ARTICLE IN PRESS

Respiratory Medicine (2005) 99, 1319–1324

KEYWORDOrganophoOccupationEnvironmeRestrictivefunction

0954-6111/$ - sdoi:10.1016/j.r

�CorrespondiE-mail addr

Low-level exposure to organophosphate pesticidesleads to restrictive lung dysfunction

Roshini Janet Peiris-Johna,�, Dawala Kusuma Ruberua, AnandaRajitha Wickremasingheb, Wim van-der-Hoekc

aDepartment of Physiology, Faculty of Medical Sciences, University of Sri Jayewardenepura,Gangodawila, Nugegoda, Sri LankabDepartment of Community and Family Medicine, Faculty of Medicine, University of Kelaniya,Ragama, Sri LankacInternational Water Management Institute, Colombo, Sri Lanka

Received 24 April 2004

Ssphate;al;ntal;lung dys-

ee front matter & 2005med.2005.02.001

ng author. Tel.: +94 1 2ess: roshi@visual.lk (R.

Summary Apart from symptomology, there are very few reports on lung functionfollowing exposure to low levels of organophosphate (OP) pesticides in man.

Twenty-five occupationally exposed farmers and 22 environmentally exposedfreshwater fishermen were evaluated between and during OP spray seasons. Fortymarine fishermen living away from agricultural areas were recruited as a controlgroup. Forced vital capacity (FVC) and forced expiratory volume in the first second(FEV1) were measured by spirometry. Haemoglobin corrected erythrocyte acet-ylcholinesterase (AChE) levels were measured during and between (baselineestimation) spray seasons using a portable WHO-approved Test-mate system (EQMResearch, Ohio).

FVC ratio was lower in the farmers as compared to the controls (Po0:001)between exposure seasons. In the farmers, FVC ratio decreased further during theexposure season (P ¼ 0:023). FEV1 was lower in the farmers as compared to thecontrols in both periods (Po0:05). In the fishermen, the decrease in ratios of FVCand FEV1 following exposure to pesticides was not significant. FEV1/FVC ratios weresimilar in the three groups between (P ¼ 0:988) and during (P ¼ 0:159) exposureperiods. Following exposure to OPs, AChE levels dropped 12.75% in the farmers(Po0:001) and 5.62% in the freshwater fishermen (P ¼ 0:001).

Occupational exposure to OP results in restrictive lung dysfunction, a phenom-enon not observed following environmental exposure.& 2005 Published by Elsevier Ltd.

Published by Elsevier Ltd.

802182.J. Peiris-John).

ARTICLE IN PRESS

R.J. Peiris-John et al.1320

Introduction

Death in organophosphate (OP) poisoning is com-monly due to respiratory paralysis.1 In high-dose OPpoisoning, both obstructive and restrictive lungdysfunction are reported. Respiratory effects ofhigh-dose OP exposure include broncho-constric-tion,2 pulmonary oedema1 and respiratory muscleparalysis.3 Short-term respiratory symptoms re-ported following low-level exposure to OP includechest pain, cough, wheezing, difficulty in breath-ing, shortness of breath, runny nose and throatirritation.4 Apart from symptomology, there arevery few reports on lung function followingexposure to low-levels of OP pesticides in man.

In Sri Lanka, pesticide imports have increased by50% from 1990 to 2001 with OP being thecommonest insecticide used.5 Pesticide poisoningis a high-priority health issue as it is among thethree leading causes of hospital deaths in thecountry.6 Low-level exposures to OP pesticides aresuspected to be considerable in agricultural areasin the country, where farmers are known toextensively and indiscriminately use pesticides.This study was conducted to determine the effectsof low-level exposure to OP on lung function.

Methods

Twenty-five farmers who regularly spray OP pesti-cides were recruited randomly from among 300farmers from the Uda Walawe irrigation scheme inSouthern Sri Lanka. Twenty-two freshwater fisher-men who live within a 25 km radius of agriculturalfields in Uda Walawe and are likely to be exposed toOP by environmental spray drift were randomlyselected from among 100 fishermen in the area.They were evaluated once between exposureperiods, when workers are not occupationallyexposed to pesticides for over 30 days as verifiedby questionnaire, and once during an exposureperiod. It is unlikely that environmental exposureto pesticides occurs between cultivation seasons ascultivation patterns in the area are determined byrelease of water for irrigation. Forty marine fish-ermen living away from agricultural areas in theWest coast of Sri Lanka were recruited as a controlgroup. Forced vital capacity (FVC), forced expira-tory volume in the first second (FEV1) and forcedmid-expiratory flow rate (FEF25–75%) were measuredusing Microspiro HI-601 spirometer (Chest M.I.Incorporated, Tokyo) following standard proceduresrecommended by the manufacturer. Ten microlitresof capillary blood were obtained using disposable

equipment under sterile conditions to measure redblood cell (RBC) acetylcholinesterase (AChE) levelsduring and between (baseline estimation) sprayseasons. Haemoglobin corrected RBC AChE level(U/g), a bio-marker of OP toxicity, was assayed inall subjects using a portable WHO-approved Test-mate cholinesterase test system (EQM Research,Ohio).

Data were double entered and analysed usingSPSSTM (Statistical Products and Service Solutions,Chicago). The percent-predicted normal and stan-dardised residuals were calculated. The residualmethod recommended by the American College ofChest Physicians,7 was used for comparing theobserved and predicted values of lung functionusing reference norms established for Sinhalese.8

Analysis of covariance was used to compare lungfunctions of farmers, fishermen and controlsbetween and during exposure seasons after con-trolling for confounding variables. Paired t-testswere used to compare values between and duringexposure in each group to assess acute low-levelexposure effects.

Ethical clearance for the study was obtainedfrom the Ethical Review Committee of theFaculty of Medical Sciences, University of SriJayewardenepura. All subjects were recruited afterobtaining informed written consent. Subjects de-tected with any abnormality were referred to aspecialist.

Results

The mean ages (years) of the farmers, freshwaterfishermen and the controls were 37.45, 37.33 and39.35, respectively (P ¼ 0:680). On average, farm-ers had a lifetime exposure to pesticides of 13.8years. Smoking was commoner among the controls(65%) than the farmers (40%) (P ¼ 0:026). Smokinghabits were similar between the farmers andfishermen (53%) (P ¼ 0:252), and the controls andfishermen (P ¼ 0:324).

All farmers used OP pesticides during spraying.Thirteen farmers (52%) used methoxy compoundsand 9 (36%) used ethoxy compounds. In three (12%)farmers, data on the type of OP used were notavailable. Lung function results did not differ withthe methoxy or ethoxy group of compound usedduring the exposure season.

Comparisons of the observed to predicted ratiosof FVC, FEV1, FEF25–75% and FEV1/FVC ratio, andAChE levels between subjects, and between andduring exposure periods within study groups aresummarised in Table 1.

ARTICLE IN PRESS

Table 1 Lung function and AChE levels (U/g) in the farmers, fishermen and controls.

Parameter Period Mean (7standard deviation) of values in P-value�

Farmers (n ¼ 25) Fishermen (n ¼ 22) Controls (n ¼ 40)

FVC ratio Between 75.95 (15.31)a 81.40 (15.40)a 87.02(7.34)b o0.001Exposure 71.09 (13.21)a 79.79 (19.76)b 87.02(7.34)b o0.001P-valuey 0.023 0.743

FEV1 ratio Between 87.83 (19.24)a 92.94 (17.59)a 102.49(33.42)b 0.001Exposure 84.04 (17.08)a 89.80 (19.99)a 102.49(33.42)b o0.001P-valuey 0.433 0.551

FEF25–75% ratio Between 108.5(31.95)a 116.08(32.99)a 123.36(33.42)a 0.172Exposure 111.45(37.24)a 109.03(37.13)a 123.36(33.42)a 0.209P-valuey 0.733 0.474

FEV/FVC Between 0.951(0.08)a 0.941(0.06)a 0.941(0.67)a 0.988Exposure 0.973(0.06)a 0.938(0.06)a 0.941(0.67)a 0.159P-valuey 0.060 0.431

AChE (U/g) Between 30.43(3.87)a 28.28(2.87)b 27.61(2.89)b 0.013Exposure 26.55(3.82)a 26.69(3.30)a 27.61(2.89)a 0.367P-valuey o0.001 0.001

Values having the same superscripts are not significantly different at P ¼ 0:05:�ANOVA comparing means of farmers, fishermen and controls, during each period (values remained significant at P ¼ 0:05

after correcting for differences in smoking status).yPaired t-test comparing values between and during exposure periods within each group.

1

1.5

2

2.5

3

3.5

Time (seconds)

Controls

Fishermen

Farmers (between exposure periods) Farmers (during exposure period)

FV

C (

L)

p=0.001

p=0.023

Figure 1 Forced vital capacity (FVC) in the controls andfishermen and in the farmers between and duringexposure periods. Controls, fishermen, farmers (betweenexposure periods), farmers (during exposure period).

ControlsFishermen Farmers (between exposure periods) Farmers (during exposureperiod)

1

1.5

2

2.5

3

3.5

Time (seconds)

p=0.003

p=0.079

1

FE

V1

(L)

Figure 2 Forced expiratory volume in the first second(FEV1) in the controls and fishermen and in the farmersbetween and during exposure periods. Controls, fisher-men, farmers (between exposure periods), farmers(during exposure period).

Lung dysfunction in low-level OP exposure 1321

FVC was significantly lower in the farmers ascompared to the controls, both between and duringexposure periods (Po0:001) (Fig. 1). The decreasein FVC during acute exposure to OP was significantin the farmers (P ¼ 0:023), but not in the fishermen.FEV1 ratio was significantly lower in the farmers ascompared to the controls, both between and during

exposure periods (Pp0:001) (Fig. 2). The decreasein FEV1 following acute exposure to OP, as comparedto between exposure, was not significant in thefarmers or the fishermen. FEF25–75% and FEV1/FVCwere similar in the three groups and did not changesignificantly following exposure to OP either in thefarmers or in the fishermen.

ARTICLE IN PRESS

R.J. Peiris-John et al.1322

Discussion

The reduction of FVC and FEV1 with a normal FEV1/FVC ratio among farmers occupationally exposed toOPs in this study suggests that low-level, occupa-tional exposure to OP results in restrictive lungdysfunction, a phenomenon not observed followingenvironmental exposure. In obstructive lung dys-function the reduction in FEV1 is usually greaterthan FVC, which results in a reduction in the FEV1/FVC ratio.

The findings of this study are dependent on thevalidity and reliability of measurements made.Spirometry measurement of FVC, FEV1, and theratio FEV1/FVC are established as the single besttest to assess airflow limitations.9 FEV1 is relativelyeffort independent, very reproducible, sensitive tothe narrowing of all classes of airways and is themost widely used of all lung function indices.9

However, both FEV1 and FVC are dependent on thedepth of the preceding inspiration. FVC is alsodependent on the expiratory effort.

FEF25–75%, the average flow rate over the middlehalf of forced expiration, is sensitive to mildobstruction and small airways disease. FEF25–75%

measurements are less reproducible than FEV1.10

FEF25–75% may be reduced in restrictive lung diseasebecause of loss of lung volume.11

OPs affect the respiratory system by peripheralmuscarinic actions on the airways, nicotinic actionson the muscles of respiration, effects on themedullary center in the brain and direct toxiceffects on the alveolar-capillary membrane. Aeset al.12 reported inhibition in the activity of AChE inthe bronchial smooth muscle accompanied byan increase in affinity to acetylcholine (ACh),after both acute and subacute inhalation of OP inWistar rats. Pulmonary toxicity resulting fromphosphorothioate impurities found in OP insecti-cides was reported by Imamura and Gandy.13

Animal studies have shown that the lung is involvedin phosphorothioate-induced delayed toxicity.The cause of death in phosphorothioate-treatedanimals was attributed to hypoxia or anoxiaresulting from insufficient uptake of oxygen acrossthe alveolar-capillary membrane. Neuropathicinvolvement of the diaphragm and phrenic nerve,and toxic pneumonitis with injury to the trachealepithelium were observed in rats exposed toOP.14 Morphologic changes in the tracheal epithe-lium, hyperplasia, thickening of the alveolar-capillary membrane, degeneration in alveoli, andductus alveolaris were seen on histopathologicinvestigation.

A restrictive lung dysfunction pattern on spiro-metry may occur in restrictive parenchymal lung

disease or neuromuscular disease.11 A reduction inthe amplitude of action potentials following repe-titive nerve stimulation together with a decrease inhandgrip force following low-level OP exposurewas found in a parallel study investigating theneurological effects of low-level exposure toOP.15 Just as parenchymal disease may result inlimited expansion of the thorax, decrease inrespiratory muscle power may produce similarabnormalities in lung function tests. The resultantlung function test pattern resembles restrictiveparenchymal lung disease.11 Neuromuscular diseaseaffecting the lung can be evaluated by measuringmaximal inspiratory and expiratory pressureswhich are, for the most part, unaffected byunderlying lung disease.11 This study may havebenefited from measurement of force of inspiratoryand expiratory muscle contraction. Yet, the findingthat chronic occupational exposure to OP leads tolimited expansion of the thorax and/or reducedlung compliance possibly due to thickening ofalveolar-capillary membrane leading to a restric-tive pattern on spirometry, is an important findingby itself.

Rhinorrhoea, bronchorrhoea and pulmonary oe-dema occur in high-dose OP poisoning and arebelieved to interfere with respiratory function, asmay bronchoconstriction. The primary cause ofdeath in high-dose poisoning is usually respiratoryfailure with a contributory cardiovascular compo-nent. Persistent asthma after high-dose exposure todichlorvas, an OP, has been reported.2 Pulmonaryfunction (FVC, FEV1, FEF50–75%) 20 days after high-dose exposure showed bronchial obstruction. Localtoxicity to the bronchial epithelium leading tonon-immunological asthma, as described by Brookset al.16 was speculated to be the cause.

OPs irreversibly bind to cholinesterase, causingphosphorylation and deactivation of AChE. Theclinical effects are secondary to ACh excess atcholinergic junctions (muscarinic effects), in thecentral nervous system, at the skeletal neuromus-cular junction, and at autonomic ganglia (nicotiniceffects). AChE is also found in RBCs, lung andspleen17 and the measurement of AChE activity inRBCs is used to monitor occupational exposure toOP.18 RBC AChE levels, as measured in this study, isthe best way to evaluate toxicity of OP pesti-cides.19 Analytical methods of determination ofplasma levels of OPs are difficult and expensive.20

Given the extensive use of OPs in the country, itwas not practical nor feasible to measure bloodlevels of all types of OPs used by the farmers in thecurrent study. Baseline cholinesterase levels areimportant in monitoring workers exposed to OPs21

and it is recommended that such samples should be

ARTICLE IN PRESS

Lung dysfunction in low-level OP exposure 1323

taken with the worker not been exposed to OP andcarbamate pesticides for at least 30 days.22 Higherbaseline AChE levels as seen in the farmers in thisstudy results from increased production of AChEconsequent to long-term OP exposure as suggestedby Marrs.1

WHO guidelines for interpreting RBC AChE mea-surements state that a 20–30% inhibition indicatesevidence of exposure, 30–50% inhibition indicateshazard, and 50% or greater suggests poisoning.19 Anincrease in the prevalence of toxic symptoms wasreported among Kenyan agricultural workers atcholinesterase activities considered to be non-adverse.4 The average inhibition level of AChEfollowing OP exposure in the Kenyan study was 35%.In chronic poisoning, the significance of a givenpercentage inhibition of AChE may be difficult toassess. It is believed that the variation in thedegree of inhibition required to produce symptomsis related to the rate of inhibition rather than thedegree of inhibition.23 In this study, AChE inhibitionfollowing exposure to OP was 12.8% in the farmersand 5.6% in the fishermen. Though AChE inhibitionis low in this group as compared to WHO recom-mended levels of exposure, the rate of inhibition isnot known and adverse effects may be due tohigher rates of inhibition.

The clinical picture after poisoning depends onthe rate at which the pesticide is absorbed.There is significant variability in the affinity ofvarious OPs for AChE. Lipophilicity of OPs canalter the course and severity of poisoning asthe enhanced lipid solubility of some OPspermit its storage in fat resulting in delayedtoxicity due to late release. Some OPs need to bemetabolised to a more active form to producetoxicity. The reaction between an OP and AChEresults in the formation of a transient intermediatecomplex which partially hydrolyses resultingin a stable, phosphorylated and largely non-reactive inhibited enzyme. The phosphorylatedenzyme, under normal circumstances, can bereactivated only at a very slow rate. While theactive site of the enzyme is phosphorylated, it isunavailable for hydrolysis of ACh resulting inaccumulation of ACh at receptor sites. OPs aremade up of two alkoxy groups, an oxygen (orsulphur) atom, and a leaving (or acid) groupattached to a phosphorus atom. The alkoxy group(methoxy/ ethoxy) in OPs is important in determin-ing the rate at which AChE is released and availablefor interaction with its natural substrate, ACh.Methoxy compounds reactivate AChE faster thanethoxy compounds and, therefore, poisoning withmethoxy compounds is short-lived as compared toethoxy compounds.1

Occupational exposure to OP occurs predomi-nantly via the dermal route. Though most farmersin Sri Lanka are aware of protective measures to beused when applying pesticides, there was nosignificant positive relationship between awarenessand use of protective measures.24 The hot tropicalclimatic conditions make the use of recommendedprotective clothing impractical and add to workersdiscomfort. When exposure to OP occurs by inhala-tion, absorption is rapid and complete and reachesthe systemic circulation bypassing the liver, whereit is metabolised.22 The effects of occupationalexposure to OP on lung functions could be dueeither to absorption via skin, ingestion (eating withcontaminated hands) or inhalation and the extentof the contribution of each route to the overalleffect cannot be ascertained in this study. It ispossible that other constituents in the compoundsprayed may also have direct effects on lungfunction.

Although the farmers and the controls may beexposed to agents that may affect lung function,the changes in the test values with exposure to OPpesticides are consistent. Therefore, it is highlylikely to be related to OP exposure. The smokingstatus of the participants of the study wasdocumented and corrected for in the analysis ofthe data.

From this study, it is difficult to concludewhether exposure to an OP or a particularcombination of OPs or the solvent(s) used or all ofthese resulted in lung dysfunction and this needsfurther investigation. It is unlikely that seasonalfactors other than OPs would have affected lungfunction in the farmers as the fishermen who livewithin a radius of 25 km of the farmlands, beingexposed to the same seasonal effects, were foundto be unaffected.

Although evidence of neurophysiological effectsfollowing both, occupational and environmentalexposure to OPs is reported,25 this study did notfind evidence of respiratory dysfunction followingenvironmental exposure to OPs.

The present study shows the significance ofoccupational exposure to OPs on the health offarmers and emphasises the need for implementa-tion of control measures.

Acknowledgement

This study was supported by a grant from theInternational Development Research Centre, Otta-wa, Canada, through the International WaterManagement Institute, Colombo, Sri Lanka.

ARTICLE IN PRESS

R.J. Peiris-John et al.1324

References

1. Marrs TC. Organophosphates: history, chemistry, pharmacol-ogy. In: Karalliedde L, Feldman S, Henry J, Marrs T, editors.Organophosphates and health. London: Imperial CollegePress; 2001.

2. Deschamps D, Questel F, Baud FJ, Gervais P, Dally S.Persistent asthma after acute inhalation of organophosphateinsecticide. Lancet 1994;344:1712.

3. Senanayake N, Karalliedde L. Neurotoxic effects of organo-phosphate insecticides: an intermediate syndrome. N EngJ Med 1987;316:761–3.

4. Ohayo-Mitoko GJA, Kromhout H, Simwa JM, Boleij JSM,Heederik D. Self reported symptoms and inhibition ofacetylcholinesterase activity among Kenyan agriculturalworkers. Occup Environ Med 2000;57:195–200.

5. Food and Agricultural Organisation FAOSTAT, 2000. Availablefrom URL: http://apps.fao.org/default.htm.

6. Ministry of Health. Annual Health Bulletin. Colombo: TheMinistry; 1999.

7. American College of Chest Physicians statement on spiro-metry: a report of the section on pathophysiology. Chest1983;83:547–50.

8. Udupihille M. Respiratory function tests: reference normsfor Sinhalese. Cey Med J 1995;40:53–8.

9. Kumar P, Clark M. Clinical medicine. London: WB Saunders;1999.

10. Cotes JE, editor. Assessment of bellows and mechanicalattributes of the lung. Lung function assessment andapplication in medicine. London: Blackwell Scientific Pub-lications; 1993.

11. Kelley MA. The physiological basis of pulmonary functiontesting. In: Grippi MA, editor. Pulmonary pathophysiology.Philadelphia: JB Lippincott Company; 1995.

12. Aes P, Veiteberg TA, Fonnum F. Acute and subacuteinhalation of an OP induce alteration of cholinergicmuscarinic receptors. Biochem Pharmacol 1987;36:1261–6.

13. Imamura T, Gandy J. Pulmonary toxicity of phosphorothioateimpurities found in organophosphate insecticides. Pharma-col Ther 1988;38:419–27.

14. Atis S, Comelekoglu U, Coskun B, Ozge A, Ersoz G, Talas D.Electrophysiological and histopathological evaluation ofrespiratory tract, diaphragm, and phrenic nerve afterdichlorvos inhalation in rats. Inhal Toxicol 2002;14:199–215.

15. Peiris-John RJ, Ruberu DK, Wickremasinghe AR, van-der-Hoek W. Effects of low-level exposure to organophosphateson motor nerve conduction and neuromuscular transmission.J Neurochem 2004;40:84.

16. Brooks SM, Weiss MA, Bernstein IL. Reactive airwaysdysfunction syndrome: persistent asthma after high levelirritant exposures. Chest 1985;88:376–84.

17. Swaminathan R, Widdop B. Biochemical and toxicologicalinvestigations related to organophosphorus compounds. In:Karalliedde L, Feldman S, Henry J, Marrs T, editors.Organophosphates and health. London: Imperial CollegePress; 2001.

18. Plestina R. Prevention, diagnosis and treatment of insecti-cide poisoning. Geneva: World Health Organisation; 1984WHO/VBC/84.889.

19. Lotti M. Cholinesterase inhibition: complexities in inter-pretation. Clin Chem 1995;41:1814–8.

20. Moretto A, Lotti M. Monitoring occupational exposure toorganphosphorus compounds. In: Karalliedde L, Feldman S,Henry J, Marrs T, editors. Organophosphates and health.London: Imperial College Press; 2001.

21. Jeyaratnam J, Lun KC, Phoon WO. Blood cholinesteraselevels among agricultural workers in four Asian countries.Toxicol Lett 1986;33:195–201.

22. Extension Toxicology Network, 2003. Available from URL:http://ace.orst.edu/info/extoxnet.

23. O’Malley M, McCrdy S. Sub-acute poisoning with theorganophosphate insecticide phosalone. West J Med 1990;153:619–24.

24. Sivayoganathan C, Gnanachandran S, Lewis J, Fernando M.Protective measure use and symptoms among agropesticideapplicators in Sri Lanka. Soc Sci Med 1995;40:431–6.

25. Peiris-John RJ, Ruberu DK, Wickremasinghe AR, Smit LAM,van-der-Hoek W. Effects of occupational exposure toorganophosphate pesticides on nerve and neuromuscularfunction. J Occup Environ Med 2002;44:352–7.