Environmental Research Section A 81, 87^91 (1999)Article ID enrs.1999.3958, available online at http://www/idealibrary.com on

Subclinical Health E¡ects of Environmental Pesticide Contaminationin a Developing Country: Cholinesterase Depression in Children1

Rob McConnell,*,2 Feliciano Pacheco,{KÔreWahlberg,{Willy Klein,{ Omar Malespin,{ Ralph Magnotti,}Malin Ðkerblom,} and Douglas Murray||

*Department of Preventive Medicine, University of Southern California School of Medicine, Los Angeles, California 90033;{NicaraguanMinistry of Health; {SwedishVoluntary Services and National Autonomous University, Leon, Nicaragua; }EQMResearch,

Cincinnati, Ohio; }International Science Programs, Uppsala University, Uppsala, Sweden; and ||Department of Sociology,Colorado State University, Fort Collins, Colorado

Received June 27, 1997

The e¡ect of exposure to pesticides among chil-dren in a Nicaraguan community in the path of rainwater runo¡ from a large crop-dusting airport wasevaluated by measuring plasma cholinesterase. Meancholinesterase activity in 17 children in the path of run-o¡ was 2.4 international units/ml blood/min, lowerthan the 2.9 IU/ml/min measured in a group of 43 chil-dren from an unexposed community (di¡erence= 0.49IU/ml/min; 95% C.I. 0.24, 0.76). Six (35%) of the 17 ex-posed children had abnormally low cholinesterase le-vels. A possible explanation for this physiologicale¡ect of exposure to pesticides is the dermal absorp-tion which may have occurred among children playingbarefoot in puddles grossly contaminated by runo¡fromthe airport. Drinking water from awell in the ex-posed community demonstrated low level residues ofcholinesterase-inhibiting pesticides, although con-tamination with toxaphene (not a cholinesterase inhi-bitor) exceeded by over 8-fold the United StatesEnvironmental Protection Agency maximum permis-sible concentration in drinking water.The di¤culty inmeasuring health e¡ects resulting from environmen-tal pesticide contamination, and in controlling expo-sure resulting from the rapidly increasing use ofpesticides, is a growing problem for developing coun-tries like Nicaragua. # 1999 Academic Press

Key Words: insecticides; cholinesterase inhibitors;child; developing countries; organophosphorus com-pounds.

1This study was supported by the NicaraguanMinistry of Health,American Friends Service Committee, Care Nicaragua, SwedishVoluntary Services, and by the National Institute of EnvironmentalHealth Science (Grant 5P30ES07048-02).

2To whom reprint requests should be addressed at Department ofPreventive Medicine, 1540 Alcazar Street, Suite 236, Los Angeles,California 90033. E-mail: rmcconne@hsc.usc.edu.

87

INTRODUCTION

The use of pesticides is increasing rapidly in devel-oping countries at a time when their use in developedcountries is stable or declining (World Health Organi-zation, 1990). Pesticide contamination of the environ-ment, especially of ground water, is of increasingconcern in the developed countries (World Health Or-ganization, 1984). However, except for descriptions ofepidemics (Moses, 1986), the literature evaluatingeither the public health or the environmental impactof the increasing use of pesticides in the developingworld is limited (Wesseling et al., 1997).

Studies of the epidemiology of pesticide poisoninginNicaragua have demonstrated that acute poisoningis a major public health problem which primarily af-fects workers (McConnell and Hruska, 1993; McCon-nell et al., 1990; Cole et al., 1988). As in most of LatinAmerica, cholinesterase-inhibiting organophosphateinsecticides are responsible for most acute poisoning.Either plasma or erythrocyte cholinesterase can bemonitored as an indicator of the physiologic e¡ectof overexposure to these insecticides. Althoughdi¡erent organophosphates preferentially inhibitplasma or erythrocyte cholinesterase (McConnell,1994), it has been suggested that plasma cholinester-ase may be a more sensitive indicator of low-levelcommunity exposure (Richter et al., 1986). Plasma cho-linesterase may re£ect more recent exposure to inhi-bitors compared to erythrocyte cholinesterase, whichis replaced more slowly after inhibition. In this study,the e¡ects of environmental pesticide contaminationon plasma cholinesterase of exposed children and ondrinking water quality were investigated in a typicaldeveloping country setting in Nicaragua.

0013-9351/99 $30.00Copyright 1999 byAcademic Press

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88 MCCONNELL ETAL.

METHODS

A cross-sectional study was conducted of childrenaged 5 through 12 living in three sites: (1) a commu-nity in Northern Paci¢c Nicaragua adjacent to a largecrop-dusting airport (and through which drains run-o¡ from the airport during the rainy season fromJuneto December), (2) two blocks of a community facingthe airport but protected from runo¡ by a road, and(3) two blocks from a community on the other side ofthe city from the airport (presumably unexposed topesticides). Each house was visited, and parents wereasked to bring all children of the appropriate ages to aliving room in a house in the corresponding commu-nity, where ¢nger-stick blood samples were obtainedand processed.

Because organophosphate insecticides mixed andloaded in large quantities at the airport inhibit choli-nesterase, plasma cholinesterase was measured in allparticipating children, using Ellman's method asadapted for use with a battery-operated colorimeterwith a light-emitting diode sourcewith nominal wave-length of 440 nm (Magnotti et al., 1988).The Model 176of this instrument, whichwas used in this study, had asilicon photodiode detector (Instruction Manual IM-29: Cholinesterase kit for the ¢eld determination ofpesticide exposure. EQMResearch, Cincinnati, Ohio,1988). Drift was reported to be less than 0.002 Ð/h and£uctuation was less than 1% of absorbance values.The unit was blanked with distilled water in a 10-mm-path-length polystyrene cuvette. Blood was col-lected using an air-displacement pipetter by lancingan alcohol-swabbbed ¢nger. Five microliters weremixed with 0.2 ml of bu¡er and added to 2 ml of a re-agent solution containing butyrylthiocholine sub-strate and DTNB indicator.The reagent formulationshave been described previously (Magnotti et al., 1988).The change in absorbance at 440 nM over 2 min wasrecorded. Results were converted to internationalunits/ml blood/min and adjusted for ambient tem-perature (in degrees centigrade) according to themanufacturer's instructions:

Each child or the accompanying older sibling oradult was asked about the following seven symptomscompatible with cholinesterase inhibition during theweek prior to the examination: headache, blurred vi-sion, diarrhea, nausea, vomiting, stomach ache, or ex-cessive sweating.

Samples of well water were collected from threewells adjacent to the airstrip. The ¢rst sample came

from a well on land adjacent to the airport. This wellis the source of drinking water for the children in the¢rst community (through which drains runo¡ fromthe airport). The second sample was from anotherwell, located approximately 75 m from the airstrip,which serves children living across the street fromthe airport and (to a lesser extent) the unexposed chil-dren living across the city.This well is one of the sevenprincipal pumping stations serving a major popula-tion center. A third sample was collected from a wellinside the airport.Water from this well has been usedto dilute pesticide concentrate since the airportopened in 1953.

Water was collected according to U.S. Environmen-tal Protection Agency recommendations (UnitedStates Environmental Protection Agency, 1980). Fromeach sample 1800 ml were extracted twice with di-chloromethane (40 ml, then 20 ml), and the samplewas evaporated in an ampule with 0.25 ml of 10% car-bovax for shipment.These ampules were analyzed for126 common pesticides by the Swedish National La-boratory for Agricultural Chemistry, according to apreviously published protocol (Andersson and Ohlin,1986). The samples were analyzed for most pesticideswhichwere reported to have beenmixed and loaded atthe airport during the previous month. These in-cluded acephate, chlordimeform, chlorpyrifos, delta-methrin, methamidophos, mephosfolan, methomyl,methyl and diethyl parathion, propargite, propicona-zole, and toxaphene. Samples were not analyzed forJupiter, Dominex, Bendocet, or lambda cyhalothrin(not registered in Sweden at the time of the study) orfor bacillus thuringiensis, maneb, or mancozeb,although these pesticides also were used at the air-port.

RESULTS

Demographic characteristics of the children fromthe three communities and plasma cholinesterase ac-tivity are presented in Table 1. The participation rateamong the children present in each of the three com-

Temperature: 25 26 27 28 29 30 31 32 33 34 35 36 37 38Adjustment factor: 1.00 0.96 0.92 0.89 0.88 0.87 0.86 0.84 0.81 0.77 0.73 0.70 0.68 0.67

munities was greater than 65%. Although the meanlevels of cholinesterase were similar among childrenin the unexposed community and in the communityprotected from runo¡ from the airport by the inter-vening road, the community where children can playin the runo¡ had mean levels well below those ofthe unexposed community (di¡erence of means 0.49international units/ml blood/min; 95% C.I. 0.24,0.76).

TABLE 1Demographic Characteristics and CholinesteraseActivity

Community

Exposed Exposedto runo¡ across road Unexposed

Characteristic N=17 N=22 N=43

Mean age (S.D.) 7.3 (2.2) 7.8 (2.4) 8.2 (2.2)SexNo. boys (%) 11 (65) 10 (46) 24 (56)No. girls (%) 6 (35) 12 (55) 19 (44)

Meancholinesterasea,b (S.D.) 2.4 (0.49) 2.8 (0.47) 2.9 (0.38)

Mediancholinesterasea,b (range) 2.4 (1.6^3.3) 2.8 (1.8^3.8) 2.8 (2.1^3.7)

Symptom prevalencec (%) 4 (24) 6 (27) 14 (33)

aIn international units/ml blood/min.bThree outlying cholinesterase measurements were not included

in the analysis because theycame fromthree siblingswho had levelsmore than six standard deviations greater than the mean.

cTwo or more symptoms suggestive of mild overexposure to choli-nesterase inhibitors (headache, nausea, or blurred vision).

FIG. 1. Percentage of childern with low cholinesterase by com-munity exposure to pesticide runo¡ from airport.

CHOLINESTERASE DEPRESSION INCHILDREN 89

A nonparametric evaluation of these data (Mann±WhitneyU test) also demonstrated a statistically sig-ni¢cant di¡erence between these groups (P=0.003).

Inadequate adjustment for ambient temperature isa source of error in the analysis of cholinesterase(Amaya et al., 1996). In order to determine whethersuch error may have accounted for the di¡erencesbetween communities, mean cholinesterase activity(adjusted for temperature according to recommenda-tions of the manufacturer of the cholinesterase kit)was modeled in a multiple linear regression as afunction of temperature and two dummy variables re-presenting residence in each of the exposed commu-nities. The resulting mean cholinesterase activity ofchildren in the community exposed to runo¡ was still0.39 international units/ml blood/min less than in theunexposed community (95% C.I. 0.17,0.61).

Based on the distribution of cholinesterase activityamong children in the unexposed community, abnor-mal low levels were calculated to be less than 2.2 IU/ml blood/min (1.65 S.D. below the mean for children inthis community). Nine children in this study had lowlevels of cholinesterase; two lived across the road fromthe airport (both of whom reported playing on theother side of road, where they could walk in pesti-cide-contaminated runo¡); one lived in the unex-posed community. This child reported no contactwith pesticides. The other six children lived in thepath of runo¡ from the airport. The distribution ofthese children as a proportion of all children testedin each community is demonstrated graphically inFig. 1. The relative risk of low cholinesterase activity

(with respect to the unexposed community) was 4.6for children in the community across the road fromthe airport (95% con¢dence interval 0.53,39), and therelative risk was 15.2 for the children in the commu-nity exposed to runo¡ from the airport (95% C.I.3.4,68).

There was little di¡erence between communities inthe proportion of childrenwith two or more symptomssuggestive of mild overexposure to cholinesterase in-hibitorsöheadache, nausea, or blurred vision (Table1). The statistically nonsigni¢cant trend observed isthe opposite of that expected, that is, there were moresymptomatic children in the less-exposed commu-nities.The distribution of each of the seven individualsymptoms was also examined, and none was statisti-cally signi¢cantly associated with community ofresidence. In the children from the community con-taminated by pesticide runo¡, there also was no dif-ference between the mean cholinesterase level amongchildren with more than two symptoms and thosewith two or fewer symptoms.

Pesticides found in any of the three water samplesare listed inTable 2. Concentrations of detectable pes-ticides were uniformly higher in the well water fromthe community through which runo¡ £ows than inwater from the well inside the airport or from the citypumping station. Toxaphene exceeded by over 8-foldthe 3 mg/L maximum which would be permitted inthe United States by the U.S. Environmental Protec-tion Agency (O¤ce of Water, United States Environ-mental Protection Agency). Concentrations ofcholinesterase-inhibiting organophosphate insecti-cides were not high in any of the three samples. The

TABLE 2Pesticide Contamination ofWellWater

Other twoWell: Community communities Insideexposed to Runo¡ (city water) Airport

Pesticide Concentration (mg/l)

Chlordimeforma 2.6 n.d.* n.d.Chlorpyrifosb 0.3 n.d. n.d.Diazinonb 0.06 n.d 0.08Fenthionb 4.5 n.d. n.d.Fenthion

sulfoxideb 1.4 n.d. n.d.Mephosfolanb 1.2 n.d. 0.2Methyl parathionb 1.0 n.d. 0.7DDE PPc 0.06 n.d. n.d.DDD PPc 0.1 n.d. n.d.DDT OPc 0.2 n.d. n.d.DDT PPc 1.2 n.d. n.d.Beta HCHc n.d. n.d. 0.7Lindane(gamma HCH)c n.d. n.d. 0.02Toxaphenec 25 0.2 5.0

aOvicide.bOrganophosphate.cOrganochlorine.*Not detectable.

90 MCCONNELL ETAL.

low-level residues of chlorpyrifos, diazinon, andmethyl parathionwerewithin the range of contamina-tion likely tobewithout appreciable health risk if con-sumed over a lifetime. EPA does not have speci¢cdrinking water health advisories for fenthion, me-phosfolan, chlordimeform, or DDT.

DISCUSSION

The low levels of cholinesterase among children inthe community exposed to airport runo¡ suggeststhat there was contamination of the childrens' livingenvironment by organophosphate pesticides. Choli-nesterase is a relatively crude measure of exposureand would not be a¡ected by the low levels of contam-ination by pesticides found in the water samples (ofwhich only the organophosphates would have inhib-ited cholinesterase). A possible explanation for thisphysiological e¡ect of exposure to pesticides is dermalabsorption occurring among children playing bare-foot in puddles grossly contaminated by runo¡ fromthe airport. Additional possible contributing routesof exposure included the inhalation of pesticide-im-pregnated dust from the airport or pesticide residuesin food.

These results are consistent with another studyof acommunity surrounded by cotton ¢elds sprayed withpesticides, where mean cholinesterase activity was

also low (Keifer et al., 1996). Unlike that study, symp-toms in the present study were not correlatedwith ex-posure. However, symptoms depend on the percentageinhibition from an individual's baseline and on theabruptness of the decline (the acuteness of exposure),rather than the absolute level of cholinesterase activ-ity (Coye et al., 1986). Therefore, the results of thisstudy are consistent with chronic exposure to resi-dues and a slow decline in mean cholinesterase activ-ity.

Lowered cholinesterase is a physiological e¡ect ofexposure to organophosphate insecticides, whichmay have chronic health e¡ects. Recent study, for ex-ample, suggests that adults chronically exposed to or-ganophosphate insecticides at work have de¢cits insustained attention and speed of information proces-sing (Stephens et al., 1995). Although the relevance forchildren is not yet known, exposure of neonatal ratsto an organophosphate cholinesterase inhibitorcauses de¢cits in DNA synthesis and severe cell lossin the brainstem and forebrain, e¡ects which theauthors suggested may contribute to behavioral ab-normalities (Dam et al., 1998; Campbell et al., 1997). Inaddition, cholinesterase inhibition may be consideredan indicator that exposure also occurred to other non-cholinesterase-inhibiting insecticides used at the air-port (for which direct measurements of exposurewerenot available). Other insecticides used at the airporthave known chronic health e¡ects. Chlordimeform,for example, is a human bladder carcinogen (Stasik,1988), and toxaphene is an animal carcinogen (Inter-national Program on Chemical Safety, 1984).

The presence inwell water of some environmentallypersistent organochlorine pesticides no longer usedat the airport, for example DDT, may have re£ectedcontamination resulting from use in prior years andmay be indicative of ground water contaminationrather than contamination of individual wells fromruno¡. Diazinon and fenthion contamination may re-£ect either previous use at the airport or householdapplication for vector control.

Possible sources of bias in this study include thepossibility that children from the communities nextto the airport were di¡erentially selected by their par-ents for participation in this study because of a per-ceived exposure to pesticides. This bias may haveexaggerated the di¡erences observed between ex-posed communities and the comparison group. Theparticular model of ¢eld colorimeter used in the studyis no longer commercially available, which may limitthe reproducibility of the methods described. Finally,there mayhave been some residual confounding of therelationship between cholinesterase activityand com-munity by ambient temperature at the time of the

CHOLINESTERASE DEPRESSION INCHILDREN 91

examination, but this source of bias did not explainthe di¡erences between communities.

Many Latin American countries do not have labora-tory facilities capable of performing analyses of low-level pesticide residues, either in the environment orin human tissues. Although environmental conditionssimilar to those around the airport exist elsewhere inNicaragua and throughout the cotton-growing re-gions of Central America and the developing world,in onlya relatively few instances has expertise existedto measure accurately subtle cholinesterase depres-sion and other e¡ects of exposure to organo-phosphates. Unfortunately, at present there isinadequate research infrastructure in most develop-ing countries to evaluate the e¡ect of carcinogenicpesticides, such as chlordimeform and toxaphene, oncancer mortality.

Developing countries often lack the regulatory in-frastructure or the resources to preventÐmuch lessto clean upÐenvironmental contamination. Pesti-cides like toxaphene, which have been severely re-stricted in the developed countries, were usedfrequently until recently in the cotton ¢elds of CentralAmerica. Although one result of this study was a gov-ernment proposal to relocate the airport, the cost ofrelocating either the airport or the exposed commu-nity has been considered prohibitive. In order to pro-tect drinking water consumed by the adjacent city,pesticide residues from operations at the airportshould be collected and contaminated water ¢lteredprior to release into the environment. The only long-term solution to the problem of environmental pesti-cide contamination is the implementation of non-chemical alternatives to control pests.

ACKNOWLEDGMENTS

We are grateful to Carmen Ibania Kuan, Donald Roque, and Fre-sia Morales Cabrera for their collaboration in the collection andanalysis of data. We also appreciate the thoughtful comments ofDrs. Philip Landrigan and StevenMarkowitz.

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