1 Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC.
2 Department of Epidemiology and Preventive Medicine, University of California, Davis, CA.
3 Present address: California Environmental Protection Agency, Sacramento, CA.
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ABSTRACT |
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environmental exposure; fetal death; pesticides; pregnancy outcome; survival analysis
Abbreviations: CI, confidence interval; HR, hazard ratio; TRS, township, range, section
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INTRODUCTION |
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Both epidemiology and toxicology studies have shown that the susceptibility of the fetus is dependent upon the timing of exposures (6, 16
). The best understood period of susceptibility in humans is organogenesis, which occurs from the third to the eighth weeks of gestation; birth defects, whether fatal or not, are most likely to originate in this time window (17
). For fetal death due to causes other than congenital anomalies, susceptible time periods are not well characterized. While the organ systems are in place by the end of the eighth week, factors that could influence growth; neural, metabolic, and immunologic development; and, ultimately, survival to birth may have a greater effect during the last two trimesters of gestation (18
).
In a previous analysis of fetal death due to congenital anomalies, we found an increased risk among women living near pesticide applications during the period of organogenesis (19). For the 60 percent of women who responded to a self-administered questionnaire, the residential proximity results were not explained by use of pesticides on the job or in the home. A separate analysis of fetal deaths from all causes among questionnaire respondents showed an increased risk among mothers who reported occupational or home use of pesticides (20
). Unlike our previous reports, this study evaluated residential proximity to agricultural pesticide applications (not home or occupational use) in relation to fetal death due to causes other than congenital anomalies.
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MATERIALS AND METHODS |
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Selection of noncases
Noncases consisted of a stratified random sample of normal livebirths occurring in 1984. They were frequency matched to all cases by maternal age (in 5-year age groups) and county of maternal residence recorded on the fetal death or birth certificates to allow for more efficient control of urbanization and potential differences in health care services. Normal births were defined as those with no congenital malformations recorded on the birth certificate. A total of 642 noncases were selected, 31 of which were subsequently excluded due to missing information on the date of last menstrual period.
Data extraction
Fetal death, death, and birth certificates were obtained from the California State Vital Statistics Registry. From these, we abstracted delivery information (date of delivery, gender, and plurality), parental information (age, race, ethnicity, and mother's address), cause of death, and medical data (date of last menses, month prenatal care began, and birth weight). Information on additional risk factors (i.e., smoking status, occupational exposures, alcohol consumption, etc.) was gathered from a self-administered questionnaire returned by 55 percent of the study population, as previously described (22).
Exposure ascertainment
The California Pesticide Use Report database for the years 19831984 contains information on applications of all restricted use pesticides, including the chemical used, amount applied, and date and location of each application (23). Location is specific to the level of township, range, and section (TRS). The Public Land Survey System from the US geologic survey imposes a grid on the entire United States that divides it into units of 1 square mile (1.6 km), each identified as a unique TRS. We used county maps to locate the TRS for each maternal address (maternal TRS). Pesticide exposure was determined by linking the maternal TRS to the TRS of each pesticide application.
Two levels of exposure were identified for this study population (figure 1). First, the pregnancy was considered to be exposed to a particular pesticide if the TRS of a pesticide application was the maternal TRS or any of the surrounding eight TRSs ("broad definition"). Second, a "narrow definition" limited exposure to within the maternal TRS.
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Exposure classification
Since separate analyses of all individual pesticides would be unwieldy, pesticides were categorized into classes based on their chemical structure and biologic action. Five "initial" categories were selected for analysis: phosphates, carbamates, pyrethroids, halogenated hydrocarbons, and endocrine disruptors. These categories were chosen on the basis of their high use and potential adverse effects indicated in animal and epidemiologic studies (5, 25
). The individual pesticides within each of these five categories are listed in table 1. In addition, a recent article by Sonnenschein and Soto (26
) described several pesticides that are estrogenic xenobiotics. We analyzed the two estrogenic pesticides that were in our database (endosulfan and methoxychlor).
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Since relevant biologic time periods for exposure were not known, separate analyses were completed for each trimester and each month of gestation. Women were classified as exposed if a pesticide application took place at least once in the gestational period of interest. While this definition potentially lumps women exposed only once with those exposed many times during that time period, multiple pesticide applications occurred during any given gestational month for the vast majority of women classified as exposed.
Statistical analysis
Exposure prevalence among noncases and the distribution of covariates by case status were determined for the following: race, gender of fetus/infant, trimester prenatal care began, season of conception, and prior fetal loss. The covariate-exposure association was examined separately for all pesticide classes and trimesters. No covariates were associated with both exposure and case status (i.e., odds ratios were not less than 0.7 or greater than 1.5), indicating that they did not need to be considered as confounders. The final models included maternal age and county of residence (the two matching variables).
Stratified odds ratios were examined to screen for potential effect modifiers. Our inclusion criterion for meaningful effect modifiers required that stratum-specific odds ratios differ by 100 percent or more. No variable met this criterion.
Given that exposure opportunity (length of observation or, in this case, length of gestation) was also associated with case-status in our data, adjustment was necessary (20, 22
, 27
). We therefore used survival analysis to control for gestational age and to simultaneously analyze time-dependent exposures. Since the data were originally collected in a "standard" case-control design based on cumulative risk of death at 1 day after birth, survival analysis (i.e., estimation of failure probabilities over time) required knowledge of the sampling probabilities. These sampling probabilities were p = 1 for cases and p = 0.01 for noncases (67,915 livebirths occurred in the 10 counties in 1984). To convert standard (cumulative incidence) case-control data to a case-cohort design, the controls and a random sample of cases (selected with the same probability as the controls) can be combined to create a subcohort that is a random sample of the original cohort from which the cases arose. In this study, random sampling of controls was conducted within strata of maternal age and county. At the time of failure, the case is compared with a risk set composed of those from the subcohort still eligible to be a case, i.e., those who have not yet delivered. Adjusted hazard ratios (HRs) and 95 percent confidence intervals were calculated by using multivariate proportional hazards models, with time-dependent exposures for each trimester and each month of gestation.
Analyses were completed separately for the broad and narrow exposure definitions. Because of the small number of persons exposed to only one pesticide class, the analyses for each pesticide class were completed without adjusting for or removing those exposed to other pesticide classes.
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RESULTS |
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DISCUSSION |
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Several epidemiologic studies have found occupational and environmental exposures to pesticides to be associated with fetal death, with estimated risk ratios ranging from 1.4 to 3.6 (7, 9
11
, 13
). Specific pesticide classes were not examined in any of these studies, and the exposure assessments were based on broad job classifications or self-reported use of pesticides. For the same study population, Pastore et al. (20
) evaluated self-reported occupational and home pesticide use for all cases and controls with completed questionnaires. Occupational pesticide exposure during the first two trimesters of gestation was associated with all causes of fetal death (HR = 1.4, 95 percent CI: 1.0,1.7) and fetal death due to complications of the placenta, cord, and membranes (HR = 1.6, 95 percent CI: 1.1, 2.3). In our evaluation of residential proximity to applications of agricultural pesticides, however, adjustment for self-reported use of pesticides on the job or in the home among those who returned the questionnaires did not alter the findings.
We found that subjects were more likely to be exposed to multiple pesticide classes than to one specific pesticide class, making it difficult to examine the impact of exposure to only one pesticide class. In a previous analysis of these data, an association with fetal death due to congenital anomalies was observed for exposure to three or more pesticide classes during the period of organogenesis. A much weaker association was observed for those exposed to one to two classes (19). In both the trimester and the monthly analyses presented here, we observed increased HRs for carbamates, direct and carbamate acetylcholinesterase inhibitors, and halogenated hydrocarbons. Several animal studies have shown specific pesticides within these classes to be fetotoxic, but at exposure levels not likely for humans. Thus, plausible mechanisms involving maternal, fetal, or placental units have yet to be identified. Human case studies have shown that pesticides can cross the placenta and accumulate in fetal organs (28
, 29
). In addition, decreased levels of placental acetylcholinesterase have been observed in pesticide- exposed rodents (30
, 31
). However, the effect of decreased acetylcholinesterase on the fetus is not well understood.
In many respects, exposure assessment was a strength in this study. Exposure was determined from state-maintained computer databases covering all applications of the pesticide classes examined here. Hence, its ascertainment was independent of birth outcome, a distinct advantage over case-control studies that rely on self-reports and therefore are subject to differential recall by case status. Exposure assessment was also improved over previous studies in that information was specific for each day of pregnancy. The daily exposure data enabled trimester- and month-specific analyses and control of differential exposure opportunity between cases and controls. The availability of data on specific pesticides and proximity of the pesticide application to maternal residence as defined in the TRS allowed us to refine the exposure definition further.
Despite these strengths, several limitations pertaining to exposure assessment were still present. The smallest unit of the TRS system is 1 square mile. Hence, the exact distance of the pesticide application from the home (e.g., a few feet or >1 mile) could not be determined. In addition, daily activity patterns, home monitoring, and biologic samples were not available. Wind and weather conditions, the hour of application, and the location of the mother at the time of the application are all factors that would determine actual exposure. For example, mothers who worked away from the home (and who were not exposed to pesticides at work) would have a lower likelihood of being exposed compared with mothers at home. More generally, whether absorption occurs by inhalation, dermal absorption, contamination of ground water, or ingestion of residues on foodstuffs or by a combination of these remains unclear.
In addition, since maternal residence at the time of delivery was used, misclassification of exposure could occur for those mothers who moved during pregnancy. Residential history was available for those mothers who returned questionnaires. For those who reported having moved, the updated address reported by the mother was used to determine exposure for the corresponding days of pregnancy. For mothers who did not return questionnaires, incorrect addresses may have been used to assign exposure. Exposure misclassification would be greater for the early months of pregnancy because women would have had more time to change residences. Since there is a greater probability of a bias toward rather than away from the null, our specific findings for the third to fifth months of pregnancy may be underestimates of the true effect. Estimated associations for later months may have also been underestimated, but probably less so.
Another limitation was the lack of questionnaire data on potential confounders for almost 45 percent of the study participants. Twenty-five percent were not locatable (i.e., they had moved, and no forwarding addresses were obtainable), and 20 percent did not respond to the questionnaire. These two groups did not differ from respondents by case status. Both groups, however, were more likely to be younger, to be Hispanic, and to have sought prenatal care later in pregnancy compared with those who returned questionnaires. Smoking status, alcohol consumption, income, education, and occupational and home use of pesticides did not confound the observed associations among the questionnaire group, but our inability to adjust for these factors in the entire study cohort may have led to some bias in the HRs.
Fetal deaths may have been underascertained and/or misclassified regarding their causes. In California, fetal deaths at 28 weeks or more gestation are more likely to be reported than are those that occur between 20 and 27 weeks gestation (32). If environmental exposure to pesticides influences the risk differentially for the earlier versus the later fetal deaths, our results may not represent effects for earlier events. In addition, uncertainties in assigning cause make it difficult to assemble etiologically homogenous groups, particularly since causes are frequently unspecified (as was true for 25 percent of our cases). We attempted to address the problem of heterogeneity by excluding those fetal deaths not likely to be caused by environmental exposures; however, our cases were still very heterogeneous.
Finally, because fetal death rates are higher for young and old maternal ages, the matching by maternal age resulted in a study population age distribution that was not representative of the full cohort (all pregnant women who delivered after 20 weeks in 1984 in the study counties). If maternal age is an effect modifier, our summary effect estimates could differ slightly from those in the total cohort. The likelihood that matching on county constituted overmatching with respect to exposure is extremely low, given that the controls were not matched on date of birth (pesticide applications occur in specific seasons) and that the counties are large (1,4266,017 square miles) (33).
In summary, in 10 agricultural counties of California, residential proximity to application of several pesticide classes during pregnancy was, on the whole, not associated with fetal death. Slightly elevated risks were observed for women who lived near applications of halogenated hydrocarbons in the fourth and fifth months of gestation and of carbamates and carbamate inhibitors in the third and fourth months of gestation. However, despite our attempts to improve exposure assessment and case definition, the increased HRs observed in this study were small (HR < 1.5); thus, we are not able to rule out the possibility that our observations were the result of some unmeasured factor. Nevertheless, the consistency of the time period for these associations suggests that months 35 of gestation may be a period of fetal vulnerability to some pesticide exposures.
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ACKNOWLEDGMENTS |
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The authors acknowledge Dr. Andrew Olshan, Dr. David Savitz, and Dr. Ernest Hodgson for their comments on earlier versions of this paper and Lawrence Park for his computer programming assistance. The authors also thank Dr. Steven Samuels, James Singleton, and Susan Lutzenhiser for their contributions to study design, data collection, and database development.
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NOTES |
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REFERENCES |
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