold for convulsions

(89) as well as increased gait abnormalities

and tremors (90) and deficits in delayed

alternation on mazes (91).

Some studies suggest that early gestation

may be a critical period for the neurodevelopmental effects of certain pesticides. Muto

et al. (86) studied the effects of exposure to

chlorpyrifos in rats occurring both during

gestation (gestation days 0-7 and 7-2 1) and

the postnatal period. They reported that

early prenatal exposures were more likely to

result in poorer performance on the rotorod

test than exposures during later gestation,

which was in turn, more likely to result in

deficits than those occurring postnatally.

A number of mechanisms have been

proposed to explain the observed neurobehavioral effects in animals. Chanda and

Pope (88) found that repeated exposure of

rodents to low levels of chlorpyrifos during

gestation was related to inhibited levels of

AChE and downregulated muscarinic receptors in the fetal brain. Transient brain

AChE inhibition also has been consistently

reported in neonates postnatally exposed to

chlorpyrifos (90-94). Other effects of

chlorpyrifos that, in part, could explain the

neurobehavioral impairment include

decreased muscarinic receptor binding

(90,94,95), altered brain RNA concentrations (96), and inhibition of brain DNA

synthesis (84,85,97). For example, after

treating rats on postnatal days 1-4 with a

dosage that produced minimal AChE inhibition (1 mg/kg), Dam et al. (85) reported

large deficits in DNA synthesis in the brain

stem and forebrain, with lesser effects on the

cerebellum. Similar deficits in DNA synthesis were observed after a single early postnatal exposure but at a slightly higher dose

(97). Early postnatal exposure to chlorpyrifos (postnatal days 1-4 or 11-45) also

altered RNA concentrations in the brain

stem and forebrain of rats (96). By targeting

RNA, the macromolecule that controls

postmitotic processes of cell differentiation

and growth, the chemical may evoke alterations in cell function and number in

developing organisms (96).

The results of these studies indicate that

OP pesticides could contribute to behavioral

abnormalities in young animals by producing cellular deficits in the developing brain.

Recently, Campbell et al. (84) concluded

that regions rich in cholinergic projections,

such as the brain stem and forebrain may be

more affected than the less cholinergic

regions such as the cerebellum. However,

the maturational timetable of each region

(brain stem then forebrain then cerebellum)

may be an important factor in determining

relative vulnerability. Nevertheless, there is

reasonable evidence that even subtoxic exposure to chlorpyrifos during the critical

period of brain development could produce

cellular, synaptic, and neurobehavioral

aberrations in animals (97).

Odter OP Pesticides

OPs other than chlorpyrifos have been

associated with lowered AChE in the brain

of rodents exposed prenatally [bromophos

(98), dichlorvos (99), dimethoate (100),

methyl parathion (101), quinalphos

(102)]. Studies in which animals were

exposed early in the postnatal period to

these other organophosphate pesticides

have also reported inhibition of brain

AChE [dichlorvos (103), diisopropylfluorophosphate (104), quinalphos (105),

parathion (92,93,95,106)] and downregulation of muscarinic receptors [diisopropylfluorophosphate (104,107), parathion

(95,106)]. In addition, evidence from a

single in vitro study suggests that prenatal

exposure to organophosphates could alter

human fetal brain AChE levels. For example, Banerjee et al. (108) reported a dosedependent inhibition of cerebellar AChE

activity in human fetal brain cells (8-10

weeks gestation) treated with 5 x 10-11 -

5 x 10-8 M diisopropylfluorophosphate.

Further research in rodents has found

reductions in brain weight, most pronounced in the cerebellum and brainstem,

following OP exposure during gestation

[dichlorvos (109), trichlorfon (109-111)].

Neurobehavioral deficits such as

impaired maze performance [dichlorvos

(99), diazinon (112)], decreased open-field

activity [sumithion (113)], impaired locomotion or swimming [trichlofon (110),

diazinon (112)], and reduced time on the

rotorod test [diazinon (112), sumithion

(113)] have also been associated with prenatal organophosphate exposure. In addition, permanent alterations in spontaneous

motor behavior (i.e., locomotion, rearing,

and total activity) have been observed in

mice exposed to a single subsymptomal

dose of diisopropylfluorophosphate early in

the postnatal period (107).

Other Potential

Developmental Effects

of OP Pesticides

Desed Birth Weight and

Altered Growth

A number of the animal studies reported

above have demonstrated a decrease in

birth weight or body weight in developing

animals exposed to OPs. Anticholinesterase

agents such as OPs may have a nonspecific

regulatory effect on growth, perhaps by an

influence on placental transport of nutrients (112,114) or by altering the activity

and reactivity of the adenylyl cyclase

412 Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999
CHILDREN AND PESTICIDES

Table 1. Review of the literature of the effects of organophosphate pesticides on neurobehavioral functioning in developing animals.a

Agent

(dose mg/kg)

Species Route Exposure period Neurodevelopmental effects

Mouse Diazinon Throughout gestation Lower birth weight

(0.18, 9)

Oral

Rat Methyl parathion

(1.0)

Gavage

Rabbit Dichlorvos

(9.0)

Gavage

Rat Methyl parathion

(1.0, 1.5)

Oral

Guinea pig Trichlorfon

(100)

Gavage

Pig Trichlorfon

(60)

Oral

Rat Parathion

(1.3, 1.9)

Subcutaneous

Rat Sumithion

(5, 10, 15)

Gavage

Rat METASYSTOX-R

(Methyl demeton)

(0.5, 1.5, 4.5)

Oral

Rat Parathion

(0.882)

Subcutaneous

Rat Methyl parathion

(7.8) (adult: 18)

Parathion

(2.1) (adult: 18)

Chlorpyrifos

(45) (adult: 279)

Subcutaneous

Rat Chlorpyrifos

(0.03, 0.1, 0.3)

(0.1, 0.3)

Intraperitoneal

Rat Methyl parathion

(< 7.8) (adult: < 18)b

Parathion

(< 2.1) (adult: < 18)b

Chlorpyrifos

(< 45) (adult: < 279)b

Subcutaneous

GD 7-15

PND 6-16 or 16

GD 6-12

GD 36-38 or 51-53

GD 55 or 55 and 70

PND 5-20

GD 7-15

GD 6-15

PND 5-20

PND 7 and adult

GD 0-7 or 7-21

PND 3, 10 or 12, 6-10

PND 7 and adult

Decreased rate of postnatal weight gain

Balance (rotorod), swimming, and maze (speed) effects

No differences in auditory startle, visual cliff response, or

open-field motor activity

Slight changes in learning ability as measured by simple two-choice maze

Effects on open-field activity

Inhibited AChE activity in all brain regions tested; recovery

slower in animals exposed over 10 days than after a single dose

Decreased serotonin concentration in brainstem (22%),

mesencephalon (26%), and hippocampus (59%) after prolonged exposure

Altered postnatal development of brain cholinergic neurons

Reduced AChE activity in all brain regions (1.5 mg/kg)

Increased choline acetyltransferse activities in all brain regions (1.5 mg/kg)

Subtle alterations in selected behaviors: impaired cage emergence,

accommodated locomotor activity, and operant behavior in a mixed paradigm

No morphologic changes in hippocampal or cerebellar tissue

Disturbances of locomotion

Reduced brain weight, particularly cerebellum, hippocampus, thalamus,

and colliculi

Dose-related cerebellar hypoplasia

Ataxia not observed in neonates

Dose-dependent reductions in AChE activity and muscarinic receptor binding

in the cortex

No differences in most reflex measures, eye opening, or incisor eruption

during the preweanling period

Small deficits in tests of spatial memory in both the T-maze and the radial

arm maze during the postweanling period

Dose-related decrease in open-field activity and motor coordination (rotorod)

Alterations in acquisition and extinction of a conditioned escape response

Increased social interactions

No significant behavioral effects at lowest dose (5 mg/kg)

No differences in fetal brain AChE

No differences in neonatal survival, growth, and development

No alteration of sensory or reflex functions, maze learning ability, or openfield activity

Cellular disruption and necrosis in the dentate gyrus and CA4 regions of the

hippocampus

Depressed hippocampal AChE (73%) and muscarinic [3H1 QNB binding (36%)

at PND 12

ChE inhibition in whole brain, plasma, and erythrocytes

(transient)

Maximal brain ChE inhibition (>78%) similar in both age groups

ChE activity recovered faster in neonate

Lower body weight

Balance effects (rotorod)

Effects on open-field motor behavior

Effects early gestation > late gestation > postnatal

Inhibition of brain and plasma ChE activity in both neonate and adult

Good correlation between brain ChE (r= 0.93) or plasma

ChE (r= 0.99) inhibitory potency and acute toxicity

(Continued)

Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999

Author, year

Spyker and Avery,

1977 (112)

Crowder et al.,

1980 (132)

Maslinska et al.,

1981 (103)

Gupta et al.,

1985(101)

Berge et al.,

1986(110)

Pope et al.,

1986 (133)

Stamper et al.,

1988 (95)

Lehotzky et al.,

1 989 (113)

Clemens et al.,

1990 (134)

Veronesi and

Pope, 1990(106)

Pope et al.,

1991 (92)

Muto et al.,

1992(86)

Pope and

Chakraborti,

1992(93)

413
ESKENAZI ETAL.

Table 1. Continued.

Agent

(dose mg/kg)

Author/Year Species Route Exposure period Neurodevelopmental effects

Srivastava et al., Rat Quinalphos GD 6-20

(0.5, 1.5)

Gavage

Rat Diisopropylfluorophosphate

(0.5-1.0)

Subcutaneous

Rat Chlorpyrifos

(0.3-10)

Subcutaneous

Rat Chlorpyrifos

(40)

Subcutaneous

MehI et al., Guinea pig Dichlorvos

1994 (109) (15-30)

Trichlorfon

(125)

Subcutaneous

Santhoshkumar Rat Bromophos

and Shivanandappa, (500)

1994 (98) Gavage

PND 4-9 or 4-20

PND 16 or 17

PND 7-1 0

GD 42-46

GD 18

Reduced AChE activity in fetal brain (0.5-1.5 mg/kg) and placenta (1.5 mg/kg)

No differences in fetal weight or anomalies

Inhibition of AChE

Downregulation of muscarinic receptor recognition sites

These alterations may delay the maturation of the cholinergic system and

may account for some long-lasting neurotropic effects observed after

developmental exposure

More rapid amygdala kindling in treated animals

Proconvulsant effect was dose related

Increased excitability of the amygdala

AChE activity 55-60% controls

Less inhibition of AChE in neonate relative to adult

Muscarinic [3H] QNB receptor binding in cortex,

hippocampus, and striatum marginally affected (5-11% reduction) in neonate

Basal motor activity levels not affected

Reduction in brain weight

Most pronounced in cerebellum, medulla, thalamus, hypothalamus, and

quadrigeminal plate

AChE inhibition in fetal brain started at 2 hr and reached a maximum

at 16 hr postexposure (transient)

Recovery almost complete by PND 1

Sensitivity of ChE inhibition in vivo: maternal serum > maternal brain >

fetal brain

Rat Chlorpyrifos

(90, 120, 240)

Subcutaneous

Mouse Diisopropylfluorophosphate

(1.5)

Gavage

Rat Chlorpyrifos

(200)

Subcutaneous

Rat Dimethoate

(7, 10.5, 14, 28)

Dichlorovos

(1, 1.5, 2, 3.9)

Methyl parathion

(0.2, 0.3, 0.4, 0.9)

Gavage

Rat Dichlorvos

(0.97-3.88)

Oral

Rat Chlorpyrifos

(2.0)

Subcutaneous

Rat Chlorpyrifos

(0.1, 3, 15) gavage

(0.1, 1, 5) oral

PND 21

PND 3, 10, or 19

GD 12

Three generations

Males and nonpregnant females:

5 days/week

Pregnant females

throughout gestation

and lactation

Throughout gestation

and lactation

PND 1

GD 6-15

Two generations:

5 days/week

Signs of severe poisoning prevented behavioral testing at highest dose

Transient memory impairment on maze (120 mg/kg)

Dose-related inhibition of brain AChE but transient

Reduced muscarinic binding of [3H]QNB in frontal cortex (240 mg/kg)

Altered spontaneous motor behavior (increased locomotion, rearing, and

total activity) observed at adult age of 4 months

Decreased muscarinic receptor density at adult age

Persistent effects found in adult mice exposed to single subsymptomal dose

on PND 3 or 10; not in animals exposed on PND 19

Decreased brain AChE activity in both dams (85-88%) and fetuses (42-44%)

By PND 3, brain AChE still inhibited in pups (30%); less than for

dams (82%)

In vitro inhibition of maternal and fetal brain AChE activity indicated that

prenatal AChE activity was somewhat more sensitive

Righting reflex time was increased in PND 1 pups

No differences in righting reflex at PND 3

Altered electrophysiological function in primary somatosensory, visual,

and auditory cortex

Increased mean frequency and EEG index, and decreased mean amplitude

dose dependent

Changes more expressed in second and third generations

AChE inhibition in brain (significant at highest dosages)

Increased maze running time and errors

AChE in brain 40-65% control

Changes were dose related

Inhibition of DNA synthesis within 4 hr of treatment and at 8 days of age in

all brain regions

Concluded that low doses target the developing brain during critical period in which

cell division is occurring, effects that may produce eventual cellular, synaptic,

and behavioral aberrations after repeated or prolonged subtoxic exposures

No teratogenic effects found in animals exposed on gestational days 6-15

Two generation study: AChE inhibition in brain (52% control) and decreased

body weight in Fl litters (5 mg/kg)

(Continued)

Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999

1992 (102)

Balduini et al.,

1993 (104)

Wurpel et al.,

1993 (89)

Chakraborti

et al., 1993 (94)

Stanton et al.,

1994 (91)

Ahlbom et al.,

1995 (107)

Chanda et al.,

1995 (87)

Nagymajtenyi

et al., 1995 (135)

Schulz et al.,

1995 (99)

Whitney et al.,

1995 (97)

Breslin et al.,

1996 (115)

414
CHILDREN AND PESTICIDES

Table 1. Continued.

Agent

(dose mg/kg)

Author/Year Species Route Exposure period Neurodevelopmental effects

Chanda and Rat

Pope, 1996(88)

Srivastava and Rats

Raizada, 1996

(100)

Campbell et al., Rat

1997(84)

Song et al., Rat

1997(82)

Dam et al., Rat

1998(85)

Gupta et al., Rat

1998(105)

Hjelde et al., Guin

1998(111)

Johnson et al., Rat

1998(96)

Moser and Pedilla, Rat

1998(90)

Chlorpyrifos GD 12-19

(6.25-25)

Subcutaneous

Dimethoate

(3.75, 7.5, 15, 30)

Gavage

Chlorpyrifos

(1,5)

Subcutaneous

Chlorpyrifos

(1,5)

Subcutaneous

Chlorpyrifos

(1.0)

Subcutaneous

Quinalphos

(0.5)

Oral

nea pig Trichlorfon

(25-200)

Gavage or

subcutaneous

Chlorpyrifos

(1,5)

Subcutaneous

Chlorpyrifos

(15, 80)

Gavage

GD 6-20

PND 1-4 or

11-14

PND 1-4 or

10-14

PND 1-4

PND 10-21 or

10-45

GD 42-44 or

34-53

PND 1-4 or

11-14

PND 17 and adult

AChE inhibition

Righting reflex and cliff avoidance affected

Dose-related downregulation of muscarinic receptors

Low-level, repeated exposures caused extensive neurochemical and

neurobehavioral changes

Dams exposed to 15 and 30 mg/kg produced 20 and 60% mortality,

respectively; highest dosage group not considered for toxicity evaluation

Inhibition of AChE activity in fetal brain and placenta dose dependent;

less than for dams

Reduced fetal weight

No teratogenic effects

Significant mortality and severe cell loss in brainstem at higher dose (PND 1-4)

Cell loss in forebrain (PND 11-14); loss occurred after treatment

DNA levels elevated in cerebellum after exposure, then subnormal

Deficits in adenylyl cyclase cascade in forebrain, cerebellum, and heart

Disrupted signaling function in transduction of both cholinergic and

adrenergic signals

Concluded that disruption of cell development is not restricted to cholinergic

targets, nor even to the central nervous system

Decreased DNA synthesis in brainstem and forebrain on PND 5 (24 hr after

last treatment)

No effects seen 4 hr after treatment

Smaller deficits in cerebellum

No differences in RNA or protein synthesis

Decreased AChE in the brain and blood but transient

Increased superoxide radical generation in brain: 43% by PND 21 and

59% by PND 45

Enhanced brain lipid peroxidation: 28% by PND 45

Produced cerebral oxidative stress, which may affect central nervous

system function

Reduced weight in almost all regions of the brain, especially cerebellum,

medulla, and hypothalamus

Minimum dose required for effect was 1 00 mg/kg over 3 consecutive days

Suggested alkylation of DNA or effects on its repair capability as possible

mechanisms

Altered RNA concentrations in the brain stem and forebrain at subtoxic doses

Targeted macromolecules that control cell differentiation during critical

postmitotic period; may elicit delayed developmental neurotoxicity as a result

Concluded that the developing brain is a selective target for chlorpyrifos

Behavioral effects (lowered activity, gait abnormalities, tremors, smacking)

and ChE inhibition at 5-fold lower dose than adult

Maximal effects occurred at 6.5 hr after dosing

Blood and brain ChE recovery by PND 24

Decreased muscarinic receptor (QNB) binding

Abbreviations: AChE, acetylcholinesterase; ChE, cholinesterase; GD, gestational day; PND, postnatal day; QNB, quinuclidinyl benzilate. 'All reviewed studies contain an unexposed control group for

comparison. hAnimals were treated with graded doses up to the maximum tolerated dose (MTD).

signaling cascade, which would disrupt cell

development in all areas of the body, not

only those cholinergically regulated (82).

Muto et al. (86) reported lower body

weight in rats exposed during the first 7 days

of gestation to chlorpyrifos (0.03 mg/kg);

with higher doses they found a decrease in

the length of the limbs (0.1 and 0.3 mg/kg)

and head circumference (0.3 mg/kg). Other

studies have reported a decrease in pup

weight (115) and a decrease in weight gain

postnatally following exposure to chlorpyrifos (88) or diazinon (112) during gestation.

Spyker and Avery (112) also reported lower

birth weight and a slower rate of postnatal

weight gain in mice exposed to diazinon (9

mg/kg) throughout gestation. In rats, low

levels of two carbamates and a triazine herbicide administered postnatally interacted to

increase thyroxine levels and alter levels of

somatotropin, hormones that regulate

growth (116).

Potential Respiratory Health Effects

Much of the animal literature reviewed here

has focused on the central nervous system

effects of organophosphate exposure.

Because OP pesticides exert their pharmacologic effects through inhibition of

AChE, both short- and long-term effects on

autonomic regulation are prominent features in the toxicology of this class of pesticides (76,117). No previous work has

addressed the autonomic sequelae of pesticide exposure per se, yet disorders of autonomic regulation may be one of the earliest

and most sensitive measures of long-term

physiologic effects of exposures in infants

and young children.

Environmental Health Perspectives * Vol 107, Supplement 3 * June 1999

3

415
ESKENAZI ET AL.

Similarly, the parasympathetic nervous

system provides the principal neural control

of lung airway tone. There are considerable

data indicating that dysregulation of both

parasympathetic (cholinergic) and sympathetic autonomic control of airways, such as

by pesticide exposure, may be important in

the occurrence of asthma and its severity

(118). Dysregulation of parasympathetic

function, as measured by respiratory sinus

arrhythmia, predicts the onset of wheezing

in adults (119). Although there are few

direct studies of the effects of OP and carbamate pesticide exposure on asthma risk,

farmworkers' exposure to carbamate pesticides has been associated with the occurrence of asthma after adjustment for other

relevant factors (120). Professional fumigators reportedly have an increased occurrence of allergy and asthma in parallel with

a higher risk of a > 20% decrease in red

blood cell AchE (121). Exposure to chlorpyrifos has also been associated with an

increase in the occurrence of atopic conditions (122). Although none of these studies

involved children, they raise the prospect

that pesticide exposure could be important

etiologic and morbidity-modifying factors

in the occurrence of childhood asthma.

Biologic Plausibility for the

Effects of Low-Level Chronic

Pesticide Exposure in Children

Tests on young rodents demonstrate a

progressive decrease in susceptibility to OP

pesticides with increasing age (13,123-125).

In some cases, the lethal dose in immature

animals is only 1% of the adult lethal dose

(92,93,97). A study of rats found that animals 1 and 7 days of age tolerated only 4%

and 17% of the adult dose, respectively

(92,93,97). Seven-day-old rats were 2.3,

8.6, and 6.2 times more sensitive than

adults to the acute toxicity of methyl parathion, parathion, and chlorpyrifos, respectively (92). In humans, children have had

higher fatality rates than adults in several

cases of OP poisoning (13).

Young animals may be more susceptible

to the toxic effects of organophosphates

due to lower activity of detoxifying enzymes

such as paraoxonase that deactivate active

OP metabolites (e.g., paraoxon, chlorpyrifos-oxon) (123,126-131). For example,

Mortensen et al. (126) reported markedly

lower plasma and liver chlorpyrifos-oxonase

levels in neonate compared to adult rat tissue. They concluded that the higher sensitivity of young rats to acute chlorpyrifos

toxicity may not be explained by increased

sensitivity of the target enzyme, brain

AChE, but it may be partially explained

by a deficiency of chlorpyrifos-oxonase

activity (126).

Although young animals are more

sensitive than adults to the acute toxic

effects of chlorpyrifos, some researchers

have suggested that lower level chlorpyrifos

exposures may produce more extensive

neurobehavioral effects in the adult rat

than in the neonate (94). In addition,

more extensive changes have been found in

cholinergic parameters in the maternal

brain compared to the fetus or neonate

(88). Developing animals also appear to

recover more quickly from cholinesterase

inhibition than the adult (92,98), and may

be less susceptible to developing OPIDN

(71). However, repeated low-dose chlorpyrifos exposure during gestation has been

associated with persistent neurochemical

and neurobehavioral changes in developing

rodents (88).

In summary, young children may be

especially vulnerable to pesticides because

of the sensitivity of their developing organ

systems combined with a limited ability to

enzymatically detoxify these chemicals

(13,123,126-131). According to the

National Academy of Sciences (13), children's OP exposures are of special concern

because "exposure to neurotoxic compounds at levels believed to be safe for

adults could result in permanent loss of

brain function if it occurred during the

prenatal and early childhood period of

brain development" (13). Because there is

so little information available on the levels

and routes of children's pesticide exposure,

it is not feasible to conduct a risk assessment predicting the likelihood of adverse

effects based on animal studies. Thus far,

there are no data in children to support or

refute the hypothesized health effects of

chronic low-level pesticide exposure.

Future Directions

There is clearly a lack of information on

the sources, pathways, and levels of pesticide exposures of children, and in particular, of those children at highest risk because

they live in agricultural communities.

Similarly, there is a dearth of information

on whether low-level chronic exposure

to pesticides is associated with adverse

health effects. The goal of the Center for

Children's Environmental Health Research

at the University of California, Berkeley is

to address these questions by conducting a

longitudinal cohort study of approximately

500 pregnant women and their children

who live in a rural agricultural community

in the Salinas Valley of Monterey County,

California. The specific aims of this Center

for the Health Assessment of Mothers and

Children of Salinas, or CHAMACOS

(which means "little child" in Chicano

Spanish), are to a) characterize OP exposure levels and pathways in pregnant

women and their children; b) determine

the predictors of OP levels in the body and

home; c) describe the exposure-prone

behavior of children at different developmental stages using time-activity analysis;

and d) follow up the children to 3 years of

age to determine whether exposure in utero

and/or during the postnatal period is

associated with poor neurodevelopment

(assessed by tests of the central and autonomic nervous system), slower and stunted

growth, and increased prevalence of respiratory symptoms and disease. Our ultimate

goal is to involve community partners in

planning, coordinating, and conducting an

intervention to reduce pesticide exposures to

young children in this agricultural

community and to evaluate the efficacy and

sustainability of the intervention. To

accomplish our goals, we have established a

multidisciplinary partnership comprised of

farmworkers, health care providers, growers,

journalists, scientists, educators, and representatives of community groups and state

and county health and agricultural departments. We are hopeful that the results of

this study will benefit this community and

agricultural communities, in general, and

will directly contribute to the information

necessary for the implementation of the

Food Quality Protection Act.

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