APPENDIX 6

Detailed Reviews of Children’s Health Epidemiology Studies
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Table of Contents

 

Article 1. Whyatt et al. (2004)....................................................................................... 3

Article 2. Berkowitz et al. (2004)................................................................................... 9

Article 3. Eskenazi et al. (2004) ................................................................................... 16

Article 4. Harley et al. (2011)....................................................................................... 21

Article 5. Engel et al. (2007) ........................................................................................ 29

Article 6. Young et al. (2005)....................................................................................... 37

Article 7. Rauh et al. (2006)......................................................................................... 42

Article 8. Lovasi et al. (2010)....................................................................................... 48

Article 9. Engel et al. (2011) ........................................................................................ 54

Article 10. Eskenazi et al. (2007) ................................................................................... 63

Article 11. Eskenazi et al. (2010) ................................................................................... 69

Article 12. Marks et al. (2010)....................................................................................... 75

Article 13. Rauh et al. (2011)......................................................................................... 82

Article 14. Bouchard et al. (2011) ................................................................................. 90

Article 15. Whyatt et al. (2007)..................................................................................... 98

Article 16. Whyatt et al. (2009)................................................................................... 101
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Article 1. Whyatt et al. (2004)

Prenatal Insecticide Exposures and Birth Weight and Length among an Urban Minority Cohort. Robin 

M. Whyatt, Virginia Rauh, Dana B. Barr, David E. Camann, Howard F. Andrews, Robin Garfinkel, Lori A. 

Hoepner, Diurka Diaz, Jessica Dietrich, Andria Reyes, Deliang Tang, Patrick L. Kinney, and Frederica P. 

Perera. Environmental Health Perspectives. 112:1125–1132 (2004).

Study Summary

In this report, Whyatt et al. report on the relationship between pesticide exposures and developmental 

outcomes in newborns in the Columbia Mother’s and Newborn Study to include additional insecticides 

(the organophosphate diazinon and the carbamate propoxur), a larger sample size (n = 314 mother–

newborn pairs), and insecticide measurements in maternal personal air sampled in the home, as well as 

in umbilical cord plasma sampled at delivery. 

Study participants were recruited during early pregnancy (≤20th week) among African-American and 

Dominican women age 18-35 years, and registered at New York Presbyterian Medical Center and 

Harlem City. Women who smoked, had a history of drug abuse, diabetes, hypertension, or HIV infection 

were excluded from participation in the study, as were women who resided in New York City for less 

than 1 year. The study sample presented in this report was recruited between 1998 and 2002, a period 

which straddles the December, 2000 voluntary cancellation by registrants of indoor residential 

chlorpyrifos use. 

Insecticides, including chlorpyrifos, were measured in 48-hr personal air samples collected during the 

third trimester of pregnancy. Analyses of air samples for pesticide levels were carried out at the 

Southwest Research Institute. 

Chlorpyrifos levels in umbilical cord blood samples were available for 256 newborns, sampled as close to 

the time of delivery as possible, and within 2 days post-partum. Cord blood plasma chlorpyrifos levels 

were imputed from maternal blood levels for 31 newborns for whom no cord blood sample was 

obtained. Quantification of chlorpyrifos levels in plasma were conducted by the Centers for Disease 

Control and Prevention (CDC). 

Insecticide levels in both maternal personal air and blood samples were available for 259 of the 314 

mother–newborn pairs (82%). Measures in either personal air or blood were available for the remaining 

55 pairs (18%). Pesticide levels in personal air and blood samples below the level of detection (LOD) 

were assigned a value of half the LOD; levels were then log2-transformed for statistical analysis. For 

pesticides in blood, 31% of chlorpyrifos levels were below the LOD. Infants with below LOD cord blood 

chlorpyrifos levels were used as a comparison group and compared to groups with increasing levels, 

categorized by tertile of above LOD levels. 
Page 4 of 105

Infants’ weight, length, and head circumference were assessed at birth and obtained for this study 

through medical record abstraction. 

Multiple linear regression analyses were performed to evaluate associations between prenatal 

insecticide exposure and the indicators of infant development at birth. Covariates were selected from 

variables known to be associated with insecticide exposure or fetal growth. Effect modification by 

race/ethnicity was evaluated, but not observed. 

Controlling for potential confounders, the researchers found no association between maternal personal 

air insecticide levels and birth weight, length. Head circumference was not significantly associated with 

maternal personal air or cord blood chlorpyrifos levels. However, for each log unit increase in cord 

plasma chlorpyrifos levels, birth weight decreased by 42.6 g (95% CI: –81.8 to –3.8) and birth length 

decreased by 0.24 cm (95% CI: –0.47 to –0.01). Combined measures of (ln)cord plasma chlorpyrifos and 

diazinon (adjusted for relative potency) were also inversely associated with birth weight and length (p < 

0.05). Birth weight averaged 186.3 g less (95% CI: –375.2 to –45.5) among newborns with the highest 

compared with lowest 26% of exposure levels (combined p-value = 0.01). When stratified by date of 

birth prior to or after 2001 (before and after the voluntary cancellation by registrants of indoor 

residential chlorpyrifos use was implemented), associations between birth weight and length and cord 

plasma chlorpyrifos were statistically significant (p ≤ 0.007) among newborns born before the 

cancellation. Among newborns born after January 2001, exposure levels were substantially lower, and 

no associations with fetal growth outcomes were observed (p > 0.8). Results suggest that prenatal 

chlorpyrifos exposures may have impaired fetal growth among this inner-city, low income cohort.

Study Review

This was a very well conducted study with numerous strengths, and some noted limitations. The 

prospective nature of the study limits the influence of systematic errors in the exposure assessment on 

the association observed, such that mismeasurement of chlorpyrifos exposure would likely be nondifferential with respect to the indicators of fetal growth. 

A notable strength of this study, relative to investigations in other cohorts, is the direct measurement of 

chlorpyrifos in cord blood and personal air samples, rather than non-specific markers of 

organophosphate pesticide exposure. Errors in estimation of cord blood chlorpyrifos levels were likely to 

have been non-differential with respect to the indicators of fetal development. However, authors did 

not formally assess the variability in the exposure estimate resulting from the imputation of missing 

cord blood chlorpyrifos levels. 

A limitation of the specific biomarker exposure indicators in this study is the single sampling period (at 

delivery). It is not clear to what extent this single measurement reflects exposures over critical windows 

of fetal growth and development during pregnancy. If the exposure is chronic, a biomarker measured at 

a single time point can provide a representative dosimeter, even if the toxicant has a short half-life, as is 
Page 5 of 105

the case for chlorpyrifos. However, if pesticide exposures are sporadic or otherwise vary over short time 

scales, the biomarker measurement will not be representative of “usual” exposure, or of the exposure 

during critical periods of fetal development. An observation effect could also influence the validity of 

the 48-hour personal air sampling if, for example, the act of wearing the dosimeter inhibited 

participants from using pesticides or otherwise altered their behavior patterns in a manner that affected 

exposure levels. These limitations are assessed in subsequent validation studies conducted within this 

cohort (Whyatt, 2007 and Whyatt, 2009). 

The health outcomes in this study, birth weight, birth length, and head circumference were obtained 

through direct linkage with the mothers’ and infants’ computerized medical records, and were likely to 

have been accurately measured. Any errors in outcome measurement would have occurred in the clinic, 

and are likely to be small and likely to be non-differential with respect to pesticide exposure.

The investigators collected data on, and adjusted for, important potential confounders. Covariate 

selection proceeded appropriately by assessing relationships between potential confounders and the 

indicators of fetal growth, change-in-estimate of the main effect of chlorpyrifos exposure, and 

assessments of model fit. Covariates included in final models were race/ethnicity, gestational age, 

parity, maternal pre-pregnancy weight, and net weight gain during pregnancy, maternal self-reported 

environmental tobacco smoke in the home, sex of the newborn, and season of delivery. Models for head 

circumference also included an indicator for delivery type (vaginal versus cesarean section). 

Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) during pregnancy was not included in 

the final models because it did not improve model fit. However, to ensure that PAH exposure did not 

confound the pesticide associations with fetal development, models including PAHs were evaluated. The 

authors’ consideration of other pesticides and environmental contaminants as covariates in their 

multivariate models is a notable strength of this study. 

Confounding by maternal smoking behaviors was controlled by restriction (i.e., excluding smokers from 

the study sample). Confounding by unmeasured factors may have been similarly controlled by design, 

this being a relatively homogeneous population with respect to determinants of fetal growth, compared 

to the general population. For example, the authors found it unnecessary to adjust for maternal alcohol 

consumption (which was measured), partly because few women in the study drank alcohol during 

pregnancy.

Bias due to confounding by unmeasured determinants of fetal growth is possible in this observational 

study, for example, confounding by factors related to socioeconomic condition. Annual household 

income, maternal education, maternal marital status, material hardship during pregnancy, and degree of 

housing disrepair were evaluated as possible markers for socioeconomic status but were not included in 

final models because they were not associated with birth outcomes, did not improve the model fit, and 

did not substantially alter parameter estimates, confidence intervals (CIs), or significance levels of 

variables included in the final models. This observation that indicators of socioeconomic condition were 
Page 6 of 105

not associated with the health outcomes is somewhat unexpected, although perhaps explainable by the 

relative homogeneity of these factors in this study population (control through restriction of study 

population). 

In categorical analyses, infants with the lowest tertile of observable cord blood levels had, as a group, 

larger heights and weights at birth, compared to infants with chlorpyrifos levels below the level of 

detection, although the increases were not statistically significant. Among those with higher cord blood 

levels, increasing tertiles of chlorpyrifos were significantly associated with decreasing birth weight and 

height. This is suggestive of a threshold effect (a hockey stick dose-response curve). Departures from 

linearity in the exposure-response relationship were not assessed in models including (ln)chlorpyrifos 

entered as continuous variables. 

The authors observed an association between chlorpyrifos and fetal growth only when the biomarkers 

were used as indicators of prenatal exposure and not when environmental levels (in maternal 48-hr 

personal air sampling conducted during the third trimester) were used. Researchers note several 

possible explanations for this discrepancy, including the notion that the biomarkers are integrating 

dosimeters of exposure. This may be particularly important for insecticide exposure by routes other 

than inhalation in the home (e.g., dermal, ingestion, and occupational exposures). In addition, the 

authors speculate that the biomarkers may provide better dosimeters of the dose to the developing 

fetus, relative to the airborne exposures.

The voluntary cancellation by registrants of indoor residential chlorpyrifos use introduces exogenous 

variation in the exposure of interest. No associations were observed between the fetal growth 

indicators and chlorpyrifos levels among infants born after the implementation of the voluntary 

cancellation of chlorpyrifos containing in home pesticide products. This finding is similarly suggestive of 

a possible threshold effect (decreasing dose-levels before and after voluntary cancellation), although it 

could also be due to a lack of statistical power to assess associations at lower levels of exposure in the 

relatively small subsample (n=77). 

The statistical analysis appeared to be conventional and appropriate. However, the authors did not 

present nor discuss regression diagnostics to assess the degree to which their models met or violated 

the assumptions implicit in linear models (i.e. homoscedasticity, normality of error term distribution, 

independence of error terms, and linearity of the exposure-response relationship), other than to state 

that investigators log transformed chlorpyrifos levels. As discussed previously, there is some evidence in 

the data to suggest a non-linear exposure response relationship (i.e., threshold) between the natural log 

of cord blood chlorpyrifos levels and birth weight and length, but no formal statistical testing of this 

possibility was presented or discussed. 

Bias due to differential errors in the selection of study participants is unlikely to have occurred in this 

prospective study with low loss to follow-up. Appropriate exclusion criteria were applied, although 
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these design decisions may reduce external validity (generalizability) when making inference to other 

populations using the associations observed in this narrowly defined study population. 

The results presented in this expanded analysis confirm the authors’ earlier findings of an inverse 

association between chlorpyrifos levels in umbilical cord plasma and birth weight and length in this 

study population of non-smoking African-American and Dominican women, age 18-35 years, living in 

New York City (Whyatt et al., 2003). Systematic errors in the selection of study participants or in the 

measurement of key analytic variables (exposure and outcome) are unlikely to explain this observed 

association. The possibility of unmeasured or poorly measured potentially confounding variables cannot 

be completely excluded, although study authors thoroughly evaluated this possibility in the data 

available. Data suggest a threshold effect may be possible, although authors did not formally evaluate 

this characteristic of the data.
Page 8 of 105

Works Cited

Whyatt, R. M., Barr, D. B., Camann, D. E., Kinney, P. L., Barr, J. R., Andrews, H. F., . . . Perera, F. P. (2003). 

Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery 

among urban minority mothers and newborns. Environ Health Perspect, 111(5), 749-756. 

Whyatt RM, Garfinkel R, Hoepner LA, Holmes D, Borjas M, Williams MK, Reyes A, Rauh VA, Perera FP, 

Camann DE. Within- and Between-Home Variability in Indoor-Air Insecticide Levels during Pregnancy 

among an Inner-City Cohort from New York City. Environmental Health Perspectives. 115:383–389 

(2007).

Whyatt RM, Garfinkel R, Hoepner LA, Andrews H, Holmes D, Williams MK, Reyes A, Diaz D, Perera FP, 

Camann DE, Barr DB. A Biomarker Validation Study of Prenatal Chlorpyrifos Exposure within an InnerCity Cohort during Pregnancy. Environal Health Perspectives. 117:559–567 (2009).
Page 9 of 105

Article 2. Berkowitz et al. (2004)

In Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Gertrud S. 

Berkowitz, James G. Wetmur, Elena Birman-Deych, Josephine Obel, Robert H. Lapinski, James H. 

Godbold, Ian R. Holzman, Mary S. Wolff. Environmental Health Perspectives. 112:388–391 (2004)

Study Summary

To assess the relationship of exposure to pesticides during pregnancy and subsequent risk of impaired 

growth and neurodevelopment, Berkowitz and colleagues (Mount Sinai Child Development Study) 

investigated the relationship between prenatal pesticide exposure and infant growth and 

neurodevelopment in a prospective, multiethnic cohort study of mothers and infants delivered at Mount 

Sinai Hospital in New York City. 

The study population was drawn primarily from East Harlem and consisted largely of young Hispanic 

women (predominantly Puerto Rican), but also included African-American and Caucasian women. 

Mothers were enrolled during early pregnancy from a prenatal clinic and two private practices at Mount 

Sinai Hospital from March 1998 to March 2002. The study included only primiparous with singleton 

births. Mothers were excluded if they had any of the following: an initial prenatal visit after 26 weeks of 

gestation; serious chronic diseases; a serious pregnancy complication that could affect fetal growth and 

development; alcohol consumed greater than two alcoholic beverages per day; or, illicit drug use. 

Mothers and infants were also excluded if the child was born with a congenital malformation or severe 

prematurity. A total of 479 prenatal patients were recruited; 75 were excluded because of medical 

complications, premature birth, an infant with birth defects, inability to collect biologic specimens 

before birth, change of hospital or residence outside New York City, or refusal to continue to participate. 

Pesticide exposure was assessed by a prenatal questionnaire and measurement of urinary concentration 

of 3,5,6-trichloro-2-pyridinol (TCPy), a metabolite of chlorpyrifos exposure. The survey was 

administered, and maternal urine and blood specimens were collected, during the third trimester. Cord 

blood samples were also obtained at birth. Measurement of paraoxanase-1 (PON1) genotype and 

phenotype was made using maternal and infant cord blood samples. PON1 is an enzyme that can 

detoxify chlorpyrifos before it can inhibit acetylcholinesterase in the peripheral and central nervous 

systems. The investigators assessed birth weight, length, head circumference, and gestational age of 

the newborns for 404 participating mother-child pairs.

Almost half of the participants in the study, (46%) reported that they, or a household member, had used 

indoor pesticides during the pregnancy; 72% were classified as exposed using a composite index defined 

as indoor pesticide use in the household or exterminator application, fumigation, or pesticide use in 

common areas. Detectable levels of urinary 3,5,6-trichloro-2-pyridinol (TCPy; a metabolite of 

chlorpyrifos) were observed in 44% of the study participants. 
Page 10 of 105

The authors constructed generalized linear models to evaluate the associations among pesticide 

exposure and PON1 activity with average birth weight, birth length, head circumference, and gestational 

age, adjusting for selected covariates (race/ethnicity, infant sex, and gestational age). The authors 

found no statistically significant associations between pesticide exposure based either on questionnaire 

responses or biomarkers of pesticide exposure (TCPy) on fetal growth or gestational age. 

Researchers also evaluated the potential effect modifying role of PON1 genotype and phenotype in the 

relation of interest. Stratifying participants by presence of PON1 polymorphisms and tertiles of maternal 

PON1 activity, researchers reported a small but statistically significant reduction in head circumference 

among children of mothers with levels of chlorpyrifos above the limit of detection and also in the lowest 

tertile of PON1 activity, i.e., slowest metabolic activity. However, the interaction between TCPy level and 

PON1 activity was not statistically significant. In the subgroup of infants whose mothers had TCPy levels 

greater than the level of detection, those with low maternal PON1 had an average (SD) head 

circumference of 33.3cm (1.5cm) which was significantly smaller than those with medium (34.0cm 

(1.5cm)) and high (34.1cm (1.6cm)) maternal PON1 activity after adjusting for race/ethnicity, infant sex, 

and gestational age (p = 0.014). A similar trend was observed with head circumference when maternal 

PON1 activity was considered alone; the adjusted means were 33.5 cm for the low PON1, 33.9 cm for 

medium PON1, and 34.1 cm for high PON1 activity (p = 0.004). Maternal PON1 genetic polymorphisms 

were not associated with reduced head size. No trends were seen for birth weight or birth length for 

TCPy or the other metabolite levels when maternal paraoxonase level was taken into account. Infant 

paraoxonase activity had no association with any of the fetal growth measures. 

Because small head size has been found to be predictive of subsequent cognitive ability, the authors 

conclude that chlorpyrifos may have a detrimental effect on fetal neurodevelopment among mothers 

who exhibit low PON1 activity.

Study Review

The largely null study had notable strengths and some limitations; notably authors did not discuss any 

limitations of their study in the published manuscript. The prospective nature of the study limits the 

influence of errors in the self-reported exposure classification on the association observed; as such 

errors would likely be non-differential with respect to determinants of the health outcome under 

investigation.

The authors assessed pesticide exposure using both subjective questionnaire-based methods and 

biomarkers of exposure which are objective, sensitive and specific surrogates of pesticide exposure. 

Biomarkers of specific pesticides (i.e., TCPy) were assessed in a single maternal urine sample provided 

during the third trimester. The authors do not discuss the impact of policies aimed at reducing use of 

chlorpyrifos on the prevalence and magnitude of pesticide exposures during the time period of this 

study; it is likely that use of chlorpyrifos exposure was decreasing over the time period of the study, as 

noted in other similar studies of the same time period. The authors do not deliberate on the degree to 
Page 11 of 105

which the biomarkers assessed represent usual exposure to pesticides versus recent, acute exposure(s), 

nor do they discuss the relative importance of assessment of exposure during the third trimester vis-àvis fetal growth and development. The critical window of exposure for fetal growth and 

neurodevelopment health effects is not fully characterized. If sources and patterns of exposure remain 

static, a single measurement would be a good indicator of pesticide exposure during pregnancy, even if 

the pesticides have short half-lives in vivo. If, however, sources and exposure patterns change over short 

time-spans, perhaps on the order of days to weeks, the biomarker assessment would not necessarily 

provide a good indicator of the relevant exposure. The authors classified biomarker levels according to 

the assays’ levels of detection (above versus below). Falsely negative exposure characterization would 

occur if mothers were exposed only early in pregnancy, and the pesticides were fully metabolized prior 

to the third trimester. Incorrect classification of participants as exposed, when in fact they were not 

exposed, was unlikely to have occurred using the biomarkers. Like other sources of measurement error 

in this study, errors in the biomarker indicators of exposure in this study are likely to have resulted in a 

non-differential misclassification, with respect to fetal growth and development. 

The health outcomes in this study, birth length, birth weight, head circumference, and gestational age 

were likely to have been accurately measured in this study, as they were obtained through direct linkage 

with the computerized medical record. Any errors in outcome measurement would have occurred in the 

clinic, and are likely to be small and certainly non-differential with respect to pesticide exposure. 

To some extent, the recruitment process resulted in a population that was likely homogeneous with 

respect to confounders of the in utero pesticide exposure-fetal growth relationship (compared to the 

general population), and the potential for confounding was thus limited by design. The authors were 

also able to collect data on, and adjust for, important covariates, including race/ethnicity, infant sex, and 

gestational age. The authors also controlled for birth weight or birth length in their assessment of head 

circumference and pesticide exposure. Covariates used in the analyses were selected, according to the 

authors, from variables known to be associated with either pesticide exposure or fetal growth. 

However, factors related to socioeconomic condition of those assessed and co-contaminants to which 

the study participants may have been exposed were not addressed in detail in the article. To the extent 

that such factors may be determinants of fetal growth and development and correlated with (but not 

caused by) pesticide exposure, they may confound the associations of interest in the study. If these 

factors are positively associated with both pesticide exposure and diminished fetal development, they 

would bias the true association upward (towards larger mean differences associated with exposure). 

However, this is unlikely to have occurred in this largely null study.

Only factors that were statistically significant (p < 0.05) were included in the final models. The use of a 

statistical test as a means to select covariates from a larger set of potential confounders is discouraged 

in some epidemiologic circles (Miettinen 1976, Breslow and Day 1980, Greenland 1989). The 

assessment of confounding is generally not considered to be a statistical endeavor, in part because the 

association between the confounder and the outcome is just one factor that determines the degree of 

confounding, and also because the statistical significance of an association is a function not only of the 
Page 12 of 105

magnitude of the association but also of other factors (e.g., sample size, and sample variability) which 

are irrelevant to bias due to confounding. Moreover, the use of significance testing in the selection of 

confounders treats false negative errors (i.e. the deleting a confounder) as secondary to false positive 

error (i.e., inclusion of a non-confounder). This implicit ranking of error is backward, as deleting a 

confounder introduces bias and is justified only if the bias induced by its exclusion is tolerably small or 

deemed to be worth the precision gain, whereas including a non-confounder only (potentially) reduces 

precision by increasing the size of the model. This is a small consideration in this study, and different 

approaches are not likely to qualitatively change the study findings or conclusions. Notably, smoking was

not included as a potentially confounding variable in this analysis as the prevalence of smoking was low 

in this cohort. According to the authors, the inclusion of smoking in final models did not alter risk 

estimates and added to variance as stated by the authors.

Bias due to differential errors in the selection of study participants is unlikely to have occurred in this 

prospective study with no loss to follow-up at the time of this study. Appropriate exclusion criteria were 

applied, including mothers with no prenatal medical visit prior to 26 weeks of gestation, mothers with 

serious chronic diseases (e.g., diabetes, hypertension, thyroid disease), mothers who developed a 

serious pregnancy complication that could affect fetal growth and development, and mothers who 

consumed more than two alcoholic beverages per day or who used illicit drugs, mothers with multiple 

births, and infants born with a congenital malformation or severe prematurity. The subjective, surveybased pesticide exposure assessment was fortified by objective indicators of exposure (i.e. urinary 

biomarkers). 

The generalizability of the study may be limited as consequence of the particulars of the study 

population assessed and the numerous exclusion criteria applied. Making valid inference using these 

results requires an assumption that the pesticide exposure-fetal development association is the same in 

this population as the population to which the results are being generalized. This study population 

consisted of primiparous women, recruited early on in their pregnancies from the prenatal clinic and 

two private practices at Mount Sinai Hospital in New York from March 1998 to March 2002, consisted 

predominantly of young Hispanic women with low education. Sixty-eight percent were less than 25 

years old. Just under half were Hispanic (49.8%), although substantial numbers of White (21.0%) and 

African-American (27.7%) women also participated. Just under half (46.8%) classified themselves as 

being either single, divorced, widowed, or separated, and just over half (50.1%) had no more than a high 

school education. The exclusion criteria applied to potential study participants further focused the 

population assessed, relative to the general population of pregnant women. In the likely situation that 

the prevalence of important modifiers of the exposure-fetal development association in this focused 

study population is different from that in other populations of concern, generalizability will be limited. 

Important modifiers to consider may include time spent in the home, patterns of food preparation and 

consumption, health consciousness, the baseline health status of the mother (and father), and a host of 

socioeconomic determinants. The investigators finding of effect modification by PON1 activity is 

illustrative of limits on the generalizability of the study; populations who have high levels of 

paraoxonase activity may vary by genotypic and phenotypic determinants. These factors were not 
Page 13 of 105

discussed in the report, but it has been noted previously that there is 10- to 40-fold inter-individual 

variability in rates of paraoxon hydrolysis (Playfer et al 1976). The potential limitation vis-à-vis 

generalizability is offset by a strength; the study has increased statistical efficiency, relative to one set in 

a general population. 

Because small head size has been found to be predictive of subsequent cognitive ability, the authors 

conclude that chlorpyrifos may have a detrimental effect on fetal neurodevelopment among mothers 

who exhibit low PON1 activity. Implicit in this conclusion is the notion that small head size is a good 

surrogate for fetal neurodevelopment. To support their conclusion, the authors note that head 

circumference has been shown to correlate with brain weight, and that brain size and head 

circumferences are both predictive of IQ and cognitive ability. They additionally cite research associating 

brain volume with attention-deficit/hyperactivity disorder. Although head circumference was 

significantly associated with cognitive ability, the differences in head size across tertiles of exposure 

were notably small (i.e. less than one centimeter); the clinical relevance of such small changes in the 

context of even a small amount of measurement error in assessment of head size is unclear. 

The authors were not surprised that infant paraoxonase levels in their study were not associated with 

birth outcomes because PON1 is not well expressed until after birth, and infant PON1 levels are much 

lower during the first year of life than maternal levels. The expression of PON1 in infants is 

approximately one-third that of their mothers (Chen et al. 2003). They speculate that lower PON1 

activity in infants may make them more susceptible to PON1-related toxicity, including exposure to 

organophosphate pesticides, relative to adults.

The net effects of confounding, selection bias, and information bias, are unlikely to have qualitatively 

changed the results of this well conducted, prospective cohort study. Non-specific biomarkers of 

exposure, as were assessed in this study, are likely more objective, sensitive, and likely also more 

accurate and reliable indicators of pesticide exposure, relative to exposure assessment methodologies 

that rely on self-report. However, to the extent that prenatal chlorpyrifos is the true exposure of 

interest, the assessment of non-specific organophosphate pesticide metabolites in a single sample of 

prenatal urine is a primary potential source of exposure measurement error. Almost certainly, such 

biases would not have resulted in the observation of a falsely positive association between pesticide 

exposure and fetal growth. More likely to have occurred is an attenuation of the true (unobserved) 

associations, toward (observed) associations that are relatively smaller, or null. Indeed, the findings of 

this study are either null, or associations of small magnitude. To contextualize these findings, the 

authors cite literature describing the finding that even small increases in prenatal or early childhood 

exposures to other environmental toxicants (lead and polychlorinated biphenyls) are associated with 

significant decrements in IQ and academic achievement up to 11 years of age. The validity of this 

analogy is speculative. The external validity of the study findings may be limited if the organophosphate 

pesticide exposure-fetal development association is modified by factors that are more, or less, prevalent 

in this young, urban, minority population, relative to the population(s) to which inference is being made. 

That said, the decision to focus on this population is reasonable, as it is a population likely to be highly 
Page 14 of 105

exposed, and perhaps also at high risk of cognitive and behavioral deficits enhancing the likelihood of 

observing an association if an association exists. 
Page 15 of 105

Works Cited

Breslow NE, Day NE. Statistical Methods in Cancer Research: Volume II: The Design and Analysis of 

Cohort Studies. Lyon: IARC, 1987. 

Chen J, Kumar M, Chan W, Berkowitz G, Wetmur J. Increased influence of genetic variation on PON1 

activity in neonates. Environtal Health Perspectives. 2003;111:1403–1409.

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL, Siskind J, Meisel SJ, Wetmur JG, Wolff MS. Prenatal 

Organophosphate Metabolite and Organochlorine Levels and Performance on the Brazelton Neonatal 

Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. American Journal of Epidemiology. 

2007;165:1397-1404.

Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canfield RL, Wolff MS. Prenatal Exposure to 

Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Environmental Health 

Perspectives 2011;119:1182-1188 

Greenland, S. Modeling and Variable Selection in Epidemiologic Analysis. American Journal of Public 

Health. 1989; 79:340-349.

Miettinen OS. Stratification by a multivariate confounder score. American Journal of Epidemiology 

1976;104:609- 620. 

Playfer J, Eze LC, Bullen MF, Evans DAP. Genetic polymorphism and inter-ethnic variability of plasma 

paraoxonase activity. J Med Genet. 1976;13:337–342.
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Article 3. Eskenazi et al. (2004)

Association of in Utero Organophosphate Pesticide Exposure and Fetal Growth and Length of 

Gestation in an Agricultural Population. Brenda Eskenazi, Kim Harley, Asa Bradman, Erin Weltzien, 

Nicholas P. Jewell, Dana B. Barr, Clement E. Furlong, Nina T. Holland. Environmental Health 

Perspectives. 115:792–798 (2007).

Study Summary

In this article by Eskenazi et al, the authors report on the relationship between prenatal urinary 

organophosphate metabolite levels and fetal growth and gestational duration in the CHAMACOS cohort 

of Mexican-American mothers and their children living Salinas Valley, an agricultural region of California.

Enrollment of the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) 

cohort took place at community clinics. A total of 601 out of 1,130 eligible women agreed to participate 

and were enrolled between October, 1999 and October, 2000, with 538 being followed through to 

delivery of a live born infant, whose birth weight was measured. The study populations consisted largely 

of low-income, agricultural workers. Women considered eligible for the study were less than 20 weeks 

gestation, aged 18 years or older, Medi-Cal eligible, fluent in English and/or Spanish, and planning to 

deliver at Natividad Medical Center.

Excluded from analyses were women with gestational or preexisting diabetes (n = 26), hypertension (n = 

15), twin births (n = 5), or stillbirths (n = 3). One additional woman for whom birth weight information 

was out of range (< 500 g) was also excluded. The final analytic sample consisted of 488 mother-infant 

pairs.

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of 

organophosphate dialkyl phosphate metabolites (DAPs) and seven different pesticide-specific 

metabolites in maternal urine during pregnancy, including 3,5,6-trichloro-2-pyridinol (TCPy), a 

metabolite specific to chlorpyrifos. Cholinesterase (ChE) in whole blood and butyryl cholinesterase 

(BChE) in plasma collected from mothers during pregnancy, at delivery, and from umbilical cord blood 

was also assessed. Maternal urine samples were collected between 5 and 27 weeks gestation and again 

between 18 and 39 weeks. The dialkyl phosphates measured were dimethylphosphate, 

dimethyldithiophosphate, dimethylthiophosphate (DMPs), diethylphosphate, diethyldithiophosphate, 

and diethylthiophosphate (DEPs). Total DAPs and DMPs were available for 485 women; DEP metabolite 

levels were available for 486 women. Quantification of organophosphate metabolites using gas 

chromatography-tandem mass spectrometry and isotope dilution calibration was conducted by the 

Centers for Disease Control and Prevention (CDC) labs. 

Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP 

metabolites, dimethylphosphate (DMP) metabolite level as the sum of the molar concentrations of the 
Page 17 of 105

three dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of 

the molar concentrations of the three diethylphosphate metabolites. Total DAP, DMP, and DEP levels 

were determined for each participant for each of the two pregnancy urine samples. Individual 

metabolite levels below the limit of detection (LOD) were assigned a value of the LOD divided by the 

square root of two, and this value was included in each sum. Total DAP, DMP, and DEP metabolite levels 

were log10 transformed. DAP, DMP, and DEP values were created by averaging the two log-transformed 

pregnancy measures. For specific metabolites, if a woman was below the LOD for both measurements, 

she was classified as below LOD for the average. If she was above the LOD at either or both 

measurements, her measurements were averaged and classified in relation to the median. For 19 

women with only one DAP measurement in pregnancy, the single measure was used.

Similar to the analytic treatment of dialkyl phosphate metabolite, authors assigned a value of the LOD 

divided by the square root of two for TCPy measurements below LOD. Because a large proportion of 

women had non-detectable levels of TCPy, the chlorpyrifos-specific metabolite was categorized into 

three groups: TCPy less than LOD for both pregnancy measurements, and, for those with at least one 

detectable level, subdivided below and above the median of the average pregnancy level.

Infant birth weight, crown–heel length, and head circumference were obtained from hospital delivery 

logs and medical records. Infant ponderal index, a measure of proportionality of growth, was calculated 

as (birth weight in grams × 100)/(length in centimeters). Gestational age was obtained from medical 

records and was based on ultrasound procedures for 25% of women. Low birth weight was defined as < 

2,500 g. Preterm delivery was defined as birth at less than 37 completed weeks of gestation. A “small for 

gestational age” (SGA) birth was defined as birth weight less than the 10th percentile for gestational age 

according to ethnicity, parity, and infant sex.

Multiple linear regression models were developed to assess associations between the markers of 

pesticide exposure and length of gestation, birth weight, length, head circumference, and Ponderal

index. Multivariate logistic regression was used to evaluate associations between exposure 

measurements and low birth weight, preterm delivery, and SGA births. Covariates in the models 

included including maternal age, pregnancy weight gain, week of initial prenatal care, parity, infant sex, 

mother’s country of birth, body mass index, and family income. All models of birth weight, length, head 

circumference, and Ponderal index were adjusted for linear and quadratic terms for gestational age.

The women in this study averaged 25 years of age (SD = 5); two-thirds were multiparous, 80% were 

married, 79% had not graduated from high school, 58% were overweight or obese, 88% preferred to 

speak Spanish, and 84% were born in Mexico, with more than half residing in the United States for < 5 

years. Almost all of the women were living within 200% of the poverty level. Very few women reported 

smoking (6%), drug use (2%), or alcohol consumption (1%) during pregnancy. Approximately 28% of the 

women had worked in the fields during the pregnancy, and another 14% had worked at other jobs in 

agriculture. Eighty-five percent of the women in the study had agricultural workers living in their homes 

during their pregnancy. The median total DAP, DMP, DEP and total DAP metabolite levels for the study 
Page 18 of 105

population were 136 nmol/L, 101 nmol/L, and 22 nmol/L, respectively. Only one woman had no 

detectable DAP levels and TCPy was detected in 77% of maternal urine samples. The median level for 

TCPy was 3.3 μg/L.

The mean (± SD) duration of gestation was 38.9 ± 1.7 weeks; mean birth weight was 3,449 ± 516 g; mean 

body (crown–heel) length was 50.2 ± 2.7 cm; mean head circumference was 34.1 ± 1.5 cm; and mean 

Ponderal index was 2.7 ± 0.3 g/m3. A total of 3.7% (n = 18) of children were born of low birth weight; 

4.8% (n = 23) were SGA births, and 6.6% (n = 32) were preterm.

The authors did not observe an adverse relationship between fetal growth and any measure of in utero

organophosphate pesticide exposure. On the contrary, they found positive associations between body 

length and head circumference associated with some exposure measures, but not chlorpyrifos. They did 

observe decreases in gestational duration associated with two measures of in utero pesticide exposure: 

urinary DMP metabolites (β = –0.41 weeks per log10 unit increase; 95% CI: –0.75––0.02; p = 0.02), which 

reflects exposure to dimethyl organophosphate compounds such as malathion, not chlorpyrifos. 

Study Review

The study has numerous strengths; most notably the prospective design the longitudinal assessment of 

both exposure and outcomes. Strengths in the study design also include the use of multiple exposure 

biomarkers including quantification of non-specific (DAPs), chlorpyrifos-specific (TCPy) metabolites, 

cholinesterase (acetyl and butyl), and other environmental co-exposures. 

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. 

Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more 

accurate and objective indicators of organophosphate pesticide exposure, relative to other 

ascertainment methods such as self-report. Although they are sensitive indicators of exposure, it is 

difficult to infer chlorpyrifos effects specifically from urinary DAPs. Urinary metabolite levels may reflect 

exposure not only to a host of organophosphate parent compounds, but also to potentially less toxic 

environmental metabolites. Also, because exposure to pesticides varies considerably from day to day, 

DAPs from spot urine samples may not represent average exposure over time. However, the prenatal 

exposure measures were, with some exceptions, the average of two measurements, and thus may 

better reflect chronic exposure during the pregnancy than a one-time exposure measure.

Errors in the assignment of exposure in this prospective study will likely have resulted in attenuation of 

observed associations. The likelihood of these errors is high, and the magnitude of the bias induced 

difficult to assess. Under an assumption of no other biases, the negative findings, those for the 

association between children DAP levels and fetal development outcomes, for example, could have 

resulted from exposure measurement error.
Page 19 of 105

To identify “critical windows” of fetal development when exposure may have a greater impact, the 

authors analyzed the associations of outcomes and metabolite levels measured during moving 6-week 

windows of pregnancy (e.g., 5–10 weeks, 6–11 weeks, 7–12 weeks) using a series of multiple regression 

analyses. No period of greater impact was observed in this largely null study.

The indicators of fetal growth and development (infant birth weight, length, and head circumference, 

Ponderal index, and gestational age) were obtained from hospital delivery logs and medical records, or 

calculated using data from these records. They are likely to have been accurately and objectively 

measured. Although partially self-reported, any errors in gestational age are unlikely to be associated 

with pesticide exposures in this prospective study. 

The statistical analysis used to assess the associations between the markers of exposure and fetal 

growth and development were appropriate. However, the authors did not present nor discuss 

regression diagnostics to assess the degree to which their models met or violated the assumptions 

implicit in linear models (i.e. homoscedasticity, normality of error term distribution, independence of 

error terms, and linearity of the exposure-response relationship), other than to state that they log 

transformed exposure marker levels. The authors also conduct secondary and sensitivity analyses to 

assess influence of modeling decisions on their findings, and generally found their results to be robust. 

A reasonable set of exclusion criteria were applied which likely resulted in recruitment of a study 

population that was relatively homogeneous with respect to confounders of the in utero pesticide 

exposure-fetal growth relationship (compared to the general population), and the potential for 

confounding was thus limited by design. For example, smoking, alcohol use, and illicit drug use were not 

included in the models because very few women reported use and controlling for these variables did not 

alter the results.

Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for 

impaired fetal growth and development that are potentially associated with pesticide exposure, 

including maternal age, pregnancy weight gain, week of initial prenatal care, parity, infant sex, mother’s 

country of birth, body mass index, and family income. Variables were selected as potential confounders 

for the multivariate models based on associations reported in the literature, and included in final models 

if they changed the coefficient associated with the exposure by 10% or more. Inclusion of indicators for 

environmental tobacco smoke exposure, consumption of caffeinated beverages, history of low birth 

weight and history of preterm delivery, did not alter the relationship of pesticide metabolites and birth 

outcome and were not included in final models. In addition to restriction of confounding by design, the 

selection of the CHAMACOS population, which consists mostly of children from low-income families, 

also served to increase the relative statistical efficiency of the study, as this population is at high risk of 

developmental deficits, compared to the general population.

The results of this study by Eskenazi et al (2004) in the CHAMACOS cohort failed to demonstrate an 

adverse relationship between fetal growth and in utero organophosphate pesticide exposure as 
Page 20 of 105

assessed by multiple measures of prenatal pesticide exposure. The prospective study design and 

reasonable methods used protect from certain false-positive inducing biases, although it is possible that 

(non-differential) exposure measurement error attenuated the associations. Misclassification of the 

fetal growth outcomes was unlikely to have biased the results. Residual confounding by mismeasured or 

unmeasured factors is possible in this observational study, although it is unlikely that such confounding 

would have resulted in the null finding. 
Page 21 of 105

Article 4. Harley et al. (2011)

Association of Organophosphate Pesticide Exposure and Paraoxonase with Birth

Outcome in Mexican-American Women. Kim G. Harley, Karen Huen, Raul Aguilar Schall1, Nina T. 

Holland, Asa Bradman, Dana Boyd Barr, Brenda Eskenazi. PLoS ONE 6(8)1-10 (2011).

Study Summary

In this article by Harley et al, the authors present a follow-up assessment of the relationship between 

prenatal urinary organophosphate metabolite levels and fetal growth and gestational age in the 

CHAMACOS cohort of Mexican-American mothers and their children living Salinas Valley, an agricultural 

region of California.(Eskenazi et al, 2004) They evaluate whether paraoxonase (PON1), a key enzyme 

involved in detoxification of organophosphate pesticides, could be an effect modifier of the association 

between indicators of maternal organophosphorus pesticide exposure (DAP metabolite concentrations) 

and poorer fetal growth previously reported in this cohort. They additionally examine the main effects 

of maternal and child PON1 genotypes and PON1 activity on birth outcomes. 

Enrollment of the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) 

cohort took place at community clinics. A total of 601 out of 1,130 eligible women agreed to participate 

and were enrolled between October, 1999 and October, 2000, with 538 being followed through to 

delivery of a live born infant, whose birth weight was measured. The study populations consisted largely 

of low-income, agricultural workers. Women considered eligible for the study were less than 20 weeks 

gestation, aged 18 years or older, Medi-Cal eligible, fluent in English and/or Spanish, and planning to 

deliver at Natividad Medical Center.

Excluded from analyses were women with gestational or preexisting diabetes (n = 26), hypertension (n = 

15), twin births (n = 5), or stillbirths (n = 3), and one infant with a birth < 500 grams. In addition, motherinfant pairs with missing PON1 genotype and activity levels were excluded (n =21), leaving a final sample 

size of 467. Of these, PON1 genotype information was available for 451 mothers and 436 infants, and 

PON1 activity data were available for 371 mothers and 324 infants. 

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of 

organophosphate dialkyl phosphate metabolites (DAPs). Maternal urine samples were collected 

between 5 and 27 weeks gestation and again between 18 and 39 weeks. The dialkyl phosphates 

measured were dimethylphosphate, dimethyldithiophosphate, dimethylthiophosphate (DMPs), 

diethylphosphate, diethyldithiophosphate, and diethylthiophosphate (DEPs). 

Quantification of organophosphate metabolites using gas chromatography-tandem mass spectrometry 

and isotope dilution calibration was conducted by the Centers for Disease Control and Prevention (CDC) 

labs. Total DAP metabolite level was defined as the sum of the molar concentrations of the six DAP 

metabolites, dimethylphosphate (DMP) metabolite level as the sum of the molar concentrations of the 
Page 22 of 105

three dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of 

the molar concentrations of the three diethylphosphate metabolites. For eight women, missing 

metabolite values were imputed using linear regression to predict the missing metabolite level as a 

function of the other known metabolites for a given participant at a given time point. 

The variables for total DAP, DMP, and DEP concentrations were log-transformed to reduce the impact of 

outlier points. Metabolite measurements from the two sampling time points were averaged to create a 

summary measure of in utero organophosphate pesticide exposure.

The primary analyses were conducted using DAP concentrations unadjusted for creatinine, although 

sensitivity analyses included creatinine as a covariate in the models. Total DAPs and DMPs were 

available for 485 women; DEP metabolite levels were available for 486 women.

Maternal and umbilical cord blood samples were collected at the time of delivery. Genotyping of the 

PON1-108 and PON1192 SNPs was performed using genomic DNA extracted from blood clots and Taqman 

PCR methods.

Two PON1 enzyme activity assays (arylesterase and paraoxonase) were performed using 

spectrophotometric methods. Arylesterase activity measures the rate of hydrolysis of the substrate 

phenyl acetate, and reflects the quantity of PON1 enzyme. Paroaxonase activity measures the rate of 

hydrolysis of paraoxon, and thus reflects a combination of the catalytic efficiency and quantity of the 

PON1 enzyme. 

Birth outcome data, including infant birth weight, length, head circumference, and gestational age, were 

abstracted from medical records by a registered nurse.

Linear regression models were constructed to examine the main associations of PON1 genotype and 

activity with length of gestation, infant birth weight, birth length, and head circumference, independent 

of pesticide exposure. Maternal and child PON1 genotypes were entered into models as categorical 

variables; for PON1-108, the CC genotype was the reference, and for PON1192, the RR genotype was the 

reference. Arylesterase and paraoxonase activity in maternal and umbilical cord blood were examined 

as continuous variables, normalized by dividing by the standard deviation.

The associations of PON1 activity with birth outcomes were assessed within each PON1192 genotype by 

stratifying by genotype and by testing for interaction between activity and genotype. 

The presence of effect modification of associations between maternal DAP metabolite and birth 

outcomes by PON1 genotype and activity was assessed by stratification, and use of interaction terms. 

PON1 genotype (as a categorical variable) or activity (as a continuous variable) and cross-product terms 

were included in models previously used to assess pesticide associations in the 2004 report by Eskenazi 

et al. (Eskenazi et al, 2004) For PON1 genotype, two cross-product terms were included in the model 
Page 23 of 105

(e.g. QR*DAP concentrations and RR*DAP concentrations). Similar cross-product terms were generated 

using tertiles of PON1 activity. Statistical significance for interaction was set at p=0.1. In addition, effect 

modification by PON1 was assessed by conducting linear regressions on data stratified by PON1

genotype and by tertiles of enzyme activity.

Models of PON1 genotype included maternal pre-pregnancy BMI and maternal weight gain during 

pregnancy as covariates. Models of PON1 enzyme activity also included maternal age and country of 

birth, assay temperature, and PON1192 genotype. In models of birth weight, length and head 

circumference, the authors also controlled for linear and quadratic gestational age to examine 

associations independent of gestational duration.

As previously summarized, women in this cohort were predominantly Spanish-speaking and born in 

Mexico; 41% of the women worked in agriculture during pregnancy. More than 60% of families were 

living below the federal poverty threshold, and 89% of women had never completed high school. The 

mean maternal age was 25.5 years (SD = 5.0). All women had detectable levels of organophosphate 

pesticide metabolites in their urine during pregnancy. The geometric mean for the DAP concentrations 

during pregnancy (for the average of the two sampling periods) was 146 nmol/L (95% CI: 133, 160); of 

this, a larger proportion was DMP metabolites (GM = 109 nmol/L; 95% CI =98, 120) than DEPs (GM= 23 

nmol/L; 95% CI = 21, 25). The allele frequencies of the PON1192 Q allele and the PON1-108 T allele were 

50% and 46%, respectively, and did not differ between mothers and newborns. The mean arylesterase 

and paraoxonase activity was 33.6 U/mL (SD = 16) and 256.6 U/L (SD = 165), respectively, for infants and 

136.6 U/mL (SD = 44) and 989.0 U/L (SD = 616) for mothers.

In this study, infants’ (but not mother's) PON1 genotype and PON1 activity were associated with 

gestational age and head circumference: Infants with the susceptible PON1-108 TT genotype had shorter 

gestational age ( =-0.5 weeks, 95%CI: -0.9, 0.0) and smaller head circumference ( =-0.4 cm, 95% CI: -

0.7, 0.0) than those with the PON1-108 CC genotype. Infants’ arylesterase and paraoxonase activity were 

positively associated with gestational age. Maternal DAP concentrations were associated with shorter 

gestational age among infants of the susceptible PON1-108 TT genotype, although only the interaction 

between PON1-108 genotype and DEP metabolite concentrations was statistically significant (p-value for 

interaction = 0.09). However, maternal DAP concentrations were associated with larger birth weight (pvalue for interaction = 0.06) and head circumference (p-value for interaction =0.01) in infants with nonsusceptible genotypes. The authors conclude that infants with certain PON1 genotypes (e.g., PON1-108

TT) may be more susceptible to effects of in utero organophosphate pesticide exposure. 

Study Review

This study improves on the work by Eskenazi et al (2004) by including an assessment of effect 

modification of the association between prenatal pesticide marker levels and birth outcomes by PON1

genotype and activity. Additional strengths of this study include the prospective design, the assessment 
Page 24 of 105

of pesticide markers at two time-points during pregnancy, and the assessment of PON1 activity by two 

different methods. 

The authors speculate that a possible reason that they did not observe an association between DEPs and 

gestational age in the earlier study by Eskenazi is that it was “masked” by the effects of PON1. They note 

that the observation of an interaction only for DEP concentrations is plausible, since the known 

biologically relevant action of PON1 is with two DEP pesticides (chlorpyrifos and diazinon).

The finding of an independent effect of PON1 genotype on birth outcomes is consistent with finding of 

reductions in average gestational age and head circumference at birth associated with the PON1-108 TT 

genotype in the Mount Sinai cohort, as reported by Berkowitz et al. (Berkowitz et al, 2004). The 

association between infant PON1 (arylesterase) activity and gestational age was null in the Mount Sinai 

cohort. This is in contrast to the positive association reported by Harley et al. Maternal PON1 

(arylesterase) activity was not associated with birth outcomes in the Haley study, but positively 

associated with head circumference in the Berkowitz et al study. Finally, the interactions observed in the 

two studies differed. Berkowitz et al reported a decrease in head circumference associated with 

chlorpyrifos marker levels among newborns of mothers with low arylesterase activity in the Mount Sinai 

cohort. Harley et al, in contrast, observed a statistically significant interaction between DEP levels and 

gestational age among infants in the UC Berkeley CHAMACOS cohort with the TT PON1 genotype, but 

they did not observe significant interaction between PON1 activity and birth outcomes. In addition to 

measuring different indicators of pesticide exposure (TCPy), the PON1 genotypes and activity assessed 

for the Mount Sinai cohort differed somewhat from those assessed in the UC Berkeley cohort; 

Berkowitz et al did not assess PON1192 (only PON1-108) and paraoxonase activity (only arylesterase 

activity)

In the Columbia Mothers and Newborn Study (Whyatt et al, 2004), organophosphate pesticide levels in 

maternal blood were associated with decreased fetal growth, while in the UC Berkeley CHAMACOS 

study, modest increases in birth weight and head circumference were associated with in utero DAP 

concentrations. In fact, stratification by PON1 status seemed to enhance these positive associations, 

such that in the non-susceptible groups (e.g. PON1-108 CC, PON1192 RR, and high arylesterase activity), 

increasing DAP concentrations were significantly associated with increased birth weight and head 

circumference. 

The finding of a positive association between DAPs and DMPs and both head circumference and birth 

weight among those with the CT or CC PON1 genotypes, and a positive association between head 

circumference and DAPs among infants with RR PON1 genotypes is perplexing.

The authors speculated that it is possible that, among the less susceptible groups, high urinary DAP 

concentrations may be an indication of rapid detoxification and excretion of organophosphate 

pesticides, rather than a marker of high exposure.
Page 25 of 105

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. 

Quantification was conducted at the CDC, using published methods. However, the use of DAP 

metabolites to measure exposure has some limitations, and these were discussed by the authors. 

Although they are sensitive and relatively specific indicators of organophosphate exposure, it is difficult 

to infer chlorpyrifos effects specifically from urinary DAPs. Urinary metabolite levels may reflect 

exposure not only to a host of organophosphate parent compounds, but also exposure to the less toxic 

metabolites themselves. Because exposure to pesticides varies considerably from day to day, DAPs from 

spot urine samples may not represent average exposure over time. However, the prenatal exposure 

measures were, with some exceptions, the average of two measurements taken early and late 

pregnancy, and thus may better reflect chronic exposure during the pregnancy. Additionally, DAP 

metabolites in urine may overestimate organophosphate pesticide exposure if they reflect exposure to 

both pre-formed DAPs in the environment and the parent organophosphate compounds. 

Errors in the assignment of exposure in this prospective study will likely have resulted in attenuation of 

observed associations. The likelihood of these errors is high, and the magnitude of the bias induced 

difficult to assess. Under an assumption of no other biases, the negative findings, those for the 

association between children’s DAP levels and attention outcomes, for example, could have resulted 

from exposure measurement error. 

The indicators of fetal growth and development (infant birth weight, length, and head circumference, 

and gestational age) were obtained from hospital medical records, and ascertained by a registered 

nurse. They are likely to have been accurately and objectively measured, although the potential for 

errors in their measurement was not assessed. Although partially self-reported, any errors in gestational 

age are unlikely to be associated with pesticide exposures, PON1 genotype, or PON1 activity in this 

prospective study. 

The statistical analysis used to assess the associations between the markers of exposure and fetal 

growth and development were appropriate. However, despite a reasonable sample size for assessing 

main effects, the Harley et al study likely lacked sufficient statistical power to assess effect modification 

by PON1 genotype and PON1 activity, as the CHACAMOS Cohort study was not originally designed to 

assess interactions between prenatal pesticide exposures and these co-factors. As a result, the analysis 

is at risk of failing to detect effect modification if effect modification is truly present. 

The authors did not present nor discuss regression diagnostics to assess the degree to which their 

models met or violated the assumptions implicit in linear models (i.e. homoscedasticity, normality of 

error term distribution, independence of error terms, and linearity of the exposure-response 

relationship), other than to state that they log-transformed DAP levels to reduce the impact of outlier 

observations. The authors did conduct secondary and sensitivity analyses to assess influence of 

modeling decisions on their findings, including adjustment for creatinine. 
Page 26 of 105

A reasonable set of exclusion criteria were applied which likely resulted in recruitment of a study 

population that was relatively homogeneous with respect to confounders of the relationship between in 

utero pesticide exposure and fetal growth (compared to the general population), and the potential for 

confounding was thus limited by design. For example, smoking, alcohol use, and illicit drug use were not 

included in the models because very few women reported use and controlling for these variables did not 

alter the results.

The authors reported that factors known, a priori, to be associated with birth outcomes in this 

population were considered potential confounders, although they did not enumerate these variables. 

Covariates were retained in final models if they were also associated with PON1 genotype or activity (in 

the study population, presumably) or if their exclusion from the model changed the main effect 

coefficient by more than 10%. The authors’ final models for birth weight and head circumference 

adjusted for timing of urine collection, timing of entry into prenatal care, maternal age, parity, country 

of birth, household income, pre-pregnancy body mass index, maternal weight gain, infant sex, and 

gestational age using both linear and quadratic terms. Gestational age models adjusted for timing of 

urine collection, timing of entry into prenatal care, maternal age, parity, country of birth, and household 

income.

The authors controlled for gestational age in models of birth weight, length, and head circumference to 

examine associations independent of gestational duration. It is possible that gestational age is an 

intermediate on the causal pathway. If this is the case, the coefficients from these models can be 

interpreted as estimates of the ‘direct-effect’ of prenatal urinary DAPs and fetal growth outcomes due 

to causal pathways other than those mediated by gestational age. These pathway-specific analyses have 

a potential limitation that was not discussed by the authors. Namely, there is the potential for inducing 

bias if gestational age and fetal growth have unmeasured common causes (i.e. an unknown coexposure) (Cole and Hernan, 2002). 

In addition to restriction of confounding by design, the selection of the CHAMACOS population, which 

consists mostly of children from low-income families, also served to increase the relative statistical 

efficiency of the study, as this population is at high risk of neurodevelopmental deficits, compared to the 

general population.

The results of this study by Harley et al in the CHAMACOS cohort demonstrate modest adverse 

associations between some measures of PON1 and fetal growth, as well as providing some evidence of 

effect modification of the relationship between organophosphate pesticide exposure markers and fetal 

growth. The prospective study design and reasonable methods used protect from certain false-positive 

inducing biases, although it is possible that exposure measurement error attenuated the associations. 

Misclassification of the fetal growth outcomes was unlikely to have biased the results. Residual 

confounding by mismeasured or unmeasured factors, including bias induced by adjusting for an 

intermediate variable (gestational age), is possible in this study, and may account for the observed 

associations.
Page 27 of 105
Page 28 of 105

Work Cited

Berkowitz GS, Wetmur JG, Birman-Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS. In 

Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Environmental 

Health Perspectives. 2004;112:388–391.

Cole SR and Hernan MA. Fallibility in Estimating Direct Effects. Int. J. Epidemiol. 2002; 31 (1): 163-165.

Eskenazi B, Harley K, Bradman A, Weltzien E, Jewell NP, Barr DB, Furlong CE, Holland NT. Association of 

in Utero Organophosphate Pesticide Exposure and Fetal Growth and Length of Gestation in an 

Agricultural Population. Environmental Health Perspectives. 2004;115:792–798.

Whyatt RM, Rauh V, Barr DB, Camann DE, Andrews HF, Garfinkel R, Hoepner LA, Diaz D, Dietrich J, Reyes 

A, Tang D, Kinney PL, Perera FP. Prenatal Insecticide Exposures and Birth Weight and Length among an 

Urban Minority Cohort. Environmental Health Perspectives. 2004;112:1125–1132.
Page 29 of 105

Article 5. Engel et al. (2007)

Prenatal Organophosphate Metabolite and Organochlorine Levels and Performance on the Brazelton 

Neonatal Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. Stephanie M. Engel, 

Gertrud S. Berkowitz, Dana B. Barr, Susan L. Teitelbaum, Jodi Siskind, Stefanie J. Meisel, James G. 

Wetmur and Mary S. Wolff. American Journal of Epidemiology. 165:1397-1404, 2007.

Study Summary

Engel et al conducted a study leveraging the Children’s Environmental Health Study cohort data to 

investigate the impact of pesticide exposure on pregnancy outcomes and child neurodevelopment in an 

inner-city multiethnic cohort of women recruited during pregnancy. The study enrolled primiparous 

women presenting for prenatal care with singleton pregnancies at the Mount Sinai prenatal clinic and 

two private practices and who delivered their infants at Mount Sinai Hospital between May 1998 and 

July 2001. Participants in this study were predominantly young, unmarried, Hispanic and African 

American women with low educational attainment. 

This is the second of three published analyses of prenatal pesticide exposure and child growth and 

development in the Mount Sinai Hospital Children’s Environmental Health Study. The first (Berkowitz et 

al 2004) reported on assessments of birth weight, length and head circumference. The third (Engel et al 

2011) reports on assessments of infant development at age 12 and 24 months and child development at 

ages 6-9 years. 

The study population was drawn primarily from East Harlem and consisted largely of young Hispanic 

women (predominantly Puerto Rican), but also included African-American and Caucasian women. 

Mothers were enrolled during early pregnancy from the Prenatal Clinic and two private practices at 

Mount Sinai Hospital from May 1998 to July 2001. The study included only primiparous with singleton 

births. Mothers were excluded if they had any of the following: an initial prenatal visit after 26 weeks of 

gestation, serious chronic diseases, a serious pregnancy complication that could affect fetal growth and 

development; alcohol consumed greater than two alcoholic beverages per day, illicit drug use. Mothers 

and infants were also excluded if the child was born with a congenital malformation or severe 

prematurity. A total of 479 prenatal patients were recruited; 75 were excluded because of medical 

complications, premature birth, an infant with birth defects, inability to collect biologic specimens 

before birth, change of hospital or residence outside New York City, or refusal to continue to participate. 

The authors assessed the relationship between total diethylphosphates (DEPs), total 

dimethylphosphates (DMPs), total dialkylphosphate metabolites (DAPs), as well as malathion 

dicarboxylic acid (MDA) and indices of abnormal neonatal behavior and/or neurological integrity using 

outcome measures derived from the Brazelton Neonatal Behavioral Assessment Scale (BNBAS), which 

was administered to the infants of participating mothers within days after birth and before they left the 
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hospital. The authors also assessed modification of the associations between pesticide metabolite levels 

and metrics of neurodevelopment by maternal paraoxonase 1 activity.

Environmental exposures, sociodemographic characteristics, medical history, and lifestyle factors were 

assessed by a prenatal questionnaire administered to the mothers during the third trimester. 

Participating mothers provided urine samples during the 3rd trimester prenatal visit which were 

provided to the CDC in order to assay six dialkylphosphate metabolites and malathion dicarboxylic acid 

(MDA). The investigators imputed values for samples with one or more missing values using methods 

described by Eskenazi et al. (2004), extending the method to also include imputation of metabolite 

values for women who were missing two metabolites in a class. Diethyl- and dimethylphosphate 

metabolites were summed on a molar basis (as nm/liter) to obtain total diethylphosphates (DEPs) and 

total dimethylphosphates (DMPs), respectively, and together to obtain total dialkylphosphates (DAP) 

levels. All biomarkers except MDA were included in analyses as log10 linear terms in the initial model 

selection stage. Results were reported for DEPs dichotomized at the median level. MDA was 

dichotomized at the limit of detection because only 21.6% of the population had detectable values. 

Maternal plasma paraoxonase 1 activity was classified in tertiles.

A high prevalence of pesticide use was reported in this cohort of pregnant women residing in New York 

City. The study cohort was enrolled between 1998 and 2002, which, the authors point out, overlaps 

with a period when chlorpyrifos and diazinon were being phased out of residential use. However, the 

authors note that in 1997, chlorpyrifos was the most heavily used insecticide by pest control operators 

at the New York City Housing Authority. The authors noted that all of the women who participated in 

personal air monitoring of their home had detectable levels of diazinon, chlorpyrifos, carbamate 

propoxur, and o-phenylphenol. 46% of women in the cohort reported that pesticides had been applied 

in their home during their pregnancy. Total diethylphosphates were calculated as the sum of 

diethyldithiophosphate, diethylphosphate, and diethylthiophosphate. In this cohort, the DEPs were 

detectable in 88.8% of the mothers, with a median (IQR) of 24.7 nm/liter (8.9–52.8 nm/liter). Total 

dimethylphosphates were calculated as the sum of dimethyldithiophosphate, dimethylphosphate, 

dimethylthiophosphate. DMPs were detectable in 90.2% of the mothers, with a median (IQR) of 47.8 

nm/liter (15.6–149.2 nm/liter). Total dialkylphosphates were detectable in 96.5% of the mothers, with a 

median (IQR) of 82.0 nm/liter (35.2–194.7 nm/liter).

To assess infants’ neurodevelopment, the investigators conducted a behavioral assessment using the 

Brazelton Neonatal Behavioral Assessment Scale (BNBAS). All BNBAS examinations were completed 

within 5 days of delivery, and 86.1% were conducted within two days of delivery. The BNBAS includes 

28 behavioral items and 18 primitive reflexes. The exam does not yield a single score but instead 

assesses the infant across different developmental areas; infants' behaviors were scored according to 

seven domains: 

1) Habituation - the ability to respond to and inhibit discrete stimuli while asleep; 

2) Orientation -attention to visual and auditory stimuli and quality of overall alertness; 
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3) Motor - motor performance and quality of movement and tone; 

4) Range of state - a measure of infant arousal and state lability; 

5) Regulation of state - the ability to regulate state in the face of increasing levels of stimulation; 

6) Autonomic stability - signs of stress related to homeostatic adjustments of the central nervous 

system;

7) The number and type of abnormal primitive reflexes 

Of the 308 infants evaluated, all 18 primitive reflexes assessed in the BNBAS, 64% had all reflexes within 

the normal range, 18% had one abnormal reflex, 8% had two abnormal reflexes, and 10% had three or 

more abnormal reflexes. The majority of abnormal reflexes were due to hypotonicity (which includes a 

reflex not elicited after stimulation). Authors do not discuss the prevalence of other common causes of 

hypotonicity such as hypothyroidism, hypoglycemia, hypoxic encephalopathy. 

The authors observed an association between both generic and pesticide-specific biomarkers of prenatal 

organophosphate pesticides and an increased number of abnormal primitive reflexes which are 

considered a critical marker of neurologic integrity. Notably, no statistically significant adverse 

associations were found for the dialkylphosphate metabolites and any measured behavior in the 

author’s primary analyses. 

They did observe, however, an interaction between paraoxonase 1expression levels and total 

dimethylphosphates on risk of abnormal reflexes, such that infants born to women in the first 

(interaction p = 0.002) and second (interaction p = 0.01) tertiles (slower metabolizers) had a greater risk 

of abnormal reflexes than infants of those in the highest tertile (fast metabolizers). In the first tertile of 

paraoxonase 1 expression, the relative risks (RR) of having abnormal reflexes were 1.78 (1.01, 3.14) 

associated with total diethylphosphates, 1.96 (1.27, 3.03) for total dialkylphosphates, and total 

dialkylphosphates 2.38 (1.37, 4.15). In the second tertile of paraoxonase 1 expression, the 

corresponding relative risks (RR) were 1.42 (0.85, 2.35) for total diethylphosphates, 1.66 (1.03, 2.65), for 

total dialkylphosphates, and total dialkylphosphates 1.75 (0.96, 3.17). No increased risk of abnormal 

reflexes with increasing exposure was found for women in the highest tertile of paraoxonase 1 

expression, nor was a similar interaction observed between total diethylphosphates and paraoxonase 1.

Higher levels of total diethylphosphates and total dialkylphosphates, too, were associated with an 

increase in the risk of having abnormal reflexes. Subjects with prenatal total diethylphosphates levels 

above the median delivered infants who were 2.3 times more likely to have at least two abnormal 

reflexes (95% CI: 1.1, 5.0). Among women with lower tertiles of paraoxonase 1 expression (i.e., slow 

organophosphate pesticide metabolizers) the authors also observed a significantly increased risk of 

abnormal reflexes with increasing exposure.

Relative to the first quartile, quartiles 2–4 of total diethylphosphates, total dimethylphosphates, and 

total dialkylphosphates were associated with an increased proportion of abnormal reflexes, although 
Page 32 of 105

the associations did not increase monotonically and varied in their strength and precision. The authors 

speculated that this may due to a threshold effect.

With the exception of MDA, there appeared to be stronger associations between metabolite levels and 

abnormal reflexes for examinations performed after the first day of life, although only the total 

dimethylphosphates interaction p-valueinteraction= 0.10. In contrast, an interaction (pinteraction = 0.10) with 

MDA showed that, for infants examined before day 2, the relation between MDA and abnormal reflexes 

was stronger (RR= 2.51, 95% CI: 1.61,3.90) compared with that for infants examined later (RR= 1.34, 

95%CI: 0.72, 2.49).

The authors noted that their findings were remarkably consistent with those of Young et al. 

(Neurotoxicology, 2005) who reported that for each log10 unit increase in total dialkylphosphates, there 

was a 26% increased risk of abnormal reflexes, (compared to 32% increased risk in this study). The 

magnitudes of association were also similar for total diethylphosphates (RRYoung = 1.25, RRcurrent study = 

1.49) and total dimethylphosphates (RRYoung = 1.20, RRcurrent study) = 1.13). Both studies reported stronger 

associations between dialkylphosphate metabolites and abnormal reflexes for older children, although, 

in the present study, the interaction with day of administration was not statistically significant.

The association between MDA, a malathion-specific metabolite, and abnormal reflexes appeared to be 

age dependent in this study, with the most pronounced effect observed among infants evaluated within 

the first day of life. The authors noted that this finding conflicts with the suggestion of later effects for 

dialkylphosphate metabolites, including dimethylphosphates, which encompasses nonspecific 

metabolites of malathion. The authors concluded that it is unclear what pesticides may be driving this 

disparity.

Study Review

This was a well conducted prospective study conducted in a young, predominantly minority population. 

The study design, analytic approach, and statistical analyses were appropriate. The authors used a 

sensitive albeit non-specific marker of chlorpyrifos pesticide exposure (DEP/DAP). While the analytic 

methods appear sound, authors assessed these markers using only a single maternal urine specimen 

taken during the third trimester. However, in their discussion, the authors note that organophosphate 

pesticide metabolites have short half-lives. If sources and patterns of exposure remain static, a single 

measurement would be a good indicator of pesticide exposure during pregnancy. If, however, sources 

and exposure patterns change over short time-spans, perhaps on the order of days to weeks, such an 

exposure assessment would not necessarily provide a good indicator of the relevant exposure. Like the 

many other sources of error possible in this study, this, too is likely to result in a non-differential 

misclassification of the exposure, with respect to the disease outcome. Serial measurements of urinary 

biomarkers of pesticide exposure in the third trimester would shed light on these patterns, although the 

benefits of such information are offset by the added cost, logistical complexity expense, and burden on 

the study participants. 
Page 33 of 105

The Neonatal Behavioral Assessment Scale (NBAS), used to ascertain the health endpoints in this study, 

is designed to examine individual differences in newborn behavior (http://www.brazeltoninstitute.com/intro.html). Examinations took place in a quiet, semi-darkened, warm room adjacent to 

the neonatal nursery, or in the mother’s private room. Examiners in this study were either trained and 

certified by the Brazelton Institute or trained by a certified examiner. It was not specified how many 

examinations were conducted by the certified examiners, nor how recently the certified examiners had 

received their certification. Procedures for tracking the quality of the assessment process were also not 

discussed in the report. The health outcome assessment methodology used in this study is less than 

objective; assessments rely on substantial interaction between the infant and the examiner. Procedures 

for ensuring that the examiners were blinded to the participants’ exposures were not discussed in the 

article. However, authors did include examiner as an indicator variable in statistical analyses to control 

for slight differences in examiner methodology.

Moreover, the behavioral assessment was administered before hospital discharge only on a subset of 

children in the cohort (n =311/404). The BNBAS was not administered if the infant was admitted to the 

NICU (n = 21); if the infant was delivered and discharged over a weekend (n = 43); if the parent refused 

(n = 5); if the infant was not testable (n = 2); or if study personnel were unavailable (n = 22). Factors 

related to weekend delivery (e.g., fewer inductions) would be underrepresented among the tested 

subjects. This missing data has the potential to result in a selection bias, reduce the degree of precision 

with which associations were estimated, and limit the generalizability of the study findings. However, 

the authors’ explanation for the incomplete ascertainment is presented and seems appropriate and 

largely logistical in nature.

The authors employed a reasonable set of exclusion criteria that likely resulted in recruitment of a study 

population that was relatively homogeneous with respect to confounders of the in utero pesticide 

exposure-neurodevelopment relationship (compared to the general population). The potential for 

confounding was thus limited by design (restriction). Bias due to confounding was also controlled in the 

analysis, by assessing and adjusting for risk factors for neuromuscular functioning that are potentially 

associated with pesticide exposure, including maternal age, race, marital status, education, cesarean 

delivery, delivery anesthesia, infant age at examination, infant gender, infant jaundice, and smoking, 

alcohol consumption, caffeine consumption, or illicit drug use during pregnancy, and the examiner 

conducting the BNBAS. 

The use of an automated statistical procedure (backward elimination and change of effect measure) as a 

means to eliminate potential covariates is discouraged in some epidemiologic circles on the basis that 

assessment of confounding is generally not considered to be a statistical endeavor, although they 

continue to be used by others, largely on the grounds that it is ‘objective’. The authors used a change-inestimate approach, rather than a reliance on the statistical significance (p-value) associated with a 

covariate; their variable selection rule has been shown to produce more reliable models than variable 

selection methods based on statistical significance (Greenland, 1989). Because they used a backward 

elimination procedure, they considered confounding by a given risk factor in the context of adjustment 
Page 34 of 105

for other covariates. The approach to covariate selection is a minor consideration in this study, and 

different approaches are not likely to qualitatively change the study findings or conclusions. A robust 

discussion of covariate selection concepts can be found in Chapter 15 of Modern Epidemiology (Kenneth 

J. Rothman, Sander Greenland, Timothy L. Lash).

The authors noted that post-entry exclusions/loss-to follow-up in the study may have biased their 

findings if they were associated with both organophosphate pesticide levels and neonatal behavior. 

Missing data has the potential to induce a selection bias if the reasons for missingness are determined 

by exposure (or a predictor of exposure) and are also not independent of neurodevelopment. Most of 

the excluded women were those who moved out of the study area, were lost-to-follow up, or lacked 

prenatal biologic specimens, which largely reflect socioeconomic condition. Participants included and 

excluded differed slighted with regard to maternal age and race; however, these factors were not 

associated with number of abnormal reflexes in this study. These considerations may, however, limit the 

generalizability of the study findings. 

The statistical analysis used in the study was appropriate, although several subtleties in the analytic 

procedures are worthy of mention. The main finding of the study was a statistical interaction between 

biomarker levels of organophosphate pesticides and paraoxon activity. It was not clear whether this 

effect modification was consideration a priori. Also, the authors imputed missing data, and this may 1) 

affect the precision of association estimates and 2) result in attenuated effect estimates as a result of 

exposure measurement error. The description of the imputation procedure used was insufficient for 

drawing strong conclusions regarding its validity with respect to the magnitude of measurement 

error/bias, and its effect on estimates of precision. 

The generalizability of the study may be limited as consequence of the particulars of the study 

population assessed and the numerous exclusion criteria applied. This study population consisted of 

primiparous women, recruited early on in their pregnancies from the prenatal clinic and two private 

practices at Mount Sinai Hospital in New York from March 1998 to March 2002, consisted 

predominantly of young Hispanic women with low education. Sixty-eight percent were less than 25 

years old. Just over half were Hispanic (51.1%), although substantial numbers of White (20.3%) and 

African-American (27.3%) women also participated. Just under half (47.6%) classified themselves as 

being single. The exclusion criteria applied to potential study participants further focused the population 

assessed, relative to the general population of pregnant women. In the likely situation that the 

prevalence of important modifiers of the exposure-fetal development association in this focused study 

population is different from that in other populations of concern, generalizability will be limited. 

Important modifiers to consider may include time spent in the home, patterns of food preparation and 

consumption, health consciousness, the baseline health status of the mother (and father), and a host of 

socioeconomic determinants including access to health care. The investigators finding of effect 

modification of the biomarker-fetal growth relationship by PON1 activity is illustrative of limits on the 

generalizability of the study; populations who have high levels of paraoxonase activity may vary by 

genetotypic and phenotypic determinants. These factors were not discussed in the report, but it has 
Page 35 of 105

been noted previously that there is 10- to 40-fold inter-individual variability in rates of paraoxon 

hydrolysis (Playfer et al 1976). 

The primary finding was an interaction between high paraoxonase 1expression levels and 

organophosphate pesticide exposure, whereby fast metabolizers had lower risk of abnormalities 

associated with prenatal pesticide metabolite levels in maternal urine samples. As with their previous 

report, bias due to confounding, and selection of study participants is unlikely to have large and 

qualitative impact on the association observed. To the extent that prenatal chlorpyrifos is the true 

exposure of interest, the assessment of non-specific organophosphate pesticide metabolites in a single 

sample of prenatal urine is a primary potential source of exposure measurement error. Because of the 

prospective design, information bias is expected to result in attenuation of the true (unobserved) 

associations, toward (observed) associations that are relatively smaller, or null. 
Page 36 of 105

Works Cited

Berkowitz GS, Wetmur JG, Birman-Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS. In 

Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Environmental 

Health Perspectives. 2004;112:388–391.

Breslow NE, Day NE. Statistical Methods in Cancer Research: Volume II: The Design and Analysis of 

Cohort Studies. Lyon: IARC, 1987. 

Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canfield RL, Wolff MS. Prenatal Exposure to 

Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. Environmental Health 

Perspectives 2011;119:1182-1188 

Greenland, S. Modeling and Variable Selection in Epidemiologic Analysis. American Journal of Public 

Health. 1989; 79:340-349.

Miettinen OS. Stratification by a multivariate confounder score. American Journal of Epidemiology 

1976;104:609- 620. 

Playfer J, Eze LC, Bullen MF, Evans DAP. Genetic polymorphism and inter-ethnic variability of plasma 

paraoxonase activity. J Med Genet. 1976;13:337–342. 

Rothman KJ, Greenland S, Lash TL. Modern Epidemiology. Third Edition. Lippincott, Williams, & Wilkins, 

2008.
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Article 6. Young et al. (2005)

Association Between In Utero Organophosphate Pesticide Exposure and Abnormal Reflexes in 

Neonates. Jessica G. Young, Brenda Eskenazi, Eleanor A. Gladstone, Asa Bradman, Lesley Pedersen, 

Caroline Johnson, Dana B. Barr, Clement E. Furlong, Nina T. Holland. NeuroToxicology. 26:199–209 

(2005).

Study Summary

Young et al. assessed the relationship between in utero and early postnatal organophosphate pesticide 

exposure and neonatal neurobehavior in the Center for the Health Assessment of Mothers and Children 

of Salinas (CHAMACOS) cohort. Urinary levels of dialkylphosphate (DAP) metabolites, including dimethyl 

and diethylphosphate metabolites, measured at approximately fourteen and twenty-six weeks 

gestation, and once post-delivery, measured at approximately seven days postpartum were assessed as 

markers of exposure to organophosphate pesticides. Cognitive development was assessed using the 

Brazelton Neonatal Behavioral Assessment Scale (BNBAS). 

The CHAMACOS study is a longitudinal birth cohort study of environmental exposures and the health of 

pregnant women and their children living in the Salinas Valley, California. The Salinas Valley is a major 

center of agricultural production in the United States with approximately 500,000 pounds of 

organophosphate pesticides applied annually, according to the CDC. (California EPA. Pesticide Use 

Reporting 2001 Summary Data, 2002. (www.cdpr.ca.gov/docs/pur/pur01rep/01_pur.htm) Pregnant 

women initiating prenatal care at the Natividad Medical Center, a county hospital located in the city of 

Salinas, were recruited to participate in the Center for the Health Assessment of Mothers and Children 

of Salinas (CHAMACOS) project. Both the source and study populations consisted largely of low-income, 

Latina agricultural workers. Women considered eligible for the study were less than 20 weeks gestation, 

aged 18 years or older, Medi-Cal eligible, fluent in English and/or Spanish, and planning to deliver at 

Natividad Medical Center. A total of 601 women were enrolled between October, 1999 and October, 

2000, with 528 followed through delivery of a live born infant. The BNBAS was performed on 421 

infants. Eight twins and 27 preterm infants were excluded from analysis.

Exposure was assessed by measurement of organophosphate dialkylphosphate (DAP) metabolites in 

maternal urine samples collected between 5 and 27 weeks gestation and again between 18 and 39 

weeks. Maternal urine specimens were generally collected at the time of the prenatal and post-delivery 

interviews. The post-delivery urines were collected within 1 week of delivery for 73% of the sample, 

with the remainder obtained up to 176 days afterwards. Quantification of metabolites was conducted 

by Centers for Disease Control and Prevention (CDC) labs. Total DAP metabolite level was defined as the 

sum of the molar concentrations of the six DAP metabolites, dimethylphosphate (DMP) metabolite level 

as the sum of the molar concentrations of the three dimethylphosphate metabolites only, and 

diethylphosphate (DEP) metabolite level as the sum of the molar concentrations of the three 

diethylphosphate metabolites. Total DAP, DMPT, and DEP levels were determined for each participant 
Page 38 of 105

for each of the two pregnancy urine samples and for the post-delivery urine sample. Individual 

metabolite levels below the limit of detection (LOD) were assigned a value of the LOD divided by the 

square root of two, and this value was included in each sum. A small number of missing metabolite 

values were imputed using regression to predict the missing metabolite level as a function of the other 

known metabolites for given participant at a given time point. The total DAP, DMP, and DEP metabolite 

levels were then log10 transformed.

For 30% of participating infants, developmental outcomes were assessed prior to discharge from the 

hospital; the remainder was assessed at the CHAMACOS research office or in the participant’s home. 

Assessments were performed away from the mother in a private room with low-level light and noise 

control. The BNBAS, consists of 28 behavioral measurements scored on a nine-point scale and 18 reflex 

items scored on a four-point scale. These scores were classified into seven clusters: habituation, 

orientation, motor performance, range of state, regulation of state, autonomic stability, and reflex. 

Higher scores on the behavioral clusters indicate more optimal functioning while higher scores on the 

reflex cluster indicate less optimal functioning. Missing individual BNBAS items for the six behavioral 

clusters were imputed as the median value of known scores within the cluster associated with the 

missing item. If all items in a particular cluster were missing, then the cluster score was considered 

missing. Missing individual reflex items comprising the reflex cluster were not imputed.

Separate linear regression models were fit for six of the seven clusters of the BNBAS. Poisson regression 

was used to model the association between urinary metabolite levels and number of abnormal reflexes. 

Independent variables included the average of prenatal metabolite levels, the postnatal level, and 

relevant covariates. Models relating metabolite levels to each cluster score were initially fit including 

the postnatal metabolite level and the average of the two prenatal metabolite levels. For eighteen 

mothers missing one of the two pregnancy urine samples, the one remaining was used to represent the 

average. All models adjusted for covariates, and were rerun with creatinine-adjusted metabolite levels. 

Analyses were performed for the entire study sample and stratified by the median age at BNBAS 

assessment (3 days).

Of the 378 infants included in this analysis, 47.5% were male. The mean maternal age was 26±5 years. 

Most mothers lived at or below the poverty line (64%), were born in Mexico (85%), had previous live 

births (68%), were married (81%), and had not completed high school (81%). Few women smoked (5%) 

or drank alcohol (16%) during pregnancy, although most reported caffeine use (75%). Only 1% of the 

sample reported any illicit drug use during pregnancy. 

The authors observed a statistically significant association between organophosphate pesticide 

exposure and the reflex cluster of the BNBAS, both for the entire sample and for infants assessed at 

greater than 3 days of age. Among the >3 day old infants, increasing average prenatal urinary metabolite 

levels were associated with both an increase in number of abnormal reflexes (total DAP: β = 0.53, 95% CI 

= 0.23, 0.82; DMP: β = 0.41, 95% CI = 0.12, 0.69; DEP: β = 0.37, 95% CI = 0.09, 0.64), and the proportion 

of infants with more than three abnormal reflexes (total DAP: OR = 4.9, 95% CI = 1.5, 16.1; DMP: OR = 
Page 39 of 105

3.2, 95% CI = 1.1, 9.8; DEP: OR = 3.4, 95% CI = 1.2, 9.9). No other associations between either prenatal or 

postnatal DAPs with any other developmental outcomes were observed. 

Study Review

Overall, authors performed a well-conducted study of pre- and post-natal pesticide exposure and 

adverse neurodevelopmental outcomes. The prospective nature of the study, use of multiple 

biomarkers of exposure, and extensive data collection allowing for adjustment of potentially 

confounding variables are key strengths of this work. For the purpose of informing the nature of the 

relation between chlorpyrifos per se and these outcomes, use of non-specific OP metabolites is a 

disadvantage. Other limitations of this work are also noted. 

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. 

Quantification was conducted at the CDC, using published methods. DAP metabolites are sensitive albeit 

non-specific markers of organophosphate pesticide exposure. They are likely more accurate and 

objective indicators of organophosphate pesticide exposure, relative to other ascertainment methods 

such as self-report. In their discussion, the authors note that, “presently, there are no better methods of 

exposure assessment to capture exposure to multiple organophosphate compounds.” It is difficult to 

infer chlorpyrifos effects specifically using metabolite levels, as they also indicate exposure to other 

organophosphates. The study population was likely sustained many other potentially relevant exposures 

to multiple chemicals and other pesticides which may affect development. It is not possible to 

disentangle the various effects from these potential co-exposures and chlorpyrifos in this study. 

Moreover, the degree to which metabolites in prenatal maternal urine samples reflect ‘usual’ levels, and 

thus usual exposure, is uncertain. The authors’ observation of large within-mother variability in pesticide 

marker levels between the two prenatal sampling periods is evidence that a single sample may not be 

representative of longer-term levels in this population, particularly when exposures are transient and 

highly variable. Averaging of the two prenatal exposure measures may ameliorate this issue somewhat.

Metabolite levels in postnatal urines likely reflect early postnatal, rather than in utero exposure, given 

the short half-life of organophosphate pesticides. This is particularly likely for the 27% of postnatal 

samples collected greater than one week after delivery. The effect of errors in the assessment of 

organophosphate pesticide exposure, whether due to mismeasurement of the metabolites, imperfect 

correlation between metabolite levels and pesticide exposure, or imputation of missing data, is likely to 

be an attenuation of the association (a bias toward no association) in this prospective study.

The BNBAS was appropriately administered by one of four examiners trained by a BNBAS certified 

trainer. BNBAS is considered to provide an appropriate evaluation of newborn development when 

administered up to the second month of life, and is considered a valid tool, when properly conducted. 

The cluster approach to classification of BNBAS scores has precedence in the literature. Missing BNBAS 

data points were imputed, if possible. Indeed, there was a substantial amount of missing outcome data, 

due largely to the particular requirements of the assessment (e.g. that the infant be asleep immediately 

prior to the assessment of habituation and that the infant by crying prior to assessment of consolability 
Page 40 of 105

and self-quieting). For example, fifty-four percent of the sample was missing all habituation items and 

an additional 22% required at least one imputation. Forty percent of the sample required at least one 

imputation of the four items comprising the regulation of state cluster, with 13, 25, and 0.5% of the 

sample requiring one, two, and three items imputed, respectively. Individual items with the most 

missing values for the regulation of state cluster were consolability and self-quieting. Two infants were 

missing all items comprising the orientation cluster score. The number of abnormal reflexes was 

calculated based on non-missing values only. Reflexes with the most missing values included incurvation 

(9%), nystagmus (7%), and tonic neck reflex (7%). Missing values did not exceed 4% for the remaining 

reflex items. Again, the effect of errors in the ascertainment of developmental outcomes is likely to 

result in attenuation of observed associations as it is unlikely that missingness in outcome measure is 

related to pesticide exposure, as examiners were blind to the infant’s exposure status and the 

imputation procedures were unlikely to induce bias. 

The analyses presented were appropriate to the data collected and the research question posed. The 

authors averaged participants’ pesticide marker levels from the two prenatal urine samples (if available), 

and, as a separate variable, the postnatal metabolite level. Sixteen mothers were missing the postdelivery urine. In order not to exclude those with missing post-delivery urines from the statistical 

models, results were re-examined with the variable representing post-delivery metabolite level 

excluded from all models. The effect of the missing exposure data is a decrease in the precision, and also 

likely attenuation of observed associations due to measurement error. 

Potential confounders considered in this investigation included primarily predictors of BNBAS 

performance, including: maternal age, pre-pregnancy body mass index, self-reported smoking, alcohol, 

caffeine, and illicit drug use during pregnancy, vitamin use during pregnancy, gestational age at which 

prenatal care was initiated, total number of prenatal care visits, average blood pressure during 

pregnancy, parity, method of delivery, general anesthesia use during delivery, breastfeeding initiated 

after delivery, and poverty level. Other covariates included infant sex, age in days at BNBAS assessment, 

minutes since last fed at BNBAS, and BNBAS examiner. Missing covariate values were imputed by 

random selection of an individual among all participants with known values for the covariate and 

assigning the missing value the selected participant’s value. 

Covariates were selected for inclusion in final models if they were statistically significantly associated 

with each of the seven cluster scores in univariate models, (alpha = 0.15). The authors used a less 

conservative alpha level as a cut-off for selection of covariates, a somewhat more conservative approach 

to covariate selection, relative to use of the conventional alpha level of 0.05. However, the use of a 

statistical test as a means to select covariates from a larger set of potential confounders is discouraged 

in some epidemiologic circles (Miettinen 1976, Breslow and Day 1987, Greenland 1989). The 

assessment of confounding is generally not considered to be a statistical endeavor, in part because the 

association between the confounder and the outcome is just one factor that determines the degree of 

confounding, and also because the statistical significance of an association is a function not only of the 

magnitude of the association but also of other factors (e.g., sample size, and sample variability) which 
Page 41 of 105

are irrelevant to bias due to confounding. Moreover, the use of significance testing in the selection of 

confounders treats false negative errors (i.e. the deleting a confounder) as secondary to false positive 

error (inclusion of a non-confounder). This implicit ranking of error is backward, as deleting a 

confounder introduces bias and is justified only if the bias induced by its exclusion is tolerably small or 

deemed to be worth the precision gain, whereas including a non-confounder only (potentially) reduces 

precision by increasing the size of the model. This is a small consideration in this study, and different 

approaches are not likely to qualitatively change the study findings or conclusions. Birth weight and 

gestational age were considered potential causal intermediates in this relation, and not confounders.

Regardless of the confounder evaluation conducted by the investigators, residual confounding due to 

poorly measured or unmeasured confounders is likely to have influenced the observed associations. 

Missing covariate data were imputed. The resulting analysis is relatively more precise, owing to the 

larger sample size analyzed, but this statistical efficiency comes at the trade off of bias due to residual 

confounding by imperfectly measured covariates. Socioeconomic conditions and other difficult to 

measure determinants of early life development may further confound the reported associations in the 

direction of more deleterious effects associated with the organophosphate metabolites and with 

unknown magnitude. The potential for this bias may be mitigated somewhat due to restriction in the

variability of these confounding factors in this relatively homogeneous study population. 

The generalizability of the findings to other populations may be limited, due to the particular nature of 

this study population with respect to factors which may modify the association between 

organophosphate exposures and infant development. 

The results of this investigation are suggestive of a detrimental association between prenatal 

organophosphate exposure, as measured by maternal urinary metabolite levels, and abnormal reflexes, 

as measured by the BNBAS, particularly in infants assessed after the first 3 days of life. This study may 

be limited in its direct contribution to the literature base regarding the specific association between 

chlorpyrifos and developmental outcomes; although approximately 500,000 pounds of 

organophosphate pesticides are applied annually in Salinas Valley, chlorpyrifos constitutes only a small 

percentage of the pesticides applied. Although the prospective study design and reasonable methods 

protect from certain false-positive inducing biases, there remains the potential of residual confounding 

in the direction of an adverse effect on development by unmeasured factors. 
Page 42 of 105

Article 7. Rauh et al. (2006)

Impact of Prenatal Chlorpyrifos Exposure on Neurodevelopment in the First 3 Years of Life Among 

Inner-City Children. Virginia A. Rauh, Robin Garfinkel, Frederica P. Perera, Howard F. Andrews, Lori 

Hoepner, Dana B. Barr, Ralph Whitehead, Deliang Tang, Robin W. Whyatt. Pediatrics. 18(6): 1835-

1859. 

Study Summary

To evaluate the affect/impact of prenatal exposure to ambient and indoor pollutants including 

pesticides, the investigators conducted a prospective cohort study among inner-city (NYC) mothers and 

their babies born between February 1998 and May 2002. A major aspect of the protocol was to assess 

pesticide exposure, specifically chlorpyrifos, and its impact on pregnancy outcome, neurodevelopment, 

and behavior. Previous publications presented outcomes of this cohort such as birth weight and length 

findings (Whyatt, et al, 2004 and Perera et al, 2003.) This publication presents findings focused on the 

neurodevelopmental assessments made at the 12, 24, and 36 month time points. 

The women enrolled into the study were identified during their pregnancy, through the New York 

Presbyterian Medical Center and Harlem Hospital. To be eligible for the study they were required to be 

between the ages of 18 and 35 years, non-smokers (later verified by blood cotinine levels,) and without 

a history of diabetes, hypertension, HIV infection, or drug abuse. They must have sought prenatal care 

by the 20th week of pregnancy and self-identified as African American or Dominican. At the time of this 

report, the authors explain that out of the original 648 pregnant women consented, 536 (83%) of them 

and 254 children were still actively enrolled at the 3 year milestone. Of these children, 228 completed 

evaluations at 12 months of age, 227 at 24 months, and 228 at 36 months, with 189 children having full 

data at all 3 time points. 

The primary source of chlorpyrifos measurements were samples of cord blood collected at delivery as 

well as samples of maternal blood collected shortly after (within 2 days of) delivery. These serum levels 

were supplemented by maternal self-report of residential pesticide use during the third trimester of 

pregnancy. Using findings from previous analyses of chlorpyrifos effects on birth weight (Whyatt, et al, 

2004 and Perera et al, 2003,) the investigators chose a cord blood chlorpyrifos limit of 6.17pg/g to 

define the exposure groups. Children were categorized as having either high (>6.17pg/g) or low 

(<6.17pg/g) prenatal exposure. 

Two instruments were used to evaluate neurodevelopment and child behavior. The Bayley Scales of 

Infant Development II (BSID-II) was used to generate a Mental Development Index (MDI) and a 

Psychomotor Development Index (PDI) at 12, 24, and 36 months of age. Second, the behavior of the 

children was described by the mother using the 99-item Child Behavior Checklist (CBCL) and the 

resulting syndrome scale scores. Finally, the Home Observation for Measurement of the Environment 

(HOME) instrument was used to assess the quality of the child-care environment. This instrument 
Page 43 of 105

collects data regarding the physical and interactive qualities of the child’s home as a measure of mental 

stimulation and interactions.

The maternal and cord blood samples also provided cotinine levels to confirm maternal report of 

environmental tobacco smoke (ETS) exposures and lead levels to understand the role lead exposure may 

play interacting with the outcomes of measure. During their third trimester of pregnancy, the women 

completed a 45-minute questionnaire to collect demographics, education and occupational history, 

income, active and passive smoking, alcohol and drug use during pregnancy, and residential pesticide 

use. From this self-report questionnaire and additional analyte measurement in biological sample, 

authors obtained information regarding potentially confounding variables. 

Multivariate linear regression and logistic regression were used to estimate associations between 

prenatal chlorpyrifos exposure and developmental outcomes. General linear modeling of the repeated 

measures (12, 24, 36 month evaluations) was also used to estimate the effect over time that 

chlorpyrifos had on neurodevelopment. All models included prenatal ETS exposure, gender, ethnicity, 

gestational age at birth, quality of home care-taking environment, maternal education, and maternal IQ. 

Other measured co-variables including blood lead levels were evaluated as to the potential confounding 

influence on the main effect, and were not included in final models due to lack of statistical or biological 

evidence of confounding bias.

The descriptive statistics present the cohort as relatively homogenous with respect to socioeconomic 

variables but showing a wide range of chlorpyrifos exposure. Chlorpyrifos levels ranged from 

undetectable to 63 pg/g with 20.6% of the cord blood results falling into the high exposure category. 

Significant differences in chlorpyrifos exposure were seen across ethnic categories with 24.2 % of African 

American participants and 14.9% of Dominican participants classified as highly exposed (p=.02). Birth 

weight and prenatal exposure to ETS were also significantly associated with the exposure categories 

with infants in the high exposure group weighing approximately 211g less than babies in the low 

exposure group (p <.05) and 56% of those in the high exposure group reporting prenatal ETS versus 32% 

in the low exposure group (p = .002).

When the BSID-II results were analyzed in terms of exposure group, no significant differences were seen 

at the 12 and 24 month time points. At the 36-month milestone, although MDI scores did not show a 

significant difference across the exposure groups, PDI scores were significantly lower among children in 

the highly exposed group (95.69 vs. 101.63, p = .006). Statistically significant differences were also seen 

at the 36 month milestone, in mental (cognitive) and psychomotor delays (p = .048 and .002 

respectively.) 

When analyzing the 36-month child behavior checklist (CBCL) (behavioral) scores, results from the 12 

and 24 month milestones were not significantly associated with exposure status. At 36 months, 

significant differences were observed in the general category of attention-problems (p=.010) and in the 

more specific DSM-IV scale for ADHD problems (p = .018). 
Page 44 of 105

The multivariate regression models included gender, ethnicity, gestational age at birth, home 

environment, prenatal ETS exposure, maternal education, and maternal intelligence. The authors 

reported that the difference in MDI scores was “marginally significant” (p = .06) with the exposed group 

scoring an average of 3.3 points lower. Among the significant covariants were race/ethnicity (at 24 and 

36 months), gender of infant (at 12, 24, and 36 months), gestational age (at 24 and 36 months), low 

maternal education (at 36 months), and HOME score (at 36 months) with Dominicans, male children, 

low gestational age, low maternal education (< high school,) and low HOME score all linked to lower 

MDI scores. It was also reported that environmental exposures (chlorpyrifos and ETS) and 

sociodemographic covariables accounted for 25% of the variance seen in the 36-month MDI scores. 

When the same multivariate regression models were calculated regarding the PDI scores, none of the 12 

or 24 month PDI scores showed significant effects. The 36 month scores did show significant effects for 

chlorpyrifos exposure, race, and gestational age. 

Next, investigators calculated estimates of adjusted risk for developmental delays (MDI and PDI) related 

to chlorpyrifos exposure. Adjustments were made for gestational age, gender, ethnicity, maternal 

education, maternal intelligence, and home environment. The findings showed that before 36 months 

of age, delays were no more likely in the highly exposed group. But, at the 36 month milestone, the 

likelihood of highly exposed children developing mental delays were 2.4 times greater (95% CI: 1.12-

5.08, p = .02) and motor delays were 4.9 times greater (95% CI: 1.78-13.72; p = .002) than those with 

lower prenatal exposure. 

General linear models (GLM) were fit to describe longitudinal developmental trajectories for high and 

low chlorpyrifos exposure groups, adjusting for race/ethnicity, gender, gestational age, maternal

educational level, HOME score, and ETS exposure. The GLM results were consistent with the 12, 24, and 

36 month linear regression analyses indicating that associations between developmental scores and 

chlorpyrifos were most pronounced at 3 years of age. The authors describe an initial decrease in scores 

in both groups between the ages of 12 and 24 months, but remark that such decreases are commonly 

observed among low-income groups followed by a rebound in scores after 24 months of age. The rate 

of increase in the MDI scores after 24 months was greater among the low exposure group than the high 

exposure group but was not significantly greater (p = .23). The GLM analysis for PDI scores showed a 

significant effect of chlorpyrifos exposure over time with an estimated deficit of approximately 7 points 

by age 36 months (p = .01). 

The authors summarize three main findings from the study: 1) by age 3, the more highly exposed 

children demonstrated mental and motor delays; 2) the observed developmental trajectories for PDI 

and MDI scores confirmed that the adverse impact on cognitive and motor development increased over 

time; and 3) by age 3, highly exposed children were more likely to demonstrate clinically significant 

attention problems.

Study Review
Page 45 of 105

The investigators conducted a well-designed prospective cohort study and retained a high percentage 

(83%) of their participating mothers over the three year follow-up period. With very narrow inclusion 

criteria, the investigators selected a homogenous cohort of minority, inner-city women, most of whom 

fall into low income and low education categories. This homogeneity serves to equalize the study 

comparison groups with regard to potential measured and unmeasured confounders, but may also limit 

the generalizability of the findings. 

While the high retention rate of the mothers provides as stable study population, the authors do not 

address that only 53% of the children from these mothers reached the three year milestone with study 

data collected. It is unclear what, if any, percentage of these children did not survive, were lost to 

follow-up, or were too sick/affected by neurodevelopmental delays to participate. Authors did not 

provide information concerning the degree of missingness by chlorpyrifos exposure category in the 

published analyses, however in supplemental analyses suggested by the 2008 FIFRA SAP on chlorpyrifos 

authors did provide this information (See Appendix 4-1). In these additional analyses, authors concluded 

the degree of missingness was relatively equal by exposure category and therefore not likely to have 

imposed a selection bias. 

As discussed by the authors, one weakness of the study is the reliance on a single exposure level 

(prenatal/cord blood.) It is acknowledged that this snapshot of exposure may not accurately reflect 

both pre- and post-natal exposure levels, although in general, for chronic exposures with a high 

prevalence and consistent frequency of use as indicated in this study population, a single measure may 

be sufficient to accurately rank participant exposure levels. The authors further explain that previous 

analyses of this cohort showed that exposures to chlorpyrifos did not show significant variation 

throughout pregnancy (Whyatt et al., 2007) and therefore concluded that this single measure can 

accurately represent prenatal exposure. While this may accurately represent prenatal exposure, there is 

no control for exposure over the subsequent 3 years which may be critical, especially as the authors 

note that their GLM analyses show a critical developmental re-bound period between 24 and 36 

months. From this study, it is unclear if an increase or decrease in exposure during the period of early 

childhood could also influence the neurodevelopment of these children. Finally, the creation of a 

dichotomous exposure variable also brings limitations to the interpretation of the study results due to 

the amount of within-group variation likely present using this method of exposure classification. By 

setting the cut-off at 6.17pg/g, there are members of their “low exposure” group who may approximate 

high exposure levels and vice versa, resulting in non-differential measurement error. 

The authors also address limitations to the sensitivity and predictive validity of the developmental tests, 

especially among children less than 3 years of age. Although, these tests are considered to be most valid 

among school-aged children they are also used as indicators for early intervention to prevent continued 

developmental delay. The authors describe a statistically significant reduction in scores associated with 

high exposure levels but they do not discuss whether this 7-point deficit is clinically relevant in terms of 

diagnosing or treating a child at this stage of development. 
Page 46 of 105

Also complicating this analysis is the pervasive, non-specific nature of neurological effects and the 

difficulty in attributing causal pathways related to just one environmental exposure. Although the 

authors site previous research linking chlorpyrifos exposure to inhibition of neural growth (Dam, 1998) it 

is still difficult to attribute developmental and behavioral effects to a single exposure variable measured 

at one point in time. In future studies it would be advantageous to include other potential covariates 

such as family history of developmental delay or attention disorders (especially among parents and 

siblings) as well as other co-morbidities not adjusted for by design (i.e, restriction using eligibility criteria 

to exclude co-morbidities). Not only is the etiology of attention and related behavioral disorders not 

well understood but also the diagnosis of these disorders is often influenced by complex outside forces.

This well-designed study provides suggestive findings linking prenatal chlorpyrifos exposure with 

neurodevelopmental deficits at the three years of age. The lack of outcome information for a high 

percentage of cohort children is a concern which is not directly addressed by the authors. The use of a 

single exposure estimate (which likely led to non-differential exposure misclassification bias in this 

study) and an imprecise outcome characterization (although a clinical tool, neurodevelopmental 

milestones are ‘soft’ outcomes to measure) may have an accumulative effect to underestimate the true 

effect (bias results toward to null value), if a true effect exists. As mentioned previously, at the time of 

this publication, these analyses covered only a snapshot of the findings of the ongoing cohort study. The 

authors allude to the fact that they will use the subsequent evaluations, especially the 7-year scores to 

further describe the effects of chlorpyrifos exposure on neurodevelopment and behavior. 
Page 47 of 105

Works Cited

Whyatt RM, Rauh V, Barr DB, et al. Prenatal insecticide exposure and birth weight and length among an 

urban minority cohort. Environ Health Perspect. 2004;112:1125–1132

Perera F, Rauh VA, Tsai WY, et al. Effects of transplacental exposure to environmental pollutants on 

birth outcomes in a multi-ethnic population. Environ Health Perspect. 2003;111: 201–205

Whyatt, R. M., Garfinkel, R., Hoepner, L. A., Holmes, D., Borjas, M., Williams, M. K., . . . Camann, D. E. 

(2007). Within- and between-home variability in indoor-air insecticide levels during pregnancy among an 

inner-city cohort from New York City. Environ Health Perspect, 115(3), 383-389. doi: 10.1289/ehp.9546

Dam K, Seidler FJ, Slotkin TA. Developmental neurotoxicity of chlorpyrifos: delayed targeting of DNA 

synthesis after repeated administration. Brain Res. 1998;108:39–45
Page 48 of 105

Article 8. Lovasi et al. (2010)

Chlorpyrifos Exposure and Urban Residential Environment Characteristics as Determinants of Early 

Childhood Neurodevelopment. Gina S. Lovasi, James W. Quinn, Virginia A. Rauh, Frederica P. Perera, 

Howard F. Andrews, Robin Garfinkel, Lori Hoepner, Robin Whyatt, Andrew Rundle. American Journal 

of Public Health. 101(1):63-70 (2005).

Study Summary

Lovasi et al evaluated associations between neighborhood characteristics and early-life 

neurodevelopment in the Columbia Mother’s and Newborn Study Cohort, and assessed whether such 

characteristics confound the association between exposure to chlorpyrifos and neurodevelopment 

previously reported. US Census data (2000) were used to estimate neighborhood-level measures of 

physical infrastructure, socioeconomic status, crowding, demographic composition, and linguistic 

isolation, augmenting previously reported prenatal chlorpyrifos exposure, psychomotor development, 

and mental development data on a New York City birth cohort. 

Study participants were recruited during early pregnancy (≤20th week) among African-American and 

Dominican women age 18-35 years, and registered at New York Presbyterian Medical Center and 

Harlem Hospital, in New York City. Women who smoked, had a history of drug abuse, diabetes, 

hypertension, or HIV infection were excluded from participation in the study, as were women who 

resided in New York City for less than 1 year.

Cord blood plasma chlorpyrifos levels were assessed, and dichotomized as high (greater than the third 

tertile level of 6.17 pg/g) or low (<6.17pg/g). Maternal blood levels of chlorpyrifos were used to 

estimate cord blood levels for the 12% of infants for whom cord blood was not obtained. 

The Bayley Scales of Infant Intelligence–Revised (BSID-II) were used to assess cognitive and psychomotor 

development at age 36 months. Development was reported using a Psychomotor Development Index 

(PDI) and a Mental Development Index (MDI), which served as the continuous outcome measurements 

in the study.

Participants’ neighborhoods were defined using a 1-kilometer network buffer. Using 2000 US Census 

data, network buffer characteristics were determined, including: the percentage of housing units 

without complete plumbing, the percentage of vacant housing units, the percentage of residents below 

the federal poverty line, the percentage of residents older than 25 years of age who completed high 

school, the percentage of households receiving public assistance, the percentage of housing units with 

one or more residents per room, racial composition, the percentage of residents born outside the 

United States, the percentage of Spanish-speaking residents, and the percentage of residents who were 

linguistically isolated (all household members aged 14 years and older have at least some difficulty with 

English).
Page 49 of 105

Indicators of building disrepair were self-reported by mothers during prenatal interviews, and summed 

to create an index of disrepair that included one point for each of the following problems: holes in 

ceilings or walls, peeling or flaking paint, water damage, leaking pipes, and lack of gas or electricity in 

the prior 6 months. 

Generalized estimating equation models were created for the continuous outcomes (PDI and MDI) and 

Bayley Scores of Intelligence at 36 months. Robust standard errors were used to correct for possible 

autocorrelation within community district areas. All models included the following potential 

confounders: gender, gestational age at birth, Dominican versus African American ethnicity, maternal 

education, maternal intelligence quotient, the presence of secondhand smoke in the home during 

pregnancy, and an index of the quality of the home environment with respect to caretaking. Using 

hierarchal regression techniques which allow for control of individual level as well as group-level 

variables, authors were able to further clarify the nature of the association between mental and 

psychomotor development (Bayley scales of infant development) and prenatal chlorpyrifos exposure. 

Other variables including blood lead, other environmental exposures among other factors were not 

considered confounders in the prenatal chlorpyrifos and Bayley score association.

Of 266 children included as participants, 47% were male, 59% were Dominican, and 41% were African 

American. After adjusting for potentially confounding variables, high chlorpyrifos exposure (greater than 

6.17 pg/g) was associated with a 6.5-point decrease in PDI and a 3.3-point decrease in MDI at age 36 

months, as previously reported. These associations remained statistically significant and similar in 

magnitude in models additionally adjusting for dilapidated housing and neighborhood characteristics.

Study Review

This study helps clarify the nature of the relation between prenatal chlorpyrifos exposure and childhood 

neurodevelopment by controlling for the potential influence of socioeconomic status (SES) using 

advanced statistical techniques. Unique to this research, authors were able to measure variability due to 

SES using several different variables measured at both the individual-level and the group-level. This 

work enhances the precision and accuracy of the epidemiologic measures previously reported. 

The authors note that participants lost to follow-up were not significantly different from continuing 

participants with respect to maternal age, ethnicity, marital status, education, income, or gestational 

age and birth weight of the newborn; authors cited a retention rate at the 3-year follow-up of 83%. How 

they arrived at that number is unclear. Originally, 648 pregnant women consented to participate and 

327 children completed a developmental assessment at approximately 36 months of age. In this report, 

analyses were conducted among 266 children with available chlorpyrifos exposure data, yielding an 

effective retained proportion of 41%. This leaves open the possibility of selection bias resulting from 

loss to follow-up; one may only speculate as to the magnitude and direction of such bias. 
Page 50 of 105

The direct measurement of cord blood chlorpyrifos in this study is a substantial strength, compared to 

the assessment of non-specific markers as assessed in similar studies of organophosphate pesticide and 

child neurodevelopment. Umbilical cord blood was obtained at delivery for 88% of the children in the 

study. For the remaining 12% for whom no cord blood was obtained, cord blood plasma chlorpyrifos was 

estimated from maternal plasma levels using blood sampled two days prior to delivery. Any chlorpyrifos 

measurement error resulting from this estimation is almost assuredly non-differential with respect to 

cognitive development outcomes. Children excluded for missing chlorpyrifos exposure data were similar 

to the analytic sample except that excluded children had more indicators of building dilapidation, so the 

likelihood of selection bias of large magnitude is unlikely. The decision to dichotomize cord blood 

chlorpyrifos levels using the highest tertile as a cut-point reduces the ability to assess exposure-response 

relationships, and may somewhat decrease statistical efficiency relative to using a continuous measure. 

If a large number of participants had levels of chlorpyrifos below the level of detection, however, using a 

categorical approach dispenses with the need to estimate or otherwise define exposure at low levels. 

The prenatal exposures assessed in this study are likely correlated with other prenatal exposures and 

factors in the postnatal environment which themselves predict cognitive development. This presents a 

challenge for a causal interpretation of prenatal chlorpyrifos exposure per se, as the agent and time 

period responsible for observed cognitive deficits.

The Bayley Scales of Infant Development–Revised (BSID-II) is a widely used norm-referenced 

developmental test for young children, it can be used to diagnose developmental delay, and it is known 

to be sensitive to the effects of toxic exposures such as low-level intrauterine lead. The assessment was 

appropriately administered, with each child being evaluated under controlled conditions by a trained 

bilingual research assistant. Inter-observer reliability was assessed but data were not presented in this 

report. The authors note that, when administered at age 3 years, the BSID-II demonstrates only

moderate predictive power for subsequent intelligence and school performance, but cite previous 

literature as evidence that the test is nevertheless clinically useful for children performing in the 

subnormal range.

The statistical analyses were generally appropriate. Missing data on covariates were estimated using 

multiple imputations, and the variance estimates presented appropriately reflect the degree of 

uncertainty caused by missing covariate data. Robust standard errors were estimated to account for 

potential autocorrelation. The authors presented multiple model results, each with a different indicator 

of neighborhood character. Only neighborhood covariates representing socioeconomic conditions (i.e.

percentage of individuals living in poverty, and percentage with high school education) were statistically 

significant predictors of PDI and MDI. Interestingly, and without explanation, the authors did not 

present the results of a model which simultaneously included all of the neighborhood characteristics as 

covariates. Presumably, this was done for the sake of parsimony, as one could argue against the 

inclusion of covariates considered not to be predictors of cognitive development. Alternatively, these 

factors were, a priori, considered potential confounders, and the use of statistical significance tests in 

the assessment of confounding is discouraged in many epidemiologic circles. 
Page 51 of 105

The observation that neighborhood characteristics did not confound the observed association between 

chlorpyrifos levels and cognitive development in this study has several possible explanations. This was a 

relatively homogenous study population living in the New York City Area, and it is possible that 

confounding is limited by the small degree of variability in neighborhood characteristics between those 

with and without high exposure levels of chlorpyrifos. It is also possible that the neighborhood 

characteristics are measured with error, such that they fail to capture aspects of socioeconomic 

condition, neighborhood composition, and neighborhood condition that effect cognitive development, 

even within this context of measurement of several individual-level as well as group-level SES measures. 

This residual bias, if it exists, could (at least partially) explain the decrement in cognitive development 

associated with chlorpyrifos exposure, assuming that poorer neighborhood conditions are both 

predictors of reduced cognitive function and comparatively more prevalent among those with high 

chlorpyrifos exposure. The first of these two explanations has no effect on internal validity; under the 

assumption of no other bias, the observed association is unbiased, but external validity may be limited 

due to the restricted study population. The investigators’ methods to assess neighborhood 

characteristics were well conceived and well implemented, and reflective of commonly utilized variables 

to measure important aspects of the multi-dimensional characteristic of socio-economic status. 

However, such exposures are notoriously difficult to measure well even using these advanced statistical 

methods and approaches. 

The authors did not assess effect modification of the association between prenatal chlorpyrifos 

exposure and cognitive development by neighborhood conditions, which is an equally, if not more, 

interesting area of inquiry than assessment of confounding. The present study simply does not have the 

sample size/statistical power to address these effects, but, in the discussion, the authors speculate that 

large studies that collect both neighborhood context and exposure data could reveal patterns of effect 

modification. The relative homogeneity of the study population may have precluded statistically robust 

examination of effect modification by factors related to SES in this study.

The setting of the investigation in a sample drawn from low-income African American and Dominican 

communities is both a strength and a limitation of the study. The population is likely at high risk of 

developmental deficits, which increases the power of the study relative to a similar study set in a 

population at low risk. The assessment of confounding by demographic and neighborhood 

characteristics is limited by the restriction of the study sample to low-income communities (a 

weakness), but so too is the bias induced by these potential confounders (a strength). The 

generalizability of the study results may also be limited because participants were recruited from a 

restricted range of neighborhoods in New York City (a weakness), but the study population is likely 

among the most highly exposed and also at risk for cognitive impairment (Landrigan, 1990a, 1990b; 

Landrigan et al., 1999). 

This study represents an extension of previously published work with additional adjustment for multiple 

neighborhood characteristics. The work was well conceived and conducted, representing perhaps the 

best assessment of the association between chlorpyrifos and cognitive development in the 
Page 52 of 105

epidemiologic literature. Chief among its strengths is the direct assessment of cord blood plasma 

chlorpyrifos, the prospective design, and the assessment and control of potential confounders. Using 

the methods of data collection, statistical analyses, and enhanced methods to evaluate the role of SES as 

a potentially confounding variables, authors report no substantial difference in the reported association 

between prenatal chlorpyrifos exposure and child neurodevelopment as previously reported (Rauh et al. 

2006).
Page 53 of 105

Works Cited:

Landrigan, P. J. (1990a). Health effects of environmental toxins in deficient housing. Bull N Y Acad Med, 

66(5), 491-499. 

Landrigan, P. J. (1990b). Housing and health: conclusions and challenges for the future. Bull N Y Acad 

Med, 66(5), 587-591. 

Landrigan, P. J., Claudio, L., Markowitz, S. B., Berkowitz, G. S., Brenner, B. L., Romero, H., . . . Wolff, M. S. 

(1999). Pesticides and inner-city children: exposures, risks, and prevention. Environ Health Perspect, 107 

Suppl 3, 431-437. 
Page 54 of 105

Article 9. Engel et al. (2011)

Prenatal Exposure to Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood. 

Stephanie M. Engel, James Wetmur, Jia Chen, Chenbo Zhu, Dana Boyd Barr, Richard L. Canfield, and 

Mary S. Wolff. Environmental Health Perspectives 2011; 119:1182-1188

Study Summary

This is the third of three published analyses of prenatal pesticide exposure and child growth and 

development in the Mount Sinai Hospital Children’s Environmental Health Study. The first (Berkowitz et 

al 2004) reported on assessments of birth weight, length and head circumference. The second (Engel et 

al 2007) reported on assessments of infant neurocognitive development and reflexes shortly after birth.

In this study, Engel and colleagues extended their 2007 to include analysis of PON1 activity, PON1 

polymorphisms, and cognitive development at ages 12 and 24 months and 6–9 years. 

Of the 404 women originally enrolled between May, 1998 and July, 2001 (n = 404), a subset (n = 360) 

had organophosphate metabolite levels quantified in urine samples collected during the third trimester. 

Prenatal maternal blood and cord blood samples were analyzed for PON1 activity and genotype. 

Children of these mothers returned for neurodevelopment assessments at ages 12 months (n = 200), 24 

months (n = 276), and 6–9 (n = 169) years of age. Participants in this study were predominantly young, 

unmarried, Hispanic women with low educational attainment. Most women delivered term infants of 

normal birth weight, as prematurity and very low birth weight were exclusion criteria for study 

enrollment. 

The study population was drawn primarily from East Harlem and consisted largely of young Hispanic 

women (predominantly Puerto Rican), but also included African-American and Caucasian women. 

Mothers were enrolled during early pregnancy from the Prenatal Clinic and two private practices at 

Mount Sinai Hospital from May 1998 to July 2001. The study included only primiparous with singleton 

births. Mothers were excluded if they had any of the following: an initial prenatal visit after 26 weeks of 

gestation, serious chronic diseases, a serious pregnancy complication that could affect fetal growth and 

development; alcohol consumed greater than two alcoholic beverages per day, illicit drug use. Mothers 

and infants were also excluded if the child was born with a congenital malformation or severe 

prematurity. A total of 479 prenatal patients were recruited; 75 were excluded because of medical 

complications, premature birth, an infant with birth defects, inability to collect biologic specimens 

before birth, change of hospital or residence outside New York City, or refusal to continue to participate. 

A questionnaire was administered to participants during their third trimester of pregnancy to obtain 

information on environmental exposures, sociodemographic characteristics, obstetrical and medical 

history, and lifestyle factors.
Page 55 of 105

Generalized linear models were used to analyze the relationship between total diethylphosphates 

(DEPs), total dimethylphosphates (DMPs), total dialkylphosphate metabolites (DAPs), and subsequent 

cognitive development evaluated at age 12 and 24 months using the Bayley Scales of Infant 

Development, 2nd edition (BSID-II) and using the Wechsler Preschool and Primary Scale of Intelligence, 

3rd edition (WPPSI-III) at approximately age 7 years. The authors also assessed modification of pesticide 

metabolite - cognitive development associations by maternal and child PON 1 activity and PON 1 

polymorphisms.

The authors presented their results stratified by level of race/ethnicity and PON1 genotype, having 

observed apparent effect modification by these variables. The authors found that prenatal total 

dialkylphosphate metabolite level was associated with a decrement in mental development at 12 

months among blacks and Hispanics. The associations appeared to be strongest among children of 

mothers who carried the PON1 Q192R QR/RR genotype. In later childhood, increasing prenatal total DAP 

and DMP metabolites were associated with decrements in perceptual reasoning in the maternal PON1

Q192R QQ genotype (imparting slow catalytic activity for chlorpyrifos oxon), with a monotonic trend 

consistent with greater decrements with increasing prenatal exposure. The authors concluded that their 

findings are suggestive of an association between prenatal exposure to organophosphates and 

decrements in cognitive development, particularly perceptual reasoning, with evidence of effects 

beginning at 12 months and continuing through early childhood, with PON1 being a potentially 

important potential susceptibility factor for these deleterious effects. The authors did not observe any 

effect modification by PON1 enzyme activity.

The authors noted that the associations they observed between the organophosphate pesticide 

metabolites and cognitive development are similar to those reported in a similar prospective cohort 

study set in an agricultural area of California (the Center for the Health Assessment of Mothers and 

Children of Salinas Study, Eskenazi et al. 2010). 

Urine samples obtained during the 3rd trimester prenatal visit were provided to the CDC to quantify 

levels of six dialkylphosphate metabolites. In cases where individual dialkylphosphate metabolite levels 

were missing because of analytic interference, metabolite levels were imputed using regression analysis 

to predict the missing metabolite on the basis of the other non-missing metabolites measured for that 

woman within the group of correlated metabolites (Eskenazi et al. Environ Health Perspect, 2004). 

Samples below the limit of detection (LOD) were defined as LOD/ √–2. Diethyl- and dimethylphosphate 

metabolites were then summed on a molar basis (as nm/liter) to obtain total diethylphosphates (DEPs) 

and total dimethylphosphates (DMPs), respectively, and together to obtain total dialkylphosphates 

(DAP) levels. Twenty-six very dilute urine samples with < 20 μg/dL creatinine were excluded from 

organophosphate metabolite analyses. Approximately 97%, 89%, and 90% of the cohort had detectable 

levels of DAP, DEP, and DMP metabolites, respectively. Prenatal organophosphate marker levels were 

dichotomized using median exposure as the cut-off. 

Plasma was separated from samples of prenatal maternal peripheral blood and cord blood 
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and used to measure PON1 activity. PON1 polymorphisms in both maternal and child DNA 

were also assessed using the blood samples provided by participants. Prenatal and cord blood PON1 

activity levels were classified in tertiles for analysis of effect modification of the relationship between 

pesticide marker levels and subsequent cognitive development. 

The Bayley Scales of Infant Development, 2nd edition (BSID-II), was administered to participating 

children at 12 and 24 months of age, approximately, to evaluate mental and psychomotor development. 

The Mental Development Index (MDI) portion of the assessment rates a child’s cognitive ability (i.e.

memory, habituation, problem solving, early number concepts, generalization, classification, 

vocalizations, language, and social skills). The Psychomotor Development Index (PDI) rates fine and gross 

motor coordination. Between the ages of 6 and 9 years of age, a subset of participating children was 

again assessed using psychometric intelligence tests. Before age 7, children were administered Block 

Design, Information, Matrix Reasoning, Vocabulary, Picture Concepts, Symbol Search, Word Reasoning, 

and Coding subtests of the Wechsler Preschool and Primary Scale of Intelligence, 3rd edition (WPPSI-III). 

Composite Verbal, Performance, Processing speed, and Full-Scale IQ (FSIQ) scores were derived using 

age-standardized WPPSI-III norms. Children between the ages of 7 and 9 years were administered the

Block Design, Similarities, Digit Span, Picture Concepts, Coding, Vocabulary, Letter-Number Sequence, 

Matrix Reasoning, Comprehension, and Symbol Search subtests of the Wechsler Intelligence Scale for 

Children, 4th edition (WISC-IV). Working Memory, Processing Speed, and FSIQ scores were derived using 

age-standardized WISC-IV norms. 

The BSID-II was administered at Mount Sinai Hospital. Interviews and examinations were conducted in 

English or Spanish. The WPPSI-III was administered in English or Spanish by one of four examiners, as 

was the WISC-IV. by one of four examiners Both the WPPSI-III and the WISC-IV were administered in a 

private room, without the parent being present.

Of the 200 children who were administered the BSID-II at approximately 12 months of age (mean ± SD, 

13.1 ± 1.6 months), one was excluded from analyses because they refused to compete a substantial 

portion of the examination; two children were excluded from the 12-month analysis because their 

parent reported a diagnosis of pervasive developmental disorder and 20 children were excluded 

because their corresponding maternal urine samples were deemed to dilute to analyze. Of the 177 

eligible children who completed 12-month exams, 174 had organophosphate metabolites measured in 

prenatal urine. At the 24-month BSID-II (mean ± SD, 27.4 ± 4.5 months), 276 children completed the 

exam. Two were excluded from the 24-month analysis because the parent reported a diagnosis of 

pervasive developmental disorder. Children of mothers who submitted very dilute samples of urine (less 

than 20 μg/dL creatinine) were also excluded from analysis (n = 23). Of the remaining 251 eligible 

observations, 247 had organophosphate metabolites measured in prenatal urine. MDI scores for 10 

children were not usable due to extensive refusals; they were included in the PDI analyses because their 

scores were valid. The authors reported an association between prenatal total dialkylphosphate 

metabolite level and a decrement in mental development at 12 months among blacks and Hispanics. 

These associations appeared to be enhanced among children of mothers who carried the PON1 Q192R 
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QR/RR genotype. In later childhood, increasing prenatal total dialkyl- and dimethylphosphate 

metabolites were associated with decrements in perceptual reasoning among those with maternal 

PON1 Q192R QQ genotype, which imparts slow catalytic activity for chlorpyrifos oxon, with a monotonic 

trend consistent with greater decrements with increasing prenatal exposure.

Study Review

This was a well conducted prospective study conducted in a young, predominantly minority population. 

The study design, analytic approach, and statistical analyses were appropriate. Differential 

measurement error of exposure and outcomes is likely to be minimal, although other sources of bias 

may have influenced the study findings. 

The dramatic loss-to-follow-up over time is a possible cause of selection bias in this study. The authors 

noted that mothers returning for follow-up assessments tended to be older, with a disproportionately 

low fraction of women in the youngest age category; they also had disproportionately higher level of 

education, on average, relative to the original cohort. However, the subset of participants who returned 

for follow-up visits did not differ substantially from the originally enrolled cohort with respect to 

racial/ethnic composition, breast-feeding behaviors, and self-reported alcohol use during pregnancy.

The authors employed a reasonable set of exclusion criteria that likely resulted in recruitment of a study 

population that was relatively homogeneous with respect to confounders of the in utero pesticide 

exposure - cognitive development relationship (compared to the general population), and the potential 

for confounding was thus limited by design (restriction). Bias due to confounding was also controlled in 

the analysis, by assessing and adjusting for known risk factors for cognitive development that are 

potentially associated with pesticide exposure, including maternal age, race/ethnicity, marital status, 

education, breast-feeding, child sex, alcohol, smoking, or drug use during pregnancy, maternal IQ, a 

score based on assessment of the home environment (HOME), season of urine collection, language 

spoken in the home, age at testing, examiner and urinary creatinine level. The authors considered 

gestational age at delivery and birth weight, not as confounders, but as potential causal intermediates. 

They used a change-in-estimate approach to covariate selection, beginning with a full model, which 

allowed them to consider confounding by a given risk factor in the context of adjustment for other 

covariates. Given the limited sample size in this study, a full model with many covariates may be 

unstable, although it is unclear from the article whether such instability was observed. 

Residual confounding by unmeasured or poorly measured confounders is likely in this study. 

Socioeconomic determinants, which are notoriously difficult to measure well, are likely predictive of 

pesticide exposure, and risk factors for poor cognitive development. The authors adjusted for some 

factors which may be surrogates for socioeconomic status (e.g., race/ethnicity, marital status, maternal 

education), but adjustment for these factors is unlikely to have fully accounted for confounding by 

socioeconomic status. If such residual confounding exists, it is likely to result in effect estimates which 

overestimate the effect of organophosphate pesticide exposure. However, the extent of this 
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confounding was likely limited by the recruitment of a relatively homogenous study population, vis-à-vis 

socioeconomic conditions. 

Presence of environmental contaminants in the home, either during pregnancy or after birth, may 

confound the relationship under study. The authors do not discuss co-contaminants present in the home 

during the pregnancy; such factors would likely result in the observation of a greater than effect of 

pesticide exposure on cognitive development if they are positively associated with pesticide exposure 

and predictive of deficits in cognitive development. The authors stated that they considered a “Home 

Observation for Measurement of the Environment” (HOME) score as a potential confounder, and, in 

fact, they included this score in their multivariate models. The degree to which this “HOME” score is a 

good proxy for relevant co-exposures is unclear, and leaves open the possibility of residual confounding 

by environmental contaminants in the home. 

The authors observed apparent effect modification by maternal race/ethnicity. For example, among 

non-whites, increasing DAP and DMP tertiles of exposure were associated with a decrease in the MDI. 

However, among whites, the opposite was observed; higher metabolite levels were associated with an 

increase in MDI scores. Such an observation may be indicative of a true interaction between 

race/ethnicity and prenatal pesticide exposure, but it may also be explained by residual and differential 

confounding by co-contaminants that are correlated with pesticide exposure and race. Indeed, in this 

study population, race/ethnicity was strongly associated with housing type (i.e. public versus private 

housing), and the authors did, indeed, observe similar trends in the effect estimates when stratifying by 

housing type rather than race/ethnicity. The authors speculated that this observed heterogeneity may 

indicate that whites, or those living in private or owner-occupied housing, may have experienced a 

different source of exposure to pesticides or their metabolites that may have contributed substantially 

to their urinary concentrations, and identify pesticide residues from fresh fruit and vegetable 

consumption as one such source. The authors adjusted for seasonal variation in exposure, although they 

note that other time-related variability may influence exposure assessment.

Although they observed heterogeneity by PON1 genotype, the authors did not observe any effect 

modification by PON1 enzyme activity in blood sampled during the third trimester, as they had in their 

earlier assessments of pesticide metabolites and cognitive development assessed shortly after birth. 

They speculated that this was because genotype is a more stable, long-term predictor of metabolism 

potential, relative to prenatal phenotype as measured by PON1 activity.

The metabolites assessed in this study are sensitive, but non-specific, markers for chlorpyrifos. The 

analytic methods appear sound. However, they assessed these markers using only a single maternal 

urine specimen taken during the third trimester – a critical period for neurodevelopment. If sources and 

patterns of exposure remain static, a single measurement would be a good indicator of pesticide 

exposure during pregnancy. If, however, sources and exposure patterns change over short time-spans, 

perhaps on the order of days to weeks, such an exposure assessment would not necessarily provide a 

good indicator of the relevant exposure. Like the many other sources of error possible in this study, this, 
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too is likely to result in a non-differential misclassification of the exposure, with respect to the cognitive 

outcome. Serial measurements of urinary biomarkers of pesticide exposure in the third trimester would 

shed light on these patterns, although the benefits of such information are offset by the added cost,

logistical complexity expense, and burden on the study participants.

The instruments used to assess neurodevelopment in this study (i.e., the Bayley Scales of Infant 

Development, 2nd edition (BSID-II), the Wechsler Preschool and Primary Scale of Intelligence, 3rd 

edition (WPPSI-III), and the Wechsler Intelligence Scale for Children, 4th edition (WISC-IV)) are 

commonly used in both a clinical and research setting and are widely accepted tools.

In this study, prenatal maternal DEP urinary metabolite concentrations were associated with slight 

decrements in FSIQ, Perceptual Reasoning, and Working Memory between the ages of 6 and 9 years. 

Among children of QQ mothers, DAP and DMP urinary metabolite concentrations were associated with 

poorer scores on Perceptual Reasoning and FSIQ. The authors reported stronger Q192R interactions for 

DAP and DMP urinary metabolites compared to that for the DEP metabolites, although the explanation 

is unclear and may have been influenced by small sample size. The authors note that PON1 Q192R 

exhibits substrate specificity, but the strongest interactions were observed for DMP, not DEP (into which 

chlorpyrifos and diazinon both metabolize). Interestingly, researchers state that malathion spraying for 

mosquitoes carrying West Nile virus was a well-publicized source of exposure during the period of 

enrollment which may have increased the levels of DMP in this population.

The statistical analyses used in the study appear to have been appropriate, although they are not 

explained in detail. The authors stratified their findings by both race/ethnicity and PON 1 genotype, 

although it is unlikely that the study was sufficiently powered to provide optimal statistical power to 

assess pesticide effects in the presence of such effect modification. To maximize the number of 

participants included in the analysis, the investigators defined a combined population of children who 

came for at least one of the Wechsler psychometric intelligence exams. The WISC-IV composite scores 

were included preferentially, and WPPSI-III composite scores substituted if the child did not return for 

the later exam. Regarding the use of a composite population for the outcome evaluations at 6-9 years, 

the authors report ‘convergent validity’ between the WISC-IV and WPPSI-III. In a previous report, the 

correlation between WPPSI-III and WISC-IV FSIQ scores was 0.89; WPPSI-III Verbal IQ and WISC-IV Verbal 

Comprehension, 0.83; and WPPSI-III Performance IQ and WISC-IV Perceptual Reasoning, 0.79. In the 

present study, correlations between the composite scores on the WPPSI-III and WISC-IV were likewise 

strong: FSIQ = 0.83, Verbal IQ/Verbal Comprehension = 0.84, and Performance IQ/Perceptual Reasoning 

= 0.78. In their statistical modeling, the authors included a variable to indicate whether the WISC-IV and 

WPPSI-III was used. 

Regarding the measurement of developmental outcomes using the Bayley Score, in general, associations 

observed during the 12-month follow-up were either attenuated or non-existent at the 24-month visit, 

including the observation of race-based heterogeneity. The authors speculated that, although exposures 

likely decreased over time, the 0- to 12-month exposure window may have included less hand-to-mouth 
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childhood exposure (from crawling exposure to house dust, and/or fresh fruit and vegetable 

consumption) than in the 12- to 24-month window; thus, more childhood exposure in the 12- to 24-

month window may modify the effect of prenatal exposure.

The prospective nature of the study is a primary strength. Pesticide exposure indicators and outcomes 

metrics are likely to have been measured with some error, but these errors are likely to be nondifferential. On average, such non-differential measurement error results in attenuation of the measures 

of association, and are thus unlikely to result in false-positive findings. A further strength attributable in 

part to the prospective design was the investigators’ ability to consider potential confounders and 

effect-modifiers a priori, measure them by design, and adjust for/assess their influence in the analysis. 

Importantly, this study was conducted at a time during which important regulatory changes in 

residential use of chlorpyrifos were being implemented (occurring approximately midway through the 

study). As a result, the participating children’s exposures to chlorpyrifos may have decreased over time. 

The investigators reported that they did not observe any time-period interactions, but they did not 

present data on the presence or absence of temporal changes in participants’ pesticide metabolite 

levels (i.e. comparing those that were enrolled earlier versus later in time). In the discussion, they did 

mention that it was likely that childhood exposure to chlorpyrifos and diazinon was lower post-2000 

(following the voluntary cancellation of indoor residential chlorpyrifos use). To the extent that postnatal exposures were largely reduced, the associations observed can be more definitively linked to 

prenatal exposures. If however, pesticide exposures persisted into childhood, it would be less clear 

whether deficits in cognitive functioning observed in this study were caused specifically by pre-natal 

exposures, rather than subsequent exposure early in life, assuming that pesticide exposure is causally 

associated with such decrements. 

In this third study of organophosphate pesticide markers in maternal urine samples collected during the 

third trimester and subsequent cognitive development in children participating in the “Mt. Sinai Child 

Growth and Development Study”, the authors report on findings out to age nine. The specter of 

selection bias induced by loss-to-follow-up is a marked weakness in an otherwise well conducted study. 

Unfortunately, it is difficult to assess the magnitude and direction of such bias. Confounding by 

unmeasured or poorly measured factors is yet another limitation, specifically with reference to the 

measurement of socio-economic factors and other environmental co-exposures. Non-specific 

biomarkers of exposure, as were assessed in this study, are likely more objective, sensitive, and likely 

also more accurate and reliable indicators of pesticide exposure, relative to exposure assessment 

methodologies that rely on self-report. However, to the extent that prenatal chlorpyrifos is the true 

exposure of interest, the assessment of non-specific organophosphate pesticide metabolites in a single 

sample of prenatal urine is a primary potential source of exposure measurement error. Because of the 

prospective design, information bias is expected to result in attenuation of the true (unobserved) 

associations, toward (observed) associations that are relatively smaller, or null. The finding of apparent 

effect modification by maternal race/ethnicity in this study could result from differential loss to follow-
Page 61 of 105

up, measurement error in the assessment of actual pesticide exposure, and/or differential residual 

confounding. 
Page 62 of 105

Works Cited

Berkowitz GS, Wetmur JG, Birman-Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS. In 

Utero Pesticide Exposure, Maternal Paraoxonase Activity, and Head Circumference. Environmental 

Health Perspectives. 2004;112:388–391.

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL, Siskind J, Meisel SJ, Wetmur JG, Wolff MS. Prenatal 

Organophosphate Metabolite and Organochlorine Levels and Performance on the Brazelton Neonatal 

Behavioral Assessment Scale in a Multiethnic Pregnancy Cohort. American Journal of Epidemiology. 

2007;165:1397-1404.

Eskenazi B, Huen K, Marks A, Harley KG, Bradman A, Barr DB, Holland N. PON1 and neurodevelopment 

in children from the CHAMACOS study exposed to organophosphate pesticides in utero. Environmental 

Health Perspectives 2010;118:1775–1781. 
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Article 10. Eskenazi et al. (2007)

Organophosphate Pesticide Exposure and Neurodevelopment in Young Mexican-American Children. 

Brenda Eskenazi, Amy R. Marks, Asa Bradman, Kim Harley, Dana B. Barr, Caroline Johnson, Norma 

Morga, Nicholas P. Jewell. Environmental Health Perspectives. 115:792–798 (2007).

Study Summary

In this article by Eskenazi et al, the authors report on the relationship between prenatal and child 

urinary organophosphate metabolite levels and child neurodevelopment up to two years of age in the 

CHAMACOS cohort.

Enrollment for the CHAMACOS took place from 1999–2000 at community clinics, and included women 

who were ≥ 18 years old, pregnant at < 20 weeks gestation, Spanish- or English-speaking, eligible for 

low-income health insurance, and planning to deliver at the local public hospital. Researcher followed 

531 women to delivery of a live-born, surviving, neonate. Excluded from the analysis were children that 

did not have a neurodevelopmental assessment, (n=71), who did not have prenatal and relevant 

concurrent DAP metabolite measurements (n = 3), were not singletons (n=8), had a medical condition 

that could affect performance on the neurobehavioral assessment (n = 3; deafness, Down syndrome, 

hydrocephalus). Also excluded from analysis were Bayley results with raw scores which were too low for 

standardization (n=3, 1, and 1, for the 6-month, 12-month, and 24-month assessments, respectively). 

Maternal/fetal exposure was assessed by measurement of six non-specific organophosphate 

dialkylphosphate (DAP) metabolites in maternal urine samples collected between 5 and 27 weeks 

gestation and again between 18 and 39 weeks. Maternal urine was also analyzed for 3,5,6-trichloro-2-

pyridinol (TCPy), a metabolite specific to chlorpyrifos. Post-natal early childhood urine samples were 

also collected, and DAP metabolites quantified, for the participating children at the time of the 

neurodevelopmental assessments. Quantification of organophosphate metabolites using gas 

chromatography-tandem mass spectrometry and isotope dilution calibration was conducted by the 

Centers for Disease Control and Prevention (CDC) labs. 

To asses exposure, investigators measured six nonspecific organophosphate DAP metabolites in 

maternal and child urine: three dimethyl (DMP) phosphate metabolites (dimethylphosphate, 

dimethylthiophosphate, dimethyldithiophosphate); and three diethyl (DEP) phosphate metabolites 

(diethylphosphate, diethylthiophosphate, and diethyldithiophosphate) (Bradman et al. 2005). These six 

metabolites represent the by-products of approximately 80% of OPs used in the Salinas Valley. Values 

below the limit of detection (LOD) were assigned a value of LOD/(√2). The urine analysis was completed 

by the CDC. Maternal prenatal urine was also analyzed for specific metabolites including TCPy, a 

metabolite specific chlorpyrifos. Because a large proportion of women had non-detectable levels of 

TCPy, the chlorpyrifos-specific metabolite was categorized into three groups: TCPy less than LOD for 
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both pregnancy measurements, and, for those with at least one detectable level, subdivided below and 

above the median of the average pregnancy level.

The outcome, child neurodevelopment, was assessed using the Bayley Scales of Infant Development 

(Mental Development (MDI) and Psychomotor Development (PDI) Indices) (Bayley, 1993) and the Child 

Behavior Checklist (CBCL). The Bayley Scales of Infant Development were completed at the 6 months (n 

= 396), 12 months (n = 395), and 24 months (n = 372) of age. Both scales were administered in Spanish 

and/or English by psychometricians blinded to exposure. Psychometricians were trained using these 

standardized protocols and were supervised for quality assurance by a clinical neuropsychologist. 

Assessments were performed in a private room at the CHAMACOS research office or in a recreation 

vehicle (RV) modified to be a mobile testing facility. Scores > 1 SD below the mean (i.e., < 85) indicate 

possible developmental delay.

Mother’s completed the Child Behavior Checklist (CBCL) at the 24 month visit (n = 356). From the CBCL 

report, the authors assessed three scales as indicators of child neurodevelopment: The Attention 

Problems syndrome scale, which includes such items as “can’t concentrate” and “can’t sit still”; the 

DSM-oriented Attention-Deficit/Hyperactivity Disorder (ADHD) scale, which additionally includes such 

items as “gets into everything”; and the DSM-oriented Pervasive Developmental Disorder (PDD) scale, 

which includes such items as “avoids eye contact,” “rocks head, body,” and “unresponsive to affection”. 

Experts consider a score greater than the 98th percentile of a national normative sample of clinical 

significance; they consider a score greater than the 93rd percentile of borderline clinical significance.

Many confounders were ascertained including, intelligence of the mother, maternal depression, a 

measure of how stimulating the child’s environment is, and prenatal exposure to some known or 

suspected neurotoxins. Maternal intelligence was assessed via the Peabody Picture Vocabulary Test 

(PPVT). Maternal depression was assessed using the Center for Epidemiologic Studies Depression Scale 

(CES-D). To measure the quality and extent of stimulation available to a child in the home environment, 

the Infant-Toddler HOME (Home Observation for Measurement of the Environment) inventory was 

completed at the 6-month, 12-month, and partially at the 24-month visit. The following known or 

suspected neurotoxicants were measured: polybrominated diphenyl ethers (PBDEs), polychlorinated 

biphenyls (PCBs), p,p’-dichlorodiphenyltrichlorethane (DDT), p,p’- dichlorodiphenyltrichlorethylene 

(DDE), and lead. Lead was measured in cord blood. PBDEs, PCBs, DDT, and DDE were measured in 

maternal serum samples collected at 26 weeks gestation. 

Statistical analyses of the relationship between metabolite levels and Bayley performance were 

conducted using multiple linear regression models and generalized estimating equations (i.e., 

longitudinal models) for MDI and PDI were developed for each of the three assessment time points (i.e., 

6, 12, and 24 months). Neurodevelopmental scores were modeled as continuous variables. Covariates in 

the models included indicators for the psychometrician conducting the assessment and the location of 

assessment, age at assessment, sex, duration of breast-feeding, HOME score, and household income, 

parity and indicator of maternal intelligence (PPVT score). 
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Other covariates that were considered but not included in the analysis were: maternal age, education, 

depressive symptoms, active/passive smoking exposure during pregnancy, regular alcohol use during 

pregnancy, marital status, father’s presence in home, housing density, maternal work status, ≥ 15 hours 

out-of-home childcare/week, birth weight, gestational age, abnormal reflexes, PCBs, lead, DDT, βhexachlorocyclohexane, and hexachlorobenzene. Furthermore, the authors examined interactions 

between child DAPs and child sex and, because they previously observed an association with maternal 

DDT and 24-month MDI, between maternal DAPs and DDT. Finally, authors also reran the models using 

log-transformed creatinine-adjusted metabolites for comparative purposes.

Child behavior checklist score at the 24-month assessment was modeled using separate multivariate 

logistic regression models for the five child behavior checklist indices, dichotomized at the clinical 

significance level and at the borderline clinical significance level. The same covariates that were included 

in the final Bayley models were used for the CBCL models with three exceptions: maternal depression 

was added, and psychometrician and assessment location were dropped, because scores were based on 

maternal report.

Of the 348 infants included in this analysis, 49.4% were male. Most mothers were less than 30 years old 

at baseline (75.6), born in Mexico and Spanish-speaking (89%), had household income below the 

poverty line (62%). Many of the participating mothers worked in agriculture during pregnancy (43%) and 

had symptoms of depression (51%). Most of the women were married, non-smokers, and did not drink 

alcohol during pregnancy. 

In this study population, prenatal DAP levels were adversely associated with MDI, while early life DAP 

levels were positively associated with MDI. At 24 months of age, these associations reached statistical 

significance (per 10-fold increase in prenatal DAPs: β = –3.5 points; 95% CI: –6.6 to –0.5; child DAPs: β = 

2.4 points; 95% CI: 0.5 to 4.2). Neither prenatal nor child DAPs were associated with PDI or child 

behavior checklist attention problems. Both prenatal and postnatal DAPs were associated with risk of 

pervasive developmental disorder (per 10-fold increase in prenatal DAPs: OR = 2.3, p = 0.05; child DAPs 

OR = 1.7, p = 0.04). TCPy was not associated with any neurodevelopment outcome in this study. 

Study Review

This study tested whether in utero or postnatal exposures to organophosphate pesticides have a 

detrimental impact on neurodevelopment, as assessed by the Bayley Scales of Infant Development and 

the child behavior checklist (CBCL). The study population was appropriate to the hypothesis, given that 

there were a range of exposure levels to OPs and there was some homogeneity in the population, e.g., 

mostly low SES, Hispanic, agricultural workers. The homogeneity of the population restricted 

confounding by design. For example, another paper reported that smoking, alcohol use, and illicit drug 

use were rare the CHAMACOS cohort. The homogeneous population also increased the relative 

statistical efficiency of the study because it is likely at higher risk of deficits in neurodevelopment than 
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the general population (Landrigan, 1990; Landrigan et al., 1999). However, the exclusion of children who 

lacked a neurodevelopmental assessment (n = 71, approximately 13% of the study population), has the 

potential to induce a selection bias if the reasons for the missing data are determined by exposure (or a 

predictor of exposure) and are also not independent of neurodevelopment. For example, participants 

with high pesticide exposures could have missed neurodevelopmental assessments for reasons related 

to uncontrolled determinants of exposure which are also related to neurodevelopment. Comparisons of 

maternal DAP metabolite levels between those included in this analysis and those not included in the 

analysis were not presented.

The exposure assessment methods in this study were good. Quantification of the metabolite levels was 

conducted at the CDC, using published methods. The study measured the organophosphate biomarker, 

DAP metabolites, in maternal and child spot urine samples, and the chlorpyrifos specific biomarker, 

TCPy, in maternal urine only. Measurement of biomarkers in urine is a more objective and likely more 

accurate indicator of organophosphate pesticide exposure than other ascertainment methods such as 

self-report. However, these biomarkers do present a few limitations, because study participants could 

be exposed to less toxic preformed DAP or TCPy metabolites (Lu et al. 2005; Wilson et al. 2003; Morgan 

et al. 2004), and exposure to pesticides varies over time and the half-life of chlorpyrifos in the body is 

short. However, the prenatal exposure measures were, with some exceptions, the average of two 

measurements, and thus may better reflect chronic exposure during pregnancy than in studies where 

only a single spot urine sample was collected. Under an assumption of no other biases, the negative 

findings could have resulted from exposure measurement error. 

The outcome measurements, the Bayley Scales of Infant Development and the CBCL, were also 

appropriate. The Bayley Scales of Infant Development is considered a gold standard assessment. The 

assessments were reportedly appropriately administered. Both the MDI and PDI scales were 

administered in Spanish and/or English by psychometricians masked to exposure status of the children. 

Psychometricians were trained using standardized protocols and were supervised for quality assurance 

by a clinical neuropsychologist. Assessments were performed in a private room. The influence of interobserver reliability was controlled by inclusion for an indicator for the assessed but data were not 

presented in this report.

The Child Behavior Checklist (CBCL) has been widely used in cross-cultural research and collects data on 

a range of behavior problems, yielding scores for several syndrome scales and five scales designed to be 

consistent with Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnoses. The three scales 

used in this assessment were selected by the investigators a priori because the effects they measured 

were similar to effects seen in relation to DAP exposure in animal models. The child behavior checklist is

limited in that it is reliant on maternal report and the authors note the findings from the child behavior 

checklist are not directly equivalent to a DSM diagnosis.

The statistical analysis used to assess the associations between the markers of exposure and 

neurodevelopment were appropriate. However, the authors did not present nor discuss regression 
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diagnostics to assess the degree to which their models met or violated the assumptions implicit in linear 

models (i.e. homoscedasticity, normality of error term distribution, independence of error terms, and 

linearity of the exposure-response relationship), other than to state that they log transformed DAP 

levels. The authors also conducted a host of sensitivity analyses to assess influence of modeling 

decisions on their findings, and generally found their results to be robust. For example, they controlled 

for birth weight, gestational age, and abnormal reflexes which were considered causal intermediates, reran models excluding low birth weight and preterm infants, and ran models with and without 

adjustment of biomarkers for creatinine levels. The authors also assessed effect modification by gender 

and DDT exposure because population of primarily immigrant women who may have been exposed to 

DDT and other U.S. banned substances in country of origin; associations between pesticide markers and 

the indicators of neurodevelopment did not differ by either of these co-variables.

To increase the statistical efficiency of the study by preserving the size of the analytic population, 

missing covariate values were imputed by randomly selecting a value from participants with non-missing

values. Maternal depression had the largest percentage of values requiring imputation (5%). Of 

remaining covariates, between 0% and 1.8% of values were imputed. Although a reasonable approach, 

poorly measured confounders (i.e. residual confounding) may also be influenced by imputed covariate 

data, although perhaps to a lesser degree because imputed data may still be preferable to no data at all. 

Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for 

neurodevelopment that are potentially associated with pesticide exposure, including age at assessment, 

sex, duration of breast-feeding, HOME score, and household income, parity and indicator of maternal 

intelligence. In addition to the variables included in the final models, the authors examined potential 

confounding by several other variables suggested by the literature (i.e., maternal age, education, 

depressive symptoms, active/passive smoking exposure during pregnancy, regular alcohol use during 

pregnancy, marital status, father’s presence in home, housing density, maternal work status, childcare), 

but they did not markedly alter the observed associations. Covariates were selected for inclusion in the 

final analyses if they were related to conditions of testing, related to neurodevelopment in the literature 

and associated (p < 0.10) with most outcomes or consistently related to neurodevelopment in the 

literature, even if not in the data. This is a reasonable approach to assessment of confounding. 

The authors’ quantification of environmental co-exposures (DDT, DDE, β-hexachlorocyclohexane, 

hexachlorobenzene, PCBs, and lead) is a significant strength of this study. Children and mothers, 

especially those from economically impoverished environments and émigrés from home countries in 

which these compounds were frequently use in the recent past, may be exposed to other environmental 

agents that may affect neurodevelopment. Potential confounding by these exposures, as well as by 

indicators of fetal development (shortened gestation and poorer reflexes) was assessed, and observed 

associations in these models were consistent with the primary findings.

This study has apparent inconsistencies in the findings which are worth noting. First, while DAPs were 

associated with an increased risk of PDD in this population, post-natal DAPs were positively associated 
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with MDI. No explanation was presented by the authors. One possible explanation is that children with 

higher cognitive functioning may be more interactive with their environment, leading to higher exposure 

to pesticide residues, an example of “reverse causation”, whereby the temporal order of the exposure 

and outcome are reversed. The measurements of prenatal pesticide markers do not suffer from the 

same issues of unknown temporal order; they clearly preceded the measures of post-natal child 

development. Another possible explanation suggested by the authors is confounding by diet. Children 

who eat more fruits and vegetables may have higher DAP levels, but because of better diets have higher 

functioning. Second, although the investigators observed associations between neurodevelopment and 

the non-specific metabolite markers of organophosphate pesticides (DAPs), no such associations were 

observed for metabolites specific to chlorpyrifos (TCPy), nor for a malathion-specific metabolite (MDA). 

The organophosphate metabolites (DAPs) cannot be traced back to individual pesticides, and the 

observed associations with DAPs may be attributed to other pesticides, oxydemeton-methyl for 

example, for which no specific metabolite is measureable, or may be due to the short-half life and lower 

storage stability of the pesticide specific metabolites as compared to the non-specific metabolites.

The study has numerous strengths, most notably the prospective design the longitudinal assessment of 

both exposure and outcomes. Strengths in the study design also include the quantification of nonspecific (DAPs), chlorpyrifos-specific (TCPy) metabolites, and other environmental co-exposures. The 

results of this investigation by Eskenazi et al in the CHAMACOS cohort are suggestive of a detrimental 

association between prenatal organophosphate exposure, as measured by maternal urinary metabolite 

levels and neurodevelopment up to 24 months of age. Although the prospective study design and 

reasonable methods protect from certain false-positive inducing biases, there remains the potential of 

residual confounding in the direction of an adverse effect on development by unmeasured factors 

leading to the observation of a false-positive association. The authors reasonably suggest that their 

results should be interpreted with caution because, although they observed adverse associations 

between prenatal DAPs and mental development and pervasive developmental problems at 24 months 

of age, they also observed a positive relationship between neurodevelopment and postnatal DAPs.
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Article 11. Eskenazi et al. (2010)

PON1 and Neurodevelopment in Children from the CHAMACOS Study Exposed to Organophosphate 

Pesticides in utero. Brenda Eskenazi, Karen Huen, Amy Marks, Kim G. Harley, Asa Bradman, Dana Boyd 

Barr, and Nina Holland. Environmental Health Perspectives. 118:1775-1781 (2010).

In this article by Eskenazi et al. (2010), the authors report on the relationship between PON1 genotype 

and PON1 activity level and child neurodevelopment, and the potential effect modifying role of PON1 

status in the relation between prenatal and child urinary organophosphate metabolite levels and child 

neurodevelopment up to two years of age in the CHAMACOS cohort.

Methods and analyses employed in this investigation were similar to Eskenazi et al. 2007, with the 

exception of the measurement of PON1 status in association with this outcome, and assessment of 

interaction between PON1 and DAPs with child neurodevelopment. Enrollment for the CHAMACOS took 

place from 1999–2000 at community clinics, and included women who were ≥ 18 years old, pregnant at 

< 20 weeks gestation, Spanish- or English-speaking, eligible for low-income health insurance, and 

planning to deliver at the local public hospital. Researcher followed 528 women to delivery of a liveborn, surviving, neonate. Excluded from the analysis were children that did not have a 

neurodevelopmental assessment, (n=71), who did not have prenatal and relevant concurrent DAP 

metabolite measurements (n = 3), were not singletons (n=8), had a medical condition that could affect 

performance on the neurobehavioral assessment (n = 3; deafness, Down syndrome, hydrocephalus). 

Also excluded from analysis were Bayley results with raw scores which were too low for standardization 

(n=3, 1, and 1, for the 6-month, 12-month, and 24-month assessments, respectively), and participants 

for whom PON1 information was not available. The study included 371 mother-infant pairs.

Maternal/fetal exposure was assessed by measurement of six non-specific organophosphate 

dialkylphosphate (DAP) metabolites in maternal urine samples collected between 5 and 27 weeks 

gestation and again between 18 and 39 weeks. Specifically, investigators measured six nonspecific 

organophosphate DAP metabolites in maternal and child urine: three dimethyl (DMP) phosphate 

metabolites (dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate); and three diethyl 

(DEP) phosphate metabolites (diethylphosphate, diethylthiophosphate, and diethyldithiophosphate) 

(Bradman et al. 2005). These six metabolites represent the by-products of approximately 80% of OPs 

used in the Salinas Valley. Chlorpyrifos and diazinon were the most commonly used organophosphates 

in the region that metabolize to the diethyl phosphate metabolite. Values below the limit of detection 

(LOD) were assigned a value of LOD/(√2). Quantification of organophosphate metabolites using gas 

chromatography-tandem mass spectrometry and isotope dilution calibration was conducted by the 

Centers for Disease Control and Prevention (CDC) labs using published, validated, and accepted 

methods. 

Maternal and umbilical cord blood samples were collected at the time of delivery. Genotyping of the 

PON1-108 and PON1192 SNPs was performed using genomic DNA extracted from blood clots and Taqman 
Page 70 of 105

PCR methods. Two PON1 enzyme activity assays (arylesterase and paraoxonase) were performed using 

spectrophotometric methods. Arylesterase activity measures the rate of hydrolysis of the substrate 

phenyl acetate, and reflects the quantity of PON1 enzyme. Paroaxonase activity measures the rate of 

hydrolysis of paraoxon, and thus reflects a combination of the catalytic efficiency and quantity of the 

PON1 enzyme. Using triplicate samples, quality assurances methods illustrated the coefficient of 

variation was within the acceptable range (<10%).

The outcome, child neurodevelopment, was assessed using the Bayley Scales of Infant Development 

(Mental Development (MDI) and Psychomotor Development (PDI) Indices) (Bayley, 1993) and the Child 

Behavior Checklist (CBCL). The Bayley Scales of Infant Development were completed at the 6 months (n 

= 396), 12 months (n = 395), and 24 months (n = 372) of age. Both scales were administered in Spanish 

and/or English by psychometricians blinded to exposure. Psychometricians were trained using these 

standardized protocols and were supervised for quality assurance by a clinical neuropsychologist. 

Assessments were performed in a private room at the CHAMACOS research office or in a recreation 

vehicle (RV) modified to be a mobile testing facility. Scores > 1 SD below the mean (i.e., < 85) indicate 

possible developmental delay.

Mother’s completed the Child Behavior Checklist (CBCL) at the 24 month visit (n = 356). From the CBCL 

report, the authors assessed three scales as indicators of child neurodevelopment: The Attention 

Problems syndrome scale, which includes such items as “can’t concentrate” and “can’t sit still”; the 

DSM-oriented Attention-Deficit/Hyperactivity Disorder (ADHD) scale, which additionally includes such 

items as “gets into everything”; and the DSM-oriented Pervasive Developmental Disorder (PDD) scale, 

which includes such items as “avoids eye contact,” “rocks head, body,” and “unresponsive to affection”. 

Experts consider a score greater than the 98th percentile of a national normative sample of clinical 

significance; they consider a score greater than the 93rd percentile of borderline clinical significance.

Many confounders were ascertained including, intelligence of the mother, maternal depression, a 

measure of how stimulating the child’s environment is, and prenatal exposure to some known or 

suspected neurotoxins. Maternal intelligence was assessed via the Peabody Picture Vocabulary Test 

(PPVT). Maternal depression was assessed using the Center for Epidemiologic Studies Depression Scale 

(CES-D). To measure the quality and extent of stimulation available to a child in the home environment, 

the Infant-Toddler HOME (Home Observation for Measurement of the Environment) inventory was 

completed at the 6-month, 12-month, and partially at the 24-month visit. The following known or 

suspected neurotoxicants were measured: polybrominated diphenyl ethers (PBDEs), polychlorinated 

biphenyls (PCBs), p,p’-dichlorodiphenyltrichlorethane (DDT), p,p’- dichlorodiphenyltrichlorethylene 

(DDE), and lead. Lead was measured in cord blood. PBDEs, PCBs, DDT, and DDE were measured in 

maternal serum samples collected at 26 weeks gestation. However, these variables were not included in 

final models. The following variables were adjusted in the interaction analysis: age at assessment, sex, 

parity, breast-feeding duration, HOME score, maternal PPVT, and household poverty status. For the MDI 

and PDI models an indicator variable for the psychometrician was included, and maternal depression 

included in the model for PDD. 
Page 71 of 105

Statistical analyses of the relationship between PON1 status and Bayley performance were conducted 

using multiple logistic regression models; interaction terms and stratified analyses were presented to 

evaluate the potential effect modifying role of PON1 status in the DAPs and child neurodevelopment 

association. Neurodevelopmental scores were modeled as continuous variables. PON1 genotype was 

modeled as a three-level variable, using the wild type as the referent. PON1 enzyme levels were 

categorized as tertiles. Authors log10 transformed DAP levels, and averaged the two measures of urinary 

DAPs taken during pregnancy in the analyses, when available (n=22 for which only one sample 

available). Covariates in the models included indicators for the psychometrician conducting the 

assessment and the location of assessment, age at assessment, sex, duration of breast-feeding, HOME 

score, and household income, parity and indicator of maternal intelligence (PPVT score). Authors 

imputed missing values to preserve sample size, particularly in the stratified analyses. Statistical 

significance of the main effect was considered a p-value of 0.05, and significance of an interaction term 

was considered a p-value of 0.15. 

Among the selected participants, mothers were generally young age (average 26.4 years, sd 5.2), born in 

Mexico, were predominantly married or living as married, and were poor, 60% living at or below the 

poverty line. Forty-four percent of the women reported working in agriculture during pregnancy, 82% 

resided with a household member who works in agriculture, and 23% live near an agricultural operation. 

Most mothers reported breast feeding, and the duration of breast feeding was on average 6 months. 

Authors detected dimethyl phosphates more frequently than diethyl phospahtes. The average score for 

the MDI was 85 and PDI 98; 14% of children evaluated exceeded the concern level of the CBCL.

Overall, infants with the variant PON1108TT allele who produced lower amounts of PON1 enzyme had 

reduced scores on the MDI and PDI, and increased risk of PDD using the CBCL. Authors observed no 

association with PON1192QQ genotypes in the relation with MDI, PDI or PDD. Similarly, authors observed 

no association between enzyme levels and any measure of child neurodevelopment in this study. The 

interaction between maternal DAP and PON1 genotype with the Bayley assessed outcomes was nonsignificant (p=0.98), however researchers observed a trend of lower scores by the number of PON1108

variants alleles. For each 10-fold increase in total DAPs, the MDI score reduced by 3.2 PON1108CC, 3.7 

PON1108CT, and 5.5 PON1108TT (p-interaction 0.98). A similar trend was observed for the diethyl 

phosphates, but neither strata-specific estimates nor trend tests were significant. No similar trend was 

apparent across the number of variant alleles at the PON1192 genomic locus. No associations were

observed between DAPs and PDI by level of the PON1108 genotype. However, there was limited evidence 

of heterogeneity by PON1192 genotype in the relation between DAPs and PDI, but results were not 

significant. 

Study Review

Overall, researchers offer some evidence of a link between infant PON1 status and child 

neurodevelopment state, and authors’ state they cannot conclude the study provides evidence of effect 
Page 72 of 105

modification by PON1 genotype or phenotype status in the relation between DAPs and MDI, PDI or PDD 

in this study. Similarly to the accompanying investigation (Eskenazi et al. 2007), the study population 

was appropriate to the hypothesis, given that there were a range of exposure levels to OPs and there 

was some homogeneity in the population, e.g., mostly low SES, Hispanic, agricultural workers. The 

homogeneity of the population restricted confounding by design. For example, another paper reported 

that smoking, alcohol use, and illicit drug use were rare the CHAMACOS cohort. The homogeneous 

population also increased the relative statistical efficiency of the study because it is likely at higher risk 

of deficits in neurodevelopment than the general population (Landrigan, 1990; Landrigan et al., 1999). 

Authors did not share whether DAP levels were similar in mothers of infants for whom the MDI and PDI 

scores were not available; the potential for selection bias therefore cannot be assessed. The 

measurement of both urinary DAPs and PON1 status were well conducted, however limitations 

previously noted regarding the non-specific nature of a DAP measurement to the question of 

chlorpyrifos toxicity and the use of one- or two measures during the latter part of pregnancy as an 

estimate for gestational exposure apply equally to this investigation.

The outcome measurements, the Bayley Scales of Infant Development and the CBCL, were also 

appropriate. The Bayley Scales of Infant Development is considered a gold standard assessment. The 

assessments were reportedly appropriately administered. Both the MDI and PDI scales were 

administered in Spanish and/or English by psychometricians masked to exposure status of the children. 

Psychometricians were trained using standardized protocols and were supervised for quality assurance 

by a clinical neuropsychologist. Assessments were performed in a private room. The influence of interobserver variability was controlled by inclusion of an indicator variable for the psychomatrician. 

The Child Behavior Checklist (CBCL) has been widely used in cross-cultural research and collects data on 

a range of behavior problems, yielding scores for several syndrome scales and five scales designed to be 

consistent with Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnoses. The three scales 

used in this assessment were selected by the investigators a priori because the effects they measured 

were similar to effects seen in relation to DAP exposure in animal models. The child behavior checklist is 

limited in that it is reliant on maternal report, and the authors note the findings from the child behavior 

checklist are not directly equivalent to a DSM diagnosis.

Again, the statistical analysis used to assess the associations between the markers of exposure and 

neurodevelopment were appropriate. However, the authors did not present nor discuss regression 

diagnostics to assess the degree to which their models met or violated the assumptions implicit in linear 

models (i.e. homoscedasticity, normality of error term distribution, independence of error terms, and 

linearity of the exposure-response relationship), other than to state that they log transformed DAP 

levels. The authors also conducted a host of sensitivity analyses to assess influence of modeling 

decisions on their findings, and generally found their results to be robust. For example, investigators 

controlled for maternal depression in the relation between pesticide exposure and PDD, and ran models 

with and without adjustment of biomarkers for creatinine levels. 
Page 73 of 105

To increase the statistical efficiency of the study by preserving the size of the analytic population, 

missing covariate values were imputed by randomly selecting a value from participants with non-missing 

values. Maternal depression had the largest percentage of values requiring imputation (5%). Of 

remaining covariates, between 0% and 1.8% of values were imputed. Although a reasonable approach, 

poorly measured confounders (i.e. residual confounding) may also be influenced by imputed covariate 

data, although perhaps to a lesser degree because imputed data may still be preferable to no data at all. 

Bias due to confounding was also controlled in the analysis, by assessing and adjusting for risk factors for 

neurodevelopment that are potentially associated with pesticide exposure, including age at assessment, 

sex, duration of breast-feeding, HOME score, and household income, parity and indicator of maternal 

intelligence. In addition to the variables included in the final models, the authors examined potential 

confounding by several other variables suggested by the literature (i.e., maternal age, education, 

depressive symptoms, active/passive smoking exposure during pregnancy, regular alcohol use during 

pregnancy, marital status, father’s presence in home, housing density, maternal work status, childcare), 

but they did not markedly alter the observed associations. Covariates were selected for inclusion in the 

final analyses if they were related to conditions of testing, related to neurodevelopment in the literature 

and associated (p < 0.10) with most outcomes or consistently related to neurodevelopment in the 

literature, even if not in the data. This is a reasonable approach to assessment of confounding. 

The authors’ quantification of environmental co-exposures (DDT, DDE, β-hexachlorocyclohexane, 

hexachlorobenzene, PCBs, and lead) is a significant strength of this study. Children and mothers, 

especially those from economically impoverished environments and émigrés from home countries in 

which these compounds were frequently use in the recent past, may be exposed to other environmental 

agents that may affect neurodevelopment. Potential confounding by these exposures, as well as by 

indicators of fetal development (shortened gestation and poorer reflexes) was assessed, and observed 

associations in these models were consistent with the primary findings.

Similarly to the previous investigation by Eskenazi et al. (2007), the study has numerous strengths, most 

notably the prospective design the longitudinal assessment of both exposure and outcomes. Strengths 

in the study design also include the quantification of non-specific (DAPs), PON1 status, and other 

environmental co-exposures. The results of this investigation by Eskenazi et al. (2007) in the CHAMACOS 

cohort were suggestive of a detrimental association between prenatal organophosphate exposure, as 

measured by maternal urinary metabolite levels and neurodevelopment up to 24 months of age, and 

this investigation presents some additional, albeit limited evidence of potential effect modification by 

PON1 status. While results were not significant, sample size within strata of PON1 genotype and 

question as to the relevance of measurement of just PON1 among the suite of enzymes involved in 

organophosphate metabolism remain. Although the prospective study design and reasonable methods 

protect from certain false-positive inducing biases, there remains the potential of residual confounding 

in the direction of an adverse effect on development by unmeasured factors leading to the observation 

of a false-positive association, however less so with respect to PON1 status as these factors would not 
Page 74 of 105

likely be correlated. The authors reasonably suggest that their results should be interpreted with caution 

as the evidence for true effect modification in this relation is weak.
Page 75 of 105

Article 12. Marks et al. (2010)

Organophosphate Pesticide Exposure and Attention in Young Mexican-American Children: The 

CHAMACOS Study. Amy R. Marks, Kim Harley, Asa Bradman, Katherine Kogut, Dana Boyd Barr, 

Caroline Johnson, Norma Calderon, and Brenda Eskenazi. Environmental Health Perspectives. 

118:1768–1774 (2010).

Study Summary

Using data collected as part of the CHAMACOS Study, Marks et al conducted a study to investigate the 

association between urinary dialkyl phosphate (DAP) metabolites in pregnant women and their children, 

as a marker of organophosphate exposure, and attention-related outcomes among Mexican-American 

children living Salinas Valley, an agricultural region of California.

Enrollment for the CHAMACOS took place from 1999–2000 at community clinics, and included women 

who were ≥ 18 years old, pregnant at < 20 weeks gestation, Spanish- or English-speaking, eligible for 

low-income health insurance, and planning to deliver at the local public hospital. The investigators 

followed 526 women to delivery of a live born, surviving singleton. Excluded from analyses were 

children who did not have a prenatal DAP metabolite measurement (n = 2), had a medical condition that 

could affect performance on the neurobehavioral assessment (n = 3; deafness, Down syndrome, 

hydrocephalus), were lost to follow-up, or did not participate at the 3.5- or 5-year study visit (n = 173) 

were excluded from the study. The study population consisted of the remaining 348 children who had 

available data at 3.5 and/or 5 years. 

Exposures were assessed by measurement of organophosphate dialkylphosphate (DAP) metabolites in 

maternal and child urine samples. Maternal urine was collected between 5 and 27 weeks gestation and 

again between 18 and 39 weeks. The post-delivery urines were collected within 1 week of delivery for 

73% of the sample, with the remainder obtained up to 176 days afterwards. Urine samples were also 

collected from participating children at the 3.5 year and 5 year visits. Quantification of organophosphate 

metabolites was conducted by Centers for Disease Control and Prevention (CDC) labs. Total DAP 

metabolite level was defined as the sum of the molar concentrations of the six DAP metabolites, 

dimethylphosphate (DMP) metabolite level as the sum of the molar concentrations of the three 

dimethylphosphate metabolites only, and diethylphosphate (DEP) metabolite level as the sum of the 

molar concentrations of the three diethylphosphate metabolites. Total DAP, DMP, and DEP levels were 

determined for each participant for each of the two pregnancy urine samples and for the children’s 

urine samples. Individual metabolite levels below the limit of detection (LOD) were assigned a value of 

the LOD divided by the square root of two, and this value was included in each sum. Total DAP, DMP, 

and DEP metabolite levels were log10 transformed. “Pregnancy” DAP, DMP, and DEP values were created 

by averaging the two log-transformed pregnancy measures. For 19 women with only one DAP 

measurement in pregnancy, the single measure was used. Several children were missing DAP measures 

at 3.5 years (n = 58) and 5 years (n = 14).
Page 76 of 105

Attention-related outcomes were assessed in three ways: 1) maternal report of child behavior at 3.5 and 

5 years of age; 2) direct assessment of the child at 3.5 and 5 years; 3) and by a psychometrician’s report 

of the behavior of the child during testing at 5 years. 

Mothers completed the Child Behavior Checklist (CBCL) for 1.5–5 years of age as part of the maternal 

interview at the 3.5- and 5-year visits to assess emotional/behavioral problems and competencies of the 

children. Attention was evaluated as continuous raw scores on these scales, and by the proportion 

above a cutoff score greater than the 93rd percentile (a borderline clinical range). 

Children completed a battery of neurodevelopmental assessments at each visit: The visual attention 

subtest of the NEPSY-II at the 3.5-year visit, and the Conners’ Kiddie Continuous Performance Test (KCPT), during the 5-year neurodevelopmental assessment. The NEPSY-II was administered to children by 

trained psychometricians. Children were asked to circle specific images on a page of pictures that 

included distracters. For data analysis, the authors used continuous scores scaled to a normative sample 

of U.S. children; the age-standardized mean ± SD for this subtest is 10 ± 3. The K-CPT is a 7-min 

computerized test that assesses reaction time, accuracy, and impulse control. Briefly, children were 

instructed to press the space bar when they saw any image on the computer screen except a ball. The 

computer program yields T-scores age-standardized to a general U.S. population (mean ± SD = 50 ± 10) 

for errors of commission (i.e., the child responds when he or she should not), errors of omission (i.e., the 

child fails to respond when he or she should), and hit reaction time. The authors examined T-scores 

continuously and categorically using the cutoff of T-score > 65, which is reportedly considered markedly 

atypical. The program used also combined measures to generate a clinical ADHD Confidence Index score 

(range, 0–100). For statistical analyses, the authors used continuous Confidence Index percentiles and 

selected a cut-point of > 70th percentile (meaning that 70% of children performing similarly on the test 

could be correctly classified as having clinical ADHD).

Following the 5-year neurodevelopmental assessment, psychometricians blinded to exposure status 

answered several subjective questions evaluating the behavior of the child during the 2-hr visit, 

including four questions derived from the seven-item Hillside Behavior Rating Scale. The authors 

summed responses to two questions assessing motor activity and distractibility to create an adapted 

ADHD symptoms scale. The Hillside Scale is reportedly associated with parent and teacher ratings and 

has been found to add significantly to the clinical prediction of ADHD. For analysis the authors created a 

dichotomized Hillside outcome variable with scores ≥ 7 of 12 possible points (representing < 10% of 

children) to flag children displaying a higher degree of attention problems based on psychometricians’ 

observation.

Statistical analyses were conducted using multivariate linear and logistic regression models to evaluate 

the associations between the markers of organophosphate pesticide exposure and the indicators of 

attention deficits summarized above. Separate models were run with maternal DAPs only, child DAPs 

only, and the two together in the same model. Odds ratios greater than one and β-estimates greater 
Page 77 of 105

than zero indicate positive associations between exposure and increased risk of adverse outcome, 

except for the NEPSY visual attention subtest, where a β-estimate greater than zero indicates better 

performance. All models adjusted for the following covariates: an indicator of the evaluating 

psychometrician (not included in CBCL models), age at assessment, sex, maternal education, depressive 

symptoms, maternal intelligence, out-of-home child care, and breast feeding duration. The covariates 

maternal age, parity, marital status, active/passive smoking exposure and regular alcohol use during 

pregnancy, presence of father in home, maternal work status, and household income were also 

considered but not included in the final models because they did not markedly alter the observed 

associations and were therefore not used in final models. To preserve the size of the analytic 

population, remaining missing covariate values (six instances for maternal depression and three for 

maternal PPVT) were imputed by simple random selection of a value from participants with non-missing 

values.

The authors also conducted a number of sensitivity analyses. They reran all models using creatinineadjusted DAP metabolite concentrations. In addition, they added to final models some factors 

potentially on the causal pathway (i.e., birth weight, gestational duration). The authors also considered 

whether controlling for lead (log10-transformed), a known neurotoxicant, altered results for DAP 

concentrations in the subsamples with cord (n = 229) or 2-year (n = 296) lead values. Finally, because 

rates of ADHD vary considerably by sex in the general population (Pastor and Reuben 2008) and effects 

of other toxicants have been found to vary by sex (Delaney-Black et al. 2004), the authors tested for 

interactions between DAP concentrations and child sex, using p < 0.15 for the interaction term to 

determine whether associations of DAP concentrations with measures of attention differed for boys and 

girls.

Of the 378 infants included in this analysis, 47.5% were male. The mean maternal age was 26.5±5.2 

years. Most mothers were born in Mexico (86%), spoke only Spanish in the home (90%), had low income 

(97% within 200% of the federal threshold for poverty; 63% within 100%), had not completed high 

school (79%), were nonsmokers (96%), had previous live births (66%), and were married (82%). Many of 

the participating mothers had symptoms of depression (44%). 

In this study population, higher concentrations of organophosphate metabolites in the urine of pregnant 

women were associated with increased odds of attention problems and poorer attention scores in their 

children at age 5 years. Prenatal DAPs were non-significantly associated with maternal report of 

attention problems and ADHD at age 3.5 years but were significantly related at age 5 years (CBCL 

attention problems: β = 0.7 points; 95% CI: 0.2–1.2; ADHD: β = 1.3; 95% CI: 0.4–2.1). Prenatal DAPs were 

associated with scores on the K-CPT ADHD Confidence Index > 70th percentile (OR = 5.1; 95% CI: 1.7–

15.7 and with a composite ADHD indicator of the various measures (OR = 3.5; 95% CI: 1.1–10.7). Some 

outcomes exhibited evidence of effect modification by sex, with associations found only among boys. 

Children’s concurrent total DAP and DMP metabolite levels at 3.5 years and 5 years were unrelated to 

attention outcomes, and but child DEP concentrations at 5 years were adversely associated with the 

composite measure of attention (OR = 2.0; 95% CI:1.1–3.6).
Page 78 of 105

Study Review

This study tested whether in utero or postnatal exposures to organophosphate pesticides have a 

detrimental impact on attention. The study population was appropriate to the hypothesis, given that 

there were a range of exposure levels to OPs and there was some homogeneity in the population, e.g., 

mostly low SES, Hispanic, agricultural workers. The homogeneity of the population restricted 

confounding by design. For example, another paper reported that maternal smoking, alcohol use, and 

illicit drug use were rare this cohort. The homogeneous population also increased the relative statistical 

efficiency of the study because it is likely at higher risk of deficits in neurodevelopment than the general 

population. The exclusion of children who did not complete the 3.5- or 5-year assessment (n = 173), has 

the potential to induce a selection bias if the reasons for the missing data are determined by exposure 

(or a predictor of exposure) and are also not independent of the development of attention disorders. 

However, the authors stated that the levels of DAPs were similar in the mothers of the children which 

were not included relative to those included in the analysis. The authors also report that distributions of 

other covariates measured before 3.5 years were also similar, except that more boys than girls dropped 

from the study. This provides some indication that selection bias may not be a large factor in this 

analysis. 

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. 

Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more 

accurate and objective indicators of organophosphate pesticide exposure, relative to other 

ascertainment methods such as self-report. Although they are sensitive indicators of exposure, it is 

difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also indicate 

exposure to other organophosphates. The study population likely sustained many other potentially 

relevant exposures to multiple chemicals and other pesticides which may affect development. The six 

organophosphate metabolites cannot be traced back to individual pesticides but, according to the 

authors, they represent the breakdown products of approximately 80% of the total organophosphate 

pesticides used in the Salinas Valley. It is not possible to disentangle the various effects from these 

potential co-exposures and chlorpyrifos in this study. 

Because organophosphate pesticides have short half-lives, the degree to which metabolites in urine 

samples reflect ‘usual’ levels, and thus usual exposure, is uncertain. The prenatal exposure measures 

were, with some exceptions, the average of two measurements, and thus may better reflect chronic 

exposure during the pregnancy. The absence of findings associated with child levels may be due, in part, 

to the fact that the child’s levels are poor indicators of exposure during critical windows of exposure 

with respect to development of attentional difficulties. 

Errors in the assignment of exposure in this prospective study will likely have resulted in attenuation of 

observed associations. The likelihood of these errors is high, and the magnitude of the bias induced 

difficult to assess. Under an assumption of no other biases, the negative findings, those for the 
Page 79 of 105

association between children’s concurrent DAP levels and attention outcomes, for example, could have 

resulted from exposure measurement error. 

The outcome in this study is very difficult to measure. Instead of opting for a single best 

instrument/indicator of attention deficits, the investigators chose to use multiple instruments, and also 

to combine the results of these various instruments to form a composite indicator of attentional 

difficulties. There is no evidence presented in the report that would suggest that the outcome 

assessments were improperly conducted. Nevertheless, outcome measurement error is likely. As with 

the exposure assessment, errors in the assignment of attention deficits in this prospective study will 

likely have resulted in attenuation of observed associations. Again, the likelihood of these errors is high, 

and the magnitude of the bias induced difficult to assess. The authors did not report on an assessment 

of changes in attentional difficulties between the 3.5 and 5 year visits. If such changes did occur, it 

would have been interesting to the reader to present results of a longitudinal analysis between this 

change in the outcome, and the child DAP levels over time. 

The statistical analysis used to assess the associations between the markers of exposure and attention 

deficits were appropriate. Model diagnostics were performed and, according to the authors, found 

acceptable. The results of the model diagnostics were not presented. The authors also conducted a host 

of sensitivity analyses to assess influence of modeling decisions on their findings, and generally found 

their results to be robust. 

In addition to those included in final models, the following covariates were considered, but not included 

in multivariate models: maternal age, parity, marital status, active/passive smoking exposure, regular 

alcohol use during pregnancy, presence of father in the home, maternal work status, and household 

income at the time of assessment. Confounding by blood lead levels was also assessed in a subset of 

children with measurements of cord blood (n = 229) and 2-year (n = 296) lead levels, and found not to 

influence the DAP and attentional outcomes. 

Covariates were selected for these analyses if they were related to conditions of testing or related to 

attention deficits in the literature and/or associated with more than one outcome (p < 0.10 or changes 

to the main effect coefficient by ≥ 10%). The authors used a less conservative alpha level as a cut-off for 

selection of covariates, a somewhat more conservative approach to covariate selection, relative to use 

of the conventional alpha level of 0.05. However, the use of a statistical test as a means to select 

covariates from a larger set of potential confounders is discouraged in some epidemiologic circles 

(Miettinen 1976, Breslow and Day 1980, Greenland 1989). The assessment of confounding is generally 

not considered to be a statistical endeavor, in part because the association between the confounder and 

the outcome is just one factor that determines the degree of confounding, and also because the 

statistical significance of an association is a function not only of the magnitude of the association but 

also of other factors (e.g., sample size, and sample variability) which are irrelevant to bias due to 

confounding. Moreover, the use of significance testing in the selection of confounders treats false 

negative errors (i.e. the deleting a confounder) as secondary to false positive error (inclusion of a non-
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confounder). This implicit ranking of error is backward, as deleting a confounder introduces bias and is 

justified only if the bias induced by its exclusion is tolerably small or deemed to be worth the precision 

gain, whereas including a non-confounder only (potentially) reduces precision by increasing the size of 

the model. This is a small consideration in this study, and different approaches are not likely to 

qualitatively change the study findings or conclusions. Despite the confounder evaluation conducted by 

the investigators, residual confounding due to mismeasured or unmeasured confounders is likely to 

have somewhat biased the observed associations.

To preserve the sample size the authors appropriately imputed or replace data for missing covariates. 

Data on maternal depression were missing for 5% of observations and maternal intelligence scores for 

less than 1%. Values of maternal depression measured when the child was 1 year old were available and 

substituted for 13 instances where maternal depression when the child was 3.5 years was missing. 

Other missing covariate values (six instances for maternal depression and three for maternal PPVT) were 

imputed by simple random selection of a value from participants with non-missing values. The resulting 

analysis is relatively more precise, owing to the larger sample size analyzed, but this statistical efficiency 

comes at the tradeoff of bias due to residual confounding by imperfectly measured covariates. 

Socioeconomic conditions and other difficult to measure determinants of early life development may 

further confound the reported associations in the direction of more deleterious effects associated with 

the organophosphate metabolites and with unknown magnitude. The potential for this bias may be 

mitigated somewhat due to restriction in the variability of these confounding factors in this relatively 

homogeneous study population.

The generalizability of the findings to other populations may be limited, due to the particular nature of 

this study population with respect to factors which may modify the association between 

organophosphate exposures and infant development. The organophosphate pesticide exposure markers 

and the prevalence of attention deficits in this low-income Mexican-American population sampled from 

an agricultural area are almost certainly not generalizable to the U.S. population, although exposure 

levels are within a comparable range to the U.S. general population (see Section 5.0). 

The study has numerous strengths, most notably the prospective design and the longitudinal 

assessment of both exposure and outcomes. The positive association between organophosphate 

metabolites in the urine of pregnant women and increased odds of attention problems and poorer 

attention scores in their offspring was relatively consistent using different assessments of attentional 

difficulties (i.e. by maternal report, psychometrician observation, and neuropsychological testing). 

Associations were stronger at age 5 years compared to age 3.5 years, and there was evidence of a 

stronger association among boys, compared to girls. The results of this investigation by Marks et al in 

the CHAMACOS cohort are suggestive of a detrimental association between prenatal organophosphate 

exposure, as measured by maternal urinary metabolite levels, and attentional difficulties at age 5 years. 

This study may be limited in its direct contribution to the literature base regarding the specific 

association between chlorpyrifos and developmental outcomes because the assessment of non-specific 

organophosphate pesticide metabolites (DAPs) is a primary potential source of exposure measurement 
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error. Because of the prospective design, information bias is expected to result in attenuation of the 

true (unobserved) associations, toward (observed) associations that are relatively smaller, or null. 
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Article 13. Rauh et al. (2011)

Seven-Year Neurodevelopmental Scores and Prenatal Exposure to Chlorpyrifos, a Common 

Agricultural Pesticide. Virginia Rauh, Srikesh Arunajadai, Megan Horton, Frederica Perera, Lori 

Hoepner, Dana B. Barr, and Robin Whyatt. Environmental Health Perspectives. 119:1196–1201 (2011).

Study Summary

Having previously reported an association between prenatal exposure to chlorpyrifos and 

neurodevelopmental problems at three years of age in the Columbia Center for Children’s 

Environmental Health, Rauh et al now evaluate the relationship between prenatal chlorpyrifos exposure 

and neurodevelopment among cohort children at age seven. 

Study participants were recruited during early pregnancy (≤20th week) among African-American and 

Dominican women age 18-35 years, and registered Hospital, in New York at New York Presbyterian 

Medical Center and Harlem City. Women who smoked, had a history of drug abuse, diabetes, 

hypertension, or HIV infection were excluded from participation in the study, as were women who 

resided in New York City for less than 1 year. The study sample presented in this report was recruited 

between 1998 and 2002, a period which straddles the January, 2001 implementation of a voluntary 

cancellation by registrants of indoor residential chlorpyrifos use. Of originally 725 consenting women, 

535 were active participants in the ongoing cohort study at the time of this report. This report uses data 

from 265 of their children who had reached the age of 7 years and had a complete set of data including 

prenatal maternal interview data, prenatal chlorpyrifos marker levels from maternal and/or cord blood 

samples at delivery, postnatal covariates, and neurodevelopmental outcome data.

Chlorpyrifos levels in umbilical cord blood samples were available for 256 newborns, sampled as close to 

the time of delivery as possible, and within 2 days post-partum. In cases where the umbilical cord blood 

sample was not collected (12% of subjects), cord blood chlorpyrifos levels were imputed from mothers’ 

values. Quantification of chlorpyrifos levels in plasma were conducted by the Centers for Disease 

Control and Prevention (CDC).

Neurodevelopment at age 7 years was assessed using the Wechsler Intelligence Scale for Children, 4th 

edition (WISC-IV). The instrument measures four areas of mental functioning: the Verbal 

Comprehension Index, the Perceptual Reasoning Index, the Working Memory Index, and the Processing 

Speed Index. These indices are associated with, but distinct from, overall intelligence quotient (IQ) and 

are sensitive to cognitive deficits related to learning and working memory, which have been linked to 

chlorpyrifos exposure in rodent studies. A Full-Scale IQ score combines the four composite indices. A 

General Ability Index score is a summary score of general intelligence, similar to Full-Scale IQ, but 

excludes contributions from both Working Memory Index and Processing Speed Index.
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Multivariate linear regression models were used to estimate associations. Chlorpyrifos exposure level 

(picograms per gram) was entered as a continuous variable. The WISC-IV Composite Index scores were 

natural log (ln) transformed to stabilize their variances and to improve the linear model fit, based on 

regression diagnostics. Linear and non-linear functional relationships between chlorpyrifos exposure 

and each of the log-transformed WISC-IV indices were assessed and compared. 

Participating mothers were predominantly from low income households, with 31% failing to complete 

high school by child’s age 7 years, and 66% were never married. The children in the study were largely 

full term and included very few low-birth-weight infants through study design. Chlorpyrifos exposure 

levels as measured in cord blood ranged from non-detectable to 63 pg/g. 

Unadjusted correlations showed significant inverse associations between chlorpyrifos levels and 

Working Memory (r=-0.21, p=<0.0001); and Full-scale IQ (r=-0.13, p=0.02). Because cord lead levels 

were not significantly correlated with chlorpyrifos levels, and in an effort to avoid excluding those with 

missing lead levels, lead was not included in the regression models. ETS was significantly correlated with 

chlorpyrifos levels (Spearman coefficient 0.113, p=0.01) but not with any of the WISC-IV indices. In 

addition, birth weight was not significantly associated with any of the WISC scales and not included in 

the final models.

The investigators used both fully-adjusted linear regression modeling and the Least Absolute Shrinkage 

and Selection Operator (LASSO) technique, described as a more parsimonious selection of covariates 

that addresses unstable estimates while avoiding overfitting (Houwelingen 2001; Tibshirani, 1996.) Both 

methods yielded very similar estimates, finding associations between chlorpyrifos exposure and FullScale IQ as well as the Working Memory Index. For Full-Scale IQ both techniques generated a 

coefficient (B) of -0.003 with matching CIs of -0.006, 0.001 but the P-values were calculated as 0.064 for 

the LASSO and 0.048 for the fully-adjusted model. The fact that both CI’s contain zero while the fullyadjusted model’s P-value is lower, suggests that the LASSO technique does in fact shrink unstable 

estimates towards zero. For the Working Memory Index, both techniques generated a coefficient (B) of 

-0.006 with the LASSO calculating a CI of -0.009 to -0.002, p<0.001 and the fully-adjusted model 

calculating a CI of -0.010 to -0.002, p=0.003. The investigators articulated these results as showing that 

a 1 pg/g increase in chlorpyrifos exposure was associated with a 0.006 point decrease in the logtransformed Working Memory score and a 0.003 point decrease in the log-transformed Full-Scale IQ 

score. In order to understand the magnitude of these effects, the investigators calculated the 

neurodevelopmental deficit associated with the increase in exposure and concluded that for each 

standard deviation increase in exposure (4.61pg/g) there is a 1.4% reduction in Full-Scale IQ and a 2.8% 

reduction in Working Memory. Authors explain that their full model results did not show any significant 

interactions between ETS or polycyclic aromatic hydrocarbons (PAH) with measures of prenatal 

chlorpyrifos exposure during the prenatal period and intelligence measures in this study. 

The investigators conducted a sensitivity analysis to determine if the association between chlorpyrifos 

levels and Working Memory Index was partially due to the effect on general intelligence. To do this, 
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they looked at the General Ability Index of the WISC-IV, a general intelligence scale calculated without 

the inclusion of the items included in the Working Memory Index or Processing Speed Index. When the 

General Ability Index was added to the linear regression model, the estimated chlorpyrifos effect did not 

change and they did not observe an interaction between chlorpyrifos and General Ability Index. From 

this they concluded that the Working Memory Index is a targeted measure, independent of general 

intelligence. 

The investigators further explored their findings by conducting a supplemental analysis to evaluate 

whether the observed associations between chlorpyrifos and the Working Memory Index were related 

to underlying behavioral problems. They used the results of the Child Behavior Checklist and its 

clinically-oriented behavioral scales and concluded that there was no evidence of mediation caused by 

behavioral issues. 

Lastly the investigators addressed the possible influence of their method and frequency of use of LOD

imputations. To do this they recalculated all of the association estimates using only those records that 

had detectable chlorpyrifos levels and explained that this method would provide an unbiased estimate 

of the association (Little, 1992.) They did not find any consistent differences in estimates between the 

full sample (including imputed exposure levels) versus those calculated without those imputed levels.

Study Review

This was a very well conducted study with numerous strengths including selection of a relevant and 

appropriate cohort, the prospective study design, direct measurement of chlorpyrifos, comprehensive 

consideration of confounding, and analytic design choices which maximize statistical efficiency. 

However, some limitations are also noted.

The direct measurement of chlorpyrifos in cord blood and personal air samples, rather than selfreported indirect exposure assessment or use of biomarkers of organophosphate pesticide exposure 

that are not specific to chlorpyrifos exposure is a notable strength of the Columbia Mother’s and 

Newborn Study cohort. Unlike previous analyses among this cohort that used a dichotomous variable 

(low vs. high exposure) to classify exposure status, these findings employ a continuous chlorpyrifos level, 

which provides a more meaningful look at the data including the ability to assess potential threshold 

effects and linear and non-linear dose-response trends. The investigators rigorously evaluated their 

methods for imputing values for undetectable chlorpyrifos levels; analyses illustrated imputation 

methods were valid. However, regarding the LOD estimations, the authors did not describe the 

methodology used (e.g. bootstrap estimation), if any, to incorporate the added uncertainty (i.e. added 

imprecision to estimates) inherent in the use of imputed data into the estimation of the corresponding 

variance estimates. However, errors in estimation of cord blood chlorpyrifos levels were likely to have 

been non-differential with respect to the indicators of childhood neurodevelopment and intelligence. 
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A limitation of the specific biomarker exposure indicators in this study is the single sampling period (at 

delivery). It is not clear to what extent this single measurement reflects exposures over critical windows 

during pregnancy. If the exposure is chronic, a biomarker measured at a single time point can provide a 

representative dosimeter, even if the toxicant has a short half-life, as is the case for chlorpyrifos. 

However, if pesticide exposures are sporadic or otherwise vary over short time scales, the biomarker 

measurement will not be representative of “usual” exposure, or of the exposure during critical periods 

of fetal neurodevelopment. In previous publications from this same cohort, the investigators explain 

that analyses were performed showing that, among these women, exposures to chlorpyrifos did not 

show significant variation throughout pregnancy (Whyatt et al, 2007) and therefore concluded that this 

single measure can accurately represent prenatal exposure. This assumption is further assessed in 

subsequent validation studies conducted by Whyatt et al (2007 and 2009) within this cohort. 

Whether or not these cord blood measurements accurately represent prenatal exposure, there is no 

control for exposure over the subsequent 7 years which may be critical, especially as the process of 

neurocognitive development is fluid and rapid during these early childhood years. From this study, it is 

unclear if an increase or decrease in exposure during this influential postnatal period, caused by agerelated or seasonal dietary changes or changes in pesticide application, could also impact the 

neurodevelopment of these children. Because this imprecise measure of exposure is unlikely to vary 

differentially, this error would bias towards the null.

Another influence unaccounted in the analysis is the effect of increased awareness of the risks of 

pesticide exposures over the time period of cohort follow-up in this study (1998-2004) and the potential 

for differential exposure or outcome (CBCL) measures. It is feasible to assume that those parents of 

children with suspected or diagnosed neurodevelopmental issues in early childhood would be more 

aware of issues concerning organophosphate exposure, and thus more likely to subsequently reduce 

their child’s exposures, especially through a reduction in pesticide or dietary sources in childhood. If this 

is true, and post-natal exposures play a role in childhood neurodevelopment measured in this study, 

those children with neurodevelopmental deficits may be less likely to experience post-natal exposures 

than those not diagnosed, thus making the pre-natal effects appear weaker. Similarly, mothers of 

infants in the high exposure category may differentially report behavioral issues on the maternalreported child behavior checklist . Either type of differential measurement error may lead to information 

bias, with unknown consequences on the estimate of risk in these studies. It would be meaningful to 

assess the mother’s awareness of the effects of organophosphate exposures as well as details regarding 

dietary exposures and how they may have changed over the 7-year period.

The prospective nature of the study limits the influence of errors in the exposure assessment on the 

association observed, such that mismeasurement of chlorpyrifos exposure would likely be nondifferential with respect to the indicators of neurodevelopment and intelligence at age 7 years. The 

voluntary cancellation of the residential use of chlorpyrifos introduces exogenous variation in the 

exposure of interest. After the cancellation, levels of chlorpyrifos in personal and indoor air samples in 

this cohort decreased by more than 65%, and plasma blood levels dropped by more than 80%.
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The Wechsler Intelligence Scale for Children (WISC-IV) instrument measures four areas of mental 

functioning: the Verbal Comprehension Index is a measure of verbal concept formation, a good 

predictor of school readiness; the Perceptual Reasoning Index measures nonverbal and fluid reasoning; 

the Working Memory Index assesses children’s ability to memorize new information, hold it in shortterm memory, concentrate, and manipulate information; and the Processing Speed Index assesses 

ability to focus attention and quickly scan, discriminate, and sequentially order visual information. The 

authors explain that the sensitivity of the WISC-IV among 6 to 7.5 year old children has been validated in 

previous neurodevelopmental studies of environmental lead exposure, even at low-dose levels (Chiodo 

et al. 2004). However, WISC-IV scores are also known to be influenced by socioeconomic background 

and/or child behavior problems, particularly those related to anxiety (Wechsler, 2003). The narrowly 

restricted cohort and evaluation of behavioral issues, which may in part reflect anxiety issues, within this 

study somewhat limits this potential bias of the outcome assessment tool in this study.

One potential weakness is that the investigators do not give the details of the administration of the 

WISC-IV; specifically the credentials of the interviewers, whether or not they were blinded to the child’s 

exposure status, any trainings or certifications, and whether or not intra- or inter-interviewer reliability 

or quality assurance was conducted. These types of administration issues can result in less-sensitive 

measures and misclassification, but there is no reason to believe that there would be a differential effect 

based on exposure level. 

Also complicating this analysis is the pervasive, non-specific nature of neurological effects and the 

difficulty in attributing causal pathways to one agent. In future studies it would be meaningful to 

include other potential co-variants such as family history (especially among parents and siblings) of 

developmental delay or attention disorders as well as other co-morbidities. Not only is the etiology of 

attention and related behavioral disorders not well understood but also the diagnosis of these disorders 

is often influenced by complicated outside forces. Because the study population is relatively 

homogenous with respect to socio-economic variables, it is unlikely that differences in co-morbidities or 

family history would be associated with exposure status, thus biasing the association towards the null.

The authors describe an elegant and methodologically sound statistical analysis, addressing many of the 

potential shortcomings of their exposure data and co-variables. Out of the five WISC-IV scores, two 

were described as being significantly influenced by chlorpyrifos exposure. After they conducted both 

full-adjusted linear regression and LASSO techniques, they concluded that both Full Scale IQ and 

Working Memory index were affected by prenatal chlorpyrifos exposure. For each standard deviation 

increase in exposure (4.61pg/g), there was a 1.4% reduction in Full-Scale IQ and a 2.8% reduction in 

Working Memory. The Working Memory Index conclusions seem reasonable, but the associations for 

Full Scale IQ were not statistically significant (LASSO: -0.006, 0.001, p=0.064; fully-adjusted: -0.006, 

0.001, p=0.048.) In addition, the authors do not address the clinical relevance of the 1.4% and 2.8% 

reductions and how this may or may not influence a child or his/her psychological or educational 

success. 
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In their confirmatory analyses they used the General Ability Index (the intelligence score calculated after 

removing Working Memory and Processing Speed items) to verify that the association seen between 

chlorpyrifos exposure and Working Memory was not an artifact of the association with Full Scale IQ. 

With the General Ability Index included in the linear regression model, the estimated chlorpyrifos effect 

did not change and they did not observe an interaction between chlorpyrifos and General Ability Index. 

Subsequently they concluded that the deficits in Working Memory Index were associated with 

chlorpyrifos exposure, independent of general intelligence but analyses could have also taken an 

additional step. These results could also be used to conclude that since the General Ability Index was 

not found to be affected by chlorpyrifos exposure, the Full Scale IQ findings were caused by the 

influence of the Working Memory module of the instrument. The influence of chlorpyrifos exposure on 

the working memory aspect of the Full Scale IQ score was strong enough to overcome the nonsignificant results from the other modules of the instrument.

Confounding by maternal smoking behaviors was controlled by restriction (i.e., excluding smokers from 

the study sample). Confounding by unmeasured factors may have been similarly controlled by design, 

this being a relatively homogeneous population with respect to determinants of neurodevelopment, 

compared to the general population. Authors evaluated several potentially confounding variables 

including exposure to other environmental chemicals including blood lead, and only included those 

variables which displayed evidence of confounding.

While controlling confounders by restriction, the choice to study low income, urban dwellers likely 

resulted in a relative increase in statistical efficiency (i.e. increased power) because this population likely 

experiences a greater variability of pesticide exposure and is at higher risk of neurodevelopmental 

deficits, relative to the general population.

Although blood lead levels were not included as covariates in the multivariate models, the authors 

reported that umbilical cord lead was not significantly correlated with chlorpyrifos level or WISC-IV 

scores among the subgroup of children in this population for whom blood lead was assessed (n=89). 

Polyaromatic hydrocarbons were weakly correlated with chlorpyrifos in this cohort but were not 

significantly associated with WISC-IV scores. Birth weight was not significantly associated with any of 

the WISC-IV indices and was not included in the final models.

Bias due to confounding by unmeasured determinants of neurodevelopment is possible in this 

observational study, for example, confounding by factors related to socioeconomic condition. The urban 

dwelling, minority children assessed in this study may experience other unmeasured exposures or 

underlying health problems that could potentially confound (or modify) associations with pesticide 

exposure. However, both adjustment through restriction of study population, as well as control for 

several measures of SES in multi-variable models, as well as additional methodological analyses of the 

role of SES in this association (Lovasi et al. 2011), does not suggest a significant additional role of 

unmeasured confounding due to variation in SES within this study population. Effect modification by all 
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of the covariates was assessed, including for environmental tobacco smoke and polycyclic aromatic 

hydrocarbons, but no interactions were observed.

The authors state that the retention rate for the full cohort was 82% at the 7-year follow-up, and 

reported that there were no significant sociodemographic differences between subjects retained in the 

study and those lost to follow-up, although these data were not provided. However, the 265 children 

included in this analysis were born to a 36.6% subsample of the 725 women who originally consented to 

participate in the study. The resulting large proportion of missing data has the potential to induce a 

selection bias if the reasons for missingness of outcome data are determined by exposure (or a predictor 

of exposure) and are also not independent of neurodevelopment. For example, participants with high 

pesticide exposures could have missed neurodevelopmental assessments for reasons related to 

uncontrolled determinants of exposure which are also related to neurodevelopment. This was not 

discussed. 

A comprehensive set of sensitivity analyses and regression diagnostics were conducted, including 

examination of residuals for normality and homoscedasticity, influential observations, and the linearity 

of the exposure-response relationship, and assessment of the influence of design choices (e.g.

imputation) on observed results. Models were determined to be consistent with modeling assumptions. 

Non-linearity of the exposure response curves were assessed by superimposing smoothed cubic spline 

regression curves over scatterplots of the data. The log-transformed Working Memory Index and FullScale IQ were approximately linear, whereas the other functions demonstrated some curvature across 

exposure levels, with sparse observations at the highest exposures. However, formal goodness of fit 

testing did not demonstrate that non-linear models provided a better fit to the data, compared to 

models that assume a linear exposure-response relationship (i.e. no observed threshold effect). 

Results of this study showed that higher prenatal chlorpyrifos exposure, as measured in umbilical cord 

blood plasma, was associated with decreases in cognitive functioning on two different WISC-IV indices, 

in this cohort of urban minority children at 7 years of age. For each SD increase in exposure (4.61 pg/g), 

Full-Scale IQ declined, on average, by 1.4% (0.94–1.8 points) and Working Memory Index scores declined 

by 2.8% (1.6–3.7 points). These results are consistent with earlier findings of an inverse association 

between chlorpyrifos levels in umbilical cord plasma and neurodevelopment in the same cohort. The 

study represents perhaps the most rigorous study design and analysis of the studies reviewed. 
Page 89 of 105

Works Cited

Bradley RH and Corwyn RF. Socioeconomic Status and Child Development. Annual Review of Psychology. 

53:371-399 (2002).

Chiodo LM, Jacobson SW, Jacobson JL. 2004. Neurodevelopmental effects of postnatal lead exposure at 

very low levels. Neurotoxicol and Teratol, 26:359-371.

Houwelingen JCv. 2001. Shrinkage and penalized likelihood as methods to improve predictive accuracy. 

Stat Neerlandica. 55(1):17-34.

Lovasi, G. S., Quinn, J. W., Rauh, V. A., Perera, F. P., Andrews, H. F., Garfinkel, R., . . . Rundle, A. (2011). 

Chlorpyrifos exposure and urban residential environment characteristics as determinants of early 

childhood neurodevelopment. Am J Public Health, 101(1), 63-70. doi: 10.2105/ajph.2009.168419

Little RJA. 1992. Regression with missing X’s: a review. J Amer Stat Assoc. 87:1227–1237.

Wechsler D. Wechsler Intelligence Scale for Children. 4rd Edition. San Antonio, Texas: Psychological 

Corporation. 2003.

Whyatt RM, Rauh V, Barr DB, et al. Prenatal insecticide exposure and birth weight and length among an 

urban minority cohort. Environ Health Perspect. 2004;112:1125–1132

Whyatt RM, Garfinkel RS, Hoepner LA, Borjas M, Camann DE. Within and between home variability in 

indoor-air insecticide levels during pregnancy [abstract W2A–3]. Presented at the 15th Annual 

Conference of the International Society of Exposure Analysis; October 30–November 3, 2005; Tucson, AZ
Page 90 of 105

Article 14. Bouchard et al. (2011)

Prenatal Exposure to Organophosphate Pesticides and IQ in 7-Year-Old

Children. Maryse F. Bouchard, Jonathan Chevrier, Kim G. Harley, Katherine Kogut, Michelle Vedar, 

Norma Calderon, Celina Trujillo, Caroline Johnson, Asa Bradman, Dana Boyd Barr, Brenda Eskenazi. 

Environmental Health Perspectives. 119:1189–1195 (2011)

Study Summary

Researchers with the Center for the Health Assessment of Mothers and Children of Salinas study 

(CHAMACOS study) published an analysis which investigated the relationship between prenatal and 

early life exposures to organophosphate pesticides (OPs) and cognitive development. The CHAMACOS 

study is a prospective birth cohort study of environmental exposures and the health of pregnant women 

and their children living in the Salinas Valley, an agricultural area of California. 

A 2007 report from this cohort suggested that prenatal, but not postnatal, exposure to OPs was 

associated with increased odds of pervasive developmental disorder and lower scores of mental 

development at two years (Eskenazi B 2007). Additionally, a 2010 paper reported an association 

between organophosphate pesticide exposure and poorer attention skills as well as hyperactive 

behaviors at 5 years (Marks AR 2010). This 2011 study by Bouchard et al was conducted to determine 

whether cognitive deficits associated with prenatal exposure to organophosphate pesticides are 

persistent, since previous cohort studies have not followed children to school-age, when cognitive 

deficits may have greater implications for school performance. This paper reports the association 

between prenatal and postnatal exposure to organophosphate pesticides, indicated by urinary dialkyl 

phosphate (DAP) metabolite concentrations, and cognitive abilities of 7-year olds.

Enrollment took place from 1999–2000 at community clinics, and included women who were ≥ 18 years 

old, pregnant at < 20 weeks gestation, Spanish- or English-speaking, eligible for low-income health 

insurance, and planning to deliver at the local public hospital. The initial 601 women who were enrolled 

into the study delivered 526 infants. From this analysis, 2 participants with missing DAP concentration 

measurements were excluded, 4 children with a medical condition that would affect the assessment, 

children who were lost to follow-up or who did not participant in the 7-year visit were excluded (72 

moved, 59 refused, 24 unable to trace, 21 unable to schedule, 2 deceased), and children missing the 

cognitive assessment at the 7-year visit (13) were also excluded.

Prenatal urine was collected between 5 and 27 weeks gestation and again between 18 and 39 weeks. 

Urine was collected from the children at 6 months and 1, 2, 3 1⁄2 and 5 years of age. Six nonspecific 

organophosphate DAP metabolites (3 DEP metabolites and 3 DMP metabolites) were measured in 

maternal and child urine which represent the breakdown products of about 80% of the total 

organophosphate pesticides used in the Salinas Valley (CDC 2009). The most commonly used 

organophosphate pesticides in the Salinas Valley include chlorpyrifos and diazinon (which are 
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metabolized to DEP) as well as malathion and oxydemeton-methyl (which devolve to DMP). 

Concentrations below the limit of detection (LOD) were randomly imputed based on a log-normal 

probability distribution whose parameters were estimated using maximum likelihood estimation. The 

DAP metabolite concentrations were expressed on a molar basis and summed to yield total DEP, DMP, 

and DAP concentrations.

Cognitive abilities were measured at the 7-year study visit using the Wechsler Intelligence Scale for 

Children - Fourth Edition (WISC-IV) (Wechsler 2003). Scores for four domains were calculated based on 

the following subtests: Verbal Comprehension (comprised of the Vocabulary and Similarities subtests), 

Perceptual Reasoning (Block Design and Matrix Reasoning subtests), Working Memory (Digit Span and 

Letter-Number Sequencing subtests), and Processing Speed (Coding and Symbol Search subtests). All 

subtests were administered in the dominant language of the child, which was determined through 

administration of the Oral Vocabulary subtest of the Woodcock-Johnson/Woodcock-Muñoz Tests of 

Cognitive Ability in both English and Spanish (Woodcock and Johnson 1990). Ultimately, 67% of children 

were tested in Spanish, and 33% in English. WISC-IV scores are standardized against U.S. populationbased norms for English and Spanish-speaking children.

Many confounders were measured, including intelligence of the mother, measures of how stimulating 

the environment is, and known or suspected neurotoxins were measured prenatally. Maternal 

intelligence was assessed via the Peabody Picture Vocabulary Test (PPVT). To measure the quality and 

extent of stimulation available to a child in the home environment, the Infant-Toddler HOME (Home 

Observation for Measurement of the Environment) inventory was completed at the 6-month, 1, 2, 3.5, 

5, and 7 year visits. The following known or suspected neurotoxicants were measured: polybrominated 

diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), p,p’-dichlorodiphenyltrichlorethane (DDT), 

p,p’- dichlorodiphenyltrichlorethylene (DDE), and lead. Lead was measured in maternal blood at 26 

weeks gestation, in cord blood for a subset of participants, and children’s blood a 2 years of age. PBDEs, 

PCBs, DDT, and DDE were measured in maternal serum samples collected at 26 weeks gestation. 

Nonspecific total DAP, DEP, and DMP metabolites (nmol/L) were log10-transformed. The authors 

examined the association between non-creatinine adjusted urinary DAP concentrations and cognitive 

scores using multiple linear regression, with point estimates representing the change in cognitive scores 

for each 10-fold increase in DAP concentrations. For prenatal exposure, they examined associations with 

the DAP concentrations measured separately for urine collected during the 1st and 2nd half of 

pregnancy (≤20 vs. >20 weeks gestation). However, since they found similar relations between cognitive 

scores and DAPs measured earlier and later than 20 weeks gestation, they averaged the two DAP 

measures for further analyses. For 20 children (6%) only one prenatal measure was available for the 

analyses. Because DMP and DEP metabolites might have different relationships to the outcomes, the 

authors examined them separately. For postnatal exposure, they examined the cross-sectional 

association of cognitive scores with DAP concentrations measured in children’s urine collected at 

different ages in separate models.
Page 92 of 105

They also calculated the cumulative DAP level between 6 and 60 months using the area under the curve 

(AUC), calculated using the trapezoidal method. For 46 children with 1 missing DAP measurement at 1, 

2, or 31⁄2 years, they imputed the mean of the 2 measures closest in time for the AUC calculation. Fortynine children who were missing DAP measures at either the 6-month or 5-year visit, or missing more 

than 1 DAP measure from the 3 other time points were excluded from the AUC analysis. For comparison 

with prenatal exposure, they calculated the mean urinary DAP concentrations measured during 

childhood for children with at least 3 out of 5 measures (taken at 6 months, 1, 2, 3 1⁄2, and 5 years); this 

excluded 20 children.

The authors compared effect estimates for urinary DAPs measured in early vs. late pregnancy and in the 

prenatal vs. postnatal periods using seemingly unrelated estimation and they used the mean postnatal 

DAP concentrations (as opposed to AUC) for these analyses in order to compare metrics with similar 

units. They used generalized additive models with 3-degree-of-freedom cubic splines to evaluate the 

shape of dose-response curves, test the linearity assumption, and investigate potential thresholds while 

controlling for covariates. They did not observe evidence of departure from linearity, or threshold for 

effect, thus, they retained the simpler models based on linear regression. For illustration purposes, they 

grouped DAP concentrations into quintiles, entered this categorical variable in the multiple regression 

model with the same covariates described above, and obtained the mean IQ score for each quintile.

The authors explored potential confounding effects for the following variables: maternal intellectual 

abilities, maternal education, HOME score at 6 months, 1, 2, 3 1⁄2, and 5 years, breastfeeding duration, 

maternal age, birth order, poverty category, marital status, children’s age at WISC testing, and maternal 

levels of PBDEs, PCBs, DDE, DDT, and lead during pregnancy. They added each of these variables added 

individually to the final model. However only maternal intellectual abilities (PPVT score, continuous), 

maternal education (3 categories), and continuous HOME score at 6 months were retained in the final 

models. Other variables were dropped from the final models because they did not change the 

magnitude of the coefficient for urinary DAP concentrations by >10%. 

In separate analyses, the authors also investigated potential confounding and effect modification by 

variables possibly on the causal pathway (i.e. birth weight and gestational age, assessed continuously). 

Because the majority of children (67%) were tested in Spanish, the authors reran the analyses restricted 

to this subset. The authors also examined the interaction between sex and DAP concentrations, based 

on previous findings in this cohort (Marks AR, 2010).

To explore possible synergistic effects between pre- and postnatal DAP concentrations, the authors 

included an interaction term for mean prenatal DAP concentrations×AUC. However, since this term was 

not statistically significant (p > .15) they did not include it in the final models.

The authors observed evidence of an association between prenatal exposures to organophosphate 

pesticides as measured by urinary DAP metabolites in women during pregnancy, and decreased 

cognitive functioning in children at age 7. This finding was seen for total DAP, DMP, and DEP 
Page 93 of 105

metabolites. Children in the highest quintile had of maternal DAP concentrations had an average deficit 

of 7.0 IQ points compared with those in the lowest quintile. The associations were linear with no 

threshold. Urinary DAP concentrations in childhood were not associated with cognitive scores in this 

cohort of children.

Study Review

This study tested whether in utero or postnatal exposures to organophosphate pesticides have a 

detrimental impact on cognitive abilities at age seven years. The study population was appropriate to 

the hypothesis, given that there were a range of exposure levels to OPs and there was some 

homogeneity in the population, e.g., mostly low SES, Hispanic, agricultural workers. The homogeneity of 

the population restricted confounding by design. For example, another paper reported that maternal 

smoking, alcohol use, and illicit drug use were rare this cohort. The recruitment of an inner-city cohort 

may also have increased the relative statistical efficiency of the study if the study population is at higher 

risk of neurodevelopmental deficits compared to the general population. Selection bias may have been a 

factor in this analysis as only 329 families out of 601 who were originally enrolled into the study were 

included. The authors state that the families included in this analysis did not differ on most attributes, 

including urinary DAP concentrations during pregnancy, maternal measures of cognitive ability, 

maternal education, marital status, poverty category, and child’s birth weight. However, mothers of 

children included in the present study were slightly older and breastfed longer than the initial cohort. 

Differences between maternal blood lead and other environmental exposures which are potential 

confounders were not discussed.

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. 

Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more 

accurate and objective indicators of organophosphate pesticide exposure, relative to other 

ascertainment methods such as self-report. Although they are sensitive indicators of exposure, it is 

difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also indicate 

exposure to other organophosphates. The study population likely sustained many other potentially 

relevant exposures to multiple chemicals and other pesticides which may affect development. The six 

organophosphate metabolites cannot be traced back to individual pesticides but, according to the 

authors, they represent the breakdown products of approximately 80% of the total organophosphate 

pesticides used in the Salinas Valley. It is not possible to disentangle the various effects from these 

potential co-exposures and chlorpyrifos in this study. 

The IQ test used the WISC-IV is very widely utilized in both clinical and research setting, and has a long 

history of use, and was appropriately administered by psychometrician. Additionally, the test was 

administered at age 7 years, which is above the floor for administration (age 6) (Wechsler 2003). 

However, IQ tests are notoriously susceptible to possible biases due to differences in cultural and/or 

ethnic composition of the population, although the homogeneity of the population may ameliorate the 

effect of this potential bias. The authors did perform the analysis both by looking at only the tests that 
Page 94 of 105

were administered in Spanish and with the Spanish and English included. However, language may not 

have controlled for all potential biases which are inherent in IQ testing, e.g., degree of acculturation. 

The statistical models used are appropriate and well powered. For exposure measures that were less 

than the laboratory analytic limit of detection, the measures were imputed. Imputation is a better 

method than substitution of the LOD/√2 or LOD/2, which is the method used in many studies. It appears 

that the investigators selected parsimonious models by only including terms which changed the 

outcome measure and were statistically significant. Additionally, the authors tested the linearity 

assumption which is inherent in linear regression using cubic splines. 

Residual confounding by unmeasured or poorly measured confounders is possible in this study. The 

authors did measure a large number of potential confounders, but not all were evaluated notably, 

childhood exposure to neurotoxins, and maternal drug used during pregnancy. A number of 

environmental chemicals were considered, although only measurements of exposure during pregnancy 

were included in the analysis. Additionally, exposure to carbamates, which are also potentially 

neurotoxic, was not included in the analysis. The authors also did not consider maternal alcohol, 

tobacco, or drug use in this analysis. However, another study with this cohort reported that these 

confounders are very rare in this study population.

The authors did not discuss paternal intelligence or intelligence of other caregivers. This is probably 

because only the mothers and children were enrolled into the study, and it is difficult to enroll fathers 

and other caregivers into birth cohort studies. Although it is understandable that these measures were 

not addressed, it seems highly possible that intelligence of the fathers and other caregivers could be 

associated with the exposure, organophosphate pesticides, and the disease, cognitive impairment of the 

child. Thus these factors could have confounded the study results, and it is a limitation that they were 

not included in the measurement or analysis. 

A potential limitation with respect to generalizability is the high level of exposures observed in the study 

cohort, compared to the US population. The mean level of urinary DAP metabolites in this cohort is 

higher than the mean which has been seen among women of child bearing years in NHANES. However, 

the investigators report that 25% of the US population have urinary DAP levels exceeding the median in 

this study. So, the concentrations in this cohort are not out of the range of normal for the US as a whole. 

Generalizing beyond the range of exposure observed in this study requires an assumption that the linear 

exposure-outcome relationship observed in the study is applicable to populations with different levels of 

exposure.

This study has many strengths, the longitudinal design, the measurement of urinary DAP at many time 

points and following children to age seven when tests of cognitive function are reportedly more reliable. 

The authors were able to adjust for or consider many factors related to cognitive function, such as 

prenatal exposure to other environmental agents, socioeconomic indicators, maternal intelligence and 

education, and child stimulation. The cohort had a relatively homogenous socioeconomic profile, 
Page 95 of 105

reducing the potential for uncontrolled confounding. Additionally, the authors noted their findings were 

consistent with findings from previous reports from this cohort (Eskenazi, 2007) and other investigations 

of adverse associations between prenatal exposure to organophosphate pesticides and cognition (Rauh,

2006). They also noted that although they did not see an association between childhood exposures to 

organophosphate pesticides, the other studies that have differed in the measurement of exposure 

and/or the specific outcomes found to be associated with exposure to organophosphate pesticides 

(Lizardi, 2008, Ruckart, 2004). The many advantages of this study, the plausibility of the findings, and 

that similar association have been seen in other studies support the hypothesis that prenatal exposure 

to OPs reduces cognitive function of children. However, to the extent that prenatal chlorpyrifos is the 

true exposure of interest, the assessment of non-specific organophosphate pesticide metabolites is a 

primary potential source of exposure measurement error. Because of the prospective design, 

information bias is expected to result in attenuation of the true (unobserved) associations, toward 

(observed) associations that are relatively smaller, or null. 
Page 96 of 105

Works Cited

Bradman A, Barr DB, Claus Henn BG, Drumheller T, Curry C, Eskenazi B. "Measurement of pesticides and 

other toxicants in amniotic fluid as a potential biomarker of prenatal exposure: a validation study." 

Environ Health Perspect, 2003: 1779-1782.

Bradway DE, Shafik TM, Lores EM. "Comparison of cholinesterase activity, residue levels, and urinary 

metabolite excretion of rats exposed to organophosphorus pesticides." J Agric Food Chem, 1977: 1353-

1358.

CDC. "Fourth National Report on Human Exposure to Environmental Chemicals. Chemical Information. 

Organophosphorus Insecticides: Dialkyl Phosphate Metabolites." 2009. 

http://www.cdc.gov/exposurereport/data_tables/OP-DPM_ChemicalInformation.html (accessed March 

29, 2011).

Eskenazi B, Marks AR, Bradman A, Harley K, Barr DB, Johnson C, et al. "Organophosphate pesticide 

exposure and neurodevelopment in young Mexican-American." Environ Health Perspect, 2007: 792-798.

Lizardi PS, O'Rourke MK, Morris RJ. "The effects of organophosphate pesticide exposure on Hispanic 

children's cognitive and behavioral functioning." J Pediatr Psychol, 2008: 91-101.

Lu C, Kedan G, Fisker-Andersen J, Kissel JC, Fenske RA. "Multipathway organophosphorus pesticide 

exposures of preschool children living in agricultural and nonagricultural communities." Environ Res, 

2004: 283-289.

Marks AR, Harley K, Bradman A, Kogut K, Barr DB, Johnson C, et al. "Organophosphate pesticide 

exposure and attention in young Mexican-American children." Environ Health Perspect, 2010: 1768-

1774.

Muto MA, Lobell F Jr, Bidanset JH, Wurpel JN. "Embyrotoxicity and neurotoxicity in rats associated with 

prenatal exposure to DURS-BAN." Vet Hum Toxicology, 1992: 498-501.

Rauh VA, Garfinkel R, Perera FP, Andrews H, Barr D, Whitehead D, Tang D, Whyatt RM. "Impact of 

prenatal chlorpyrifos exposure on neurodevelopment in the first three years of life among inner-city 

children." Pediatrics, 2006: 1845-1859.

Ruckart PZ, Kakolewski K, Bove FJ, Kaye WE. "Long-term neurobehavioral health effects of methyl 

parathion exposure in children in Mississippi and Ohio." Environ Health Perspect, 2004: 46-51.

Wechsler, D. Wechsler Intelligence Scale for Children-IV: Administration and Scoring Manual. San 

Antonio, TX: Harcourt Assessment Inc, 2003.
Page 97 of 105

Whyatt RM, Garfinkel R, Hoepner LA, Andrews H, Holmes D, Williams MK, et al. "A biomarker validation 

study of prenatal chlorpyrifos exposure within an inner-city cohort during pregnancy." Environ Health 

Perspect, 2009: 559-567.
Page 98 of 105

Article 15. Whyatt et al. (2007)

Within- and Between-Home Variability in Indoor-Air Insecticide Levels during Pregnancy among an 

Inner-City Cohort from New York City. Robin M. Whyatt, Robin Garfinkel, Lori A. Hoepner, Darrell 

Holmes, Mejico Borjas, Megan K. Williams, Andria Reyes, Virginia Rauh, Frederica P. Perera, and David 

E. Camann. Environmental Health Perspectives. 115:383–389 (2007).

Study Summary

Whyatt et al conducted a study to assess within- and between-home variability in indoor-air insecticides 

over the final 2 months of pregnancy among a subset of participants in the Columbia Mother’s and 

Newborn Study, a cohort of women from New York City. Participants (n = 102) assessed in this 

investigation were enrolled between February, 2001 and May, 2004, after the January, 2001 

implementation of a voluntary cancellation by registrants of indoor residential chlorpyrifos use. 

Expectant mothers were recruited during early pregnancy (≤20th week) among African-American and 

Dominican women age 18-35 years, and registered at New York Presbyterian Medical Center and 

Harlem Hospital, in New York City. Women who smoked, had a history of drug abuse, diabetes, 

hypertension, or HIV infection were excluded from participation in the study, as were women who 

resided in New York City for less than 1 year or, for this validation study, who were employed outside 

the home at the time of enrollment. Most of the mothers (96%) in the present study lived in residential 

apartment buildings; the remaining lived in multifamily housing (n = 3) or in combined 

residential/commercial buildings (n = 1).

Nine insecticides (the organophosphates chlorpyrifos, diazinon, malathion, and methyl parathion; the 

carbamates propoxur, bendiocarb, and carbofuran; and the pyrethorids cis and trans-permethrin) and 

piperonyl butoxide, an adjuvant that is frequently added to pyrethroid formulations, were measured in 

48-hr personal air samples and 2-week integrated indoor air samples collected sequentially for 7.0 ± 2.3 

weeks (n = 337 air samples). Indoor air monitoring occurred around the 32nd week of pregnancy, and air 

samples were collected every 2 weeks throughout the monitoring period. Personal air samples were 

obtained for mothers over a 48 hour period during the 32nd week of pregnancy.

Arithmetic means ± SDs were calculated for pesticides detected in greater than 45% of samples. Before 

statistical analyses, pesticide values were log-transformed to normalize their distributions. Values below 

the level of detection (LOD) were assigned a value of half of the LOD. Pearson correlation coefficients or 

Spearman’s correlation coefficients, as appropriate, were calculated to assess the relationship between 

pesticide levels in indoor air and personal air samples. Analysis of variance (ANOVA) was used to test 

whether pesticides levels varied significantly by season and year of monitoring and among the following 

groups: women not using any pest control methods; women using “nonspray” methods only; and 

women using higher-toxicity pesticides. If levels differed significantly among the groups, the least 

significant difference test was used to determine which groups varied significantly. T-tests were 

employed to compare differences in mean pesticide levels by ethnicity. Chi-square analyses were used 
Page 99 of 105

to test whether the proportion of women using pest control measures over the final 2 months of 

pregnancy was related to pest sightings in the home. Finally, a mixed-effects model was used to assess 

within-home and between-home variability in repeat indoor air samples, after controlling for season and 

year of delivery. Except for the Spearman’s rank and chi-square analyses, all statistical testing was 

conducted on log-transformed data. 

Sixty-one percent of the participating women reported using pest control during the air samplings. 

Chlorpyrifos, diazinon, and propoxur were detected in 99–100% of personal and indoor samples (range, 

0.4–641 ng/m3

). Piperonyl butoxide (a pyrethroid adjuvant) was detected in 45.5–68.5% (0.2–608 

ng/m3

). There was little within-home variability and no significant difference in air concentrations within 

homes over time (p ≥ 0.2); between-home variability accounted for 88% of the variance in the indoor air 

levels of propoxur, 92% in chlorpyrifos, 94% in diazinon, and 62% in piperonyl butoxide (p < 0.001). 

Indoor and maternal personal air insecticide levels were highly correlated (r = 0.7–0.9, p < 0.001). 

Diazinon and chlorpyrifos levels declined 5-fold between 2001 and 2004 but were detected in all homes 

at 1.5 and 2.5 years, respectively, after implementation of a voluntary cancellation by registrants of 

indoor residential chlorpyrifos use. No previously unreported associations between chlorpyriphos and 

health outcomes were presented in this report. The authors concluded that the insecticides were 

persistent in the home with little variability in air concentrations over the 2 months and likely 

contributed to chronic maternal inhalation exposures during pregnancy.

Study Review

Although the Columbia Mother’s and Newborn Study enrolled participants in years prior to the 

implementation of the 2001 voluntary cancellation by registrants of indoor residential chlorpyrifos use, 

only data for participants recruited in the post-cancellation period were considered in this validation 

study analysis. Explanation for this design choice was not provided, but is likely that appropriate 

repeated sampling was not conducted during earlier study periods. The authors noted that the mothers 

in the present study were comparable with women in the larger cohort in terms of maternal age, 

ethnicity, marital status, annual household income, and reported use of pest control methods during 

pregnancy. However, a greater proportion of women in the present study compared with those in the 

larger cohort reported having less than a high school education, which could be due to the fact that, for 

these analyses, the investigators excluded women who worked outside the home. 

The exposure assessment in this study was well conducted using appropriate and previously published 

methodology. The semivolatile insecticides were readily detected in indoor air after residential use. 

Indoor air levels of these chlorpyrifos and the other organophosphate pesticides in the 2-week 

integrated indoor air samples remained remarkably stable within most homes over 6–8 weeks of 

sequential sampling. The two-week integrated sampling methodology was targeted towards evaluation 

of chronic exposure during the latter pregnancy period, and thus precludes assessment of changes in 

concentrations in indoor air at times scales shorter than two-weeks (e.g. day-to-day changes). 
Page 100 of 105

In this study, pesticide levels in indoor air were remarkably stable. Correspondingly, almost all (90%) of 

the total variance in the indoor air levels was explained by between-home variability. The degree to 

which this finding in the post-cancellation period informs variability in airborne pesticide levels prior to 

the cancellation was beyond the scope of the investigation. 

The authors did not observe an association between indoor air levels of chlorpyrifos and maternal selfreported pesticide use, a finding which indicates that most of the chlorpyrifos concentrations detected 

in the indoor air samples likely resulted from prior, rather than current, pesticide use. However, that 

chlorpyrifos was detected in indoor air samples from all the homes assessed, including those evaluated 

2.5 years after implementation of the voluntary cancellation of indoor residential chlorpyrifos use, 

demonstrates the persistence of the compound in certain indoor environments. 

No associations between pesticide exposures and health outcomes were assessed in this validation 

study.
Page 101 of 105

Article 16. Whyatt et al. (2009)

A Biomarker Validation Study of Prenatal Chlorpyrifos Exposure within an Inner-City Cohort during 

Pregnancy. Robin M. Whyatt, Robin Garfinkel, Lori A. Hoepner, Howard Andrews, Darrell Holmes, 

Megan K. Williams, Andria Reyes, Diurka Diaz, Frederica P. Perera,1 David E. Camann, and Dana B. 

Barr. Environal Health Perspectives. 117:559–567 (2009).

Study Summary

Whyatt et al conducted a biomarker validation study within the Columbia Mother’s and Newborn Study 

to evaluate trends over time in multiple biomarkers of prenatal chlorpyrifos exposure. The group 

previously documented significant decreases in chlorpyrifos concentrations in maternal personal and 

indoor air samples among pregnant African-American and Dominican women from New York City after 

the 2001 voluntary cancellation by registrants of indoor residential chlorpyrifos use. Participants (n = 

102) were enrolled between February, 2001 and May, 2004. 3,5,6-trichloro-2-pyridinol (TCPy) was 

measured in newborn meconium (n = 83), repeat prenatal maternal urine samples (n = 253), and 

postnatal urine from mothers (n = 73) and newborns (n = 59). Chlorpyrifos was directly measured in 

postnatal maternal (n = 92) and umbilical cord (n = 65) blood plasma and in personal and indoor air 

samples. No previously unreported associations between chlorpyriphos and health outcomes were 

presented in this report.

Study participants were recruited during early pregnancy (≤20th week) among African-American and 

Dominican women age 18-35 years, and registered at New York Presbyterian Medical Center and 

Harlem Hospital, in New York City. Women who smoked, had a history of drug abuse, diabetes, 

hypertension, or HIV infection were excluded from participation in the study, as were women who 

resided in New York City for less than 1 year or who were employed outside the home at the time of 

enrollment (for the purposes of this validation study). The study sample presented in this report was 

recruited between 2001 and 2004, after the January, 2001 implementation of a voluntary cancellation 

by registrants of indoor residential chlorpyrifos use. Most of the mothers (96%) in the present study 

lived in residential apartment buildings; the remaining lived in multifamily housing (n = 3) or in 

combined residential/commercial buildings (n = 1).

Indoor air monitoring occurred around the 32nd week of pregnancy using 2-week integrated indoor air 

samples collected every 2 weeks throughout the monitoring period. Personal air samples were obtained 

for mothers over a 48 hour period during the 32nd week of pregnancy.

Repeat prenatal maternal urine samples were collected from mothers at the end of each 2-week indoor 

air sampling period, beginning at the 34th week of pregnancy and continuing until delivery. Women who 

went into labor before the end of the first 2 weeks of indoor air sampling did not provide urine samples. 

Urine samples were collected from 97 of 102 women in the study; more than half of the women 

provided three or more urine samples, 29 of 97 (30%) two samples, and 15 of 97 (15%) provided one 
Page 102 of 105

sample. An additional urine sample was obtained on the day after delivery for 73 women and 59 

newborns. 

An umbilical cord blood sample was collected from 65 newborns after delivery. A sample of maternal 

blood was obtained from 92 mothers within 2 days postpartum. Paired maternal and cord blood 

samples were available for 64 of the mothers and newborns.

Meconium samples were collected from 83 newborns during the postpartum hospital stay. In 53 (64%) 

of the births, the meconium samples were collected within 1 day of delivery.

Biologic samples were analyzed for organophosphate pesticide markers by the CDC using previously 

established methods using both positive and negative controls. The limit of detection (LOD) of 

chlorpyrifos in blood samples was 0.5–1 pg/g plasma. The LOD of TCPy in urine samples was 0.26 ng/mL 

urine. The LOD for TCPy in meconium was 0.2 ng based on a sample weighing 0.5 g.

Exposure marker levels below the LOD were given a value of half the level of detection, and were then 

log10 transformed. Nonparametric statistics were used when distributions remained non-Gaussian. 

Spearman rank correlation coefficients were used to assess relationships between levels of chlorpyrifos 

or TCPy in biological samples and to examine correlations between levels of chlorpyrifos or TCPy in the 

biological samples and chlorpyrifos levels in air samples. Multiple linear regression was employed to 

evaluate the amount of variance in prenatal maternal urine TCPy levels explained by indoor air 

chlorpyrifos levels among subjects enrolled in 2001–2002. 

Analysis of variance (ANOVA) or Kruskal–Wallis tests with post-hoc testing as appropriate, were used to 

assess whether pesticide levels varied by year of birth (between 2001 and 2004), or among the following 

groups: women not using any pest control methods; women using “nonspray” pest abatement methods 

only; and women using can sprays, pest bombs, or sprays by an exterminator, with or without nonspray 

methods. Authors performed t-tests corresponding nonparametric Mann–Whitney U-test were used to 

assess whether pesticide levels differed by ethnicity. Chi-square analyses were used to test whether the 

frequencies of detection of chlorpyrifos and TCPy in biological samples varied by year of assessment as 

well as to compare detection frequencies of chlorpyrifos in maternal and cord blood samples. A mixed 

effects model was used to evaluate the within- and between-subject variability in TCPy levels in the 

repeat maternal urine samples collected during pregnancy, and to calculate an intraclass correlation 

coefficient (ICC) for women enrolled in 2001–2002 who had three repeat urine samples (TCPy was 

detected in only one-third of samples among subjects enrolled after 2002). The ICC is a measure of 

reliability of repeated measures over time, defined as the ratio of between-subject variance to total 

variance. 

The authors did not detect TCPy in infant urine, but all other biomarkers showed a highly significant 

decrease in detection frequencies (χ2 = 7.8–34.0, p ≤ 0.005) and mean ranks (p ≤ 0.006) among subjects 

enrolled in 2003–2004 compared with those enrolled in 2001–2002. Chlorpyrifos in maternal personal 
Page 103 of 105

and indoor air declined 2- to 3-fold over the same period (p < 0.05). In 2001–2002 samples, TCPy levels 

in repeat prenatal urine samples were positively correlated (r = 0.23–0.56), although within-subject 

variability exceeded between-subject variability (intraclass correlation coefficient = 0.43); indoor air 

levels explained 19% of the variance in prenatal urine TCPy (p = 0.001). Meconium TCPy concentrations 

were positively correlated with chlorpyrifos in maternal and cord blood (r = 0.25–0.33, p < 0.05) and 

with TCPy in maternal urine (r = 0.31, p < 0.01).

Study Review

Although the Columbia Mother’s and Newborn Study enrolled participants in years prior to the 

implementation of the 2001 voluntary cancellation by registrants of indoor residential chlorpyrifos use, 

only data for participants recruited in the post-cancellation period were considered in this analysis. 

Explanation for this design choice was not provided, but is likely that appropriate repeated sampling was 

not conducted during earlier study periods. The authors noted that the mothers in the present study 

were comparable with women in the larger cohort in terms of maternal age, ethnicity, marital status, 

annual household income, and reported use of pest control methods during pregnancy. However, a 

greater proportion of women in the present study compared with those in the larger cohort reported 

having less than a high school education, which could be due to the fact that, for these analyses, the 

investigators excluded women who worked outside the home. 

The investigators’ use of nonparametric, rank-based statistics is appropriate for analysis of non-normally 

distributed data. However, the large number of ties among marker levels due to observations below the 

level of detection receiving equal rank, may be problematic. When ranked values have the same 

numeric number (e.g., half the level of detection), the rank is 0.5 times the number of ties. Whether, 

and at what point a large proportion of ties begins to limit the inferential power of rank based statistics 

is unclear, although at some point, a non-continuous classification of the data (e.g., classification of 

observations into bins at a cut-point equal to the level of detection) seems to make more sense if the 

number of below the level of detection observations is large. 

The exposure assessment conducted in this investigation is the most comprehensive of any 

epidemiologic study of chlorpyrifos to date. Strengths of the exposure assessment include the 

quantification of actual blood chlorpyrifos levels in addition to sensitive, but non-specific markers of 

organophosphate pesticide exposure in urine, the repeated sampling of urinary markers, measures in 

meconium and the environmental sampling. The exposure assessments were appropriately timed with 

respect to critical window of gestation for fetal growth and neurodevelopment outcomes which likely 

occurs, in part, during the third trimester of pregnancy, and appear to have been well conducted. 

Although inhalation exposures are likely a major route of exposure, no direct dietary assessment was 

included in this investigation. 

The study was set during a period of rapidly declining use of chlorpyrifos, and the findings are 

demonstrative of decreasing exposure over time. The investigators observed a highly significant 
Page 104 of 105

decrease in the detection frequency and mean ranks of all biomarkers among subjects enrolled during 

years 2001–2004, with the exception of TCPy in newborn urine, which was not detected. 

Maternal and umbilical cord blood plasma chlorpyrifos levels were below the LOD in all samples 

collected after 2002. The results suggest that chlorpyrifos from residential use was the main contributor 

to the chlorpyrifos levels detected in both the maternal and umbilical cord blood among study subjects 

in the larger cohort (1998-2004, both before and after voluntary cancellation of chlorpyrifos containing 

compounds).

While no association was observed between maternal and umbilical blood chlorpyrifos levels measured 

at delivery and TCPy in maternal urine samples collected through the latter third trimester, TCPy levels 

in meconium (an integrative measure week 13-delivery) were significantly correlated with chlorpyrifos 

levels in both maternal and umbilical cord blood measured at delivery, and with TCPy levels in maternal 

urine samples collected in third trimester. These results suggest that TCPy in meconium may provide a 

valid biomarker of prenatal exposure, possibly both prior to and subsequent to the cancellation period.

The finding that within-subject variability exceeded between-subject variability in maternal urine TCPy 

(intraclass correlation coefficient, ICC= 0.43), again emphasizes the point that a single prenatal urine 

sample is likely insufficient to capture ‘usual’ exposure. The authors noted that despite the significant 

within-subject variability seen in their study, the ICC observed was higher than that observed in another 

population (Meeker et al., 2005).

The authors concluded that their results suggest that pesticide biomarkers utilized in these studies are 

reliable dosimeters to differentiate between groups with prenatal chlorpyrifos exposures varying by a 

factor of 2 or more, and that cord blood measures are reasonable well correlated with exposure 

measures taken throughout pregnancy (meconium correlated with cord blood (delivery) and maternal 

urine (3rd trimester). Authors also support that these data illustrate the efficacy of the voluntary 

cancellation of indoor residential chlorpyrifos use to reduce the internal dose during pregnancy. 
Page 105 of 105

Works Cited

Meeker, J. D., Barr, D. B., Ryan, L., Herrick, R. F., Bennett, D. H., Bravo, R., & Hauser, R. (2005). Temporal 

variability of urinary levels of nonpersistent insecticides in adult men. J Expo Anal Environ Epidemiol, 

15(3), 271-281. doi: 7500402 [pii]

10.1038/sj.jea.7500402