1 Chemical Exposures and Molecular Biology Branch, Office of Program Development, Division of Extramural Research and Training, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC
2 Division of Epidemiology, Statistics, and Prevention Research, National Institute of Child Health and Human Development, Rockville, MD
3 Department of Chemistry, Warren Wilson College, Asheville, NC
4 Department of Biostatistics, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC
5 Westat, Inc., Durham, NC
6 Epidemiology Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC
Correspondence to Dr. Matthew P. Longnecker, Epidemiology Branch, National Institute of Environmental Health Sciences, MD A3-05, Research Triangle Park, NC 27709 (e-mail: longnecker{at}niehs.nih.gov).
Received for publication October 30, 2003. Accepted for publication March 3, 2005.
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ABSTRACT |
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child development; cohort studies; polychlorinated biphenyls
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INTRODUCTION |
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Epidemiologic studies of perinatal exposure to background-level PCBs in relation to cognitive functioning in children have given inconsistent results. For example, among Michigan children followed from birth, prenatal PCB exposure was associated with poorer performance on the verbal and memory scales of the McCarthy Scales of Children's Abilities at 4 years of age (n = 133) and with lower full-scale and verbal intelligence quotient (IQ) scores on the Weschler Intelligence Scale for Children-Revised at age 11 years (n = 178) (3, 13
). In a birth cohort from the Netherlands, the findings were similar on the Kaufman Assessment Battery for Children at 42 months of age. Children who had the highest level of in utero exposure to PCBs performed less well on cognitive tests (n = 395) (4
). On subsequent testing at age 6.5 years, however, the relation was no longer present. In a cohort from upstate New York, higher umbilical cord PCB levels were associated with lower scores on the McCarthy Scales of Children's Abilities at 38 months of age but not at 54 months (n
195) (14
). In a North Carolina birth cohort (15
), performance on these same scales at ages 3, 4, and 5 years was unrelated to PCB exposure. A clearer understanding of the relation between low-level PCB exposure and performance on examinations of cognitive function would improve assessment of risks associated with exposure.
Our study was designed to evaluate the association between maternal third trimester serum PCB levels and offspring cognitive test scores at 7 years of age by use of the Wechsler Intelligence Scale for Children (WISC). Subjects were participants in the Collaborative Perinatal Project.
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MATERIALS AND METHODS |
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Study data were collected at each prenatal visit, at delivery, and when the child's age was 24 hours, 4 months, 8 months, and 1, 3, 4, and 7 years. Data were collected by physicians or personnel, for example, psychologists, who were specifically trained for the job. They administered age-appropriate standardized tests that assessed cognitive, neurologic, and motor development, as well as psychological and behavioral functioning.
Of the 55,908 pregnancies recruited into the study, 46.2 percent were Black, 46.0 percent were White, 6.8 percent were Puerto Rican, and the remaining 1 percent were Asian or other race. At registration, the subjects' median socioeconomic index score was 4.5, slightly lower than the median socioeconomic index score of 5.0 for the US general population (18). The socioeconomic index calculated for subjects in the Collaborative Perinatal Project was the mean of three percentile scores (for education, occupation, and family income), where education was that of the head of the household, occupation was that of the head of the household or the chief wage earner, and the score used to calculate the percentile for an occupation was based on the percentiles of education and income among those with the same occupation. By delivery, 4 percent of the mothers were lost to follow-up. Among those women not lost to follow-up at delivery, approximately 80 percent were recruited after 20 weeks of gestation; all had at least one prenatal visit recorded, and 9,161 women had more than one pregnancy included in the study. Of the liveborn children in the Collaborative Perinatal Project, 71 percent were followed to age 7 years.
Selection criteria and sampling scheme
For the present study, eligible children met the following criteria: 1) they were liveborn, 2) they were singletons, and 3) a 3-ml third trimester maternal serum specimen was available. From the 43,628 eligible children, 1,256 subjects were selected at random. An additional 207 children were randomly selected from eligible children whose 7-year full-scale IQ score was either one or more standard deviations below the mean or one or more standard deviations above the mean. By increasing the probability of inclusion for subjects with "extreme values" of an outcome (outcome-dependent sampling), we increased the study power relative to a study of equivalent size that used simple random sampling alone (19). The institutional review board of the National Institute of Environmental Health Sciences approved the study protocol.
Exposure assessment
Beginning at registration, nonfasting maternal blood was collected every 8 weeks during pregnancy. Serum samples were stored at 20°C in glass with no recorded thaw. The variation of PCB levels across trimesters of pregnancy and after delivery has been examined using Collaborative Perinatal Project sera (20). Between any pair of serial measures of lipid-adjusted PCBs, such as the first trimester and the third trimester specimens, the correlations were high (Pearson's r = 0.77 or above). Because more subjects had a third trimester serum specimen available than for any other period, third trimester samples were analyzed. Levels of PCBs in maternal serum and in umbilical cord serum correlate fairly well; for example, Spearman's rs ranged from 0.52 to 0.74 for four congeners (21
). The 3-ml serum samples were analyzed for 11 specific PCB congeners (denoted by their International Union of Pure and Applied Chemistry (IUPAC) (22
) designation: 28, 52, 74, 105, 118, 138, 153, 170, 180, 194, and 203) and nine other organochlorines at the Centers for Disease Control and Prevention by use of solid-phase extraction, followed by dual-column gas chromatographic separation with electron capture detection (23
). Measured concentrations that were below the detection limit were reported by the laboratory and included in the analyses; in such instances, imputation was not done (24
26
). In the primary analysis, PCB exposure was characterized using the sum of the 11 measured PCB congeners, expressed as µg/liter of serum ("wet weight"). Serum cholesterol and triglycerides were measured, using standard enzymatic assays.
We included in every batch of specimens analyzed (10 specimens/batch) an aliquot from a single large pool of serum to allow an independent calculation of a coefficient of variation between batches (19.0 percent for total PCBs; mean, 3.54 µg/liter). Fifty-three percent of batches contained specimens for both subjects selected at random and those selected because of their IQ at age 7 years; all batches that included a specimen for a subject selected because of his/her IQ score had at least one specimen for a subject selected at random.
Outcome measures
Wechsler Intelligence Scale for Children.
Children were given seven of the 11 components of the 1949 WISC (information, comprehension, vocabulary, digit span, picture arrangement, block design, and coding) (27) at the 7-year visit. A verbal IQ score was based on results from the information, comprehension, vocabulary, and digit span tests, and a performance score was based on results from the picture arrangement, block design, and coding tests. The full-scale score was based on the results from all seven of the components. For this analysis, the age-adjusted standardized test scores calculated by the original investigators were used.
The Collaborative Perinatal Project investigators used great care to ensure the comparability of testing procedures among study centers. For each battery of tests, a few examiners from each center traveled to another center and readministered the test to a randomly selected subset of children (28). The correlation between the scores of two IQ tests administered 3 months apart by examiners from different study centers was high (r = 0.83). Additional information regarding the validity and reliability of the WISC has been reported elsewhere (29
).
Assessment of related outcomes.
Although our analyses focused on the WISC IQ results, other related outcome data were also available. At the 4-year examination, the Stanford-Binet IQ test was administered (28). At the 7-year visit, children were also given the Wide Range Achievement Test (WRAT) (30
). The WRAT has three subtests: spelling, reading, and arithmetic. Age-standardized WRAT scores were used in the analysis.
Data analyses
We focused on the 7-year cognitive measures, as compared with similar measures at age 4 years or achievement at age 7 years, because the persistent effects of PCB exposure on cognition were of the greatest interest to us, and because cognition has been assessed in most of the other studies of early PCB effects. For PCB congeners 28, 74, 118, 138, and 180, there were two, 23, 41, 20, and three subjects, respectively, for whom the level of the congener was missing, mainly because the measured value did not meet the quality control standards for acceptance (22). Using regression models fit to subjects with complete data, we imputed values for the missing congeners. The levels of specific congeners are highly correlated in background-exposed populations (31
, 32
).
Subjects were divided into five categories on the basis of in utero PCB exposure, as reflected by the maternal serum total PCB level. Cutpoints at 1.25-µg/liter intervals gave at least 80 subjects in the category with the fewest subjects in the crude analyses. The mean IQ showed an essentially monotonic change across categories, suggesting that representing PCBs as a continuous, linear variable adequately reflected the PCBIQ relation. We also fit quadratic models to assess whether a linear model was sufficient; all results indicated that it was.
All models in the analysis used weights. The weights were the inverse of the sampling probabilities (33). Subjects in the random sample had a weight of 35; those in the special sample with low IQ (n = 61) were given a weight of 57, and those in the high-IQ sample were given a weight of 43 (n = 101). (The numbers of subjects presented here are the number included in the final model, described below.) Use of the weights avoided bias that would have occurred had the outcome-dependent sampling been ignored in the analysis (19
). Models were fit using Proc GLM and Proc REG in SAS software (34
), with normalized weights.
Study center (12 categories), maternal age, serum cholesterol, and triglycerides (all continuous), as well as child's sex, were a priori considered to be potentially confounding factors and were included in all multivariate models. Many other factors were considered as potentially confounding, and these are listed in appendix table 1. To evaluate confounding in a model of IQ, we compared the coefficient for PCB as a continuous variable, adjusted for the a priori confounders, with the PCB coefficient from a model that additionally included each of the other potential confounders one at a time. Any variable whose inclusion changed the PCB coefficient by 10 percent or more was considered a confounder. The confounders so identified were maternal race, parity, education, socioeconomic index, housing density, smoking status, serum level of heptachlor epoxide, socioeconomic index at the 7-year follow-up, maternal or caregiver education at the 7-year follow-up, maternal or family income at the 7-year follow-up, whether the home emotional environment at the 4-year follow-up was favorable, age in months at the time of the 7-year follow-up, whether meconium was present at birth, and whether the child was breastfed during the hospitalization for delivery. In the fully adjusted model, the coefficient for PCBs changed by less than 3 percent when we deleted maternal education at registration, maternal/caregiver income at 7 years, and housing density; thus, these variables were not included. Effect modification was evaluated similarly, but by examining model fit statistics before and after the addition of the cross-product terms for all the variables listed above. Any term(s) giving an F test with a p value of 0.10 were considered potentially noteworthy. Although our focus was on PCB levels expressed on a wet-weight basis (17
, p. 321), we also fitted models with PCB levels expressed on a per unit of serum lipid basis (without cholesterol or triglycerides as covariates).
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RESULTS |
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The distribution of race across the "low IQ sample," "random sample," and "high IQ sample" reflects the previously described association between race and IQ score (29). Because of that association, we stratified by race for all the other factors in table 1, to allow a more straightforward evaluation of the relation of other factors to IQ in our data. Within each racial group, IQ showed clear relations with maternal education, socioeconomic index, and breastfeeding by the child.
For PCB congeners 118, 138, and 153, 90 percent or more of the subjects had levels that were above the detection limit for the assay (table 2). The overall median PCB level was 2.85 µg/liter (quartiles: 2.00, 4.02), similar to that in most other studies of PCBs and cognitive function (35). Women from Richmond had the highest median total PCB level (3.6 µg/liter), while those from Portland had the lowest (1.7 µg/liter).
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The studies of in utero PCB exposure showing an inverse relation with cognitive function (3, 4
) were done in populations with higher mean IQ and socioeconomic status than in our population. Therefore, we reevaluated our results after excluding subjects 1) with an IQ less than 82 and 2) with a socioeconomic status less than the median. In both cases, the results were essentially unchanged from those shown.
The association of IQ with the level of each PCB congener (continuous, unimputed values only) was examined in separate, fully adjusted models. In addition, after summing the levels of the most highly correlated PCBs (congeners 74, 105, 118, 138, 153, 170, and 180; all rs > 0.6), we included the sum and the levels of the remaining four congeners in one model. In no instance was there an indication that any congener (or group) was especially associated with IQ or that the association differed among congeners.
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DISCUSSION |
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As noted earlier, Gladen and Rogan (15), who examined McCarthy scores in 712 children aged 35 years, found no association between prenatal PCB exposure and cognitive testing results. In another study of 435 subjects from a population with PCB serum levels about three- to fourfold higher than in the Collaborative Perinatal Project (and most other studies of this relation), in utero PCB exposure was not related to WISC scores at age 7 years (36
). In the cohort of 353 Dutch children, prenatal PCB exposure was associated with decreased cognitive scores at 3.5 years of age (4
), but subsequent testing at age 6.5 years no longer supported this relation (37
). In the cohort from upstate New York, higher umbilical cord PCB levels were associated with lower scores on the McCarthy Scales of Children's Abilities at 38 months of age but not at 54 months (n
195) (14
). Only in the two remaining smaller studies of cognition was an unequivocal adverse association with PCB exposure found among the children tested after the longest period of follow-up, at ages 11 (3
) and 3 (38
) years. Although different tests were used across studies, scores among the tests are highly correlated (29
). The ages of subjects also varied across studies, but test results are correlated, especially at age 7 years or more (29
). An explanation for why results vary was not obvious, though differences in adjustment for confounding factors may have played a role (39
).
The limitations of the present data and their potential impact on our findings merit consideration. First, as a result of loss to follow-up, IQ data were missing for 321 of 1,463 subjects (22 percent). The median PCB level among those with missing IQ data (2.92 µg/liter) was similar to that for the subjects included in the final analysis (2.85 µg/liter), suggesting that follow-up was unrelated to exposure. In addition, the 208 subjects missing data for covariates were excluded. The ß coefficient for the adjusted IQPCB relation was similar in those with and without the covariate information (not shown), suggesting that limiting the analysis to those with complete covariate data did not substantially alter the findings.
Second, the sera used to ascertain exposure had been stored since the early 1960s. Despite the widely held view that PCBs resist degradation in most settings (40), few data exist regarding stability in serum during long-term frozen storage. However, pooled milk specimens from Swedish mothers, stored in 1972 at 20°C, were analyzed for PCBs after 15 and 25 years; levels showed no decline over the 10-year period between analyses (41
, 42
).
Third, we reported a slightly higher degree of measurement error, with a between-assay coefficient of variation of 19 percent for total PCBs, as compared with the value of 12 percent in one of the few epidemiologic studies for which these types of data were calculated using blinded samples and presented (40). The precision of the measurement of exposure, however, was nonetheless sufficient to detect a modest association with IQ at age 4 years, albeit in the direction opposite of that hypothesized. Additionally, if third trimester serum PCB levels were not the optimal biomarker, as compared with levels measured in cord serum or maternal milk, the cascading effect of relatively small errors at various steps affecting exposure measurement could have attenuated the measure of an effect, if any. Finally, use of a subset of the WISC components could have decreased our power.
In our data, women with higher PCB exposure had higher socioeconomic status (Spearman's r = 0.16) and were more likely to breastfeed their children (r = 0.11). If socioeconomic status and breastfeeding protected children against the adverse effect of PCBs, then failure to adequately adjust for socioeconomic status and breastfeeding could have caused a true adverse effect of PCBs on neurodevelopment to be masked. In additional analyses (not shown) that included only children who were not breastfed (n = 727), adjustment for socioeconomic status (baseline and age 7 years) and maternal education caused the "fully" adjusted estimate (change in full-scale WISC IQ per unit increased in PCB level in µg/liter) to decrease from 0.56 to 0.36. But stratification of those not breastfed according to whether the family's socioeconomic index (at child's age of 7 years) was above or below the median revealed essentially no difference in the PCBIQ relation, suggesting no adverse effect of PCBs even in the most disadvantaged children.
In developing animals, including primates, PCBs are usually neurotoxic, even at environmental exposure levels (43). While exposure to PCBs themselves could account for the inverse PCBIQ associations observed in some epidemiologic studies, the possibility exists that PCB levels serve as a biomarker for the level of another neurotoxicant or for an altogether different determinant of IQ.
As noted above, the level of total PCBs among mothers in our study was similar to those in studies in which an adverse effect was found (3, 4
, 38
). On the other hand, the mixture of PCBs in the Collaborative Perinatal Project specimens was unusual compared with that in other studies (35
); for example, the level of PCB 118 was higher than in any other study, and the level of PCB 180 may well have been proportionately lower (20
, 36
, 44
). Levels of unquantitated PCBs may also have varied in biologically significant ways. Thus, if the composition of PCBs affects neurotoxicity, we may have had a relatively benign mixture. However, our examination of congener-specific effects, to the extent that this was possible, did not suggest that any specific PCBs were related to the outcomes.
Manufacture of PCBs was banned in the United States in 1977, and background-level exposure levels appear to be decreasing (35). However, understanding the risks associated with low-level PCB exposure is nonetheless an important ongoing process, because of the possibility that present-day exposure levels have adverse effects (1
, 14
) and because of environmental management and legal questions in areas of local contamination (45
, 46
). Recently, several scientists have made the following comments: "The literature on prenatal exposure to PCBs and developmental toxicity is one of the strongest bodies of evidence of human health effects in low-level environmental exposures" (45
, p. A187), and "the weight of evidence for PCB effects on neurodevelopment is growing" (1
, p. 376). Low-level PCB exposure early in life may well have adverse effects, but the present data indicate that the long-term effects on cognition, as reflected by performance on IQ-type tests, are not so clear.
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ACKNOWLEDGMENTS |
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References |
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