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Review of the Literature Concerning Human Epidemiological Studies on the

Neurodevelopmental Effects
of Fetal in utero Exposure to Endocrine Disruptors
by Susan Kinkead-Acree, MD
May 2005

Much has been learned about the neurodevelopmental effects of in utero fetal exposure to
endocrine disruptors through toxicological models. These studies have been motivated by
evidence from wildlife studies that exposure to even low levels of endocrine disruptors at critical
points in fetal development can have profound reproductive, behavioral, and neurological
effects. While chemical sensitivities and toxicokinetics can differ widely among animal species
and between animals and humans, the potential of some chemicals for significant functional
endocrine disruption has raised concern about their impact on human health. The human
epidemiological studies carried out to date have demonstrated small but statistically significant
adverse fetal developmental effects, but because most have used different methods of chemical
analysis, measures of exposure, and statistical methodologies, it is often difficult to compare
their results. By looking to these studies, investigators can obtain guidance on ways to improve
chemical detection methods, outcome assessment tools, and statistical methodologies so as to
optimize the data quality and the inter-study comparability of future investigations.

Introduction

History of Endocrine Disruptors

Since Silent Spring was published in 1962 (Carson 1962) there has been a growing recognition
that chemicals in the environment can cause deleterious health effects on wildlife and humans
(The International Programme on Chemical Safety 2002). In Our Stolen Future (Colborn et al..
1996), the authors presented compelling scientific evidence that these ubiquitous environmental
contaminants may interfere with the endocrine hormonal signals that orchestrate fetal
development, resulting in reproductive and developmental defects. Laboratory studies have
confirmed that chemicals can affect the endocrine system in animals (U.S. EPA 2004b). These
chemicals came to be termed “endocrine disruptors.”

Although much controversy exists among scientists regarding the magnitude of the potential
impact of endocrine disruptors on human health, concern was great enough that Congress
amended the Federal Food, Drug, and Cosmetic Act with the Food Quality Protection Act of
1996, requiring that the Environmental Protection Agency Administrator “develop a screening
program, using appropriate validated test systems and other scientifically relevant information, to
determine whether certain substances may have an effect in humans that is similar to an effect
produced by a naturally occurring estrogen, or such other endocrine effect as the Administrator
may designate” (United States Congress 1996). The EPA screening program assays and testing
methods are currently under development. The President’s National Science and Technology
Council’s Committee on Environment and Natural Resources (CENR) has established a Working
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Group on Endocrine Disruptors with representatives from 14 federal agencies to assess, plan, and
coordinate research on endocrine disruptors in the United States.

International coordination of endocrine disruptor assessment is provided by the Organization for

Economic Cooperation and Development (OECD), which established in 1996 a Special Activity
on Endocrine Disrupter Testing and Assessment “to ensure that testing and assessment
approaches for endocrine disrupters would not substantially differ among countries”
(Organization for Economic Cooperation and Development 2004).

Properties of Endocrine Disruptors

Endocrine disruptors have been defined as “exogenous agents that interfere with the production,
release, transport, metabolism, binding, action, or elimination of natural hormones in the body
responsible for the maintenance of homeostasis and the regulation of developmental processes”
(Kavlock et al.. 1996; Tilson et al.. 1997; cited in Tilson 1998). Because the endocrine system
regulates all biological processes in the body from conception to death, including brain and
nervous system development, growth and function of the reproductive system, and metabolism,
endocrine malfunction caused by environmental contaminants has grave implications for human
health.

The list of potential endocrine disruptors is long and comprises many classes of compounds (The
International Programme on Chemical Safety 2002). Known endocrine disruptors include heavy
metals like methyl mercury and lead; chlorinated pesticides; natural or synthetic hormones; and
dioxin-like compounds (DLCs), a diverse group of halogen substituted multi-ring structures
including polychlorinated dibenzodioxins (PCDD's), polychlorinated dibenzofurans (PCDF's),
hexachlorobenzene (HCB), and polychlorinated biphenyls (PCB's) that exhibit similar
biochemical properties (Hauser et al. 1998).

Some degrade quickly, but most endocrine disruptors are biostable and persist in the
environment for decades. Many are lipophilic and accumulate in animal adipose tissues. In this
way, the body burden of these compounds increases exponentially up the food chain. A major
source of human exposure is thought to be consumption of fish, fish products, and animal fats
(Cicchetti et al. 2004; Ribas-Fitó et al. 2001). “With widespread contamination of the food chain,
virtually all humans have measurable quantities of PCBs in their body tissues” (Nicholson et al.
1994).

Endocrine Disruptors and Neurodevelopment

Because these compounds cross the placenta, maternal exposure to endocrine disruptors can
affect the developing fetus. Disruption of the hypothalamic-pituitary axis can result in altered
reproductive behaviors, sexual differentiation, and cognitive and psychomotor development
(Tilson 1998). Thyroid hormones play a particularly critical role in brain development, as they
regulate neuronal proliferation and migration, synaptic development and myelin formation
(Porterfield et al. 1998). “Moderate to severe alterations in thyroid hormone concentrations
during development result in motor dysfunction, cognitive deficits, and other neurologic
abnormalities” (Tilson 1998). The nature of the nervous system deficit depends not only on the
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severity of the thyroid disruption, but on the specific fetal developmental period when the
disturbance occurs (Tilson 1998). Because their molecular structure is very similar to the active
form of thyroid hormone, T3, dioxin-like compounds are of particular concern for
neurodevelopmental toxicity (Porterfield et al. 1998; Leatherland 1998). They bind with high
affinity to thyroid hormone transport proteins and T3 receptors, as well as the selenoprotein
deiodinases, 5- and 5’ monodeiodinase; these compounds thus affect thyroid hormone kinetics,
transport, and neuronal intracellular production of T3 (Leatherland 1998).

Epidemiological Studies and Reported Results

The major human epidemiological studies investigating the neurodevelopmental effects of in

utero exposure to known endocrine disruptors were reviewed, as well as the major reviews
published concerning these studies. The databases Medline, Toxnet, and PsychInfo were
searched using the following search terms: endocrine disruptors; dioxins; PCBs; pesticides;
prenatal exposure; fetus; pregnancy; neurological effects; neurodevelopment; neurotoxicity;
brain. Publications of governmental agencies and international organizations were also
reviewed. Further sources were identified from the citations in these documents. English
language documents only were reviewed.

The primary source of human data on neurobehavioral and developmental toxicity of in utero
fetal exposure to endocrine disruptors is from epidemiological studies undertaken on a number of
cohorts of women who had consumed food contaminated with dioxin-like compounds,
principally PCBs. Some of the cohorts were also evaluated for exposure to heavy metals (lead
and mercury). Laboratory and wildlife studies had demonstrated the endocrine disrupting
potential of these substances. One of the objectives of the infant and child portions of these
studies was to determine whether human fetal exposure to these compounds caused intellectual,
neuropsychological, and and/or behavioral problems in the children.

► Yusho and Yu-Cheng cohorts In 1968, thousands of Japanese were exposed to thermally
degraded PCB's that contained high concentrations of extremely toxic congeners of
polychlorinated dibenzofurans (PCDFs) and polychlorinated napthalenes through consumption of
rice oil contaminated during the manufacturing process (Schantz May-June 1996). In 1979, a
similar poisoning incident occurred in Taiwan, also traced to contaminated rice oil (Chen et al.
1992). “Rice oil disease” is called Yusho in Japan and Yu-Cheng in Taiwan.

Pregnant Japanese women suffering from Yusho gave birth to children with various physical
dysmorphologies, and a follow-up study of a subset of the children reported mental, motor, and
behavioral deficits including slowness, clumsy movement, apathy, and IQs averaging around 70
(Harada 1976; cited in Schantz 1996). Harada did not report quantitative data, nor did he use
standardized tests, and no control children were included in his study (Lai et al. 2001; Ribas-Fitó
et al. 2001). Mean PCB concentration of exposed children’s blood was 6.0 ppb (Jensen 1987,
cited in Ribas-Fitó et al. 2001).

In Taiwan, 118 children born to women with Yu-Cheng between June 1978 and March 1985
were matched with controls and tested annually for six years from 1985 to 1991 (Chen et al.
1994). The studies on this cohort are presented below.
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Measures of exposure--Serum PCB levels in the Yu-Cheng cohort were not measured at the time
of birth in either the mothers or children; blood samples were drawn and PCB levels measured
in 34 mothers and 36 children in 1991--11 years after exposure (Yuet al. 1991). Exposure indices
were estimated from the median PCB concentration in exposed children’s blood: 0.99 ppb
(Ribas-Fitóet al. 2001). However, umbilical cord PCB levels are considered to be the most direct
measure of in utero exposure (Schantz et al. 2003). Outcome measurements for the Yu-cheng
cohort included mental, motor, and behavioral deficits.

Instruments and Outcome Measures--Cognitive development was measured using Bayley Scales
for Infant Development (6-30 months of age), Stanford-Binet IQ Test (30 months-6 years), and
Wechsler Intelligence Scale for Children-Revised (WISC-R) (age > 6 years) (Yu, Hsu, Gladen
and others March-April 1991). Neurophysiological assessment was also performed at age > 6
years with auditory event-related potentials (P300), pattern visual evoked potentials (P-VEPs),
and short-latency somatosensory evoked potentials (SSEPs). P300 latencies evaluate the speed
of information processing, and P-VEP’s and SSEPs evaluate the sensory pathways of the
peripheral and central nervous systems (Chen et al. 1994). The WISC-R and neurophysiological
tests were given to 79 of the original cohort of 118 Yu-Cheng children and their controls in 1991.

Results--Compared with the controls, the exposed children exhibited statistically significant
delays in reaching developmental milestones as measured by parental report, clinical evaluation
and formal developmental testing. Interestingly, there was no relationship between maternal
serum PCB levels and developmental scores in the child, but children with detectable serum
PCBs had lower developmental scores than those without (Y et al. 1991). “Further exposure to
these chemicals through breast-feeding did not have any apparent effect” (Gladen et al. 1988,
cited in Yu et al. 1991). IQ scores of the exposed children averaged 5 points lower than the
controls, but did not correlate with either maternal or child serum PDB levels (Chen et al. 1992;
Schantz 1996).

Spatial abilities were assessed annually using the Ravin Colored and Standard Progressive
Matrices. The Yu-Cheng children scored lower than their matched control through 12 years of
age, after which they appeared to catch up to the controls (Lai et al. 2001). Interestingly, when
broken down by gender, Yu-Cheng boys showed decreased spatial abilities compared with
controls, while girls showed no differences, leading the investigators to hypothesize that PCB
exposure may have caused male-specific cognitive deficits (Schantz et al. 2003).

Behavioral development was measured using the Rutter Child Behavior Scale
(emotional/behavioral disorders) and the Werry-Weiss-Peters Activity Scale (activity level). The
exposed children consistently scored higher on both scales, indicating more behavioral problems
and higher activity levels. Similar effects on behavior and attention are seen in children born
with congenital hypothyroidism, even after early hormone replacement therapy (Porterfield et al.
1998). There was no correlation between maternal or child serum PCB levels and scale results
(Schantz 1996).

The WISC-R and neurophysiological studies showed that, compared with the controls, the Yu-
Chen group again showed lower IQ scores (average 5 points), but as before, there was no
apparent relationship between a child’s IQ score and either his or his mother’s serum PCB
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concentration. The mean P300 latencies were longer with lower amplitude in the Yu-Chen
group, indicating slow cognitive processing and attention deficits; these correlated with lower IQ
scores (Lai et al. 2001; Chen et al. 1994).There were no significant differences in P-VEP or
SSEP sensory pathway performance between exposed children and controls, nor were there
significant differences on the general neurological exams or the motor tests (Chen et al. 1994;
Schantz 1996). The authors concluded that in utero PCB exposure affected high cortical
functioning rather than sensory pathways (Chen et al. 1994).

Criticisms—The fact that Harada did not use quantitative data, standardized measuring
instruments or controls in his study precludes the possibility to compare his results with later
studies.

Because the Yu-Cheng PCB exposure measurements were based on samples drawn, not from
umbilical cord blood, but from children 11 years after exposure, it is difficult to interpret the lack
of correlation between serum PCB levels and IQ because the exposure indices used may not
accurately reflect in utero exposure (Schantz 1996). Schantz also notes that the children’s
behavioral differences might also be due to parental perception of them as damaged or to the
stress of growing up in a family traumatized by the poisoning.

Unlike later cohorts studied, the Yusho and Yu-Cheng populations were exposed to high
concentrations of PCDF congeners that are much more toxic than PCBs (Schantz 1996).
Because of this, some experts believe that the measured effects might be due more to the PCDFs
than PCBs, and therefore the study results of the rice oil disease population should not be
compared with those of later cohorts exposed only to lower background levels of PCBs in the
environment (Schantz 1996; Lai et al. 2001; Cicchetti et al. 2004).

► Michigan cohort Jacobson, Jacobson, and colleagues carried out an 11-year prospective
longitudinal study on a cohort of 313 women formed in 1980-81 relating low-level maternal PCB
exposure from Lake Michigan fish to developmental outcomes in children (Schantz et al. 2003).
In interviews one day post-partum, mothers estimated from long-term recall the total amount,
source, yearly rate of consumption and specific species of fish consumed for a number of years
(up to 16) (Schell et al. 2001). All of the exposed women had reported eating at least 26 pounds
of Lake Michigan fish over the preceding six years, while the controls had never eaten Lake
Michigan fish. The control sample was selected randomly, not matched to the exposed group
(Seegal 1996), and was one third the size of the exposed sample (Schantz 1996).

Measures of exposure--PCB exposure measurements included lifetime species-weighted

maternal fish consumption as recalled by mothers (PCB contamination levels of different fish
species were taken into account, based on EPA data), PCB concentrations in umbilical cord and
maternal blood at birth, and breast milk concentrations at birth and 5 months post-partum
(Jacobson et al. 1996; Schantz 1996). Blood samples were taken again when the children were
four and 11 years of age. However, cord serum samples were collected in only 198 of 313 (63%)
of the newborns (Schwartz et al. 1983). The umbilical cord blood samples were analyzed for
PCBs and polybrominated biphenyls, while the blood samples drawn at 4 and 11 years were also
analyzed for seven organochlorine pesticides (only DDT was detected) and lead. At 11 years
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old, the children’s serum PCB concentration was found to have decreased substantially; mercury
levels in hair were also measured at 11 years old (Jacobson et al. 1996).

PCB levels were analyzed by the Michigan Department of Public Health using packed column
gas chromatography and the Webb-McCall method. PCB levels were quantitated by summing
10 Webb-McCall peaks. No data on specific congeners are available. Values matched to the
PCB Aroclors 1016 (lower chlorinated congeners) and 1260 (highly chlorinated congeners) were
used as standards. The PCB detection limit for the 1016 standard was ≤ 5 ppb, and for the 1260
standard it was ≤ 3 ppb. The means for the more highly chlorinated congeners matched to the
1260 standard were higher than the lower chlorinated congeners matched to the 1016 standard.
Additionally, most of the cord and maternal sera PCB levels matched to the 1016 standard were
below the laboratory’s quantification limits, presumably because lower chlorinated congeners are
susceptible to more rapid environmental degradation and are metabolized more quickly by living
organisms. The investigators used the values matched to the 1260 standard (i.e., the more highly
chlorinated congeners) for their analyses because they provided a more reliable measure of body
burden (Schwartz et al. 1983).

The reported mean PCB concentration values (1260 standard) were: cord serum = 2.5 ppb;
maternal serum =5.5 ppb; and neonatal breast milk = 812.7 ng/g fat (Schwartz et al. 1983). No
explanation was found in the literature concerning how the investigators ascertained the cord
blood PCB concentration of 2.5 ppb when the reported quantification limit was 3 ppb. The PCB
concentrations were positively skewed, so they were logarithmically transformed.

To improve reliability and sensitivity of fetal exposure assessment, PCB concentrations in cord
serum, maternal serum and milk were converted to z-scores and averaged together to provide a
composite measure of prenatal exposure in the 1996 study (Jacobson et al. 1996). According to
the authors, “Because placental transfer provides the sole route of fetal exposure to these
compounds, which are in equilibrium in fat deposits throughout the body, maternal serum and
milk concentrations provide alternatives to cord serum for evaluating prenatal exposure” (Rogan
et al. 1986, cited in Jacobson et al. 1996).

No correlation was found between fish consumption and cord serum PCBs. However, significant
positive correlations were found between all three fish consumption measures and PCB levels in
maternal serum (all p < .001) and breast milk (all p < .01).

Instruments and Outcome Measures--Neuropsychological assessments were performed on the

children with the Brazelton Neonatal Behavioral Assessment Scale (NABS) at birth (Jacobson et
al. 1984a). Cognitive development was assessed with the Bayley Scales of Infant Development
at five months of age (Jacobson et al. 1990) and with the Fagan Test of Infant Intelligence (FTII)
at seven months of age (Jacobson et al. 1985). They were reassessed at four years of age
(Jacobson et al. 1990) with the McCarthy Scales of Children’s abilities, supplemented by two
standardized tests, the Beery Test of Visual-Motor Integration and the Peabody Picture
Vocabulary Test-Revised; and finally, at 11 years of age (Jacobson et al. 1996) using the WISC-
R, the spelling and arithmetic subtests of the Wide Range Achievement Test-Revised, and the
word and passage comprehension subtests of the Woodcock Reading Mastery Tests-Revised.
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Maternal intelligence was evaluated using the Peabody Picture Vocabulary Test-Revised (PPVT-
R), a measure of receptive vocabulary, that has shown little correlation with IQ (Maxwell et al.
Wise 1984). The potential confounding effect of home environment on children’s’ cognitive
development was controlled through administering the Home Observation for Measurement of
the Environment (HOME) scale (Cicchetti et al. 2004).

Control variables and analysis— The investigators used a multivariate statistical approach to
control for potential confounders that were known or suspected to be related to the outcomes
considered and controlled for variables that related to exposure at p<.10; however, it is not clear
whether all of these were controlled in all of the analyses (Schantz 1996). For example, the
infant and child visual recognition memory study reported that the potentially confounding
variables correlating with umbilical cord serum at p < .10 included socioeconomic status,
maternal age, parity, pre-gravid maternal weight, and use of acetaminophen and antacids during
pregnancy (Jacobson et al. 1985), while Jacobson et al. (1984, cited in Schantz 1996) list
maternal age, maternal weight gain during pregnancy, and use of alcohol and caffeine during
pregnancy as potential confounders that correlated with cord serum PCB level at p<.10.

For the tests on the 11 year old children, twenty-four control variables were evaluated by
correlation analysis for use in multivariate analyses to control for confounding variables, and
were chosen for relation to outcome instead of exposure (Jacobson et al. 1996). By doing this,
the authors sought to increase precision and include covariates unrelated to exposure (Kleinbaum
1998, cited in Jacobson et al. 1996). The cognitive outcomes were evaluated using four
regression analyses, one for prenatal PCB exposure and three for postnatal exposure (breast milk
PCBs, number of weeks nursing, and serum PCBs at four years of age).

Results—Brazelton Neonatal Behavioral Assessment Scale. Assessment with the NBAS at birth
revealed that increasing maternal consumption of contaminated fish predicted a linear
combination of five of the clusters (r = .28). The strongest relationships were with autonomic
maturity, reflexes, and range of state, but no correlation was found between NBAS outcomes and
cord serum PCB levels. The investigators suggested that this finding is possibly accounted for
by the fact that approximately 70% of the cord PCB levels were below the laboratory’s detection
limit, or that the effect was mediated by one or more contaminants other than PCBs (Jacobson et
al. 1984a).

Bayley Scales of Infant Development. The infants’ performance at five months of age on the
Bayley Scale MDI and PDI did not correlate with any measure of prenatal PCB exposure
(Jacobson et al. 1986).

Fagen Test of Infant Intelligence. Multiple regression analyses on the Fagan visual recognition
test scores at seven months of age showed statistically significant (p<.005) dose-dependent
correlation with prenatal PCB exposure. After controlling for confounders, poorer recognition
memory correlated with higher cord serum PCB concentration (Jacobson et al. 1985). Using the
Newman-Keuls procedure, the most highly exposed children were later compared with the least
exposed: The comparison showed that the highly exposed infants demonstrated significantly less
preference for novelty than infants in the two lesser exposed groups (p<.05) (Jacobson et al.
1985).
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McCarthy Scales of Children’s Abilities. At four years of age (n=219), a dose-dependent

association was found between increasing cord serum PCB levels and poorer performance on the
Verbal (p < .05) and Memory scales (measure of verbal and numerical short-term memory) (p =
.02), with the most highly exposed children scoring 6.6 points (0.66 SD) lower than the least
exposed children, after adjusting for confounders. The magnitude of this deficit is modest (0.66
to 0.92 on the average), and there was no evidence of mental retardation or gross impairment.
There were no significant effects on the Beery VMI or the PPVT-R, indicating no adverse effects
on visual-motor scores or long-term memory (Jacobson et al. 1990). Exposures to lead, PBBs
and seven organochlorine pesticides were also evaluated in this study, and statistically controlled
for, when necessary.

Cognitive Assessments. Higher prenatal exposure to PCBs was associated at age 11 (n=212)
with significantly lower full-scale and verbal IQ scores (6.2-6.5 points lower in the most highly
exposed group), as well as poorer verbal and numeric memory and attention span. Prenatal PCB
exposure was associated with poorer verbal comprehension, particularly on the vocabulary,
information and similarities subtests, as well as poorer performance on the freedom-from-
distractibility scale, which assesses focused attention, short-term memory, and executive
function (Jacobson et al. 1996).

Assessment with academic achievement tests also showed a dose-related association between
prenatal PCB exposure and poorer word and reading comprehension after controlling with
separate regression analyses for school district score variations, postnatal PCB and DDT
exposure, and lead exposure (Jacobson et al. 1996).

The importance of controlling for heavy metal exposure in endocrine disruptor

neurodevelopmental studies is illustrated by findings in this study: It was shown that lead and
mercury were significantly related to poorer performance after controlling for confounding
variables—a blood lead level of > 10 mcg/dl at four years of age was associated with lower IQ,
poorer verbal comprehension, and poorer reading comprehension (p< .05), while a higher
mercury concentration was associated with poorer spelling at 11 years of age (p=.006) (Jacobson
et al. 1996).

Postnatal PCB exposure from breast milk was not associated with poorer performance on any of
the tests administered, nor was the children’s serum PCB concentration at 11 years old related to
any of the IQ or achievement measures (Jacobson et al. 1996).

Criticisms—The Jacobson studies have been reviewed thoroughly by several authors (Paneth
1991; Seegal 1996; Cicchetti et al. 2004; Schell, Budinsky et al. 2001; Schantz 1996) and have
been shown to have some methodological flaws, although many of their findings have been
replicated in later studies.

The method of exposure assessment is widely considered to be inaccurate. As noted by the

Jacobsons themselves, maternal recall at one day postpartum of lifetime quantity and species of
fish consumption was subject to significant recall bias and inaccuracy (Jacobson et al. 1983,
cited in Schell et al. 2001). Indeed, the measure of self-reported fish consumption did not
correlate with the other measures of tissue PCB concentrations, confirming the inaccuracy of this
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measure (Schell et al. 2001). Schantz notes (Schantz 1996) that a later study used a similar
weighting approach (Dar et al. 1992), but women were asked to recall the type and quantity of
fish consumption for only the past 12 months. In this case, fish consumption correlated more
highly with serum PCB levels.

It should be noted, however, that the misclassification bias of recall studies does not increase the
probability of finding significant results, nor does it produce systematic patterns of data (Stewart
et al. 2004). On the contrary, it tends to decrease the likelihood of finding an association and
reduce the power of the study. Therefore, the weak positive associations found in the study may
actually represent an underestimation of the effects.

Although the investigators attempted to improve the accuracy of PCB exposure indices by
measuring PCB levels in cord serum, maternal serum and breast milk, critics hold that the
detection limitations of the gas chromatograph, the low percentage of cord serum samples
obtained, and the high attrition rate of women from the highest fish consumption category,
resulted in weak data (Schantz 1996). The validity of the composite score developed in 1996 has
been questioned because the authors failed to account for pharmacokinetic differences in the
various fluid compartments, and because the PCB levels of the tissues used (cord serum,
maternal serum, breast milk) had not always been correlated in the previous studies (Cicchetti et
al. 2004). The Jacobsons have also been criticized for their “pick and choose” approach to
applying the four exposure indicators to the various endpoints measured (Schell et al. 2001;
Schantz 1996).

The control sample has also been found to have shortcomings with respect to control for
confounding variables. According to Paneth, the fact that the control sample was only one-third
the size of the exposed sample reduced the study’s statistical power by 50% compared with
samples of equal numbers (Paneth 1991, cited in Schantz 1996). In prospective studies, the
larger the control group, the greater the ability to detect statistically significant differences
between sample groups (Mausner and Kramer 1985, cited in Schell et al. 2001). Schantz also
argues that, because exposure was not randomly assigned in this study, controls should have
been carefully matched on as many potential confounders as possible. She notes that “matching
is particularly important in studies of neurodevelopmental outcomes because they can be
influenced by a wide variety of socioeconomic and maternal factors” (Schantz 1996). Significant
differences in lifestyle variables were later found (e.g., alcohol consumption and smoking
quantity), while others were not ever considered (e.g., diabetes and gestational diabetes) (Schell
et al. 2001). Paneth notes (Paneth 1991, cited in Schell et al. 2001) that if several measured
variables differ substantially between the two sample groups then it is likely that other,
unmeasured, differences might account for the findings.

Cicchetti et al. (2004) note that the crucial variable of parental IQ was not adequately controlled
as a potentially confounder despite the fact that children’s IQ is affected by both genetic
endowment and environment. Environment was controlled through administration of the HOME
scale. Maternal intelligence was measured using the PPVT-R, a measure only of vocabulary, not
IQ (Maxwell et al. 1984), and there was no control for parents’ academic achievement. In their
opinion, “this lack of appropriate control of parents’ IQ and academic achievement, coupled with
a failure to control for chance findings, makes the conclusion of IQ and academic deficits
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associated with PCB exposure insupportable.” However, it was later shown (Stewart et al. 2004)
that the PPVT controlled adequately for maternal IQ in the Oswego cohort study. When
compared with a more direct measure of maternal IQ, the Kaufmann Brief Intelligence test
(KBIT), the PPVT controlled for maternal IQ the same or better, both by itself and when
included with other potential confounders that evidence colinearity with IQ, such as HOME
scores, maternal education, and substance use/abuse. This tends to lend credence to the
arguments supporting the validity of using PPVT to control for maternal IQ in these studies.

Environmental confounders like methylmercury were not controlled for, either, because the
Jacobsons considered the correlation between serum PCBs and methylmercury to be too low,
even though primate studies had shown that fetal exposure to low levels of methylmercury
produced similar neurodevelopmental effects to those reported in the Jacobson study (Schell et
al. 2001). In studies on the Faroese cohort, for example, neuropsychological results showing a
significant relationship to PCB exposure before adjusting for methylmercury dropped out after
controlling for methylmercury intake (Grandjean et al. July-August 2001, cited in Cicchetti et al.
2004).

Cicchetti et al. (2004) also criticize the Fagen Test for poor reliability (Cicchetti et al. 2004).
Responses (Stewart et al. 2004) to Cicchetti et al.’s criticisms of the test-retest reliability of the
FTII have acknowledged that the test-retest reliability of the FTII mean novelty preference score,
in particular, has not been as reliable over time as first suggested and that more stable and
internally consistent measures of infant visual recognition memory need to be employed. In
addition, they have even questioned whether or not psychometric assumptions of internal
consistency and stability over time apply to a measure like the FTII.

However, regardless of the validity of Cicchetti et al.’s (2004) criticisms, the central question is
whether it is possible that the PCB associations are due to chance or residual confounding. The
less reliable the test, the less likely that significant relationships will be observed and thus bias
results in favor of the null hypothesis. This makes the fact that the Michigan study found
significant associations all the more meaningful, especially since the results were later replicated
in the Oswego study (Stewart et al. 2004).

The Jacobson study series of the Lake Michigan cohort is the most recognized study reporting
neurodevelopmental effects of PCBs in humans. Its technical limitations in study design and
exposure measurements leave some uncertainties that preclude definitive conclusions regarding a
cause and effect relationship (Schell et al. 2001). However, as Stewart et al. (2004) articulately
explain,

“The reality in epidemiology is that no single study is ever definitive, as there is always
the possibility that results may be due to residual confounding or, as yet, unknown or
unmeasured sources of bias...The evaluation of a “significant finding”...is whether or not
the finding is replicated. Therefore, multiple studies are conducted and the results
compared to examine replicable findings. To the extent that an association is replicated,
it gains credibility as a scientifically defensible association. In the unlikely event that an
association is falsely judged significant in a single study, it is incredibly unlikely that
chance will operate in the same way to produce identical results in a second study. This
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is why epidemiologists are far more interested in whether an effect replicates than they
are in the number of other questions addressed in the same report.”

As will be seen, the Michigan study findings were replicated in later studies on the Oswego
cohort (Lonky et al. 1996; Stewart et al. 2000; Darvill et al. 2000; Stewart et al. 2003), which is a
more powerful argument for the validity of the Michigan findings than the criticisms levied
against them.

► North Carolina cohort In 1978, Rogan, Gladen, and colleagues initiated a 5-year
prospective PCB exposure study in North Carolina with a non-randomized cohort of 880
pregnant women recruited from three health centers in the Raleigh-Durham area exposed only to
background levels of PCB from the local food supply (Rogan, Gladen, McKinney and others
1986b). The women were not representative of the general population, being mostly white, well
educated, professional, non-smokers (Rogan et al. 1986b). Originally 931 children were to
participate; due to attrition, 807 women and 856 children participated in the first neonatal contact
(Rogan et al. 1986b). The children, born between April 1978 and October 1982, were assessed
shortly after birth, then at six-month intervals until age two, then yearly until five (Rogan et al.
1986b). Of the original 856 children, more than 600 were still available for assessment at 5 years
of age (Gladen et al. 1991).

Measures of exposure—Samples of maternal milk/colostrum, maternal blood, umbilical cord

blood and placenta were collected at time of birth and sent off on dry ice to Raltech Scientific
Services, Inc. in Madison, Wisconsin for analysis. PCBs and p,p’-DDE were measured by gas
liquid chromatography and total PCB levels were quantitated by adding two Webb-McCall
peaks. Lipid content was also measured to adjust milk contamination levels (Rogan et al.
1986b). Milk was collected until lactation ceased at 3, 6, 12 months, and an additional maternal
serum sample was collected from the mother at the 6-week visit; these samples were assessed
identically to the first set (Rogan et al. 1986b). Not all women provided each type of milk
sample, so to maximize statistical power, values from all samples from each woman were
combined and expressed as an estimated amount of chemical in milk at birth by a three-step
statistical method described in detail in the original study (Rogan et al. 1986b). The authors
reported a high correlation between the estimated amount in milk and the actual amount, when
known, of .86 for PCBs and .94 for DDE (Rogan et al. 1986b). However, Jensen (Jensen 1987,
cited in Schantz et al. 2003) estimates that this method actually overestimated the actual PCB
concentration by a factor of two.

The median fat-adjusted PCB levels in milk at birth was 1,770 ng/g milk fat; DDE levels
averaged 40% higher, with a median of 2,400 ng/g, both of which decreased substantially over
time to 1,170 and 1,510 ng/g, respectively (Rogan et al. 1986b). The median level of PCBs and
DDE in maternal serum at birth were 9.06 and 12.60 ppb; median cord serum PCB and DDE
levels were <4.27 and 3.95 ppb; and median placenta PCB and DDE levels were <12.00 and 6.77
ppb, respectively (Rogan et al. 1986b).

Instruments and Outcome Measures—The neonates were assessed using the Brazelton Neonatal
Behavioral Assessment Scale (NBAS). For logistical reasons, the test was not administered to
some infants within three days of life—the ideal--but within three weeks after birth (59% within
one week, 20% in the second week, and 16% in the third week) (Rogan et al. 1986b). The
 12

Bayley Scales of Infant Development was administered to the children at 6 months, 12 months,
18 months, and 24 months (Gladen et al. 1988; Rogan et al. 1991). The McCarthy Scales of
Children’s Abilities (cognitive ability, gross and fine motor abilities) were administered at ages
3, 4, and 5 years (Gladen et al. 1991). Tests were administered at three different centers, and
there were several examiners at each center—the authors did not carry out formal intra- or inter-
rater testing, only monitored scores and discussed any drift at staff meetings (Gladen et al. 1988).

Control variables and analysis—For the neonates, multiple regression analysis was used to
assess the correlations between BNBAS scores to PCB and DDE exposure levels (Rogan et al.
1986b). The following potential confounders with p ≤ .05 were considered for the BNBAS
multiple regression analyses: the child’s race (white, black, other), sex, birth weight, head
circumference, age at which the test was administered, number of hours since last nursing, and
jaundice status; the mother’s age, education, and occupation, tobacco and alcohol use in general,
consumption of sport fish during pregnancy, and if she had received general anesthesia (Rogan et
al. 1986b). Parental IQs and home environment were not controlled for.

For the Bayley assessments, the authors identified potential confounding variables “a priori,”
based on the analyses performed on the neonates and variables considered in other studies
(Gladen et al. 1988). These included the child’s sex, gestational age, birth weight, head
circumference, jaundice, duration of breast feeding, number of older siblings, number of
abnormal reflexes on the BNBAS, and age at which the Bayley exam was given; and maternal
age, race, education, occupation, tobacco and alcohol use (Gladen et al. 1988). Parental IQs and
home environment were not controlled for.

Covariates for the McCarthy Scales analyses included were maternal age, race, occupation,
education, smoking, drinking, child’s gender, number of older siblings, bottle feeding vs. breast-
feeding of short, medium, long, or very long duration. Statements that differences were not
significant indicated p values >.10.

Results—The NBAS assessments showed that higher PCB exposure correlated significantly with
lower muscle tone and lower activity levels, while both PCBs and DDE were associated with
hyporeflexia (Rogan et al. 1986b). When only children assessed within three days of birth were
considered, the effect of PCBs on tonicity no longer achieved statistical significance, but the
authors attribute this to insufficient sample size rather than to confounding from the age at test
administration (Rogan et al. 1986b).

The Bayley psychomotor index (PDI) showed statistically significant downward trends at six and
twelve months that correlated with increasing PCB exposure (a 0.96 point drop per 1 ppm
increase in PCB exposure) (Gladen et al. 1988). The adjusted scores on the psychomotor scales
at 18 and 24 months showed a statistically significant decrease at 24 months only (four to nine
points lower among children in the top fifth percentile of in utero PCB exposure) (Rogan et al.
1991).

These results differ from the Lake Michigan cohort, which showed no decrease in the Bayley
psychomotor scores with increasing PCB exposure. However, because the Jacobsons assessed
psychomotor function only at five months and four years of age, not between six months and two
 13

years, the discrepancy of results was thought to be due to differences in timing of the assessment
(Schantz 1996). However, later studies testing children at the same age would also show
discrepancies.

The mental index scores at six and twelve months were not related to in utero PCB exposure,
while the DDE showed a relationship at six months that disappeared at 12 months (Gladen et al.
1988). There was no evidence of an effect through in utero exposure to DDE (Rogan et al.
1991).

There was no association at any year found between in utero exposure to PCBs and any of the
McCarthy scales, including psychomotor and memory (Gladen et al. 1991). Thus, the delay in
psychomotor development measured with the Bayley scales did not persist beyond two years of
age (Schantz 1996), and the verbal and memory deficits found in the Jacobsons’ Lake Michigan
cohort were not confirmed (Schantz et al. 2003).

Criticisms—Just as with the Lake Michigan studies, North Carolina studies are criticized for
unreliable measurement of serum PCB levels due to the limitations of the packed column gas
chromatography technique used (Cicchetti et al. 2004; Schantz1996). No congener-specific data
is available for these studies because of this.

As mentioned above, the authors’ method for estimating PCB exposure based on milk PCB
levels may have overestimated the actual PCB concentration by a factor of two (Jensen 1987,
cited in Schantz et al. 2003). Due to the different analytical methods used to assess exposure
indices in the Lake Michigan and North Carolina cohorts, it is not possible to compare the
exposure levels directly, making it difficult to assess the discrepancies between the two studies
(Schantz 1996).

Like Jacobson and colleagues, Rogan and Gladen did not include as potential confounders
maternal IQ and the home rearing environment, important predictors of cognitive outcome
(Cicchetti et al. 2004). Unlike the Michigan researchers, who controlled for alcohol and tobacco
use specifically during pregnancy, the North Carolina researchers questionnaire asked only about
use of these substances in general, thus making it difficult to assess reliably the prenatal effects
of these potential confounders (Cicchetti et al. 2004).

► Dutch cohort The first study to assess the effects of specific PCB congeners was initiated in
1989 in the Netherlands by the Dutch government. Contamination of breast milk with PCBs and
dioxins in the Netherlands is among the highest in the world (WHO 1989, cited in Patandin et al.
1999). The Dutch PCB/Dioxin study is a prospective, longitudinal study designed to assess the
possible adverse effects of in utero and lactational PCB and dioxin exposure. It was carried out
in two widely separated areas in the Netherlands: Groningen, a semi-urban area; and Rotterdam,
a highly industrialized region (Koopman-Esseboom et al. 1996).

The initial cohort consisted of 418 pregnant women non-randomly recruited through their
midwife or obstetrician between 1990 and 1992; 211 of the women came from Gronigan, 207
from Rotterdam. They were visited in their homes for an explanation of the study protocol. One
half of each group planned to breast feed for at least six weeks, while the other half planned to
 14

formula feed and agreed to use the same brand from one batch as a reference (Huisman et al.
1995b). To participate in the study the women and their children had to meet certain criteria.
They were all healthy Caucasians who had suffered no complications during pregnancy or
delivery. All of the 418 children were first- or second-born via spontaneous vaginal delivery at
37-42 weeks of gestation with no forceps or vacuum extraction. Mothers agreed to provide a
maternal blood sample in the last month of pregnancy and a cord blood sample from the baby at
birth (Huisman et al. 1995b). The study reported on the children’s neurodevelopment at 3, 7, 18,
42, and 84 months of age.

Measures of exposure— In utero PCB exposure was assessed by measuring maternal and cord
blood levels of the four non-planar PCB congeners 118, 138, 153, and 180 (International Union
of Pure and Applied Chemistry (IUPAC) nomenclature) by gas chromatography with electron
capture detection (GC-ECD). The samples were centrifuged and stored at -20º until analysis
(Huisman et al. 1995b). The sum of the four congener levels in cord (∑PCBcord) and maternal
(∑PCBmaternal) blood were calculated (Koopman-Esseboom et al. 1996). These four congeners
are the predominant ones found in human tissue worldwide, typically account for around 50-60%
of the total PCB's, and are high enough to measured very accurately (Schantz et al. 2003;
Koopman-Esseboom et al. 1996). The detection limit for the four congeners was 0.01ppb
(Huisman et al. 1995b).

The breast fed group was studied using additional exposure variables generated from breast milk
PCB and dioxin measurements. A 24-hour representative breast milk sample was collected from
the nursing mothers two weeks post partum and analyzed for the 17 ubiquitous 2,3,7,8-
substituted polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofuran (PCDF) dioxin
congeners, which are usually found in biotic samples; three planar PCBs (77, 126, 169); three
mono-ortho PCBs (105, 118, 156); two di-ortho PCBs (170, 180); and 18 other non-planar PCBs
(28, 52, 66, 70, 99, 101, 128, 137, 138, 141, 151, 153, 177, 183, 187, 194, 195, 202) (Patandin et
al. 1998).

These compounds were measured in breast milk rather than blood because plasma has a
relatively low fat content compared with human milk, and too large a volume of blood would
have been necessary to measure all of the PCDD, PCDF, and PCB congeners (Huisman et al.
1995b). This approach was considered valid because dioxin levels in human milk have been
shown to correlate highly with the dioxin levels in maternal plasma and adipose tissue (Van den
Berg et al. 1994, cited in Koopman-Esseboom et al. 1996; and Schantz et al. 2003). The four
PCB congeners 118, 138, 153, and 180 were also measured in the breast milk of the nursing
mothers and served as additional indicators of prenatal exposure (∑PCBmilk ). Levels in the
formula given to bottle-fed infants were below the limits of determination (Huisman et al.
1995b). Another exposure variable for the breast fed infants was formed by summing all of the
non-dioxin-like PCB congener levels, including those in ∑PCBmilk.

The 17 dioxins and three planar PCB congeners were measured by gas chromatography-high-
resolution mass spectrometry, and the 23 non-planar PCB congeners were measured by GC-ECD
(Huisman et al. 1995b). The samples were analyzed at the Nutrition and Food Research
Institute, Zeist, the Netherlands (Vreugdenhil et al. 2002).
 15

The toxic equivalency factor (TEF) approach was used to express the toxicity, or total toxic
equivalent (TEQ), of the dioxins and dioxin-like PCBs in the breast milk. The TEF reflects a
given dioxin compound's Ah receptor-mediated toxicity relative to 2,3,7,8-TCDD, which is
assigned the maximum toxicity designation of one. Other dioxin and dioxin-like compounds are
given equal or lower numbers, with each number roughly proportional to its toxicity relative to
that of 2,3,7,8-TCDD. The TEF approach was developed by the WHO (Van den Berg et al.
1998) after experts deemed that there was sufficient evidence from both in vivo and in vitro
studies supporting the dose or concentration-additivity model for Ah receptor-mediated
responses. It is used extensively by scientists around the world.

However, as noted earlier, PCBs and dioxins have also been shown to exert effects by altering
hormone and neurotransmitter levels; thus their neurotoxicity via endocrine disruption likely
occurs by other routes than Ah receptor agonism, and the TEQ may not be the best predictor of
endocrine disruptor-mediated neurotoxicity of the dioxin-like compounds. However, the TEF
approach is currently the only method of assessing toxicity of mixtures of dioxin-like
compounds.

The 26 PCB congeners measured included dioxin-like (77, 105, 118, 126, 156, 169), and non-
dioxin-like (28, 52, 66, 70, 99, 101, 128, 137, 138, 141, 151, 153, 177, 183, 187, 194, 195, and
202), based on their Ah receptor agonist activity (Patandin et al. 1998). Interestingly, the studies
on the Dutch children are inconsistent in their classification of the di-ortho congeners 170 and
180: They are classified as dioxin-like in all studies published in 1998 or earlier, but were
classified as non-dioxin-like in the studies published in 1999 and after. It is possible that the later
studies reflected the WHO recommendation to discontinue use of the toxic equivalency factors
(TEFs) established previously for di-ortho PCBs 170 and 180 because of insufficient in vivo
evidence for Ah receptor-agonist activity (Van den Berg et al. 1998). However, no mention of
the discrepancy was found in the literature.

Using the TEF approach, the authors calculated total PCB-dioxin toxic equivalent (TEQ) values
by multiplying the milk concentrations by the TEFs of the compounds measured. They then
formed five exposure variables based on the TEQs: Total PCB-dioxin TEQs, dioxin TEQs,
planar PCB TEQs, mono-ortho PCB TEQs, di-ortho PCB-TEQs (Lanting et al. 1998). For the
studies published in 1999 and after, the di-ortho PCBs were not included in the PCB-dioxin
TEQ, but were included in the sum with the other 18 non-dioxin-like PCBs.

Although this review focuses on neurological effects of prenatal exposure, it should be noted that
postnatal exposure was also assessed using the ∑PCBmilk for PCB congeners 118, 138, 153, and
180. As mentioned above, the formula used for the bottle-fed infants was tested, and no PCBs
were detectable (Koopman-Esseboom et al. 1996; Huisman et al. 1995b). The breast milk PCB-
dioxin TEQ levels calculated for the other compounds used to estimate in utero exposure were
also used to estimate postnatal exposure through breastfeeding. Both values were multiplied by
the number of weeks of breastfeeding to give a total exposure estimate.

Only 105 milk samples were obtained, of which 80 could be measured accurately for the total
PCB-dioxin TEQ level, and 100 for the PCB milk sum, due to either inadequate sample amounts
or chromatogram interferences (Koopman-Esseboom et al. 1996).
 16

The ∑PCBmaternal averaged 2.2 ± 1 ppb, about five times higher than the ∑PCBcord, which
averaged 0.5 ± 0.3 ppb, but they were highly correlated (r=0.72, p < .001) (Koopman-Esseboom
et al. 1996). When the values were adjusted for the differences in lipid content between maternal
and cord blood, the levels were similar (Schantz et al. 2003). The mean ∑PCBmilk based on the
four congeners measured in blood was 428.5 ng/g of milk fat, whereas the mean ∑PCBs based
on all 23 congeners measured in milk was 455.9 ng/g (Koopman-Esseboom et al. 1996). This
was lower than the levels reported for Michigan (812 ng/g) and North Carolina (1770 ng/g).
However, as discussed earlier, these studies used packed-column gas chromatography to measure
PCB levels, and the analytic method used in the North Carolina study may have overestimated
the PCB levels by a factor of two, so comparisons may not be valid (Schwartz et al. 1983; Rogan
et al. 1986a; Jensen 1987, cited in Schantz et al. 2003). However, the Dutch study authors state
that, despite the differences in analytical methods in the Michigan and North Carolina studies,
the estimated levels of exposure on the basis of maternal milk samples are “reasonably
comparable” (Huisman et al. 1995b).

The total PCB-dioxin TEQs averaged 66.6 ± 24.2 pg TEQ/g milk fat, of which dioxins and
furans represented 46%, planar PCBs 24%, mono-ortho PCBs 23%, and di-ortho PCBs 7%
(Koopman-Esseboom et al. 1994a, cited in Schantz et al. 2003, Table 1). The total dioxin TEQs
are higher than in the United States, but are comparable to other highly industrialized Western
European countries such as West Germany and the United Kingdom (WHO 1989, cited in
Schantz et al. 2003).

At 42 months of age, plasma samples were obtained from the children and the current PCB body
burden was assessed using the sum of PCBs 118, 138, 153, and 180 (∑PCB42 mo). These samples,
also, were analyzed at the Nutrition and Food Research Institute, Zeist, the Netherlands
(Vreugdenhil et al. 2002).

No differences were found between the Rotterdam and Groningen groups with respect to PCB
concentrations measured in maternal, cord, and 42-month-old plasma, nor in breast milk PCB
and dioxin concentrations (Patandin et al. 1999).

Other measures-Lead and cadmium were measured at 18 months of age in a subgroup (n = 151)
to study the neurotoxic influence of these heavy metals along with PCBs and dioxins.

The effect of PCBs and dioxins on thyroid hormone status on the mother-infant pairs was also
studied in a breastfeeding subgroup of the Rotterdam cohort (n = 105) (Koopman-Esseboom et
al. 1994b). TSH, total T3, total T4, free T4 levels were measured in the mothers in the last
month of pregnancy and two weeks post-partum; and in the infants at weeks and three months of
age.

Instruments and Outcome Measures—Neurological Assessments. The neurological condition of

the infants was assessed at 10 to 21 days of age by two different observers (one in Rotterdam, the
other in Groningen) using the Prechtl neurological exam. The exam measures postural tone,
reflexes, and responses and has proven to be predictive for later major and minor neurological
dysfunction (Huisman et al. 1995b). The Prechtl leads to a diagnostic classification of normal,
suspect, or abnormal. The infants were also given a neurological optimality score (NOS) based
 17

on the infants’ cluster scores and on individual items for which an optimal range was defined
(Schantz et al. 2003).

The children’s neurological condition was assessed again at 18 months (Huisman et al. 1995a)
and 42 months (Lanting et al. 1998) by two examiners (one in each city) using the
Touwen/Hemple method, an age-specific neurological examination that yields a qualitative
appraisal of brain function (Lanting et al. 1998). It assesses motor functions like grasping,
sitting, crawling, standing, and walking (Huisman et al. 1995a). The examiners were blind to the
children’s exposure levels. The children were classified as normal, mildly abnormal, or
abnormal. As in the neonatal assessments, the neurological findings were also evaluated in terms
of optimality (NOS) in which a list of 57 neurological items, most describing the fluency of
movements as an indicator of brain function integrity, were assigned an optimal range. The
children would receive one point for each item scored as optimal.

Bayley Scales of Infant Development. The children’s mental developmental index (MDI) and
psychomotor developmental index (PDI) were measured in the Rotterdam group only (n = 207)
with the Dutch standardized version of the Bayley Scales of Infant Development (BOS 2-30) at
3, 7, and 18 months of age. All Bayley tests were performed at the infants’ homes in the presence
of the parent(s) by one examiner who was blind to the infants’ PCB and dioxin exposures
(Koopman-Esseboom et al. 1996).

Cognitive Assessments. The children’s cognitive abilities were assessed at 42 months of age by
two different examiners (one in each study center) with the Kaufman Assessment Battery for
Children (K-ABC) (n=395, 94%) (Patandin et al. 1999). The K-ABC is a standardized test of
intellectual function similar to the McCarthy Scales of Children’s Abilities. It yields an overall
cognitive score as well as scaled scores for sequential and simultaneous processing abilities. The
sequential subtests include hand movement, number recall, arithmetic and gross and fine motor
skills; and the simultaneous subtests include the Magic Window, face recognition, Gestalt
closure, vocabulary, faces and places, and riddles (Patandin et al. 1999). For logistical reasons,
only the Rotterdam sample (n=193) was assessed for verbal comprehension with the Reynell
Language Developmental Scales (RDLS). Because language ability, and verbal comprehension,
in particular, is a measure of mental ability, the RDLS is considered to be a measure of general
mental ability (Patandin et al. 1999).

At 84 months (6.5 years) of age, 376 (90%) of the original cohort of 418 children were
reassessed (Vreugdenhil et al. 2002) using the Dutch version of the McCarthy Scales of
Children’s Abilities which, as noted earlier, measures cognitive ability, as well as gross and fine
motor abilities.

Control variables and analysis—Obstetrical variables were evaluated according to an

“obstetrical optimality list” (Touwen et al. 1980, cited in Huisman et al. 1995b) consisting of 72
items that measure socioeconomic situation, and pre-, intra- and post-partum conditions. An
optimality score was calculated by counting the number of items that fulfilled preset criteria for
optimality.
 18

Potential confounders identified with univariate analyses included the study center (Rotterdam or
Groningen) to adjust for inter-rater variability, parents’ educational level (high vs. low according
to if at least secondary education was completed), profession (three categories including
student/unskilled/unemployed vs. skilled/middle class employee vs. independent middle
class/higher profession), maternal smoking and alcohol consumption during pregnancy (yes or
no), parity (first or second), maternal age, duration of gestation, birth weight, Apgar score at one
minute and five minutes, sex of infant, weeks of breast-feeding, thyroid hormone levels
measured in maternal blood in the last month of pregnancy, cord blood at birth, infant blood at
two at weeks , three months, and 18 months of age. The HOME inventory was carried out at 18
months of age to control for differences in home environment. The verbal IQ of the principal
caregiver parent (usually the mother) was assessed only for Patandin et al.’s (1999) 42-month K-
ABC study using the Information and Vocabulary subtests of the Dutch Wechsler Adult
Intelligence Scale.

For the infant Prechtl and NOS assessments, low, medium and high exposure were defined as the
5th, 50th, and 90th percentiles, respectively, of measured congener concentration. The chi-square
and Wilcox on rank sum tests were performed at the 5% level for a univariate comparison of the
results in Groningen and Rotterdam. Both univariate analysis and logistic regression analysis
were used to examine the relation between test outcome on one hand, and levels of PCB and
dioxin exposure, and obstetrical variables, on the other hand. The NOS and the Prechtl postural
tone and reflex and response clusters were dichotomized at the median of the pooled population.
The PCB and dioxin values were logarithmically transformed to normalize for positive skew.
After adjusting for obstetrical variables, the effect of each chemical compound and total TEQ
scores was examined. The results were reported as odds ratios associated with doubling of
concentrations, together with 95% confidence intervals, without any correction for multiple
hypothesis testing (Huisman et al. 1995b).

Data analysis for the Bayley Scale results was performed using the Statistical Package for the
Social Sciences (SPSS/PC, Cary, NC), and studied the relationship between mean Bayley scores
and perinatal PCB/dioxin exposure (low, medium, and high exposure) (Koopman-Esseboom et
al. 1996). Multiple regression analysis was used to study the effects of pre- and post-natal
exposure to PCBs and dioxins separately as well as combined.

The analysis for the Touwen/Hemple method and NOS assessments at 18 months used the chi-
square, Student’s t-test, and the Mann-Whitney U-test to compare groups. Multiple linear
regression analysis was used to assess the effects of PCB and dioxin exposure on brain function.
The dependent variables were the NOS and the fluency cluster score at 18 months. The NOS
score distribution was skewed to the left, so it was transformed to achieve normality. The PCB
and dioxin levels were logarithmically transformed. The other independent variables were
social, perinatal and obstetrical variables from the obstetric optimality list, and the study center
(Huisman et al. 1995a). A p value of ≤ .05 was considered significant.

Data analysis for the Touwen/Hemple and NOS neurological assessments at 42 months of age
was the same as that used for the 18 month assessment described above, including normalization
of a left-skewed NOS distribution (Lanting et al. 1998). Because the plasma PCB values were
positively skewed, they were normalized by natural logarithm (Patandin et al. 1999). The K-
 19

ABC and RDLS data analysis used the chi-squared, the Student’s t-, and the Mann-Whitney tests
to perform single variable comparisons of the two groups. Multiple regression analyses adjusted
for covariables were used to assess exposure effects. Dependent variables were scores on the
overall cognitive scale, and the sequential and simultaneous processing scales of the K-ABC, and
on the verbal comprehension scale of the RDLS. Covariables entered in the final regression
analyses are listed above, and included the HOME score and parental verbal IQ.

As in the previous assessments, the effect of prenatal PCB exposure estimated from the ∑PCBs
in maternal and cord blood was examined in the whole group. The breast-fed group was
additionally studied for the effect of prenatal exposure to dioxin TEQs, dioxin-like PCB TEQs
and 20 nondioxin-like PCBs (here including the di-ortho PCBs 170 and 180) measured from
breast milk concentrations (Patandin et al. 1999).

The data analysis on the McCarthy scale assessments at 84 months of age used the chi-square,
Student t-, and the Mann-Whitney U tests to compare groups for a single variable. PCB and
dioxin levels were normalized for positive skew by a natural logarithmic transformation. Effects
of PCB and dioxin exposure on cognitive and motor abilities were studied using multiple
regression analyses. The list of covariables in the final regression relevant to prenatal exposure
effect assessment included study center, sex, parity, maternal age at birth, parental education
level, parental verbal IQ and HOME score, and age at examination. To evaluate effects of
prenatal PCB exposure in the two feeding groups separately, an interaction variable (the product
of feeding type and ∑PCB) was included in the regression model. The authors note that no
correction for multiple testing was made because of the exploratory nature of the study, and two-
tailed p values <.05 were considered significant (Vreugdenhil et al. 2002).

Results—Neurological Assessments The Prechtl neurological exam and neurological optimality

assessments showed no correlation between the NOS and the sum of PCB 118, 138, 153, and
180 levels in maternal (∑PCBmaternal) or cord blood (∑PCBcord) after adjusting for maternal age,
study center, alcohol consumption and an interaction between age and alcohol consumption.
Plasma PCB levels had no effect on the Prechtl cluster scores for postural tone or reflexes and
responses (Huisman et al. 1995b).

However, when logistic regression analysis with covariate adjustment was performed on breast
milk data and adjusted for study center, a significant amount of hypotonia was associated with
higher exposure to planar PCB TEQ (OR 1.64, 95% CI: 1.03-2.63). In addition, after adjusting
for study center and ∑PCBcord, a reduced NOS and a higher prevalence of hypotonia was found
in the highest exposed group starting at 540 ng ∑PCBmilk /g milk fat, representing 23% of the
breast-fed infants (Huisman et al. 1995b). Schantz notes that the discrepancy between blood and
breast milk significant associations could reflect either a postnatal exposure effect, or
alternatively, that breast milk may be a more reliable measure of prenatal exposure than maternal
or cord blood because of its high fat content and the large amount of sample available (Schantz
et al. 2003).

The individual dioxin and PCB congeners negatively associated with the NOS did not appear to
be structurally or toxicologically similar: Some are Ah receptor agonists while others are not,
and there was no correlation between TEQ and neurotoxicity among the Ah receptor agonists.
 20

The significant associations could simply be due to higher prevalence of the congeners in the
sample population, or due to more reliable measurement afforded by higher concentrations
(Schantz et al. 2003). No insight into human in vivo structure-activity relationships of the dioxin
and PCB congeners was provided by this study.

Unlike the neonatal findings, the neurological assessments at 18 months (Huisman et al. 1995c)
and 42 months (Lanting et al. 1998) using the Touwen/Hemple method showed no association
between the NOS and PCB or dioxin levels in breast milk at either age. However, at 18 months,
a small two-point difference in NOS (49.9 vs. 47.9) was found between children with low cord
∑PCBcord or maternal ∑PCBmaternal blood exposure variables at the 5th percentile (e.g. 0.18 ppb)
and those with high exposure in the 95th percentile (i.e. 0.86 ppb). No adverse effects of prenatal
PCB exposure on the neurological condition found at birth and 18 months of age could be
detected at 42 months of age.

Bayley Scales of Infant Development. A significant negative association between prenatal PCB
exposure and psychomotor development was found at 3 months of age: A doubling of the
maternal blood sum ∑PCBmaternal was associated with a three point decrease in the psychomotor
development index (PDI). No effect could be found when the cord blood sum ∑PCBcord was
used, even in cases in which the ∑PCBmaternal still had a negative influence. When used as the
measure of prenatal exposure for the breastfed group, the total PCB-dioxin TEQ level also was
negatively associated with psychomotor development at three months, but did not achieve
statistical significance (β = -7.4, SE = 4.0, p = .07). There was no relationship found between
exposure and the mental development index (MDI) (Koopman-Esseboom et al. 1996).

At 7 months of age, no significant association was found between prenatal exposure and PDI or
MDI scores. The only significant result related to a strongly positive association between PDI
scores and duration of breastfeeding, the effect of which was decreased in the children with
medium and high postnatal total dioxin TEQ exposures (Koopman-Esseboom et al. 1996).

At 18 months, no relation was found between prenatal exposure and PDI or MDI scores. As
would be expected, positive relationships were found between the children’s mental development
and the HOME score, as well as with the mother’s level of education (Koopman-Esseboom et al.
1996).

The Dutch study Bayley Scale negative effect on PDI at three months cannot be compared with
Michigan or North Carolina studies because the children were not tested at three months of age
with the Bayley Scale instrument. The Dutch Bayley Scale results at seven months are consistent
with those of the Michigan Bayley Scale assessment (Jacobson et al. 1986) at five and seven
months of age, which showed no association between prenatal PCB exposure and either MDI or
PDI scores.

In contrast, the North Carolina studies (Gladen et al. 1988; Rogan et al. 1991), showed a negative
relationship between prenatal PCB exposure and PDI scores at the ages of 6, 12, and 24 months.
As noted earlier, the negative trend shown at 18 months was not statistically significant. Schantz
suggests that, because the North Carolina cohort was much larger (880 children) it provides more
 21

statistical power than the Dutch Rotterdam or the Michigan cohorts, which comprised 207 and
313 children, respectively (Schantz et al. 2003).

Cognitive assessments. The Kaufman Assessment Battery for Children (K-ABC) cognitive
assessment at 42 months of age showed that, after controlling for confounders, prenatal PCB
exposure measured from maternal blood (∑PCBmaternal ) was significantly associated with lower
scores on the overall cognitive scale, as well as the sequential and simultaneous processing
scales (p < .05). The score on all three scales in the highest exposed group (∑PCBmaternal ≥ 3 ppb)
was four points lower than that in the lowest exposed group (∑PCBmaternal < 1.5 ppb). No
negative effects on K-ABC performance were found to be related to prenatal PCB or PCB-dioxin
TEQ exposure. A negative association was also found between ∑PCBmaternal and the Reynell
Developmental Language Scales (RDLS) scores in the Rotterdam cohort, but the association was
not statistically significant (p = .08) (Patandin et al. 1999).

When the breast fed and formula fed children were assessed separately, the effects of prenatal
PCB exposure on cognitive abilities at 42 months of age were more pronounced in the formula
fed group than in the breast fed group, even though the breast fed children had higher prenatal
exposure levels. The analyses showed that the differences in vulnerability to prenatal PCB
exposure related more to differences in parental and home environmental characteristics than to
beneficial effects of breast-feeding (Vreugdenhil et al. 2002). When the results were adjusted for
these covariables, the scores of the two feeding groups no longer differed.

A discrepancy appears to exist between the neurological and 42 month cognitive assessment
results. The adverse effects of prenatal exposure on neurological condition at birth and 18
months could not be detected at 42 months of age, while cognitive abilities at 42 months were
negatively associated with in utero exposure. The authors suggest that this difference can be
explained by the testing procedures, insofar as the K-ABC measures cognitive abilities in a
quantitative fashion and the neurological examination is a qualitative measure of brain
development (Patandin et al. 1999).

The McCarthy Scales assessment at 84 months showed no relationship between prenatal PCB
exposure ∑PCBmaternal and the general cognitive index (GCI), memory, or motor skills, after
adjustment for covariables. There was no relationship when the breast fed and bottle fed were
considered separately.

Interestingly, when prenatal PCB exposure ∑PCBmaternal was analyzed with the interaction
variables of maternal age, parental IQ, and parental education level, negative effects were seen in
children born to younger mothers and to parents with lower verbal IQ scores and education level.
In formula fed children, higher ∑PCBmaternal was related with lower motor scores in children
whose parents had lower verbal IQ scores or lower HOME scores. These results further support
the evidence that favorable parental and home characteristics could compensate for or mask
developmental delays (Vreugdenhil et al. 2002).

Exposure to non-dioxin-like PCBs and ∑PCBmilk showed negative relationships, but were not
statistically significant.
 22

Other measures. Lead and Cadmium- The concentrations of lead and cadmium were very low
and not related to outcome variables (Patandin et al. 1999).

Thyroid hormone-Elevated levels of dioxins and PCBs were shown to alter the human thyroid
hormone status. Except for one mother with an elevated TSH level, all thyroid hormone levels of
the mothers and infants were within the normal limits. However, some subtle but significant (all
p < .05) effects were found. Higher total dioxin-PCB TEQs correlated significantly with lower
plasma levels of maternal total T3 and total T4, and with higher levels of TSH in the infants at
two weeks and three months of age. Infants exposed to higher TEQs also had lower total T4 and
freeT4 at two weeks of age. Higher ∑PCBmaternal and ∑PCBcord were also associated with higher
TSH levels in infants at two weeks of age (Koopman-Esseboom et al. 1994b; Schantz et al.
2003).

However, although other animal and human studies have also demonstrated that exposure to
dioxin-like compounds can alter thyroid hormone homeostasis concurrently with
neurobehavioral changes, it remains to be definitively established that changes in thyroid
function are responsible for the neurobehavioral effects of DLCs (Hauser et al. 1998).

Criticisms-- The Dutch study results are complex because many different outcomes were
assessed in relation to so many different exposure variables (Schantz et al. 2003). One criticism
that has been raised about the statistical analyses used is that there was failure to control for the
number of significant findings that could have been expected by chance alone. Cicchetti et al.
(2004) illustrate this issue, using as an example the more than 40 multiple regression analyses
Koopman-Esseboom et al. (1996) performed to relate Bayley Scale MDI and PMI scores at ages
three months, seven months, and 18 months with various prenatal and post-natal exposure
variables, resulting in only three significant findings, one of which was not toxin-related.
Cicchetti et al. (2004) argue that two toxin-related findings out of 40 analyses might very well be
chance occurrences.

However, based on the North Carolina Bayley studies, the investigators had reason to predict a
small but negative association between prenatal PCB exposure and decreased performance on the
Bayley assessments. If, as Cicchetti et al. (2004) proposed, the investigators had employed
Bonferroni adjustments to the analyses without regard to prior prediction or theory, this would
have moved the critical region from p=.05 to less than .00125, forcing the investigators to retain
the null hypothesis of no relationship.

The Bonferroni correction is a statistical adjustment for multiple comparisons in which the 0.05
alpha level is divided by the number of comparisons (n) that are being made on a data set (.05/n).
The data are then tested at the resulting alpha value to ensure that the overall chance of making a
Type I error is still less than .05. It can also be applied to the p value by multiplying it by the
number of outcomes being tested, and is rounded down to 1.0 if the adjusted p value is greater
than 1.0 (Simon 2005).

As Stewart et al. (2004) point out, this is counterproductive in epidemiological studies, because
the “truth” of any association is not evaluated in a single study, but rather, by whether or not it is
replicated. Since the effects of background levels of PCBs are thought to be small, extremely
 23

conservative alpha levels imposed by Bonferroni corrections would greatly limit the possibility
of uncovering real relationships. As noted earlier, if an association is falsely judged significant
in one study, chances are minutely small that identical results would be produced in a second
study (Stewart et al. 2004).

Another criticism raised by Cicchetti et al. (2004) is the issue of statistical vs. clinical
significance, which they illustrate with the 4-point difference in K-ABC scores found between
the highest and lowest PCB exposure groups in the Dutch cohort at 42 months of age (Patandin
et al. 1999). This difference was presented as clinically meaningful, but a difference this small
(0.27 SD) can be expected to occur in the test-retest reliability of the same child on two separate
evaluations (Cicchetti et al. 2004). Kaufman (2001, cited in Cicchetti et al. 2004) notes that to
be 90% or 95% certain that you have captured a person’s “true” IQ, the confidence interval must
be expanded to ± 5 or ± 6 points. In addition, when using standard deviation units as the basis of
effect sizes, an effect size of less than 0.50 is small and probably not meaningful (McLean 1995,
cited in Cicchetti et al. 2004). Furthermore, Patandin et al. (1999) controlled for covariates on
the whole sample, which they divided into five subgroups based on exposure levels. They then
compared only the two extreme subgroups after deleting the three middle subgroups without re-
controlling the extremes for the covariates. Thus, according to Cicchetti et al. (2004), “not only
is four points too small of a difference to be clinically meaningful, but that number of points is
not an actual outcome of the significant results they obtained for the total sample; it is based on a
greatly reduced sample that includes only the extremes.”

On the other hand, the fact that all participants in the study had some level of PCB and dioxin
exposure means that there was no true control population. This is likely to bias the results
against finding an effect (Colborn et al. 1996). Thus, the small negative associations found on the
K-ABC might have been larger if true controls had been used.

Other limitations of the study relate to the exposure estimates. Because dioxin TEQs were
measured in breast milk due to small maternal and cord blood samples, no exposure data were
available to evaluate DLC neurodevelopmental effects on the formula fed children. This reduced
the power to detect associations. Moreover, as discussed earlier, breast feeding may have
protected the nursing children from DLC neurotoxic effects and biased the results against finding
an effect. Advances in analytic techniques that allow PCB and dioxin congeners in smaller
samples will be needed to address this limitation (Schantz et al. 2003).

The European Union provided funding to expand the Dutch study into a transnational,
multicenter cooperative study, which included a German cohort in Düsseldorf and a Danish
cohort in the Faroe Islands (Schantz et al. 2003).

► German Cohort The German cohort consisted of 171 healthy mother-infant pairs from
middle and upper class families non-randomly -recruited between 1993 and 1995 from the
obstetrical wards of three Düsseldorf hospitals. Inclusion criteria included healthy first and
second born infants born at term with no complications during pregnancy or delivery, and a
minimum five-minute Apgar score of seven. The objectives of the study were to assess
independently the neurodevelopmental effects of prenatal and early postnatal PCB exposure on
 24

one hand, and effects of the home environment on neurodevelopment, on the other. Previous
studies had treated home environment only as a potential confounder (Winneke et al. 1998).

Measures of exposure— PCB exposure was based on the sum of PCB congeners 138, 153, and
180 (∑PCBcord) measured in umbilical cord blood to assess prenatal exposure and venous blood
∑PCB42 at 42 months of age to assess postpartum exposure through breast milk. Samples were
centrifuged and stored at -18º C. The first step of PCB measurement consisted of a column liquid
chromatographic sample clean up without a prior solvent extraction step, and the determination
was done by dual-column capillary gas chromatography with electron capture detector (ECD) or
gas chromatograph-mass spectrometer (GC-MS). Ring tests between two analytical laboratories
in Kiel and Düsseldorf revealed excellent comparability for ∑PCB (r > 0.95) (Walkowiak et al.
2001).

Breast milk samples were also collected at two and four weeks post partum to provide an
additional prenatal exposure variable. They were stored at -18º C until they were analyzed for
the same congeners (∑PCBmilk) (at the Institute of Environmental Toxicology in Kiel using high
resolution gas chromatography with a capillary column and ECD. The two and four week values
were combined to yield mean values. Ring tests revealed good comparability for lipid-adjusted
∑PCB (r > 0.80) (Winneke et al. 1998). The PCB congener detection limits were 0.01 ppb
(serum) and 5 ng/g fat (milk). The correlations between PCBs in cord blood and milk were
positive and highly significant (r = 0.57, p < .0001) (Walkowiak et al. 2001).

Although the cohort comprised 171 mother-infant pairs, only 169 cord blood samples and 131
breast milk samples were available for PCB analysis. The mean ∑PCBcord was 0.55 ppb, the
mean ∑PCBmilk was 426.5 ng/g fat (Winneke et al. 1998). These are comparable to the Dutch
study levels which were 0.5 ± 0.3 ppb for cord blood, and 428.5 ng/g fat for breast milk
(Koopman-Esseboom et al. 1996). Values for the 42 month levels will not be discussed, as this
review addresses only prenatal exposure effects.

The lead levels found in cord blood were very low, with a mean value of 20.3 ppb (Walkowiak et
al. 2001).

Instruments and Outcome Measures—Home Environment. An infant (0-3 yr) version of the
HOME score adapted to fit the German cultural context was administered at 18 months of age at
home in the presence of the child. The German HOME consists of 45 items. Inter-rater reliability
for two independent observers from the Düsseldorf team at two 11 month intervals, were r =
0.61-0.81 (Winneke et al. 1998; Walkowiak et al. 2001).

Psychodevelopmental tests. The children’s psychomotor and cognitive development were

assessed with the Fagan Test of Infant Intelligence (FTII) at seven months of age; the Bayley
PDI and MDI at seven, 18, and 30 months of age; and with the K-ABC at 42 months of age.
Schantz et al. (2003) state that the children were also assessed with the Heidelberg Test for
Language Development at 42 months of age, but these results have not yet been discussed in the
literature.
 25

Control variables and analysis— The children were examined by one examiner who was blind
to the children’s PCB levels. By exchanging videotapes with another experienced group inter-
rater reliabilities were calculated and found to be satisfactory for both the MDI (r = 0.85) and the
PDI (r = 0.98). The re-test reliability of the FTII novelty score for the mobile test version after
two weeks for two observers and 10 children was found to be zero and even negative (r = -0.195)
(Winneke et al. 1998).

Potential confounders included age at examination, gestational age, alcohol/smoking during

pregnancy (separately scored as yes/no), Apgar score at five minutes, neonatal illness/jaundice,
spontaneous delivery, parity, lead in cord blood, chronic diseases during past year, duration of
breastfeeding, parental education (maximum years of schooling of either parent) parental
occupation (10 categories, highest of either parent), maternal BMI, and maternal verbal IQ
measured with the German form of the Wechsler Adult Intelligence Scale vocabulary subtest
(Walkowiak et al. 2001).

From recruitment until 42 months of age, the sample size decreased from 171 to 116; however,
the only significant difference in the remaining sample with regard to the potential confounders
was that the families tended to be better educated (Walkowiak et al. 2001).

The data was analyzed with the statistical data analysis software program SAS® 8.01. The PCB
distributions were skewed so all values were logarithmically transformed to yield normalized
distributions (Walkowiak et al. 2001). Multiple linear regression analysis was used to analyze the
association of HOME and PCBs with the target variables. The initial regression model included
variables selected based on priority (e.g., lead in cord blood, maternal age) or those with
significant association with the outcome (e.g., HOME or Apgar score). In the second model,
selection of variables was data-driven. Only variables with correlations at p < .20 with both the
exposure index and two or more of the outcome variables were selected after including the initial
variables. The second variables were parity, smoking in pregnancy, and maternal BMI. All
outcome variables were uniformly adjusted for these variables. Additionally, repeated
measurements analysis was done on the Bayley Scales data. One-tailed probabilities were used
throughout. The basis of the authors’ justification for using one-tailed probabilities was that the
hypotheses were directional (Winneke et al. 1998; Walkowiak et al. 2001).

Results— There was no negative association found between cord blood PCBs (∑PCBcord) and
any of the outcome measures. However, significant negative associations were found between
outcome measures and maternal blood PCBs (∑PCBmilk).

Fagan Test of Infant Intelligence. The FTII assessment at seven months of age showed
complete lack of association between neonatal PCB exposure and visual recognition-memory
scores (Winneke et al. 1998). This result is at variance with the Michigan FTII results at seven
months, which showed a dose-dependent negative association with prenatal PCB exposure
(Jacobson et al. 1985). The authors suggest that, given the very low test-retest and inter-rater
reliabilities of the mobile FTII in this study, the results might not be valid (Winneke et al. 1998).

Bayley Scales of Infant Development. The only negative association found for the Bayley Scale
assessment at seven months after controlling for confounding variables was between the Bayley
 26

MDI and ∑PCBmilk (one-tailed p < 0.048) (Winneke et al. 1998). This result is not consistent
with the Michigan and Dutch Bayley Scale assessment results at seven months of age, which
showed no relation between prenatal PCB exposure and either the MDI or PDI (Jacobson et al.
1996; Koopman-Esseboom et al. 1996). It is also at variance with the North Carolina Bayley
Scale assessment at six months of age, in which PDI, not MDI, had a negative association with
prenatal PCB exposure (Gladen et al. 1988).

At seven and 18 months, borderline negative associations were found between ∑PCBmilk and
both the Bayley Scale MDI and PDI, but became significant at 30 months (one-tailed p = 0.035
for the 30 month MDI and p =.05 for the MDI). When the seven to 30 month age range was
treated as a time-series, prenatal PCB exposure showed significant negative overall associations
with mental and motor development that increased with age (Walkowiak et al. 2001).

The seven month German Bayley Scale result is at variance with that of the Michigan study
(Jacobson et al. 1986), which showed no association between prenatal PCB exposure and either
the MDI or PDI. The 18 month and 30 month German Bayley Scale results cannot be compared
with the Michigan study because the cohort was not tested again with the Bayley Scale
instrument until four years of age. These results are also inconsistent with the North Carolina
Bayley Scale assessment at six months (Gladen et al. 1988), which showed no association
between prenatal PCB exposure and MDI, but did show a negative impact on PDI. The Dutch
study Bayley Scale assessment at seven months showed no association with either MDI or PDI
(Koopman-Esseboom et al. 1996).

At 42 months of age, a significant negative association between ∑PCBmilk and the Mental
Processing Composite Index of the K-ABC was found (p = 0.028) (Walkowiak et al. 2001). This
finding is consistent with the Dutch K-ABC assessment at 42 months of age (Patandin et al.
1999), which found a negative association between the K-ABC scores and ∑PCBmaternal .
However, the Michigan Bayley MDI results at four years of age showed no negative association
between prenatal PCB exposure and mental developmental effects.

The HOME score analyses showed a positive impact on mental/motor development. The slopes
of both variables over the range of observed HOME scores or ∑PCBmilk are similar, but the
magnitude of the overall positive impact of the home environment on mental/motor development
was stronger than the negative effect of neonatal PCB exposure: The positive effect size of the
home environment on Bayley scale and K-ABC scores was approximately twice that of the
negative effect size of ∑PCBmilk on these measures. For example, an increase of 17.7 points
associated with increasing HOME score on the 30 month Bayley PDI contrasts with a 9.9 point
decrease associated with increasing ∑PCBmilk. These effect size differences were less
pronounced, however, when the data were treated with the time-series approach (Walkowiak et
al. 2001).

Schantz et al. (2003) note that the German study findings add to the overall weight of the
evidence that prenatal PCB exposure can cause neurodevelopmental effects.

Criticisms—The sharpest criticisms of the German studies found in the literature relate to the
investigators’ failure to control for chance results when conducting multiple comparisons, and
 27

the investigators’ use of one-tailed significance tests for interpreting the significance of their
results.

According to Cicchetti et al. (2004), one-tailed tests do not permit a valid interpretation of study
results when they occur in a direction opposite to the one predicted. Their contention is that one-
tailed tests are appropriate for studying effects that biologically can vary in only one direction,
such as survival rates associated with graded cancer stages. In addition, Cicchetti et al. (2004)
note that one-tailed tests essentially double the odds of obtaining a statistically significant
finding, as a probability level of .10 in a two tailed test will be interpreted as one of .05.

For example, Winneke et al. (1998) reported a significant negative association between ∑PCBmilk
and the Bayley Scale MDI. However, Cicchetti et al. (2004) note that, not only did the
investigators run six analyses simultaneously without controlling for chance findings, they used a
one-tailed significance test. The resulting one-tailed p value of .048 transforms to a p value of
about .10 when a traditional two-tailed test is applied to the same data—a result that does not
approach significance. In the words of Cicchetti et al. (2004), “to employ one-tailed tests,
accompanied by no control for multiple comparisons, solely in the service of maximizing the
odds of finding statistical significance, is a scientifically indefensible methodological strategy.”

In response to Cicchetti et al.’s criticisms, Winneke and colleagues replied:

“Domenic Cicchetti and Alan Kaufman suggest inappropriate testing of non-directional

hypotheses, with no adjustment for multiple testing or quality of the home environment.
Our hypotheses were directional. We tested for adversity of prenatal and neonatal PCB
exposure, because the assumption of a beneficial impact of PCBs on motor and mental
development is not biologically plausible. We also tested for a beneficial effect of the
home environment because, for psychological reasons, good parental caretaking supports
rather than impairs psychodevelopment. Thus, one-tailed testing is mandatory” (Winneke
et al. 2002).

Additionally, as has been noted earlier, the “truth” of the small associations found in the PCB
epidemiological studies lies in their replicability (Stewart et al. 2004), and should be evaluated in
the context of many studies using similar methodologies and outcome measures. When viewed
from this perspective, the tone of Cicchetti et al.’s criticisms sounds somewhat harsh and
snappish.

Schantz et al. (2003), however, believe that the German studies add to the overall weight of
evidence that prenatal exposure to PCBs can cause adverse neurodevelopmental effects.
However, they note that these studies are limited in that only three marker PCB congeners were
measured. Thus, the studies are unlikely to contribute to our understanding of the effects of
individual PCB congeners or classes of congeners.

► Oswego cohort The Oswego Newborn and Infant Development Project was the first large-
scale replication and extension of the Lake Michigan study (Daly et al. 1996; Lonky et al. 1996).
The Oswego cohort was intentionally designed to replicate and extend the Michigan cohort in
terms of cohort characteristics, exposure metric, and assessment protocols to measure the same
endpoints as the Michigan study, at similar ages. Additionally, the investigators sought to
 28

improve the quality and degree of covariate control relative to the Michigan study (Stewart et al.
2004).

New York State had issued advisories recommending restrictions in Lake Ontario fish
consumption because of the large quantities of PCBs, organochlorine insecticides, and other
chemicals found in Lake Ontario sport fish during the early 1980s. These advisories
recommended that pregnant women completely eliminate Lake Ontario fish from their diets
(Stewart et al. 1999). Despite the advisories, pregnant women continued to consume large
amounts of the fish; in a survey of over 650 pregnant women in the Oswego, New York area,
Lonky et al. (1996) found that 8.2% of pregnant women in the area had consumed over 26
pounds in the previous six years, with 46% indicating they had eaten some fish from Lake
Ontario.

To corroborate and extend the Michigan study findings suggesting that prenatal PCB exposure
exerted adverse effects on fetal neurodevelopment, a prospective longitudinal study was carried
out on mother-infant pairs recruited between June 1991 and June 1994 during the physician’s
required 20 week sonogram visit at the county’s only obstetrical practice (Lonky et al. 1996).
The original cohort consisted of 602 pregnant women, most of whom were fish eaters, but the
cohort included a randomly selected control subset of non-fish eaters. Attrition reduced the
number to 559 (395 fish eaters and 164 non-fish eaters). A subset of 309 of the women and their
children were followed longitudinally; the rest of the children were evaluated only at birth.

Most of the participants were Caucasian and, unlike previous cohorts, were of low to middle
socioeconomic status. The cohort was representative of the obstetrical clinic’s patient population
with respect to age, parity, marital status and on most aspects of labor and delivery, except that
study participants tended to have more spontaneous vaginal deliveries and their infants tended to
have significantly higher Apgar scores than non-participants (Lonky et al. 1996).

The study's purpose was to assess cognitive and behavioral effects on children of pre- and
postnatal exposure to persistent toxic chemicals, including PCB's, from maternal consumption of
contaminated Lake Ontario sport fish (Stewart et al. 1999). A congener-specific analytic
technique permitted quantitation of 68 individual PCB congeners in umbilical cord serum and
placenta, with analysis of both concentration and congener distribution patterns.

Measures of exposure—The primary measure of exposure in the Oswego studies was the
concentration of PCBs in umbilical cord blood. However, maternal fish consumption was the
measure of exposure in the first NBAS study (Lonky et al. 1996). Samples of placenta were also
collected, but placental PCB concentrations have not been published yet (Schantz et al. 2003). A
subset of 83 women provided a breast milk sample collected during the first six months after
birth. The cord blood was analyzed for the presence of 69 PCB congeners and coeluters,
hexachlorobenzene (HCB), dichlorodiphenyldichloroethene (DDE), the insecticide Mirex, and
lead. Maternal hair was analyzed twice for methylmercury (MeHg), once during the first half of
pregnancy, and again during the second half of pregnancy (Stewart et al. 2000). Dioxins, furans,
and coplanar congeners were not measured in this cohort.
 29

The fluid samples were stored at 5º C until analysis. The cord blood was centrifuged and
prepared by a CDC laboratory method and analyzed at Wadsworth Center for Laboratories and
Research on a gas chromatograph with an electron capture detector. The methodology is
described in cookbook detail in Stewart et al. (2000). Analysis of maternal hair mercury was
performed at the University of Rochester.

The average concentration of PCBs in cord blood was very low (< 1 ppb), and, like the Michigan
study, no direct relationship was found between fish consumption and total cord blood PCB
concentration: the mean total PCBs for fish eaters was 0.8 ppb, and that for the controls was
1.03 ppb (Stewart et al. 1999).

However, analysis of the pattern of PCB congener exposure revealed differences in the congener
distribution patterns in the two groups. Congener pattern analysis was made possible because of
a concurrent laboratory rat experimental project in which the brains of rats fed Lake Ontario
Salmon vs. Pacific Ocean salmon vs. rat chow were analyzed for PCB homologues. The
homologues were then classified, based on degree of chlorination, into three clusters representing
the lower tail (C11-13), the middle (C14-16), and upper tail (C17-19) of their distribution
(ecological persistence increases with degree of chlorination) (Stewart et al. 1999). The
congener-specific results have not been published yet for the Oswego cohort.

Pattern analysis showed that Lake Ontario salmon contain a significantly higher proportion
(mol%) than controls of the most persistent and highly chlorinated PCB homologues (C17-19),
and that the proportion of heavily chlorinated congeners grew larger as the total PCB
concentration in the sample increased. This analytical approach was then applied to the human
cord blood samples, with consistent results: the mol% of the highly chlorinated congener cluster
(C17-19) was significantly greater in the Lake Ontario fish eaters (p = .006); the mol% of this
cluster grew larger with total cord blood PCB concentration (p = .02); and the absolute
concentration of highly chlorinated PCB homologues was greater in fish eaters than in controls
(p < .001). When compared with breast milk samples, only the cord blood C17-19 cluster
significantly correlated with breast milk measures (r = +.29, p < .01) (Stewart et al. 1999).

On the basis of these findings, Stewart et al. (1999) argued that the most heavily chlorinated
PCBs are the most valid index of fish-borne PCB exposure in cord blood, just as Schwartz et al.
(1983) had after their study of PCB exposure from Lake Michigan fish. Indeed, when the
investigators assessed exposure on the basis of total PCBs alone, the prenatal PCB exposure of
fish eaters and controls appeared to be comparable, yet the homologue analysis revealed a far
greater proportion of the most highly chlorinated PCBs in the fish eaters which became more
easily detectable as more PCBs were detected in the sample. Why?

Stewart et al. (1999) suggest that 1) heavily chlorinated PCBs are more persistent and thus
represent a greater proportion of the bioaccumulated PCBs that contaminate food; and 2) fewer
contaminants (thus less chromatographic noise) are found at the retention times where the most
heavily chlorinated PCBs are measured, making assessment more reliable in the low lipid/low
PCB environment of cord blood. Thus, the neurobehavioral outcomes in this study were
assessed with respect to total PCBs and total highly chlorinated PCBs (Darvill et al. 2000;
 30

Stewart et al. 2000), similar to the Michigan Study, in which PCB concentration values matched
to the Aroclor 1260 standard (highly chlorinated congeners) were used (Schwartz et al. 1983).

PCB exposure levels appear to be lower in this Great Lakes cohort than in the Michigan cohort,
where the umbilical cord PCB concentration level averaged 2.5 ppb (Schwartz et al. 1983).
However, as discussed earlier, the Michigan cord blood concentration was measured using a
packed-column gas chromatograph and the Webb-McCall method, which was inadequate to
detect the PCB concentrations in 70% of the samples. As noted earlier, it is also unclear how the
cord PCB concentration level could have been accurately ascertained when the reported limit of
quantitation was 3 ppb (Schwartz et al. 1983). In the 1996 study, the investigators created a
composite measure of prenatal exposure by converting cord blood (over 3 ppb), maternal serum,
and maternal breast milk PCB concentrations, to z-scores and averaging them together. This
makes it difficult to compare the two cohorts’ exposure levels.

Instruments and Outcome Measures—The children were assessed twice at birth with the
Brazelton Neonatal Behavioral Assessment Scale (NBAS) at between 12-24 hours (Time 1) and
25-48 hours (Time 2); with the Fagan Test of Infant Intelligence (FTII) at six and 12 months of
age; and with the McCarthy Scales of Children’s Abilities at 36 and 54 months of age (Stewart et
al. 2003). The children were assessed at nine years of age with the Wechsler Intelligence Scale
(WISC-III) (Stewart et al. 2004).

Control variables and analysis—In the first NABS study by Lonky et al. (1996), all cohort
members were compared to non-volunteers served by the same obstetric practice and delivering
in the same hospital. The investigators established equivalence in the populations by analyzing
demographic, labor, and delivery variables with independent sample t-tests or chi-squared tests
of independence. No meaningful differences were found between the two groups on crucial
demographic, labor or delivery variables, enabling the results to be generalized to the entire
population of women delivering babies in the county (Lonky et al. 1996). Including the non-
longitudinal sample greatly increased both sample size and degrees of freedom available for
analysis (Stewart et al. 2000). The second NABS study (Stewart et al. 2000) was restricted to
longitudinal participants who had provided cord blood for PCB exposure analysis.

The children were evaluated twice, once between 12 and 24 hours after birth, and again between
25 and 48 hours after birth, by a member of the study’s behavioral assessment team who was
blind to the infants’ exposure histories. They were tested in a separate quiet room with controlled
lighting and temperature. The NBAS clusters employed as the preferred model of data reduction
were reflex, range of state, autonomic, motor, orientation, regulation of state, and habituation.

Fifty eight variables were constructed and submitted to principal components analyses with the
goal of reducing this set to a smaller number of components that were linear functions of the
original variables. The components were then used to evaluate potential confounds in subsequent
analyses, thereby allowing combinations of variables to be accounted for (Lonky et al. 1996).

Control for potential confounders in this study was excellent. Potential confounders included
maternal education, maternal IQ estimated with the Peabody Picture Vocabulary Test-Revised
(PPVT-R), SES (socioeconomic scale) score, Obstetric Complications Scale score, HOME
 31

scores, maternal substance use (data on alcohol, cigarettes, and caffeine use were collected
before pregnancy, during pregnancy, and after delivery), and mercury and lead levels. Inter-rater
reliabilities on all assessments was strong (Stewart et al. 2004).

A multivariate analysis of covariance (MAN-COVA) was performed on change scores reflecting

the differences in NBAS performance assessed at Time 1(12-24 hours) and Time 2 (25-48
hours), in relation to maternal fish consumption. Change scores are thought to be more
predictive of cognitive, motor, and personality outcomes (Lonky 1996). Multiple regressions
were run for each NBAS cluster, with covariates acting as multiple predictors, to investigate the
power of the covariates to adjust dependent variables. Univariate and stepdown analyses were
used to investigate the effects of the exposure group variable on the dependent variables. The
Autonomic and Reflex clusters, which had shown significant effects in the Michigan studies,
were entered last in the stepdown analysis, with the ordering of the four remaining clusters
randomly determined. This permitted adjusting the Autonomic and Reflex clusters not only for
the covariates, but also for the four remaining NBAS clusters (Lonky et al. 1996).

The Bonferroni procedure was performed on the analyses to control for chance findings. The
Bonferroni correction is a statistical adjustment for multiple comparisons in which the 0.05 alpha
level is divided by the number of comparisons (n) that are being made on a data set (.05/n). The
data are then tested at the resulting alpha value to ensure that the overall chance of making a
Type I error is still less than .05. It can also be applied to the p value by multiplying it by the
number of outcomes being tested, and is rounded down to 1.0 if the adjusted p value is greater
than 1.0 (Simon 2005). Lonky et al.’s (1996) use of the Bonferroni procedure constitutes one of
the few times endocrine disruptor researchers have controlled for multiple comparisons
(Cicchetti et al. 2004).

In the second NBAS study, Stewart et al. (2000) included only longitudinal participants who had
cord blood collected and analyzed, and investigated associations between NBAS performance
and cord blood PCB concentration. The statistical model considered as confounds variables
from the first study that had demonstrated at least a weak (p ≤ .20) association with the exposure
variable (Stewart et al. 2000).

To assess the presence/absence of dose-response relationships, covariate data were examined for
the presence of monotonically increasing or decreasing linear associations through linear trend
analysis. In addition, one-way ANOVA (Analysis of Variance) was used to test for inter-group
differences regardless of monotonicity. The investigators chose this approach because it was
conservative and adjusted the group means appropriately in the event of any nonlinear
association between a covariate and the PCB variable. Nominal/ordinal data were evaluated with
a chi-square analysis, with the same criterion level (Stewart et al. 2000).

Potential confounders used for the FTII and McCarthy Scale analyses were the same as used for
the NBAS. Those that were not normally distributed were log transformed, and those found to
be even marginally related (p < .20) were used as covariates. The statistical methodology used
was linear trend analysis while controlling potentially confounding variables in an Analysis of
Covariance, described above. Departures from linearity were assessed with Sidak reversals at
high alpha (p < .2) (Darvill et al. 2000; Stewart et al. 2003).
 32

Inter-rater reliability, though not reported in the original study (Darvill et al. 2000), were later
reported by Stewart et al. (2004) as being within three percentage points for each individual item
on a 0-100 point scale. Overall mean percent fixation to novel stimuli was within two
percentage points for all testers. Thus, inter-rater reliability for the FTII was strong.

The investigators in the FTII assessments chose to use a one-tailed significance test. The reason
given was that they were attempting to replicate the findings of Jacobson et al. (1985) (Darvill et
al. 2000).

Results—NBAS Assessment. In the first NBAS study, a significant negative association was
found between increasing maternal fish consumption and poorer change scores on the Reflex (p<
.001), Autonomic (p<.001), and Habituation (p<.001) clusters of the NBAS after controlling for
confounders. Orientation was significant in the univariate analysis (p<.01) but failed to reach
significance in the stepdown analysis where the error rate was Bonferroni adjusted (.05/6 =.008)
(Lonky et al. 1996). High fish group babies were hyporeflexive, exhibited more startles and
tremors, and had poorer habituation recovery than newborns of controls (Lonky et al. 1996;
Stewart et al. 2000). There was also some evidence of less developed attention to visual and
auditory stimuli (Orientation) in high fish group babies, but this finding did not reach the
Bonferroni adjusted alpha level of .008 (Lonky et al. 1996).

The investigators conducting the second NBAS study predicted that the most heavily chlorinated
PCBs would relate to impairments on the NBAS clusters most strongly associated with Lake
Ontario fish consumption, i.e., the Autonomic, Reflex, and Habituation clusters (Lonky et al.
1996). These predictions were based on the previous findings (Stewart et al. 1999) that highly
chlorinated PCBs may be a good index of total PCB exposure because cord blood levels
correlated significantly with both total PCB and highly chlorinated PCBs in breast milk (Stewart
et al. 2000). Indeed, excepting the Reflex cluster, these predictions were confirmed.

Linear trend analysis of PCB exposure variables and NBAS cluster scores in the second study
revealed a significant linear association between highly chlorinated PCBs and poorer habituation
scores (p < .05), as well as poorer autonomic scores (p < .05) after controlling for relevant
covariates. PCBs of light or moderate chlorination, DDE, lead, and hexachlorobenzene were not
related to the proportion of poor NBAS scores. A negative trend was observed for reflex cluster
scores, but it did not achieve significance (p = .09, two tailed) (Stewart et al. 2000). These
findings are largely consistent with the Michigan and North Carolina studies, which found
comparable effects (Jacobson et al. 1984a; Rogan et al. 1986b).

Fagen Test of Infant Intelligence. Of the original 293 infants followed longitudinally, 230
infants were tested at the ages of six months and 219 were tested at 12 months, 202 of whom
were tested at both ages. After controlling for confounders, a significant, but small, linear
association between total cord PCBs and declining FTII performance was found at both six
(effect size 2.1%) and twelve months (effect size 1.4%) of age. After controlling for potential
confounders and effects of MeHg and DDE, a linear association was also found between highly
chlorinated PCBs and poorer performance at 12 months of age (effect size 2.3%, one-tailed p=
.023; 2-tailed p=.046), but not at six months. In contrast to the NBAS study (Stewart et al. 2000),
total cord blood PCB levels, not the highly chlorinated congeners, were the better predictor of
 33

exposure effects (Darvill et al. 2000). The investigators noted this in their report, but suggested
no explanation. No departures from linearity were observed in any of the analyses.

Unlike the NBAS studies, no association was found between maternal fish consumption and FTII
performance. Non-PCB contaminants (MeHg, DDE, and Pb) were shown to have no effect on
FTII performance when included as covariates in the PCB analyses.

Both the Oswego and Michigan (Jacobson et al. 1985) studies found significant, dose-dependent
differences in infant preference for novel visual stimuli related to increasing cord blood PCB
levels at six and seven months of age, respectively, although the effect sizes were much greater
in the Michigan study (10.4%) (Jacobson et al. 1985). It was also shown that the association was
not secondary to confounding by DDE, MeHg, Mirex, HCB or lead (Darvill et al. 2000). The
Oswego results suggest that the PCB related performance deficits on the FTII reported at seven
months in the Michigan study last until at least 12 months of age (Darvill et al. 2000).

The investigators suggest that the four to five fold difference in effect size may be due to a
decline in PCB levels over the 15 years that separated the two studies, thus decreasing the
prenatal exposure levels (Darvill et al. 2000). However, as previously discussed, differences in
analytical methods make it difficult to compare the exposure levels across the two studies.

The Oswego investigators failed to find the association between maternal fish consumption and
FTII scores reported in the Michigan study, and there was no difference between the fish-eaters
and the controls with respect to their infants’ performance. The investigators suggest this might
be due to background PCB exposure from other sources than fish, such as meat and dairy
products (Darvill et al. 2000).

When performances on the NBAS and FTII were compared, it was found in both the Oswego
and Michigan studies that NBAS performance was not related to later FTII performance. The
investigators suggest that this indicates that prenatal PCB exposure may exert effects on several
independent behavioral domains (Darvill et al. 2000). Also, it may be because the FTII has been
shown to be consistently unreliable (Cicchetti et al. 2004).

In sum, the Oswego study (Darvill et al. 2000) confirmed the Michigan (Jacobson et al. 1985)
findings that cord blood PCBs are associated with poorer performance on the FTII. In addition,
the Oswego study included analysis of a much larger number of babies, included a much more
detailed array of potential confounders, and ruled out competing contaminants (Stewart et al.
2004). Although one-tailed significance tests were used, two of the three findings significant on
the one-tailed tests were also significant on two-tailed tests, including the most important
analysis, which was the association between FTII performance at 12 months and the sum of
highly chlorinated PCBs (Stewart et al. 2004).

McCarthy Scales of Children’s Abilities. The McCarthy Scales assessment at 38 months

(n=194) revealed a significant, dose-related decline in performance on the General Cognitive
Index (GCI) for both wet weight (p = .012) and lipid-adjusted (p = .008) highly chlorinated PCB
cord blood values, as well as significant negative associations with the Perceptual and
Quantitative subscales, after controlling for confounders. No significant departure from linearity
 34

was observed on any analysis. Effect size analysis showed the proportions of variance to be
3.2% for absolute and 4.0% for lipid-adjusted PCB values. Analysis for additive/interactive
effects of MeHg showed that PCBs potentiated MeHg effects on McCarthy Scale performance
(Stewart et al. 2003).

In contrast to performance at 38 months, McCarthy performance at 54 months (n=197) showed

no linear association between PCBs and the McCarthy GCI, or with any of the subscales, after
controlling for confounding variables. Neither was there a significant association found with
MeHg.

All exposure groups showed a large increase in McCarthy GCI performance from 38 to 54
months (p < .0001 for all four groups). In fact, the most highly exposed children actually caught
up to the performance level of the least exposed group by 54 months of age (Stewart et al. 2003).

Thus, the Oswego McCarthy Scale studies did replicate the Michigan McCarthy scale findings at
four years of age (Jacobson et al. 1990) and, because of improved analytical methods, were able
to demonstrate effects at lower levels of exposure, after controlling for the influence of over 50
potentially confounding variables. They also suggest that the adverse neurodevelopmental
effects caused by these exposure levels are reversible by school age (Stewart et al. 2003).

Although the North Carolina McCarthy Scale studies showed no effect at three, four, or five
years of age (Gladen et al. 1991), the Dutch (Patandin et al. 1999) and German (Walkowiak et al.
2001) K-ABC assessments showed a decline in cognitive performance at 42 months of age
associated with ∑PCBmaternal, and ∑PCBmilk .

Wechsler Intelligence Scale (WISC-III)- The WISC-III data are currently being reviewed by the
investigators’ granting agencies before submission for publication. The investigators expect to
be able to release the data sometime in the summer of 2005 (Lonky, E. Personal
communication).

Criticisms—Cicchetti et al. (2004) have criticized Lonky et al. (1996) for their control of inter-
observer variability, which was measured as the percentage of agreement, defined as
(agreements)/(agreements + disagreements). The levels of agreement reported were 92%, 90 %,
and 88% at the initial, 6-month, and 1-year reliability checks (Stewart et al. 2004). Cicchetti et
al. (2004) argue that the percentage of agreement approach does not take into account the ordinal
or interval quality of the scales, and the reliability indices should have been determined
separately for each of the six NBAS clusters used. Cicchetti et al. also suggest that using the
intra-class correlation coefficient (ICC) would have been preferable, as it would have produced
true reliability coefficients that are adjusted for the amount of inter-examiner agreement expected
on the basis of chance alone.

However, Stewart et al. (2004) rebut Cicchetti et al.’s (2004) criticism with the following
argument:

“Consider the likelihood of chance alone being responsible for obtaining absolute
agreement between two raters on just one, of the 28, nine-point behavioral items. That
 35

probability is 1/9 or 11%. Now consider that same situation occurring on all 28 items of
a single administration of the NBAS. Add to this the requirement of perfect agreement
on the 20 four-point reflex responses. The conjoint probability of obtaining this perfect
agreement by chance alone is p < .000000000000000000000000001. The probability of
obtaining at least 90% agreement by chance alone is p < .0000000000000000004. In light
of this, to cast doubt and label as suspicious the 90% + NBAS reliabilities we obtained is
simply not tenable.”

In addition, while Stewart et al. (2004) acknowledge that the intra-class correlation is an
alternative strategy for calculating reliability estimates, it is certainly not a requirement for valid
reliability measurement on the NBAS.

Cicchetti et al. (2004) criticize the Oswego investigators’ use of the PPVT-R to control for
maternal IQ. As has been previously noted, the PPVT-R measures receptive vocabulary, not IQ,
and has been shown to not correlate well with instruments designed to measure IQ (Maxwell and
Wise 1984; Cicchetti et al. 2004). However, Stewart et al. (2004) have shown that, while the
PPVT-R does not directly measure IQ, it does effectively control for the effect of maternal IQ on
outcome measures either alone or when combined with other covariates that evidence colinearity
with IQ, such as SES, the HOME environment, maternal education, and substance use/abuse.

The Oswego investigators have been supplementing their PPVT data with an assessment using
the Kaufman Brief Intelligence Test (KBIT), which assesses both verbal and nonverbal
components of intelligence. When applied to the FTII scores, neither the PPVT nor the KBIT
met the p < .20 criterion on control variable inclusion. Both the PPVT and the KBIT were
related to the McCarthy and WISC-III scores, however. Analysis showed that the variance
predicted by the KBIT had been controlled by the variables already present in the Oswego study.
The investigators concluded that the KBIT failed to control for any additional variance in the
data set; did not predict outcome measures better than the PPVT; and that the PPVT alone, but
especially in combination with other covariates measured in the Oswego study, “effectively and
convincingly controlled for maternal IQ” (Stewart et al. 2004). Given these results, Cicchetti et
al.’s (2004) criticisms regarding the use of the PPVT in this and other PCB epidemiological
studies appear to be invalid.

As noted above, the Oswego studies have also been criticized for using the FTII, which Cicchetti
et al. (2004) argue is an unreliable, poorly validated instrument. However, as noted previously
with respect to the Michigan studies, the less-than-optimal reliability of the FTII tends to bias the
results in favor of the null hypothesis, and makes it less likely to find significant associations.
Thus, the fact that the Oswego study replicated the Michigan FTII results makes the findings all
the more significant and impressive (Stewart et al. 2004).

Cicchetti et al. (2004) criticize the Oswego researchers for using one-tailed significance tests to
assess the FTII results, but fail to mention that two out of three of the results (including the most
important one relating highly chlorinated PCB’s to poor FTII performance at 12 months) retain
their significance when two-tailed statistical tests are used (Stewart et al. 2004). Thus, the
Cicchetti criticisms in this regard are irrelevant.
 36

Schantz et al. (2003) note that, while more than 60 individual PCB congeners were quantitated in
the Oswego cohort, all studies published to date relate outcomes to total PCBs or to PCB
homologue groups with no attempt made thus far to assess the importance of individual
congeners. Schantz et al. (2003) also note that analysis of the placental tissue collected should
be particularly valuable in this regard because the higher concentrations of PCBs and larger
amounts of tissue available for analysis will permit accurate measurement of a large number of
congeners.

However, as Grandjean (2003) explains, in a population with dietary exposure to PCBs from the
same sources, concentrations of different PCB congeners generally show a very high degree of
association with one another. Because of this, total PCBs (mainly based on persistent di-ortho
PCBs) correlates with the estimated weighted concentration of dioxin-equivalent concentrations
of mono-ortho substituted PCBs. Thus, epidemiological studies are unlikely to be able to
separate effects attributable to individual PCBs or congener groups, anyway.

A shortcoming of the Oswego studies is that no maternal serum samples were collected for PCB
analysis. This limits inter-study comparisons, as most other studies used maternal blood PCBs as
an exposure measure (Schantz et al. 2003).

Ongoing studies:

► Faroe Island cohorts Two cohorts that are contributing to our understanding of the
neurodevelopmental effects of concomitant human fetal exposure to methylmercury and PCBs
are currently being studied in the Faroe Islands (Grandjean et al. 1997; Steuerwald et al. 2000).
The Faroe Islands are located northwest of Scotland between the Norwegian Sea and the North
Atlantic, about halfway between Iceland and Norway. The population of the Faroe Islands is
Nordic, numbering approximately 45,000 people largely descended from Viking settlers who
arrived in the 9th century. They have been a self-governing overseas administrative division of
Denmark since 1948 (Central Intelligence Agency 2005).

The cohorts were originally recruited to assess neurodevelopmental outcomes from mercury
exposure. Mercury exposure in this population originates mainly from the lean pilot whale meat
which, according to ancient tradition, is shared in the communities where the whales are killed
(Grandjean et al. 1997). However, after the cohort was formed, the whale blubber, a traditional
food item, was found to be significantly contaminated with PCBs. Because of the different
dietary sources and biological half-lives, the investigators decided to try to separate the effects
attributable to organochlorine compounds from those related to mercury (Grandjean et al. 2001).

1986-1987 Cohort. The first cohort was comprised of 1022 mother-infant pairs recruited from
the three hospitals in the Faroe Islands over 21 months from 1986-1987 (Grandjean et al. 1997).
Because the effects of fetal methylmercury exposure are persistent, the children were examined
in detail later than those in the PCB studies, that is, at seven years of age. The children recruited
in 1986 were evaluated in Spring of 1993 (n=443), and the children recruited in 1997 were
evaluated in Spring of 1994 (n=474). A total of 917 children completed the examinations. The
following PCB congeners were measured: 118, 138, 153, 170, and 180. DDE levels and TSH/T4
levels were also measured.
 37

Measures of exposure—At birth, cord blood and about 10 cm of umbilical cord were collected
from each infant and frozen for subsequent analysis. Mercury concentrations were measured in
cord blood and maternal hair obtained at parturition; in the children’s hair at 12 months of age;
and in the children’s hair at seven years of age. Concentrations of the major PCB congeners 118,
138, 153,170, and 180, as well as DDE, were measured at seven years of age in 436 stored
samples of umbilical cords from the 443 children examined in 1994. The sum of the three PCB
congeners 138, 153, and 180 multiplied by two, i.e., 2(∑PCB 138, 153, 180 ) was used as the
surrogate for the total concentration of PCBs, as the sum of these three had been found to
represent about 50% of the total PCB concentration in milk from the cohort mothers (Grandjean
et al. 1995, cited in Grandjean et al. 2001) and in maternal serum from a subsequent Faroese
cohort (Steuerwald et al. 2000, cited in Grandjean et al. 2001). TSH and T4 were measured in
stored neonatal screening samples of capillary whole blood that had been collected on filter
paper and stored at -20º C.

In the statistical analyses performed by Grandjean et al. (2001), DDE and PCB 153 were also
used as exposure indicators.

To measure umbilical cord PCB concentrations, the umbilical cord samples were distributed
between two laboratories, the National Environmental Research Institute in Denmark, and the
U.S. Centers for Disease Control and Prevention for cord tissue PCB analysis, such that
distributions of cord blood mercury concentrations were similar in the two groups. The
methodology used for sample preparation is described in Grandjean et al. (2001). PCB levels
were measured using capillary gas chromatography with ECD. Quality control was ensured by
analyses of Community Bureau of Reference (BCR) certified reference material (CRM) 350, and
by analysis of bovine umbilical cords spiked with representative PCB congeners at two
concentration levels. The quality of the results was deemed satisfactory, and comparable
between the two laboratories (Grandjean et al. 1997; 2001). To validate the cord tissue PCB
measurements, cord blood from 50 of the cords was later given to the Department of
Environmental Toxicology of the State Agency for Nature and Environment in Schleswig-
Holstein, Germany, for analysis using high-resolution gas chromatography with an autosampler,
a 30-m DB-5 capillary column, and ECD. These analyses were then confirmed on another gas
chromatograph with a 30-5 DB-1701 capillary column. Lipid content of the blood samples was
determined by gravimetry of the condensed extract. The detection limit for PCB congeners was 5
ppb lipid. Only two cord samples had PCB concentrations below the detection limit, and they
were assumed to contain a total PCB concentration of 0.1 ppb (Grandjean et al. 2001).

The wet-weight-based and lipid-adjusted PCB measures were highly correlated, and the
correlation coefficients did not differ between the two laboratories. Also, the whole blood PCB
concentrations also correlated well with the cord tissue results. The sum of the median
concentrations of five PCB congeners (wet) was 1.88 ppb; the lipid adjusted sum of median
values was 1,020 ppb. The sum of the median (wet) concentrations of PCB congeners 138, 153,
and 180 multiplied by two was 1.92 ppb; and the sum of their median lipid concentrations
multiplied by two was 1002 ppb (Grandjean et al. 2001). The wet weight mean PCB
concentration of the 435 umbilical cords was 1.12 ppb (Grandjean et al. 1997).
 38

DDE was detectable in all samples. The median concentration (wet) was 1.29 ppb; the median
lipid value was 713 ppb (Grandjean et al. 2001). Steuerwald et al. (2000) reported that analysis
of milk samples in 1987 by Grandjean et al. (1995) showed total dioxin equivalent exposure
levels similar to the average reported from the Dutch cohorts (Huisman et al. 1995b).

Cord blood mercury concentrations were determined by cold vapor atomic absorption
spectroscopy. Cord blood mercury concentrations averaged 22.9 ppb; maternal hair mercury
averaged 4.27 µg/g; Child hair mercury at 12 months averaged 1.12 µg/g; and child hair mercury
at 7 years averaged 2.99µg/g.

The PCB concentration was significantly associated with the cord blood mercury concentration,
and correlated better for the wet weight (r=.41) than for the lipid adjusted concentration (r=.31).

Instruments and Outcome Measures—The children were extensively evaluated at seven years of
age with the following neurophysiological and neuropsychological tests (Grandjean et al. 1997;
2001):

Neurophysiological tests: Pattern reversal visual evoked potentials (VEP); brain stem auditory
evoked potentials (BAEP), postural sway, and recording of the coefficient of R-R interval
variation on ECG as a measure of autonomic nervous system function.

Neuropsychological tests: Computerized tests from the Neurobehavioral Evaluation System

(NES), including the Finger Tapping, Hand-Eye Coordination, Tactual Performance and
Continuous Performance Tests; Wechsler Intelligence Scale for Children- Revised (WISC-R)
subtests, including Digit Spans, Similarities, and Block Designs; Bender Gestalt Test; California
Verbal Learning Test –Children (CVLT-C); Boston Naming Test; and the Nonverbal Analogue
Profile of Mood States.

Control variables and analysis—Maternal cognitive function was controlled for, measured with
Raven’s Progressive Matrices. Additional covariates included sex and age of the child, medical
risk for neurobehavioral deficit, maternal and paternal education level, paternal employment, day
care, alcohol/smoking (yes or no). The computer-assisted tests were adjusted for child’s level of
computer skills. Adjustment for examiner was also included. The HOME test was not
administered.

Parametric tests were used because most of the neurobehavioral test results and the logarithmic
transformation of the exposure parameters approximated a Gaussian distribution. For some
neuropsychological outcome variables that deviated from this distribution, other transformations
were attempted. Multiple regression analyses were performed first for associations between
mercury and test outcomes. Similar regression models were calculated for the children examined
in 1993 where the PCB concentration was available for inclusion as an independent variable.
Two-sided p values were used throughout (Grandjean et al. 1997)

To further analyze any mediating role of PCBs on mercury effects, the group of children with
PCB exposure data were divided into three subgroups according to PCB exposure (Budtz-
Jørgensen et al. 1999). One outcome variable was selected to reflect each of five different
 39

domains of brain function: motor, attention, visuospatial, language, and memory. Regression
equations were then fitted to the data for each of the three PCB subgroups (Schantz et al. 2003).

Later, Grandjean et al. (2001) performed further analyses to separate the effects attributable to
PCBs from those related to methylmercury. Statistical analysis was carried out using the same
approach as previously (Grandjean et al. 1997), but with PCB included as an independent
exposure variable after logarithmic transformation. Parallel calculations were made using wet-
weight-based and lipid-adjusted PCB concentrations. The possible effect modification by
mercury exposure was investigated in regression analyses where a PCB effect was suggested.
To further exposure possible interactions, the children were divided into three groups according
to the cord-blood mercury concentration, and PCB regressions were performed similar to the
method used by Budtz-Jørgensen et al. (1999) using mercury regressions within tertile groups of
PCB exposure.

The two thyroid variables, TSH and T4, were considered as possible effect parameters affected
by PCB. A TSH above 2 mIU/l and a T4 below the fifth percentile were included as independent
variables in the regression analyses to control for abnormal brain development secondary to
perinatal thyroid function abnormalities (Grandjean et al. 2001).

Results—Among the neurophysiological tests, BAEP showed mercury-associated delays of peaks

III and V, but inter-peak latencies were not related to mercury exposure. No association was
found with PCB exposure.

Among the neuropsychological tests, mercury cord blood levels were found to be significantly
associated (p≤.05) with poorer performance on the NES2 Finger Tapping (motor speed), NES2
CPT reaction time (attention), the WISC-R digit spans (attention) and block designs (visuospatial
function), Bender Visual Motor Gestalt tests (visuospatial performance), Boston Naming Test
(Language), and the CVLT-C (verbal learning and memory), after controlling for covariates
(Grandjean et al. 1997; 2003).

Four outcomes variables showed an association with the logarithmic transformation of the wet
weight PCB concentration (p<.1) before adjustment for mercury: the CPT reaction time, Boston
Naming Test with and without cues, and the CVLT-C long-term reproduction test. The
regression analyses were repeated with mercury alone and with both of the two exposure
parameters included as independent variables to determine the role of PCBs in mediating the
effects. After adjusting for PCB exposure, only the association with the CPT reaction time
remained significant, suggesting that PCBs may have played some role in mediating the mercury
effects on the other tests (Grandjean et al. 1997; Schantz et al. 2003). An independent effect of
PCB exposure was suggested for the Boston Naming Test, as PCBs were shown to increase the
magnitude of the mercury regression coefficient (Grandjean et al. 1997).

Budtz-Jørgensen et al. (1999) conducted additional analyses to explore the issue of whether
concomitant prenatal exposure to PCBs had affected the MeHg-related neurobehavioral effects
assessed by Grandjean et al. (1997). On the basis of the 436 cord PCB analyses carried out by
Grandjean et al. (1997), children with complete data were divided into tertile PCB exposure
groups. Since the main source of increased PCB exposure is whale blubber, those mothers who
 40

were known not to eat blubber (approximately half of the cohort) served as a control group with
only a background exposure to PCBs. One outcome variable was selected to reflect each of five
different domains of brain function, motor, attention, visuospatial function, language, and
memory. A regression equation with a uniform series of confounders was then fitted to the data
for each of the three subgroups. It was shown that PCBs did not change the regression
coefficients for mercury (Budtz-Jørgensen et al. 1999).

Because the PCB exposure variable in this analysis was reduced to tertile classes only, the
investigators then explored the possible effect modification by PCBs with regression analyses
which, in addition to the confounders, also included the mercury and PCB exposure variables, as
well as a product term between the two exposure biomarkers. The p value for no effect
modification was between .21 and .75, suggesting that no interaction occurred. Just as reported
in Grandjean et al. (1997), an independent effect of PCB exposure was suggested for the Boston
Naming Test, although it affected the mercury regression coefficient only slightly (Budtz-
Jørgensen et al. 1999).

All of the calculations were based on the wet-weight PCB concentrations, which showed a better
correlation (r=.41 after logarithmic transformation) with cord blood mercury concentration than
did the lipid based PCB concentration, so the potential confounding and interaction effect were
thereby maximized. Thus, the expanded analyses do not suggest that the mercury effect can be
explained by concomitant PCB exposure or that PCB exposure potentiated the mercury-
associated effects (Budtz-Jørgensen et al. 1999).

This contrasts with the Oswego study findings, in which analysis for additive/interactive effects
of MeHg showed that PCBs potentiated MeHg effects on McCarthy Scale performance, which
measures cognitive abilities and gross and fine motor abilities (Stewart et al. 2003).

However, the association found between PCBs and performance on the Boston Naming Test, a
measure of language development, is consistent with findings from the Dutch cohort (Patandin et
al. 1999), which showed a negative impact on language development, and the Michigan cohort
(Jacobson et al. 1996), in which both word and reading comprehension were negatively impacted
(Schantz et al. 2003).

Grandjean et al. (2001) later performed a more detailed analysis of the neurotoxic interactions of
concomitant exposure to MeHg and PCBs. The statistical analysis was carried out as previously
done for mercury (Grandjean et al. 1997), but this time with PCB as the independent exposure
variable after logarithmic transformation. Parallel calculations were made using wet weight
based and lipid adjusted PCB concentrations. DDE and PCB 153 were also used as exposure
indicators for comparison purposes (Grandjean et al. 2001).

Because of the correlation between PCB and mercury exposure biomarkers, the possible effect
modification by mercury exposure was investigated in regression analyses where a PCB effect
was suggested. In addition to the confounders, these analyses also included both the mercury
and PCB exposure variables. Just as in the study by Budtz-Jørgensen et al. (1999), the children
were also divided into tertile groups, but in this case based on the cord-blood mercury
concentrations. For the tests showing a possible PCB effect, the confounder-adjusted PCB
 41

regression coefficient was calculated within the three tertile groups. As mentioned above,
abnormal TSH and T4 levels were included as independent variables in the regression analyses
as possible effect parameters affected by PCB (Grandjean et al. 2001).

This study showed a negative association between cord blood PCB concentration and the CPT
reaction time, and the two Boston Naming Test measures. A possible PCB associated deficit
was seen on the CVLT-C, as well. However, adjustment for mercury reduced the PCB
association substantially for the four tests associated with PCB. None of the PCB regression
coefficients for the tertile groups were statistically significant, but the regression coefficients for
the group with the highest mercury exposure were slightly greater than those calculated for the
total group. In addition, this study was unable to confirm any association between PCBs and
alteration of neonatal thyroid function (Grandjean et al. 2001).

The neurophysiological results did not show any clear patterns of PCB-mediated effects. Pure-
tone audiometry at eight frequencies showed that one threshold at a low frequency was
associated with the wet-weight PCB concentration, but only on the left side. No effects on
evoked potential were found in the Faroese children.

The investigators suggest that PCBs could possibly augment mercury-related neurobehavioral
deficits at increased levels of mercury exposure, but for the Faroese population, it appears that
methylmercury neurotoxicity may be a greater hazard than that associated with PCBs. This
study suggests that methylmercury exposure should be taken into account as a confounder or
effect modifier in studies investigating neurodevelopmental effects of PCB exposure (Grandjean
et al. 2001).

1994-1995 Cohort. This cohort was formed with European Union funding to expand the Dutch
study into a transnational, multicenter cooperative study, which included the German cohort in
Düsseldorf and a Danish cohort in the Faroe Islands (Steuerwald et al. 2000). The cohort was
recruited during a 12 month period in 1994 and 1995, and consists of 182 singleton term births at
the National Hospital in Tórshavn, Faroe Islands. The mothers resided in regions outside of the
capital area of Tórshavn.

Measures of exposure-Maternal serum was obtained at 34 weeks of gestation. Umbilical cord

blood was collected at parturition, as well as a maternal hair sample. Transition milk samples
were collected on day four or five after parturition. Two milliliters of all maternal serum samples
and 10% of the cord serum samples were analyzed at the National Center for Environmental
Health at the Centers for Disease Control and Prevention in Atlanta. Eighteen parent pesticides
or their metabolites and 28 persistent PCB congeners were quantified by a two-stage solid-phase
extraction method, followed by gas chromatography analysis with ECD. The results were
adjusted for total serum lipid content and reported as ng/g lipid. The detection limit varied from
0.08 ppb to 0.66 ppb. For maternal serum with an average lipid content of about 9 g/L, these
limits corresponded to 9 ng/g to 70 ng/g lipid (Steuerwald et al. 2000).

As an additional measure of perinatal exposure, 5 ml of maternal milk was analyzed at the

Institute of Environmental Toxicology, Kiel, Germany, for a similar array of organochlorine
compounds. After solid-liquid-phase extraction, analyses were performed by gas
 42

chromatography with ECD. At a 5% fat content, the detection limits were 2 and 5 ng/g lipid for
pesticides and PCBs, respectively (Steuerwald et al. 2000).

Mercury exposure was estimated from maternal hair, cord whole blood, and cord serum samples
by analysis with flow-injection cold-vapor atomic absorption spectrometry after digestion of the
sample in a microwave oven. Levels were similar to those found in the previous cohort. Levels
of selenium and fatty acids were also measured in the cord blood (Steuerwald et al. 2000). TSH,
free T3 and free T4, as well as thyroxin and resin T3 uptake were determined in maternal and
cord serum.

The ∑PCB concentration was calculated as two times the sum of the most prevalent congeners,
138, 153, and 180. Lipid based concentrations in serum and milk correlated well but were
generally higher in milk. The lipid based ∑PCB concentrations in the 19 cord serum samples
were similar to those in maternal serum (r=.97). However, volume based serum concentrations
correlated less well and reflected the 3- to 4-fold difference in lipid content (mean values were
2.5 g/L and 9.0 g/L in cord and maternal serum, respectively). Other organochlorine compounds
in the same sample correlated well with ∑PCB and DDE (Steuerwald et al. 2000).

The PCB exposure levels of the Faroese were almost three times higher than the Dutch and
German cohorts: The mean ∑PCBmilk in Faroese maternal mild was 1520 ng/g milk fat
(Steuerwald et al. 2000), compared with 428.5 ng/g lipid in the Dutch cohort (Koopman-
Esseboom et al. 1996), and 426.5 ng/g lipid in the German cohort (Winneke et al. 1998).

The frequency of whale meat dinners was the best predictor for the mercury concentrations, and
the frequency of blubber dinners during pregnancy was the best predictor of the organochlorine
concentrations. The maternal serum ∑PCB concentration correlated significantly with mercury
concentrations in maternal hair and cord blood (r=.53 and .43, respectively).

Instruments and Outcome Measures—As in the Dutch study (Huisman et al. 1995b), the infants
were assessed by for neurological optimality with the Prechtl neurological exam at
approximately two weeks of age, adjusted for gestational age. The infants were then given a
neurological optimality score (NOS) based on their functional abilities, reflexes and responses,
and the stability of behavioral status during the examination. The optimal score was 60. The
infants were assessed by the same examiner, who was blind to the children’s exposure
information (Steuerwald et al. 2000).

Control variables and analysis—Covariates included maternal age, parity, smoking and alcohol
use during pregnancy (yes or no), sex of child, age at examination, and obstetric optimality
scores.

Measurements that deviated substantially from a Gaussian distribution, especially the exposure
biomarkers, were normalized by logarithmic transformation. Parametric methods were used
whenever applicable. Regression analyses were carried out with the NOS as the dependent
variable and with potential predictors selected a priori. Individual exposure parameters were
then entered as continuous variables, with covariates that were significant at p<.1 for confounder
adjustment. Two-tailed p values were used throughout.
 43

Results—The infants had an average NOS of 49.8; 43.8% of them had an NOS less than 50. As
expected, mercury exposure was negatively associated with the NOS, such that a 10-fold
increase in mercury was associated with an NOS decrease of two.

PCB concentrations and DDE showed weak, positive associations with the NOS, and adjustment
for covariates and mercury exposure caused only minor changes. Adjustment for ∑PCB also did
not have a discernible effect on the mercury regression coefficient.

The Dutch researchers (Huisman et al. 1995b) reported that a decreased neonatal NOS was
associated with increased dioxin exposure, and PCB congeners showed a significant association
with decreased muscle tone. Although the same outcome measure was used in the Faroe Island
cohort, the findings were different. However, the investigators caution that the findings must be
interpreted in the context of the exposure settings.

Steuerwald et al (2000) suggest that the differences in the NOS results could be due either to
confounding by mercury in the Dutch studies, or possibly to confounding by dioxins in the
Faroese studies. Even though Dutch seafood consumption is lower than that of the Faroese, even
small amounts of mercury exposure may result in a decreased neonatal NOS, so an undetected
mercury effect could be responsible for the Dutch NOS results. On the other hand, even though
the scattered correlation between mercury and PCB exposure parameters suggests that
confounding between mercury and PCBs may be limited in the Faroese studies, it is possible that
a dioxin effect on the NOS could explain the mercury effect observed. If that were true,
however, the dioxin exposure level would have to be more closely associated with the mercury
concentration in cord blood than with the PCB concentration, which is unlikely. Schantz et al.
(2003) note that a recent National Research Council committee reviewed the Faroese data set and
concluded that the effects of co-exposure to methylmercury and PCBs appeared to be
independent (NRC 2000).

In addition, the Faroese studies failed to show an association between PCB exposure and thyroid
function. Thyroid function did not correlate with contaminant concentrations, except that the
resin-T3 uptake in cord serum seemed to decrease at increasing ∑PCB concentrations (r=-.21;
p=.01). The TSH concentration in cord blood and maternal serum showed a weak negative
association with ∑PCB (r=-.04, p=.66; and r=-.13; p=.09). Measured thyroid hormone levels
were all within the normal range (Steuerwald et al. 2000).

The validity of the NOS assessment itself appears to be good, as the tests were administered by
the same examiner, a pediatrician who had been specially trained and who was blind to the
exposure parameters. In addition, the test is highly standardized and has been shown to have a
high degree of predictive validity (Hadders-Algra et al. 1985, cited in Steuerwald et al. 2000).

In sum, it is unclear why no PCB neurodevelopmental effects were found on the NOS
assessment in this population of infants three times more highly exposed to PCBs in utero than
the Dutch infants, in whom negative effects were found using the same, reliable measurement
instrument (Schantz et al 2003).
 44

Grandjean et al. (2005) are carrying out an extended follow-up of the 1994-1995 cohort with the
primary objective of examining whether biomarkers of in utero exposure to endocrine disruptors
are associated with development and neurobehavioral function an children at prepuberty ages,
and whether effects of organochlorine compounds, such as PCBs, can be differentiated from
those caused by methylmercury exposure.

In the spring of 2004, the investigators began assessing the children at 10 years of age. Fifty of
the 164 children assessed at age seven have thus far been assessed. Advanced exposure
measures are also being elaborated, and the investigators are currently using stored maternal
pregnancy serum to determine the integrated estrogen activity (after removal of endogenous
hormones), as well as the dioxin-like effect on the Ah receptor. This work is scheduled for
completion in the fall of 2005 (Grandjean et al. 2005).

Criticisms—Cicchetti et al. (2004) criticize Grandjean et al. (1997; 2001) for not administering
the HOME test to control for home environmental effects on the neuropsychological
development of the 1986-1987 cohort. However, Grandjean et al. (1997) provided a reasonable
justification for their decision: “For the socially highly homogeneous Faroese society where
social status scales from other countries are not appropriate, we used vocational or professional
education of each parent and the employment status of the father as supplementary indicators of
social background.” Indeed, studies have shown a linear relationship between HOME scores and
SES (Evans and Kantrowitz 2001). Therefore, little variation in HOME scores would be
expected in this particular population, and employment status and parental educational level
would seem adequate to control for potential confounding by home environment. Furthermore,
Grandjean et al. (2001) did control for maternal IQ with the Raven’s Matrices.

Cicchetti et al. (2004) criticize Steuerwald et al. (2000) for not administering a parental IQ test or
the HOME in their study of young infants in the 1994-1995 cohort. However, the infants were
assessed at two weeks of age only, for outcome measures of postural tone, reflexes, and
responses. Since controlling for maternal IQ is most important in the context of assessing
cognitive abilities, it seems an unlikely confounder in this study. Similarly, HOME scores
reflect influences of the home environment over time on a child’s neurodevelopment; at two
weeks of age, this environment would be unlikely to have exerted an influence significant
enough to affect NOS scores. Therefore, Cicchetti et al.’s (2004) criticisms of the Faroese cohort
studies seem largely irrelevant.

►The Inuit Cohort Another population exposed to unusually high quantities of heavy metals
and organochlorine compounds through their diet of fish and sea mammal fat, primarily ringed
seal and beluga whale, is the Inuit people of Nunavik. The Nunavik region is located north of
the 55º parallel in Arctic Québec, Canada, where 7,660 Inuit reside in 14 villages scattered along
2,000 kilometers of the Hudson Bay and Ungava Bay shorelines (Ayotte et al. 2003). In 1988,
researchers found very high mean levels of PCBs and DDE in the milk of 24 Inuit women from
Hudson Bay—levels five times greater than those of 48 Caucasian women living in southern
Québec (Dewailly et al. 1989; 1993).

In 1995, the Inuit Cohort Study was initiated. The cohort consists of approximately 300 Inuit
mother-infant pairs, recruited after their first prenatal medical exam. The objectives of the study
 45

are to identify determinants of PCB and MeHg exposure in Inuit women (Muckle et al. 2001a);
to measure the magnitude of prenatal exposure to these contaminants (Muckle et al. 2001b); to
compare different biomarkers of prenatal PCB exposure and models to predict PCB plasma lipid
concentration in infants at six months of age (Ayotte et al. 2003); and finally, to investigate
adverse neurodevelopmental effects induced by in utero PCB exposure in Inuit infants. The
neurodevelopmental study has been initiated, and was designed to extend the findings reported in
earlier PCB-exposed cohorts by studying more highly PCB-exposed infants and by using new
infant assessment procedures that have been liked to specific brain regions and neural pathways.
Statistical analyses of the neurodevelopmental assessment data are currently being performed, so
results are not yet available (Muckle, G. Personal communication, cited in Schantz et al. 2003).

Measures of exposure—Maternal blood was collected at delivery or within several weeks

postpartum. Cord blood samples were collected after the umbilical cord was severed. Infant
blood samples were drawn at approximately six months of age. Blood samples were drawn again
from the children at around 5.5 years of age. Maternal breast milk samples were collected from
breast feeding mothers approximately one month after parturition. Two maternal hair samples
were collected, one at the prenatal interview and one at the one-month postal interview.

The blood was centrifuged and the plasma stored at -80ºC until analysis; the breast milk was
manually expressed and stored at -20ºC until analysis. Fourteen PCB congeners (28, 52, 99, 101,
105, 118, 128, 138, 153, 156, 170, 180, 183, 187) and 11 chlorinated pesticides or their
metabolites were measured in the plasma and milk samples by the laboratory of the Centre de
Toxicologie du Québec using a high resolution gas chromatograph equipped with dual capillary
columns and dual ECDs. The methods are described in detail in Muckle et al. (2001b). The
detection limit for plasma congeners was approximately .02 ppb for each compound, with
percent recoveries ranging from 89 to 100%. The detection limit for breast milk PCBs varied
from .08 to .16 ppb. Two certified milk reference materials (CRM 188 and 450; Community
Bureau of Reference, Brussels, Belgium) were used to assess precision and accuracy of the
results (Muckle et al. 2001b). The blood samples drawn from the children at around 5 ½ years of
age were analyzed for Hg and PCB 153 (Saint-Amour et al. 2004).

The PCB concentrations in plasma and milk were highly correlated, indicating that the
measurements are very reliable. For PCB congener 153, the correlations were cord plasma-
maternal plasma, r=.93; cord plasma-breast milk, r=.91; maternal plasma-breast milk, r=.95.
These very high correlations obtained using the dual capillary column method in the Inuit Cohort
study contrast with the more modest intercorrelations obtained in the Michigan study (cord
blood-maternal blood, r=.42; cord blood-maternal milk, r=.16; maternal blood-maternal milk,
r=.35) (Jacobson et al. 1984b). Because these measurement methods are more reliable than
those used in previous studies, any deficits associated with prenatal PCB and DCL exposures
will more likely be detected (Muckle et al. 2001b).

PCB congener 153 was highly intercorrelated with all of the moderate- to heavily-chlorinated
congeners, and with most pesticides. This suggests that congener 153 can be considered a
marker for exposure to most organochlorines in the Arctic region. The data show that the mixture
of OC contaminants persisting in the Arctic today is quite homogeneous, so that it is unlikely
 46

that the neurobehavioral effects associated with individual congeners and pesticides can be
evaluated in this population (Muckle et al. 2001b).

Because organochlorines distribute mainly in body fat, concentrations in plasma and milk
samples were reported in micrograms per kilogram lipids. The concentration of total plasma
lipids was estimated according to the formula developed by Phillips et al. (1989, cited in Ayotte
et al. 2003). An aliquot of the milk fat extract was weighed to determine the concentration of
lipids in milk samples (Ayotte et al. 2003).

To compare their data with those of other studies, the Inuit cohort investigators recalculated the
Nunavik OC concentrations according to the method used in the comparison study. For
example, to compare their data with the Faroe Islands cohorts, where total PCB exposure was
calculated as two times the sum of congeners 138, 153, and 180, they performed a similar
calculation using the Nunavik data. Cord and maternal plasma PCB concentrations in Nunavik
were found to be similar to those found in the Dutch cohort (Koopman-Esseboom et al. 1994a)
and about twice as high as those in the New Bedford cohort (Korrick et al. 2000), described
below. The Nunavik breast milk PCB concentrations are somewhat lower than those of the
Dutch cohort. Concentrations of OCs in Nunavik cord plasma and breast milk are two to three
times lower than those found in the groups of marine mammal consumers from the Faroe Islands
cohorts (Steuerwald et al. 2000). This suggests that marine mammal consumption may be less
frequent in Nunavik women than in the Faroese (Muckle et al. 2001b).

Mercury concentrations were measured in cord and maternal blood, and in the postnatal maternal
hair sample, using cold vapor atomic absorption spectrometry. The hair sample was cut into three
segments of 3 cm in length, each segment corresponding to a trimester of pregnancy. Accuracy
and precision data were obtained using certified reference material through Health Canada’s hair
mercury inter-laboratory comparison program. The detection limit for blood mercury analysis
was 1.0 nmol/L, and for hair mercury it was 1.0 nmol/g. The geometric mean cord blood
mercury concentration was 18.5 ppb, similar to that of the Faroese cohorts (22.9 and 20.4 ppb,
respectively) (Muckle et al. 2001b).

The intercorrelations between PCB congener 153 and Hg were modest: Cord PCB-cord Hg,
r=.24; cord PCB average hair Hg, r=.34; maternal PCB-average hair Hg, r=.17 (Muckle et al.
2001b).

Lead concentrations were measured in cord blood using graphite furnace atomic absorption with
Zeeman background correction. The detection limit was 50 nmol/L. Four reference specimens
were used to calibrate the analytic method for lead. The average lead concentration measured in
the Nunavik cohort was twice as high (32.6 ppb) as that of the general population of southern
Québec. However, since lead concentration was found to be unrelated to OC or Hg exposure,
lead is not likely to be a confounder that will need to be controlled in analyses of
neurodevelopmental effects of prenatal OC and Hg exposure (Muckle et al. 2001).

Selenium concentrations were measured in cord and maternal blood, and n3-PUFA was
measured in cord plasma. These data will be used to test hypotheses regarding neuroprotective
 47

effects of Se and PUFAs, as well as to control for confounding in the neurodevelopmental data
analyses (Muckle et al. 2001b).

Instruments and Outcome Measures—The infants were assessed at birth, 6 ½, 11 months, and
around 5 ½ years of age. Thyroid hormones, neurological status, physical maturity, and
anthropometric measures were assessed in the newborns. Neurobehavioral and cognitive
development were assessed at 6.5 and 11 months of age, as well as physical growth, neuromotor
and neurological functions, and overall health (Schantz et al. 2003). A Visual Evoked Potentials
study was performed on the children at the mean age of 5.44 years (Saint-Amour et al. 2004).

Tests included the Bayley Scales of Infant Development and the FTII, as well as several new
tests, such as the A-not-B object permanency test, the Haith Paradigm Test, and Teller Visual
Acuity Cards (Schantz et al. 2003).

Control variables and analysis—Five groups of potential confounding variables are being
considered in the statistical analysis of the neurodevelopmental assessment data. These include
variables relating to exposure to other toxic substances, dietary fat, perinatal medical
complications, sociodemographic status, and psychosocial risk (Schantz et al. 2003).

For the Visual Evoked Potentials test performed at around 5 ½ years of age, pattern-reversal
VEPs were recorded from occipital (Oz) derivation. The relationships between Hg and PCB 153,
and VEP components were assessed by multivariate regression analyses taking into account fish
consumption nutrients (Se and n-3 PUFAs) as covariables (Saint-Amour et al. 2004).

Results—Results of the statistical analysis of the neurodevelopmental assessments have not yet
been published.

For the VEP study, it was found that, after adjusting for n-3 PUFAs and Se, no significant
association was found between cord blood concentrations of PCB 153 and VEP results.
However, PCB 153 concentrations in blood collected at testing time was related to a delay of the
P100 and N150 latencies and a decrease of the N75/P100 amplitude. Cord blood Hg
concentrations were associated with a delay of the P100 latency. N-3 PUFAs were negatively
related to the latency of many components (P100 and N150 at 95%; N75 and N150 at 30%);
however Se was positively related to the latency of N75 and P100 (95% and 30%).

In sum, while cord blood Hg, serum PCBs at time of testing, and a high intake of Se where
shown to have an adverse impact on VEPs, n-3 PUFAs appear to be beneficial for sensory
processing either when absorbed during the pregnancy or childhood (Saint-Amour et al. 2004).
No mention was made of the investigators’ possible explanations regarding why an association
was found between PCB 153 concentration at time of testing and VEPs, whereas none was
shown between VEPs and PCB 153 concentrations in cord blood.

These results differ from the VEP study carried out on the Yu-Chen children at age > 6 years, in
which no significant differences were found in P-VEP or SSEP sensory pathway performance
between exposed children and controls (Chen et al. 1994).
 48

Criticisms—No criticism of exposure measures or statistical methodologies of the studies

published to-date were found in the literature.

►The New Bedford Cohort New Bedford harbor in southeastern Massachusetts is one of the
most highly PCB contaminated sites in the United States, a result of heavy use of PCB oil as an
insulator/dielectric fluid in the area’s two capacitor manufacturing facilities. Oily PCB waste
was dumped directly into the harbor, and indirectly via the sewerage system, from around 1940
until PCBs were banned in 1977. In 1982, New Bedford was designated an EPA Superfund site,
the largest marine Superfund site for PCB contamination. Over 100,000 people live within 3
miles of the site. Bioaccumulation of PCBs within the marine food chain has resulted in closing
the area to lobstering and fishing, and recreational activities and harbor development have been
limited by the widespread PCB problem (U.S. EPA 2004a). Surveys conducted of the area’s
adult population have suggested elevated PCB exposures in the subset of the New Bedford
population with heavy occupational or dietary (contaminated fish) exposures (Miller et al. 1991,
cited in Korrick et al. 2000).

The New Bedford cohort study was initiated in 1993 with three objectives in mind: 1) to
document the analytic sensitivity, validity, and reproducibility of congener-specific PCB and
organochlorine pesticide analyses in cord serum; 2) to characterize cord serum levels of PCBs,
DDE, and HCB; and 3) to assess the neurodevelopmental toxicities of prenatal exposure to these
compounds in infants born to mothers residing in the towns bordering the New Bedford Harbor
contaminated site (Korrick et al. 2000). The results of the serum analyses have been published
(Korrick et al. 2000), but the neurodevelopmental assessment results are not yet available.

The cohort consists of 788 infants born between March 1993 and December 1998 whose mothers
were recruited at parturition at one of the main hospitals serving the greater New Bedford area.
Participation was limited to mothers who had resided in the area for the duration of their
pregnancy (average residency was 20 years), had spontaneous vaginal deliveries, and were fluent
English speakers. Eligible infants were generally healthy. The study infants were racially and
ethnically diverse Korrick et al. 2000).

Measures of exposure—For the characterization and detection optimization studies on serum

concentrations of PCB congeners, DDE and HCB, only cord blood results were reported
(Korrick et al. 2000). However, in the ongoing neurodevelopmental studies, exposure measures
will include cord serum and/or breast milk levels of PCB, DDE, and Pb; preschool blood Pb
levels; and peripartum maternal hair mercury levels (Korrick Ongoing). Cord blood samples
were obtained from 762 (97%) of the infants, of whom 11 were excluded because of laboratory
technical problems with the samples, leaving a total of 751 infants for the analyses. The cord
blood samples were centrifuged, and the serum was stored at -20º until extraction. All sample
analyses were performed by the Harvard School of Public Health Organic Chemistry Laboratory
(Korrick et al. 2000).

Cord serum samples were analyzed for 51 individual PCB congeners and two chlorinated
pesticides (p,p’-DDE and HCB). The non-ortho-substituted congeners were not included because
their concentrations were expected to be orders of magnitude lower than other congeners, and
therefore undetectable in cord serum. However, mono-ortho-substituted congeners were
 49

measured, as they contribute significantly to the total dioxin TEQ (Van den Berg et al. 1998,
cited in Korrick et al. 2000). PCB concentrations were reported as individual congeners and as
the sum of all congeners assayed (∑PCB). Three pairs of congeners (77/110, 157/201, 196/203)
co-eluted and were reported as co-eluting. Lipid content of the serum samples was not measured
because the sample volume was too small (Korrick et al. 2000).

The PCBs, DDE and HCB were extracted using procedures developed by the Centers for Disease
Control and Prevention with modifications to conform to ultra-trace-level analyses, discussed in
detail in Korrick et al. 2000). The extracts were analyzed using gas chromatography with ECD
using an HP 5890 Series II GC with temperature and pressure programming capabilities and a
split/splitless injector, and a capillary column. Results suspicious for contamination were
checked by confirmatory analyses using a capillary column of different polarity, and the lower
value was reported if the results differed. The mean ±SD percentage recovery for the standard
reference material (SRM 1589, NIST, Aroclor 1260 in Human Serum) was 100% ± 0.8%.
Rigorous standards of quality assurance and control were maintained throughout the analyses,
and are described in detail in Korrick et al. (2000). The relative percent difference (RPD)
between the Harvard School of Public Health laboratory results and the certified value for SRMs
was <5% in most comparisons.

Method detection limits for the cord serum samples were determined on the basis of three times
the standard deviation obtained from analysis of eight aliquots of pooled cord serum spiked with
.07 ng of each target analyte. They were also estimated using three times the standard deviation
of repeat measures of each target analyte in the procedural blank specimens. The two methods
gave comparable results for most analytes (Korrick et al. 2000). The MDL for targeted individual
PCB congeners ranged from 0.002 to 0.036 ng/g serum (ppb) with most MDLs <.01 ng/g serum.
After subtraction of procedural blank values, p,p’-DDE was ≥MDL in 96% of samples, HCB
was ≥MDL in 66% of samples, and 13 of the PCB congeners were measured at values ≥MDL in
at least 50% of the samples (Korrick et al. 2000).

Inter-laboratory comparison was carried out by splitting serum reference pools and duplicate
cord blood samples between the Harvard School of Public Health laboratory and that of the
National Center for Environmental Health. They were analyzed before study sample analysis
was begun and then interspersed in the study analytic batches. In addition, the Harvard School
of Public Health laboratory participated in an international inter-laboratory comparison of
plasma PCB and pesticide analyses organized by the Institute for Quality Management in
Medicine, University of Erlangen-Nuremberg, Germany (Korrick et al. 2000). The RPD between
the Harvard and NCEH laboratories were generally within 20%. The RPDs between the Harvard
laboratory results and the reference values for two pools of plasma samples in the 1999
international inter-laboratory comparison were generally within 15%, a value well within the
tolerance range for certification (Korrick et al. 2000).

The analyses showed that the mean ∑PCBcord (118, 138, 153, 180) was 0.25 ppb, and the mean
concentration of the ∑PCBcord (Total) of all 51 congeners measured was 0.54 ppb. The median
∑PCBcord (Total) was 0.38 ppb (Korrick et al. 2000). There were moderate to strong correlations
(Spearman r=.56-.88) between ∑PCB and the 13 most prevalent PCB congeners (74, 99, 118,
 50

138, 153, 167, 177, 180, 183, 187, 194, 196/203). Correlations among commonly studied
congeners (118, 138, 153) and ∑PCB ranged from .70 to .88 (Korrick et al. 2000).

Comparatively, the New Bedford mean sum of the cord serum concentrations of congeners 118,
138, 153, and 180 (0.25 ppb) was about one-half those of the Dutch and German cohorts: the
Dutch cohort ∑PCBcord (118, 138, 153, 180) averaged 0.5 ppb (Koopman-Esseboom et al. 1996); and
the German cohort ∑PCBcord, (138, 153, 180) averaged 0.55 ppb (Winneke et al. 1998). The New
Bedford median and mean serum concentrations were approximately 30% lower than those of
the Oswego cohort: the New Bedford median cord serum concentration of all congeners was
0.38 ppb, compared with 0.52 ppb in the Oswego cohort (Darvill et al 2000). The difference
between the mean total PCBs of the two cohorts was similar: for fish eaters in the Oswego
study, the ∑PCBcord was 0.8 ppb (Stewart et al. 1999), compared with 0.54 ppb in the New
Bedford cohort.

The slightly lower cord blood serum concentrations in the New Bedford cohort may be due to the
U.S. government and Massachusetts Department of Public Health’s efforts to warn the local
population of the dangers of PCBs, thus reducing exposures. In 1982, the U.S. Coast Guard
erected signs warning the public of the presence of PCBs in the harbor; these signs have been
maintained, and newer signs erected, as needed. In 1985, 2,000 feet of chain-link fence at two
recreational facilities were erected to keep people out of contaminated areas. The Massachusetts
Department of Public Health closed the harbor to lobstering and fishing in 1979, thereby
minimizing dietary exposures of the local populace (U.S. Environmental Protection Agency
2004).

In Oswego, by contrast, local consumption of PCB contaminated fish has continued despite state
advisories recommending restrictions on consumption of Lake Ontario sport fish. As reported in
Lonky et al. (1996), pregnant women have continued to consume large amounts of the fish, after
recommendations had been issued to completely eliminate Lake Ontario sport fish from their
diets.

Instruments and Outcome Measures—According to Korrick (Ongoing), detailed developmental

assessments were performed on the children during their first six months of life. Because the
results have not yet been published, no detailed information about the assessments is available.
Schantz et al. (2003) report that the New Bedford study includes infant cognitive testing with the
FTII. A follow-up study is currently underway to evaluate these children as they achieve school
age to assess whether low-level in utero PCB, DDE, MeHg and Pb exposures are associated with
long term toxicity to memory and other necessary learning skills; poorer scores on achievement
tests; and chronic behavioral disturbances, in particular those associated with attention. A
secondary hypothesis will be to assess whether the proximity to the PCB contaminated site is
associated with adverse developmental effects independent of individual exposure measures.

As part of this research, assessments of memory, learning, language skills, general cognition, and
attention will be performed, as well as standardized achievement testing. Teacher and parental
behavioral assessments will be obtained for 500 six- to eight-year olds (Korrick Ongoing).
 51

Control variables and analysis—The descriptive statistics for the cord serum PCBs, p,p’-DDE,
and HCB analyses were generated with SAS software, and EXCEL was used for certain
laboratory summary measures.

Results—No results have been published yet from the neurodevelopmental studies on the New
Bedford cohort in infancy and at school age. Because the Oswego and New Bedford cohorts
have somewhat similar exposure levels, it will be important to compare the New Bedford results
with those of the Oswego cohort (Schantz et al. 2003). The Oswego study found impairments on
the FTII assessment at six and 12 months of age (Darvill et al. 2000). The results of the Oswego
McCarthy Scale assessments at 36 and 54 months of age will be published within the next
several months (Lonky, E. Personal communication). As mentioned above, the New Bedford
infants were also assessed with the FTII, and are being extensively assessed at school age
(Korrick Ongoing).

Because of its large sample size and extensive congener-specific PCB analyses, the New Bedford
cohort will constitute an important resource for determining whether exposure to specific
congeners or congener classes are associated with adverse neurodevelopmental effects (Schantz
et al. 2003).

Criticisms—No criticisms were found in the literature regarding the New Bedford study results
published to-date.

►The Collaborative Perinatal Project and Child Health and Development Study Cohorts

Two large-scale studies of child development were conducted during the 1960s that involved
thousands of children who were followed longitudinally from birth to school age. Several
investigators have initiated studies designed to take advantage of this archived data, including
serum samples collected during a time period when PCB exposure was much higher than it is
today. Modern congener-specific analytic techniques are being used to analyze the samples, and
the new exposure measures are being used to analyze the associations between
neurodevelopmental outcomes and prenatal PCB exposure (Schantz et al. 2003).

The Collaborative Perinatal Project (CPP). The CPP followed the growth and neurological
development of approximately 55,000 children born in 1959 through 1966. Approximately
42,000 pregnant women were recruited in to the CPP from 12 hospitals in the United States
(Baltimore, Maryland; Boston, Massachusetts; Buffalo, New York; Memphis, Tennessee;
Minneapolis, Minnesota; New Orleans, Louisiana; New York, New York (two hospitals;
Philadelphia, Pennsylvania; Portland, Oregon; Providence, Rhode Island; and Richmond,
Virginia) (Daniels et al. 2003).The group of investigators assessing the association between in
utero PCB exposure and neurodevelopmental outcomes selected subsets of children from the
database for their research.

Exposure Measures— Maternal blood samples were collected every eight weeks during
pregnancy, at delivery, and six weeks postpartum. The sera was stored in glass containers at -20º
with no recorded thaws. Third trimester samples were analyzed in 1997-1999 for 11 PCB
congeners (28, 52, 74, 105, 118, 138, 153, 170, 180, 194, 203) using solid phase extraction
 52

followed by dual column gas chromatography with ECD at the Centers for Disease Control and
Prevention. Total PCB exposure represented the sum of the 11 measured congeners, expressed
as µg/liter (ppb) of serum (“wet weight”). Serum cholesterol and triglyceride levels, which
affect serum PCB levels, were analyzed using standard enzymatic assays (Daniels et al. 2003).

Maternal serum PCB levels were detectable in 99.9 percent of the samples that met the quality
control criteria. The PCB distribution had a long right tail with a mean of 3.1 ppb and a median
of 2.7 ppb. Ninety-five percent of maternal total PCB concentrations were below 6.25 ppb. The
highest concentration measured was 16.3 ppb. Subjects for all analyses were divided into five
categories based on in utero PCB exposure as reflected by the maternal serum total PCB level,
with cut points at 1.25 ppb intervals (Daniels et al. 2003; Longnecker et al. 2004; Gray et al.
2005).

A comparative study was recently published (Longnecker et al. 2003) that attempted to express
the PCB levels measured in prior cohorts in a uniform manner. Investigators from each study of
PCBs and neurodevelopment (CPP, CHDS, North Carolina, Michigan, Netherlands, Oswego,
Germany, New Bedford, Faroe Islands, Northern Québec) contributed and followed a protocol
for parallel presentation of data. Using a combination of data from original investigators,
laboratory reanalyses, calculations based on published data, and expert opinion, the investigators
compared the PCB levels across studies and assessed the extent to which true or technical
differences accounted for the variation. The mainstay of the comparison was the median level of
PCB 153 across studies for several reasons: 1) comparing levels of one congener precluded
comparability issues; 2) PCB 153 is always present at high concentrations; 3) coelution by other
congeners, when present, is relatively minor; 4) PCB 153 constitutes a large portion of, and
consistently correlates highly with, the sum of quantitated PCBs in all studies.

This study showed that the lipid based PCB 153 levels measured 40 years ago in the CPP cohort
are similar to levels measured recently in the German cohort (median=140 ppb). The median
level measured in the Inuit and Dutch cohorts was100 ppb. By contrast, the median PCB level
measured about ten years ago in the Oswego study was only 40 ppb (Longnecker et al. 2003).

Instruments and Outcome Measures—For the CPP prenatal PCB exposure studies, both
neurophysiological and neuropsychological data were used in the analyses. The children were
assessed at birth with a neonatal neurological exam; at eight months of age with the Bayley Scale
of Infant Development; at four years of age with the Stanford-Binet IQ test; at seven years of age
with the 1949 WISC (administered seven of the eleven components: information,
comprehension, vocabulary, digit span, picture arrangement, block design, and coding) and the
spelling, reading, and arithmetic subtests of the Wide Range Achievement Tests (WRAT); and at
eight years of age with audiograms. The results of the neonatal neurological assessment have not
been published. The WISC IQ/WRAT report (Gray et al. 2005) is currently in press (data was
kindly provided prior to publication by Dr. Longnecker). [NOTE: DR. LONGNECKER HAS
REQUESTED THAT THE WISC DATA BE KEPT CONFIDENTIAL UNTIL
PUBLICATION OF THE ARTICLE, BUT GRANTED PERMISSION FOR ME TO USE IT
IN MY REVIEW.]
 53

Control variables and analysis—The covariates evaluated included study center, maternal age,
maternal IQ (continuous scale), race (White, Black, other), parity, SES (continuous scale),
education (less than high school, high school, greater than high school), marital status (married,
single, divorced, widowed), prenatal smoking (none, < 10 cigarettes per day, ≥ 10 per day);
prepregnancy BMI (low, normal, high, obese), pregnancy complications, serum DDE levels, and
third trimester serum lipids,; and child’s gestational age/age, birth order, gender, head
circumference at birth, and breast feeding (yes/no). Only the study center variable and those
covariates that changed the effect estimate for the PCB-neurodevelopment relation by more than
10 percent were included as confounders in the final models (Daniels et al. 2003; Longnecker et
al. 2004; Gray et al 2005).

Great care was taken to ensure comparability of testing procedures among CPP study centers.
For the Bayley Scale assessments (Daniels et al. 2003), Dr. Bayley made regular site visits to
monitor testing procedures. Tests were edited on site and again after the forms were received by
the NIH in Bethesda, Maryland. Raw scores were converted to age-standardized scores for these
analyses. Most infants (93%) were tested between 7.5 and 9 months of age. For each battery of
WISC tests (Gray et al. 2005), a few examiners from each center would travel to another center
and re-administer the test to a randomly-selected subset of children. For the audiometry exams
(Longnecker et al. 2004), examiners attended three standardization and training workshops on
the CPP audiometric examination. A quality control program involved ongoing exchange of
examiners across institutions who administered a second examination to subjects. repeated
examinations by a second examiner were also done within institutions. In addition, a hearing
specialist from the National Institute of Neurologic Diseases and Stroke Perinatal Research
Branch visited all study centers specifically to review audiometer calibration, background noise,
and sound-treated rooms. The hearing specialist assisted in the standardization procedures.

The Bayley Scales analyses (Daniels et al. 2003) included 1,207 children, 1065 of whom were
selected at random, plus an additional 194 children selected randomly from among the children
scoring more than one standard deviation below or above the mean Bayley Psychomotor
Development Index in the total study population. Using outcome dependent sampling (including
subjects with extreme values) increases the study power relative to a study of equivalent size
employing simple random sampling alone (Zhou et al. 2002, cited in Gray et al. 2005). In this
study, however, the investigators used standard, more straightforward, methods to account for
the sampling, because additional power would not have altered the conclusions.

The analyses used both traditional linear regression and multilevel random-effects models that
adjusted the covariance structure for the clustering of PCB levels and covariates within centers
and were run in SAS software. The investigators also fit a multilevel model that assessed the
individual- and center-level influences of race, breastfeeding, and PCB congener ratios using
BUGS software (Bayesian inference Using Gibbs Sampling, used for the Bayesian analysis of
complex statistical models using Markov chain Monte Carlo (MCMC) methods (Cambridge
University Medical Research Council Biostatistics Unit 2004).

The WISC analyses (Gray et al. 2005) included 1,256 children selected at random, plus an
additional 207 children randomly selected from children with IQ scores either one or more
standard deviations below or above the mean to increase study power. The mean IQ showed a
 54

monotonic change across categories, suggesting that representing PCBs as a continuous, linear
variable adequately reflected the PCB-IQ relation. Quadratic models were also used to assess
whether a linear model was sufficient; all results indicated that it was. All models used in the
analyses employed weights (the inverse of the sampling probabilities) to avoid bias from the
outcome dependent sampling. Models were fit using PROC GLM and PROC REG in SAS.

For the audiometry analyses (Longnecker et al. 2004) 1200 children were randomly selected, of
whom 726 had an 8-year audiometric evaluation, reflecting a 40% loss to follow-up by the 8-
year examination. An additional 200 eligible children were randomly selected from the 440
children whose 8-year audiometric evaluation showed sensorineural hearing loss (SNHL). The
prevalence of SNHL in the CPP children was 2.2%. Among the children selected, some did not
have a PCB sample or were missing audiometry data, leaving 615 from the simple random
sample and 195 selected because of SNHL.

A multivariate unconditional logistic regression model was used for the primary data analysis,
supplemented by a 1 df test for trend, based on assigning to each subject an exposure value
equal to the median PCB level among subjects in their PCB category. A weighted multivariate
linear model of hearing threshold in the worst ear was then fitted (weights were the inverse of the
subject’s probability of being in the sample). The PCB categories and trend analysis were the
same as in the logistic model. When the models of SNHL and hearing threshold were fitted with
PCB level modeled as a continuous variable, the results of the trend tests were essentially the
same. Secondary analyses were similar to the primary analyses, but focused on frequency- and
ear-specific weighted linear model results, and examined how the findings were affected by
using several different approaches to the data analysis.

Results— Bayley Scales. No association was found between maternal serum PCB level and
either the MDI (p=.71) or PDI (p=.14). Thus, these results are consistent with those studies that
did not observe an association between PCBs and the Bayley MDI: Michigan at five months of
age (Jacobson et al. 1986); North Carolina at six months of age (Gladen et al. 1988); and Dutch
at seven months of age (Koopman-Esseboom et al. 1996). However, they are inconsistent with
the German study at seven months of age (Winneke et al. 1998), which showed a negative
association with MDI, as well as with the North Carolina study, which found negative
associations between PCBs and Bayley Scale PDI.

There was some heterogeneity of PCB-PDI relation across study centers that did not correlate
with any individual-level or center-level characteristics after exhaustive analysis for possible
confounding. The difference in the PCB-PDI relation remains unexplained, but may be due to
unknown factors, such as nutrition or other environmental neurotoxicants. The investigators
suggest that it is possible that the unknown factors responsible for heterogeneity among sites in
this study may also be responsible for differences between the overall results of the CPP cohort
and those of studies showing an inverse relation (Daniels et al. 2003).

Cognitive Function Tests. Contrary to the hypothesis that PCBs would negatively affect IQ
scores, positive associations were found between increasing PCB exposure and scores on all
three cognitive function tests, although only the 4-year Stanford Binet results remained
statistically significant after full adjustment for confounders. Evaluation of the four-year
 55

Stanford-Binet IQ test scores showed the IQ-PCB fully-adjusted coefficient was 1.03 IQ units/µg
PCB/L (SE=0.29; n=758); lipid-weight basis results were similar (7.84 IQ units/µg PCB/g lipid,
SE 2.15). The confounders that changed the association the most were center, race, and grade
level at seven years of age.

The Michigan, Dutch, and German cohorts had shown an inverse relation with cognitive function
(Jacobson et al. 1996, Patandin et al. 1999, Walkowiak et al. 2001). Because these studies were
done in populations with higher mean IQ and SES that the CPP population, the CPP investigators
reevaluated their results after excluding subjects with IQ < 82 and with an SES below the median
to adjust for these factors. Neither significantly changed the outcomes (Gray et al. 2005). No
indication was shown that any congener or group of congeners was especially associated with IQ
or that the association differed among congeners (Gray et al. 2005).

In summary, the CPP cognitive assessments showed that background levels of prenatal PCB
exposure were not associated with lower IQ test scores at seven years of age. In fact, the PCB-
IQ coefficient was actually found to be positive; the investigators posit that this result is possibly
due to residual confounding by SES.

Previous studies that found an association between prenatal PCB exposure and cognitive
function were the Michigan studies, which showed decreased cognitive scores at four (n=219)
(Jacobson et al. 1990) and 11 (n=212) years of age (Jacobson et al. 1996); the Dutch children
(n=353) showed decreased cognitive function related to PCB exposure at age 42 months (3 ½ y)
but not at 6 ½ years of age; the German cohort also exhibited decreased cognitive function at 3 ½
years of age, but were not tested later; the Oswego cognitive study at 38 months (n=194) showed
poorer performance with increasing PCB exposure, but not at 54 months (4 ½ y) (n=197). Of
these studies that tested children after 3 ½ years of age, only the Michigan study showed an
effect at school age (11y).

Prior studies that also found no association at any age were the North Carolina study (Gladen et
al. 1991), in which the children’s (n=712) cognitive function was tested at three, four, or five
years of age; and the Faroese children (n=746),with prenatal exposure levels more than three
times higher those of the CPP cohort (Longnecker et al. 2003), who were tested at seven years of
age) (Grandjean et al. 2001).

Cognitive test results have not yet been published for the Oswego, New Bedford, and the CHDS.

The investigators note that, although different tests were used across these studies, scores among
the tests are highly correlated (Sattler 1992, cited in Gray et al. 2005). In addition, the ages of
subjects also varied across studies, but test results are correlated, especially at age seven years
and older (Sattler 1992, cited in Gray et al. 2005). It is unclear why the association between
prenatal PCB exposure and cognitive test results have varied across studies, but Mink et al.
(2004, cited in Gray et al. 2005) have shown that “relatively small differences (0.5 standard
deviations) in confounding variables between “exposed” and “unexposed” groups, if unmeasured
and unaccounted for in the analysis, could produce spurious differences in cognitive test scores.
The magnitude of this difference (3-10 points) has been suggested to have a meaningful impact
 56

in populations. The method of measuring confounders (e.g., maternal intelligence) could also
substantially affect the results.”

Neurophysiological Study. Nearly all of the results showed no association between prenatal PCB
exposure and performance on audiometric examination at approximately eight years of age. The
data suggested a modest direct association between total PCBs and hearing threshold at 2000 Hz
in the left ear, but this finding was not consistently observed across analytical approaches. A
direct association of PCBs with hearing level was seen for the right ear at 4000 Hz, but it was
only one result out of 96 hypotheses tested, and was of borderline statistical significance. The
investigators questioned the importance of these two findings because 1) one would expect to
find some significant associations from chance alone when making so many comparisons; 2) the
second finding was of borderline significance; and 3) an increase in hearing threshold by less
than 1 dB associated with one inter-quartile increase in exposure (1.84 ppb PCBs) if present,
would be of little clinical consequence (Longnecker et al. 2004).

The Faroe Island audiometry study at age seven years (Grandjean et al. 2001) found an increased
hearing threshold related to increasing PCB cord blood level (wet) at 250 Hz and 12,000 Hz in
the left ear. While the CPP study had no measure at 12,000 Hz, the 250 Hz finding suggested no
increase in hearing threshold with PCB level. Although the exposure levels among the Faroese
were much greater than those of the CPP cohort, the CPP study had reasonable power to detect
subtle effects on that outcome because subjects with SNHL were sampled (Longnecker et al.
2004).

In sum, while animal studies clearly indicate a potential sensorineural hearing loss hazard from
PCB exposure (Crofton et al. 2000; Goldey et al. 1998; Goldey et al. 1995, cited in Longnecker
et al. 2004), the CPP data suggest no clinically significant risk associated with background level
exposure in humans.

Child Health and Development Study (CHDS). A similar approach to that of the CPP is being
used to assess neurodevelopmental effects of in utero PCB exposure in children from the CHDS.
The CHDS was a prospective cohort study that enrolled about 20,000 women either attending
prenatal clinics or giving birth at Kaiser Foundation Health Plan Medical Centers in the San
Francisco Bay Area during the 1960’s. The cohort consisted mainly of women with stable
employment, with wealthy and very poor persons generally not well-represented. Otherwise,
members represented a broad cross-section of the population (Hertz- Piciotto et al. 2005).
Information was gathered about their reproductive histories, medication use, medical conditions,
smoking, alcohol use, demographic factors, and occupation, and maternal blood samples were
collected during each trimester of pregnancy. Subsets of the children were enrolled in specialized
developmental studies (Schantz et al. 2003).

The objectives of the CPP PCB studies are to relate exposure to specific PCB congeners with in
utero and postnatal growth, cognitive development, hearing, speech, vision, and morbidity
(Schantz et al. 2003). James et al. (2002) published a study analyzing the determinants of
maternal PCB levels in the CHDS cohort. The data on PCB effects on intrauterine growth is
currently in press (Hertz-Picciotto et al. 2005). The work on cognitive development has not yet
been published.
 57

Exposure Measures—Serum specimens collected during pregnancy were aliquoted and stored at
-20º C at NIH facilities in Frederick, Maryland. James et al. (2002) performed a study on
predictors of serum PCB and OC compounds in the CHDS cohort. They analyzed serum
concentrations from 399 women from the original CHDS cohort in relation to a number of social,
demographic, and reproductive factors. These women were selected from a subset of 3,400
women whose children underwent an extensive examination at five years of age. Detailed
information about exclusion criteria is given in James et al. (2002).

For the study, the frozen specimens were thawed, aliquoted, frozen, and then shipped in dry ice
to the University of California, Davis. The organochlorine extraction protocol is described in
detail in James et al. (2002). Serum specimens were analyzed using an HP 6890 series gas
chromatograph with ECD using and RTX-5MS and an RTX-1701 column run simultaneously in
the same GC. The columns have different polarity and therefore increased the number of
completely resolved congeners in the samples. Detailed information about quality
assurance/control and laboratory methods is described in Willman et al. (2001). Standard
enzymatic techniques were used to measure triglycerides and total cholesterol, and total lipids
were estimated with the formula of Phillips, a formula that includes factors for estimating
phospholipid contribution to total lipids (Willman et al. 2001).

Concentrations were measured of 26 PCB congeners associated with developmental or thyroid

outcomes in prior studies either individually or as part of an Aroclor mixture. The pesticides
o,p’-DDE, p,p’-DDE, o,p’-DDT, p,p’-DDT, and heptachlor epoxide were also analyzed. PCB
congeners were excluded if they were consistently below the limit of quantification, or if they
coeluted. Results were reported for nine PCB congeners: 101, 105, 110, 118, 137, 138, 153, 156,
170, 180, and 187, as well as for o,p’-DDE, p,p’-DDE, o,p’-DDT, p,p’-DDT, and heptachlor
epoxide. “Total PCBs” was calculated as the sum of the nine congeners, with fewer than 30%
below the quantification limit. The sum of DDT and its metabolites were also calculated (James
et al. 2002).

The most abundant PCB congener was 153, followed by 138, 118, and 180. The mean lipid
based ∑PCB was 696±308 ppb (median = 616 ppb) and the mean lipid based concentration of
PCB 153 was 148±67 (median=133 ppb) (James et al. 2002). This median PCB 153
concentration is similar to that found in the CPP and German cohorts (140 ppb) (Longnecker et
al. 2003).

Mean lipid-based concentration of p,p’-DDT (1,931±1,247 ppb) was more than seven times that
of o,p’-DDT, and is much higher than has been found in more recent population studies (Hoyer
et al. 2000; Wart et al 2000; Dorgan et al. 1999, cited in James et al. 2002) The mean lipid-based
concentration of p,p’-DDE (6,854±4,804 ppb) was about four times that of p,p’-DDT, a ratio
generally consistent with more recent exposure to the parent compound (DDT), since DDT’s
half-life is shorter than that of DDE. The mean lipid-based serum heptachlor level was 79±89
ppb (James et al. 2002).

Instruments, Outcome Measures, Results—Data from the CHDS studies relating prenatal PCB
exposure to cognitive development have not yet been published.
 58

►CHAMACOS: A cohort of approximately 550 children was formed from predominantly low
income Latino farmworker families living in the Salinas Valley of Monterey County, California.
The mothers were enrolled during pregnancy through the Center for the Health Assessment of
Mothers and Children of Salinas (CHAMACOS). The CHAMACOS project is funded jointly by
the National Institutes of Health and the U.S. Environmental Protection Agency. EPA Pesticide
Use Reporting data indicates that over 600,000 pounds of endocrine disruptor pesticides are used
annually in the Salinas Valley and may result in high background environmental exposures in
this population (Eskenazi et al. Ongoing).

The investigators measured the levels of 14 organochlorine pesticides in archived CHAMACOS

maternal serum samples collected at 26 weeks of gestation and 16 non-persistent ED pesticides
or their metabolites in archived maternal urine samples collected at 13 and 26 weeks of gestation.
Associations between ED biomarkers and performance on the Brazelton Neonatal Behavioral
Assessment Scale at six, 12, 24, and 42 months will be assessed. The results have not yet been
published.

►National Children’s Study : The National Children’s Study (NCS) is a very large
prospective epidemiologic study being developed by the National Institute of Child Health and
Human Development, in collaboration with the National Institute of Environmental Health
Sciences, the Environmental Protection Agency, and the Centers for Disease Control and
Prevention. The Center for Children’s Health and the Environment of the Mount Sinai School of
Medicine convened a workshop titled, “Endocrine Disruptors and Children’s Health: A
Workshop to Examine the Effects of Endocrine Disruptors on Child Development for a National
Longitudinal Study” on March 16-17, 2000 (New York, NY). The goals of the workshop were to
provide evidence-based guidance to the NCS in assessing exposures to EDs and their effects on
children’s health and development (Landrigan et al. 2003).

The goal of the NCS is to examine the influences of early exposures (environmental, behavioral,
lifestyle, and socioeconomic) on human development and on child and adult health The NCS
will follow as many as 100,000 children throughout the United States, from early pregnancy to
21 years of age. Exposure measurement will be based on infant serum and maternal serum and
breast milk levels. The large base population will provide significant statistical power to
investigate possible links between ED exposures and health outcomes. Additional advantages of
the NCS include state-of-the-art laboratory assessment of chemical exposures and genetic
fingerprinting. The latter will permit examination of genetic polymorphisms that may influence
gene-environment interactions and thus will allow assessment of genetically determined
interindividual differences in susceptibility to EDs (Landrigan et al. 2003).

Issues for future studies

As one reviews the previous epidemiological studies on adverse neurodevelopmental effects

caused by in utero exposure to endocrine disruptors, it soon becomes clear what a daunting task
it truly is to assess their impact. To assess exposures accurately, state-of-the-art laboratory
methodologies and technologies must be used, applying rigorous standards of quality control and
 59

interlaboratory validation procedures. Great care must be taken to anticipate myriads of potential
confounders including coexposures to other neurotoxicants, obstetrical complications, maternal
behaviors, breast feeding, home environment, parental intelligence and educational levels, and
possibly, even the fetal genotype. Assessment instruments must be chosen wisely to ensure that
the most recent and validated test versions are used, and that the limited numbers of outcomes
measured are the relevant ones out of the wide range possible. In addition, the assessments must
be performed at the optimal ages, and interexaminer and intertest reliabilities must be measured
and maximized. As we have seen, investigators must use an appropriate research design,
appropriate data-analytic strategies, and appropriate interpretation of the meaning of the obtained
results. At the same time, they must minimize bias and control for chance findings. Because the
truth of an association lies in its replicability, researchers must also take care to design their
studies such that their findings can be compared with previous studies.

Many uncertainties clearly remain concerning the effects, exposure, assessment, and risk
management of endocrine disruptors. Efforts must continue using toxicological models to
determine dose-response relationships and the effects of exposure to mixtures of endocrine
disruptors, including metabolites and degradation products. Models for risk assessment need to
be expanded and refined. For example, the EPA’s Endocrine Disruptor Research Initiative is
working to apply the Physiologically Based Pharmacokinetic/ Pharmacodynamic (PBPK/PD)
modeling of chemical mixtures to endocrine disruptor human risk assessment (U.S. EPA 2003).
Under the Endocrine Disruptor Screening Program (EDSP) priority setting activities, EPA
scientists are rederiving existing Quantitative Structure-Activity Relationship (QSAR) models
and supplementing them with the latest endocrine disruptor data to improve the predictive
capability of these models (U.S.EPA 2004c).

In his paper on information gaps and research needs for research in this area, Tilman (1998) also
points out that more research is needed to identify experimental variables that could affect
identification of ED-induced neurodevelopmental effects, such as the time at which effects are
measured following exposure, changes in environmental temperature, hormonal status, the phase
of nervous system maturation at the time of exposure. A chemical could affect
neurodevelopmental processes if exposure occurred during a critical period of neuroendocrine
maturation, but have little or no effect if exposure occurred at some other period of development.
Another variable that could affect neurodevelopmental outcomes of prenatal ED exposure is the
shape of the dose-response curve. It is possible that nonlinear dose-response curves could be
generated for some measures of neurodevelopment and that a chemical could have multiple
effects at different points on the dose-response curve, making it impossible to predict effects of
long-term, low-level exposures from studies using short-term, high-dose exposures. It will be
necessary to integrate epidemiological and laboratory studies to focus on critical experimental
variables, such as dose-response function, critical periods of exposure, and the use of more
sensitive or appropriate end points (Tilman 1998).

These toxicological research data and computational tools will support the development of the in
vitro and in vivo assays for use in EPA’s EDSP, an activity itself plagued by controversy. Many
of the chemicals being considered as potential endocrine disruptors are pesticides and other
chemicals associated with highly profitable agricultural and manufacturing industries. These
industries are, understandably, concerned about public perception of their products’ safety and
 60

about possible future regulatory restrictions on the use of these compounds if the EPA program
definitively establishes that they are endocrine disruptors that cause adverse human health
effects.

Another source of resistance is the large, well-organized, and politically influential animal
protection community (People for the Ethical Treatment of Animals (PETA) 2005). These well-
meaning people are concerned about the large number of animals that potentially would be used
in the in vivo EDSP assays, and argues that animal testing will not provide an assessment of
effects in humans, as is stipulated in the Food Quality Protection Act of 1996 (United States
Congress 1996). Indeed, as Tilson (1998) notes, uncertainty does exist about extrapolating
biologic findings obtained in animals to humans. The development of the endocrine system in
different species of animals and humans may be associated with the development of qualitatively
different neurodevelopmental functions. It may, therefore, be problematic to determine if a
chemical-induced change observed in an animal is relevant to human health.

However, the vast majority of the scientific community believes that, while some in vitro assays
may demonstrate that a chemical is capable of interacting with the estrogen, androgen, and/or
thyroid hormonal systems, some in vivo assays are nonetheless required to show this activity.
They also believe that in vivo assays are definitely necessary to demonstrate that the chemical
exerts these hormonal effects in living biological systems and to identify, characterize, and
quantify those effects (U.S. EPA 2004d). Given these uncertainties, data from future
epidemiological studies will be critical to confirm that an effect observed in mammalian animal
tests also occurs in humans.

A large amount of research is currently underway around the world to understand more fully the
mechanisms of action of endocrine disrupting compounds, whether they act directly or indirectly
on endocrine hormone receptors, or via other influence on the neuroendocrine systems. Our
growing knowledge and understanding will inform future human epidemiological investigators
to assess more effectively what effects are occurring in human populations, which chemicals are
of the greatest concern, and what exposure doses are associated with adverse effects. As
professional scientists, researchers will strive to do their science as best they can. As members
of the human race, researchers will endeavor to protect human health and our environment for
the generations to come.
 61

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