Pesticide Exposure and Stunting as Independent Predictors of Neurobehavioral Deficits in Ecuadorian School Children

METHODS

The town of Tabacundo has 2 public elementary schools and a medical care center that belongs to the Ministry of Public Health. Eligible subjects included all of the children in the 2 lowest grades (called second and third) of 1 of these 2 schools. Through the school headmaster, an invitation to participate was extended to the parents, with a written description of the study. One of the teachers served as contact person. An informational meeting was held at the school on one evening, where the mothers signed the informed consent form, with the teacher signing on behalf of 2 illiterate mothers.

Examinations were scheduled at the nearby medical care center during normal school hours or in the late afternoon. The aim was to complete examinations of each child within 2 hours. The results of the examination were reported to the mother after completion of the clinical tests. The protocol was developed in accordance with the Helsinki convention and was approved by the Comité de Bioética of the Centro de Biomedicina, Universidad Central del Ecuador.

Skilled personnel interviewed the mothers and filled-in detailed questionnaires with ∼150 items about the mother's own exposure history, demographic parameters, and the child's past medical history and current health. Each interview took ∼1 hour. The questionnaires were then reviewed by a specialist in public health and occupational medicine, and a score was developed in regard to the standard of family housing (traditional/contemporary, running water, and sewage drainage), nutrition (2 or 3 meals per day and extent of animal protein in the diet), and potential maternal pesticide exposure during pregnancy. These categories were determined without knowledge about the child's clinical results.

RESULTS

All 79 eligible children participated in the examinations. However, 1 child had an acute upper respiratory infection requiring antibiotic treatment. Three additional children had clinical abnormalities on the physical examination, and 3 children were older than a limit of 9 years of age considered appropriate for the school grade. These 7 children were excluded from the data analysis. The total number of children included in the data analysis was, therefore, 34 boys and 38 girls. Relevant demographic information is given in Table 1. After these exclusions, a total of 37 children were found to have prenatal pesticide exposure from their mother's work during pregnancy, whereas 35 were free from such exposure. None of the mothers had worked as a pesticide applicator, and all of them reported that they had followed normal safety precautions. None of them had ever been poisoned by pesticides, and relevant symptoms recalled were weak, nonspecific, and without clear association to occupational exposures. In regard to paternal employment, 19 of the exposed children's fathers had worked in floriculture or agriculture during the pregnancy, and the same was true for 16 fathers of the children not prenatally exposed from their mother's work. Paternal work was otherwise mainly in construction trades. The 2 groups of children were remarkably similar. The only major difference was that almost all of the exposed children had access to running water at home. In addition, although not statistically significant, more often the exposed mothers had attended secondary school, and they also had fewer children (Table 1).

The pesticide metabolite measurements indicated that the control children had slightly higher current exposure levels than the children with prenatal pesticide exposure (Table 2). A total of 21 children exceeded a summary RfD of 1 nmol/kg per day for the composite measures of dimethylphosphate or diethylphosphates metabolites; 12 of these children were controls, and 9 were prenatally exposed. The erythrocyte acetylcholinesterase activities were within expected ranges and similar in children exposed below and above the RfD level; urinary-excretion rates for individual metabolites or the 2 composite measures did not correlate with the enzyme activity.

In regard to anthropometric measures, exposed and unexposed children were quite similar (Table 3). The exposed children were slightly shorter, but their BMI was slightly higher. These tendencies were not statistically significant. Apart from 1 control child with a hematocrit of 32 in the absence of clinical anemia, all of the children had hematocrit values of ≥37 and were, therefore, considered normal. None of these parameters was related to current pesticide exposure.

The average systolic blood pressure was higher in the exposed children than in those not exposed (Table 4). An approximate 95th percentile of 113 mm Hg³⁶ was exceeded by 9 children, 7 of whom had prenatal pesticide exposure (P = .09). Diastolic blood pressure showed less clear association with prenatal pesticide exposure (Table 4), and 4 of 10 children with a diastolic blood pressure above a 95th percentile of 76 mm Hg³⁶ belonged to the exposed group (P = .44). Blood pressure was not significantly related to any of the anthropometric parameters or to current pesticide exposure.

Three children were left-handed, 2 of whom belonged to the exposed group. The neurologic examination showed normal sensitivity and normal Romberg test, but some abnormalities were observed, particularly in tendon reflexes, finger opposition, and standing on one leg for 10 seconds. At least 1 abnormality was present in 9 children from the control group and 14 from the exposed group (P = .31). The computerized posturography showed no difference between the 2 groups of children with open or closed eyes (Table 4), in agreement with the clinical Romberg test.

On the neuropsychological tests (Table 4), no difference was seen on simple reaction time, the Santa Ana form board score, and Digit Span. However, both copying test scores showed a statistically significant deficit in the exposed children compared with the controls. Similar bivariate analyses showed that stunting was also significantly associated with the copying scores but not with any of the other neuropsychological parameters.

Increased reaction time was associated with current pesticide exposure, as indicated by the urinary organophosphate metabolite excretion, but no such association was seen with the other neuropsychological tests. Reaction time correlated with both composite dimethylphosphate (r = 0.27; P = .02) and composite diethylphosphate excretion (r = 0.30; P = .01). The correlation with the sum of all metabolites was even better (Fig 1). However, no association with acetylcholine esterase was seen. These associations were affected only marginally by age, gender, prenatal exposure, and stunting.

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FIGURE 1

Average simple reaction time for right and left hands in relation to composite urinary excretion of dimethylphosphate and diethylphosphate metabolites in 72 Ecuadorian school children (r = 0.36; P = .002).

Blood pressure and the copying scores were then compared in groups stratified according to both prenatal pesticide exposure and stunting (Table 5). Stunting had no clear effect on blood pressure. The highest systolic blood pressure was seen in the exposed children, who were also stunted; prenatal pesticide exposure remained statistically significant in this subgroup.

The highest copying scores were obtained by children without signs of stunting who had also not been exposed to pesticides prenatally (Table 5). The lowest scores were seen in stunted children who had been exposed to pesticides prenatally. Both of these predictors showed statistically significant effects. Similar results were obtained with the full copying score.

Therefore, multiple regression analyses were conducted (Table 6). Age and gender were considered overall obligatory cofactors, and body weight was included as a recognized risk factor for blood pressure. Other anthropometric factors were also included in exploratory analyses but were not statistically significant and changed the regression coefficients for pesticide exposure only minimally. Height for age, also as a continuous parameter, did not approach statistical significance. After covariate adjustment, prenatal pesticide exposure was associated with a significant increase of 4.6 mm Hg in systolic blood pressure (Table 6); the parallel result for diastolic blood pressure was 2.7 mm Hg (P = .16).

The copying score was adjusted for age, gender, maternal education, and medical risk factors as a priori predictors of the neuropsychological outcome. In addition, running water at home was included as a significant covariate. The adjusted regression coefficients for both pesticide exposure and stunting were statistically significant (Table 6). When included in the regression model as a continuous variable, the height-for-age z score showed a P > 0.05 and caused a slight change in the regression coefficient for pesticide exposure to −0.92 (P = .018). As additional confounders, we examined other potential predictors of childhood neurobehavioral development, but none of them was statistically significant. Adding or removing these confounders from Table 1 only marginally affected the estimates for pesticide exposure and stunting. Virtually the same results were obtained with the copying scores for the complete series of designs. When an interaction factor between pesticide exposure and stunting was added, it showed a nonsignificant regression coefficient in the direction opposite to expectation, whereas both pesticide exposure and stunting remained significant.

DISCUSSION

In this study, prenatal pesticide exposure was associated with a higher blood pressure, an effect that seemed independent of stunting. Clinical neurologic examination showed a marginal increase in abnormalities among exposed children, and a lower score on the Stanford-Binet copying test was found in children with prenatal pesticide exposure and also in stunted children. Both risk factors remained statistically significant in adjusted analyses, and pesticide toxicity may, therefore, add to the adverse influence of malnutrition. Current pesticide exposure, as indicated by increased total excretion of dimethyl and diethyl metabolites of organophosphates, was associated with increased simple reaction time only, and this effect was independent of the other risk factors. These findings suggest that effects of prenatal pesticide exposure may be lasting and may differ from the effects of postnatal exposure.

The present study has aimed at exploring whether neurobehavioral deficits may be associated with prenatal pesticide exposure in a carefully chosen community, where social differences were thought to be minimal. Although nutritional deficiencies are widespread, pesticide exposure and stunting were not linked. The children examined attended the same school and were growing up under similar circumstances, thereby providing an opportunity to study children who were comparable in most respects, except for prenatal neurotoxicant exposure. Although employed mothers could be less capable of providing necessary parenting,⁴ those who were capable of obtaining employment in floriculture seemed to be better educated and seemed to provide slightly better living conditions for their fewer children.

A cross-sectional study like the present one cannot provide any detailed information about dose-response relationships or the time of impact of toxic exposures. The dichotomous prenatal exposure score is crude and based only on maternal interview but allows statistical comparison of children with some degree of prenatal exposure to those with limited risk of such exposure. The magnitude and exact duration of the prenatal exposure is not known. Exposure misclassification is possible, but would be random, because the maternal interviews were conducted before the mothers received the results of the examinations of the children. Such misclassification is likely to result in a bias toward the null hypothesis, and any adjustment, if possible, would strengthen the findings of this study. Also, selection bias would be unlikely, because all of the eligible children participated in the examinations. Given the similarity between the exposed children and the controls in regard to past medical history and most other respects, confounding bias also seems unlikely to be a major problem in this study.

Women who do not work in floriculture may earn an income from handicraft, cooking, and similar occupations that do not entail exposures to pesticides or other neurotoxicants. Still, because of the widespread use of pesticides, such as for household purposes, the children could potentially be exposed from other sources (eg, from contaminated food or the environment). Yet, prenatal exposure was associated with neither a decreased current acetylcholine esterase activity nor an increased urinary metabolite-excretion rate, thus indicating no confounding in this regard.

Under the cross-sectional circumstances of the present study, assessment of stunting had to rely on current anthropometric measurements without the benefit of prospective growth data. Therefore, the height-for-age z score obtained at school age may be associated with some uncertainty in regard to malnutrition otherwise determined at ages <5 years, according to international recommendations.³⁷ However, most children are likely to have maintained their relative rank due to tracking and limited compensatory growth during the next couple of years, because the nutritional mechanism involved in stunting also leaves the child unable to catch up in growth.³⁸

The standard requirement by the WHO of −2 as the stunting cutoff point for height for age³⁷ may be unreasonably strict for 2 reasons.¹⁹ Populations at altitudes >3000 m may be affected by low oxygen tension, with a proportionately greater growth of the chest in comparison with other body proportions.³⁹ In addition, the Andean physique is typically short,³⁹ with mean adult heights of <150 cm,⁸ thus suggesting that genetic factors may be of importance in regard to vertical growth. Furthermore, weight for height and BMI values in the present study deviate only minimally from expectation. Thus, the calculated height-for-age z scores likely exaggerate the extent of stunting in this population. Still, this dichotomous classification was a better risk indicator for decreased copying scores than was the continuous z score. In agreement with other research,⁵ these findings, therefore, indicate that even a minor degree of stunting can impact brain development.

The neurobehavioral tests included in the present study represent only a preliminary assessment of relevant brain functions. Although the tests were entirely feasible in this population, the results obtained must be interpreted in light of the likelihood of the tests being sensitive to neurotoxic damage. Thus, the computer-assisted tests provided excellent separation of performance results, but the traditional neuropsychological tests allowed fewer possible scores in the age range tested, thus substantially limiting the test sensitivity to small deficits.

Visuospatial performance, as assessed by the copying test, is important in regard to orientation in space and spatial performance. The adjusted regression coefficient for prenatal pesticide exposure was 4 times greater than the coefficient for age. This means that the deficit on the copying test associated with prenatal pesticide exposure corresponds to a developmental delay associated with being 4 years younger. Although the confidence interval is relatively wide, this statistically significant finding in a study of limited size suggests that the effect could be substantial. Preservation of spatial functions is likely of wider significance, because spatial perception and visuomotor coordination may be a prerequisite for other brain functions and their optimal development. Thus, increasing degrees of dysfunction on copying designs of the Rey-Osterrieth complex figure significantly predicted a greater probability of requiring remedial services in school.⁴⁰

One previous study of children with developmental pesticide exposure in Mexico¹⁵ found no exposure-related difference in growth patterns, but the exposed children showed deficits in eye-hand coordination, short-term memory, and the ability to draw a person. Although the present study found no effects on one test of motor coordination and did not use comparable memory tests, it replicated the findings in regard to spatial functions. The Mexican study used an ecological exposure classification that may have been imprecise and may have involved some degree of unmeasured confounding.¹⁵ With exposure information available on an individual level, the present study, therefore, provides strong support for the notion of a pesticide effect on visuospatial development.

Increased simple reaction time was significantly associated with increased dimethylphosphate and diethylphosphate metabolite-excretion levels. Although not associated with erythrocyte acetylcholinesterase activity, the effect on reaction time could not be explained by any relevant cofactors assessed in this study. An increased reaction time is in accordance with observations in children and adults exposed to organophosphates,^41,42 and it could, therefore, likely reflect an effect of current pesticide exposures. On the other hand, concentrations of organophosphate metabolites in the urine were similar to levels reported for US children aged 6–11 years,⁴³ and the 90th percentile for composite metabolite excretions at 574 μg/g of creatinine was not exceeded by any of the Ecuadorian children. However, these results may not be directly comparable, because the metabolites may originate from different parent organophosphates. Likewise, the cutoff level of 1 nmol/kg per day for dimethyl and diethyl metabolite excretions may be considered arbitrary and difficult to interpret. At the same time, acetylcholinesterase activities were within anticipated ranges and did not show any decrease associated with urinary metabolite-excretion levels. The apparent increase in reaction time nonetheless suggests that adverse effects may occur in children even when biomarker results are not increased beyond ranges considered normal.

In regard to blood pressure, heart action and vascular resistance are regulated by the autonomic nervous system, and ventricular arrhythmias are well known from organophosphate pesticide poisonings.⁴⁴ Neurobehavioral research has only recently begun to include cardiovascular parameters, and adverse cardiovascular effects in children are now known to occur as a result of exposures to neurotoxicants, such as lead⁴⁵ and methylmercury.^46,47 In the present study, the exposure-related increase in systolic blood pressure and the smaller, not statistically significant, increase in diastolic blood pressure were independent of stunting and other covariates. Parallel to this increase, a higher number of exposed children exceeded the expected normal range of systolic blood pressure. Nutritional factors^48–50 and maternal smoking during pregnancy⁵¹ may contribute to increased blood pressure in children. These parameters would be unlikely to explain the associations found, because stunting was of negligible importance, and only a few women smoked at all during pregnancy. Increased blood pressure, when present in childhood, is a risk factor for cardiovascular disease in later life,³⁶ and these results may, therefore, be important from a public health viewpoint.

In the present study, current pesticide exposure, which was independent of prenatal exposure, was not associated with the same outcomes that showed deficits after prenatal pesticide exposure. This observation is in accordance with the notion that prenatal and postnatal exposures may affect different nervous system functions in children.¹ Furthermore, the effects of prenatal pesticide exposure showed similarities with those associated with stunting, thus suggesting that malnutrition and toxicant exposures during early development may affect similar functions.

Within the limited literature on developmental neurotoxicity, the findings of the present study show parallels to another Mexican study.⁵² This research showed that the height-for-age index was an independent risk indicator for low IQ scores, which were also affected by exposures to the neurotoxicants lead and arsenic.⁵² In this study as well, the neurotoxicant effects seemed to be independent of the sequelae of malnutrition, although the coexistence of these factors seemed to render a child potentially more vulnerable to delayed mental development. The findings of both studies underline the need to assess chemical exposures as a cofactor when studying the adverse effects of malnutrition.

The toxicological literature has convincingly documented the neurotoxicity of many pesticides, and the increased vulnerability of the developing nervous system is also well substantiated.^{11–14,40,53} However, despite the extensive literature on pesticide poisonings in adults, very limited information is available on the effects of pesticides on the developing brain in humans.^1,2 Part of the reason for this lack of information is undoubtedly that the most widespread pesticide exposures occur in developing countries, where epidemiological studies may be difficult to carry out and where malnutrition and other potential confounders may compromise the results. On the other hand, extensive evidence has been gathered on the neurobehavioral consequences of malnutrition and stunting,^4–6,19,54 but these studies have not included pesticide exposure as a relevant cofactor that could impact on child development.

CONCLUSIONS

This study shows that maternal occupational exposure to pesticides during pregnancy is an important risk factor for the neurobehavioral development of a child. Statistically significant effects were increased blood pressure and decreased visuospatial performance. Although the latter outcome was also affected by stunting, the associations were robust to confounder adjustment. Furthermore, current pesticide exposure was associated with an increased simple reaction time, a parameter that was not affected by prenatal pesticide exposure or stunting. These results, therefore, suggest that prenatal pesticide exposure may adversely affect brain development, that the effects may resemble those caused by malnutrition, and that they differ from postnatal toxicity. Because developmental neurotoxicity is likely to cause permanent deficits, pesticide exposure during early life must be considered an important risk to human health that deserves attention by prevention authorities and the research community.