anticholinesterase pesticides (metabolism, neurotoxicity, and epidemiology) || epidemiological...

28EPIDEMIOLOGICAL STUDIES OFANTICHOLINESTERASE PESTICIDEPOISONING IN GREECE

M. STEFANIDOU, S. ATHANASELIS, C. SPILIOPOULOU, AND C. MARAVELIAS

Department of Forensic Medicine and Toxicology, University of Athens, School of Medicine, 75, Mikras Asias str., Athens 115.27, Greece

28.1 Introduction 403

28.2 Anticholinesterase Pesticides 404

28.3 Epidemiological Studies 404

28.4 Epidemiological Studies in Greece 40528.4.1 Acute and Occupational Poisonings 40628.4.2 Chronic Effects of Pesticides 408

28.4.3 Honeybee Poisoning in Greece 41128.4.4 Poisonings of Domestic and Domestic

Animals in Greece 411

28.5 Conclusions 412

References 413

28.1 INTRODUCTION

The use of pesticides has increased tremendously over thepast 60 years. The acceleration in demand for chemical pestcontrol in developing countries is due, in part, to the factthat pesticides in general, and herbicides in particular,are relatively new agricultural tools for most countries(Ecobichon, 2001; Sanborn et al., 2004). A contribution tomitigating the problem of the health of the world’s populationhas been made by the field of disease control, for example bycontrolling anopheline mosquitoes and malarial infectionsince 1945, as well as vectors for typhus, plague, andyellow fever, through the use of DDT, which has undoubtedlysaved millions of lives (Pope, 1998).

Pesticides have brought enormous benefits to public healthand to the world economy, but their use has also given rise tomany problems, including contamination of foodstuffs withresidues, development of pest resistance, development ofnew pests, and pest resurgence. The incidental destructionof wildlife, such as non-harmful insects, also occurs, becausepesticides exert their toxic action not only on undesired pests,

but also on non-target organisms that co-inhabit the sameenvironment (Atkins, 1992; Ecobichon, 2001).

The potential for pesticide exposure from agricultural andhome use, from food residues, water contamination, commu-nity spraying or any other pesticide, has precipitated majorscientific and political controversy over the last few decades(Blair et al., 1992). To understand this controversial issue,it is helpful to look at the history of pesticide use. BeforeWorld War II, pesticides as we know them did not exist.Organophosphate insecticides were developed in Germanyduring World War II as nerve-gas chemical warfare agents,and the phenoxy-herbicides, including 2,4-D (still widelyused in both developed and undeveloped countries), were cre-ated to eradicate the Japanese rice crop and were later used as acomponent of Agent Orange to defoliate large areas ofVietnam. Beginning after the war and continuing up to thepresent time, all these chemicals have been widely used inagriculture. In the 1960s, epidemiologists noted a rise in theincidence of non-Hodgkin’s lymphoma, an incidence thatparalleled the rise in pesticide use, prompting scientists tobelieve that there was a causal link. Rachel Carson’s

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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revolutionary book, “Silent Spring” (Carson, 2002), first pub-lished in 1962, triggered political and public awareness of thehazards posed to wildlife, humans, and the ecosystem by theuse of pesticides. This process continued after many yearswith “Our Stolen Future” (Colborn et al., 1996), describedby Al Gore as the sequel to “Silent Spring,” which documen-ted the health effects of endocrine-disrupting chemicals,among them the vast number pesticides, 1844 compoundscurrently in commercial use only in the United States, ofwhich some, in very small amounts, have been suspectedto operate through hormonal and/or genotoxic pathways(Perry, 2008; Sanborn et al., 2004).

28.2 ANTICHOLINESTERASE PESTICIDES

All the chemical insecticides in use today are neurotoxicantsand act by poisoning the nervous systems of the target organ-isms. Any insecticide that can bind or inhibit cholinesterase,making it unable to breakdown acetylcholine, is calleda “cholinesterase inhibitor,” or “anticholinesterase agent.”The two main classes of cholinesterase-inhibiting insecti-cides are the organophospates (OP) and the carbamates,which represent �90% of pesticides used today in pest man-agement (Ehrich, 1998). With restrictions having beenimposed on the use of most of the persistent organochlorinepesticides in the 1970s, the less persistent but highly effectiveanticholinesterase insecticides became the insecticides ofchoice. The problems of acute toxicity accordingly increasedas the problems of chronic toxicity decreased (Eddlestonet al., 2002; Reimer et al., 2007).

These agents inhibit a wide variety of cholinesterases,leading to acetylcholine accumulation at cholinergic receptorsites, and they are therefore potentially capable of producingeffects equivalent to excessive stimulation of cholinergicreceptors throughout the central and peripheral nervous sys-tems (Jones, 1998). In view of the widespread distributionof cholinergic neurons, it is not surprising that the anticholi-nesterase agents, especially the OPs as a group, have exten-sive applications as toxic agents, in the form of agriculturalinsecticides and potential chemical-warfare “nerve gases”(Kovacic, 2003).

There are two types of cholinesterase enzymes in thehuman organism, acetylcholinesterase (AChE) and butyryl-cholinesterase (BuChE; also known as pseudocholines-terase). As targets for OPs and carbamate insecticides,cholinesterases have been used for years as biomarkers ofexposure to anticholinesterase pesticides. They may also indi-cate the severity of a poisoning event (acute or chronic), andthey can measure specific effects at different organizationallevels, in other words as molecular, cellular, or whole organ-ism (Dunitz and Malden, 2000). An important role of cholin-esterase biomarkers can also be to provide a measure ofenvironmental health, because they give early warning of

environmental effects of OPs before adverse clinical healtheffects occur in humans and animals (Anwar, 1997; Scaps,2004; Stefanidou et al., 2003a; Timbrell, 1998).

28.3 EPIDEMIOLOGICAL STUDIES

In recent years, few environmental issues have aroused theconcern of the public as much as pesticides, especially inrelation to human health and particularly that of children.In spite of the many published studies on the subject ofpesticides, there remains deep controversy surrounding thisissue.

The acute effects of pesticides are well documented inthe literature, especially with respect to OP poisoning(Jeyararnam, 1990). However, the chronic effects of pesticideexposure continue to be strongly controversial to scientists,because these effects are much more difficult to assess.There are a number of reasons for this. Randomized controltrials, which are the most conclusive studies of cause andeffect, for ethical reasons, are not carried out with potentiallyharmful chemicals, so we have to rely on other type of studies,and these have marked limitations (Sanborn et al., 2004).

The epidemiological data referred to in the literature con-cern the incidence, prevalence, distribution, and severity ofthe effects of anticholinesterase pesticides in agriculturalpopulations and workers, and if present, their relation tomeasures of exposure, exposure burden, absorption, andexcretion. Findings from studies generally concern smallnumbers of workers involved either in the manufacture orapplication of OPs and suggest the need for systematic inves-tigation of industrial workers exposed to anticholinesterasepesticides (WHO, 1985).

Most epidemiological studies are carried out on occupa-tionally exposed groups, which are composed predominantlyof men, who have higher exposures than the general popu-lation; these studies have low participation rates and poorfollow-up. The subjects are usually not exposed to just onechemical at one time, but rather to a number of pesticides atthe same time in addition to other toxins. Exposure historyis often indirect and is very often dependent on the cropgrown, expenditure on pesticides, or job description ratherthan by direct evaluation of the exposed persons. This ismore prominent in ecological studies, which neither considerexposure at the individual level nor measure pesticideexposure directly (Sanborn et al., 2004). Furthermore, con-founding factors are often incompletely assessed, the causeof death taken from the death certificate can be incorrect orincomplete, and inert substances used in pesticides can bevery difficult to separate and evaluate. Last, but not least,there can never be a true control group for any relativestudy, because all humans will always have some degreeof background environmental pesticide exposure (Hillet al., 1995).

404 EPIDEMIOLOGICAL STUDIES OF ANTICHOLINESTERASE PESTICIDE POISONING IN GREECE

It is therefore understood that, although the toxicities ofmost pesticides have been studied extensively in animals, epi-demiological data concerning their effects in humans and inthe environment are limited. Therefore, each country hasthe obligation to immediately pursue not only the carefulevaluation of existing epidemiological studies, but also toplan well-constructed studies that carry over few if not noneof the limitations of earlier epidemiological studies.

The widespread use and easy accessibility to pesticidesresults in a huge number of intoxications, principally in agro-chemical population, where fatal poisoning is currently amajor problem in developing countries. In addition, acciden-tal exposure is the main cause of mild poisonings, and severecases are mainly due to suicidal use (Eddleston et al., 2002;Worek et al., 2005). Acute poisoning by pesticides amonghumans is usually the result of occupational exposure, mis-handling, misuse, or careless handling of such chemicals.Organophosphates account for most fatal poisonings. Never-theless, the World Health Organization estimates that 20,000women, men, and children die of accidental pesticide poison-ing each year; three million are non-fatally poisoned andnearly three-quarters of a million new people each yearexperience chronic effects from exposure (Dinham andMalik, 2003). Although acute pesticide poisoning is not amajor problem in western industrialized countries, it is animportant cause of mortality and morbidity in developingcountries. The pattern of poisoning, however, depends on avariety of factors such as the availability of different poisons,socio-economic status, and religious and cultural influences(Abdullat et al., 2006).

It is obvious that the numbers of poisonings from pesti-cides are so large that we have the obligation to immediatelypursue not only the careful evaluation of the existing epide-miological studies but also the planning of well-constructedstudies to minimize the limitations of previous epidemiologi-cal studies.

The evaluation of pesticides for carcinogenicity, whichbegan with animal bioassays in the 1960s and continued forthe next 40 years, has caused the International Agency forResearch on Cancer to consider several pesticides as probablehuman carcinogens, and has led scientists to perform severalepidemiological studies on cancer risk after exposure toinsecticides, in countries all over the world (Blair et al.,1983, 1992; IARC, 1991; Innes et al., 1969).

Countries that allow farmers to use insecticides heavilyhave reported a great risk for multiple myeloma, particularlynon-Hodgkin’s lymphoma, glioma, and pancreatic cancer(Alavanja et al., 1990; Cantor and Blair, 1984; Corraoet al., 1989; Lee et al., 2004; Waddell et al., 2001).Moreover, non-Hodgkin’s lymphoma has been associatedwith potential contact with chlordane and DDT (Perssonet al., 1989; Woods and Polissar, 1989).

Excessive lung cancer has also been reported (Blair et al.,1983; MacMahon et al., 1988), and recently this has been

associated with exposure to chlorpyrifos in the AgriculturalHealth Study (Lee et al., 2004). Contradicting observationshave been reported on chronic lower respiratory diseasesand an association between lung or brain cancer mortality(Hoppin et al., 2006). Specified and unspecified blood dis-orders, including coagulation defects and thrombocytopenia,have also been reported in the Agricultural Health Study onchlorpyrifos exposure (Lee et al., 2007). Pesticide exposurehas been associated with increased Parkinson disease andother neurodegenerative diseases (Friedrich, 2005; Kameland Hoppin, 2004).

Relative recent discoveries of the hormone-disruptingproperties of some pesticides have raised interest in how con-temporary pesticide exposure, which primarily takes the formof low level environmental or occupational exposures, candisrupt hormones and reduce the ability to successfully repro-duce (Bonde, 2002; Claman, 2004; Sharpe and Irvine, 2004).

In recently published epidemiological studies, depression,a frequent disease of modern mankind, is one of the many dis-orders that have been attributed to OP insecticide exposure(London et al., 2005). Depression among spouses in theAgricultural Health Study (Beseler et al., 2006) has alsobeen associated with a history of pesticide poisoning.Anxiety at higher rates than usual is also seen as well asdepression in farmers, when compared with other occu-pational exposed groups (Sanne et al., 2004). In addition,excess suicide mortality has been reported among farmersin some studies performed from the 1990s onwards (Boxeret al., 1995; London et al., 2005), a finding that has notbeen supported by others (Pickett et al., 1998; Speratiet al., 1999).

28.4 EPIDEMIOLOGICAL STUDIES IN GREECE

Most epidemiological studies are carried out on occupation-ally exposed groups, which are composed mostly of men,who have higher exposures than the general population.The studies have low participation rates and poor follow-up. The subjects are usually exposed to more than one pesti-cide at a time, in addition to other toxins, so assessment of theresults can be difficult or inconclusive.

A few epidemiological studies have been carried out inGreece, including in the island of Crete, where there aremany greenhouses and most of the population is occupation-ally exposed to pesticides, mainly OPs and carbamates. Thesestudies cover a broad spectrum of scientific interest and con-cern different topics. In this chapter, we will report epidemio-logical studies related to anticholinesterase pesticides carriedout in parts of Greece. More specifically, it will discuss theprevalence, distribution, and severity of the effects of anticho-linesterase pesticides in agricultural populations and workers,their relation to measures of exposure, exposure burden,absorption, and excretion. Furthermore, an attempt will be

28.4 EPIDEMIOLOGICAL STUDIES IN GREECE 405

made to explore the knowledge, attitudes, and practicesregarding the safety of pesticide handling among tobaccofarmers, greenhouse farmers, and so on in some rural areasof Greece, because Greece is an agricultural country andanticholinesterase insecticides are used extensively.

Despite the widespread use of pesticides, the number andextent of epidemiological studies in Greece related to thedirect or indirect, acute or chronic and occupational conse-quences of their use for human health are limited. Somehave addressed the following issues:

† occupational exposure of workers (farmers, applicators,packaging workers) to anticholinesterase pesticides;

† biomonitoring of Greek greenhouse workers for DNAdamage or alteration in the cellular response to DNAdamage;

† pesticide use among Greek tobacco farmers;† mammographic findings in relation to occupational

exposure to these pesticides;† pesticide levels in head hair samples of Cretan popu-

lation as an indicator of present and past exposure;† fatal poisonings in some rural regions of Greece;† the use of cholinesterases as biomarkers of exposure to

these pesticides;† the harmful effects of these pesticides against benefi-

cial insects, such as honeybees (the different aspects ofbee poisoning, such as the determination of exposure,the prevention of honey bee poisoning and theco-operation of pesticide applicators and beekeeperswill be discussed);

† fatal poisonings of animals with pesticides.

28.4.1 Acute and Occupational Poisonings

Tsoukali and Epivatianos (1982) reported 76 cases of poison-ing with pesticides (92% OP, 5% carbamates, 3% others) innorthern Greece. Most cases were suicides in agriculturalregions or cases of poisoning or homicide.

Tsoukali and colleagues (1986) reported that the percen-tage of deaths due to pesticides ranged from 51% to 71%.Organophosphates were implicated in 67% of cases and car-bamates in 37%. Most cases were suicides in agriculturalregions or cases of poisoning or homicide. Tsoukali andNjau (1987) reported an interesting case of poisoning thatconcerned a pregnant woman who committed suicide withmecarbam and the toxic agent was transferred to the fetusthrough the placenta.

According to the Archives of the Greek Poison ControlCenter, during the years of 1978 and 1979, 750 cases ofnon-fatal poisoning with pesticides were registered. Amongthem, 60% were due to OPs, 15% to carbamates, 5% to para-quat, and 20% to others. Almost all cases were due to the

misuse of pesticides, including 35 OP fatal suicidal cases.Organophosphates remain the major category implicated inacute pesticide poisonings, even today, when the totalnumber of OPs in use has largely decreased, most of themhaving been banned in Greece.

Vlachos and colleagues (1982) reported 1085 cases ofnon-fatal poisonings with pesticides and home insecticides.These poisonings took place in the period 1980–1982.Among these, 430 cases were caused by rodenticides.When analysed, 76% of pesticide and insecticide cases con-cerned adults, but 80% of rodenticide cases concerned chil-dren who were exposed due to negligence or poor storageconditions.

During the period 1985–1990, according to the Children’sHospital Archives in northern Greece, poisonings of chil-dren were linked to only 3.5% of children admitted to theemergency room. Among these poisonings, 7.5% were dueto pesticides. These poisonings were the result of unsafestorage and inadequate supervision of children up to 2.5 yearsof age.

Tsoukali (1990) reported that the first cause of death innorthern Greece due to poisoning was a result of the use ofpesticides. Pesticide poisonings occur primarily as a resultof suicide attempts, accidents, occupational exposure, orhomicides. According to this study, the number of recordeddeaths due to pesticides showed a decline during the period1980–1990. Tsoukali and colleagues (1991) have alsoreported a case of the fatal suicide of a psychiatric patientwith methamidophos.

Vassiliades and colleagues (1995) reported a case of fatalsuicide of a psychiatric woman farmer, 56 years of age, withcarbofuran in northern Greece. Her death from anoxia wasthe result of respiratory paralysis due to cholinesteraseinhibition.

Tsatsakis and colleagues (1996a) have reported that,over the last two decades, the use of OP insecticides in theisland of Crete, an agricultural island with many greenhouses,is widespread and intensive. In this period, a relatively smallnumber of acute poisonings have been reported; nine cases ofpoisoning with OPs (mostly suicides), of which six caseswere fatal. Among the nine poisoned persons, six were treatedin the intensive care unit, and three were found dead byrelatives. Two patients treated in the ICU recovered fullyafter 15 and 24 days, respectively, but the third survivordeveloped delayed neuropathy. OP blood levels weredetermined on admission and during therapy, and in onecase atropine and pralidoxime levels were also measured.Significant fluctuations of plasma cholinesterase activitywere observed during therapy. Postmortem analysis revealedhigher levels of pesticides in the organs (e.g., 23.1 mgfenthion/g kidney) and in fat (135.2 mg fenthion/g) thanin blood (e.g., 4.8 mg fenthion/mL) and vitreous humor.Considerable levels of pesticide were measured in the testis(e.g., 5.8 mg fenthion/g, 0.8 mg methidathion/g) and uterus


(170.5 mg malathion/g). Extracorporeal decontamination toenhance pesticide elimination was a therapeutic challenge.

Tsatsakis and colleagues (1996b) investigated fatal carba-mate poisonings in the island of Crete in southern Greece overa period of two years (1991–1993). This investigationrevealed that the studied five lethal cases concerned the inges-tion of methomyl (Lannate), a cholinesterase-inhibiting car-bamate insecticide. In this investigation, analysis of plasmaand serum samples showed more than 90% inhibition of cho-linesterase. Blood methomyl concentrations had a meanvalue of 26.7 mg/L and a range of 5.6–57.0 mg/L. Thesevalues are much higher than those previously reported insimilar cases (0.57–1.4 mg/L). Methomyl concentrationsin organs and tissues were found to be significantly lowerthan those in blood and vitreous humor.

Ntzaou and colleagues (1996) reported the death of a manwho committed suicide with carbofuran because he fearedthat he had been infected with the HIV virus. This suicideoccurred before the confirmation of the infection by bloodanalysis.

Tsoukali and Tsoungas (1996) reported the total numberof fatal poisonings that took place in northern Greeceduring the period 1990–1995. According to this record,there was a decrease in the number of pesticide poisonings,but the total number of poisonings per year did not varysignificantly. Tsatsakis and colleagues (1998) reported twonon-fatal cases of poisoning in Crete, one with omethoate(accident) and the second with fenthion (suicide attempt).The two victims received appropriate treatment for poisoningand supportive care, and survived. Blood levels weredetermined on admission (fenthion 2.9 mg/mL, omethoate1.6 mg/mL) and during hospitalization and proved to bequite high. The patient with fenthion poisoning developedcholinergic crisis 6 hours after admission and was intubatedfor 24 days, with concomitant complications. The patientwith omethoate poisoning remained clinically well and wasdischarged 3 days later.

In a study performed by Christakis-Hampsas and col-leagues (1998) concerning 761 fatal poisonings in Crete, 37cases were due to pesticides (21 cases with OP and 16 caseswith paraquat) during the period 1991–1996. Of the 37deaths, 12 cases were registered as accidents or occupationalexposure, principally via the respiratory system. Occupationalpoisonings are common but not normally severe.

According to Vlachos (1998) 1295 poisonings with pesti-cides were recorded during 1997. Of these, 608 cases (47%)were accidents, 524 cases (40.5%) were a result of occu-pational exposure, and 163 cases (12.5%) were suicideattempts. Most poisonings occurred transdermally duringoccupational work. The main pesticides responsible wereOPs and the herbicide paraquat. Occupational poisoningswere not severe.

Tsoukali-Papadopoulou (2000) recorded poisoning casesin northern Greece between 1994 and 2000. The results of

this survey showed that the percentage of poisonings frompesticides decreased significantly from 1994 to 2000.Nevertheless, the number of fatal poisonings due to othercauses has not decreased and shows a significant increaseduring 1996 and 1997. The major pesticides responsible forfatal poisonings are the OPs (OP, 67% of poisonings; carba-mates, 27%; others, 6%).

Tzola and colleagues (2000) reported 131 cases of poison-ing from pesticides in Greece. Of these, 104 cases were due tosuicide attempts, eight were a result of occupational exposure,and one was due to homicide. Of the 104 cases, 49 werecaused by anticholinesterase pesticides.

A study was carried out in northern Greece related to thesuicidal poisoning of 273 individuals admitted to the acutemedical service ward in a Greek hospital from January1998 to December 2000. The admitted patients represented3.8% of overall admissions. Pesticides were implicated in7.7% of the total and were more frequently used by patientsfrom rural areas. The following conclusions could be drawn:

† The agents ingested are similar to those in othercountries of northern Europe.

† Psychiatric diagnoses are more common in the studiedgroup than in those reported from northern Europe(Hatzitolios et al., 2001).

Tsatsakis and colleagues (2001) reported an acute fatalpoisoning by methomyl caused by inhalation and transdermalabsorption and in 2002 reported two cases of severe fenthionintoxication (Tsatsakis et al., 2002). The first of these lattercases involved a psychiatric patient who attempted tocommit suicide by ingestion of fenthion, and the secondcase was a child exposed by air spraying. Both patientswere treated in the intensive care unit with atropine andpralidoxime and finally survived. Fenthion blood levels onadmission were 2.7 and 0.95 mg/mL, respectively. Differentconcentrations of pralidoxime were added to the first patient’spoisoned serum in order to assess in vitro the effect of prali-doxime on cholinesterase reactivation.

The Greek Poison Control Center has recorded cases ofnon-fatal poisonings from pesticides in the period 1988–2003. Of the poisoning cases, 40% were due to anticholines-terase pesticides. Acute poisonings were the most commontypes of poisoning in Greece each year. According to theArchives of the Greek Poison Control Center, for 1997,12% of poisoning cases concerned suicide attempts, 47%were a result of accidents, and 41% were related to occu-pational exposure. The state of poisoning due to pesticidesis usually severe and has a high mortality rate. The principalpesticides responsible for these poisonings and which areusually lethal are the anticholinesterase pesticides and theherbicide paraquat. The Poison Control Center has concludedthat poisonings from pesticides are usually severe and occur


due to ignorance or underestimation of the danger from theiruse. The recorded non-fatal poisonings caused by pesticidesthat are registered with the Greek Poison Control Center arepresented in Table 28.1.

Bertsias and colleagues (2004) reviewed pesticide poison-ings registered at the Center of Toxicology and ForensicSciences Research at the University of Crete between 1991and 2001. Eleven poisonings were caused by paraquat and13 by OP pesticides. Pesticide blood levels upon admissionranged from 1 to 108 pg/mL, and there were six fatalities.Carbamate poisonings were caused by methomyl (initialblood levels, 1.6–57 mg/L) and resulted in death.

Vougiouklakis and colleagues (2006) investigated the for-ensic records and toxicological data of all autopsies per-formed over the period 1998–2004 in the deprived regionof Epirus in northwestern Greece. This investigation revealedthat 46 cases (2.9%) of the 1582 autopsies performed wereattributable to acute fatal poisoning. The age range was16–94 years. Pesticides were implicated in nine cases(19.6%). Fourteen of the 46 fatal poisonings were suicides(30.4%) and pesticides were the most common poisonsused for suicide purposes (64.3% of suicides). Fatal poison-ings from pesticides were similar for both genders and wererecorded for the age groups 31–50 years and 61–80 years.Organophosphate pesticides and carbamates were the mainpesticides encountered, in agreement with other studiesfrom Mediterranean countries (Teixeira et al., 2004).

The results of the above studies highlight the toxic andpotentially lethal effects of pesticides, as well as the hand-ling and use of pesticide formulations applied in Greekagriculture.

28.4.2 Chronic Effects of Pesticides

In 1988, the effects of pesticides on the health of familiesliving in the village of Tympaki in the island of Crete werestudied, because the inhabitants of this village are occupiedwith intense agriculture practices, and particularly with theuse of greenhouses (Kafatos et al., 1989). A total of 50families were randomly chosen. The members of thesefamilies worked exclusively in the greenhouses, where theysprayed the plants for 2–3 hours weekly for 9 monthsannually, with mixtures of pesticides. The concentration ofthe sprayed pesticides was two to three times higher thanthe recommended, without any use of protective measures.The sprayers changed clothes 1–2 hours after spraying,

mixed the pesticide with their bare hands, were eating withoutwashing the hands, and only 25% of the sprayers had read theinstructions manual for the pesticides. The results of thisstudy showed that there was an increase in the frequency ofdeaths due to malignant neoplasms. The children had agreater frequency of goiter and liver enlargement in compari-son to the control group. Serum transaminases were signifi-cantly higher in men, women, and children. The levels ofred blood cell cholinesterase were significantly lower inmen, women, and children over 10 years of age. The cogni-tive function indices of the children (WISC-R, Stanford-Binet, Bender-Gestalt) were significantly lower in Tympaki,and these results may be correlated with the chronic expo-sure to pesticides and the low levels of red blood cellcholinesterase.

This study showed that the conditions of pesticide appli-cation and the personal protective measures were at a basiclevel. The information given to the farmers for the formu-lations and use of the sprayed solutions was inadequate.The duration of exposure to combinations of pesticides waslong and the farmers complained about the lack of infor-mation concerning the safe use of pesticides in closedareas, such as greenhouses, and the lack of organized effortsto provide elementary measures of protection.

This survey showed certain effects of pesticides on thecholinesterase levels of red blood cells in the exposedpopulation of Tympaki. These data are well established asmarker of overexposure to anticholinesterase pesticides.Furthermore, this survey showed a clear difference in the cog-nitive function among the two compared groups as well as inthe effects on the central nervous system (CNS) and periph-eral nervous system (PNS). According to the scientists,these differences are due to the high exposure of the popu-lation to pesticides. An increase was also observed in the fre-quency of goiter and liver enlargement in the children ofTympaki. Nonetheless, a greater population sample shouldbe examined to come to more valid conclusions.

Linou and colleagues (1988) studied the mortality fromleukemia and lymphoma in the Greek population in the1990s. There are indications of an increased frequency ofleukemia and lymphoma in the agricultural population.This finding is not completely valid, because the data areinsufficient for a thorough assessment. According to theinternational literature, there is an increased risk for themanifestation of leukemia in children whose mothers havebeen exposed to pesticides (Kiamouris and Linou, 1989).

TABLE 28.1 Non-Fatal Pesticide Poisonings in Greece from 1988–2003

Year 1988 1989 1990 1991 1992 1993 1995 1996 1997 1998 1999 2000 2002 2003

OP poisonings 496 555 620 611 537 398 539 529 475 479 556 511 343 305Carbamate

poisonings29 39 61 116 60 74 88 95 62 156 142 91 81 98


Another survey found an increased frequency of chromo-some aberrations in the peripheral lymphocytes of 29 sprayerswho were exposed to mixtures of pesticides, when comparedwith the non-exposed population. Of these, most sprayers hadworked with cucumber and tomato cultures for a period of 4years (Kourakis et al., 1992). The conclusion of this studywas that the workers must take more protective measuresand that health inspectors must monitor the workers’ health.

Kourakis and colleagues (1996) studied the frequency ofchromosomal aberrations (CA) and sister chromatid exchange(SCE) in peripheral lymphocytes. The results showed anincrease of 2.6% in CA in the applicators, in comparisonwith 0.53% in the control group (the applicators did not useprotective clothes and pesticides were mixed with barehands). An increase in CA was also observed in applicatorsworking in the greenhouses when compared with workersin the open air (CA of 3.37% and 1.88%, respectively). A cor-relation of CA increase with smoking was not observed. Noinduction of SCE was observed.

Stefanidou and Pappas (1996) evaluated the exposure ofGreek consumers to pesticide residues through consumedfood and pharmaceutical products. It was estimated that con-sumers are not exposed or in danger from agricultural pro-ducts at the levels of pesticide residues found. Stephanouand Zourari (1998) carried out a study to determine thelevel of pesticides in greenhouse atmospheres and ondermal patches of sprayers. The following conclusions weredrawn:

† Exposure through the skin is higher than exposurethrough inhalation.

† The concentration of volatile particles is higher ingreenhouses than that of non-volatile particles.

† The application of pesticides is more dangerous forsprayers than for other farmers.

It was deemed necessary that pesticide levels in the urineof the sprayers should be monitored.

Tselas and colleagues (1998) carried out a study inIerapetra, Crete, where 25% of the greenhouses in Greeceare located; almost 8000 farmers are working there for anaverage of 100 hours a year. This study was carried out inthese farmers with the following aims:

† To confirm indications of the toxic effects of pesticideson the farmers’ health.

† The establishment of a program of continuous monitor-ing of the farmers.

† The enactment of rules for safer work in thegreenhouses.

In this study, carried out over one year, 450 farmers filledin a specific questionnaire, and the results were compared

with a control group of 150 non-exposed persons. The partici-pants in the study were subjected to clinical and biochemicalexamination and the results correlated with an exposureindex. The following conclusions were drawn:

† Long-term exposure to pesticides in greenhouses withno personal protective measures affects human health,especially liver function.

† The first manifestations are histological alterations anddisturbances of liver function.

† Long-term exposure increases the possibility of livermalignancy in experimental animals. In this study, noliver neoplasm was observed.

† The identification of a specific pesticide was not poss-ible, because the farmers use more than 40 differentpesticides in the greenhouses annually.

The following actions were deemed necessary:

† The development of biological agriculture.† The development of technology for the automatic appli-

cation of pesticides.† The application of a medical monitoring program for

the farmers.† Improvement in the information and education of the

farmers and the availability of protective measures.† Prohibition of the use of pesticides with high toxicity

and resistance.† Continuous recording of the health of farmers.† Continuous determination of blood cholinesterase

levels.† Determination of re-entry time into the greenhouses.

Machera and colleagues (2000) and Makropoulos andMachera (2000) investigated the exposure of farmers to pes-ticides and developed a methodology for the assessment ofthe adverse effects of pesticides on farmers’ health due tothe combined use of mixtures of pesticides This study is inprogress and estimates the synergistic, mutagenetic, immuno-depressant, and carcinogenic actions of the combinations ofpesticides used by the farmers, aiming to assess their conse-quences in public health.

Dolapsakis and colleagues (2001) carried out a mammo-graphic screening program in Crete, Greece. This screeningwas part of a study to test whether occupational exposure topesticides in greenhouses, which is a factor in the occupationsof many of the habitants, may increase the risk of malignantor premalignant findings in mammographic examinations.The pesticides mainly used in greenhouses in Crete are OPsand carbamates. A total of 1062 women (aged 40–75years) were examined between 1988 and 1993 and followedup until 1998. Of these, 522 women had been working for


at least 10 years in the greenhouses for more than 4 hoursdaily (exposed women), and 540 women had never workedin agriculture (non-exposed). Exposed women had a signifi-cantly ( p , 0.05) higher risk than non-exposed women forfibroadenoma, ductal hyperplasia, sclerotic adenosis, fibrohy-perplastic disease, cystic disease, and inflammatory mastitis.Younger women (aged 40–49 years) in the “exposed group”had a higher detection rate of malignant tumors when com-pared with older women (50–75 years). These results indi-cate that “exposed” women may have higher risks ofincidence for a number of lesions that are risk markers forsubsequent invasive breast cancer.

The European Union, in order to develop a model to deter-mine exposure of users to pesticides, funded a research pro-ject for the “Assessment of Risk of Exposure to PlantProtection Products by Operators, Bystanders and Workers,and the Environment”, in which Greece was an active partici-pant. Within the framework of this project, 25 applications ofpesticides to greenhouses, vineyards, and olive trees werestudied by using color tracers. Twenty more applicationswere studied for similar crops for the evaluation of exposureof the users. In five more studies, drift of the applied pesticidewas estimated. The efficacy of the protective clothing of theusers was also assessed (Goumenou and Machera, 2001;Machera et al., 2002, 2003).

Stefanidou and colleagues (2003b) presented a study con-cerning the determination of levels of plasma BuChE in 28workers from a factory packaging ortho-thio-phosphate,dimethoate, with an open-type packaging machine (in liq-uid form), before and after exposure to this agent. Theworkers did not wear protective clothing, masks, or anyequipment, although recommendations from supervisorswere continuously given. In the factory there was only anold, malfunctioning exhaust ventilation system. The resultsof this study showed a post-exposure mean plasma cholin-esterase inhibition of 37%, a decrease that was statisticallysignificant when compared to the pre-exposure averagelevel. None of the workers complained of any symptoms,probably due to their non-constant exposure to the anticholi-nesterase agent.

This survey suggests the necessity for determining plasmaBuChE levels in all people working with OPs. According toscientific data, the determination of red blood cell AChE is asafer marker for estimating the degree of exposure to pesti-cides, because the BuChE has a wide range of values andshows high fluctuations in the same individual. In mostEuropean countries the determination of red blood cellAChE is routine but in Greece, however, workers are notmonitored on a routine basis.

Piperakis and colleagues (2003) evaluated the exposure ofGreek greenhouse workers to different pesticides. Morespecifically, they evaluated whether occupational exposureof workers leads to an increase in DNA damage in human per-ipheral blood lymphocytes. The results of this study proved

that exposure to pesticides caused no detectable increase inDNA damage or alteration in the cellular response to DNA.

Tsatsakis and Tutudaki (2004) reviewed the work that hadbeen done in the field of pesticide and persistent organic pol-lutants hair analysis over the previous 15 years. According tothis study, the levels of diazinon and methomyl in hair werebetween 110 and 520 ng/g, and 900 and 1800 ng/g, respect-ively. These results strongly support the possibility of usinghair as a suitable indicator for the assessment of long-termexposure to pesticides.

Damalas and colleagues (2006) made an attempt toexplore knowledge, attitudes, and practices regarding safetyissues in pesticide handling among tobacco farmers of therural area of Pieria in northern Greece. The majority of thefarmers (96%) who participated in this survey by completinga questionnaire viewed pesticides as a guarantee of hightobacco yields and high product quality. Nevertheless,almost all farmers (99%) believed that pesticides haveadverse health effects. Skin contact was recognized as themost common route of exposure during pesticide use(58%). A total of 46% of tobacco farmers reported they didnot use any protective equipment when spraying with pesti-cides. Of those farmers who used protective equipment,47% admitted the use of a hat and 63% the use of boots.Only 3% used a face mask, 8% gloves and 7% a coverallon a regular basis. The reasons given for not using protectiveequipment were as follows:

† It is uncomfortable (68%).† It is too expensive (17%).† It is time-consuming to use (8%).† It is not available when needed (6%).† It is not necessary in each case (2%).

A large majority of the farmers (84%) reported that theyreplace work clothing after each use and a considerable per-centage (48%) reported that they wash clothing after severaluses.

The conclusions reached from this study are as follows.Farmers’ knowledge about potential hazards of pesticideuse is high, but the reported use of safety measures is poor.Continuous emphasis on basic safety precautions is requiredwhen using pesticides, stressing also the importance of pro-tective equipment. The habits of farmers that may be lifethreatening must be addressed.

Chatzi and colleagues (2007) performed a study in 2006 inthe island of Crete, Greece, which revealed an association ofallergic rhinitis with pesticide use among grape farmers.Grape farmers who used pesticides had higher prevalencerates of allergic rhinitis symptoms compared to grape farmerswho reported no current use of pesticides and control sub-jects. The highest risk was observed with dithiocarbamatefungicides and carbamate insecticides. Grape farmers


exposed to multiple pesticide use were more vulnerable toallergic rhinitis. The conclusion of this study was that occu-pational exposure to multiple agricultural chemicals couldbe related to allergic rhinitis in grape farmers.

An assessment of different working populations in Crete,Greece, and common currently used organophosphate pesti-cides was performed by Tsatsakis and colleagues (2008).Pesticide levels were measured in head hair samples of theCretan population. The pesticides examined were OPs (diazi-non, fenthion, methylparathion, and malathion) as well asexachlorocyclohexane and DDT. The study population (211individuals, 110 females, 101 males) was divided into threegroups: greenhouse workers, animal breeders, and farmersworking in open cultivations. Ten head hair samples were pos-itive for diazinon with a mean concentration of 2.8 pg/mg.Fenthion, methylparathion, and malathion were not detected.The results of this study demonstrated the ability to assesschronic human past pesticide exposure from hair, offeringvaluable information to epidemiological clinical studies.

28.4.3 Honeybee Poisoning in Greece

The progress in agricultural technology from the use ofmechanized operations after World War II, and the develop-ment of agricultural aviation for the treatment of larger areasof crops, has led to the increased use of chemicals for pestcontrol in ways that can cause accidental and/or intentionalpoisoning of wildlife, domestic stock, and humans. It isimportant to state that, despite modern-day development ofsecond- and third-generation derivatives of the early chemicalpesticides, all pesticides and especially the insecticides pos-sess an inherent degree of toxicity to a wide range of organ-isms. The use of insecticides has been one of the major causesof serious ecological perturbations that have characterizedagricultural areas in large parts of Greece.

Insecticides, particularly of the anticholinesterase type,have been used to control insects for many years. From the1960s onwards the use of pesticides has increased, leadingto a decrease in the number of bees and other pollinatinginsects. As apiculture is a traditional profession from theHomeric era in Greece, comprising an important source ofGreek income, bee losses have a devastating impact on thebeekeeper, who may have to relocate damaged hives or per-haps even be forced out of business. Growers of mostinsect-pollinated crops experience lower crop yields, and ulti-mately food prices increase. Another problem is the socialand economic pressure for both crop farmers and beekeepersto produce more food on fewer acres, because much fertileland has been occupied by shopping centers, housing pro-jects, highways, and other projects derived from the modern-ization of people’s life. Consequently, many beekeepers havefound themselves in areas of intensified agriculture wherepesticide exposure is greatest. If insecticide applicationbecomes too intense, the beekeeper is again excluded from

large acreages, so honey yields decrease. Insecticides thatare highly toxic to honey bees cannot be applied to bloomingcrops when bees are present without causing serious injury.Indeed, the great majority of bee deaths are due to poisoningby insecticides applied to crops while the crop is in flower(Atkins, 1992). Among these insecticides, anticholinesteraseagents are the most common, and of these the OPs are themost frequently responsible for bee death (Oomen, 2000).

As the detection of anticholinesterase insecticides in beesis difficult due to their rapid hydrolysis, the inhibition ofAChE and/or BuChE activity is a valid and sensitive bio-chemical indicator of bee exposure to anticholinesteraseinsecticides, in the same way that it is useful for the determi-nation of a relative exposure in humans. In our laboratory wehave developed two easy and sensitive methods for the deter-mination of AChE and BuChE, respectively, in honeybeeheads for the assessment of their exposure to anticholinester-ase insecticides (Stefanidou et al., 1996, 1998).

In Greece, the past application of anticholinesterase pesti-cides by aircraft for the control of olive trees dacus has causedserious ecological perturbations, including the death of hon-eybees in huge quantities each year. However, after 1990,pesticide application by aircraft was banned, which signifi-cantly reduced bee colony losses. Nevertheless, honeybeepoisonings still occur, sometimes in large quantities, due toimproper application of pesticides by Greek farmers.

28.4.4 Poisonings of Domestic and DomesticAnimals in Greece

Lethal poisonings of domestic and domestic animals are quitecommon in Greece. These poisonings are intentional or acci-dental and may be due to the excessive use of pesticides. Themost frequently responsible pesticide agents for these poison-ings are the many derivatives of carbamate and OP esters,chlorinated pesticides, strychnine, cyanides, fluoroacetamide,coumarins, and so on. The results of toxicological analyses of2421 cases of animal lethal poisonings and evidence, such aspolluted foodstuffs, baits, and water, investigated in thedepartment of Forensic Medicine and Toxicology of theUniversity of Athens, Greece, during the period 1986–1994, revealed the following results (Pappas et al., 1996).Of the 1936 animal poisonings, 25% were fatal, and ofthese, 10% were caused by OPs, 85% by carbamates, andonly 5% by other poisons. In this study the authors refer tothe complete toxicological analysis of poisoned animals,such as biological material, foodstuffs, water, and any otherevidence that was sent to the Department of ForensicMedicine and Toxicology and to the Ministry of Agriculture.

Antoniou and colleagues (1997) recorded fatal animalpoisonings in northern Greece from 1990 to 1995. A totalof 926 specimens were analyzed by chromatographic tech-niques. Of these, 78% were caused by pesticides (mainly car-bamates plus OPs) and 22% by other poisons. The animals


affected were predominantly cats, dogs, sheep, birds, andhoneybees.

28.5 CONCLUSIONS

Pesticides will continue to constitute the basic tools in pestmanagement because there are many pest situations forwhich there are no known alternative management methods.Pesticides have considerably increased food production andhave improved human health, but critically they must beused at the right place and at the right time, and in the contextof carefully established programs that include a variety ofother techniques to tip the balance against the pest.Therefore, attempts should be made not only toward the limit-ation of pesticide use, but also to look into the future invest-ment in and use of natural enemies of undesirable insects,which have great potential ecological importance andwhich do not harm beneficial insects.

The increase in pesticide use as a major tool for increasedfood production must, and undoubtedly will be closely mon-itored, due to the fact that individual pesticides, or groups ofpesticides, may have effects not yet known, a fact that shouldcontinue to demand consideration by toxicologists andgovernments.

The evaluation of the epidemiological studies showed thefollowing. Multiple pesticides are used in greenhouses inGreece. The main agents in pesticide deaths in Greece wereOPs, due to the fact that most cases are suicide cases,where the individuals have decided to die with a definitelytoxic agent. The second responsible agents in pesticidedeaths are carbamates. However, recorded deaths due to pes-ticides have shown a decline in number during the last 20years in Greece.

Although OPs have been involved in non-fatal pesticidepoisonings in Greece over the last two decades in Greece toa greater degree than carbamates, the actual number ofcases shows a decrease in numbers for OPs and an increasefor carbamates. This may be due to the fact that OP avail-ability and use has been limited in the last 10 years in Greece.

Pesticide applications by aircraft are more hazardous tobees in flight than applications by ground equipment, becausethe chemical compounds are spread over larger areas.However, if pesticide applications by aircraft are carried outat night, when bees are not foraging, bee colony losses aresignificantly reduced (Atkins, 1992).

Organophosphate and carbamate pesticides were the mainpesticides encountered in poisonings and deaths in Greece, inagreement with studies from other Mediterranean countries.The most common poisons used for suicide in Greece are pes-ticides. Deliberate self poisoning with pesticides represents asubstantial risk factor for poisoning deaths in Greece. Mostoccupational pesticide poisonings in Greece occur due to

negligence or underestimation of the dangers involved intheir use.

The measures suggested for the prevention and manage-ment of poisonings include the application of the followingparameters (Teixeira et al., 2004):

† Governmental registration of personal protectivemeasures used by farmers in Greece during agriculturalprocesses, assessed by unannounced inspections.

† Experimentation and selection of the most suitable per-sonal protective measures for the Greek conditions,taking into account several parameters including thewarm climate, aiming to assess the effectiveness ofthese measures with the use of exposure levels of theapplicators.

† Determination of pesticides levels in farmers exposedsystematically to pesticides.

† Education of farmers and agriculturists with specialseminars, including those appropriate for the lowersocioeconomic group of farmers, regarding the necessityof using protective measures as well as selecting the mostsuitable and safe pesticide formulations in Greece.

† Increasing public education programs regarding thehigh toxicity of pesticides.

† Establishment of measures and control processes forfarmers, aiming to monitor adherence to safety rules,personal hygiene and protection measures during theuse of pesticides.

† Assurance about the safety of personnel who haveintense and prolonged exposure to anticholinesterasepesticides (pilots and greenhouse workers).

† Avoidance of exposure for more than 8 hours per day ina closed area where an insecticide vaporizer is beingoperated.

† In occupational exposure, early diagnosis of BuChEdepression (.25–30%) and the early removal ofworkers from their posts, until the regeneration ofBuChE, which may prevent manifestation of acutepoisoning.

† Changing the use of pesticides in greenhouses to auto-spray systems.

† Measurement of pesticide levels in greenhouse air anddeterminating the period of time elapsed before re-entry into the greenhouse.

† Monitoring high risk populations (sprayers, applicators)for chromosomal aberrations.

† Increasing awareness among the public regarding thefirst aid management of intoxication.

† Health services, particularly in the countryside, shouldemphasize in the training of medical and nursing staffthe prompt identification and treatment of pesticide


intoxication by ensuring the availability of antidotes inmost hospitals.

† Future pesticides must be more selective and their fate inthe environment more predictable, or the ecologicalconsequences of their use may dictate acceptance ofgreater crop loss, aiming to minimize problems relatingto their injurious effects upon man and their effects onnon-target species in the environment.

† Beekeepers must maintain positive working relation-ships with farmers and applicators of pesticides.

† Applicators must pay attention to the following:W examining fields before sprayingW determining if bees are foragingW choosing short residual productsW contacting local beekeepers.

† Changing the politics and ethics that govern farmingtoday.

† Restricting the sale of pesticides, according tolegislation.

In the present study, which is the first study of specificgroups of the Greek population, there are some aspects thatenable trends to be suggested, but there is nothing else avail-able with which to compare them. Deliberate self-poisoningrepresents a substantial risk factor for poisoning deaths andshould be monitored continuously at the regional as well asthe national level. Accurate and clearly defined data on fatalpoisonings are valuable for planning, evaluating, and fundingnational policies to reduce poisoning deaths.

Although epidemiological studies have limitations, they docontribute and always will provide extremely valuable infor-mation on the long-term effects of pesticides. Well-plannedAgricultural Health Studies must continue at a more intenserate in order to further clarify health problems that havearisen from the long and often non-controlled use of pesticides

It is ironic that although Greek farmers know the potentialhazards that pesticide use can exert on humans and theenvironment, the personal safety measures they reporttaking are poor. This unfortunate fact forces all regulatorsto (1) strongly emphasize the basic safety precautionsrequired, (2) enforce strict inspections during and after pesti-cide use, (3) help Greek farmers, through surveillance, to era-dicate their bad habits concerning not only their health, butalso public health and that of the total environment, becauseit is well known by all scientists today that, as Aldo Leopoldsaid 58 years ago, “conservation is a state of harmonybetween man and land.”

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