UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, DC 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

DATE: September 15, 2015

SUBJECT: Summary Reviews from

_ 2015 Epidemiological Literature Review for Organophosphates.

PC Code: See below Decision Number: DP Barcode : D429120 Registration Numbers : NA Regulatory Action: Petition Number: Risk Assessment Type: NA TXR Number: 0057253 MRID Numbers: NA Case Number: CAS Number: See below 40 CFR:

FROM:

THRU:

TO: Khin Swe Oo
, MD
, DABT
, Toxicologist**~** £<0 

### t!.lt. Toxicology and Epidemiology Branch Health Effects Division (7509P)

Elizabeth Holman
, Physical Scientist

### ~ )J..t Risk Assessment Branch II (RAB2) Health Effects Division (HED)
, 7 f_g 509P

_ Anna Lowit, Senior Scientist 

# ti}

. ) /1

_ Immediate Office ( 

# V I
Health Effects Division (HED), 7509P

Dana Vogel Division Director Health Effects Division (HED), 7509P

As part of the 2015 epidemiological literature review of the organophosphates
, more detailed
summary reviews were completed for each study included in the review. The attached document provides these more detailed summary reviews for each study. For the synthesis of these studies and an analysis of the overall results, please see the "Literature Review on Neurodevelopment Effects & FQPA Safety Factor Determination for the Organophosphate Pesticides" (D331251, A. Lowit et al, 09115/2015). This document was also reviewed by Dr

. Danelle Lobdell from USEPA's Office of Research and Development.

Page 1of171Page 2 of 171 Chemical PC Code CAS No. Dicrotophos 035201 141

- 66

- 2 Fosthiazate 129022 98886

- 44

- 3 Coumaphos 036501 56

- 72

- 4 Terbufos 105001 13071

- 79

- 9 Profenofos 111401 41198

- 08

- 7 Bensulide 009801 741

- 58

- 2 Diazinon 057801 333

- 41

- 5 Ethoprop 041101 13194

- 48

- 4 Dimethoate 035001 60

- 51

- 5 Malathion 057701 121

- 75

- 5 Phosmet 059201 732

- 11

- 6 Chlorethoxyfos 129006 54593

- 83

- 8 Acephate/ Methamidiphos 103301/ 101201 30560

- 19

- 1/ 10265

- 92

- 6 Pirimiphos

- methyl 108102 29232

- 93

- 7 TCVP 083701 961

- 11

- 5 Tribufos 074801 78

- 48

- 8 Phorate 057201 298

- 02

- 2 Phostebupirim 129086 96182

- 53

- 5 DDVP 084001 62

- 73

- 7 Naled 034401 300

- 76

- 5 Trichlorfon 057901 52

- 68

- 6 Fenamiphos 100601 22224

- 92

- 6 AZM 058001 86

- 50

- 0 Methidathion 100301 950

- 37

- 8 Propetamphos 113601 31218

- 83

- 4 ODM 058702 301

- 12

- 2 Disulfoton 032501 298

- 04

- 4 Methyl parathion 053501 298

- 00

- 0 Temephos 059001 3383

- 96

- 8 Chlorpyrifos

- methyl 059102 5598

- 13

- 0Page 3 of 171 Table of Contents

Study Page

HIGH QUALITY STUDIES

Study 1. Barr et al. (2010)

................................ ................................ .................... A

- 1 Study 2. Bouchard et al. (2010)

................................ ................................ ............ A

- 5 Study 3. Fortenberry et al. (2014, 2014a)

................................ ............................ A

- 10 Study 4. Quirós

- Alcalá et al. (2011)

................................ ................................ .... A

- 16 Study 5. Rauch et al. (2012)

................................ ................................ ................. A

- 21 Study 6. Wolff et al. (2007), Furlong et al. (2014)

................................ .............. A

- 25

MODERATE QUALITY STU DIES

Study 7. Anderson et al. (2015)

................................ ................................ ............ A

- 35 Study 8. Guodong et al. (2012)

................................ ................................ ............ A

- 40 Study 9. Handal et al. (2007, 2007B, 2008)

................................ ......................... A

- 45 Study 10. Llop et al. (2013)

................................ ................................ .................... A

- 55 Study 11. Oulhote et al. (2013)

................................ ................................ .............. A

- 58 Study 12. Petit et al. (2010)

................................ ................................ .................... A

- 62 Study 13. Ruckart et al. (2004)

................................ ................................ .............. A

- 67 Study 14. Suarez

- Lopez et al. (2012, 2013a, 2013)

................................ ............... A

- 73 Study 15. Wang et al. (2012)

................................ ................................ .................. A

- 84 Study 16. Zhang et al. (2014)

................................ ................................ ................. A

- 88 Study 17. Shelton et al. (2014)

................................ ................................ ............... A

- 147

LOW QUALITY STUDIES

Study 1 8

. Acosta

- Maldonado et al. (2009)

................................ ............................ A

- 92 Study 19

. Dabrowski et al. (2003)

................................ ................................ .......... A

- 95 Study 20

. Grandjean et al. (2006), Harari et al. (2010)

................................ .......... A

- 99 Study 21

. Kofman et al. (2006)

................................ ................................ .............. A

- 108 Study 22

. Koutroulakis et al. (2014)

................................ ................................ ...... A

- 112 Study 23

. Lizardi et al. (2008)

................................ ................................ ................ A

- 115 Study 24

. Lu et al. (2012)

................................ ................................ ....................... A

- 120 Study 25

. Moreno

- Banda et al. (2009)

................................ ................................ ... A

- 125 Study 26

. Nevison (2014)

................................ ................................ ....................... A

- 129 Study 27

. Rohlman et al. (2005)

................................ ................................ ............ A

- 133 Study 28

. Samarawickrema et al. (2008)

................................ ............................... A

- 137 Study 29

. Savitz et al. (1997)

................................ ................................ ................. A

- 141 Study 30. Wickerham et al. (2012)

................................ ................................ ........ A

- 152Page 4 of 171 Study 1. Barr et al. (2010)

Barr DB, Ananth CV, Yan X, Lashley S, Smulian JC, Ledoux TA, Hore P, Robson MG.
Pesticide concentrations in maternal and umbilical cord sera and their relation to birth outcomes in a population of pregnant women and newborns in New Jersey. Sci Total
Enviro n. 2010;408:790

– 795.

Study Summary

Researchers published a study which evaluated the relationship between prenatal exposures to pesticides by measuring maternal and cord serum biomarkers in a New Jersey cohort of pregnant
women and the birth outcomes of their neonates.

The authors conducted a cross

- sectional study of pesticide exposures in maternal and fetal compartments of mothers and their newborns. This study was conducted within a cohort of 150 mothers and their newborns at Saint Peter's University Hospital in New Brunswick, New Jersey from July 2003 to May 2004. They enrolled a convenience sample based on availability of research personnel. Subjects eligible for recruitment included women with singleton pregnancies
and non

- anomalous fetus scheduled for an elective cesarean birth at term (≥37 weeks) and hemoglobin level was ≥8 mg/dl. Women were excluded if there was evidence for labor or rupture of membranes at the time of operative delivery and if they were taking medications that could potentially i nterfere with metabolism of environmental chemicals. Subjects were identified prior to admission from a scheduling list and the potential subjects' physicians were asked for permission to offer participation. Each potentially eligible subject was approache d to verify eligibility and to obtain informed consent.

Maternal blood samples (10 ml) were collected prior to placement of intravenous and bladder catheters in the pre

- operative holding area or from extra specimens available from pre

- operative
maternal b lood testing stored in undisturbed Vacutainer tubes. After the cesarean delivery of the newborn, the umbilical cord was clamped, and 30

– 60 ml of cord blood was aspirated directly from the umbilical vein after cleaning the cord. All umbilical vein samples w ere obtained within 15min of delivery. An investigator was present at each delivery to assure correct sampling procedures were followed. Finally, maternal pregnancy characteristics and neonatal outcome data were also recorded from the medical record prior to hospital discharge. The authors assessedPage 5 of 171 confounders such as reported pest control use and frequency of use with a maternal age, gravidity, race, maternal pre

- pregnancy BMI, infant sex, and gestational age. Serum samples were analyzed for OP insecticide s, i.e., chlorpyrifos and diazinon, and other pesticides including herbicides, repellents, and fungicides using gas chromatography

– high resolution mass spectrometry.

The authors conducted statistical analysis consisted of assessments of distributional pr operties of each metabolite, including means, medians, distribution percentiles, ranges, and correlation analyses among analytes from the various matrices. In all analyses, concentration values below the LOD were assigned a concentration equal to LOD/√2. T hey performed 3 sets of analyses. In the first, they examined the (Pearson) correlation in each pesticide compound between maternal and cord sera. In the second set of analyses, they compared differences in the mean pesticide exposure between the maternal and cord sera, and applied the t

- test. Finally, they examined the association between pesticide exposures and birth outcomes (birth weight, head circumference, abdominal circumference and birth length). For this latter analysis, they created indicators for pesticide compounds as exposures ≥75th or <75th percentile, as well as exposures ≥90th or <90th percentile. These percentiles were derived from the total cohort, but specific to exposures in the maternal and umbilical cord sera. Since results for pesticid e exposures ≥90th percentile were similar to those for exposures ≥75th percentiles, they presented results only for the latter analyses in the paper.

The authors carried out a multivariable regression analysis to minimize biases due to confounding. This was accomplished by fitting general linear regression models to estimate changes in birth weight and birth lengths in relation to pesticide exposures, and adjusted for confounders. The confounders that were adjusted in the models included maternal age, pri migravida, maternal pre

- pregnancy body

- mass index (BMI), infant sex, and gestational age. BMI was defined as weight (in kg) to squared height (in m). The authors also tested whether or not the continuous covariates (maternal age, BMI and gestational age) h ad a non

- linear relationship with the outcomes (infant anthropometrics) by including quadratic terms for these
continuous variables in the models. Since none of the variables demonstrated non

- linear relationship with the outcome, these variables were inclu ded as single linear terms in the models.

The authors observed an inverse relation to both birth weight and birth length for chlorpyrifos in both maternal and cord serum, but when controlling for confounding variables, the associations were not present. D iazinon was not detected in samples thus not included in any models.Page 6 of 171 Study Review

This study tested whether exposures to organophosphate pesticides, chlorpyrifos and diazinon, have an association with birth outcomes such as head circumference, abdominal ci rcumference, and birth length in a population of mothers and their newborn infants in New Jersey. The population in this cross

- sectional study varied in age, race, and ethnicity. Given that the study population was a convenience sample, the possibility for selection bias exists. The authors could not present evidence to the extent of this bias as no information exists on the potential participants who were not enrolled.

The ascertainment and analysis of serum pesticide markers was appropriate. Quantificat ion was conducted with consultation from the CDC pesticide laboratory. Measurement of the parent compounds chlorpyrifos and diazinon are likely more accurate and objective indicators of organophosphate pesticide exposure, relative to other ascertainment me thods such as metabolites in urine as many metabolites. Although they are sensitive indicators of exposure, it is difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also indicate exposure to other organophosphates and the TCPY a chlorpyrifos specific metabolite has also been found in the environment. The study population likely sustained many other potentially relevant exposures to multiple chemicals and other pesticides which may affect neurodevelopment. Birth

outcomes were assessed via medical record abstraction.

The statistical models used are appropriate. For exposure measures that were less than the laboratory analytic limit of detection, the measures were substituted by dividing the LOD/√2. Imputation may have be en a better method than substitution. It appears that the investigators tried to keep the models parsimonious by only including terms which changed the outcome measure and were statistically significant. Additionally, the authors tested whether the same

re lationships held for the different infant growth measures. Residual confounding by unmeasured or poorly measured confounders is possible in this study. The authors did evaluate a large number of potential confounders, but not all were evaluated notably, di et during pregnancy and exposure to other chemicals that may restrict infant growth (e.g., lead or cadmium).

This study has many strengths, including the measurement of chlorpyrifos and measurement of several factors related to infant growth, head circumf erence, abdominal circumference, and infant length. The authors were able to adjust for or consider many factors related to infant size, maternal age, primigravida, maternal pre

- pregnancy body

- mass index (BMI), infant sex, and gestational age. However, the authors noted their findings were inconsistent with findings fromPage 7 of 171 other studies, namely the Northern Manhattan cohort (Perera 2003). The socioeconomic differences between the New Jersey and Northern Manhattan cohorts are not described in the
article other than to point out that the latter was an African American and Dominican population.

This study has several limitations, including that exposures measured at birth do not necessarily precede the outcome measured. However, these chemicals are typically sho rt

- lived in the blood, thus the fact that they were measured is suggestive of a higher magnitude of exposure than was detected, a steady state exposure, or hospital exposures. The authors reported evidence of a potential hospital exposure this cohort (Yan et al., 2009), but they had little reason to believe that chlorpyrifos exposures would come from a hospital setting. Finally, many of the concentrations were near the limits of detection of the analytical method. Analytical variability can negatively impac t outcome findings, obscuring real associations, and effectively leading to the null association observed in this study for chlorpyrifos and birth outcomes.

References cited

Perera FP, Rauh V, Tsai WY, Kinney P, Camann D, Barr D, et al. Effects of transp lacental exposure to environmental pollutants on birth outcomes in a multiethnic population. Environ Health Perspect 2003;111:201

– 5.

Yan X, Lashley S, Smulian JC, Ananth CV, Barr DB, Ledoux TA, Robson MG, Ledoux TA. Pesticide concentrations in matrices co llected in the perinatal period in a population of pregnant women and newborns in New Jersey. Human and Ecological Risk Assessment 2009;15:948

– 67.Page 8 of 171 Study 2. Bouchard et al. (2010)

Bouchard MF, Bellinger DC, Wright RO, Weisskopf MG. Attention Deficit/Hyperactivity Disorder and Urinary Metabolites of Organophosphate Pesticides in U.S. Children 8

– 15 Years. Pediatrics 2010;125(6):e1270

- e1277. doi:10.1542/peds.2009

- 3058.

Study Summary

Researchers published an analysis of data collected from the N ational Health and Nutrition Examination Survey which investigated the relationship between exposures to organophosphate pesticides (OPs) and diagnosis of ADHD. The National Health and Nutrition Examination Survey (NHANES) is a population

- based, health sur vey of non

- institutionalized US residents conducted by the National Center for Health Statistics of the Centers for Disease Control and Prevention (CDC 2010).

This 2010 study by Bouchard et al. was conducted to examine the association between urinary

con centrations of dialkyl phosphate metabolites of organophosphates and attention

deficit/hyperactivity disorder (ADHD) in children 8 to 15 years of age.

The National Health and Nutrition Examination Survey (NHANES) is a population

- based health survey of non

- institutionalized U.S. residents conducted by the National Center for Health Statistics (NCHS) of the Centers for Disease Control and Prevention (CDC). NHANES uses a complex, multistage probability sampling design, with oversampling of certain subgroups. Participants completed household surveys, which included questions about demographics and health history, and blood and urine samples were collected during a physical examinations at mobile centers (CDC 2010). The authors used data from 2000

– 2004, years fo r which ADHD was
assessed in children 8

– 15 years; a diagnosis was available for 3,998 children. Urinary DAP metabolites were measured on a random subsample of the NHANES participants. From 2000 to 2002, the sampling rate was 1/2 for ages 6

– 11 years and 1/3 for ages 12

– 15 years. From 2003 to 2004, the sampling rate was 1/3 across all ages. Measurements of urinary DAPs were available for 1,481 children among those with an ADHD diagnosis. NHANES was approved by the NCHS institutional review board, and all part icipants provided written informed consent (CDC 2010).

During the physical examination, “spot” urine specimens were collected from participants, aliquoted, and stored cold (2

– 4°C) or frozen until they were shipped on dry ice to the CDC forPage 9 of 171 analysis. Six u rinary DAP metabolites, resulting from the degradation of at least 28 different OPs, were measured in urine to provide an indicator of the body burden of common OPs (NRC, 1993). The urinary DAP metabolites measured are 3 dimethyl alkylphosphate (DMAP)
mole cules (dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate) and 3 diethyl alkylphosphate (DEAP) molecules (diethylphosphate, diethylthiophosphate, diethyldithiophosphate). DMAP metabolites are derived from O, O

- dimethyl

– substituted OP pestici des such as malathion; DEAP metabolites result from the degradation of O, O

- diethyl

– substituted OPs such as chlorpyrifos. Concentrations below the detection limit were imputed a value corresponding to the value of the detection limit divided by √2.

Atten tion

- Deficit/Hyperactivity Disorder (ADHD) was assessed using the Diagnostic Interview Schedule for Children (DISC

- IV), a structured diagnostic interview designed for use in epidemiologic studies (American Psychiatric Association, 1994). The interview was conducted by telephone by trained interviewers with the mother or another caretaker, two to three weeks after the physical examination. The interview was conducted by bilingual interviewers in English or Spanish. The DISC

- IV has evidence of substantial val idity, reliability for both its English and Spanish versions, and successful use via telephone in the DSM

- IV field trials. Use of DISC

- IV data is restricted for confidentiality, thus, data was accessed through the NCHS Research Data
Center.

The DISC

- IV sc oring algorithms determine ADHD diagnostic status for the past year, as well as ADHD subtype: i) predominately inattentive subtype, ii) predominately hyperactive

- impulsive subtype, or iii) combined subtype. Diagnosis is based on the presence, during the pr ior 12 months, of symptoms related to inattention, hyperactivity and impulsivity, with significant impairment in two or more settings (e.g., at school and home). The DSM

- IV criterion that symptoms must not occur in conjunction with another neuropsychiatric disorder is not assessed by the DISC

- IV. The DSM

- IV criterion that symptoms must have been present before 7 years of age was not used because the hypothesis was that urinary DAP concentrations are associated
with increased odds of concurrent ADHD.

Many p otential confounders were ascertained for modeling

- race/ethnicity (non

- Hispanic white, black, Mexican American, or other/multiracial), PIR (the ratio of self

- reported family income to the family’s appropriate poverty threshold value, on the basis of Cens us data, recoded into 4 categories), BMI (in quartiles), blood lead concentrations (logarithmically transformed), maternal age at birth, maternal smoking during pregnancy (yes or, no), and time since lastPage 10 of 171 consuming food or drink, recorded at the time of bl ood and urine sampling (recorded on a scale of 1 [0

– 2 hours] to 7 [21

– 24 hours]).

The authors divided DAP metabolite concentrations by the respective molecular weights then summed to yield DEAP, DMAP, and total DAP concentrations. Because the distribution s of total DAP, DMAP, and DEAP concentrations were skewed, logarithmic transformations (base 10) were applied. Because they suspected DMAP and DEAP metabolites might have different relationships to the outcomes (Eskenazi 2007), they were examined separatel y. Correlates of urinary DAP metabolite concentrations were examined with a general linear model. Risk was estimated using logistic regression analyses to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for ADHD (any subtype) and ADHD subtype s, per 10

- fold increases in total DAP, DMAP, and DEAP metabolite concentrations (in nanomoles per liter), the authors present results crude analyses and adjusted analyses. To check for potential biases due to low detection frequency of the DAP metabolites, the authors conducted further analyses on the metabolite with the highest detection frequency, namely, dimethyl thiophosphate. Cases were categorized as below the limit of detection or lower or higher than the median of detectable concentrations adjusted for creatinine levels.

The authors explored potential confounding effects for all the confounders ascertained, but only sex, age, race/ethnicity, PIR, fasting time, and logarithmically transformed urinary creatinine concentrations (as recommended by Barr et al., 2005 to adjust for urine dilution) were included
in the models.

The authors observed evidence of an association between exposures to organophosphate pesticides as measured by urinary DAP metabolites and increased odds of ADHD in a representative s ample of US children age 8

- 15. This finding was seen for total DAPs, total DMAPs, and dimethyl thiophosphate metabolites, but a null result was observed for DEAPs. There was a 55% to 72% increase in the odds of ADHD for a 10

- fold increase in DMAP

concentra tion, depending on the criteria used for case identification. This association was not explained by gender, age, PIR, race/ethnicity, fasting duration, or creatinine concentration.

Study Review

This study tested whether in exposures to organophosphate pe sticides have an association with ADHD in a representative sample of US children age 8

- 15. The study population wasPage 11 of 171 appropriate to the hypothesis, given that the representativeness of the sample allows for an estimation of risk among children with “average ” exposure levels to OPs. The population in this

cross

- sectional study varied in age, race, ethnicity and geographic area. Given that the NHANES biomonitoring is conducted in a 1/3

— 1/2 subsample of the entire NHANES population, the possibility for further selection bias exists. However, the authors present evidence that it is negligible as the demographic characteristics are similar between the population included and not included in the subsample.

The ascertainment and analysis of urinary organophosphate pesticide markers was appropriate. Quantification was conducted at the CDC, using published methods. DAP metabolites are likely more accurate and objective indicators of organophosphate pesticide ex posure, relative to other
ascertainment methods such as self

- report. Although they are sensitive indicators of exposure, it is difficult to infer chlorpyrifos effects specifically from the metabolite levels, as they also indicate exposure to other organoph osphates. The study population likely sustained many other potentially relevant exposures to multiple chemicals and other pesticides which may affect neurodevelopment.

The ADHD test, (DSM

- IV, is very widely used, and was appropriately administered by a t rained interviewer and scored using modified standards appropriate to the hypothesis being tested. (The
authors choose not to use the DSM

- IV criterion that symptoms must have been present before 7 years of age, because their hypothesis was that urinary DAP concentrations would be associated with increased odds of concurrent ADHD).

The statistical models used are appropriate and well powered. For exposure measures that were less than the laboratory analytic limit of detection, the measures were substituted by dividing the LOD/√2. Imputation may have been a better method than substitution. It appears that the investigators tried to keep the models parsimonious by only including terms which changed the outcome measure and were statistically significant. Additi onally, the authors conducted a sensitivity analysis and tested whether the same relationships held for the different ADHD subtypes. Residual confounding by unmeasured or poorly measured confounders is possible in this study. The authors did evaluate a lar ge number of potential confounders, but not all possible were evaluated, e.g., family history of ADHD.

This study has many strengths, including the representative sample, the measurement of urinary DAP, and measuring ADHD and its subtypes using the DSM

- IV . The authors were able to adjustPage 12 of 171 for or consider many factors related to cognitive function, such as socioeconomic indicators. The statistical associations observed are plausible, several biological mechanisms might underlie an association between organop hosphate pesticides and ADHD, as noted by authors (Sultatos 1994, Coccini 2009). Additionally, the authors noted that their findings were consistent with findings from other studies in more highly exposed populations. The many advantages of this study, the plausibility of the findings, and that similar associations have been seen in other studies, support the hypothesis that exposure to OPs increases risk of ADHD. Because of the cross

- sectional design, the authors cannot rule out the possibility that childr en with ADHD engage in behaviors that expose them to higher levels of organophosphates.

References cited

American Psychiatric Association. Diagnostic and statistical manual of mental disorders: DSM

IV. 4. Washington, DC: American Psychiatric Association; 1994.

Barr DB, Wilder LC, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL. 2005. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environ Health Perspect 113:192

– 200.

Centers for Disease Control and Prevention. National Health and Nutrition Examination Survey: questionnaires, data sets, and related documentation. Available at: www.cdc.gov/nchs/nhanes/about_nhanes.htm.Accessed April 6, 2010.

Coccini T, Crevani A, Rossi G, et al. Reduced p latelet monoamine oxidase type B activity and lymphocyte muscarinic receptor binding in unmedicated children with attention deficit hyperactivity disorder. Biomarkers. 2009;14(7):513

– 522.

Eskenazi B, Marks AR, Bradman A, et al. Organophosphate pesticide e xposure and neurodevelopment in young Mexican

- American children. Environ Health Perspect. 2007;115(5):792

– 798.

Sultatos LG. Mammalian toxicology of organophosphorus pesticides. J Toxicol Environ Health. 1994;43(3):271

– 289.

US National Research Council, C ommittee on Pesticides in the Diets of Infants and Children. Pesticides in the Diets of Infants and Children. Washington, DC: National Academy Press; 1993.Page 13 of 171 Study 3. Fortenberry et al. (2014, 2014a)

• 2014. Urinary 3,5,6

- trichloro

- 2

- pyridinol (TCPY) in pregnant women from Mexico City: Distribution, temporal variability, and relationship with child attention and hyperactivity. GZ Fortenberry, JD Meeker , BN Sánchez, DB Barr, P Panuwet, D Bellinger, L Schnaas, M Solano

- González, AS Ettingera, M Hernandez

- Av ila, H Hu,
MM Tellez

- Rojoj. International Journal of Hygiene and Environmental Health 217 (2014): 405

– 412.

• 2014a. Paraoxonase I polymorphisms and attention/hyperactivity in school

- age children from Mexico City, Mexico. GZ Fortenberry, JD Meeker , BN Sánchez, DB Barr, K Peterson, D Bellinger, L Schnaas, M Solano

- González, AS Ettinger, M Hernandez

Avila, H Hu, MM Tellez

- Rojoj. Environmental Research 132:342

– 349.

Study Summary

Fortenberry et al. looked at mother

- child pairs from a prospective birt h cohort study called the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) to evaluate the relationship between pesticide exposure and Attention Deficit Hyperactivity Disorder (ADHD) in school age Mexican children from Mexico City, Mexic o. The aim of the first report (Fortenberry et al., 2014) were to define the distribution of TCPY concentrations among a population of pregnant women in Mexico, to assess between

- and within

- individual variability of urinary TCPY levels over the course of pregnancy, and to explore the relationship between third trimester maternal urinary TCPY concentrations and child neurodevelopment, particularly attention and hyperactivity, using subscales from psychometric assessments in children 6

- 11years of age. The se cond report (Fortenberry et al., 2014a) examined whether maternal and/or child PON1 genotypes (PON1 R192Q and PON1 L55M ) were associated with Attention Deficit Hyperactivity Disorder

- Like Phenotypes (ADHD

- LP).

Participants of the ELEMENT study came from th ree different cohorts; 1994

– 1997 (cohort1),
1997

– 2000 (cohort 2), and 2001

– 2005 (cohort 3). All three ELEMENT cohorts enrolled homogenous, low

- to

- moderate income, pregnant women from the National Institute of Perinatology, Hospital General Dr. Manuel Gea G onzalez, or clinics affiliated with the Mexican Social Security Institute. Mother

- child pairs from the three cohorts (N = 827) were re

- invited between 2007 and 2011 to examine childhood and adolescent neurodevelopmentalPage 14 of 171 characteristics. Exclusion character istics for all ELEMENT cohorts included: plans to leave the area within the next 5 years; daily consumption of alcoholic beverages; addiction to illegal drugs; continuous use of prescription drugs; diagnosis of multiple pregnancy, preeclampsia, renal or
he art disease, gestational diabetes; a history of infertility, diabetes, or psychosis; diagnosis of high risk pregnancy; or suffering from seizures requiring medical treatment.

In the overall ELEMENT study, participants provided second morning void urine sa mples at each trimester of pregnancy. For the first study (2014), third trimester urine samples from
mother

- child pairs that had completed psychometric assessments for children (N = 187) from cohorts 2 and 3 were utilized. In a subset of women randomly sel ected from the 187 mother

child pairs (N = 21), urinary TCPY in samples collected during all three trimesters of pregnancy was measured. TCPY concentrations in maternal urine samples were measured by enzyme hydrolysis. Metabolites were quantified using iso tope dilution calibration. The LOD was 0.10 ng/ml for TCPY.

For the second report (2014a), blood samples from mother

– child pairs who were re

- invited for additional studies (N = 591) were analyzed for coding PON1 polymorphisms (Q192R and L55M). For PON1 Q19 2R , there were N = 531 mothers and N = 532 children with genotype
information. For PON1 L55M , 458 mothers and 478 children had genotype information. The PON1 genotypes were determined using the Sequenom Mas

- sARRAY iPLEX Platform. The three psychometric ass essments used to evaluate behavioral characteristics of children 6

– 13 years of age were same as what was used for the first study.

Three psychometric assessments were used to evaluate behavioral characteristics of children 6

– 11 years of age: Conners’ Paren tal Rating Scales

- Revised (CPRS

- R)

- tool for parents used to
determine a child’s behavior for children and adolescents 3

– 17years; Behavior Assessment System for Children

- Parental Rating Scales (BASC

- PRS)

– tool to assess attention problems and hyperactivi ty in children 6

- 11 years of age; and Conners’ Continuous Performance Test (CPT)

- 14

- minute computer test that measures sustained attention and impulsivity and compares the participant’s answers to a reference group and results in a confidence index of a clinical profile. Scores from these psycho

- metric instruments assess ADHD

- related symptoms and are not designed as diagnostic tools, but rather for screening. The instructions and prompts were translated into Spanish by a researcher who also trained and su pervised the personnel who administered the assessments. Standardization and quality control checks were conducted by reviews of videotaped evaluations.Page 15 of 171 Fortenberry et al. (2014)

Multivariable regression models were created using variables found to be as sociated with
ADHD

- related behaviors and/or TCPY in bivariate analyses (p < 0.05). Variables were also considered from a priori suspicion of being potential confounders of the association between prenatal CPF/chlorpyrifos

- methyl exposure and psychometric o utcome. Covariates included: maternal IQ, maternal education, income, maternal urine specific gravity, breast feeding, maternal blood lead one month after delivery, season of sample collection, child’s age at testing, child’s sex, birth length, and head ci rcumference at birth. Birth length and head circumference at birth were determined by a nurse at the time of delivery and were used as continuous variables. Maternal IQ was calculated on the basis of a mother’s scores on the Information, Comprehension, Sim ilarities, and Block Designs scales of the Spanish Wechsler Adult Intelligence Scale.

TCPY was detected in over 90% of urine samples in the present study. Geometric mean TCPY concentration among ELEMENT women was significantly higher than pregnant women i n NHANES (p

- value = 0.03).Among the subset of women with repeated measures, there were no significant differences in geometric mean TCPY concentrations between trimesters. However, there was significant within

- woman variability across trimesters.

Based on the multivariable regression analysis, no statistically significant associations were observed in models that considered all children combined and models stratified by sex. However, a suggestive trend for increasing Hit RT Block Change in relation to in creasing TCPY tertiles (p

value = 0.09) was found. In models stratified by sex, this relationship appeared to be stronger among boys than girls. In the sex

- stratified analysis, a suggestive trend for increased ADHD Index in relation to TCPY tertiles (p

- val ue = 0.06) was also found. In this model, the highest TCPY tertile was associated with an ADHD index score that was 5.55 points higher than children in the lowest tertile (p

- value = 0.06). Among girls, there was a suggestive increase inattention problems s core in relation to exposure, but only for the middle TCPY tertile (p = 0.08).

So the authors conclude that there is no statistically significant associations between tertiles of maternal third trimester urinary TCPY and measures of attention and hyperact ivity in children. However, there was suggestive evidence for increases in the ADHD index in relation to TCPY tertiles among boys. This suggests that fetal exposure to increased quantities of chlorpyrifos,Page 16 of 171 chlorpyrifos

- methyl, or TCPY during the last trime ster of pregnancy may influence the display
of ADHD characteristics in childhood.

Fortenberry et al. (2014a)

Multivariable regression models were created using variables found to be associated with
ADHD

- LP, and/or PON1 genotypes in bivariate analyses (p< 0.05). Variables were also considered from a priori suspicion of being potential con

- founders of the association between prenatal exposure to OP pesticide exposure and psychometric outcome. In PON1 main effects models, covariates included continuous matern al IQ, education, socioeconomic status and blood lead one month after delivery, breast feeding (yes/no), child's sex, continuous age at testing, birth length and head circumference at birth.

The distribution of genotype frequencies for PON1Q192R and PON1L 55M were similar for both populations. For mothers, allelic frequencies for PON1Q192R were 41% for A and 59% for G while children's allelic frequencies 40% and 60%, correspondingly. For PON1L55M, mothers and children both had allelic frequencies of 88% and 12%, for A and T, respectively.

Results from the PON1 main effects multivariable regression analyses showed a 2.32

- point higher score (95% CI (0.26, 4.38)) in DSM IV Hyperactivity/Impulsivity for children with mothers having PON155MM in comparison to PON 155LM+LL. No other significant associations were found in models fitted separately by either mother or child genotype information. In models further stratified by child sex, no statistically significant associations were observed with PON1 genotypes in eit her boys or girls. However, in boys, there were suggestive associations of 2.00, 2.61 and 2.22 point higher scores, respectively, for ADHD Index (p = 0.09), DSM IV Hyperactivity/Impulsivity (p = 0.06), and DSM IV Total (p = 0.07) in
relation to mothers' PO N155MM. Also, in males who had mothers with PON1192QQ, there was a borderline significant association of 2.81

- point higher score (p=0.05) for DSM IV Hyperactivity/Impulsivity.

The authors concluded that there is a significant relation between ADHD

- LP and mother’s PON1 genotype. Because maternal PON1 polymorphisms were associated with child ADHD

- LP, this may be a viable biomarker of susceptibility for ADHD

- LP.Page 17 of 171 Study Review

This was a well conducted study with notable strengths and few weaknesses. The stu dy population was homogenous with participants from diverse socio

- economical background from Mexico City. The fact that this study looked into TCPY concentrations among a population of pregnant women to assess between

– and within

- individual variability of urinary TCPY levels over the course of pregnancy makes this study unique. The analytic methods, analytic approach, and statistical analysis were appropriate. The author’s approach to evaluating and adjusting for covariates were prudent.

The authors emplo yed a reasonable set of inclusion/exclusion criteria that likely resulted in
recruitment of an inner

- city study population that was ethnically diverse. The investigators prospectively collected maternal and child information (potential confounders and effe ct modifiers).

Selection bias is unlikely to have occurred in the study.

Fortenberry et al (2014)

The urine sampling and quantification of TCPY in maternal urine samples appears to have been conducted appropriately. This method is more objective, sens itive, and likely also more accurate and reliable. The psychometric instruments used to assess the behavioral characteristics of children seem to be appropriate. Measures were taken to ensure that the tests were administered correctly. However, it should b e noted that these instruments have their own strengths and weaknesses and are screening, not diagnostic tools.

The analytic methods, analytic approach, and statistical analysis were appropriate. A relatively modest sample size may have reduced the statis tical significance of the study. The author’s approach to evaluating and adjusting for covariates were prudent. The investigators prospectively collected maternal and child information (potential confounders and effect modifiers. On the whole, the investig ators handling of confounding was reasonable. It was interesting that TCPY was detected in over 90% of the samples and the geometric means of TCPY concentrations were significantly higher than that of the pregnant women in NHANES. This shows that the women in Mexico are exposed to pesticide during pregnancy. History of maternal and paternal exposure to pesticides was not collected in the study. Also, the use of a single urinary measure to estimatePage 18 of 171 exposure may not have provided the true level of exposure. I n addition, potential for differential exposure misclassification is also possible because mothers were likely aware of the neurobehavioral status of their children, and it is plausible that this knowledge of outcome status may have impacted the psychometr ic assessments to assess behavioral characteristics of children.

Statistical analysis showed that there is no significant association between tertiles of maternal
TCPY concentrations and ADHD

- related outcomes in children. The authors rightly conclude their paper by stating that considering the continued widespread agricultural and possible residential use of chlorpyrifos and chlorpyrifos

- methyl in Mexico and the educational implications of cognitive and behavior deficits, these relationships deserve fu rther study.

Fortenberry (2014a)

This was a well conducted prospective birth cohort study with notable strengths and few weaknesses. The objective of this study was to investigate the relationship between maternal and/or child PON1 genotypes (PON1R192Q a nd PON1L55M) and ADHD

- LP in children aged
6

- 13 years of age in Mexico City, Mexico. The review comments above also apply to this report. The fact that maternal and child PON1 genotype was taken into consideration make this study unique.

The analysis of m aternal and child blood for PON1 genotype appears to have been conducted appropriately. This method is more objective, sensitive, and likely also more accurate and reliable. The psychometric instruments used to assess the behavioral characteristics of chil dren seem to be appropriate. However, it should be noted that these instruments have their own strengths and weaknesses and are screening, not diagnostic tools.

The analytic methods, analytic approach, and statistical analysis were appropriate. A large

sa mple size may have increased the statistical significance of the study. The author’s approach to evaluating and adjusting for covariates were prudent. The investigators prospectively collected maternal and child information (potential confounders and effec t modifiers). On the whole, the investigators handling of confounding was reasonable. However, potential for differential exposure misclassification is also possible because mothers were likely aware of the neurobehavioral status of their children, and it is plausible that this knowledge of outcome status may have impacted the psychometric assessments to assess behavioral characteristics of children.Page 19 of 171 Statistical analysis showed that there is an association between maternal PON1 genotype and parent reported ADHD

- LP in children. These findings have important implications as potential susceptibility factors for ADHD

- LP in children, and add to the growing body of evidence that maternal PON1 polymorphisms may affect child neurodevelopment. The authors rightly

co nclude their paper by stating that this study provides platform for further analysis.Page 20 of 171 Study 4. Quirós

- Alcalá et al. (2011)

Maternal prenatal and child organophosphate pesticide exposures and children’s autonomic function. Lesliam Quirós

- Alcalá, Abbey D . Alkon, W. Thomas Boyce, Suzanne Lippert, Nicole V. Davis, Asa Bradman, Dana Boyd Barr, Brenda Eskenazi. NeuroToxicology 32:646

– 655, 2011.

Study Summary

The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) longitudinal birth cohort study was conceived to evaluate the health effects of pesticides on maternal and child health in a population living in the agricultural community of Salinas Valley, California. In this report, Quirós

- Alcalá et al. leverage data from the CHAMA COS cohort to evaluate whether urinary biomarkers of prenatal and childhood organophosphate (OP) pesticide exposure are associated with autonomic nervous system dysregulation at ages 6 months, 1 year, 3.5 years, and 5 years.

The original study populatio n consisted of pregnant mothers recruited in 1999

- 2000 who were in the first half of their pregnancy (<20 weeks), qualified for government assistance, were receiving prenatal care at a specific set of community clinics, and who planned to deliver at the Sa linas County hospital. Of the 601 women originally enrolled, 531 were followed to delivery of their child. A subset of participating infants with an even identification number were eligible to participate in the autonomic nervous system reactivity protocol s at 6 months, 1 year, and 3.5 years of age. The autonomic nervous system reactivity protocol was offered to all children at the
5

- year follow

- up. After excluding children with missing or otherwise insufficient pesticide metabolite or outcome data, twins, and those taking medications that may affect autonomic nervous system responses, the study sample consisted of 149 children who provided data at the
6

- month follow

- up, 149 at 1 year, 97 at 3.5 years and 274 at 5 years.

Participating mothers were intervie wed at approximately weeks 14 and 26 of their pregnancy, at delivery, and again at the time of each child assessment to obtain demographic and potential
covariate information. Information was also abstracted from the child’s birth records and pediatric med ical records.Page 21 of 171 The investigators assessed both prenatal OP pesticide exposure and OP pesticide exposure at the time of the outcome assessment. To estimate prenatal exposures, dialkylphosphate (DAP) metabolite concentrations, including three diethylphosphates (DEP, DETP, DEDTP) and three dimethylphosphates (DMP, DMTP, DMDTP) were quantified in maternal spot urine samples collected at the time of the prenatal interviews (i.e. at 14 and 27 weeks gestation, on average). Childhood OP pesticide exp osures were estimated by measuring the same set of DAP metabolites in spot urine samples collected from the children at ages 6 months, and at age 1,2,3.5, and 5 years of age. All urine samples were processed for shipping by study staff and shipped to the C enters for Disease Control and Prevention for quantification of DAP
concentrations. Individual DAP metabolite concentrations below the lower level of detection (LOD) were imputed as LOD/√2. DAP, DEP, and DMP concentrations were summed then averaged over th e two prenatal sampling periods.

Assessments of autonomic nervous system dysregulation under resting and challenge conditions were conducted using a standardized protocol, administered by trained, bilingual research staff. The resting condition at 6 month s and 1 year was listening to lullabies and at 3.5 and 5 years, listening to a story. The challenge conditions at 6 months and 1 year were watching a jack

- in

the

- box being wound up and jumping out of the box (social/startle), listening to a recording of a baby crying (emotion), and feeling a vibrator on the leg (physical). At 3.5 and 5 years, the challenge conditions included asking the participants to answer questions (social), watch a scary video (emotion), taste concentrated lemon juice (physical), and r epeat a series of numbers (cognitive). An electrocardiograph was used to obtain continuous measures of the participants’ sympathetic and parasympathetic autonomic responses (respiratory sinus arrhythmia, pre

ejection period, and heart rate) during the rest ing and challenge conditions.

Covariates were selected if they were potentially related to the outcome measures (age of the child at the time of the outcome assessment; location of the assessment, investigator conducting the outcome assessments) or if th ey were significantly associated with OP pesticide exposure and outcome (α= 0.1) (breastfeeding duration). The child’s sex was also included as a covariate in all models, and interactions between sex and OP pesticide exposure were examined. The investigato rs also considered prenatal maternal smoking status, prenatal maternal alcohol use, income, maternal education, birth weight and gestational age though these were not included in final models because they were not significantly associated with outcomes or OP pesticide exposure at any age. Six separate linear regression models were constructed for the three autonomic nervous system outcomes (during resting and challenge conditions) at each time pointPage 22 of 171 (6 months, and 1, 3.5, and 5 years of age). Log

- transforme d prenatal and current (cross

sectionally sampled) child DAP concentrations and their interaction were included in each model, along with covariates. Cumulative prenatal and child OP pesticide exposure was also estimated using an area

- under

- the

- concentrati on

– time

- curve methodology. (Kaplan et al., 1997) Models were constructed using unadjusted urinary DAP metabolite concentrations and repeated using creatinine

- adjusted DAP metabolite concentrations, and (also with creatinine level entered
as a covariate in the multivariate models. Mixed

- effects models of the longitudinal relationship between DAP concentrations and autonomic nervous system responses were also constructed, but results of these models were reported to be similar to the cross

- sectional modeling results and were not presented in the report.

Statistically significant relationships were not observed between reactivity patterns and prenatal or child DAP concentrations. Child total dimethylphosphates and total DAP concentrations were significantly n egatively associated with resting respiratory sinus arrhythmia at 6 months and maternal prenatal dimethylphosphates and child total diethylphosphate concentrations were significantly positively associated with resting pre

- ejection period at age 1 year. How ever, no associations with resting autonomic system parameters were observed in 3.5 year old or 5 year old children, nor with reactivity (i.e. autonomic nervous system response to challenge conditions) at any age. Cumulative maternal total diethylphosphate s were associated with lower average heart rate, but only at the 5

- years follow

- up. No statistically significant differences in autonomic nervous system parameters were observed for 5

- year

- olds who had consistently high total DAP levels, relative to those who had consistently low DAP concentrations. Statistically significant interactions between prenatal and child DAP concentrations, nor did associations between the OP pesticide biomarkers and autonomic nervous system profiles vary by sex of the
child.

Al though some evidence of autonomic nervous system dysregulation under resting condition in early childhood (≤1 year of age) was reported in this study, associations between non

- specific biomarkers of maternal prenatal OP pesticide exposure and child OP pest icide exposure, and children’s autonomic nervous system regulation during resting and challenging conditions up to age 5 years were not consistently observed.Page 23 of 171 Study Review

This was a well

- conducted prospective study of prenatal and postnatal OP pestic ide exposure effects on autonomic nervous system regulation among children up to age 5 years of age participating in the CHAMACOS birth cohort study. The discussion of the biologic plausibility underpinning the study hypothesis was robust. The analytic met hods, exposure assessment, outcome assessment, and analytic approach were appropriate. Strengths of the study include the reasonable large sample size, the prospective design, the measurement of prenatal maternal urinary DAP concentrations at two time poin ts, and the repeated measurements of child DAP urinary DAP concentrations and objective measurements of autonomic nervous system profiles at four time points in the first 5 years of life.

The CHAMACOS study population was selected from a low income, agri cultural community. The participants in this study are a subset of the original study sample, but the selection of this subsample was likely unbiased. The exclusion criteria (i.e. missing exposure or outcome data, use of medications that may affect autono mic nervous system responses, and twins) were reasonable and unlikely to have adversely affected the generalizability of the study results. The reasons for having missing DAP biomarker or outcome data were not discussed in detail, but the missing data is a possible source of selection bias. Only 27 children had urinary DAP concentrations available for all four time periods, which limited the investigator’s ability to evaluate cumulative exposure

- response associations.

The collection of urine samples and q uantification of maternal prenatal and postnatal child urinary DAP concentrations appears to have been well conducted, and errors in these measurements, to the extent they exist, are almost certainly non

- differential with respect to autonomic nervous syste m outcomes. The repeated sampling of DAP biomarker concentrations during pregnancy and during early childhood is a strength of this study. The use of non

- specific
biomarkers of OP pesticide exposure, as were assessed in this study, are likely more objectiv e, sensitive, and likely also more accurate and reliable indicators of OP pesticide exposure, relative to exposure assessment methodologies that rely on self

- report. That said, urinary DAP metabolite levels may reflect exposure not only to OP parent compou nds, but also to the potentially nontoxic pre

- formed metabolites in the environment.

The investigators collected participant information via questionnaire, and also collected birth and pediatric medical records, allowing for a comprehensive assessment of potential confounders andPage 24 of 171 potential effect modifiers. Strong residual confounding by unmeasured or poorly measured confounders is unlikely in this study.

The statistical analysis was appropriate. The authors presented results only from their cross

sectional statistical models, though they also constructed longitudinal models and noted that the estimated associations from these more complex models were gener ally similar to those of the simpler models. Though it is likely that the study was not statistically powered to evaluate effect modification, several interactions were assessed in this study. Perhaps owing to the limited power, no statistically significan t effect modification was observed. The authors constructed many models, but did not adjust their statistical results for multiple comparisons.

The methods used to model resting and challenge conditions appear to have been reasonable and

were age

- appropr iate. The assessments of autonomic nervous system function (respiratory sinus arrhythmia and preejection period) appear to have been similarly appropriate, valid, and well

conducted.

While several associations were observed, no consistent associations be tween biomarkers of prenatal and child OP pesticide exposure and autonomic nervous system function were observed. The findings of this well

- conducted study were largely null and thus generally not consistent with the hypothesis that prenatal and child OP p esticide exposures are associated with autonomic nervous system dysregulation. Though the authors adequately convey the biological plausibility and potential mechanism underlying the study hypothesis, this is the first reported study of OP pesticide exposu re and indicators of autonomic nervous system function. As such, replication of the study in other study samples is needed before a well

- supported conclusion can be reached regarding the potential association between OP pesticide exposure and aut onomic ner vous system function. In addition, t here is not a body of literature to compare these ANS outcome results against, making it difficult to put them into context. Furthermore, this study did not focus on neurodevelopmental outcomes, wh ich is the focus of t he OP epidemiological literature
review . Consequently, this Qu irós

- Alcalá et al. 2011 study was not evaluated further in the synthesis of the OP epidemiological literature review ( add citation )

.

References cited

Kaplan B, Meier

- Kriesche H, Napoli K, Kahan B. A limited sampling strategy for estimating sirolimus area

- under

- the

- concentration curve. Clin Chem 1997;43(3):539

– 40.Page 25 of 171 Study 5. Rauch et al. (2012)

Associations of Prenatal Exposure to Organophosphate Pesticide Metabolites with Gestational Age a nd Birth Weight. Stephen A. Rauch, Joe M. Braun, Dana Boyd Barr, Antonia M. Calafat, Jane Khoury, M. Angela Montesano, Kimberly Yolton, and Bruce P. Lanphear. Environmental Health Perspectives. 120:1055

– 1060 (2012).

Study Summary

To assess the relations hip of exposure to pesticides during pregnancy and its association with gestational age and birth weight, Rauch and colleagues (EHP 2012) used the data collected from
mothers and newborns enrolled in the Health Outcomes and Measures of the Environment (HOM E) study, an ongoing prospective birth cohort study. They also examined whether these associations differed according to maternal race and infant PON1 genotypes status.

The study population was drawn primarily from Cincinnati, OH and consisted largely of women attending seven prenatal clinics in the Cincinnati metropolitan area between March 2003 and January 2006. Eligibility criteria to be enrolled in the study included being at least 18 years of age, living in a home built before 1978, and ≤ 19 weeks of gestation, being HIV negative, living within five surrounding counties; and not receiving thyroid or seizure medications, or chemotherapy or radiation treatments. Of 1,263 eligible women, 468 were enrolled in the study. Sixty

- seven women dropped out of the study before delivery. The remaining 401 women gave birth to 389 live singleton babies, as well as 9 sets of twins and 3 still

- born infants. Of those delivering singletons, 344 women provided urine at both 16 and 26 weeks gestation and they made up the pr imary study sample. In addition, 38 mothers were excluded in the final model; one mother because of a missing creatinine value, 22 because of missing covariate values, and 15 in the “other race” category. Thus, the final sample size was 306.

Pesticide ex posure was assessed by measuring the concentrations of six urinary nonspecific dialkyl phosphate (DAP) metabolites in two maternal spot urine samples collected at 16 and 26 weeks of gestation using a modification of the analytical method of Bravo et al. (2 004) that employs gas chromatography

- tandem mass spectrometry with isotope dilution calibration. DNA was extracted from frozen archived blood for PON genotyping using the TaqMan assays. Genotype data was available for 312 of 344 (90.7%) of infants, with 30 0 (87.2%) having identifiable single nucleotide polymorphisms (SNPs) at both PON1 sites. Birth weight wasPage 26 of 171 abstracted from medical records and gestational age was calculated from the mother’s self

reported date of last menstrual period. When this informatio n was not available, the results of an ultrasound (n = 7) was used or a Ballard examination performed just after delivery (n = 3) to determine gestational age. Demographic factors (maternal age, education, race/ethnicity, parity, income, marital status) we re obtained from subject interviews.

Multivariable linear regression was performed to examine associations between the sum of six DAP concentrations (ΣDAP) with gestational age and birth weight. In addition, difference in associations according to infant PON1_192 and PON1

– 108 genotypes and mother’s race were also examined. To examine gene

– environment interactions between PON1 genotype and OP pesticides, several PON

- stratified analyses were conducted, stratifying by PON1192 or PON1

– 108. Because race appeare d to be an effect modifier and PON1 genotype can vary across racial groups, authors also ran the PON1

- stratified analyses separately for black and white mothers whenever possible given sample sizes.

A 10

- fold increase in ΣDAP concentrations was associated with a 0.5

- week decrease in gestational age (95% CI:

– 0.8,

– 0.1) and 151 grams decrease in birth weight (95% CI:

– 287,

– 16). In race

- stratified models, ΣDAP concentrations were associated with shorte ned gestation in white newborns (

– 0.7 weeks; CI:

– 1.1,

– 0.3), but not in black newborns (

– 0.1 weeks; 95% CI:

– 0.9, 0.6). Associations with ΣDEAP and ΣDMAP concentrations were weaker, but ΣDMAP
concentrations were associated with a 0.4

- week decrease in gest ational length (95% CI:

– 0.7, 0.0). Birth weight was also inversely associated with ΣDAP concentrations and ΣDMAP concentrations (

– 124 g, 95% CI:

– 245,

– 2), but not ΣDEAP concentrations. PON1 genotype was also associated with maternal race/ethnicity in the study sample. Infants born to white mothers were more likely to have PON1192QQ (51.4% vs. 12.7%) and PON1108CT genotypes (45.7% vs. 20.0%) than infants born to black mothers. Infants with black mothers were more likely to have PON1192RR (44.3% vs. 10.4%) and PON1

– 108CC (76.3% vs. 29.8%) genotypes (all chi

square p

- values < 0.001).

In summary, prenatal OP insecticide exposure was associated with shorter gestation and lower birth weight in this cohort. The magnitude of these associations differed according to maternal race and infant PON1 genotype status. In infants born to white mothers (mostly PON1192QQ),
OP pesticide exposure was associated with shortened gestation and in infants born to black mothers (mostly PON1108CT), OP pesticide exposure was associa ted with lower birth weight.Page 27 of 171 Study Review

This was a well conducted study with notable strengths and few weaknesses. Study population included racially and demographically mixed birth cohort. The fact that two urine samples were collected for exposure as sessment and the infants PON1 genotype was taken into consideration to assess the magnitude of exposure between OP insecticide and birth outcomes, make this study unique. The analytic methods, analytic approach, and statistical analysis were appropriate. T he author’s approach to evaluating and adjusting for covariates were prudent.

Biomarkers of pesticide (DAPs, DEAPs, and DMAPs) were assessed in two maternal urine sample provided during 16 and 26 weeks gestation. So, this study was able to account for so me of the temporal variation in urinary DAP concentrations by combining the concentrations of two samples taken months apart. However, because of the short elimination half

- life, episodic nature of exposures to OP pesticides, and individual variation in me tabolism, , it is unclear the extent to which the biomarker assessment necessarily provided a good “level” of exposure. A urine sample was also collected within 24 hours of delivery but the data was not used to avoid confounding due to metabolic changes th at occur around the time of delivery.

Birth weight was likely to have been accurately measured, since it was abstracted from medical records. However, gestational age may have recall errors as that was obtained from the mother’s
self

- reported date of las t menstrual period. When this information was not available, the results of an ultrasound (n = 7) was used or a Ballard examination performed just after delivery (n = 3) to determine gestational age.

According to the study, decrements in birth weight and gestational age associated with ΣDAP concentrations were greatest among infants with PON1192QR and PON

– 108CT genotypes. The study did not measure serum levels of PON and other OP

- detoxifying enzymes, or maternal PON1 genotype, which may be more relevant du ring gestation and fetal development. Also, OP insecticide was associated with shorter gestation in white newborns but not in black newborns
and the reason of this association is not clear.

The authors were also able to collect data on, and adjust for, im portant covariates, including income, education, maternal depressive symptoms, maternal IQ, insurance status, area of residence, prenatal care, PON1 genotype, and gestational exposure to alcohol, lead, and tobacco. Covariates used in the analyses were sele cted, according to the authors, from variables known to be associated with gestational age and birth weight.Page 28 of 171 The authors also conducted sensitivity analyses. They found that mother’s medical conditions were associated with birth outcomes and DAP urinary c oncentrations. Mothers with medical conditions had shorter gestations and lower birth weight newborns than the rest of the study population.

Since this study evaluated the association between exposure to OP pesticides during pregnancy and gestational age and birth weight by maternal race, this study may be used in other studies in which race of a population is taken into consideration.

Authors concluded that maternal exposure to OP insecticide is inversely associated with gestation age and birth weight and the magnitude of these associations differed according to maternal race and infant PON1 genotype status.

References cited

Supplemental Material, Table 1 and Figures 1 and 2, http://dx.doi.org/10.1289/ehp.1104615Page 2 9 of 171 Study 6. Wolff et al. (2007), Furlong et al. (2014)

• 2007. Prenatal Pesticide and PCB Exposures and Birth Outcomes. MS Wolff, S Engel, G Berkowitz, S Teitelbaum, J Siskind, DB Barr, J Wetmur. Pediatric Research 61(2):243

- 250.

• 2014. Prenatal exposure to organophosphate pesticides and r eciprocal social behavior in childhood. Melissa A. Furlong, Stephanie M. Engel, Dana Boyd Barr, Mary S. Wolff.
Environment International 70:125

- 131.

Study Summary

In these reports on the Mt. Sinai Environmental Health Study, a multi

- ethnic prospective b irth cohort study in New York City, USA, Furlong et al. evaluate the relationship between prenatal organophosphate (OP) pesticide exposure and impaired social behavior during infancy among participant mothers and their children (Engel et al. 2007), while W olff et al. evaluate biomarkers of maternal exposure to DDE, PCB, and OP metabolites in the third trimester of pregnancy among 404 mothers in a multiethnic cohort in New York City and their effects on fetal growth.

Primiparous women with singleton pregnancies who delivered at the Mount Sinai Hospital between May, 1998 and July, 2001 were recruited at either an East Harlem clinic serving a predominantly minority population or at either of two private practices on the Upper East Side of
Manhattan. Of 479 mother

– infant pairs recruited, 75 were excluded, resulting in a final sample
of 404 mother

– infant pairs for whom birth data were available.

Maternal blood and urine samples were collected between 25 and 40 weeks of ge station (during the third trimester of pregnancy). Cord blood samples were obtained at the time of birth. Maternal blood plasma was used to analyze DDE and PCB levels in a random subset of women (n 194). Maternal urine samples collected before January 2001 (n 386) were analyzed by the Centers for Disease Control and Prevention (CDC) for six dialkylphosphate metabolites (DAPs) and malathion dicarboxylic acid (MDA) by GC

- tandem MS. Metabolite levels below the lower LOD were assigned a value equal to the LOD / √ 2 . Molar concentrations of diethylphosphate metabolites (DEP) and dimethylphosphate metabolites (DMP) were then summed to obtain total DEP and total dimethylphosphates, respectively. DEP and DMP totals were then together summed to obtain total dialkylphosp hates (DAP).Page 30 of 171 Lead levels (PbB) were measured in cord blood (n 102) or maternal whole blood (n 192) if a cord blood sample was not available.

To ascertain data on social and demographic characteristics, medical history, and lifestyle factors, a questionn aire was administered to the expectant mothers at the time of the urine specimen collection. Birth outcomes were obtained from a perinatal medical database.

Wolff et al (2007)

The authors modeled pesticide exposure and birth outcomes using linear models; covariates included environmental biomarker values, maternal race/ethnicity (white versus nonwhite), infant sex, and, except for those analyses predicting gestational age, gesta tional age. Models with total DAPs included PON1 phenotype as tertiles or PON192 genotype (RR, RQ, QQ), or BuChE. OC models were adjusted for maternal age. Additional covariates were identified using bivariate analyses for biomarkers (Spearman correlation for continuous and χ 2 or Mantel

- Haenszel (M

- H) for categorical variables with p < 0.15), including those reported previously for OCs (Wolff et al. 2005). Race, education, and marital status were highly correlated, and therefore only race was
included in bi rth outcome models (Berkowitz 2004). Continuous biomarker values were log

transformed, and 0.5 was added to total DAPs to remove zero values. Potential covariates were added to the base model for each birth outcome, and backward stepwise elimination was us ed to remove covariates that altered biomarker betas by <15%. Final alkylphosphate models did not have entered any variables besides the base covariates. The authors evaluated the effect of PON1 activity by comparing the least squares means for PON1 by exa mining the interactions between APs and PON1 or PON192 genotype for those models where a main effect at p< 0.1 had been seen for urinary APs, PON1, or PON192.

The authors observed maternal total DAPs exhibited weak, non

- significant inverse associations
wi th three birth outcomes: total DEP with birth weight, total DEP with Ponderal index, and total DAP with head circumference (adjusted; Table 3). MDA was not associated with any birth outcomes (p <0.15).

Furlong et al (2014)

This study was conducted on a subset of the initial cohort children (n= 136) who returned for the
7

– 9 year follow

- up visit. The investigators used regression analysis of non

- missing values toPage 31 of 171 impute six (of 136) metabolite levels lost due to “analytic interference” (Engel et al. 2007). Overall, approximately 97%, 89%, and 90% of the cohort had detectable levels of DAP, DEP and DMP metabolites, respectively.

Impaired reciprocal social behavior in childhood was measured by the Social Responsiveness Scale (SRS). The SRS is a caregiver su rvey of social behaviors originally designed to quantify impairments in reciprocal social behaviors in evaluating Autism Spectrum Disorders (Constantino and Gruber, 2005), but was used by the investigators of this study as a general indicator of impaired s ocial responsiveness common to a wider set of with neuropsychiatric conditions, including as ADHD, language problems, maladaptive behavior, social anxiety, and
mood disorders. Each of the 65

- items rates the frequency of a specific behavior on a Likert scal e, with higher scores indicating greater impairment.

Levels of DAP, DEP, and DMP (nm/L) were examined continuously (after Log10

- transformation) and using creatinine

- corrected tertiles (nm/g). Multivariate linear regression was used to analyze relationshi ps between the Log10

- transformed OP metabolite exposure biomarkers (independent variables) and continuous SRS total scores (the dependent variable). The investigators initially identified potential confounders (maternal age, maternal education,
race/ethnic ity, marital status, child sex, smoking during pregnancy, breastfeeding, housing type, child age at the time of testing, and urine creatinine) after constructing and evaluating a directed acyclic graph (DAG) to identify confounding pathways (Rothman et al. 2008). They then used backward elimination and a change

- in

- estimate criteria of 20% to construct a parsimonious
model. The authors also assess modification of the association between SRS score and DAP levels (independently) by child sex and race/ethnicity

.

Linearity of the exposure

– response curve was evaluated by estimating associations between SRS
score and tertiles of creatinine

- adjusted DAP levels as well as by constructing restricted cubic splines of the dose

– response function in exploratory analyses , although results of the smoothed association curves were not presented by the authors.

In their primary analyses, Furlong et al. did not observe statistically significant associations between prenatal maternal total DAPs, DEP, or DMP and total SRS scor es. Among all participants who returned for the 7

- 9 year visit (n=139), poorer social responsiveness as measured by the SRS was not associated with a 10

- fold increase in any of the OP metabolitesPage 32 of 171 metrics (DAP: β = 0.3, 95% CI:

- 2.3, 2.9); DEP: β = 1.8, 95% CI:

- 0.7, 4.3; DMP: β =

- 0.2, 95%
CI:

- 2.4, 2.0) in multivariate models.

Although overall, no statistically significant associations were observed between non

- specific prenatal OP metabolite (DAP, DMP, DEP) levels and impaired social responsiveness in ea rly childhood in this population of urban, inner

- city cohort, the investigators did observe some evidence of modification of the association between prenatal OP pesticide exposure and impaired social responsiveness in early childhood by race/ethnicity and child sex, with an association observed (only) among black participants and boys. Among black participants, a 10

- fold increase in DEP was associated with poorer social responsiveness (β = 5.1; 95% CI: 0.8, 9.4). There was no association among whites or His panics, or for DAP or DMP biomarker levels. A negative association between DEP and SRS score was observed among boys (β = 3.5; 95% CI: 0.2, 6.8), with no notable association observed among girls. The study did not have sufficient sample size (and correspon ding statistical power) to analyze the interaction between OP pesticide and the joint effect of race/ethnicity and child gender.

Furlong et al. report an association between one (of three) biomarkers of prenatal OP pesticide exposure (DEP) and poorer sco res on the Social Responsiveness Scale, indicating impairment of social responsiveness among blacks and possibly among boys in an urban, inner

- city cohort. It is unclear whether the association observed among blacks is due to differences in OP pesticide

ex posures or susceptibility factors associated with race/ethnicity. These results provide relatively weak evidence of a relationship between prenatal OP pesticide use and subsequent social impairment at age 7

- 9 years of age.

Study Review

These were well

- co nducted prospective cohort analyses of prenatal OP pesticide exposure and subsequent birth outcomes or social functioning at age 7

- 9 years of age in an urban setting in the US. The analytic methods, questionnaire, and analytic approach were appropriate. Th e statistical analysis was well specified and appropriate.

Wolff et al (2007)

This study tested whether exposures to organophosphate pesticides, as measured by diakylphosphate metabolites have an association with birth outcomes such as headPage 33 of 171 circumferenc e, birth weight, birth length, ponderal index, and gestational age in a population of mothers and their newborn infants in New York City

. This study presents very similar information with respect to pesticide exposure and birth outcomes as the previous rep ort from this cohort (Berkowitz et al. 2004) and doesn’t really add much to the literature on OP pesticide exposure and neurodevelopment. The strengths and limitations of this work are largely the same as the previous study.

Although the study was largely null, with respect to a relationship between the birth outcomes and the pesticide metabolites, the stratification by PON1 status provided some plausible information on individual susceptibility to pesticide exposure. As with any study, there are
notable s trengths and several weaknesses. Of note, no limitations were enumerated in the discussion of the study by the authors.

The prospective nature of the study provides assurance the OP pesticide exposure precluded the outcome measurement. Biomarkers of pesti cide were assessed in a single maternal urine sample provided during the third trimester. The authors do not discuss the impact of policies aimed at reducing use of chlorpyrifos on the prevalence and magnitude of pesticide exposures among the study populat ion; it is likely that use of chlorpyrifos exposure was decreasing over the time period of the study, as noted in other similar studies. The authors do not deliberate on the degree to which the biomarkers assessed represent usual exposure to pesticides ver sus recent, acute exposure(s), nor do they discuss the relative importance of assessment of exposure during the
third trimester vis

- à

- vis fetal growth and development. The critical window of exposure for pesticide effects is not fully characterized, but is likely occur either early (e.g., for effects on brain mass and volume) or late in gestation (e.g., for effects on birth weight and length). If sources and patterns of exposure remain static, a single measurement would be a good indicator of pesticide expo sure during pregnancy, even if the pesticides have short half

- lives in vivo. If, however, sources and exposure patterns change over short time

- spans, perhaps on the order of days to weeks, the biomarker assessment would not necessarily provide a good indic ator of the relevant exposure. The authors classified biomarker levels according to the assays’ levels of detection (above versus below). Falsely negative exposure characterization would occur if mothers were exposed only early in pregnancy, and the pestic ides were fully metabolized prior to the third trimester. Incorrect classification of participants as exposed, when in fact they were not exposed, was unlikely to have occurred using the biomarkers. Like other sources of measurement error in this study, errors in the biomarker indicators of exposure in this study are likely to have
resulted in a non

- differential misclassification, with respect to fetal growth and development.Page 34 of 171 The health outcomes in this study, head circumference, birth weight, birth lengt h, ponderal index, and gestational age were likely to have been accurately measured in this study, as Berkowitz et al. 2004 states they were obtained through direct linkage with the computerized medical record. Any errors in outcome measurement would have occurred in the clinic, and are likely to be small and certainly non

- differential with respect to pesticide exposure.

To some extent, the recruitment process resulted in a population that was likely homogeneous with respect to confounders of the in utero pesticide exposure

- fetal growth relationship (compared to the general population), and the potential for confounding was thus limited by design. The authors were also able to collect data on, and adjust for, important covariates, including race/ethnicity, infant sex, and gestational age. The authors also controlled for birth weight or birth length in their assessment of head circumference and pesticide exposure. Covariates used in the analyses were selected, according to the authors, from variables known t o be associated with either pesticide exposure or fetal growth. The authors did not include data on other environmental contaminants in their multivariate models, e.g., metals, organochlorine pesticides, polychlorinated biphenyls. To the extent that such c ontaminants are determinants of fetal growth and development and correlated with (but not caused by) pesticide exposure, they may confound the associations of interest in the study. If these co

- exposures are positively associated with both pesticide exposu re and diminished fetal development, they would bias the true association upward (towards larger mean differences associated with exposure). This is unlikely to have occurred in this largely null study, especially given that the effects of these compounds were largely null as well.

The authors chose to keep parsimonious models and only include factors that altered biomarker betas by <15% in the final models. This is preferable to using a statistical test as a means to select covariates from a larger set of potential confounders.

Bias due to confounding by unmeasured determinants of fetal growth and development is possible in this observational study. Most worrisome is the potential for confounding by factors
related to socioeconomic condition of those ass essed and co

- contaminants to which the study participants may have been exposed. Neither of these potential confounders is addressed in detail
in the article.Page 35 of 171 Bias due to differential errors in the selection of study participants is unlikely to have occu rred in this prospective study with no loss to follow

- up. Appropriate exclusion criteria were applied, including mothers with no prenatal medical visit prior to 26 weeks of gestation, mothers with serious chronic diseases (e.g., diabetes, hypertension, thy roid disease), mothers who developed a serious pregnancy complication that could affect fetal growth and development, and mothers who consumed more than two alcoholic beverages per day or who used illicit drugs, mothers with multiple births, and infants bo rn with a congenital malformation or severe prematurity.

The generalizability of the study may be limited as consequence of the particulars of the study population assessed and the numerous exclusion criteria applied. Making valid inference using
these r esults requires an assumption that the pesticide exposure

- fetal development association is the same in this population as the population to which the results are being generalized. This study population consisted of primiparous women, recruited early on in their pregnancies from the prenatal clinic and two private practices at Mount Sinai Hospital in New York from March 1998 to March 2002, consisted predominantly of young Hispanic women with low education. Sixty

- eight percent were less than 25 years old. Ju st under half were Hispanic (49.8%), although substantial numbers of White (21.0%) and African

- American (27.7%) women also participated. Just under half (46.8%) classified themselves as being either single, divorced, widowed, or separated, and just over ha lf (50.1%) had no more than a high school education. The exclusion criteria applied to potential study participants further focused the population assessed, relative to the general population of pregnant women. In the likely situation that the prevalence o f important modifiers of the exposure

- fetal development association in this focused study population is different from that in other populations of concern, generalizability will be limited. Important modifiers to consider may include time spent in the hom e, patterns of food preparation and consumption, health consciousness, the baseline health status of the mother (and father), and a host of socioeconomic determinants. The investigators finding of deferring effects by PON1 activity is illustrative of limit s on the generalizability of the study; populations who have high levels of paraoxonase activity vary by genetotypic and phenotypic determinants. The potential
limitation vis

- à

- vis generalizability is offset by a strength; the study has increased statistic al efficiency, relative to one set in a general population, as the infants are likely at relatively higher risk of sustaining cognitive and behavioral deficits. Furthermore, the population assessed in this study is of particular public health interest due to its high baseline risk of developmental challenges.Page 36 of 171 An adverse effect of OP pesticides on infant growth and gestational age is biologically plausible, as is modification of the pesticide

- fetal growth association. OP pesticides are a potential

neurotox ins detoxified by PON1. The finding that maternal PON1 activity modifies the association between markers of pesticide exposure and fetal growth and development is intriguing.

The net effects of confounding, selection bias, and information bias, are unlik ely to have qualitatively changed the results of this well conducted, prospective cohort study. Non

- specific biomarkers of exposure, as were assessed in this study, are likely more objective, sensitive, and likely also more accurate and reliable indicators of pesticide exposure, relative to exposure assessment methodologies that rely on self

- report. Almost certainly, such biases would not have resulted in the observation of a falsely positive association between pesticide exposure and fetal growth. More lik ely to have occurred is an attenuation of the true (unobserved) associations, toward (observed) associations that are relatively smaller, or null. Indeed, the findings of this study are either null, or associations of small magnitude.

Furlong et al. (201 4)

Inferences from the results of the investigation are constrained by limitations of the study design, including substantial loss to follow

- up, the exposure assessment (levels of DAP, non

- specific
biomarkers of OP pesticide exposure in a single prenatal urine sample), the assessment of social impairment (the SRS), and the methods used to select, and subsequently adjust for, confounders.

The authors employed a reasonable set of inclusion/exclusion criteria that likely resulted in
recruitment of an inner

- c ity study population that was ethnically diverse. The investigators prospectively collected maternal and child information (potential confounders and effect modifiers).

Selection bias due to substantial loss to follow

- up is likely to have occurred in the study. In this report, analyses were conducted among 136 mother

- child pairs, representing only a third of the 404 sets constituting the original cohort. This limited the study’s statistical power (efficiency) and more concerning, leaves open the possibili ty of selection bias resulting from loss to follow

up. The authors noted that the loss to follow

- up appeared only to be associated with marital status. However, it is not possible to evaluate the extent to which determinants of selection (outPage 37 of 171 of the study) also affected the children’s social impairment. One can, therefore, only speculate as to the magnitude and direction of bias induced by the loss to follow

- up.

The use of non

- specific biomarkers of prenatal OP pesticide exposure, as were assessed in this study, are likely more objective, sensitive, and likely also more accurate and reliable indicators of pesticide exposure, relative to exposure assessment methodologies that rely on self

- report. However, to the extent that in utero OP pesticide exposure is the true exposure of interest, the
assessment of non

- specific organophosphate pesticide metabolites in a single maternal sample of urine is a primary potential source of exposure measurement error. Levels of non

- specific OP metabolites in a single urine sample, as was conducted in this study, reflect very recent exposure, i.e., in the 24 to 48 hour period prior to the collection of the urine sample, and may or may not represent individual’s typical OP pesticide exposure. M oreover, urinary DAP metabolite levels may reflect exposure not only to OP parent compounds, but also to potentially nontoxic pre

formed metabolites in the environment. The use of a single urine sample to estimate maternal OP pesticide exposure is likely t o have resulted in an attenuation of associations, relative to those that would have been observed had the investigators used a time

- integrated marker of OP pesticide exposure.

On the whole, the investigators handling of confounding resulted was reasonab le. Aspects of the investigator’s approach to evaluating and adjusting for confounding were prudent, though other aspects of their approach, while conventional, have limitations. In particular, their use of DAG’s, avoiding adjustment for hypothesized inter mediates in the causal pathway between OP pesticide and social impairment as well as and “colliders,” their prospective ascertainment of covariate information from questionnaires and medical records, and their intention to construct a parsimonious model gi ven the limited sample size are all positive aspects of their methods. However, the use of a backward elimination strategy and covariate inclusion/exclusion based on a change

- in

- estimate criteria lack strong theoretical underpinnings. Although notionally l ess than ideal, these later methods were unlikely to have had a major negative impact on the study. Notable, there were large differences between beta coefficients estimated from unadjusted,
relative to the covariate

- adjusted models. This is evidence of st rong (net) confounding for which the authors appropriately adjusted.

The investigators evaluated a priori hypotheses regarding effect modification of the association
between OP pesticide and social impairment by race/ethnicity and sex of the child. Howev er, thePage 38 of 171 small sample size limited the statistical power of the tests of these hypotheses, and precluded the joint interaction between OP, race/ethnicity, and sex.

The instrument used to assess neurodevelopment in this study is commonly used, widely

accep ted, and has been validated. The authors noted that the SRS has good test

– retest temporal stability, parent

– parent and parent

– teacher interrater agreement, and discriminate and concurrent validity (Constantino and Gruber, 2005).

References cited

Berkowitz GS, Wetmur JG, Birman

- Deych E, Obel J, Lapinski RH, Godbold JH, Holzman IR, Wolff MS 2004 In utero pesticide exposure, maternal paraoxonase activity, and head circumference. Environ Health Perspect 112:388

– 391.

Constantino JN, Gruber CP. Social Responsiveness Scale. Western Psychological Sciences; 2005 [Los Angeles, CA].

Engel SM, Berkowitz GS, Barr DB, Teitelbaum SL, Siskind J, Meisel SJ, et al. Prenatal organophosphate metabolite and organochlorine levels and performance on the Brazelton Neona tal Behavioral Assessment Scale in a multiethnic pregnancy cohort. Am J Epidemiol 2007;165:1397

– 404.

Rothman KG, Greenland S, Lash T. Modern epidemiology. Philadelphia, PA: Lippincott Williams & Wilkins; 2008.

Wolff MS, Deych E, Ojo F, Berkowitz GS 2005 Predictors of organochlorines in New York City pregnant women, 1998

– 2001. Environ Res 97:170

– 177.Page 39 of 171 Study 7. Anderson et al. (2015)

Occupational pesticide exposure in early pregnancy associated with sex - specific neurobehavioral deficits in the children at school age. Helle R. Andersen, Fróði Debes,
Christine Wohlfahrt

- Veje, Katsuyuki Murata, Philippe Grandjean. Neurotoxicology and Teratology 47:1

- 9, 2015.

Study Summary

Anderson et al. reported on r esults of a study evaluating maternal occupational pesticide exposure during early pregnancy and impaired neurodevelopment in children at school age in a
birth Cohort.

This prospective birth cohort study, set in Denmark between 1996 and 2000, recruited w omen working in greenhouse horticultural occupations at the beginning of their pregnancy in order to study the potential health effects of prenatal pesticide exposure. Specifically, the investigators evaluated performance on a battery of neurobehavioral te sts among children age 6 to 11 years of age, comparing children of women considered pesticide exposed during early pregnancy, to those from mothers who were defined as not having pesticide exposure during early pregnancy.

At the start of the study, pregn ant women working in greenhouses were recruited for participation and evaluated by one of two toxicologists at Odense University Hospital in Denmark with expertise in greenhouse working conditions in order to categorize them with respect to pesticide expos ure. A detailed occupational history was collected via interview to elucidate the working conditions in the previous three months, to include job function, personal handling of pesticides, types of pesticides handled, frequency of pesticide application, pr otocols for reentering greenhouses where pesticides had been recently applied, and use of personal protective equipment. These interviews were conducted prior to any examinations of the women’s children. The women were defined as having occupational pestic ide exposure if pesticides were applied in their work area more than once per month and the women handled pesticide

- treated plants within one week after treatment and/or the women were directly involved in applying pesticides (only 6% of women applied pest icides). Women who met none of the above criteria were categorized as occupationally unexposed to pesticides cases. The women deemed pesticide

- exposed, either went on paid leave or were transferred to work functions withPage 40 of 171 less or no pesticide exposure short ly after enrollment in the study. The occupational pesticide exposure was thus confined to the early weeks of the first trimester of pregnancy.

Initially, 168 children categorized as having had prenatal pesticide exposure and 35 children categorized as n ot having had prenatal occupational pesticide exposure were enrolled in the study. Of this original cohort of children, 133 accepted an invitation to participate in this follow

up study at age 6

- 11 years old, 112 of whom had prenatal pesticide exposure, an d 21 of whom were prenatally unexposed. An additional 44 unexposed children were recruited from among friends, relatives, and neighbors of the participating families to supplement the unexposed group.

A questionnaire was completed prior to the neurobehavi oral examination to solicit information on medical history, education, occupation, lifestyle, and diet. Additional information on medical history, lifestyle, and behaviors to include diet, smoking, alcohol use, and medication use was
collected at base

- line , or prior to the neurobehavioral assessment for the new families enrolled in the study to supplement the number of unexposed children.

A standardized clinical exam was conducted on all children, to include the following neurologic functions: social resp onse, spontaneous motility, involuntary movements, Romberg’s sign, walking on a straight line, standing on one leg, hopping, reflexes, patellar reflexes, finger opposition test, diadochokinesis, finger

- to

- nose coordination, hearing and vision. Heart rate

v ariability and brainstem auditory evoked potential latencies were assessed to evaluate neurophysiological status. Neuropsychological tests included manual motor speed (finger tapping test), simple reaction time, attention (Conners' Continuous Performance T est II), short

term memory (Wechsler Intelligence Scale for Children

- Revised Digit Span Test), language function and long

- term memory (Woodcock

– Johnson III Tests of Cognitive Abilities, Verbal Comprehension test), and visuospatial performance and memory fu nctions (Raven's Colored Progressive Matrices).

The authors selected potential covariates from a list of determinants for each clinical, neurological, neuropsychological, neurocognitive, and neurobehavioral outcome considered a priori. These included age at examination, gender, maternal ethnicity, maternal smoking and maternal alcohol consumption, and social class. The statistical analysis consisted of multivariate linear regression models of the numerous neurobehavioral outcomes, followed by construction of a structural equation model to analyze the interaction between child sex and prenatal pesticide exposure on general intellectual ability parameterized as a latent variable.Page 41 of 171 After evaluating a large number of potential associations between prenatal pest icide exposure and neurobehavioral outcomes, Anderson et al. observed largely null associations between maternal occupational pesticide exposure early in pregnancy and their child neurodevelopment as assessed at age 6

- 11 years of age. A few associations we re observed that were consistent with a hypothesis that maternal occupational pesticide exposure early in pregnancy effects their children's neurodevelopment, as assessed at age 6

- 11 years of age. Specifically, prenatal pesticide exposure was significantly associated with prolonged brainstem auditory evoked potential latencies and, among girls, also with impaired neuropsychological function. Indicators of language development and motor speed functions were also inversely associated with maternal pesticide e xposure during pregnancy among girls only.

Study Review

The interpretation of the results of this prospective birth cohort study of prenatal maternal occupational pesticide exposure and neurobehavioral deficits conducted among Danish women working in gre enhouses and their children is constrained by substantial loss to follow up of the original cohort. The potential for selection bias, and, to a lesser extent, residual confounding, further limit the study. Some degree of both differential and non

- different ial errors in recall of exposures and outcomes is probable. Assessment of the interview

- based proxy indicator of pesticide exposure was well conducted, though no measurements of pesticide levels or biomarkers were made. Similarly, the assessment of neurobe havioral outcomes was comprehensive.

Selection of participants was reasonable; however, the sample size of this study was likely not sufficient (consisting of too few study participants), given the investigators' objective to evaluate a large number of a ssociations between prenatal pesticide exposure and subsequent neurobehavioral outcomes, especially considering their decision to evaluate effect modification by child gender. The small sample size was, in part, due to the loss of more than 30% of the orig inal study sample, this occurrence reduced statistical power and potentially induced selection bias. Mothers who had greater exposure, or greater concern regarding their occupational pesticide exposure and subsequent effects on their child's health may hav e been more likely to continue their participation in the study. The author's decision to address the attrition by augmenting the original non

- exposed group with additional children not sourced from the original cohort in order to increase the statistical power of the comparisons between exposed andPage 42 of 171 non

- exposed with respect to neurobehavioral status is also a potential source of selection bias. For example, the authors noted that the families of the newly recruited non

- exposed children were more likely to b elong to lower social classes, compared to children from the original birth cohort. The investigators appropriately conducted sensitivity analyses of the neuropsychological outcomes that were limited to children from the original birth cohort, observing as sociation estimates similar to those in their primary analysis.

The pesticide exposure assessment was well conducted, though exposure classification was made based on occupational history rather than actual measurements of pesticide levels or biomarkers of exposure. Very few (~6%) of the mothers actually applied pesticides as part of their job duties, and it is possible that actual exposure was very low, especially considering that personal protective equipment was available and was reported to have been used (by an unspecified proportion of the mothers). The specific pesticides classes or formulations to which the women were exposed during early pregnancy were not elucidated. The authors noted that, “the exposure situation was complex,” with use of specif ic pesticides varying with time and location, between and within companies, and according to production requirements and the type of pests being controlled. The authors noted that approximately 200 different pesticide formulations, with 124
different activ e pesticide ingredients, were used in the greenhouses. Organophosphates (e.g., dichlorvos, dimethoate, and chlorpyrifos), were used frequently in the working areas.

The transfer of women classified as having been occupationally exposed to pesticides to du ties with less or no pesticide exposure, or to paid leave, after enrollment in the study has several implications. Though not stated explicitly in the report, the transfers were likely conducted for ethical considerations. Primarily, this restricted the ev aluation of occupational pesticide exposures to those that occurred during early pregnancy (in contrast to many other investigations included in this review, which focused on exposures occurring during late pregnancy (e.g., during the third trimester). The influence of these transfers on other behaviors

- particularly those that influence fetal development

- is unknown. It is plausible that the women’s awareness of their occupational exposure and the change in job duties motivates them to make healthy choic es which positively influence fetal development. Such an influence could potentially explain the lack of associations between occupational pesticide exposure and subsequent neurodevelopmental status.

The selection and consideration of covariates for incl usion in multivariate regression models was appropriate. The acquisition of covariate information by questionnaire may have resulted inPage 43 of 171 residual confounding due to recall errors, particularly for mothers of the children enrolled to augment the non

- exposed group, who were asked to recall exposures during their pregnancy (e.g., smoking behaviors and alcohol use) which occurred 7

- 11 years prior to completion of the survey. The author's classification of social class into broad categories, a design decision mad e in light of the relatively small sample size, leaves open the possibility of residual confounding by
socioeconomic status.

Neuropsychological tests were given and scored by the same neuropsychologist. The same pediatrician performed all clinical examina tions. The same examiner conducted all BAEP latency and heart rate variability calculations. The neuropsychological tests used in the study chosen for their validity, and demonstrated sensitivity to neurotoxicity of environmental pollutants, in addition to their brevity.

The authors evaluated numerous hypotheses (>20) regarding the association between prenatal pesticide exposure and neurobehavioral deficits without adjusting for multiple comparisons. Under a null hypothesis of no effect of pesticide exposu re on these outcomes, one can expect to observe a 5% error rate, i.e. 'false positives' due to chance, and one cannot discount chance as a determinant of the observed associations in this study. Similarly, one cannot discount the possibility that the findi ngs of apparent effect modification by gender were also due to chance. The finding of associations only among girls is unique to this investigation. That said, the investigator's use of a structural equation model avoided specification of an even larger nu mber
of statistical models.

The authors' conclusion that their findings suggest an adverse effect of prenatal occupational pesticide exposure on their children's neurodevelopment overstates the study findings which were 1) largely null, and 2) largely obs erved only among girls who represented less than half of the study population.

Furthermore , given that a proxy exposure assessments was used in this study , the pesticides used may have included not only OPs, but also pyrethroids, fungicides, and growth r egulators. Given the uncertainty about whether OP exposure actua lly occurred in this studies and whether observed outcomes are associated with OP exposure or with other pesticides, this study was excluded from further analysis in the final synthesis of th e OP epidemiological literature review ( add citation )

.Page 44 of 171 Study 8. Guodong et al. (2012)

Organophosphate Pesticide Exposure and Neurodevelopment in Young Shanghai Children. Ding Guodong, Wang Pei, Tian Ying, Zhang Jun, Gao Yu, Wang Xiaojin, Shi Rong, Wang Guoquan, and Shen Xiaoming. Environmental Science and Technology 46:2911

- 2917, 2012.

Study Summary

In this report, Guodong et al. evaluate the relationship between organophosphate (OP) pesticide exposures and neurodevelopment in two

- year old healthy children living in Shanghai, China using a cross

- sectional study design with a single assessment of urinary OP metabolites and a single assessment of neurodevelopment in conjunction with a questionnaire to ascertain demographic and additional expos ure information from the children’s mothers.

Study participants were recruited during a nine

- month period in 2008. The investigators invited healthy children visiting the child healthcare departments of two community hospitals for routine physical examin ations. Children were considered eligible for inclusion in the study if they were between 23 and 25 months of age, were capable of completing the neurocognitive assessment, and had no history of any of the following: intrauterine distress, pathologic jaund ice, intrauterine infection, intracranial infection, or congenital disease. The parents of 301 of 310 children identified as having met these inclusion criteria provided consent, allowing their children to participate in the study.

Urine samples were coll ected from each participant at intervals throughout the day of their study assessment. Collected samples were stored at 2−4 °C, or frozen until shipment (on dry ice) to the CDC in Shanghai China, where they were stored at −70 °C until analysis. Participant s’ urine samples were analyzed for five non

- specific diakyl phosphate (DAP) metabolites (the dimethyl metabolites dimethylphosphate (DMP) and dimethylthiophosphate (DMTP), and the diethyl metabolites diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP)) using gas chromatography with flame photometric detection (GC

- FPD). The limit of detection (LOD) was calculated for each of the five DAP metabolites from the instrument response factor corresponding to a concentration havin g a peak area three times that of blank measurement. The LOD was 1.0 μg/L for all analytes except for DMP which had a LOD of 2.0 μg/L. All metabolite levels below the LOD were assigned a value equal to the LOD / √ 2 . The urinary DAP metabolite concentrations were creatinine

- adjusted. The creatinine

- adjustedPage 45 of 171 geometric means (GMs) for the five urinary DAP metabolites were 11.27 μg/g for DMP; 6.99 μg/g for DMTP; 7.96 μg/g for DEP; 14.19 μg/g for DETP; and 4.55 μg/g for DEDTP. These are high relative to those comm only observed in studies conducted in developed countries: Urinary concentrations (μg/L) of the nonspecific OP metabolites were also converted to summed molar concentrations (nmol/L) of the total DAPs, the two dimethyl metabolites, and the three diethyl

me tabolites were to provide summary measures of OP pesticide exposure.

A questionnaire was administered to the mother of each child by a trained interviewer in order to garner demographic and socioeconomic information as well as exposure characteristics,

e nvironmental exposures, child characteristics and maternal characteristics. The children’s medical records were also used to derive anthropomorphic characteristics.

Neurodevelopment was assessed using the Gesell Developmental Schedules (GDS). The partici pating children were administered a revised version of the GDS for 0

- 3 year

- old children, adapted for use in the Chinese population and conducted by a pediatrician trained and certified in the administration of the GDS (Beijing Mental Development Cooperati ve Group, 1985). The items in the GDS capture four domains of neurodevelopmental functioning: motor behavior, adaptive behavior, language behavior, and social behavior. Each child was assigned a developmental quotient (DQ) in each of the four domains.

Th e creatinine

- adjusted DAP metabolites were summed and Log10

- transformed prior to being entered into multivariate linear regression models predicting each of the four domain

- specific GDS DQ. All models were adjusted for covariates associated with neurodevel opment in the literature and significantly associated with 2 or more GDS DQ in this population: child sex, maternal education level, and household income. The authors did not assess modification of the association between GDS and DAP levels by other factor s.

The investigators did not observe any statistically significant associations between the children’s urinary DAP metabolite levels and neurodevelopment as measured by the four domain

- specific GDS DQ quotients. Regression coefficients (β) and correspond ing 95% confidence intervals (95% CI) and p

- values for log10

- tranformed total non

- specific DAP metabolites were as follows, for each of the GDS functional areas. Total dialkyl phosphates: Motor Area: 0.30 (95% CI:

− 1.40, 1.99), p=0.73; Adaptive Area: 1.71 (95% CI: −1.15, 4.57), p=0.24; Language Area: 2.79 (95% CI: −1.01, 6.60), p=0.15; Social Area: −0.66 (95% CI: −2.12, 0.79), p=0.37. Results forPage 46 of 171 log10

- transformed molar concentrations of dimethyl phosphates were as follows, for each of the
GDS functional ar eas: Motor Area: −1.25 (95% CI: −2.98, 0.47), p=0.1; Adaptive Area: 2.53 (95% CI: −0.05, 5.10), p=0.06; Language Area: 2.83 (95% CI: −0.60, 6.26), p= 0.11; Social Area: −0.48 (95% CI:

− 1.93, 0.97), p= 0.51. Regression coefficients for log10

- transformed mo lar concentrations of diethyl phosphates were as follows, for each of the four GDS functional areas: Motor Area: 0.32 (95% CI: −1.37, 2.01), p= 0.71; Adaptive Area: −0.41 (95% CI: −3.22, 2.39), p= 0.77; Language Area: −0.29 (95% CI: −4.02, 3.P244), p= 0.88 ; Social Area: −0.93 (95% CI: −2.40, 0.54), p= 0.22. No other associations between DAP levels and GDS DQ were presented in the paper.

Study Review

This was a well

- conducted study of OP pesticide effects on neurodevelopment conducted in a population of h ealthy, young (2

- year old) Chinese children. The analytic methods, questionnaire, and analytic approach were appropriate. The statistical analysis was not specified in great detail, but appears to be appropriate. That said, inferences to be garnered from t he results of the investigation are constrained by limitations of the study design (cross sectional), the exposure assessment (levels of DAP, non

- specific biomarkers of OP pesticide exposure in multiple urine samples collected on a single day), and the ass essment of neurodevelopment (the GDS).

The authors employed a reasonable set of inclusion/exclusion criteria that likely resulted in recruitment of a study population that was relatively homogeneous and characterized by normal neurodevelopment, on the whole; greater than 97% of the population w as considered normal in three of the four GDS neurodevelopmental domains, with 93.7% scoring in the normal range in the language area. The relative homogeneity of the population may have limited statistical power, but it also limited confounding by design. The investigators collected participant information corresponding to an almost exhaustive list of potential confounders (summarized in table 1 of the paper), and found only three factors (the child’s sex, maternal education level and household income) tha t were significantly associated with two or more of the GDS DQ, even at a relaxed alpha level of 0.10. Strong residual confounding by unmeasured or poorly measured confounders is unlikely in this study. The investigators did not discuss observed relationsh ips between the potential confounding factors and the children’s DAP biomarker levels.

Selection bias is unlikely to have occurred in the study.Page 47 of 171 A limitation of the cross

- sectional design is the reliance on the untestable assumption that (even valid) b iomarkers of OP pesticide exposure measured in urine sampled at the time of the neurodevelopmental assessment are indicative of ‘usual’ OP pesticide exposure, or, more to the point, accurately reflect OP pesticide exposure at the critical time

- window(s) fo r exertion of deleterious effects on neurodevelopment.

The use of non

- specific biomarkers of OP pesticide exposure, as were assessed in this study, are likely more objective, sensitive, and likely also more accurate and reliable indicators of pesticide

e xposure, relative to exposure assessment methodologies that rely on self

- report. However, to the
extent that OP pesticide exposure during early childhood is the true exposure of interest, the
assessment of non

- specific organophosphate pesticide metabolites in a single sample of urine is a primary potential source of exposure measurement error. Although considered by the authors to be the current standard for characterizing exposure to multiple OP pesticides that originate from different sources, the authors concede that urinary DAP metabolite levels may reflect exposure not only to OP parent compounds, but also to the potentially nontoxic pre

- formed metabolites in the environment. The geometric mean DAP concentrations observed in this investigation are highe r than those observed in the US (CDCP, 2009; Barr et al. 2004), a German reference sample (Becker et al., 2006), and a study of child biomarker levels conducted in Italy (Aprea et al., 2000). This difference between DAP biomarker levels observed in childre n living in Shanghai could be due to higher levels of OP pesticide exposure, compared to those of Western populations, though the difference noted could also be due to differences in laboratory methods; the lower limit of detection in this study was higher than those used by Barr et al., and similar to those of the German and Italian studies. The authors concluded that their relatively high LODs did not influence the essential findings of their study, though it is possible that the high end of the OP pestic ide exposure distribution is higher than that of the US exposure distribution and the investigators’ laboratory methods could not resolve the low end of the exposure distribution, resulting in a higher overall observed average concentration, than that whic h would have been estimated using DAP quantification methods similar to those employed in the US.

The collection of multiple maternal urine samples throughout the day makes for a more robust estimate of OP pesticide biomarker levels on the day of the samp le collection, but, nevertheless, the use of a single day’s urine samples to estimate the participant’s ‘usual’ OP pesticide exposure is likely to have resulted in an attenuation of associations, relative to those that would have been
observed had the inve stigators used a time

- integrated marker of OP pesticide exposure.Page 48 of 171 The investigators did not report having assessed effect modification by any factors.

The instrument used to assess neurodevelopment in this study (i.e., the GDS for 0

- 3 year olds, adapte d to the Chinese population) is commonly used, widely accepted, and has been validated in the Chinese population.

The authors rightly conclude their paper by stating that a large, longitudinal study with repeated measurement of urinary biomarkers of OP pesticide exposure is needed to further examine the relationship between OP pesticide exposure and child neurodevelopment in this Chinese population. The findings of this well

- conducted cross

- sectional study of children living in Shanghai are not consis tent with the hypothesis that OP pesticide exposure adversely affects child neurodevelopment at two years of age.

References cited

Barr, D. B.; Bravo, R.; Weerasekera, G.; Caltabiano, L. M.; Whitehead, R. D. Jr.; Olsson, A. O.; Caudill, S. P.; Schober, S. E.; Pirkle, J. L.; Sampson, E. J.; Jackson, R. J.; Needham, L. L. Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the U.S. population. Environ. Health Perspect. 2004, 112,186−200.

Becker, K.; Seiwert, M.; Angerer, J.; Kolossa

- Gehring, M.; Hoppe, H. W.; Ball, M.; Schulz, C.; Centers for Disease Control and Prevention. Fourth National Report on Human Exposure to Environmental Chemicals, December 2009. http://www.cdc.gov/exposurereport/ (accessed June 06, 2015)

Thumulla, J.; Seifert, B. GerES IV pilot study: assessment of the exposure of German children to organophosphorus and pyrethroid pesticides. Int. J. Hyg. Environ. Health 2006, 209, 221−233.

Aprea, C.; Strambi, M.; Novelli, M. T.; Lunghini, L.; Bozzi, N. Biologic monitoring of exposure to organophosphorus pesticides in 195 Italian children. Environ. Health Perspect. 2000, 108, 521−525.Page 49 of 171 Study 9. Handal et al. (2007, 2007B, 2008)

• 2007

- Effect of Community of Residence on Neurobehavioral Development in Infants

an d Young Children in a Flower

- Growing Region of Ecuador. Environmental Health Perspectives. 2007; 115(1):128

- 133.

• 2007B

- Neurobehavioral Development in Children With Potential Exposure to Pesticides. Epidemiology. 2007; 18(3):312

- 320.

• 2008

- Occupational Exposure to Pesticides During Pregnancy and Neurobehavioral Development of Infants and Toddlers. Epidemiology. 2008. 19(6):851

- 859.

Study Summary

Handal et al. examined neurobehavioral development in children from the EcoSalud project,
launched in 2001 in the Cayambe

- Tabacundo region, Ecuador in two communities: 1) lower altitude communities dominated largely by cut

- flower production, with high potential for exposure to organophosphate (OP) and carbamate pesticides, and 2) a higher altitude community
wit h low potential for exposure to pesticides. The first report (Handal et al., 2007) compared neurobehavioral development in infants and young children 3 to 61 months of age living in two communities. The second report (Handel et al., 2007b) considered house hold and community risk factors for pesticide exposure and their potential effects on neurobehavioral development of young children aged 24 to 61 months. The third report in the series (Handal et al., 2008) looked at potential effects of maternal occupatio n in the cut

- flower industry during pregnancy on neurobehavioral development in children 3 to 23 months of age.

In 2003

- 2004, a census of the three communities was taken to construct the sampling frame. All mothers living at least one year in the communit y with and one or more children, age 3

- 61 months were eligible. In total, 219 mothers (91.3% of those eligible) and 283 children participated in the study. Mothers were interviewed to obtain information on socioeconomic status, maternal occupational histor y, maternal and child health characteristics, and child’s socialization and exposure profiles. Examinations were conducted by trained testers.

• Children’s height, weight, and head circumference were measured.Page 50 of 171 • The child’s hemoglobin was measured from single finger

- stick blood samples (HemoCue hemoglobin blood testing kit).

• Neurobehavioral development was assessed using the Ages and stages Questionnaire (ASQ), a widely

- used developmental screening test, composed of 19 age

- specific questionnaires that co ver 5 broad developmental dimensions: communication, fine and gross motor skills, problem solving, and personal

- social skills. Each domain is scored from 0

– 60 points, with 60 being a perfect score. A continuous score is calculated for each age

- specific q uestionnaire, with scores summarized for each developmental domain.

• Visual Motor Integration (VMI) Test (ages 48

- 61 mos)

- Beery

- Buktenica, 4th Ed.

• Reach

- and

- grasp, bi

- manual coordination) abilities, videoed (ages 9

- 18 mos), 1 trained tester

• Visual acuity skills (ages 9

- 18 mos): UC Berkeley Preferential Looking Test Cards (WHO visual impairment classifications), 1 trained tester

Instruments were pre

- tested to ensure clarity and comprehension and researchers performed pilot

- tests to ensure high percent agree ment between testers.

2007 analysis of community of residence on neurobehavioral development (ages 3

- 61 months)

The associations between community of residence and delayed development (via ASQ) were evaluated using multiple linear regression, adjusting f or child health status and other characteristics of the home environment. Anthropometric measures included child and mother height and weight; child’s birth weight from child’s vaccine record card, or mother report; chronic malnutrition (stunting) from chi ld’s height

- for

- age z

- score (2 standard deviations below the median); presence of anemia (via hemoglobin), after taking into account the child’s age and the altitude of the community of residence; stimulation by two variables

- attendance at child

care ce nter and type and frequency of stimulating activities mother engaged in at home with each child in the preceding 3 days (6 questions adapted from UNICEF multi

- country survey); home
environment

- relationship with husbands, or their families in the case of single mothers; maternal/paternal education, mother’s ethnicity and predominant language preference, marital status, age; monthly household income; housing construction characteristics (roof composition, floor composition, wall composition, type of water u sed in home, bathroom type, and access to electricity were summarized into a housing scale); social position (job of principal economic provider of family). To handle correlations due to sibling pairs, one sibling was randomlyPage 51 of 171 removed from each pair. Pairw ise t

- tests and chi

- square were used to assess the mean difference in ASQ score between community comparison groups. The magnitude of the effect of community residence on ASQ scores was analyzed using Cohen's d.

Younger children (3

– 23 months of age) who resided in high

- exposure communities scored lower in all 5 domains, but significantly lower on gross motor (8.8 points, p = 0.002), fine motor (5.0 points, p = 0.06), and socio

- individual (5.8 points, p

- value = 0.02) skills, compared with children
in the l ow

- exposure community. In children 24

– 61 months of age residing in high

- exposure communities, developmental delays were present in 2 of 5 domains, significantly lower scores on gross motor skills compared with children of similar ages residing in the low

- e xposure community (3.8 points, p = 0.06).

In younger children (3

– 23 months of age), the effect of high

- exposure community on gross motor skill development was greater for stunted children compared with non

- stunted children (17 points lower, p = < 0.001). Children who attended child care in high

- exposure communities scored significantly better in their fine motor skills.

This preliminary study suggests poorer neurobehavioral development in infants and children residing in high

- exposure communities, especi ally in the development of gross motor skills, even after controlling for major determinants of delayed development such as low stimulation at home, presence of anemia, and stunting. Findings also suggest that there may be a double burden of pesticide expo sure and poor nutrition (i.e., stunting) on the neurobehavioral development in infants and young children, but that child care and increased stimulation at home may attenuate
these effects.

2007B analysis of neurodevelopment in children with exposure to p esticides (age 24

- 61 months)

Distribution of key risk factors (distance home to flower farm, maternal employment, father's current work status, pesticide use on domestic crops, pesticide use within home, use of potentially

- contaminated plastics/wood from flower farm, child play activities) was assessed via

cross

- tabulation for collinearity, and chi

- square tests for differences across risk factor categories.
ANOVA and t

- tests were used to assess differences in mean developmental scores among risk
factors. M ultiple linear regression models for each domain were constructed to assess effects of household environmental exposures and development, after controlling for key confounders. DuePage 52 of 171 to limited sample size, only variables found to be associated with each ASQ domain and the exposure variable of interest were included in each model.

Mother’s current employment in the flower industry was associated with better scores among their children for all 5 ASQ domains. Specifically, current employment in flowers was mo re strongly associated with better communication (beta 4.13, SE 2.23) and problem

- solving (beta 5.03, SE 2.20) skills. Employment in the flower industry during pregnancy, however, showed a positive association only with the problem

- solving skills domain (b eta 3.57, SE 2.11).

Pesticide use on domestic crops was also associated with better gross motor (beta 4.86, SE 2.18) and personal

- social (beta 4.12, SE 2.46) scores, while conversely, pesticide use within the home
was associated with lower communication scores (beta

- 4.51, SE

- 2.15, effect size: d=0.2). More hours spent outdoors (5+ hrs/d) was associated with lower gross (beta

- 2.52, SE

- 1.94) and fine motor (beta

- 2.12, SE

- 2.41) and problem solving (beta

- 3.28, SE

- 2.16) skills. Occasional or frequent p laying in irrigation water was associated with poorer fine motor (beta

- 4.92, SE

- 2.22) and problem solving (beta

- 4.39, SE

- 2.03) scores. On average, a child who played with irrigation water scored approximately 5 points lower on fine motor skills (8.2% decrease; 95% CI 9.3 to 0.5; effect size, d=0.4), 4.4 points lower on problem solving skills (7.3% decrease; 8.4 to 0.4; effect size, d=0.3), and 5.5 points less on the VMI test (3.4% decrease; 12.0 to 1.1; effect size, d=0.2) compared with children who ra rely or never played with irrigation water.

Current maternal employment in the flower industry was associated with better developmental scores. Longer hours playing outdoors were associated with lower gross and fine motor and problem solving skills. Children who played with irrigation water scored lower on fine motor skills (8% decrease; 95% CI

- 9.31 to

- 0.53), problem

- solving skills (7% decrease;

- 8.40 to

- 0.39), and VMI test scores (3% decrease;

- 12.00 to

- 1.08).

Current employment in the flower in dustry, longer hours spent outdoors playing, and contact with irrigation water were all associated with lower VMI scores; however, estimates are imprecise, as the VMI was administered only to a subset of the study, thus reducing the sample
size.

Certain e nvironmental risk factors for exposure to pesticides may affect child development, with contact with irrigation water of particular concern. However, the relationships between these risk factors and social characteristics are complex, as corporate agricult ure may increase risk throughPage 53 of 171 pesticide exposure and environmental contamination, while indirectly promoting healthy development by providing health care, relatively higher salaries, and daycare options.

2008 analysis of occupational exposure to pesticide s during pregnancy and neurodevelopment of infants and toddlers (age 3

- 23 months)

For this third report, multiple linear regression models were constructed for three domains to assess effects on neurobehavioral development in 3

- 23 year old children of mate rnal occupational exposures during pregnancy, after controlling for potential confounders. Maternal exposure variables for problem

- solving and personal

- social skills were excluded as none was associated. Logistic regression models were constructed for the associations with prehension tests (pellet test, box test); generalized estimation equations (GEE) were used to account for dependence between tests. Proxy exposure measures included maternal employment during pregnancy, total months worked during pregnanc y, total hours worked per week during pregnancy, primary job responsibility, child plays outdoors, and pesticides used on domestic crops and inside the home.

On average, children whose mothers worked in the flower industry during pregnancy scored 8%
lowe r on communication skills (

- 4.6 points, 95% CI

- 9.6 to 0.3) and 13% lower on the fine motor skills (

- 8.0 points;

- 13 to

- 3) compared with children whose mothers did not work, after adjusting for key confounders. Inclusion of low birth weight in the regres sion models did not change the estimates of the effect of maternal work in the flower industry during pregnancy on neurobehavioral development (data was not shown).

Researchers observed similar results for use of pesticides in the workplace, including low er fine motor score (13% decrease in score, 95% CI

- 21% to

- 5%), lower communication (6% decrease in score, 95% CI

- 15% to 2%), and poorer visual acuity (OR 3.9; 0.94

– 16) scores among children whose mothers reported pesticide use in the workplace during p regnancy compared with those whose mothers did not. Paid work, which included many flower workers and some non

flower workers, also yielded similar results, with lower fine motor scores (12% decrease in score, 95% CI

- 20% to

- 4%) and poorer visual acuity ( OR 3.3; 0.80

– 13 ) among those who worked outside the home during pregnancy compared with those who did not work outside the
home.

Greater total weekly hours worked during pregnancy (>45 h/wk) and pesticide use at home during pregnancy were associated wit h better gross motor scores. All measures of exposurePage 54 of 171 except for pesticide use at home during pregnancy were negatively associated with fine motor

scores.

For the large

- pellet test, higher frequencies of poorer grasp scores (<3) were found for work in
th e flower industry during pregnancy, use of pesticides in the work place during pregnancy, more hours worked per week during pregnancy, and working 6 months or more of the pregnancy. For a greater number of children with poor visual acuity, maternal work in the flower industry during pregnancy was reported. The odds of succeeding in grasping the large pellet upon first contact were approximately half for those children whose mother worked in the flower industry during pregnancy compared with those whose moth ers did not work (95% CI = 0.22

– 0.93). The odds of poor visual acuity in infants whose mothers worked in the flower industry during their pregnancy were 4.7 times higher compared with those children whose mothers did not work in the flower industry during pregnancy (95% CI = 1.1

– 20). However, estimates are imprecise due to the limited size of the infant subsample.

This study, one of few conducted in a developing country setting, provides preliminary evidence of the potentially harmful effects of maternal occupation in the cut

- flower industry during pregnancy (with its potential for in utero exposure to organophosphate and carbamate pesticides) on neurobehavioral development. Maternal occupation in the cut

- flower industry during pregnancy may be associated with delayed neurobehavioral development of children aged 3

– 23 months, particularly with fine motor skills and visual acuity. Possible hazards associated with working in the flower industry during pregnancy include pesticide exposure, exhaustion, and job

s tress.

Study Review

This was a well

- conducted study, in three reports, of proxy pesticide (OP and carbamates) exposure on infants and young children 3 to 61 months of age living in two communities in Ecuador. The study population was based on a census sa mpling frame, and so selection bias is unlikely to have occurred. The exposed and unexposed groups had somewhat different education level, child care and stimulation in the home, etc., which could impact neurodevelopment performance. The researchers looked at correlated and uncorrelated (due to sibling pairs)
estimates

– and found similar results.Page 55 of 171 The analytic methods, questionnaire, and analytic approach were appropriate in all three reports. Inferences to be garnered from the results are constrained by l imitations of the cross

- sectional study design (cannot assess temporality between exposure and outcome due to cross

- sectional design), the exposure assessment (proxy measures), and the assessment of neurodevelopment outcomes (screening measures). None of t he exposure measures in this study allow differentiation of the contribution of specific pesticides or pesticide groups (OP or carbamate) on
results.

Unlike formal psychometric assessments such as the Bayley Scales of Infant Development, the Ages and Stag es Questionnaire (ASQ) is a first

- level comprehensive screening instrument. Some sections depend on mother's recall, e.g. social interactions, and it is not validated for Andean populations.

While the use of a parent

- report screening test has been shown to be an effective and valid way in which to assess a child’s developmental progress (Glascoe 2000), general screening tools such as the ASQ may not capture subtle delays that may be present. F or most sections of the ASQ and especially for the fine and gross motor sections, children were directly tested by having them attempt various tasks such as crawling or walking and grasping blocks or toys. These activities
were easier to test and score bec ause they were observed directly. Other questions depended on mother’s recall of events at home. Specifically the social skills section of the ASQ involved questions relating to how the child reacts to other children or siblings or how the child plays with his or her toys at home. Given this, it is thus even more striking that differences were observed.

The retrospective, questionnaire

- based assessment of some confounders (including birthweight with many non

- response/missing values) is subject to imperfec t recall. Errors in recall may not be independent of exposure status, i.e., the potential for recall bias exists. Potential for bias is away from the null; magnitude of potential bias is judged to be small.

However , given that a proxy exposure assessments was used in this study , the pesticides used may have included not only OPs, but also pyrethroids, fungicides, and growth regulators. Given the uncertainty about whether OP exposure actua lly occurred in this studies and whether
observed outcomes are assoc iated with OP exposure or with other pesticides, this study was excluded from further analysis in the final synthesis of the OP epidemiological literature review ( add citation )

.Page 56 of 171 2007 analysis of community of residence on neurobehavioral development (age s 3

- 61 months)

In this first report, community of residence is the exposure measure, resulting in potential for exposure misclassification. Children were grouped into two age categories (3

– 23 months and
24

– 61 months) to account for some of the variability in development associated with age. These age groups were evaluated separately in subsequent reports. The sample size is acceptable for the main analysis, but small for breaking into smaller age groups. The researchers stated that ideally ages would be gr ouped in 1

- year categories or, with infants, in months. The effect of community of residence on gross motor skill development in younger children was greater for stunted
children than for non

- stunted children; however, because cell sizes were small, these results should be interpreted cautiously.

Other factors such as attendance in child care and stimulation in the home may modify the effect of community of residence on neurobehavioral development. Children who attended child care in high

- exposure communit ies scored significantly better in their fine motor skills. It may be that these children are exposed to more stimulating activities at child care that aid in fine motor development, compared with children who do not attend child care or who are more isola ted and
live in the rural sectors.

A larger percentage of mothers reporting low birth weight (LBW) lived in the high

- exposure communities. LBW may also be associated with work in or proximity to the cut

- flower industry. However, because there was consider able nonresponse to questions about birth weight, this potential association could not be explored. Future studies in this region should consider including LBW as an additional outcome.

There was information on types of pesticides used in the cut

- flower i ndustry, but data on quantities and possible mixtures were not available. Additionally, domestic pesticide use is reportedly common in this region. The researchers did not obtain information on the type or quantity of pesticides used, either in the work en vironment or at home, therefore leading to another potential source of measurement error. Future investigations should incorporate the use of biomarkers and environmental sampling to supplement information gathered by questionnaire.

2007B analysis of neu rodevelopment in children with exposure to pesticides (age 24

- 61 months)Page 57 of 171 Given the correlations among several of the exposure variables, this analysis focused on those variables that describe distinct exposure domains (e.g., maternal employment, pesticide use within and around the home, and child play activities). Such measures are all proxy exposure measures, gathered via a questionnaire

– thus there is potential for exposure misclassification.

Pesticide use on domestic crops and their proximity to child ren’s outdoor play area were either not associated with developmental delay or were associated with better developmental outcomes. The researchers considered several explanations for these seemingly anomalous findings. The risk of children living and playi ng in close proximity to the flower farms might be offset by the benefits of having a mother employed in the flower industry, which corresponds to a higher monthly household income, higher parental educations, and better access to health care and daycare.

Alternatively, there may be unmeasured confounders. For example, domestic pesticide use may correlate with economic security; pesticides are an added expense for the family and only those who can afford the chemicals may be using them at home.

Current m aternal employment in the flower industry and maternal employment in the flower industry during pregnancy were associated with better communication and problem

- solving
skills. Previous studies have found that take

- home exposures via contaminated parental w ork clothing and equipment contribute to household pesticide residue contamination. In this population, however, it is uncommon for flower farm workers to take home their work clothes and equipment. Therefore, assessing take

- home pathways for exposure via parental work clothing may be less relevant for this population, whereas other aspects of maternal employment in the flower industry (i.e., increased monthly income, more education, better access to daycare and health services) may benefit the child’s heal th and development.

The results highlight the importance of social relationships and opportunities that may interact with adverse exposures to affect neurobehavioral development. Neurobehavioral development is a complex and dynamic process that is affected by numerous factors , including parent

- child interaction, household relationships, maternal health, physical environment, the child’s physical and mental health, and social organization. Associated social opportunities that increase access to daycare or health care for the ch ild may counteract the effects of pesticide exposure. InPage 58 of 171 researching effects of pesticide exposure on child neurobehavioral development, recognition of other environmental factors within a larger ecosystem is critical

2008 analysis of occupational exposur e to pesticides during pregnancy and neurodevelopment of infants and toddlers (age 3

- 23 months)

In this final report, the researchers examined maternal

- prenatal exposure and neurodevelopment. The review comments above apply to this report. Primarily, meas ures of pesticide exposure are proxy, the sample size was limited, and the ASQ is a screening tool. Domestic pesticide use is widespread in this region of Ecuador, and they were not able to obtain information on the type or quantity of pesticides used on d omestic crops. It is possible that mothers who did not work in the flower industry were exposed through domestic pesticide use.

The attenuation of the effect of maternal occupation during pregnancy on neurobehavioral development when any work outside the home was considered suggests that work in the flower industry (as opposed to other employment) may explain the observed developmental delays. However, there was limited power to examine the independent effects of work in the flower industry compared with o ther types of paid work; the majority of the women who reported working outside of the home in a paid job during pregnancy reported working in the cut

- flower industry and the majority of working women reported pesticide use at their workplace. Developmenta l differences observed between children whose mother did or did not work in the flower industry during pregnancy may be due to factors other than pesticides. Approximately half of the mothers who worked in the flower industry during pregnancy reported work ing more than 45 hours per week, and almost three

- quarters of those mothers reported working 6 months or more of their pregnancy. Long work days, job stress, and difficult work responsibilities where women are on their feet most of the day could contribute to adverse pregnancy outcomes. Furthermore, flower laborers work in a greenhouse setting where heat and exhaustion may also play a role in maternal and fetal health.

Protective equipment and clothing aid in reducing the harmful effects of exposure to pes ticides on health of agricultural laborers. In the present study, mothers were asked about current use of protective gear and clothing in the workplace. The majority of working mothers reported using protective gloves and rubber boots. However, only about half reported having access to masks and plastic aprons. In the flower industry, the majority of women work either in harvest or packaging, where masks may help reduce exposure to pesticides by inhalation. There arePage 59 of 171 limitations in the assessment of the imp act of using protective gear, data specific to the pregnancy period was not available.

References cited

Glascoe FP. 2000. Evidence

- based approach to developmental and behavioural surveillance using parents’ concerns. Child Care Health Devel 26:137

– 149.Page 60 of 171 Study 10. Llop et al. (2013)

Prenatal and postnatal insecticide use and infant neuropsychological development in a multicenter birth cohort study. Sabrina Llop, Jordi Julvez, Ana Fernandez

- Somoano, Loreto Santa Marina, Esther Vizcaino, Carmen Iñiguez, Ner ea Lertxundi, Mireia Gascón, Marisa Rebagliato, Ferran Ballester. Environment International 59:175

– 182, 2013.

Study Summary

The INMA study is a multicenter birth cohort study initiated to assess the effect of environmental exposures and diet during pregnancy on fetal and child development set in four regions in Spain. Llop et al. reported on an assessment that leveraged indoor pesticide use during and after pregnancy obtained from the INME study cohort and psychomotor development in infants during the second year of life.

The original study population (n=2,644) was enrolled between 2003 and 2008, during which time pregnant wom en were recruited during their first trimester of pregnancy. Of these, 2,506 women were followed until delivery of their children, who were enrolled in the study at birth. Pesticide use during pregnancy and during the first year of the child’s life was que ried using a questionnaire delivered by trained interviewers during the third trimester, and again when the child was between 11 and 30 months of age. This evaluation was conducted on a further subset of 1,980 mother

- child pairs for whom complete data on p renatal and postnatal insecticide use information as well as neuropsychological test scores at age 11

- 23 months were available.

Use of indoor pesticides and method of application (pesticide spray or use of a plug

- in device) were used to define exposure status. Fifty

- four percent of the participating women used indoor insecticides at home during pregnancy; 47% used them postnatally. Covariate information, including socioeconomic, environmental, and lifestyle behaviors was queried via questionnaires delivered during the first trimester, again during the third trimester, and again when the infant
was between 11

- 30 months of age. Covari ate data ascertained by the first two of these questionnaires included maternal age, education, place of birth, body mass index prior to pregnancy, smoking status during pregnancy, maternal employment status, parity, having a garden at home, and residentia l proximity to agriculture. Duration of breast feeding, primary
child

- care provider, participation in day care, number of siblings, and maternal employmentPage 61 of 171 status, and smoking status were obtained during the follow

- up interview conducted when the
child was between 11 and 30 months of age.

Infant neuropsychological development was assessed at an average age of 14 months using the Bayley Scales of Infant Development (BSID

- 1). The BSID

- 1 consists of a mental scale and a psychomotor scale. All outcome testing was conducted by specially trained psychologists at the regional health center, in the presence of the mother.

Multivariate linear regression models of mental and psychomotor scores were constructed to
assess associations with the mothers’ self

- reported prenatal and postnatal indoor pesticide use.
The model for the mental BSID

- 1 score adjusted for BMI prior to pregnancy, maternal age, education level, country of birth, social class, sex, low birth weight, weeks of gestation, main
child

- care provider, and psychologist. The psychomotor score model included BMI prior to pregnancy, maternal age, country of birth, social class, sex, low birth weight, weeks of gestation, and number of siblings, small length for gestational age, nursery attendance, paternal smoki ng during the child’s first year of life, and psychologist. The investigators also evaluated interactions between indoor pesticide exposure and child’s gender, breast feeding status, blood lead and PCB levels, socioeconomic status, and pesticide applicatio n method.

Overall, Llop et al. observed a small increase in mental development score in association with
self

- reported prenatal use of indoor pesticides, and a small decrease in psychomotor development score of the children at age 11

- 30 months, neither of which was statistically significant. Prenatal use of pesticide sprays was associated with an average decrease in psychomotor score on the
BSID

- 1 that was statistically significant. A smaller observed increase in the psychomotor score
associated with use o f plug

- in devices was not statistically significant. They observed no consistent or statistically significant associations between postnatal indoor pesticide use and either mental or psychomotor development. Statistically significant effect modification wa s observed between prenatal pesticide exposure and child gender, with decrements observed in psychomotor scores in association with prenatal and postnatal pesticide exposure among girls, but not among boys, and also with social class, with decrements in ne uropsychologic test scores associated with indoor prenatal and postnatal pesticide exposure noted among those in the lowest social class (manual laborers), but not in the other social class strata.

Study ReviewPage 62 of 171 The INMA study is one of the first in Euro pe to explore the relationships between prenatal and early

- life exposure to indoor insecticides and infants’ neurodevelopment. This prospective birth cohort study was well conducted, although it relied on self

- reported exposure assessment and did
not inclu de objective measurements of pesticide levels or biomarkers, and did not address exposures to specific pesticide types (e.g. organophosphates).

The recruitment and retention of participants in this multi

- center study was noteworthy; the study sample was large relative to most similar assessments to date, and almost 80% of the children
enrolled in the cohort at birth were followed

- up at an average age of 14 months (range: 11

- 23 months). Nevertheless, a degree of selection bias due to loss to follow

- up is p ossible if drop out or retention are associated with both pesticide exposure and neuropsychological status.

Objective measurements of indoor pesticide exposures and biomarker assessments were not conducted in this study. The prospective, questionnaire

- ba sed pesticide exposure assessment was well conducted, though subject to recall errors and other sources of misclassification, all of which are likely to be non

- differential with respective to neuropsychological development.

Though residual confounding is possible, the assessment of covariates and adjustment for potential confounders was reasonable, with information on a large set of likely determinants of neuropsychological development collected via questionnaire both prospectively, and at the time
of the neuropsychological testing. The authors also assessed several modifiers of the association between prenatal and postnatal indoor pesticide exposures and neuropsychological outcomes. The assessment of interaction between indoor pesticide use and other envi ronmental toxicants (PCBs and lead) was particularly noteworthy, though interaction effect estimates for these toxicants were not statistically significant. It appears from the report that these interaction effects
were considered a priori , rather than as post hoc exploratory analyses. In general, the statistical modeling was appropriate, though no adjustment was made for multiple comparisons.

Llop et al. conclude that their study provides some evidence of adverse effects of indoor insecticide spray use d uring pregnancy on the psychomotor development of children at 2 years of age in the INMA cohort. While true, the associations were small and not consistent. Moreover, postnatal pesticide use was not associated with decrements in neuropsychological developm ent in this study.Page 63 of 171 Furthermore , given that a proxy exposure assessments was used in this study , the pesticides used may have included not only OPs, but also pyrethroids, fungicides, and growth regulators. Given the uncertainty about whether OP exposure actua lly occurred in this studies and whether observed outcomes are associated with OP exposure or with other pesticides, this study was excluded from further analysis in the final synthesis of the OP epidemiological literature review ( add citation )

.Page 64 of 171 Study 11. Oulhote et al. (2013)

Urinary Metabolites of Organophosphate and Pyrethroid Pesticides and Behavioral Problems in Canadian Children. Youssef Oulhote and Maryse F. Bouchard; Environmental Health Perspectives. 121:1378

- 1384 (2013)

Study Summary

In this report, Oulhote et al. evaluate the association between the recent exposure to organophosphate and pyrethroid pesticides, indicated by urinary metabolites, and parentally reported behavioral problems in children in Canadian children aged 6

- 11 yea rs.

For this study, data from the Canadian Health Measures Survey (CHMS; cycle 1, 2007

– 2009) was used. For cycle 1 of CHMS, data were collected from approximately 5,600 Canadians 6

– 79 years of age, including 1,081 children 6

– 11 years of age, in 15 sites spread across Canada. Children were predominantly white (75%) and had parents with relatively high education levels (80% with at least a high school degree). The percentage of parents reporting using chemicals in
the month before the interview for controll ing roaches, ants, termites, or other insects indoors was 3.8%, and 4.3% reported using chemicals to treat head lice on family members or fleas and ticks on pets. In addition, 7.3% reported that their lawn or yard, or the surrounding fields, woods,
or orch ards, had been treated with chemicals to kill insects or weeds or control plant diseases. Overall, 14% of the children lived in a household where pesticides had been used for any of those purposes in the previous month. Out of the 1,081 children from cycle 1, children who were missing data on some of the covariates (sex, age, race/ethnicity, income, parental education, blood lead levels, maternal smoking during pregnancy, and others) were excluded from the study. The sample size for this study was 779 child ren.

Each parent was asked three questions about pesticide use in the preceding month, specifically, chemicals used indoors to control roaches, ants, termites, or other insects; chemicals used to control head lice on family members or fleas on pets; and chemicals used on the lawn, yard, or the surrounding fields, woods, or orchards to kill insects or weeds, or to control plant diseases. A spot urine sample was collected from each child within 2 weeks of survey questionnaire completion and was analyzed at the Institut National de Santé Publique du Québec. In addition, urinary creatinine concentration was measured to account for urine dilution. Six urinary dialkyl phosphate (DAP) metabolites that result from the degradation of different organophosphatesPage 65 of 171 were measured in urine, and concentrations were summed to provide an indicator of total exposure. The urinary DAP metabolites measured were three dimethyl alkylphosphate (DMAP) molecules [dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiopho sphate (DMDTP)] and three diethyl alkylphosphate (DEAP) molecules [diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP)].

Behavioral problems were assessed using the parent version of the Strengths and Difficulties Questionn aire (SDQ). The SDQ is an internationally used, acceptable to parents, and validated screening questionnaire designed to assess the mental and behavioral difficulties and strengths of
3

- to 16

- year

- old children.

Blood lead levels (as a continuous value) and maternal smoking during pregnancy (parent report of smoking regularly during any stage of pregnancy) were used as a priori in models since they are considered important risk factors for behavioral problems in children. In addition, variables
such as se x, age, race/ethnicity, family income, parental education, birth weight, maternal age at child’s birth, current exposure to environmental tobacco smoke (parent report of daily tobacco smoking inside the home), child’s body mass index (BMI; as a continuous value), and fasting status (fasting duration at urine collection > 10 hr/≤ 10 hr) were examined as potential confounders. Each variable was examined in a bivariate analysis for its association with high SDQ total difficulties score, and with concentrations of urinary pesticides metabolites (logarithmically transformed). Variables that predicted both the total difficulties score and the exposure with p < 0.2 (chi

- square test, one

- way analysis of variance, or Spearman correlation depending on whether variable s were categorical or continuous) were retained as model covariates. In addition, urinary creatinine concentration was included as a covariate in models to account for urine dilution. This approach was chosen rather than standardizing urinary
concentration s for creatinine to reduce potential bias that may be introduced by age, race/ethnicity, and sex

- related variation in creatinine excretion.

Sensitivity analysis was also performed in three different ways; adjusting for the ratio of pesticide metabolite to creatinine level, adjusting for blood lead levels, and adjusting for variables used to define the weights (i.e. age, sex, and ethnicity).

At least one urinary metabolite for organophosphates was detected in 91% of children, and for pyrethroids in 97% of children. Organophosphate metabolites were not significantly associated with high SDQ scores. The pyrethroid metabolite cis

- DCCA [3

- (2,2

- dichlorovinyl)

- 2,2

-Page 66 of 171 dimethylycyclopropane carboxylic acid] was significantly associated with high scores for total
diffi culties on the SDQ (OR for a 10

- fold increase = 2.0; 95% CI: 1.1, 3.6), and there was a non

significant association with trans

- DCCA (OR = 1.6; 95% CI: 0.9, 3.0).

In contrast with previous studies, this study did not find any association between exposure t o organophosphate pesticides and behavioral scores in children. However, some pyrethroid urinary metabolites were associated with a high level of parent

- reported behavioral problems. According to the author, this is the first study to suggest that exposure to certain pyrethroids might be associated with behavioral problems in children.

Study Review

This cross sectional study of recent OP pesticide exposure in Canadian children 6

- 11 years of age and behavioral problems has some strengths and few weaknesses . The study population was not mixed. There is a reference to analytic methods but is not described in the study. The statistical analysis was well specified and appropriate. The author’s approach to evaluating and adjusting for covariates were prudent. Ho wever, inferences from the results of this investigation are constrained by the limitations in study design and exposure assessment methodology as it is based on the assessment of single urine sample.

Characteristics of the study participants (n=779) is not provided in the study but according to the author, there was no significant difference (p > 0.05) between any of the characteristics for the whole group (n=1030) and the subset (n=779) included in th e analysis. The study population was not mixed (79% white vs. 21% non

- white) and only 14% (n=151) of the children were exposed to pesticides in the previous month.

The exposure assessment method for OP pesticides and pyrethroids is not described in the a rticle and refers to Institut national de santé publique du Québec (INSPQ) 2009a, 2009b] for laboratory methods. Exposure was assessed by measuring the metabolites in a spot urine sample. The use of a single urine sample to estimate exposure is likely to h ave resulted in an attenuation of associations, relative to those that would have been observed had the investigators used a time

integrated marker of OP pesticide exposure. Also, the short elimination half

- life, episodic nature of exposures to OP pesticid es, and individual variation in metabolism, may contribute to variability in exposure measurement.Page 67 of 171 The outcome assessment method seems to be appropriate because according to the author, the SDQ is validated and accepted by parents. However, it should be noted that SDQ is a screening questionnaire only. On the whole, the investigators handling of confounding resulted was reasonable and aspects of the investigator’s approach to evaluating and adjusting for confounding were prudent. The statistical analysis appears to be appropriate. Sensitivity analysis was also performed and seems to be presented in the supplemental materials, but the references are not provided in the article.

This study showed that there was no association between the recent exposure t o organophosphate pesticides and behavioral scores in children which is in contrast with other similar studies. However, this study suggested that exposure to certain pyrethroids, at levels common in Canadian children, may be associated with behavioral pro blems. According to the author, the use of pyrethroids is increasing, but to date there is little information on potential health effects in children. The authors rightly conclude their paper by stating that longitudinal studies with a larger study populat ion should be conducted to verify these findings.Page 68 of 171 Study 12. Petit et al. (2010)

Impact on fetal growth of prenatal exposure to pesticides due to agricultural activities: a prospective cohort study in Brittany, France. Claire Petit, Cécile Chevrier, Gaël Durand, Christine Monfort, Florence Rouget, Ronan Garlantezec, Sylvaine Cordier. Environmental
Health 9:71

- 83, 2010.

Study Summary

PELAGIE is a prospective birth cohort study designed to examine the role of environmental pollutants on prenatal and child developments in Brittany, France. Using this cohort, Petit et al. evaluated the relationship between level of urbanization, and the p roportion of an area devoted to agricultural activity as indicators of prenatal exposures to pesticides, and three indicators of fetal growth: birth weight, head circumference, and fetal growth restriction.

Enrollment of pregnant women into PELAGIE took place from the spring of 2002 through the winter of 2006 in three Breton districts. Women were recruited by their gynecologists, ultrasonographers, midwives, and obstetricians during routine prenatal care visits prior to 19 weeks gestation. During these vi sits, women were informed of the study objectives, invited to participate, and consented, then provided a questionnaire which they were asked to complete at home and return. The authors estimated the participation proportion to be approximately 80%; 3,592 women agreed to participate. They subsequently excluded 171 women who returned the questionnaire after 19 weeks of pregnancy and 22 women for whom birth outcomes were unavailable. The investigators restricted the study sample to singleton live births among women who lived within the three

- district catchment area. They also excluded 42 women who were farmers, resulting in a study sample size of 3,159 women.

The authors used population size to characterize each mother’s municipality as either rural or
urban using a cut point < 20,000 inhabitants to define areas as rural. Data on agricultural activity in the three study districts were obtained from the 2000 national general census collected and maintained by the statistics department of the Ministry of Agricul ture. These Census data, originally provided by individual farmers, were aggregated at the level of municipality, providing an estimate of the percentage of the total area of each municipality used for agricultural activities, specific to crop, including c orn, wheat, colza, peas, potatoes, fresh vegetables, strawberries and melons. These municipal

- level data on agricultural usage werePage 69 of 171 matched to each of the participating mother’s municipalities of residence during early pregnancy.
The authors characterized the percentage of municipal area used for agricultural into low, medium, and high categories using tertiles (0

- 63%, 63

- 74%, and >74%). Municipalities were similarly characterized by tertile with respect to the percentage of area used to grow corn (0

- 13%, 1 3

- 20%, >20%) and wheat (0

- 9%, 9

- 15%, >15%). Because of the small percentage of municipal areas devoted to colza, peas, potatoes, and other vegetables, municipalities were characterized by a dichotomous indicator for presence/absence of each of these crops.

Indicators of fetal growth (weight, length, and head circumference) were obtained from hospital records. Live born singleton children of the PELAGIE study without major malformations were classified with fetal growth restriction (FGR) if their birth wei ght was below the 5th percentile of the distribution of expected birth weight. Small head circumference was defined as having a head circumference below the 5th percentile of the head circumference distribution at birth for a given gestational age and sex, using recent French reference curves.

Multivariate linear and logistic regression models were built to evaluate associations between indicators of fetal development and level of urbanization. Using data only from the subset of women living in rural areas, the authors similarly evaluated the re lationship between fetal development and agricultural activities in the municipality of residence. The authors considered the following as potential confounders: year of inclusion, district of residence at inclusion, maternal age, maternal educational, mat ernal alcohol consumption and tobacco smoking status at inclusion, marital status, season in early pregnancy, high blood pressure during pregnancy, and maternal diabetes (gestational or preexisting). Maternal pre

- pregnancy body mass index, maternal height, parity, child’s sex, quadratic gestational age were additionally considered as covariates for weight and head circumference at birth. For the small head circumference outcomes, the authors also considered adjustment by pre

- pregnancy body mass index, mater nal height, and parity, and cesarean delivery. Additional analyses adjusting for shellfish and fish intake were conducted, but not presented in the author’s report. Model building and covariate selection was conducted using backward selection, using a p

- va lue for exclusion of 0.2.

The authors estimated associations between many exposure metrics and several birth outcomes, and the findings of this study where largely negative (i.e. no observed association). Statistically significant associations were obser ved for head circumference and level of urbanization, with
rural

- dwelling mothers giving birth to children with statistically significantly smaller head circumference (0.1 cm, on average), and having 1.8x greater odds of having small headPage 70 of 171 circumference. Pr esence of pea crops in the municipality where the mother lived during early pregnancy was also significantly associated with smaller head circumference and a statistically significant 2.2x increase in the odds of having small head circumference.

Study Rev iew

This was a large, well

- conducted, but ultimately limited study that sought to evaluate the effects of pesticide exposure on fetal growth using a community

- level, proxy indicator of pesticide exposure and birth outcome data obtained as part of a prospe ctive birth cohort study conducted in a population of healthy mother

- child pairs living in Brittany, France. The analytic methods, questionnaire, and statistical analysis were appropriate. However, inferences to be garnered from
the results of this investi gation are constrained by substantial limitations related primarily to the assessment of (pesticide) exposure.

The authors employed a reasonable set of inclusion/exclusion criteria. Interestingly, the authors excluded farmers from the study, i.e. those w ith perhaps the greatest opportunity for exposure to agricultural pesticides. Although they did not justify this decision, presumably this was done to avoid the problem of highly exposed farm women living in areas with a low percentage of area used for agr iculture; percent of area devoted to agricultural activity is almost certainly not a good proxy for pesticide exposure among farmers. The estimated proportion of women invited to participate was high (approximately 80%), and indication that the study resul ts, if valid, are generalizable to the target population of pregnant French women. Only 22 of 3,421 women (0.64%) were lost to follow

- up, indicating that selection bias in this prospective cohort is unlikely to have biased the study findings.

The investi gators used a questionnaire to prospectively collect demographic and other covariate information from participants. Covariate selection and model building proceeded appropriately; strong residual confounding by unmeasured or poorly measured confounders is unlikely in this study. Multiple comparisons were conducted for this evaluation, and the authors did not adjust for multiple testing. Given the number of associations estimated in this study, one should expect there to be a number of “false positive” findi ngs due to chance alone.

In summarizing the motivation for their investigation, the authors noted that, in 2004, France led all of Europe in pesticide sales (~76,000 tons of active ingredients sold), with 90% of these pesticides used in agricultural acti vities ( Pingault et al., 2009). Moreover, 70% of the land area inPage 71 of 171 Brittany is devoted to agricultural activities. In short, the potential exists for exposure to agricultural pesticides. Nonetheless, the study design relies on the untested assumption that t he percentage of a municipality’s area dedicated to agricultural activities is predictive of a fetus’
individual

- level pesticide exposures if its mother lived in that municipality.

The potential for a large degree of non

- differential misclassification of pesticide exposure is a potential explanation for the largely null findings of this study. The assessment of residential proximity to crops is an aggregate exposure measurement on the scale of the municipality, whereas actual exposure occurs (or does not occur) on the level of the individual mother

- child pair. A great deal of individual

- level pesticide exposure is possible among pregnant women living in areas with little or no agricultural activity. Similarly, it is equally possible that very little pestic ide exposure is sustained by women living in areas with a great deal of agricultural activity. This potential misclassification was likely the motivation for the investigators’ decision to exclude farmers from the study sample, despite farmers having perha ps the greatest opportunity for exposure to agricultural pesticides.

There were, in fact, additional sources of potential exposure misclassification, including the two

to

- six year interlude between the conduct of the agricultural census in the year 2000 and the enrollment of pregnant women as participants in PELAGIE in the years 2002

- 2006.

The investigators did not report having assessed effect modification by any factors.

The fetal growth indicators (outcomes) were obtained from medical records and ar e unlikely to
be misclassified.

The authors conclude their report by stating that the study findings suggest that some adverse effects on cranial growth during fetal development are caused by prenatal exposure to agricultural activities in the municipali ty of residence, especially pea crops. They go on to state that their findings may be reason for concern, as the cranial growth may be predictive of future intelligence and cognitive ability. Although possible, an equally likely explanation for the few

non

- null studies is that they were false

- positive associations expected to occur when conducting multiple comparisons as the authors have done. Although unstated by the authors, the primarily null findings may be similarly suggestive of there being no associa tion between pesticide exposure and other indicators of fetal growth evaluated in the study. In fact, however, attenuationPage 72 of 171 of observed associations due to non

- differential misclassification is an equally plausible explanation.

Furthermore , given that a proxy exposure assessments was used in this study , the pesticides used may have included not only OPs, but also pyrethroids, fungicides, and growth regulators. Given the uncertainty about whether OP exposure actua lly occurred in this studies and whether observed outcomes are associated with OP exposure or with other pesticides, this study was excluded from further analysis in the final synthesis of the OP epidemiological literature review ( add citation )

.

References cited
Pingault N, Pleyber E, Champeaux C, Guichard L, Omon B: Produits phytosanitaires et protection intégrée des cultures: l’indicateur de fréquence de traitement. Notes et études socio

- économiques 2009, 32:61

- 94.Page 73 of 171 Study 13. Ruckart et al. (2004)

Long

- Term Neurobehavioral Health E ffects of Methyl Parathion Exposure in Children in Mississippi and Ohio. Environmental Health Perspectives. 2004; 112(1):46

- 51.

Study Summary

In this report, Ruckart et al. examine the association between methyl parathion (MP) exposure
and neurobehavioral development in children exposed at age 6 years or younger in Mississippi (MS) and Ohio (OH) when their homes were sprayed with MP using a retrospective cohort study design with a single assessment of urinary para

- nitrophenol (PNP) and vario us assessments of neurobehavioral development

- cognitive, motor, sensory, and affect domains, child and family dysfunction, child's personality, communication, and daily living skills.

Potential participants were identified using data files provided to A TSDR by the MS and OH state health departments. These states were selected because environmental data collection and urine testing were available. Testing was done in OH in 1994 and in MS from late 1996 through
mid

- 1997. Children residing in MS and OH who were 6 years or younger when their homes were illegally sprayed with MP were eligible for inclusion in the study. In one OH county, the spraying occurred in a multifamily, subsidized housing facility that was last sprayed in 1994. In MS, the spraying was m ore widespread and included 29 counties with residences sprayed as recently as late 1996.

Results of environmental wipe samples for MP taken from residences (household MP) and urine testing for creatinine

- adjusted PNP, a metabolite of MP, were provided b y the state health departments. Household MP samples were analyzed by laboratories approved by the U.S. EPA employing the same analytical methods, and PNP samples were analyzed by the CDC. Exposure
status was defined on the basis of the test results. Both household MP and urinary PNP levels were used to define exposure status. In MS, exposure was defined as at least one household MP sample ≥150 μg/100 cm2 or urinary PNP ≥100 ppb for at least one person in the household. For OH, exposure was defined as house hold MP ≥132.9 μg/100 cm2 or urinary PNP ≥100 ppb for at least one person in the household. To include enough exposed children in OH, it was necessary to set a lower cutoff value for household MP. Exposure was dichotomized into two groups: Highly exposed: household MP >1000 ug/100c cm2 or urinary PNP >300 ppb, and

- Moderately exposed: all other exposed children.Page 74 of 171 Comparison groups of unexposed children residing in the same communities as the exposed children were also identified. Local comparison groups wer e chosen to minimize confounding from sociocultural factors (e.g., regional variations in education, IQ, race, and cultural factors). In MS, unexposed children were selected through state records from houses that tested <25 μg/100 cm2 for household MP; no urine testing was done for children at these levels of MP. In OH, unexposed children were selected in two ways: 1) houses that tested <35 μg/100 cm2 for household MP and where no one in the household had a urinary PNP level >25 ppb (the cutoff
value for ho usehold MP in OH was increased to include enough unexposed children), and 2) a special census was done in the sprayed complex after it was remediated and in a nearby housing complex that was not sprayed, to identify additional unexposed children.

A total of 365 children in MS (147 exposed and 218 unexposed) and 287children in OH (104 exposed and 183 unexposed) were enrolled (range 1.9

- 12.5 years old at testing). The researchers attempted to enroll two unexposed children for every exposed child to ensure a n adequate number of unexposed children for analysis in subsequent study years. All children who participated in year 1 (summer 1999) were invited to be retested in year 2 (summer 2000) to see whether any differences in neurobehavioral testing between expo sed and unexposed children persisted or disappeared

Trained interviewers administered a CAPI questionnaire to obtain information on confounders

– parent’s and child’s medical history, pregnancy history, parental occupational histories, workplace chemical use, and child’s residential history. Neurobehavioral development was assessed using the following:

Performance

- based tests (scored as expected, equivocal, below expected, undetermined):

• Pediatric Environmental Neurobehavioral Test Battery (PENTB): cogn itive, motor, sensory, and affect domains (Zeitz et al., 2002)

o Developmental Test of Visual

- Motor Integration (VMI) measures the integration of visual and motor skills.

o Kaufman Brief Intelligence test (K

- BIT) test (K

- BIT) measures general intelligence, ve rbal ability, and nonverbal reasoning.

o Purdue Pegboard tests visual

- motor coordination, manual dexterity, and motor speed.Page 75 of 171 o Story Memory and Story Memory

- Delay from Wide Range Assessment of Memory and Learning tests verbal memory; immediate and delayed reca ll of both specific and general items.

o Trail

- Making test, Part A and Part B assesses multistep processing involving more than one cognitive function area (visual perception, motor speed, sequential skills, and symbol recognition), administered to children 9 years and older.

o Verbal Cancellation test The Verbal Cancellation test measures sustained selective attention.

Informant

- based tests:

• Parenting Stress Index (PSI)

- Child domain, parent domain, total stress scores

• Personality Inventory for Children (PI C)

- child’s personality

• Vineland Adaptive Behavior Scales (VABS)

- Communication, daily living skills

To assess the relationship between MP exposure and performance on neurodevelopment tests, continuous test scores were analyzed using linear regression, a nd dichotomized test scores (worst 10% for a test compared with other 90%) were analyzed using logistic regression. Raw (nonstandardized) test scores were adjusted for age. Unadjusted odds ratios (ORs) and 90% CIs were computed.

Performance

- based tests. F or overall PENTB outcome, a similar number of exposed and unexposed children were classified as below expected, while more exposed children were classified as expected, and fewer exposed children were classified as undetermined. When comparing overall PENT B score for year 1 vs year 2, 33% of exposed children classified as below expected did not improve in year 2 compared with 60% of unexposed children. These results indicated that MP exposure was not associated with persistent deficits in year 2 among
child ren who performed lower than expected in year 1. This suggests that if there are neurobehavioral effects from subacute exposure to low levels of MP, they may be transient in children who were 6 years or younger when they were exposed. A previous study foun d major impairment of memory and concentration in workers who had organophosphates for several years, but the impairments disappeared 12 months after exposure ceased (ATSDR 1999).

No effects were seen for the K

- BIT, VMI, or Trail

- Making tests (ATSDR 2003) . For the Verbal Cancellation test, an effect was seen for the mean difference only in OH (highly exposed ordered form adjusted β =

– 6.02, 90% CI,

– 11.27 to

– 0.78; highly exposed nonordered form adjusted β =Page 76 of 171 – 8.09, 90% CI,

– 13.41 to

– 2.78), and there was a trend with exposure. When test scores were dichotomized, an effect was seen only in MS (ordered form OR = 2.27, 90% CI, 0.80

– 6.45; nonordered form OR = 10.29, 90% CI, 2.23

– 47.41). For the Story Memory tests, the only effect was seen for dichotomized Story Memory Difference scores (OR = 1.27, 90% CI, 0.61

– 2.67), and the effect was strongest in OH (OR = 1.71, 90% CI, 0.50

– 3.37). For the Purdue Pegboard test, the findings were not consistent across subtests or across sites. In OH, the only effect was
seen for the nonpreferred hand (adjusted β =

– 1.82, 90% CI,

– 10.08 to 6.44; OR = 1.20, 90% CI,
0.42

– 3.49). In MS, the only effect was seen for the dichotomized preferred hand score (OR = 1.39, 90% CI, 0.66

– 2.94).

Informant

- based tests. For informant

- based tests, exposed children performed worse than unexposed children in year 1. The PSI was administered only in MS because no children among those tested in the study in OH were younger than 4 years at the time of testing. Effects were
seen for mean difference scores on all domains. Effects were also seen when child domain and total stress scores were dichotomized (child domain OR = 1.90, 90% CI, 0.24

– 15.24; total stress OR = 4.22, 90% CI, 0.62

– 28.71). However, an OR could not be computed for the Parent domain
because there were no unexposed children who scored in the worst 10% for this subtest. For the VABS, an effect was seen for the motor skills domain (highly exposed β =

– 9.05, 90% CI,

– 15.50 to 2.60; OR = 3.10, 90% CI, 0.90

– 10.63), and there was a trend with expos ure for the mean difference scores. In MS, an effect was seen for the mean difference and dichotomized test scores for all other subtests. For the mean difference, a trend with exposure was seen for all subtests except the communications domain; for dichot omized test scores, a trend with exposure was seen for the daily living skills and social skills domains. For the PIC, an effect was seen for the mean difference score for PIC factor 1, undisciplined/poor self

- control (β = 2.73, 90% CI,

– 1.16 to 6.62), and the effect was strongest among the highly exposed children in MS. In MS, an effect was seen among highly exposed children for the dichotomized test scores for PIC factor 4, cognitive development (OR = 1.79, 90% CI, 0.52

– 6.25). In OH, an effect was seen fo r PIC factor 1 when scores were dichotomized and for PIC factor 3, internalization/somatic symptoms. A trend was present for the mean difference scores for PIC factor 3.

Overall, exposure to MP was not associated with poorer performance on most neurobehavioral tests, and effects were not consistently seen in both sites. Effects were seen for tasks that involve
short

- term memory and attention, and parents of exposed childre n reported that their children had more behavioral and motor skills problems than did parents of unexposed children. ThePage 77 of 171 reported behavioral problems included children misbehaving, acting on impulse, having problems with anger, being sad or shy, and having problems relating to other children. Study Review

This was a moderate quality study. Sample size was sufficient, and having two different sites for comparison improved generalizability. Basic demographic information for participating and non

participati ng children was available (both groups were similar with respect to age and gender, participation rate in year 1 was 76% for exposed children and 57% for unexposed children), thus
selection bias is deemed to be small.

The use of two different types of ex posure measures

- environmental wipe samples for MP and urine testing for PNP

- to quantify the children’s individual exposure to MP is generally considered to be a strength of the study. However, the percentage of participants in each eligibility category was not provided

- urinary PNP levels are considered the better measure, as it is less certain whether those who lived in homes with elevated MP levels were actually exposed. It is also unclear if samples were collected, handled, and analyzed in the same manner in both sites (states). Though not explicitly stated that urine was sampled only once, the use of a single urine sample to estimate the participant’s OP pesticide exposure is likely to have resulted in an attenuation of associations, relative to tho se that would have been observed had the investigators
used a time

- integrated marker of OP pesticide exposure. PNP is a metabolite for methyl parathion, malathion, and other compounds, so there could be some misclassification if participants were exposed t o the other compounds; however this is considered to be a less important concern in the study.

The statistical methods used seem appropriate, and as mentioned above the sample size is adequate. This study obtained information on and included appropriate potential covariates in the
models

- demographic and personal characteristics such as parents’ and child’s medical history, mother’s pregnancy history with regard to the index child, parental occupational histories, workplace chemical use, and child’s resi dential history. Use of different exposure cut

- off levels to get enough children in OH in exposed and unexposed groups seems questionable, but is not considered a major concern.

The test battery (PENTB) used was reportedly developed by a group of experts to examine the neurobehavioral effects of environmental exposures specifically in children. Extensive trainingPage 78 of 171 of PENTB examiners who were blinded to the exposure status of the child and a thorough review
of the collected data also contributed to the stren gths of this study.

The authors note limitations of the study include the fact that the length of time between the last MP spraying, sample collection, and neurobehavioral testing was not known and was different in the two study sites. The frequency and d uration of spraying were also unknown. In this study, the time between the last spraying of the pesticide and the testing was at least 2.5 years in MS and at least 4.5 years in OH. Inconsistencies in the results between MS and OH may be due to the
differen ces in length of time between the spraying and testing in the two sites (the last spraying in MS was in late 1996 and the last spraying in OH was in 1994; children in both sites were initially tested in 1999). Additionally, children in OH were older than c hildren in MS at the time of testing, and the older children may have outgrown any neurobehavioral effects that were caused by MP. Also, the exposure assessment of MP in household samples and in urine for each child represents only a snapshot in time and m ay not reflect the total exposure received by the child. There were also inconsistencies in results within subtests of some tests. Additionally, there was a lack of trend with exposure for most tests.

Results for a particular test could be lacking becaus e the neurobehavioral effects are transient and were no longer measurable when the children were tested. It might also be possible that the PENTB was not the appropriate test battery to examine neurobehavioral effects from exposure to MP. For example, even with a large sample size, the standard errors for several tests were quite large, making it difficult to find subtle effects.

The findings suggest that MP might cause subtle changes to short

- term memory and attention and might contribute to problems wit h motor skills and some behaviors. However, the results of the study are not conclusive because these effects were not seen consistently in both sites. Although some domains essential to neurobehavioral development appear to have been affected by exposure to MP, the results are largely inconsistent. Furthermore, it is noted that s amples were collected in locations from OH and MS where illegal spraying of methyl parathion is known to have occurred during the 1994

- 1996 time period. Based on the OP Cumulativ e Risk Assessment (USEPA, 2006), methyl parathion is known to be among the more potent OPs. It is unknown whether study participants were exposed to MP levels that would have induced cholinergic effects. Therefore, given the uncertainty around this illeg al use combined with the high potency for cholinergic toxicity, the agency is not emphasizing th is study further in the final synthesis of the OP epidemiological literature review ( add citation )

.Page 79 of 171 References cited

ATSDR. 1999. Toxicological Profile for Methyl Parathion (update). Atlanta, GA: Agency for Toxic Substances and Disease Registry.

Zeitz P, Kakolewski K, Imtiaz R, Kaye W. 2002. Methods of assessing neurobehavioral development in children exposed to methyl parathion in Mississippi and Ohio. Envi ron Health Perspect 110(suppl 6):1079

– 1083.Page 80 of 1 71 Study 14. Suarez

- Lopez et al. (2012, 2013a, 2013)

• 2012

- Lower acetylcholinesterase activity among children living with flower plantation workers. Environmental Research. 2012; 114:53

– 59.

• 2013a

- Acetylcholinesterase Activity, Cohabitation with Floricultural Workers, and Blood Pressure in Ecuadorian Children. Environmental Health Perspectives. 2013; 121(5): 619

- 624.

• 2013

- Acetylcholinesterase Activity and Neurodevelopment in Boys and Girls.
Pedia trics. 2013; 132(6):e1649

- 1658.

Study Summary

Suarez

- Lopez et al. looked at participants of the Secondary Exposure to Pesticides among Infants, Children and Adolescents (ESPINA: Estudio de la Exposición Secundaria a Plaguicidas en Infantes, Ninos y Adole scentes) residing in Pedro Moncayo County, Pichincha, Ecuador,
where there is an active fresh

- cut flower industry, with the overarching aim to evaluate cross

sectionally the effects of secondary post

- natal exposure to pesticides on child development. The

a im of the first report (Suarez

- Lopez et al., 2012) was to evaluate the association between flower worker cohabitation and children’s AChE activity. The second report (Suarez

- Lopez et al., 2013a) examined whether lower AChE activity from secondary pesticide exposure and cohabitation with a flower plantation worker was associated with changes in blood pressure and heart rate among children. The third report in the series (Suarez

- Lopez et al., 2013) looked at whether lower AChE activity is associated with lowe r neurodevelopment (particularly attention, inhibitory control, and memory) among children living in floricultural communities, with stronger associations among boys.

In 2008, Suarez et al. contacted 419 of 922 children who had participated in the 2004 Su rvey of Access and Demand of Health Services in Pedro Moncayo County, which had obtained socio

economic status, demographic, occupational and health information (of all members in the household) of 18,187 residents (71% of Pedro Moncayo County’s population ) and collected both height (or length) and weight of children younger than 5 years (27% of Pedro Moncayo County’s children younger than 5). Most losses were due to invalid or unlocatable addresses or because participants had moved to a different house. To supplement recruitment, new volunteers (childPage 81 of 171 and parent) living in Pedro Moncayo County were also recruited through community announcements and word of mouth. A total of 124 new volunteers registered to participate.

Children were eligible if they met t he following criteria: exposed group: (1) cohabitation with a flower worker for at least one year and (2) unexposed group: never cohabited with an agricultural worker, never inhabited a house where agricultural pesticides were stored and having no previous direct contact with pesticides. Multiple children per household were allowed to participate. Parents of 343 children agreed to participate: 259 child participants surveyed in 2004 and 84 new volunteers. Twenty seven participants (47% of whom were cohabita nts with a flower worker) did not show up for the examination appointments, 1 child did not assent to be examined, and two children refused their examination. In 2008, a total of 313 children 4

– 9 years of age were examined; interview information was obtain ed from 451 adults who cohabited with the examined children (at least one parent of each child). Of these children, 277 had all covariates of interest and were included in the 2012 report of cohabitation and AChE activity (57% cohabited with a flower plant ation worker); 271 had all information for the 2013a report of cohabitation and heart rate and blood pressure; and 307 had all information for the 2013 report of AChE activity and neurodevelopment in boys and girls.

In

- person home interviews of children’ s parents and other adults were used to obtain information on socioeconomic status, demographics, health, and direct and indirect pesticide exposures of household members (e.g., pesticide usage at home, child pesticide exposure history and duration
of coha bitation with a flower worker, information regarding practices associated with secondary occupational exposures to pesticides). The distance from each child’s residence to the nearest flower plantation was calculated using ArcGIS 9.3.

Examinations were c onducted in seven schools centrally located to participants’ residences in all five parishes in Pedro Moncayo County. Examiners were unaware of exposure status of the participants.

• Children’s height was measured using a height board, following recommendations by the World Health Organization (WHO 2008), and weight (standing) was measured using a digital scale. Height

- for

- age z

- scores were calculated using the WHO growth standards. No examiner training was described.

• AChE activity was measured fr om single finger

- stick blood samples using EQM AChE Erythrocyte Cholinesterase Assay Kit 470). Blood samples were analyzed immediately at ambient temperatures between 15.6 and 28.4 1C, which are withinPage 82 of 171 the recommended range. Mean AChE activity was 3.14 U/m l, SD 0.49. No data examiner training was described.

• Hemoglobin was measured from single finger

- stick blood samples. No examiner training was described.

• Resting heart rate was measured by a 30 sec auscultation, before the blood pressure measurement. No exa miner training was described; however, AHA procedures were cited.

• Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured with a pediatric aneuroid sphygmomanometer (model BF683W; Omron Healthcare Inc.), following protocols recommend ed by the American Heart Association (Pickering et al., 2005). Measurements were taken after 3

– 5 min of rest. Children were in the seated position with the antecubital fossa supported at heart level, with uncrossed legs and both feet on the floor. Blood pr essure was measured twice, and averages of the two SBP and DBP values were used in analyses. No examiner training was described; however, AHA procedures were cited.

• Neurobehavioral development was assessed by trained examiners using the NEPSY

II general as sessment battery of examinations that included 11 age

- appropriate subtests in 5 domains: attention and executive functioning (subtests: auditory attention and response set (AARS), inhibition, statue), language (comprehension of instructions, speeded naming ), memory and learning (memory

- for

- faces immediate and delayed, narrative memory), sensorimotor (visuomotor precision), and visuospatial processing (design copying, geometric puzzles). Most children completed the general assessment battery within 50 to 80 minutes. The NEPSY

- II assesses different subtests or different items within a subtest at different ages, which explains the varying sample sizes for each subtest. Three subtests required translation into Spanish (AARS, comprehension of instructions, and na rrative memory). Translation of the NEPSY test has been found to be relatively unaffected by language and culture (Garratt & Kelley, 2008).

2012 analysis of flower worker cohabitation and children’s AChE activity

The associations of flower worker cohabita tion, duration of cohabitation and number of flower workers living at home with AChE activity among children were calculated using multiple linear regression (AChE continuous) and polychotomous logistic regression (AChE a 4 level polychotomous variable). I n addition, a dose

– response association of secondary pesticide exposure with AChE activity was obtained by analyzing duration of cohabitation both as a
continuous variable and as a 3

- level categorical variable: 0 = no cohabitation never, 1 = >0

– 4.9

years, 2 = ≥5 years; number of flower workers concurrently living at home was treated continuously and categorically: 0 = none, 1 = 1 flower worker, 2 = ≥2 flower workers; andPage 83 of 171 flower worker practices that would likely influence the amounts of pesticide inadverten tly brought home were coded 1

- 4 [the worst practice among all workers in the home (e.g., always bringing work clothes home) coded 1 and the best practice (e.g., never bringing work clothes home) coded 4, 0 assigned to children of non

- flower workers]; and a 4 category variable was created for the number of “bad” worker practices: 0, 1

– 2, 3, and 4

- 6]. The association of the 4 level AChE variable as predicted from the number of bad practices was analyzed using polychotomous logistic regression. Associations ar e presented according to levels of adjustment for pertinent covariates: (1) a minimum adjustment model included general confounders between secondary exposure and AChE activity (age, gender, race, hemoglobin concentration, and examination date); (2) a mult iple adjustment model to account for chronic nutritional status and additional potential sources of pesticide exposure (minimum adjustment + z

- score height

- for

age, income, pesticide use within household lot and pesticide use by contiguous neighbors); (3) a full adjustment model to control for potential pesticide drift from flower plantations (multiple adjustment + residence distance to the nearest flower plantation edge).

Using the 4 level polychotomous AChE activity variable, flower worker cohabitation (vs. not) had an OR 3.39 (95% CI 1.19, 9.64) for being <15th percentile compared to the highest tertile. Children cohabitating for ≥5 years (vs. never) had an OR of 4.11 (95% CI: 1.17, 14.38) of AChE activity within <15th percentile compared to the highest tertile. AChE was lower by 0.09 U/ml (95% CI

- 0.19,

- 0.001) in children of flower workers (57% of participants) than non

- flower workers’ children, after adjustment for gender, age, height

- for

- age, hemoglobin concentration, income, pesticide use within hou sehold lot, pesticide use by contiguous neighbors, examination date, and residence, distance to the nearest flower plantation. Flower worker cohabitation and AChE did not differ by gender, race, age, height

- for

- age, or number of flower workers cohabitating

.

Duration of exposure: Every year of cohabitation with a flower worker was associated with a fully adjusted mean difference of

- 0.02 U/ml (95% CI:

- 0.03,

- 0.002). Every year of cohabitation was associated with an adjusted OR of 1.22 (95% CI: 1.02, 1.45) for AChE activity in the lowest 15th percentile compared to the highest tertile. Children who cohabited with a flower worker for 5 years or more (vs. never) had an OR of AChE within the lowest 15th percentile (compared to the highest tertile) of4.11 (95% C I: 1.17, 14.38) and within the 15th

– 33rd percentiles of 3.86 (95% CI: 1.44, 10.37).Page 84 of 171 Among the 277 children, worker practices information for 233 children: parents of 52% of non

flower worker children had 0 “bad” practices, 17% had 1

– 2 “bad” practices, 18% had 3 “bad” practices, and 13% had 4

– 6 “bad” practices 13%. The most common bad work practices were: washing work clothes at home (95%), never showering at work (45%), infrequent removal of (38%) and bringing work shoes (18%) home, and bringing tools (16% ) home. The fully adjusted AChE activity means among children for the 4 level bad practices variable was: 0=3.19 U/ml (95% CI: 3.13, 3.26), 1

– 2=3.09 U/ml (95% CI: 2.97, 3.21), 3=3.09 U/ml (95% CI: 2.97, 3.20)
and 4

– 6=3.02 U/ml (95% CI: 2.90, 3.15). Childre n whose parents had 4

– 6 bad practices had a lower mean AChE activity by 0.17U/ml (95% CI:

- 0.31,

- 0.02) compared to children with no bad practices.

Overall conclusions were that living with a flower worker was related to lower AChE activity in children, l onger duration of cohabitation was associated with lower AChE activity; and
cohabitation with workers with 4

– 6 bad work practices was associated with lower AChE activity. The findings support the hypothesis, i.e., that the amount of secondary pesticide exp osure from cohabitation with a flower plantation worker is sufficient to decrease AChE activity in children. The study also demonstrated the utility of field AChE measurements as a marker of pesticide exposure in children.

2013a analysis of AChE activity from secondary pesticide exposure and cohabitation with a flower plantation worker and changes in blood pressure and heart rate

Multiple linear regression models were used to examine the association of AChE activity (continuous) with SBP, DBP, standardiz ed values for SBP and DBP, and HR (significance a=0.05). Model 1 included examination date, age, sex, race, and height

- for

- age z score, and hemoglobin concentration. Model 2 included the Model 1 covariates, HR (when not the outcome of interest), income, nu mber of smokers living at home, distance from residence to nearest flower plantation, pesticide use in the home or on the household lot, and pesticide use by contiguous neighbors. When the predictor was AChE activity, flower worker cohabitation was
added t o model 2. The researchers also plotted BP as linear function of AChE, adjusted least square means SBP and DBP to octiles AChE activity, for a visual of model fit.

AChE activity was positively associated with resting heart rate, but estimated decreases i n mean values were not statistically significant. AChE activity was positively associated with blood pressure (SBP and DBP) even after adjustment for resting heart rate. In model 2, every 1

- U/mLPage 85 of 171 decrease in AChE activity was associated with a 2.86 mmHg dec rease in mean SBP (95% CI:

– 5.20,

– 0.53, p<0.05) and a 2.89 mmHg decrease in mean DBP (95% CI:

– 5.00,

– 0.78, p<0.05), after adjustment for potential confounders. Each 1

- U/mL decrease in AChE activity was
associated with decreases in z

- scores for SBP and DB P of

– 0.26 SD (95% CI:

– 0.48,

– 0.04) and

– 0.22 SD (95% CI:

– 0.40,

– 0.03), respectively. Adjusted least square means for SBP and DBP estimated for each octile of AChE activity were closely aligned with the estimated linear trends, thus providing visual evid ence of good model fits. Findings suggest that subclinical secondary exposures to pesticides may affect vascular reactivity in children.

Children living with flower workers and longer duration of cohabitation had lower SBP than other children, and practi ces that might increase exposure also were associated with lower SBP. This suggests that the amount of take

- home pesticide exposure by flower workers may be sufficient to induce physiological changes in children. Additional evidence in support of this hypo thesis includes the negative associations of SBP with the number of flower workers living at home and the number of “bad practices” that may increase the likelihood of take

- home pesticide exposures by flower workers.

2013 analysis of AChE activity and neurodevelopment (particularly attention, inhibitory control, and memory) among children living in floricultural communities, with stronger associations among boys.

For this third report, associations between AChE level and neurodevelopment were analyzed using logistic models (dichotomous and polychotomous) and linear regression models. Effect modification according to gender was assessed among statistically significant associations by testing an interaction term and through stratification followed by form al statistical testing of the difference of the stratified coefficients. The models were adjusted for flower worker cohabitation status to control for potential secondary occupational exposures to other chemicals that might affect neurodevelopment but that do not inhibit AChE activity. A few variables were imputed for this report

- a “missing” race category (n=14), maternal education (n=15), income (n=17), and residential distance to the nearest flower plantation (n=3). The model was defined a priori to
inc lude hemoglobin (to account for varying red blood cell compositions) and potential confounders (age, gender, race, height

- for

- age z score [to estimate long

- term nutritional status], household income, maternal education, and flower worker cohabitation statu s).Page 86 of 171 AChE activity had positive (although mostly non

- significant) associations with all neurodevelopmental domains and subtests except with statue and visuomotor precision (non

significant and negative associations). There was significant effect modificati on according to gender in the associations between AChE activity and attention and executive functioning domain (P = 0.01) and AARS

– response set (P =0 .002). Girls had higher mean scores than boys
in all but 1 of the subtests within the attention and execu tive functioning domain, but only attention and executive functioning domain and AARS

– auditory attention scores were significantly different. Lower AChE activity was associated with lower total neurodevelopmental summary score among boys (

– 0.70 points per 1

- U/mL [ ∼ 2 SD] AChE decrease [95% confidence interval (CI):

– 1.36 to

– 0.04]) but not girls (0.17 point per 1

- U/mL decrease [95% CI:

– 0.53 to 0.88]).

In boys only, there were increased ORs of low (<9th percentile) neurodevelopment among those
in the lowest tertile versus the highest tertile of AChE activity (ORs: total neurodevelopment: 5.14 [95% CI: 0.84, 31.48]; attention/executive functioning domain: 4.55 [95% CI: 1.19, 17.38], memory/learning domain: 6.03 [95% CI: 1.17, 31.05]). Within these domains, at tention, inhibition and long

- term memory subtests were most affected.

Overall, the associations between AChE activity and neurodevelopment were negligibly different when flower worker cohabitation status was not included in the adjustment model.

Low AChE activity, reflecting OP and carbamate pesticide exposure, was associated with deficits in neurodevelopment, particularly in attention, inhibition, and memory in boys but not in girls. These are critical cognitive skills that affect learning and academic p erformance. The findings suggest that boys have greater sensitivity than girls for neurodevelopmental delays from subclinical pesticide exposures.

Study Review

This was a well

- conducted study, in three reports, of pesticide (OP and carbamates) exposure on AChE level, heart rate, blood pressure, and neurodevelopment conducted in a population of healthy, young (4

- 9 year old) children in Ecuador. The study included most of current eligible population, 4

- 9 years of age (from 2004 census survey, plus new comm unity volunteers), and so selection bias is unlikely to have occurred. A few more flower workers did not show up for thePage 87 of 171 examination than non

- flower workers, but was a small proportion of overall population. The exposed and unexposed groups had comparable demographics, SES, etc.

The analytic methods, questionnaire, and analytic approach were appropriate in all three reports. Inferences to be garnered from the results are constrained by limitations of the cross

- sectional study design (cannot assess temporal ity between exposure and outcome due to cross

- sectional design), the exposure assessment, and the assessment of developmental and neurodevelopment outcomes. None of the exposure measures (cohabitation with flower worker, distance to fields, AChE activity) in this study allow differentiation of the contribution of specific pesticides or pesticide groups (OP or carbamate) on results.

In addition, t he focus of this epidemiological literature review is on the neurodevelopmental outcomes from exposures to low l evels of OPs (i.e. below exposures which would result in 10% or more AChE inhibition). These t hree studies conducted in Ecuador focused on child AChE inhibition and the potential association of AChE inhibition with other measures including parental occupation (Suarez

- Lopez et al, 2012), as well as clinical autonomic nervous system (ANS) outcomes such as blood pressure and heart rate (Suarez

- Lopez et al, 2013a), and neurodevelopmental outcomes (Suarez

- Lopez et al, 2013). The range of AChE activity levels are lower in the 1st tertile (range of 1.44 to 2.93 U/mL) compared to the 3rd tertile (range from
3.33 to 4. 69 U/mL). Therefore, due to the outcomes assessed and the potentially toxic cholinergic effects that were associated with these outcomes, these studies are not considered to
be relevant to this review and were not included in the final synthesis of the ep idemiological
literature review ( add citation )

.Page 88 of 171 2012 analysis of flower worker cohabitation and children’s AChE activity

In this report, the exposure measure was flower worker cohabitation which does not prove that a child was exposed. The outcome

- AChE level in a single blood sample

– is a non

- specific
biomarker of OP pesticide exposure. However, the duration of cohabitation findings
demonstrated a dose

- response association. As this could be due to accumulating pesticides in the
home over time and/o r decreased safety practices with longer work experience, the inclusion of household distance to the nearest flower plantation in the analysis allowed researchers to disentangle the mechanisms of pesticide introduction into the home between pesticide drift from flower plantations and flower worker associated ‘‘take

- home’’ pathways. The authors note that they did not ask about consumption of ‘‘organic’’ (pesticide

- free) products as a possible source of reduced pesticide exposure given that such products are rarely available or advertised by local growers in the county. There was no reason to believe that consumption of non

- ‘‘organic’’ products would differ by flower worker cohabitation status.

The authors point out that no ‘‘unexposed’’ baseline was availab le to calculate AChE suppression because most of the children who lived with a flower

- worker were born into families with a flower worker (72%) and were potentially even exposed to pesticides in

- utero. In addition, there is a lack of multiple AChE measurem ents to allow for tracking activity over time and provide more robust conclusions on the associations of AChE and flower worker cohabitation. However, the finding that duration of cohabitation was inversely related to AChE is internally
valid and consisten t with cumulative exposures.

It is not known how much clinical impact AChE suppression equivalent to the ones observed can have on children in the short and long term neurodevelopment. Chronic pesticide exposures during childhood could effect multi

- systemic developmental impairments including neurobehavioral delays; this is a gap in the literature that needs to be addressed and is a further aim of the ESPINA study.

2013a analysis of AChE activity from secondary pesticide exposure and cohabitat ion with a flower plantation worker and changes in blood pressure and heart rate

In this second report, the researchers treated AChE activity, along with cohabitation with a flower plantation worker, as the exposure measure. While AChE measurement is an appropriate method to assess past exposures to carbamate and OP pesticides because it can reflect longer

-Page 89 of 171 term exposures than plasma cholinesterase (butyrylcholinesterase) or pesticide metabolite quantification, AChE is less sensitive in detecting pesticide exposures. Furthermore, AChE activity has a low intra

- individual variability, in contrast with urinary metabolites of pesticides
that have more intra

- individual than inter

- individual variability (Lefkowitz et al., 2007; Griffith et al., 2011).

The model s assumed that more exposed children have a more pronounced reduction in AChE than less exposed children, i.e., a larger effect size within lower levels of AChE activity than within higher levels. To the authors’ knowledge, no AChE standards exist for chil dren. To assess this, they divided the sample into tertiles, then divided the lowest tertile of AChE activity into two categories: an upper half (33rd

– 15th percentiles, 2.68

– 2.93U/ml) and a bottom half (<15th percentile, 2.68 U/ml ~<10th percentile of chil dren who do not live with a flower worker).

Because children were examined during the months of July and August and AChE activity can reflect exposures to organophosphates within the previous 82 days, there was potential that
children who were examined e arlier in July may have lower AChE activity (from secondary sources) due to heightened fumigations for the Mother’s Day (May) surge of flower production. This exposure gap may be differential by exposure status given that the proportion of examined
childre n who lived with a flower worker in early July does not necessarily represent the distribution of the overall group. Examination date was not an effect modifier for the association between flower worker cohabitation and AChE (p=0.12), and was controlled fo r in all models.

Factors other than exposure to cholinesterase inhibitor pesticides may contribute to lower SBP among children who lived with a flower worker, given that associations persisted after adjusting for AChE activity. Agricultural workers are t ypically exposed to a mix of pesticides, and it is possible that non

- cholinesterase inhibitor pesticides may also contribute to (or be responsible for) the decrease in SBP, because AChE inhibition could be acting as a proxy indicator of overall pesticide e xposure. There is also an inability to account for factors related to BP such as prenatal toxicant exposures, concurrent and previous dietary patterns (e.g., salt and fat consumption), and perinatal health history.

The results of this investigation sugges t a physiological disturbance associated with cohabitation with agricultural workers. This concept is corroborated by the previous finding in this study population, where children living with flower workers had lower AChE activity compared withPage 90 of 171 nonagricult ural workers, and lower AChE was associated with longer duration of cohabitation and a greater number of bad practices (Suarez

- Lopez et al., 2012).

Although there was a statistically significant interaction with race in the associations of flower
worker c ohabitation, duration of cohabitation, and number of flower workers living with DBP, the authors did not believe this association is important. With complementary and related information, these three exposure variables represent one exposure construct: coh abitation with a flower worker. Given the genetic similarities between indigenous and mestizos and the fact that there was evidence of effect modification of, in essence, one exposure

– outcome relation, it is likely that the observed difference by race may be spurious.

The estimated average BP decreases were relatively small, but the amount of cholinesterase inhibitor pesticide exposure is also likely small due to the secondary nature of the exposure. The
estimated association of cohabitation with a flower worker and blood pressure may be conservative because participants were examined in July and August, which are months of decreased flower production. The finding that lower AChE activity was associated with lower blood pressure and a small, non

- significant decrease in heart rate suggests that cholinesterase inhibitor pesticides may primarily induce vasodilation in children. The researchers speculated that the blood pressure decreases may reflect a predominant stimulation of muscarinic M3 receptors in the va scular endothelium.

The researchers note that flower worker children may have similar exposures as in similar communities in the US and other developed countries. For example, the mean AChE activity for children living with flower workers were consistent with mean levels reported for Hispanic children living in agricultural (3.0 U/mL) and nonagricultural families (3.1 U/mL) in Oregon (USA) (Higgins et al., 2001). Generally, lower BP is associated with better cardiovascular outcomes, but long

- term cardiovas cular effects of chronic low

- dose pesticide exposures are unknown, and the possibility of any physiological effects of low

- dose pesticide exposures in
children is a concern. Additional research is needed to determine whether the associations that
observed are evident in other populations.

2013 analysis of AChE activity and neurodevelopment (particularly attention, inhibitory control, and memory) among children living in floricultural communities, with stronger associations among boys.Page 91 of 171 In this final report , the researchers examined AChE activity and neurodevelopment. The review comments above apply to this report. In addition, while NESPY

- II seems to be an appropriate neurodevelopment tool and was administered by trained examiners, scores were overall lower
than the NESPY

- II normative sample. This could be due to differences in cultural and social practices, test

- taking abilities, nutrition, or environmental exposures. This analysis did control
for nutrition and anemia.

The findings support the hypothesis that AChE inhibition is an important mechanism of neurotoxicity of cholinesterase inhibitor pesticides. Other studies of children (Marks et al., 2010; Horton et al., 2012), as well as animal studies, have reported greater detrimental neurochemical
and beha vioral effects, including working memory, among males after early postnatal exposure to OP pesticides. However, AChE inhibition is an indicator of exposure, and other neurotoxic mechanisms may also be present but unidentified. The reason for the gender int eraction is not understood and is a topic for future research. However, for the research question of interest, i.e.,
does OP pesticide exposure affect neurodevelopment, these findings are less relevant than studies which measure OP pesticides directly or t heir exposure biomarkers.

References cited

Garratt LC, Kelly TP. To what extent does bilingualism affect children’s performance on the NEPSY? Child Neuropsychol. 2008;14(1):71

– 81.

Griffith W, Curl CL, Fenske RA, Lu CA, Vigoren EM, Faustman EM. 2011. Organophosphate pesticide metabolite levels in pre

- school children in an agricultural community: within

and between

- child variability in a longitudinal study. Environ Res 111(6):751

– 756.

Horton MK, Kahn LG, Perera F, Barr DB, Rauh V. Does the home enviro nment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory? Neurotoxicol Teratol. 2012;34(5):534

– 541.

Lefkowitz LJ, Kupina JM, Hirth NL, Henry RM, Noland GY, Barbee JY Jr, et al., 2007. Intraindi vidual stability of human erythrocyte cholinesterase activity. Clin Chem 53(7):1358

– 1363.

Marks AR, Harley K, Bradman A, et al. Organophosphate pesticide exposure and attention in young Mexican

- American children: the CHAMACOS study. Environ Health Perspec t. 2010;118(12):1768

– 1774.

Richardson JR, Chambers JE. Effects of repeated oral postnatal exposure to chlorpyrifos on cholinergic neurochemistry in developing rats. Toxicol Sci. 2005;84(2): 352

– 359.Page 92 of 171 World Health Organization, 2008. Training Course on Chi ld Growth Assessment. WHO, Geneva.Page 93 of 171 Study 15. Wang et al. (2012)

Organophosphate pesticide exposure and perinatal outcomes in Shanghai, China. Pei Wang, Ying Tian, Xiao

- Jin Wang, Yu Gao, Rong Shi, Guo

- Quan Wang, Guo

- Hua Hu, Xiao

- Ming Shen. 4 Environment International 2:100

– 104, 2012.

Study Summary

In this report, Wang et al. evaluate the relationship between maternal prenatal OP pesticide exposure and perinatal outcomes in a cross

- sectional birth cohort study set in Shanghai, China using both questionna ire

- derived and biomarker

- based pesticide exposure assessments.

To evaluate associations between indicators of fetal growth and prenatal OP pesticide exposure, healthy pregnant women between the ages of 18 and 45 years were recruited from the obstetric

w ards of two major hospitals between September, 2006 and the end of January, 2007. Women with diabetes (gestational or preexisting), hypertension, HIV/AIDS, or who had used illicit drugs in the preceding year were identified during a brief screening exam an d excluded from participation. Of 193 eligible women, 187 consented to participate in the study.

After delivery, the participating women received a questionnaire

- guided interview conducted by a specially trained nurse to solicit information on pesticide exposure, to include whether pest control measures were used by the woman herself or other househol d members during pregnancy, the types of pesticides used in the home, and the frequency of use. Paternal OP pesticide exposures and occupational history were also queried. Demographics, medical history, and socioeconomic indicators including maternal age, education and income level, reproductive history, smoking history, occupational history, and alcohol and drug consumption during pregnancy were also obtained.

A single urine sample was collected from the participating mothers at the onset of labor. Speci mens were processed and shipped to the Shanghai Centers for Disease Control and Prevention for quantification of dialkylphosphate (DAP) metabolites. DAP metabolites including dimethylphosphate (DMP), dimethyldithiophosphate (DMTP), diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP) were quantified using gas chromatography. DAP observations below the (unspecified) limit of detection (LOD) werePage 94 of 171 assigned a value equal to the LOD/√2. DAP concentrations were estimated with an d without creatinine adjustment, and were log transformed for analysis. Data on the neonates were ascertained from the infants’ medical records, to include gestational age at birth, sex, birth weight, birth length, presence of birth defects, Apgar score, p regnancy and delivery complications, as well as maternal anthropometric measures such as height, weight, and weight gain during pregnancy. LBW was conventionally defined as birth weight less than 2,500 grams; only six infants met this criterion. Preterm de livery was defined as birth at less than 37 weeks of completed gestation; only four neonates met this criterion.

Multivariate linear regression models were constructed to examine the relationship between exposure to OP pesticides and birth outcomes adjus ted for potential confounders. The following covariates were included in final models: maternal height, pregnancy weight gain and family income. Smoking, alcohol use, illicit drug use, maternal education, reproductive history, and other information about p revious pregnancies, paternal OP pesticide exposure and paternal occupation were found not to be significant predictors of outcomes (alpha=0.1) and were not included in final models. To evaluate effect modification by gender, separate models were

construct ed for boys and girls. The authors also conducted a sensitivity analyses excluding preterm infants form the analytic sample.

No associations between any of the OP pesticide exposure measures and gestational age, birth weight and birth length outcomes were observed in primary analyses. The authors observed a statistically significant 1.79 week average decrease in gestational age associated with a log increase in urinary DEP among girls, but did not observe a similar association among boys.

Study Review

T his was a well

- conducted study of prenatal OP pesticide exposure effects on birth outcomes in a population of healthy, young Chinese mothers and their newborn children. The analytic methods, questionnaire, and analytic approach were appropriate. The study was limited by the cross

sectional ascertainment of pesticide exposure and birth outcomes, a small sample size, and low prevalence of LBW and PTD outcomes (precluding their analysis).

The authors employed a reasonable set of inclusion/exclusion criteria. Almost all (97%) of the eligible women recruited for participation in the study agreed to participate and were enrolled. Selection bias is unlikely to have occurred.Page 95 of 171 The investigators collected participant OP pesticide exposure and a large set of potent ial covariate information via a questionnaire

- guided interview which was conducted after the delivery. It is therefore possible that self

- reported exposures were not independent of birth

outcomes. Objective biomarkers of OP pesticide exposure were quantified in a single maternal spot urine sample obtained at the onset of labor. Specimen collection and biomarker quantification methods appear to be appropriate. DAP concentrations were noted to be higher in this Chinese study population, relative to those observed in other Western populations. The use of urinary DAP metabolite biomarkers of exposure likely provide a more objective, sensitive, and likely also more accurate and reliable indicators of OP pesticide exposure, relative to the ex posure assessment relying on maternal self

- report after delivery. The biomarker based prenatal OP pesticide exposure assessment also has limitations. Because they are non

- specific, urinary DAP concentrations can be considered an integrated biomarker of exp osure to many OP pesticides. Because of the often intermittent nature of pesticide exposure, urinary DAP metabolite levels can exhibit considerable temporal variability (i.e. day

- to

- day fluctuations) which could not be evaluated in this study in which a ur ine sample was collected at one time point. Urinalysis for DAP metabolites, therefore provides a biomarker of very recent exposure, i.e., in the 24 to 48 hour period prior to the collection of the urine sample. In this study, that equates to the day or

two just prior to delivery. Exposures in this short time period late in gestation may not be representative of a participant’s usual prenatal OP pesticide exposure or exposures during more
relevant time

- window(s) for exposure effects. Finally, OP pesticides c an break down into DAPs in the environment, and urinary concentrations may therefore also reflect exposure to preformed (non

- toxic) environmental DAPs.

Collection of the fetal development outcomes was straightforward and unlikely to have been
conducted w ith error. To the extent that they did occur, outcome measurement errors were likely independent of DAP concentrations but, as mentioned above, may not be independent of self

reported exposures.

The statistical methods used in this study were conventiona l and appropriate. Covariate adjustment was also appropriate, though residual confounding by mismeasured or unmeasured determinants of fetal development is possible. Correction of hypothesis test results for multiple comparisons was not conducted.Page 96 of 171 The on ly findings observed in this report where within strata of gender, and specifically among girls. It was unclear whether the hypothesis of interaction between prenatal OP pesticide exposure and child gender was developed a priori , or if stratification by ge nder was an ad hoc exploratory analysis conducted after associations were not observed in the study population as a whole. However, the small size of the study sample is an indication that the decision to stratify by gender was not driven by an a priori hy pothesis.

The results of this study were largely null. Errors in the estimation of prenatal OP pesticide exposure are a possible explanation for the lack of observed association. The authors’ conclusion that high pesticide levels might adversely affect du ration of gestation is not well supported, given the solitary finding of an association between increasing DEP concentrations and decreasing gestational age only among girls, the potentially exploratory nature of this evaluation of effect modification by g ender, and the small sample size. The findings of this investigation are generally not consistent with the hypothesis that prenatal OP pesticide exposure is associated with adverse perinatal outcomes.Page 97 of 171 Study 16. Zhang et al. (2014)

Prenatal exposure to organophosphate pesticides and neurobehavioral development of neonates: A birth cohort study in Shenyang, China. Ying Zhang, Song Han, Duohong Liang, Xinzhu Shi, Fengzhi Wang, Wei Liu, Li Zhang, Lixin Chen, Yingzi Gu, Ying Tian. PlosOne 47:80

– 85, 2014.

S tudy Summary

In this report, Zhang et al. evaluate the relationship between maternal prenatal OP pesticide exposure and neonatal neurodevelopment prospective birth cohort study set in Shenyang, China using urinary markers of OP pesticide exposure.

In ord er to investigate the effects of prenatal OP pesticide exposures on neonatal neurodevelopment, 249 healthy pregnant women between the ages of 18 and 45 years were recruited from the hospital associated with the Shenyang Medical College. Women living in
She nyang for less than 3 years, and those with hypertension, diabetes, thyroid disorders, heart disease, or other non

- specified chronic diseases prior to pregnancy were excluded from participation in the study. Women with “heavy” pregnancy complications inclu ding diabetes, anemia, and hypertension were also excluded, as where those with a family or medical history of mental retardation, phenylketonuria, or Pompe’s syndrome. Infants with traumatic brain injury, meningitis, or severe illnesses were also excluded from analyses. Of 307 eligible pregnant women, 249 consented to participate and were enrolled in the study with their neonates.

After delivery, the participating women received a questionnaire

- guided interview conducted by a specially trained nurse to so licit information on pesticide exposure, to include whether pest control measures were used by the woman herself during pregnancy, or by a family member, the types of pesticides used in the home, and the frequency of use. Demographic information, and
the i nformation regarding home characteristics, residential history, reproductive history, smoking history, diet, and alcohol and drug consumption during pregnancy were also obtained.

A single urine sample was collected from the participating mothers. The tim e of the urine sampling relative to gestational age or delivery was not specified in the report. Urine specimens were processed and shipped to the Shanghai Centers for Disease Control and Prevention for quantification of five dialkylphosphate (DAP) metabol ites; Dimethylphosphate (DMP),Page 98 of 171 dimethyldithiophosphate (DMTP), diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP) were quantified using gas chromatography. The lower limit of detection (LOD) was 2.0 μg/L for DMP and 1.0 μg/L for DMTP, DEP, DETP, and DEDTP. DAP observations below the limit of detection (LOD) were assigned a value equal to the LOD/√2. DAP concentrations were estimated with and without creatinine adjustment, and were log transformed for analysis.

Infant neurodevelopment was assessed three days after birth using a neonatal behavioral neurological assessment conducted by two trained examiners who were blinded to the exposure status of the children. (NBNA, Bao et al., 1991) The NBNA assesses function al abilities, reflexes and responses, and stability of behavioral status using five scales: Behavior, Passive, Tone, Active Tone, Primary Reflexes, and General Assessment. The items from each of these
scales were summarized in a total score with a maximum score of 40. Neonates with a total score greater than 37 were considered well developed, those with a total score lower than 34 were classified as abnormal, and those with scores in between were considered as acceptable range.

Pearson Correlations were c alculated to summarize the univariate linear relationship between maternal prenatal urinary OP pesticide metabolites neonatal neurobehavioral assessments scores. Multivariate linear regressions were then constructed to estimate the association between

mate rnal DAP concentrations during pregnancy and the neurodevelopment of neonates, adjusted for potential confounders. Separate models were built with each of the scores of the five NBNA scales and the total NBNA score as dependent variables, and each of the l og

- transformed concentrations of total DAP, total DMP and total DEP as continuously distributed predictor variables. The following covariates were evaluated for inclusion in these models using a stepwise modeling approach: maternal age, maternal education, gestational age, prenatal BMI, neonatal bodyweight, and cord blood lead concentration. Statistical significance (alpha) level was set at 0.10 for inclusion of covariates and 0.20 for exclusion in the stepwise process. Potential

exposure

- response relations hips between maternal prenatal OP pesticide exposure and neonatal neurodevelopment outcomes were assessed by categorizing DAP concentrations by quintile and comparing mean neurodevelopment scores from the lowest quintile of DAP concentration (as the
refere nce group) to those in the second through fifth quintiles. Multivariate analyses were also stratified by gender.

Maternal prenatal urinary DAP, DMP, and DEP concentrations were significantly negatively correlated with summary NBNA scores. In multivariate analyses, higher prenatal DAPPage 99 of 171 concentrations were associated with lower scores in all NBNA scales. A 10

- fold increase in total DAP concentration was associated with an average decrease in NBNA Summary scores of 1.78 points (95% CI:

- 2.12 to

- 1.45). No evid ence of departure from linearity of the exposure

response relationship between maternal DAP concentrations and NBNA scores was observed. Statistically significant effect modification by gender was not observed, though associations were generally stronger a mong girls.

Study Review

This was a well

- conducted study of prenatal OP pesticide exposure effects on neonatal neurodevelopment in a population of healthy, young Chinese mothers and their newborn children. The analytic methods, questionnaire, and analytic approach were appropriate . Study strengths include the prospective study design, objective measurement of OP pesticide exposure, and use of a sensitive instrument for assessing neonatal neurodevelopment.

The authors employed a reasonable set of inclusion/exclusion criteria. A hi gh percentage (81%) of the eligible women recruited for participation was enrolled in the study. Selection bias is unlikely to have occurred.

Objective biomarkers of OP pesticide exposure were quantified in a single maternal spot urine sample. The timing of the specimen collection was not clearly reported. The biomarker quantification methods appear to be appropriate. The use of urinary DAP metabolite biomarkers of exposure likely provide a more objective, sensitive, and likely also more accurate and reli able indicators of OP pesticide exposure, relative to assessments relying on self

- report of pesticide

exposure. However, the biomarker

- based prenatal OP pesticide exposure assessment also has limitations. Because they are non

- specific, urinary DAP concentr ations can be considered an integrated biomarker of exposure to many OP pesticides. In humans, DAPs have a half

- life of less than 24 hours, so urinary concentrations reflect only recent exposure. The combination of
the short elimination half

- life and the e pisodic nature of exposures to OP pesticides can result in a high degree of day

- to

- day variability in urinary DAP concentrations. Also, urinary DAP concentrations cannot be used to quantify exposure to a specific pesticide. Exposures in this short time per iod late in gestation may not be representative of a participant’s usual prenatal OP pesticide exposure or exposures during more relevant time

- window(s) for exposure effects. Finally, OP pesticides can break down into DAPs in the environment, and urinary c oncentrations may therefore also reflect exposure to preformed (non

- toxic) environmental DAPs.Page 100 of 171 Errors in the estimation of prenatal OP pesticide exposure are likely to be non

- differential with respect to the neurodevelopment outcome scores, and thus are not a likely explanation for the
observed associations.

The neonatal behavioral neurological assessment used to evaluate infant’s neurodevelopment at three days of age has been used previously in several cohort studies conducted in China, and
demonstrate d “distinct stability and reliability” in these settings. It appears to be a highly sensitive indicator of neurodevelopment.

Covariate adjustment appeared appropriate, though residual confounding by mismeasured or
unmeasured determinants of fetal neurode velopment is possible. Correction of hypothesis test results for multiple comparisons was not conducted.

Consistent statistically significant associations were observed between all of the quantified urinary biomarkers of prenatal OP pesticide exposure an d neonatal neurodevelopment assessed 3 days after birth. Higher DAP concentrations were associated with lower summary NBNA scores (indicating increasing neurodevelopmental deficits). The associations observed in this study by Zhang et al. are consistent wi th the hypothesis that prenatal OP pesticide exposure affects fetal neurodevelopment.

References cited

Bao XL, Yu RJ, Li ZS, Zhang BL (1991) Twenty

- item behavioral neurological assessment for normal newborns in 12 cities of China. Chin Med J. 104:742

– 746

.Study 17. Shelton et al. (2014)

Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: The CHARGE Study. Janie F. Shelton, Estella M. Geraghty, Daniel J. Tancredi, Lora D. Delwiche, Rebecca J. Schmidt, Beate Ritz, Robin L. Hansen, and Irva Hertz

Picciotto. Environmental Health Perspectives 122:1103

– 1109, 2014.

Study Summary

The CHARGE study is a population

- based case

- control study of child development specifically focused on evaluating potential risk f actors for autism spectrum disorders and developmental delay. In this paper, Shelton et al. report on an evaluation of the association between residential proximity to agricultural pesticides during pregnancy and neurodevelopmental outcomes, specifically t he risks of autism spectrum disorders and developmental delay among children of families eligible for inclusion in the California Department of Developmental Services (DDS) system. An estimated 75

- 80% of all children with these diagnoses living in Californ ia are enrolled in the DDS system (Croen et al., 2002). Beginning in 2003, the CHARGE study has recruited more than 1,600 child participants whose parents completed extensive questionnaires concerning potential environmental exposures during pregnancy. Cas es (children with diagnosed
with full

- syndrome Autism Spectrum Disorders and Developmental Delay) were recruited either from one of several regional centers of the DDS or through referrals from other clinics, self

referral or unspecified “general outreach. ” The Autism Diagnostic Observation Schedule (Lord et al. 2000, 2003) was used to either confirm the ASD diagnosis or reclassify the children for the purposes of the study.

Control participants were recruited from the general California population, having been identified through birth records maintained by the State of California. Children were eligible to participate in the CHARGE study as controls if they were between two and five years of age, were born in California, live with a biological parent who s peaks either English or Spanish, and reside in a CHARGE study catchment area (mainly within a 2

- hour drive from the Sacramento area). Children potentially eligible to participate in the study in either the developmental delay group or the general populatio n control group were administered the Social Communications Questionnaire (Rutter et al., 2003) to rule out an autism

- spectrum disorder diagnosis.

Shelton et al. used publically available commercial agricultural pesticide application data from
the Califor nia Department of Pesticide Regulation's annual Pesticide Use Report (CDPR 2014)to estimate prenatal pesticide exposures. This report includes agricultural pesticide applications in the State (in pounds of active ingredient applied, by date) for square

- mi le areas known as
meridian

- township

- range

- sections (MTRS). The authors used GIS

- mapping software to create a geographic centroid for each MTRS. The investigators then mapped the residential addresses of participant's mothers during pregnancy, collected on the CHARGE questionnaire and created three buffers with radii of 1.25 km, 1.5 km, and 1.75 km around each residence. Pregnancies of participants' mothers where then assigned a pesticide exposure profile corresponding to the pesticides applied to the MTRS n earest the mother's home and during her pregnancy on which the pesticide applications occurred. Pesticides were grouped into organophosphate, carbamate, pyrethoid, or organochlorine classes. Chlorpyrifos was examined independently because of previous evide nce of its associations with neurodevelopmental outcomes. Most of the participants’ homes received a pesticide exposure level of zero, as no pesticides had been applied within the buffer zone; approximately a third of the study participants' mothers lived within 1.5km of an agricultural pesticide application during their pregnancy.

Controls were frequency matched to autism cases on sex, age, and catchment area. Potential additional confounders were initially identified using directed acyclic graphs (DAGS, Rothman et al., 2008) as potential determinants of autism spectrum disorders or developmental delays and which may influence exposure to pesticides (without consideration of statistical associations in univariate assessments). A 10% change

- in

- estimate crit erion was established to exclude potential confounders from the final models. The final models included adjustment for paternal education, home ownership, maternal country of birth, child race/ethnicity, maternal prenatal vitamin use, and year of birth. Th e authors additionally constructed models adjusting pesticide class

- specific associations for indicators of exposure to the other pesticide classes considered in this investigation.

The independent associations between risk of the neurodevelopmental outco mes (i.e. autism spectrum disorder and development delay) and the different classes of pesticides were examined, as well as potential interactions between the different classes of pesticides.

Multinomial multivariate logistic regression models (with appro priate survey weights) were constructed for each developmental outcome. Agricultural pesticide exposure was defined as a dichotomous (yes/no) variable, according to presence or absence of pesticide applications within the buffer zone during the pregnancy t ime period.Proximity to OP pesticide application at some time during pregnancy was associated with a 60% increase in the risk of autism spectrum disorder (OR=1.6; 95% CI: 1.02

– 2.51), compared to those whose mothers who those who did not live in close pro ximity (within 1.5 km) to any agricultural pesticide applications during their pregnancy. Elevations in risk were greater for exposures that occurred specifically in the third

- trimester (OR=2.0; 95% CI: 1.1

- 3.6). No statistically significant associations w ere observed between proximity to OP pesticide applications during pregnancy and risk of developmental delay.

Study Review

This was a largely well conducted case

- control study that leveraged publically available agricultural pesticide application data ag gregated to one

- square mile areas, in combination with questionnaire

- based residential location information and GIS mapping techniques to evaluate risk of autism spectrum disorder and developmental delay in a population based case

- control study set in Cali fornia.

The study was large, with a case sample of 486 children with autism spectrum disorder, 168 children with developmental delay, and a study base drawn from the catchment area of the CHARGE study (mainly the area within a two hour drive from the Sacr amento area, and also included a portion from Southern California), represented by 316 normally developing control participants.

No actual environmental pesticide measurements or biomarkers of pesticide exposure where evaluated in this study. The exposur e assessment used distance from the mother’s home during pregnancy to areas associated with specific agricultural pesticide applications as a proxy for pesticide exposure. The investigators took care to minimize misclassification of addresses in the study. However, misclassification of actual pesticide exposure is likely to have occurred, as it is possible that those living near locations with agricultural pesticides applied where not, in fact, exposed to the pesticides while those living far from sites whe re agricultural pesticides where applied may have been exposed. The exposure assessment did not consider many potential sources of pesticide exposure, including non

- agricultural institutional applications, residential indoor and outdoor pesticide use, and dietary sources. Other sources of potential exposure errors discussed by Shelton et al. in their report include errors in reporting in the CDPR annual Pesticide Use Report, the assumption of homogeneity of pesticide exposures a buffer zone, and potential G IS geocoding errors. All of these sources of exposure classification errors were likely independent of case/control status.The autism and developmental delay case statuses were well defined. Outcome misclassification was likely minimized by the study protocol which included using validated instruments to provide diagnostic confirmation at the time of enrollment.

Control sele ction was appropriate and likely represented the study base from which the cases arose. The authors mentioned having difficulty with recruitment of general population controls in Southern California early in the study, due to “logistical difficulties,” and thus terminated recruitment in this area. The impact of these difficulties and the halting of recruitment in Southern California on the representativeness of the controls selected from this region (and corresponding selection bias) were not discussed in t he report. Inverse probability of participation weights were used in an attempt to adjust the multinomial models for differential probabilities of enrollment in the study by case or control groups. The authors argued that the weighted exposure frequency di stributions and regression models more accurately represent findings that are generalizable to the study base. This weighting also likely decreased the potential for selection bias that could be induced if the estimated pesticide exposure distributions

amo ng the control participants do not represent those of the population (or more formally, the study base) that gave rise to the autism spectrum disorder and developmental delay cases.

The statistical analyses and evaluation of potential confounding factors were appropriate. In addition to the multivariate modeling, control subjects were frequency matched to autism spectrum disorder cases on sex, age, and catchment area, and this matching was appropriately addressed in the statistical analysis. Matching of co ntrols to developmental delay cases was not discussed in the report and, presumably, was not conducted.

The results of the study support the authors’ conclusion that children exposed to OP pesticides during gestation may be at increased risk for neurodeve lopmental disorders. However, selection bias, residual confounding, and chance remain alternative explanations of the associations observed in the study.

References cited

CDPR (California Department of Pesticide Regulation). 2014. Pesticide Use Reportin g (PUR). Available: http://www.cdpr.ca.gov/docs/pur/purmain.htm

Croen LA, Grether JK, Hoogstrate J, Selvin S. 2002. The changing prevalence of autism in California. J Autism Dev Disord 32(3):207

– 215.Lord C, Rutter M, DiLavore PC, Risi S. 2000. The Autis m Diagnostic Observation Schedule (ADOS). Los Angeles: Western Psychological Services.

Lord C, Rutter M, DiLavore PC, Risi S. 2003. Autism Diagnostic Observation Schedule Manual. Los Angeles: Western Psychological Services.

Rothman KG, Greenland S, Lash T. Modern epidemiology. Philadelphia, PA: Lippincott Williams & Wilkins; 2008.

Rutter M, Bailey A, Berument SK, Lord C, Pickles A. 2003. Social Communication Questionnaire (SCQ). Los Angeles: Western Psychological Services.Study 18

. Acosta

- Maldonado et al. (2009)

Effects of exposure to pesticides during pregnancy on placental maturity and weight of
newborns: A cross

- sectional pilot study in women from the Chihuahua State, Mexico.
Brenda Acosta

- Maldonado, Blanca Sánchez

- Ramírez, Sandra Reza

- López, and M argarita
Levario

- Carrillo. Human and Experimental Toxicology 28(8) 451

– 459, 2009.

Study Summary

Acosta

- Maldonado et al. reported on a study evaluating the association between third

- trimester pesticide exposure and an index of placental maturity based on microscopic assessment of placental tissue morphology. The study was set among pregnant women in Chihuahua, Mexico.

The primary objective of this cross

- sectional pilot study was to assess the effects of cholinesterase inhibitor pesticides during pregnancy and placental maturity. The investigators compared placental morphology of nine women determined to have 3 rd

- trimester pesticide exposure to forty

- five women without explicit pesticide exposure. The authors excluded women with multiple pregnancie s and those “with diseases, either previous or developed during gestation.”

Acytelcholinesterase (AChE) activity was quantified in a venous blood sample obtained from the women at the time of admission to the hospital for delivery. The authors defined AC hE ≤ 4.96 U/mL as a marker of pesticide exposure effect. Participating women were asked to complete a previously validated structured questionnaire to garner information on clinical characteristics and pesticide exposure history.

Women (n=9) were defined as exposed to pesticides if they presented with more than one of the following: 1) residence in an eminently agricultural community in which pesticides had continuously been applied during agricultural cycles; 2) home located less than five kilometers
fro m a pesticide application zone; 3) or a spouse or any person cohabitating at subject's address who worked with pesticides or in agricultural labor. Although the specific pesticide(s) to which the women were exposed was not assessed, the investigators noted that the most commonly used pesticides were organophosphates, and of these, methylparathion was the most common. Forty

five mothers of full

- term infants without an explicit pesticide exposure served as the primary comparison group. To investigate the rela tionships between gestational age, birth weight, and placental maturity, the placentas of an additional thirty

- one mothers of pre

- term babiesdetermined not to have had pesticide exposure during the third trimester of pregnancy were also
included in the co mparison group.

The sampling and processing of placental tissue was done in an appropriate and consistent manner for all women. The morphometric analysis was conducted by two investigators who were blinded to the exposure status of the women. The outcome , a placental maturity index (PMI) was calculated by dividing the number of epithelial plates in terminal villi to their thickness. PMI was calculated in central and peripheral regions of the placenta.

For their primary analyses, the authors built an init ial multivariate adjusted linear regression model of PMI, predicted by pesticide exposure status and adjusted for gestational age, number of terminal villi per 1 mm 2 , number of capillaries, diameter of capillaries, diameter of the sinusoids, placenta weigh t, and weight of the newborn. Covariates that were not significantly associated with PMI (α=0.05) were removed from the model. Collinearity of the placental characteristics covariates was assessed by evaluating variance inflation, and p

- values were adjuste d for multiple comparisons.

There was a statistically significant relationship observed between pesticide and PMI in central region, with PMI of pesticide

- exposed women being, on average, 7.38 units higher than those of the women not exposed to pesticide s (p=0.01), after adjusting for number of terminal villi, number of terminal capillaries, terminal capillary diameter, sinusoids diameter of terminal villi and placental weight. Among pesticide exposed women, the PMI was greater in samples from
the central region, relative to the peripheral region, of the placentas, while the estimated PMI were the same in central and peripheral regions for non

- pesticide

- exposed women. In this study, pesticide exposure was associated with higher placental maturity index, on average. Although not assessed explicitly, the implication of the finding is that pesticide exposure may affect nutrient transfer across the placenta by disrupting placental morphology

. The implication of a lack of observed differences in PMI in periphera l areas of the placenta were not discussed in
detail.

Study Review

The interpretation of the results of this study of pesticide exposure and placental maturity among pregnant women living in Chihuahua City, Mexico, is constrained by the small sample size , the cross sectional study design, and the imprecise nature of the definition of pesticide exposure.The rationale and specifics of participant selection and exclusion criteria were not detailed in the report. Authors excluded women with multiple pregnancies and women with prevalent diseases was most likely because both of these affect placental morphology, although this was not stated specifically.

AChE enzyme activity was evaluated using conventional, and appropriate methods , in a single venous blood sample obtained at the time of delivery. The definition of pesticide exposure using a questionnaire and AChE activity likely resulted in misspecification of pesticide exposure that

was non

- differential with respect to PMI. Invest igators did not correlate the two measures; however, AChE concentration in women residing in agricultural areas was lower than levels detected in women residing in urban areas (4.29 + 0.54 vs 5.26 + 1.07 U/mL, p = .01, respectively).

Though the processing of the placentas can affect the quality of the specimens obtained, it appears that the sampling and subsequent morphometric analysis was well conducted. The placentas assessed in the study were all processed in a similar and appropriate manner.
Classifica tion of placental maturity involves counting and evaluating the average diameter of terminal villi in an area of the slide selected by the researcher; although the multistep process is subject to errors, intraclass correlation coefficients in the range of 0.82

- 1.0 indicate a high degree of agreement across the two reviewers. Errors in assignment of the placental maturity index are likely to have been independent of exposure status, as the investigators conducting the PMI assessment were blinded to the pesti cide exposure status of the women.

The statistical methods were appropriate. Selection and assessment of covariates and subsequent model building was appropriate. The authors evaluated their final linear regression models for
violation of OLS model assum ptions (i.e. normality of residuals and homoscedasticity). They also appropriately corrected their hypothesis test results (i.e. p

- values) for multiple comparisons. However, the modeling results were not adequately presented in the paper; they were not

pre sented in tabular form, nor in figures. Confidence intervals were not presented, nor were the model results comparing PMI for peripheral placental regions between pesticide exposed and

non

- exposed women.

The generalizability and inference to be garnered from the study results is limited by the small sample size of the study, as well as the cross

- sectional evaluation of the pesticide

- PMI relationship. Due to sampling variability, the nine pesticide

- exposed women may not be representative of pesticide

- exposed women generally. Overall, this study provides some evidenceconsistent with the hypothesis that third

- trimester OP pesticide exposure is associated with placental maturity, particular in central regions of the placenta.Study 19

. Dabrowski et al. (2003)

Pesticide exposure and birthweight: An epidemiological study in Central Poland. Sławomir Dąbrowski, Wojciech Hanke, Kinga Polańska, Teresa Makowiec

- Dąbrowska, and Wojciech Sobala. International Journal of Occupational Medicine and Environmental Health 16(1): 31

- 39, 2003.

Study Summary

In this report, Dabrowski et al. evaluate the relationship between maternal exposure to pesticides
in the 1 st and 2 nd trimesters of pregnancy and low birth weight (LBW) in a case

- control study set in the Łódź region of Poland.

In order to investigate the prevalence of risk factors for poor pregnancy outcomes, the authors originally invited 389 Polish women who delivered at one of 25 maternity hospitals on a randomly sel ected set of 70 days between February 1998 and the end of December, 2000 to participate in their research project. Noting the high prevalence of pesticide exposure in the area, the investigators subsequently hypothesized that pesticide exposure may be asso ciated with low birth weight in this rural population and constructed a case

- control study to evaluate this hypothesis. To increase the number of cases, enrollment in the study was extended to mothers who delivered LBW infants in two maternity hospitals du ring an additional six month period (January, 2001 through June 2001). The study population was restricted to women involved in farm activities for at least seven days during their pregnancy. Other exclusion criteria included complicated pregnancies, mater nal diabetes, hypertension, infectious diseases, and “other pathologies” that increase the risk of fetal growth restriction or preterm delivery.

A questionnaire

- guided interview was conducted by a physician 1

- 2 days after delivery in order
to ascertain d emographic information as well as occupational and other exposure histories from the mothers. During this interview, pesticide use in each trimester of pregnancy was reported by each participant. Although trimester

- specific pesticide exposure was queried, the investigators found that most of the exposed women (83.6%) reported exposure in both trimesters; the authors therefore combined these two groups. Thus, self

- reported pesticide exposure during the 1 st and/or
2 nd trimesters was the primary exposure varia ble analyzed in this study. Confirmation of pesticide exposure was conducted 6

- 12 months after delivery, during which time an interviewer visited the husbands of the 49 women who had previously reported pesticide use on their farms during pregnancy. During this follow

- up interview, specific active ingredients were identified forboth occupational and residential exposures. Pesticide use most frequently reported included: phenoxyacetic acid derivatives, organophosphates, ureas, triazines, synthetic pyrethroi ds and N

phenylamides (anilides). Associations between specific pesticides and LBW were not estimated, however, reportedly due to the small number of participants reporting exposure to specific pesticides.

LBW case status was conventionally defined as a child with a birth weight of less 2,500 grams. 117 infants in the study met this definition, and the remaining 377 infants with birthweight ≥ 2,500 grams served as controls.

The authors ascertained covariate information during the questionnaire

- based int erview, and adjusted LBW models for maternal age, marital status, pre

- pregnancy maternal weight, parity, maternal smoking, physical work during pregnancy, and place of residence.

In a univariate (unadjusted) model, the authors observed a greater than 5

- fold increase in the odds of LBW among women exposed to pesticides during the 1 st and 2 nd trimesters, relative to women not exposed to pesticides during pregnancy (OR: 5.84; 95% CI: 3.61

- 9.47), the association was markedly attenuated, and not statistical ly significant, after adjustment for potential confounders (OR: 1.29; 95% CI: 0.63

- 2.65). The authors also constructed a multivariate linear model of infant birthweight and pesticide exposure, adjusted for other determinants of birth weight and pregnancy d uration. Average birth weight of children of women reporting pesticide exposure was 100 grams lower than that of children born to women who did not report pesticide exposure. The authors also noted that women reporting pesticide exposure during the
first o r second trimester delivered, on average, half a week earlier than women without reported pesticide exposure. Average birth weight among women with organophosphate exposure was 35 grams less than expected, although this difference was not statistically sig nificant (p=0.19).

Study Review

This investigation was originally designed to evaluate the prevalence of risk factors for adverse birth outcomes in a population of pregnant women living in a rural region of Central Poland. To conduct this investigation, the authors attempted to recruit all women giving birth on randomly selected days. In this manner, they conducted a population

- based study. They then adapted and augmented the study in order to evaluate a specific hypothesis regarding pesticide exposure
du ring early pregnancy and (low) birth weight. The design was thus a bit confused, with theauthors switching between methodologic approaches appropriate for cross

- sectional retrospective cohort study designs, and those appropriate for a case

- control study.

The recruitment of study subjects occurred in two distinct phases and, importantly, from two different source populations. Pregnant mothers and their children who participated in the initial implementation of the study were recruited from 25 hospitals, a nd included all women delivering on randomly selected days. The second set of women were recruited during a later time period, from two hospitals, and included only women delivering low birth weight infants. The inclusion/exclusion criteria were appropriat e, given the study’s objectives.

The pesticide (and covariate) exposure assessment occurred by means of a questionnaire

- guided interview, conducted by a physician 1

- 2 days after the delivery of the child. The investigators attempted to validate these self

- reported exposures by visiting the homes of the mothers, albeit 6

- 12 months after delivery (i.e. up to 18 months after the relevant time window of exposure

– the
first and secon d trimesters of pregnancy). Considerable misclassification of pesticide exposure is therefore likely to have occurred, both due to the retrospective self

- recall of exposure by the mothers, and potentially complicated by the timing of the exposure ascertain ment with respect to the birth of the child. No biomarkers of pesticide exposure were evaluated in this study.

The assessment of the birth weight and LBW outcomes based on medical records was straightforward, and was likely to have been conducted without substantial error.

A small degree of residual confounding is likely in this study. The covariates included in multivariate models appear to have been appropriate, i.e. determinants of birth weight that may be associated with pesticide exposure, although covariate selection and model building processes were not discussed in detail. The authors included an analysis adjusting the association between first or second trimester pesticide exposure and birth weight for length of gestation; the
association between pesticide exposure and (lower) birth weight persisted, though the association
was attenuated.

Several biases are likely to have influenced the associations being evaluated in this study. The controls in this study (women who delivered children with birt h weights ≥ 2,500 grams) were recruited only during the first phase of the study. While LBW cases were recruited during both periods. Notably, LBW cases enrolled during the case

- augmentation portion of the study were
recruited from different areas than the controls. As such, it is possible that the prevalence of pesticide exposures among the control participants was not representative of the exposuredistribution in the population s that gave rise to the cases. In fact, the authors noted that participants we re recruited from “different source populations with different prevalence of pesticide exposure.” Selection bias is thus likely to have occurred, to the extent that the controls did not reflect pesticide exposure in the study base. In an attempt to offset this potential bias, the authors included place of residence as a covariate in their multivariate models. Nonetheless, some degree of selection bias in this study is probable. The direction and magnitude of this bias are not easily determined, could be sub stantial, and were not discussed by the authors.

Information bias due to differential errors in the self

- reporting of pesticide exposure during pregnancy was also possible, as the exposure was ascertained by maternal self

- report shortly after birth (mothe rs were likely aware of the LBW status when recalling exposures). The attempt by the investigators to validate self

- reported pesticide exposures is a potential strength of the study, though the validation was conducted up to a year after the birth and 1

- 1. 5 years after the

exposure

- time window of interest.

Therefore, it is unclear that the sampling of cases and controls was independent of exposure status. The statistical analysis included both linear regression, as might be appropriate in a cross
sectiona l or cohort study, and logistic regression which is appropriate for analysis of case

- control study data, in addition to data from cross

- sectional and cohort designs.

The sample size and corresponding statistical power of this study was adequate, although the investigators were compelled to expand enrollment in the study to increase the number of participating cases, at the potential expense validity (i.e., increasing the potential for selection bias, as discussed above).

The study results, if valid, are likely generalizable to populations of rural families and farmers exposed to similar farm activities and risk factors as those participating in the study. The contribution of this study to the overall evidence of pesticide exposure during pregnancy on
adv erse birth outcomes, LBW in particular, is limited by the likely effects of selection bias and (differential) information bias. The study was largely null, in that a statistically significant increase in the odds of LBW, after adjusting for length of gesta tion, was not observed in relation
to self

- reported pesticide exposure during early pregnancy. It is difficult to determine from the report whether the true association (in the absence of residual confounding, information bias, and selection bias) is large r or smaller than the small observed increase in the odds of LBW among the pesticide exposed participants cannot be determined empirically.Study 20

. Grandjean et al. (2006), Harari et al. (2010)

• 2006. Pesticide exposure and stunting as independent predictors of neurobehavioral deficits in Ecuadorian school children. Philippe Grandjean, Raul Harari, MD, Dana B. Barr, PhD, Frodi Debes. Pediatrics 117(3):e546

- e556.

• 2010. Neurobehavioral Deficits and Increased Blood Pressure in School

- Age Children
Pren atally Exposed to Pesticides. Raul Harari, Jordi Julvez, Katsuyuki Murata, Dana Barr, David C. Bellinger, Frodi Debes, and Philippe Grandjean. Environmental Health Perspectives 118(6): 890

- 896.

Study Summary

In these reports, Grandjean et al. evaluate th e relationship between reported prenatal and estimated recent organophosphate pesticide (OP) exposure, as well as a measure of physiologic stunting, in relation to a large set of neurobehavioral, clinical, and anthropomorphic outcomes among school

- age children participating in a cross

- sectional study in Tabacundo, Ecuador.

Children in the lowest two grades of one of the two elementary schools in Tabacundo were
invited to participate in the study. All eligible children were allowed to participate in the examinations: In Grandjean (2006) report, 1 child with an acute respiratory infection, 3 children with prevalent (unspecified) clinical abnormalities, and 3 children older t han 9 years of age were excluded from the analysis, resulting in an analytic subset of 72 participating children. In the Harari (2010) report, 3 children were excluded from the study because 1 child had clinical history of meningitis and 2 were older than 9 years of age, resulting in an analytic subset of 84 participating children.

Examinations, scheduled to be completed in 2 hours, were conducted at a nearby health clinic, and included the neurobehavioral testing and clinical assessment. To ascertain dat a on occupational exposure history, social and demographic characteristics, the child’s medical history and current health status, a 150

- item questionnaire was administered to the mothers at the
time of the examination. Mothers were not aware of the neurob ehavioral and clinical examination results at the time they completed the questionnaire. A public health and occupational medicine specialist blinded to the children’s’ clinical statuses reviewed the mothers’ completed questionnaires and scored their respo nses for potential maternal pesticide exposure during pregnancy. In Grandjean (2006), p renatal exposure to pesticides was determinedby mothers’ self

- reporting and treated as a dichotomous (yes/no) variable. In Harari (2010), prenatal exposure to pesticide was separated into three categories (no parental exposure, paternal exposure only, and maternal exposure irrespective of paternal status).

Spot urine samples were collected from the children at the time of the examination in order to quantify urinary OP metabolites as indicators of recent OP pesticide exposure. Collected samples were shipped on dry ice to the CDC in Atlanta, USA, where they were analyzed for six dialkylphosphate (DAP) metabolites

- three dimethylphosphate (DMP) metabolites (dimethylphosph ate, dimethyldithiophosphate, dimethylthiophosphate) and three diethylphosphate (DEP) metabolites (diethylphosphate, diethyldithiophosphate, diethylthiophosphate)

- using gas chromatography

– tandem mass spectrometry. The level of detection was 0.3 μg/L for dimethylphosphate and dimethylthiophosphate and 0.1 μg/L for all of the other DAP metabolites. Urinary DAP metabolite concentrations were creatinine

- adjusted and log

- transformed; children’s individual exposures were then estimated using calculated daily

cr eatinine

- excretion levels. Metabolite excretion rates below the lower LOD were assigned a rate of 0.05 nmol/kg per day. Molar concentrations of the DMP metabolites were summed together, as were the DEP metabolites, to create composite indicators of recent pesticide exposure. Recent
OP pesticide exposure was then defined as a dichotomous variable by assuming that summed DMP or DEP excretion rates greater than 1 nmol/kg per day were indicative of recent OP pesticide exposure, based on a reference dose of 0.3 μg/kg per day, and a molecular weight of approximately 300 g/mol.

In the Harari study, in addition to a sport urine sample, a blood sample (one drop from finger prick) was obtained to measure erythrocyte acetylcholine esterase (AChE) activity using Test

mate equipment and test solutions from EQM Research, Inc. (Cincinnati, OH, USA).

Anthropometric measurements and clinical outcomes, including age, height, weight, head circumference, hematocrit, acetylcholinesterase, and blood pressure were taken during the physical examination.

Neurobehavioral status was assessed on several domains, using multiple instruments, including intelligence (the Wechsler Intelligence Scale for Children

- Revised Digit Span), simple reaction time (Catsys equipment), motor coordina tion (Santa Ana Form Board), and visuospatial and visuoconstructional function (Stanford

- Binet Copying Test). In the Harari study, attention (Conners’s Kiddie Continuous Performance Test (K

- CPT, version 5) and general intelligence (Raven’s Colored Progress ive Matrices) were also included.Granjean et al. (2010)

Tests of differences in mean outcomes (i.e., t

- tests) and their non

- parametric equivalents (i.e., comparison of difference in medians outcomes, Mann

- Whitney tests), and multivariate linear regression were used to compare children determined to have had pre natal pesticide exposure due to their mother’s occupation to those determined not to have had prenatal pesticide exposure. A similar set of analyses was conducted comparing those defined as having had recent exposure
to OP pesticides to those without recen t exposure, based on DAP metabolite urinary excretion rates. For their multivariate regression analysis, the investigators initially identified potential confounders by a priori consideration of factors which may influence nervous system development in thi s Ecuadorian community. Factors observed to be statistically associated with pesticide exposure in this population were included in multivariate regression analyses. Child’s age and sex were obligatory covariates in the models of neurobehavioral outcomes; body weight was a mandatory covariate in the blood pressure models. Trauma, other injury, and meningitis in the past medical history were considered as a joint and mandatory covariate of the neuropsychological outcomes, along with maternal education and an indicator of current nutrition. Socioeconomic variables considered by the investigators included maternal race, housing type, presence of running water in the home, sewage drainage, number of siblings, and access to health care. Maternal smoking and alcoh ol use during pregnancy was also considered as potential confounders.

Based on maternal self

- report of occupational exposure to pesticides during pregnancy, thirty

seven children were considered to have had prenatal pesticide exposure, and thirty

- five wer e considered to not have had prenatal pesticide exposure. Similarly, Grandjean et al. reported over twenty associations between prenatal pesticide exposure and neurobehavioral or clinical outcomes, and found very little evidence of significant or substanti al associations; the children deemed to have been prenatally exposed and those deemed not exposed to pesticides were quite similar to each other in most respects. Prenatal pesticide exposure was associated with a higher systolic blood pressure (though bloo d pressure did not differ significantly by recent pesticide exposure status). Score on the Stanford

- Binet copying test was lower for copying designs among children with prenatal pesticide exposure, compared to the reference group with no reported prenatal exposure to pesticides.

Anthropometric measures were quite similar comparing the children classified as exposed to those not unexposed to prenatal pesticides children.Recent pesticide exposure, as indicated by increased total DEP and DMP excretion was associated only with a statistically significant increase in simple reaction time. The investigators examined associations between the neurobehavioral and clinical outcomes among subgroups of children defined as having, or not having stunted growth. Syst olic blood pressure was highest among the children with both stunting and history of prenatal pesticide exposure. The authors constructed multivariate models including an interaction term for the joint effect of pesticide exposure and stunting, though the results were not presented in tabular form. In no instance were the interaction variables statistically significant.

Grandjean et al. reported an association between prenatal pesticide exposures and increased systolic blood pressure in this population o f 2 nd and 3 rd grade schoolchildren living in northern Ecuador. On average, systolic blood pressure was significantly, higher, by 4.6mmHg, among those with prenatal pesticide exposure, compared to those without prenatal pesticide exposure (p=0.02). Recent e xposure to OP pesticides was significantly associated only with a slight increase in mean reaction time of 74 milliseconds, on average (p=0.01). However, with respect to most of the neurobehavioral and clinical outcomes, the children identified as having prenatal exposure to pesti cides due to their mother’s occupation, and children identified as having recent
OP pesticide exposure as defined by urinary excretion of DAP metabolites were similar to their counterparts classified as non

- exposed. No other significant differences in the clinical and neurobehavioral outcomes were observed by reported (maternal) occupational exposure to pesticides during pregnancy or recent pesticide exposure. These results provide weak evidence of a relationship between prenatal OP pesticide use and subseq uent neurobehavioral and clinical impairment at age 7

- 9 years of age.

Harari et al. (2010)

In the Harari study, 35 children were exposed to pesticides during pregnancy via maternal occupational exposure, 23 children had indirect exposure from paternal wo rk, and 22 children had detectable current exposure irrespective of their prenatal exposure status. Only children with prenatal exposure from maternal greenhouse work showed consistent deficits after covariate adjustment, which included stunting and socioe conomic variables. Exposure

- related deficits were the strongest for motor speed (Finger Tapping Task), motor coordination (Santa Ana Form Board), visuospatial performance (Stanford

- Binet Copying Test), and visual memory (Stanford

Binet Copying Recall Test) . These associations corresponded to a developmental delay of 1.5

– 2 years. Prenatal pesticide exposure was also significantly associated with an average increase of 3.6 mmHg in systolic blood pressure and a slight decrease in body mass index of 1.1 kg/m 2

. Theinvestigators initially identified potential confounders by a priori consideration of factors which may influence nervous system development in this Ecuadorian community. Factors observed to be statistically associated with pesticide exposure in this p opulation were included in the analyses. Child’s age, sex, and school grade were obligatory covariates in the models of neurobehavioral outcomes; body weight was a mandatory covariate in the blood pressure models. Other potential cofactors were child’s tra uma, other injury, meningitis in the past medical history, current hematocrit, having repeated one school grade, parental education (primary school only or above), current nutrition (number of meals per day), and socio

demographic covariates that comprised race, housing (traditional or other), running water at home, house connected to a central sewer line, number of siblings, marital status, maternal access to health care (i.e., childbirth taking place at home or at the hospital), and maternal smoking and a lcohol use during pregnancy.

Reaction time (milliseconds) on the CPT was the only neuropsychological outcome on which the scores of children with current exposure and no exposure appeared to differ, although only with a borderline statistical significance (p = 0.098). Neurophysiologic measurements, including evoked potential latencies and heart variability, were not associated with either prenatal or current

exposures.

Thus, the authors conclude that that prenatal exposure to pesticide even at levels not producing adverse health outcomes in the mother can cause lasting adverse effects on brain development in children contributing to a “silent pandemic” of developmental neurotoxicity.

Study Review

Grandjean et al. (2006)

This cross

- sectional study of prenatal and recent pesticide exposure and subsequent neurobehavioral and clinical status of children at age 7

- 9 years of age in a town in northern Ecuador has substantial limitations, the most serious of which relate to the a ssessment and classification of exposure. Correspondingly, inferences from the results of this investigation are constrained by these limitations in the study design and exposure assessment methodology.

The authors employed a reasonable set of exclusion criteria (having other diseases or being older than age 9 years). The investigators cross

- sectionally collected maternal and child covariate
information.Selection bias is unlikely to have occurred in the study.

Errors in classification of prenatal pes ticide exposure status are likely to have occurred. Prenatal pesticide exposure was evaluated by maternal self

- report at the time of the clinical and neurobehavioral exam (i.e. when the children where in 2 nd or 3 rd grade). Although 37 children
were defined as having prenatal pesticide exposure due to the mother's occupation, none of the mothers had worked as a pesticide applicator; all mothers reported that they had followed normal safety precautions in handling of pesticides, and none of the participating mothers reported having been poisoned by pesticides. Furthermore, paternal occupation and pesticide exposure was not a consideration in characterizing prenatal pesticide exposure status, although the authors
noted that 19 fathers of the children considered to have prenatal exposure and 16 fathers of the children considered not prenatally exposed to pesticides had worked in floriculture or agriculture during the pregnancy. Recall of symptoms potentially associated with pesticides was “weak, nonspecific, and without clear association to occupational exposures.” Thus, it is probable that children both of mothers classified as exposed, and mothers classified as not exposed may have had very similar prenatal pesticide exposure profiles. As a consequence of these sources of non

differential exposure misclassification, observed associations with clinical and neurobehavioral outcomes are likely biased toward the null. Potential for differential exposure misclassification is also possible because, although mothers wer e blinded to the clinical and neurobehavioral test results, they were likely aware of the neurobehavioral status of their children, and it is plausible that this knowledge of outcome status may have impacted the questionnaire

- guided recall of prenatal pest icide exposure in this cross

- sectional study, although there are no data to substantiate this possibility.

The urine sampling and quantification of pesticide metabolites in the children’s urine samples appears to have been conducted appropriately. The use of non

- specific biomarkers of prenatal
OP pesticide exposure, as were assessed in this study, are likely more objective, sensitive, and likely also more accurate and reliable indicators of pesticide exposure, relative to exposure assessment methodologies that rely on self

- report. However, to the extent that OP pesticide exposure is the true exposure of interest, the assessment of non

- specific organophosphate pesticide metabolites in a single maternal sample of urine is a primary potential source of

exposur e measurement error. Urinalysis of non

- specific OP metabolites, as was conducted in this study, is a marker only of very recent exposure, i.e., in the 24 to 48 hour period prior to the collection of the urine sample (Walker & Nidiry, 2002). The single meas urement of urinary DAP metabolite levels may reflect exposure not only to OP parent compounds, but also to potentiallynontoxic pre

- formed metabolites in the environment. The use of a single urine sample to estimate
maternal OP pesticide exposure is likely to have resulted in an attenuation of associations, relative to those that would have been observed had the investigators used a time

- integrated
marker of OP pesticide exposure. Interestingly, children classified as not having had prenatal pesticide expos ure had slightly higher current exposure levels than those classified as having had prenatal pesticide exposure.

On the whole, the investigators handling of confounding resulted was reasonable. Confounders
were identified from the authors’ a priori consi deration of determinants of nervous system development in this target population. Age and sex were included as covariates in models for all clinical and neurobehavioral outcomes. The study population was relatively homogeneous with respect to medical histo ry and most other characteristics evaluated by the authors. This homogeneity

– a weakness of the study with respect to pesticide exposure profile

– is a strength of the study in that is likely that confounding was, to some extent, controlled by design.

T he statistical analysis was appropriate largely appropriate, although the authors did not include the results of multivariate analysis in the report, except those for two outcomes (blood pressure
and Stanford

- Binet Copying Scores) for which statistically s ignificant associations were observed in univariate comparisons of means between children with, and without, prenatal pesticide exposure. Also, the authors evaluated a large number of associations between pesticide exposure and clinical and neurobehavioral outcomes; as a result, false positives due to multiple comparisons are to be expected, though the authors did not adjust the results of their hypothesis tests for multiple comparisons.

The investigators evaluated a priori hypotheses regarding effect mod ification of the association
between OP pesticide exposure and neurobehavioral and clinical outcomes by “stunted” growth. However, the small sample size limited the statistical power of the tests of these hypotheses; the authors did not report observing an y statistically significant effect modification (as evidenced by interaction term p

- values in multivariate models). The results of this model were not presented in
tabular form.

The tests and tools used to assess neurobehavioral status are easy

- to implem ent, and rapidly conducted screening tools. It is possible that these tools do not have the sensitivity to resolve subtle differences caused by pesticide exposure, even if such effects were in fact present.The authors rightly conclude their paper by sta ting that a large, longitudinal study with repeated measurement of urinary biomarkers of OP pesticide exposure is needed to further examine the relationship between OP pesticide exposure and child neurodevelopment in this population. Due
to limitations of the study design and exposure assessment, this largely null study provides little evidence supporting the hypothesis that prenatal (or recent) OP pesticide exposure is associated with neurodevelopment in children attending 2 nd or 3 rd grade. Harari et al. ( 2010)

Researchers examined the child neurobehavioral function and blood pressure in school

- age children prenatally exposed to pesticides. The review comments above apply to this report.

Unlike the Grandjean study, paternal occupation and pesticide expos ure was considered in characterizing prenatal pesticide exposure status. However, errors in classification of prenatal pesticide exposure status are likely to have occurred since it was based on the maternal self

report and although 35 children were define d as having prenatal pesticide exposure due to the mother's occupation, none of the mothers reported any adverse effects that could be linked to pesticide exposures in the greenhouses. The consequences of this misclassification and mothers likely being awa re of the neurobehavioral status of their children are discussed under the Grandjean study.

The child current exposure to pesticides was ascertained by analyzing a spot urine sample for pesticide metabolites and blood to measure erythrocyte acetylcholine esterase (AChE) activity. The strengths and weaknesses of a spot urine sample for exposure to OP pesticide are described under Grandjean study. But unlike Grandjean study, this study also included AChE measurement which represents a longer

- term average ex posure. Interestingly, current exposure, whether based on individual metabolites or groups of metabolites, did not differ between children with and without prenatal exposure.

The instruments used for blood pressure measurements and neurophsychologic measures seem appropriate. Based on the authors, the instruments used for neurophsychologic measures were
validated to avoid cross

- cultural influences.

On the whole, the investigator’s approach to evaluating and adjusting for confounding were prudent as d iscussed above. This study included child’s age, sex, and school grade as covariates in models for all clinical and neurobehavioral outcomes. School grade was not included as acovariate in the Grandjean study. The small sample size reduces the statistical power of the study.

Due to limitations of the study design and exposure assessment, a large longitudinal study with repeated measurement of urinary biomarkers of OP pesticide exposure is needed to further confirm the findings of this study.References cited

Walker, B. Jr., & Nidiry, J. (2002). Current concepts: Organophosphate toxicity. Inhalation Toxicology, 14, 975

– 990.Study 21

. Kofman et al. (2006)

Motor Inhibition and Learning Impairments in School

- Aged Children Following Exposure to Organopho sphate Pesticides in Infancy. Ora Kofman, Andrea Berger, Ali Massarwa, Alon Friedman, and Abed Abu Jaffar. Pediatric Research. 60:88

- 92, 2006.

Study Summary

Kofman et al. conducted a study to investigate the relationship between organophosphate pesticid e exposure during infancy and child neurodevelopment in school aged children who were 6 to 12 years of age at the time of study. In this study, authors report a neuropsychological assessment of healthy school

- aged children, who had been hospitalized in inf ancy (before age 3) following exposure to organophosphate pesticides. They were also compared with children exposed to other toxins such as kerosene, and age

- and sex

- matched non

- exposed children.

All children enrolled in this study (total of 52) were nat ive speakers of Arabic and from the Bedouin population of the Negev region of Israel, attending regular schools. The exposed children were located after screening hospital records of children who were hospitalized for acute poisoning in Soroka Medical Cent er and treated with standard emergency procedures. Exposed children were further divided into two categories; exposed to organophosphates and exposed to kerosene or similar substances such as paint thinner (referred to as the kerosene group).Children from the unexposed control group were recruited through friends and relatives of the exposed children in the same community, matched according to age and sex.

OP poisoning was confirmed by low serum butyrylcholinesterase activity in all but one child,
who was reported to have inhaled insecticide. The mean butyrylcholinesterase level for the children exposed to OP poisoning was (Mean ± SD) 3,631 ± 3,701, and the median value was 1,769 IU/Lit. (normal range ± 6,100

– 15,000 IU/Lit).

The outcome, child neurodevel opment, was assessed using neuropsychological evaluation conducted by the examiners who were native speakers of Arabic, B.A. or M.A. level psychologists who had experience working in the educational psychology service of the Negev Bedouin community. Evalua tion included long

- term verbal memory test, working memory and attention span test, and inhibitory control/motor inhibition test. First they performed trials one through seven of the long

- term verbal memory test, followed by the Statue, Knock Tap, Digit Sp an and Corsi Blocks tests. Last, they performed trials eight and nine of the memory test(Delayed recall of List A, and Recognition). The list of words for long

- term verbal memory test was based on an Arabic translation of the word list in the NEPSY (20), with some culture

relevant changes for a desert

- dwelling community. The children were tested individually in a quiet room. Some of the children from all groups were tested within the local medical clinic in
Rahat and the others were tested in their homes. Each child was tested on the same day and in the same place as his matched control.

Parents were given a structured interview by the testing psychologist, asking if the child was on medication or had chronic health problems, if they had been hospitalized since the index incident, had repeated a grade, had trouble in any particular subject at school, or had conduct problems in school. In addition, questions concerning the child’s pastimes and issues not related to learning problems were included and the chi ld’s general demeanor was noted. The interview was not meant to be diagnostic, but was intended to alert to overt signs of serious developmental delay.

Confounders such as age, sex, background (cultural and demographic) between the exposed and unexposed groups were matched. Data analysis showed that there was no difference between the exposed and unexposed groups based on age and sex, confirming that the matching was appropriate. Family rearing practices and perinatal complications were not included in th e study but the fact that the exposed and unexposed groups were chosen from similar backgrounds mitigates the likelihood that these and other uncontrolled factors contributed to the differences between the groups.

Statistical analyses of the relationship between exposure to organophosphate pesticides in infancy and motor inhibition and learning impairments in school were conducted using
ANCOVA.

The structured interview did not reveal any conduct disorders, health or academic problems. The
WISC

- R scores w ere compared between the seven children who had been poisoned (three kerosene, four OP) and the matched comparison group. No differences were found between these exposed children and their matched controls. Analysis of the verbal memory indicated that both groups of children who had been exposed to toxic substances were impaired in verbal learning. No difference between the groups was found for the recognition test or the delayed recall test. No differences between the exposed children and controls were fou nd in the Digit Span and Corsi Blocks test. The effects of poison exposure appeared only in the repeated free recall butnot in the delayed or recognition tasks, suggesting that the children did eventually learn the list, even though they had trouble retri eving words in the acquisition phase.

The authors concluded that although the children seem to have overcome the acute one

- time exposure and they all attend regular schools, a finer assessment of specific cognitive abilities indicates impairment in the ve rbal learning and motor inhibition tasks in the exposed compared with matched control children. The long

- term effects of OP poisoning on cognitive behavior in children suggests that subtle behavioral changes may occur, even if they go undetected in school.

Study Review

This retrospective cohort study of pesticide exposure in infancy and subsequent neurobehavioral status of children aged 6 to 12 in Negev region of Israel has multiple limitations, the most serious of which is sample size.

The time period when the study was conducted is not provided in the article. This study was limited to children aged 6 to 12 at the time of study, who were victims of poisoning before age three. There is no discussion on how the participant age was decided but the age ran ge seems to be appropriate for the assessment of neurodevelopment in children. This is because in another similar study (Oulhote et al, 2013), that examined the association between exposure to organophosphate and pyrethroid pesticides, and parentally repor ted behavioral problems in children, the participants were children aged 6

- 11 years of age. Based on the author of that study, the analysis was limited to these children because information on early

- life factors important for neurodevelopment was available only for children in this age range.

The fact that both the exposed (exposed to OP and kerosene) and unexposed groups were matched and chosen from similar backgrounds mitigates the likelihood that other factors (family rearing practices, environmental f actors etc.) contributed to the differences between the groups. However, because of the life style of this community, psychologists who administered the tests were blind to the type of poisoning (OP or kerosene) but knew which children were exposed.
This m ay have introduced some bias in the study.

Exposed population that was divided into two groups (exposed to OP and exposed to Kerosene) had its corresponding control group. It is not clear from the article if both unexposed groups (unexposed to OP and unex posed to Kerosene) were not exposed to either OP or kerosene. For example, there were 9 participants who were exposed to OP and 9 participants who were notexposed to OP pesticides . However, it is not clear from the article whether or not the participants who were not exposed to OP pesticides were also not exposed to kerosene.

Errors in classification of pesticide exposure are unlikely to have occurred since OP poisoning was confirmed by the hospital records. However, as mentioned above, sample size was s mall (n=9) for both the exposed and control groups for OP. This low sample size reduces the statistical power of this study. The statistical analysis (difference in means) used to assess the associations between the exposure and neurodevelopment were appro priate given the small sample size.

The methods used for neuropsychological evaluation seems appropriate. Outcome of the exposure was assessed by the neuropsychological evaluation of the children and by conducting structured interview of the parents. Erro rs in outcome assessment were minimized as the psychologists were qualified individuals and had experience working in the educational psychology service of the Negev Bedouin population, language and cultural differences were
taken into considerations when administering the tests, and each child was tested on the same day and in the same place as his matched control.

Overall, the study showed that there was no difference between the exposed and control groups. Only a finer assessment showed differences between exposed and control groups in regards to verbal learning and motor inhibition. The article suggests that these associations were significant but it is not clear if the same will be true if the study is conducted with a large sample size.

Inference s from the results of this study are constrained by the limitations in sample size and also because of the culture of the study population which is specific to their community.

The authors rightly conclude their paper by stating that a large long

- term fo llow

- up prospective study including a more specific battery of tests focusing on memory and regulation of behavior is
needed.

References cited

Urinary Metabolites of Organophosphate and Pyrethroid Pesticides and Behavioral Problems in Canadian Children. Youssef Oulhote and Maryse F. Bouchard; Environmental Health Perspectives. 121:1378

- 1384 (2013).Study 22

. Koutroulakis et al. (2014)

Dialkyl phosphates in amniotic fluid as a biomarker of fetal exposure to organophosphates in Crete, Greece; associati on with fetal growth. D. Koutroulakis, S. Sifakis, M.N. Tzatzarakis, A.K. Alegakis, E. Theodoropoulou, M.P. Kavvalakis, D. Kappou, A.M. Tsatsakis. Reproductive Toxicology 46 (2014) 98

– 105.

Study Summary

In this prospective cohort study by Koutroulakis et al., the authors evaluate fetal exposure to OP pesticides by measuring the non

- specific dialkyl

- phosphate metabolites (DAPs) in a single amniotic fluid (AF) sample, and examine the potential association between prenatal exposure and fetal growth in pregna nt women living in Crete, Greece.

The study population consisted of 415 women with singleton pregnancies, who were permanent residents of Crete for at least two years. Women were recruited at the time of referral for
amniocentesis to the Fetal

- Maternal Un it, Department of Obstetrics and Gynecology, University Hospital of Heraklion, Crete, Greece. The indications for referral included increased risk for chromosomal abnormalities (based on advanced maternal age or the results of the first trimester
combined test for fetal aneuploidies, or sonographically detected markers or abnormalities between 18 and 22 weeks of gestation), suspicion of genetic syndromes or increased risk for single gene dis

- orders (based on history or sonographic findings), abnormal fetal growth, and suspicion of congenital infection. Mean maternal age was 32.7 (SD = 5.7) and mean gestational age at amniocentesis was 18.5 weeks (SD = 2.5).

Maternal/fetal exposure to organophosphate pesticides was assessed by measurement of organophosphate dialkyl phosphates in a single AF collected at either 16th or 20th weeks of gestation. The dialkyl phosphates metabolites (DAPs) measured were dimethylphosphate, dimethyldithiophosphate, dimethylthiophosphate (DMPs), diethylphosphate, diethyldithiophosphat e, and diethylthiophosphate (DEPs). Total DAPs, DMPs, and DEPs were available for 406, 298, and 401women, respectively. Quantification of organophosphate metabolites was done by using gas chromatography mass spectrometry. In addition, the participants were asked to complete a detailed questionnaire about their medical history, demographic data, socioeconomic status, occupational and residential status and exposure to other potentially embryotoxic factors.The association between the levels of ΣDMPs, ΣDEPs and ΣDAPs and birth weight and head circumference was evaluated by using multiple linear regression models. The association was adjusted for the confounding effect of neonatal gender, maternal age, agricul tural activities, and gestational age at amniocentesis. The authors found that ΣDMP was positively related with birth weight centile and the remaining DAPs were not correlated with biometric indices. Similar results were found after missing data imputation with LOD/2 values.

Based on the results, the authors concluded that DAPs in AF may be considered as a potential biomarker for the assessment of fetal exposure to OP. The findings add to the growing body of literature regarding the sensitivity of develop ing fetus to OP pesticide exposure.

Study Review

This was a well

- conducted but poorly written study. The aim of this study was first, to determine the presence of OP non

- specific metabolites (DAPs) in amniotic fluid (AF) as an index for in utero exposur e to these contaminants and second, to evaluate any potential link between prenatal exposure to OPs and fetal growth. The study was conducted in a population of women who were identified as having high risk pregnancies. According to the article “82.5% of t he amniocenteses were carried out between 16th and 20th weeks of gestation and an additional volume of 8

– 10 ml of AF was obtained during each procedure” which suggests additional burden and risk was placed on the participants even though the participants c onsented for the study and the ethics committee of the University Hospital of Crete approved the study’s protocol. The analytic methods, questionnaire, analytic approach, and statistical analysis were appropriate. However, it is unclear how the outcome (bi rth weight and head circumference) information was obtained and
the rationale for confounder selection.

This study had a novel biomarker (AF) for exposure assessment. The exposure measurement methods employed for the measurements of OP pesticides in AF w ere appropriate and are defined in the study. However, there are not many literatures on using AF as a potential biomarker. According to the author, there is only one study prior to this study that evaluated OP metabolite levels in AF. Therefore, comparing the exposure measurements in AF against known biomarker (e.g., OP metabolite levels in urine) would have validated AF as the potential biomarker for OP exposure.The authors do not provide information on how the data on birth weight and head circumferen ce was assessed. There are also some ethical concerns with the large volume of amniotic fluid
collected.

Results of multiple linear regressions to evaluate the relationships between ΣDMPs, ΣDEPs and ΣDAPs levels by biometric indices after adjustment for maternal age, agricultural activities, gestational week at sampling, and neonatal gender showed that there is a positive linear association between ΣDMP and birth weight centile, which is contrary to what other studies have shown, i.e., higher exposure to OP metabolite results in lower birth weight. In addition, the rationale for confounder selections is not defined in the study. It may be worthwhile to note that the smoking status of the participants (48.2% of participants smoked before pregnancy and
31.9% of participants smoked during pregnancies), participants being women with high risk pregnancies (reason for referral for amniocentesis), gestational diabetes (information not available), PON1 enzyme activity in the fetuses (information not available) coul d also have
contributed to the outcome.

The statistical analysis appears to be appropriate. The large sample size (n = 415) compared to other similar studies increased the statistical power.

The authors conclude that DAPs in AF may be considered as a po tential biomarker for the assessment of fetal exposure to OPs and sum levels of DMPs appear to be associated with birth weight. The authors do point out the need for further studies to determine the persistence of these associations and their generalizabil ity to other populations and to address concerns about the potential health effects of pre

- and postnatal exposure to pesticides and develop suitable regulatory reference doses.Study 23

. Lizardi et al. (2008)

The effects of organophosphate pesticide ex posure on Hispanic children’s cognitive and behavioral functioning. Patricia Sanchez Lizardi, PHD, Mary Kay O’Rourke, PHD, and Richard J. Morris, PHD. Journal of Pediatric Psychology 33(1): 91

– 101, 2008.

Study Summary

Lizardi et al. selected a subset of children from the Children Pesticide Survey (CPS) of southern Arizona (O’Rourke et al., 2000) to evaluate whether recent organophosphate (OP) pesticide exposure affects the cognitive and behavioral functioning of Hispani c children (average age=7 years old) living in an agricultural community in Southern Arizona. The CPS, conducted in
1998

- 2000, was itself built upon two studies previously conducted in Yuma, Arizona: the National Human Exposure Assessment Survey and the Ar izona Border Survey. Child participants who had provided a urine sample during the screening stage of the CPS study were eligible to participate in the present study. The data for this investigation was collected in 2002.

A first

- void urine sample was col lected from each child on the day of the neurobehavioral assessments. Gas chromatography with flame photometric detection (GC

- FPD) was used to
estimate non

- specific OP metabolites (dialkylphosphate) concentrations. These included dimethylphosphate (DMP), d imethylphosphorothioate, dimethylphosphorodithioate, diethylphosphate, diethylphosphorothioate, diethylphosphorodithioate. The sum of the six OP metabolite concentrations was used for all data analyses. Absence of recent OP pesticide exposure was defined a s having urinary metabolite concentrations below the level of detection limit (25 μg/l).

In the original CPS study, children were selected for participation based on the presence or absence of OP pesticide metabolites in urine samples collected during the screening phase. However, all of the children (n=48) from the original PCS study (both exposed and non

- exposed to pesticides) that participated in the current study had detectable DMP levels in urine samples provided in 2002. The authors did observe that the exposed group, as defined in the CPS, had significantly higher levels of urinary DMP measured in 2002 (and therefore DAP concentrations), relative to the original non

- exposed group.

Neurocognitive and behavioral assessments were selected by the invest igators based on their demonstrated validity and reliability. Cognitive assessments included a short form of theWechsler Intelligence Scale for Children, Third Edition, the Children’s Memory Scale; Wisconsin Card Sorting Test; and Trail Making Test A & B. The behavioral measures were the
Child Behavior Checklist/4

- 18 (CBCL/4

- 18) and The Teacher Report Form (TRF). The tests instructions were administered in the child’s preferred language

– Spanish or English. In addition to the neurobehavioral assessments, a structured interview was conducted with parents of all participating children, in order to ascertain additional information about the child and the family.

The authors presented box plots to compare urinary OP metabolite (DAP) concentrations among
the children and correlation coefficients to parameterize the associations between DAP metabolite levels and cognitive test performance.

DAP concentrations were presented primarily in figures (boxplots, stratified by the original non

exposed and exposed stat us). The mean DMP in urine sampled at the time of the neurocognitive assessments was 65.5 mcg/l (95% CI: 43

– 88 5 mcg/l). The authors contrasted this finding with the corresponding average DMP concentration at the time of the original CPS screening (110.8

m cg/l; 95% CI: 56

– 164 mcg/l); the mean national value estimated by NHANES (21.7 mcg/l;
95% CI: 10

– 41 mcg/l), and the NHANES average for the Mexican

- American population ages 6

– 59 (15 mcg/l; 95% CI: 10

– 23 mcg/l). (DHHS, 2003)

The authors observed only one st atistically significant difference between children grouped by presence or absence of pesticide exposure as originally determined by DAP urinalysis during the screening phase of the CPS and cognitive and behavioral performance at age 6

- 8 years of age. A

st atistically significant difference between the groups were observed for the TMTB, with children in the pesticide

- exposed group taking more time to complete the measure (average: 283 seconds; 95% CI: 224

– 341 seconds), relative to the non

- exposed children (a verage: 204 seconds;
95% CI: 172

– 236 seconds). No other differences were reported by the authors, other than noting that statistically significant differences were not observed for the other cognitive and behavioral

measures.

The authors observed very few statistically significant correlations between DAP concentration levels measured in urine sampled on the day of the cognitive assessment and performance of
children on the WISC

- III SF, the CMS, the TMTA, or the TMTB. They did note statistically significan t correlations with some of the measures of the Wisconsin Card Sorting Test, specifically with the total number of errors made (r=0.31, p=0.03); the number of perseverative responses (r=0.34, p=0.01); the number of perseverative errors (r=0.35, p=0.01); th e conceptual level responses provided (r=0.38, p=0.01); and the failure to maintain set (r=0.38, p=0.02).Study Review

This study was originally conceived to evaluate a primary hypothesis that cognitive and behavioral test performance of children prenatally and recently exposed to OP pesticides would be diminished relative to the test performance of children who had not be en exposed to OP pesticides. The investigators also hypothesized that higher levels of recent OP pesticide exposure would be associated with poorer performance on the neurocognitive assessments. Their study sample included a roughly equal number of childre n with, and without, prenatal OP pesticide exposure. However, contrary to their expectations, they found that all children participating in the current study had detectable levels of OP pesticide metabolites in urine samples collected just prior to the out come assessment. As a result, they were not able to evaluate their primary hypothesis. They also chose not to evaluate associations between urinary OP pesticide biomarker levels and behavioral test performance. Thus, their report was limited to a cross

- sec tional evaluation of the association between urinary DAP concentrations and cognitive test performance.

Use of urinary DAP metabolites as a marker of OP pesticide exposure is both a strength and a limitation of the study. Considering the prohibitive logi stical effort required to ascertain serial urine samples in a large epidemiologic study of children, the lack of sensitive and specific pesticide biomarker assays (such assays are available only for a few pesticide

- specific urinary metabolites, e.g., TCPy for chlorpyrifos), and a lack of biomarkers for time

- integrated pesticide exposures, the exposure assessment used in this study was reasonable, and certainly logistically expedient. Because they are non

- specific, urinary DAP concentrations can be considere d an integrated biomarker of exposure to many OP pesticides. But urinalysis and quantification of DAP metabolite concentrations in a single urine sample, as was done in this study, may not be representative of a participant’s usual pesticide exposure over an extended period of time. Because of the often intermittent nature of pesticide exposure, urinary DAP metabolite levels can exhibit considerable temporal variability (i.e. day

- to

- day fluctuations). OP pesticides can break
down into DAPs in the environmen t, and urinary concentrations may therefore also reflect exposure to preformed environmental DAPs. Urinalysis of the non

- specific OP metabolites, as was conducted in this study, may be a marker only of very recent exposure, i.e., in the 24 to 48 hour perio d prior to the collection of the urine sample.(Walker & Nidiry, 2002) That said, DAP concentrations in a single urine sample have been demonstrated to correlate reasonable well with
90

- day average levels in adult men (Meeker et al., 2005). In this study, t he exposure assessment was conducted one to two days prior to the conducting of the neurocognitive and behavioralassessments. It is unlikely that exposures in this time frame are representative of exposures during the critical time window for neurocogniti ve development, unless exposures occur uniformly over time. These aspects of urinary DAP biomarkers of OP pesticide exposure are all potential sources of exposure misclassification.

The authors noted that the instruments used to assess cognitive (and be havioral) status in this study were selected because of their frequent use in previous studies of adult populations. Although perhaps standard tools to evaluate adult populations, they may not be the most sensitive or appropriate tests to detect difference s neurocognitive and behavioral functioning of primarily Spanish

- speaking, Hispanic children living in a rural, primarily agricultural, community.

Statistical adjustment for potential confounders of the relationship between pesticide exposure and neurocog nitive status was not conducted. The behavioral outcomes were not included in this analysis. The authors justified this omission by stating that the behavioral measures refer to a 6

- month period (prior to the assessment).

The statistical methods used in the study were not well described in the report. The authors presented only correlation coefficients and associated p

- values to parameterize the association between urinary OP metabolites and performance on cognitive tests. The authors did not identify
wha t type of correlation coefficients were estimated (e.g. Pearson or Spearman). The investigators excluded two outliers with suspiciously high OP metabolite concentrations from their analyses. They did not specify how inclusion of these outliers would have a ltered the
observed associations.

The authors noted that the two groups of children (with and without detectable urinary OP metabolite concentrations at the time of the original CPS screening) had “nearly identical demographic characteristics.” However, t his observation is not evidence that the profile of determinants neurocognitive performance was similar between those with varying degrees of

recent pesticide exposure. Confounding is thus a possible explanation for the few associations
observed in this st udy. That said, it is likely that the study population is relatively homogeneous.

The pesticide exposure misclassification discussed above was very likely non

- differential with respect to the cognitive outcomes assessed in this study. One may thus expect that, on average, associations observed in the presence of such OP pesticide exposure measurement error areattenuated (i.e. closer to the null correlation of zero), relative to the true association, assuming no other bias or confounding, and apart from r andom error. Such errors are a potential explanation of the null findings with respect to WISC

- III SF, CMS, TMTA, and TMTB performance.

The study findings, if valid, are likely generalizable to rural, primarily agricultural communities of Hispanic childre n. However, the study findings were largely null; the findings are not consistent with the hypothesis that recent OP pesticide exposure adversely affects cognitive and behavioral functioning at seven years of age. This could be due to considerable misclass ification of true pesticide exposure during the relevant time period for cognitive development.

References cited

Meeker JD, Barr DB, Ryan L, Herrick RF, Bennett DH, Bravo R, Hauser R. Temporal variability of urinary levels of nonpersistent insecticides i n adult men. J. Expo. Anal. Environ. Epidemiol. 2005;15:271

– 281.

Walker, B. Jr., & Nidiry, J. Current concepts: Organophosphate toxicity. Inhalation Toxicology. 2002;14, 975

– 990.Study 24

. Lu et al. (2012)

Assessing the association between pesticide e xposure and cognitive development in rural Costa Rican children living in organic and conventional coffee farms. Int J Adolesc Med Health. 2009; 21(4): 609

- 621

Study Summary

In this report, Lu et al. examine the association between urinary concentrations of OP and pyrethroid metabolites and cognitive development in Costa Rican children whose parents worked in organic or conventional coffee farms using a cross

- sectional study design with a single assessment of selected urinary OP, pyrethroid, and other pesticide metabolites and a single assessment of cognitive development.

A convenience sample was recruited in June

- August, 2007. The investigators invited parents (primarily fathers) at an organic coffee farm (La Amistad group) to participate. Participants (primarily mothers) from conventional coffee farms (Las Mellizas group) were recruited at community meetings and through referral. A total of 17 La Amistad children and 18 Las Mellizas children between the ages of 4 and 10 years were enrolled, age and gender matched.

Two spot urine samples were collected by each parent/child participant, before bedtime the evening of Day 1 and the first morning void of Day 2. Collected samples were stored in a refrigerator or cooler with ice packs until picke d up by researchers on Day 2. Samples were transported on ice packs to the La Amistad Lodge for total volume and specific gravity measurement and aliquotting. Aliqoutted samples were kept at −4 °C, until shipment (on frozen gel packs) to the CDC/NCEH in At lanta. All samples arrived partially frozen at CDC, and were analyzed for specific metabolites of selected OPs, pyrethroids, herbicides, and DEET by LC

- MS

MS (Olsson et al., 2004). Urinary OP, herbicide and pyrethroid metabolite concentrations (ug/L)
in ea ch sample and daily volume weighted average (DVWA) urinary OP, herbicide and pyrethroid metabolite concentrations (ug/L) for each sample set were calculated.

Urinary pesticide levels did not vary considerably in La Amistad and Las Mellizas children. Of
2 0 analytes detected, only 7 were found at greater than the LOD. OP metabolites detected above
the LOD were:• 4

- nitrophenol (PNP

- malathion, methyl parathion, others) (Most frequently detected metabolite, Freq detection 99%, no levels reported)

• IMPY (diazi non) (Freq detection 23%, range 0

- 7.79 ug/L, higher in Organic farm/La Amistad children, paired t

- test p=NR)

• TCPy (chlorpyrifos) (Freq detection 14% (x4), range 0

- 6.71 ug/L (x5), marginally higher in Conventional farms/Las Mellizas children, paired t

- test p=0.065)

Other urinary pesticide metabolites detected were 2,4

- D (Freq detection 71%), d, N,N

- diethyl

- m

toluamide (DEET) (Freq detection 56%), and 3

- BPA (pyrethroids) (Freq detection 16%).

Cognitive development was assessed using the following:

• Computer ized Behavioral Assessment and Research System (BARS) (Rohlman et al., 2001), Spanish version: Digit Span, Finger Tapping, Match

- to

- Sample, Continuous Performance; Divided Attention (17 total measures)

– A 9

- button response unit overlaying the keyboard sim plified response for young children and those unfamiliar with computers. Detailed instructions and a practice session preceded each test. Instructions were spoken by the researcher and the computer, and could also be read on the computer screen. Interactio n was provided as needed, especially with younger children. A 97% response rate was attained.

• Figure drawing task: To assess child's perception and dexterity, children were asked to draw a picture of their family. Scores were assigned by a researcher with no knowledge of the participants’ exposure status. Scores were rated 1

- 11 (one point for eyes, ears, nose, mouth, hair, head, legs, feet, arms, hands, and trunk; averaged if more than one person in drawing).

• Long

- term memory test: At the beginning of a te st session, the child was told they would receive a pencil as a prize at the end. After the BARS and figure drawing, the child was asked what prize they were to receive.

The testing usually took place at the child’s home. It took about 40 minutes to admin ister.

For 8 of 17 neurobehavioral measures, performance did not differ between groups. Conventional farms/Las Mezillas children performed better on 9 measures, 3 significantly better (Long

- term memory, Continuous performance percent hits, Continuous per formance average false alarm latency) and also had higher scores in the Figure Drawing Test (average 15% more complete drawings or 1.6 more body parts than La Amistad children).To assess the relationship between pesticide exposure and performance on cogn itive development tests, linear mixed effect models for 4 significant test outcomes from paired t

- test analyses (Figure drawing score, Long term memory, Continuous performance test percent of correct responses to target, Continuous performance test average false alarm latency) were used. Significance was set at <0.10 due to small sample size. Models included age, gender, group (conventional, organic), handedness, and grade.

Age (p=0.094) and group (organic vs conventional farm) (p=0.018) were significant c ontributors to figure drawing scores. (Note: Conventional farm/Las Mellizas children performed better).

Gender (p=0.039) and group (organic vs conventional farm) (p=0.016) were significant contributors to Continuous performance percent hits. (Note: Conventional farm/Las Mellizas children performed better).

Exposures were low for both groups of children. Children in rural Costa Rica are exposed at lower levels than US children. Performance on the neurobehavioral tests was about equal
between the two groups, with Conventional farms/Las Mezillas children generally performing better. Pesticide exposure did not affect cognitive development. The researchers concluded that differences in the groups that would lead to cognitive development disparities may li kely result from families' SES and demographic characteristics.

Study Review

The study lacked a clear design and analytic strategy. The basic assumption of a difference in exposures between the groups was flawed as the Conventional/Las Mellizas farms in reality used very little pesticides due to ideal growing conditions, having less money, small farms, different pest management agricultural practices, etc. In addition, the assumption of similar demographic characteristics, schooling, nutrition, and lifest yle between groups was also flawed

– the La Amistad families lived in shacks provided by the employer, some without electricity or refrigerators, the parents (about 2/3) were usually illiterate, and children started school at 5

- 6 years old, while the Las M ellizas families owned farms, homes had basic appliances, most owned vehicles, only about 1/3 parents were illiterate, and children started preschool at 3

- 4 years old. Therefore no conclusions can be drawn with respect to the relationship between OP pestic ide exposure and cognitive development from this research.Recruitment methods were different for the two groups and no discussion was presented as to participation rate differences or selection bias between the groups.

The exposure measurement methods employed were appropriate. In fact, collection of an evening and next day first morning void urine sample is considered a good approach to a spot collection as it allows for calculation of a weighted average concentration. Measured pesticide
metabolites ar e not listed

- only those found above LOD. In Table 1, ‘CIT’ is listed but never defined or discussed. While the researchers measured specific gravity, it is not indicated whether urinary values were adjusted for specific gravity. The potential for contami nation due to collection by participants is not discussed and appears not to have been evaluated.

As the authors point out, a limitation of the cross

- sectional design is the reliance on the untestable assumption that (even valid) biomarkers of OP pestici de exposure measured in urine sampled at the time of the neurodevelopmental assessment are indicative of ‘usual’ OP pesticide exposure, or, more to the point, accurately reflect OP pesticide exposure at the critical time

window(s) for exertion of deleterio us effects on neurodevelopment. Moreover, the use of a single day’s urine sample to estimate the participant’s OP pesticide exposure is likely to have resulted in an attenuation of associations, relative to those that would have been observed had the
inves tigators used a time

- integrated marker of OP pesticide exposure.

The instrument used to assess cognitive development (i.e., the BARS) has been used in Spanish in a number of farmworker populations in US and other countries, including pre

- school and
schoo l age children. Adequate instructions, a practice session, and interaction to motivate younger children were employed. However, differential interaction with children without clear guidelines may bias results, usually towards better performance. This is no t evaluated. No training of data collectors is described.

The figure drawing and long term memory tests are not cited seem full of potential, unmeasured biases, e.g., whether the child had ever drawn a picture of their family before and feedback given
if they had, whether a pencil was a desirable prize, etc. The authors consider the 11

- point scale for the figure drawing test to allow a more detailed assessment than a 5

- point scale used previously (Guillette et al., 1998). The long term memory test is not cited in the methods section.

The statistical analysis was not specified in great detail, but appears to be appropriate. Confounders such as possible dietary exposure or pesticide use in homes are not considered. The small sample size reduces statistical power.The authors rightly conclude that covariates such as maternal education, years working in agriculture, pesticide handling, breastfeeding, nutritional breastfeeding information, SES (such as having computers at home) may all affect performance on c ognitive tests. Unfortunately they conclude they found a positive association between family SES and cognitive development in children living in rural Costa Rica, when in fact they do not actually report any SES
measurement method or data other than after

- the

- fact observations. For example, they reported estimated mother illiteracy rates based on whether the mother asked to be read the consent form. In conclusion, this study does not add the weight of evidence that OP pesticide exposure affects neurological development.

References cited

Guillette, EA, Meza, MM, Aquilar, MG, et al. (1998). An anthropological approach to the evaluation of preschool children exposed to pesticides in Mexico. Enviro Health Perspect 106:347

- 53.

Olsson AO, Baker, SE, Nguyen JV, et al. (2004). A liquid

- chromatography tandem mass spectrometry multiresidue method for quantification of specific metabolites of organophosphorus pesticides, synthetic pyrethroids, selected herbicides, and DEET in human ur ine. Anal Chem 76:2453

- 61.

Rohlman, DS, Anger, WK, Tamulinas, A, et al. (2001). Development of a neurobehavioral battery for children exposed to neurotoxic chemicals. Neurotoxicology 22:657

- 65.Study 25

. Moreno

- Banda et al. (2009)

Maternal exposure to floricultural work during pregnancy, PON1 Q192R polymorphisms and the risk of low birth weight. G. Moreno

- Banda, J. Blanco

- Muñoz, M. Lacasaña, S.J. Rothenberg C. Aguilar

- Garduño, R. Gamboa, O. Pérez

- Méndez. Science of the Total
Environ ment 407: 5478

– 5485, 2009.

Study Summary

Moreno

- Banda et al. reported on the results of a cross

- sectional study assessing the association between floricultural work during pregnancy, as proxy for prenatal pesticide exposure, and prevalence of low birth weight among women living in two states in Mexico. They evaluated whether this association is modified by the maternal paraoxonase (PON1) Q192R polymorphism.

Between January and July, 2005, floricultural workers and their spouses were recruited to partic ipate in the study if they had worked for at least one year in floricultural activity. Female workers and female spouses of male workers who reported having had at least one pregnancy between 1995 and 2005 were eligible to participate in the study.

Of 58 5 floricultural workers or spouses who completed a screening interview to determine eligibility, 219 were ineligible. The vast majority (>90%) of these workers/spouses were not eligible because they had not had a pregnancy in the 10 years prior to the inte rview. Of the 366 women who were eligible, 38 declined the invitation to participate without giving a specific reason. In total, 328 (90%) women agreed to participate, and reported a total number of 658 pregnancies in the 10 years prior to the study, 467 o f which were included in subsequent analyses. The 191 excluded pregnancies were not analyzed for one of the following reasons: pregnancy ended in a miscarriage or abortion, premature birth, twins, presence of congenital malformations, or missing exposure, outcome, or genetic polymorphism data.

Study participants completed a structured questionnaire collecting retrospective information on sociodemographic characteristics, exposure to chemical substances at home and at work,
characteristics of their work ac tivities, reproductive history, medical history, reproductive history, and maternal alcohol and tobacco consumption. Separately, the women completed a food frequency questionnaire. Paternal occupation, alcohol use, and tobacco use were also queried.The f ollowing two exposure groups were defined for the purpose of this analysis: Exposed, defined as women who worked in floriculture during the studied pregnancy; and not exposed, defined as women who did not work in floriculture during studied pregnancy.

Bir th weight data were derived from birth certificate (if available) or by maternal report. Low birth weight was defined conventionally as birth weight less than 2,500 grams.

No differences in birth weight were observed between QQ and QR genotypes. Maternal PON1 Q192R polymorphism (QQ, QR and RR) genotypes were therefore collapsed into two groups: QQ or QR genotype and the RR genotype.

The authors evaluated the risk of low birth weight associated with prenatal occupational pesticide exposure using GEE (General Estimating Equations) models with a logit link function (equivalent to logistic regression model, but accounting for correlation due to repeated measures). Modification of LBW risk by maternal PON1 polymorphism (gene

– environment interaction) was a ssessed with an interaction variable. Models were also constructed within the two strata of the PON1 polymorphism. Multivariate models were adjusted for infant sex, and presence of a history of adverse reproductive outcomes. Maternal smoking and alcohol

co nsumption during pregnancy were also considered as potential confounders but, appropriately, were not included in final models, due to an observed low prevalence of these factors in this study population.

After adjustment for potential confounders, Moreno

- Banda observed a statistically significant interaction between maternal exposure to flower growing work during pregnancy and PON1 Q192R polymorphisms on risk of low birth weight. Among mothers with QQ or QR PON1 genotype, those exposed to floricultural wo rk during their pregnancy had a lower risk of having a child with LBW than those who were not exposed (OR: 0.42; CI 95%: 0.14

- 1.24). Among mothers with the RR PON1 genotype, those exposed to floricultural work during pregnancy had, on average, twice the ri sk of having a child with LBW, relative to those who were not exposed (OR: 2.62, CI 95%: 0.89

- 7.69). Although the stratum

- specific odds ratios were not statistically significant, the interaction effect in a multivariate adjusted model of LBW including the gene by environment interaction and the main effects of floricultural occupation status (exposed or not) and maternal PON1 polymorphism was statistically significant.

In summary, a gene by environment interaction was observed between maternal exposure to floricultural work during pregnancy and maternal PON1 genotype: the group with the greatestprobability of having a child with LBW was women exposed to floricultural work and who had
PON1 192RR.

Study Review

This study relied on a proxy indicator of prenatal occupational exposure to pesticides to evaluate differences in risk of low birth weight comparing exposed to unexposed women

- infant pairs, and additionally evaluated modification of this association by PON1 polymorphism status. This was

a cross

- se ctional study with retrospective recall of occupational exposure during pregnancies up to 10 years prior to the conduct of the study. The investigators also relied on retrospective self

reporting of birth weights for a portion of the study participants for whom birth certificates were unavailable. The study design, particularly the use of an imperfect proxy for OP pesticide exposure, limits the strength of the evidence contributed by this investigation.

The recruitment procedures and inclusion/exclusion cr iteria were appropriate, though a large proportion of potential participants were not included, or did not agree to participate in the study. This is a potential source of selection bias. Twelve of the workers who declined to participate agreed to complete a brief questionnaire for nonparticipants; these individuals were similar to participants with respect to sociodemographic characteristics (e.g. age, education and income), although the data were not included in the report. The exclusion of pregnancies oc curring more than 10 years prior to the initiation of the study is also a potential source of selection bias. In sensitivity analyses, the authors attempted to evaluate potential for selection bias by comparing
the distribution of characteristics of newbor ns for whom complete (exposure, outcome, covariate, and maternal PON1 Q192R polymorphism) information was available with those of newborns for whom this information was incomplete. Also, they specifically compared infants for whom maternal genotype informa tion was available with those without this information.

No objective (bio)markers of pesticide exposure were evaluated in this study. Non

- differential misclassification of pesticide exposure is likely in this study. For example, spouses who report

not wor king in floriculture, often help with fieldwork, live near fields, wash spouse's clothing, etc., and may therefore experience para

- occupational exposure to pesticides, to varying degrees. Such errors in exposure classification would likely bias observed as sociations towards the null (OR of 1).

The assessment of birth outcome (in part) by maternal self

- report leaves open the possibility errors in classification of the LBW outcome that are differential with respect to exposure. Theinvestigators validated th e self

- reported birth weight information using a subsample of 107 pregnancies for which birth weight data were available from both birth certificates and self

report. From this analysis, they concluded that there was adequate agreement between the data
obt ained by maternal self

- report and the information obtained from the birth certificate (rho=0.93). Additionally, the classification of birthweight into a dichotomous indicator of low birth weight likely attenuated errors in birth weight, as LBW is likely to be correctly classified even in the presence of small errors in birth weight reporting.

The evaluation of the gene by environment interaction was limited by small sample size. Effect estimates from stratum specific models were not statistically significa nt, though statistically significant interaction was observed in a multivariate model including an interaction variable.

The assessment and adjustment for potential confounders was appropriate, but not particularly strong. There is potential for residual confounding due to mismeasured or missing confounders. Risk factors for LBW that are related to floricultural work/pesticide exposure would bias the results in this study, for example by differences in the ethnic makeup of the exposed and unexposed groups. Other such factors may be related to socioeconomic status of the study participants, a cofactor which is difficult to define, no less measure in an epidemiologic study. The magnitude of potential bias due to residual confounding is judged to be small.

T he finding of effect modification of pesticide exposure effects by PON1 was dramatic in this study, with qualitatively different results observed by polymorphism status (i.e. observed OR>1 among those with RR genotype, and an observed OR<1 among those with QQ or QR genotype). If replicated in a large, high

- quality study, this gene

- specific relationship may add substantially to the understanding of pesticide exposure effects. Unfortunately, the study limitations (information bias, selection bias, confounding , and inadequate sample size) limit the inference to be drawn from the study findings.Study 26

. Nevison (2014)

A comparison of temporal trends in United States autism prevalence to trends in suspected environmental factors. Environmental Health. 2014; 13(73): 16 pp

Study Summary

In this ecological study, Nevison tests the hypothesis that autism is a constant

- prevalence condition that simply has been underdiagnosed in the past. The author does this by systematically reviewing temporal trends in the t op ten environmental compounds list (and some other exposures) and identifying those that are most and least consistent with the temporal trends in autism. The implicit assumptions are: 1) environmental exposures around the time of birth (± 1.5 years) are most important, since autism by definition is either present from birth or develops within the first few years of life, and 2) that the rise in autism is driven by one or more environmental influences whose collective temporal trend resembles the trend in autism.

Temporal exposure trends were derived for the top 10 environmental compounds suspected of causing autism or related pervasive developmental delay (PDD) with increased exposure (and other factors). The top 10 toxins were compiled during an extensiv e literature survey: lead, methylmercury, PCBs, OP pesticides, organochlorine pesticides, endocrine disruptors, automotive exhaust, PAHs, polybrominated diphenyl ethers, and perfluorinated compounds. This list was expanded to include other suspected factor s: general air pollution indices, a broad range of mercury exposures, including fish, high fructose corn syrup, atmospheric mercury, and
vaccine

- administered thimerosal, maternal obesity, vaccine

- related indices, and autoimmune
disorders.

OP pesticide “ex posure” was estimated at 25

- 95 million lbs/year (5 major food crops) for 1965

- 2005, with peak use 1975

- 1980, from EPA data for U.S. annual agricultural pesticide consumption data, partitioned into total insecticides, total herbicides, etc., supplemented wi th USDA survey data on insecticide use dating back to 1964 for 5 major crops (corn, cotton, wheat, soybean and potatoes) plus various additional fruits and vegetables. The total insecticide use data were multiplied by % share of OP insecticides (compiled f or the 5 major crops only) to roughly estimate the time trend in U.S. agricultural OP consumption. OP pesticide usage over time is mixed but recently decreasing. Overall, OP pesticide usage is negatively (but not significantly) correlated with autism preva lence. (Note: Glyphosate, an OP herbicide, “exposure” was estimated and presented separately.)Temporal trends in autism for birth years 1970

– 2005 were derived from a combination of data from the United States Individuals with Disabilities Education Act (I DEA, 50 states plus DC) and California Department of Developmental Services (CDDS) reports.

• Using IDEA data, autism prevalence vs. birth year trends were calculated independently from snapshots of data from the most recent annual report, and by tracking prevalence at a constant age over many years of reports. The ratio of the snapshot:tracking trend slopes was used to estimate the “real” fraction of the increase in autism.

• From CDDS, autism prevalence data for birth years 1970

– 1997, effectively a 2002 age

- resolved snapshot of individuals 5 years of age or older receiving services for autism, and autism prevalence data tracking 5 year

- olds from 1995

– 2006, corresponding to birth years 1990

– 2001, were obtained. The CDDS tracking data set overlaps with Califor nia IDEA data tracking 5 year

- olds for birth years 1995

– 2001, providing a means for assessing whether the IDEA definition of autism has expanded from the CDDS definition to include milder ASDs. Since children in private schools are included in IDEA autism counts but not in NCES total public school population data, the IDEA/NCES ratio will tend to overestimate autism prevalence, by underestimating the denominator. To correct for this effect in all figures in which CDDS and California IDEA data are combined, the NCES total populations for California were revised upward by 14% based on available U.S. census data.

A monotonic “composite” curve was constructed by combining CDDS 2002 snapshot data for birth years 1970

– 1994 with California IDEA 5 year

- old tracking data for birth years 1995

– 2005. These data sets blend continuously, albeit for somewhat fortuitous reasons. The primary motive for combining them is to allow examination of concurrent trends in toxins and autism over the longest birth year window possible, 1970

– 2005. The California data were assumed to be broadly representative of the rest of US.

The composite curve and the temporal trend in each suspected causal agent were compared by visual inspection. The correlation coefficient was also computed betwee n the temporal trend in each suspected agent and the composite autism prevalence curve using the longest overlapping time period possible. However, these quantitative statistics are not emphasized, due to the multiple uncertainties involved in defining the long

- term trend in autism.

The author concludes that the increase in diagnosed autism prevalence is a true occurence and has occurred mostly since the late 1980s. The slope ratios for the California IDEA age

- resolved snapshot and constant

- age tracking tr ends suggest that about 80% of the tracked increase in California autism is “real” as opposed to due to better diagnosis, both for birth year intervals1988 to 1994

– 1997 and 1993 to 1999

– 2002. The CA results are similar to and within the
standard deviation of the mean results across the US. The latter suggest that 77 ± 8% of the tracked increase over birth years 1988 to 1994

– 1997 is real and that about 74 ± 8% of the tracked increase over birth years 1993 to 1999

– 2002 is real. In nearly all states, the snap shot slopes are flatter than the tracking slopes, indicating an ongoing, retroactive expansion in the diagnosis of autism among older students who were not identified as younger children. CDDS data probably includes at least some milder ASDs, despite repor ts to the contrary, while the IDEA data do not appear to include all ASDs.

In contrast to the increase in autism rates, children’s exposure to most of the top ten toxic compounds has remained flat or decreased over this same time frame. Some, including le ad, organochlorine pesticides and vehicular emissions, have strongly decreasing trends. Among the suspected toxins surveyed, polybrominated diphenyl ethers, aluminum adjuvants, and the herbicide glyphosate have increasing trends that correlate positively t o the rise in autism. OP pesticide usage over time is mixed but recently decreasing. Overall, OP pesticide usage is negatively (but not significantly) correlated with autism prevalence.

Study Review

As an ecologic study, this study was well performed an d has merits; however it suffers from the ecological fallacy in that it is not possible to link exposure (or timing or level of exposure) to individual children, i.e., those with autism may not be the same children who are exposed. Further, the exposure me asure

– OP pesticide usage per year, based on crops, not specified by specific OP or application location

- is a proxy for actual exposure and may vary geographically. Additionally, home use of OP pesticides is not considered. While chlorpyrifos was banned for residential use in 2001, other OPs continue to be used in household applications, e.g., as pet flea products, with temporal trends that are not resolved by this study. The potential confounders examined are exposures to nine other suspected environmen tal contaminants (and a few other exposures) but again these are proxy exposure measures which cannot be linked to individual children, and there is no statistical control for these exposures. Therefore no conclusions can be drawn with respect to the relat ionship between OP pesticide exposure and autism from this
research.

The author explains in clear detail the differences between the two databases used, how constant age snapshots are generated (most common and straightforward way to quantify trend), and what the snapshot

- based trend means (i.e., if autism is truly a consta nt

- prevalence condition, fullydiagnosed at all ages in the snapshot, the slope ratio will be 0). The author clearly discusses bias
correction for under

- ascertainment of autism in younger children, discarding the youngest ages: 5, 6 and 7, in calculating t he snapshot trend slopes, thus considering a range of start ages from 8 to 11 years old. Further she describes slope errors in the data, using < 10% (tracking) and < 15% (snapshot) error as criteria to filter out states with erratic data and to identify th ose for which the temporal trend could be approximated well as a linear slope. CDDS data are one of the most reliable, long

- term U.S. autism records available (include only verified cases of full

- syndrome autistic disorder (AD), the most severe and unambig uous ASD). Although she points out that some older children may remain undiagnosed, her conclusion that autism is increasing seems
well

- supported. The finding of qualitative consistency between two data sets, CDDS and IDEA, further suggests a strong increa se in autism prevalence over recent decades. She also compares this study results to Finnish data, which shows similar increases.

While epidemiology has linked ASD and PDD in children to both prenatal and postnatal exposure to cholinesterase

- inhibiting OP insecticides, the biological plausibility of these insecticides as a cause of autism has been described, and wheat and corn have been identified as the most important sources of OP pesticide exposure among U.S. children

– according to data provided in the article, this study shows that the temporal trend in total OP insecticide use does not correlate well to the trend in autism. It is possible that other compounds or a mixture of compounds is responsible for the autism increase (the top 10 compounds compri se only a small subset of the > 80,000 synthetic chemicals developed over the past 50 years, many of which have never been assessed for potential toxicity).

It should be noted that this study is limited to autism and does not address other possible

neuro development effects of the compounds.Study 27

. Rohlman et al. (2005)

Neurobehavioral performance in preschool children from agricultural and non

agricultural communities in Oregon and North Carolina. Diane S. Rohlman, Thomas A. Arcury, Sara A. Quandt, Michael Lasarev, Joan Rothlein, Rachelle Travers, Alys Tamulinas, Jennifer Scherer, Julie Early, Antonio Marín, Jacki Phillips, Linda McCauley. Neurotoxicology 26:895

- 598, 2005.

Study Summary

Rohlman et al. reported on the results of a community

- based r esearch project comparing neurobehavioral test performance of Latino children of immigrant parents living and working in agricultural communities to that of Latino children of immigrant parents not living or working in

non

- agricultural communities. Their c ross

- sectional study examined 48

- 71 month old children living in Oregon and North Carolina. The primary objective of the study was to compare the two groups’ performance on neurobehavioral tests. The investigators also compared the groups’ ability to learn test instructions and complete neurobehavioral test components.

The investigators were interested in the effects of low

- level chronic exposures to OP pesticides on neurobehavioral development in an understudied population of immigrant farm workers. They used a proxy indicator of OP pesticide exposure; children living in communities in highly agricultural regions of Oregon (Hood River County) and North Carolina (Johnston, Harnett, Sampson Counties), were defined as ‘exposed’. The children recruited from th e two agricultural communities had at least one parent working in agriculture at the time of the study. The comparison population was comprised of Latino children born to immigrant parents living in

non

- agricultural communities in Oregon (Lincoln County) o r North Carolina (Wilkes County). Neither parent of these children worked in agriculture in the year prior to the study. No objective markers of pesticide exposure were obtained.

Enrollment took place during the summers of 2002 (Oregon) and 2003 (North C arolina), during which times the authors attempted to recruit every child that met eligibility criteria and that were referred by community members. The target population is described by the investigators as a “difficult to access population of immigrant f amilies.” The children living in the agricultural locales were recruited from Migrant Head Start facilities. In Oregon, referent children were recruited from a local community program, Centro de Ayuda, while in North Carolina, referent
children were recrui ted from the community and from the Smart Start preschool program. In all,78 children were enrolled in the study; 43 from the agricultural communities, and 35 from the

non

- agricultural areas.

Neurobehavioral tests were administered to each child particip ant during two testing periods scheduled approximately 1 month apart. Comparisons of neurobehavioral test performance between children from agricultural and non

- agricultural areas were reported only for results from the second testing session. The authors stated that the repeat testing permitted examination of differences in learning between the two groups, and also maximized the number of children who were able to complete the tests. In addition, parents were asked to complete a questionnaire to
collect in formation on parental age, education, years in the United States, and work characteristics. Information on the children’s computer and video game experience was also collected for the subset of children living in North Carolina.

The neurobehavioral tests evaluated the following domains of neurobehavioral development: memory and attention, response speed and coordination, visual memory, attention, divided attention, recall and recognition memory, dexterity, hand

- eye coordination. The neurobehavioral
evalua tion consisted of five computerized tests from the Behavioral Assessment and Research System (BARS) and three non

- computerized tests (Object Memory, Purdue Pegboard, and Visual Motor Integration). The authors noted that the BARS was developed for use with a broad range of working populations of varied educational levels and diverse cultural backgrounds. The BARS tests used were the Digit Span, Finger Tapping, Match

- to

- Sample, Continuous Performance, and Divided Attention. Group test performance in all domai ns was reported as standardized means and associated standard errors, except the Continuous Performance and Divided Attention tests, which were omitted from analyses due to observed difficulties in completion of these tests.

The authors compared differe nces in average neurobehavioral test performance between the two groups of children using separate multivariate linear regression models for each test, adjusting for child age and maternal education. Because they were interested in evaluating whether

avera ge performance of children from agricultural communities was lower than, rather than simply different from, performance of children from non

- agricultural communities, the investigators reported one

- tailed p values corresponding to one

- sided hypothesis test s of the adjusted differences in mean neurobehavioral test performance.In addition to adjusting performance results for differences in age and maternal education, the authors additionally adjusted specific associations for, and examined effect modificati on by, location (Oregon versus North Carolina) and gender. The authors did not observe any statistically significant differences in any of the tests of neurobehavioral performance when comparing all children from agricultural communities to
those from non

- agricultural communities. Two statistically significant differences were observed in comparing sex

- subgroups of exposed and non

- exposed boys: Male children from agricultural communities in Oregon performed significantly worse than the male children fr om non

agricultural communities on right hand Finger Tapping. The male children from agricultural communities in North Carolina had significantly longer latencies on the Match

- to

- Sample test than boys from non

- agricultural communities. Although percent com pletion improvements were seen in children from both agricultural and non

- agricultural communities, the children from non

agricultural areas showed greater improvement in test completion between the two neurobehavioral performance testing sessions.

Study Review

This was a small cross

- sectional study conducted using community

- based research practices to engage with members of the participating communities of immigrant Latino families. The study design, particularly the use of an imperfect proxy for OP pest icide exposure, limits the strength of the evidence contributed by this investigation. The conduct and presentation of only a subset of the planned comparisons in the paper was not ideal, as was the choice of statistical methods. This poorly statistically powered and flawed study contributes little evidence consistent with the hypothesis that chronic, low

- level OP pesticide exposure effects neurobehavioral status at 48

- 71 months of age among Latino children of immigrant parents.

The authors noted that the target population of immigrant Latino families was “difficult to access.” Despite their efforts to enroll every child meeting the reasonable set of study criteria, they managed to recruit only 78 children in total, 37 of whom were defined as exposed; the total eligible population was not enumerated. This small sample size limited the statistical power of the study. The inclusion/exclusion criteria were not clearly reported; presumably, they were being Latino, age 48

– 71 months of age, and, having parents wh o were immigrants to the United
States.

The authors used living in an area with a high concentration of agricultural activity as a proxy indicator of OP pesticide exposure. There is likely some degree of misclassification of true OPpesticide exposure th at is likely independent of neurobehavioral test performance (i.e. non

differential). The authors did not provide estimates of the prevalence of OP pesticide use in the area with a high concentration of agricultural activity, though they did reference an e arlier report by McCauley et al. (2003). The outcome assessment was flawed in several respects. The authors did not present comparisons of test performance from the first test session. Two of the five planned BARS neurobehavioral tests (Continuous Performa nce and Divided Attention) were not analyzed. A varying subset of children from both the agricultural and the non

- agricultural communities were not able to complete all of the tests, despite BARS being designed for use in similar populations.

The authors did not account for multiple comparisons. Without sufficient justification, they
evaluated one

- sided hypotheses and presented corresponding one

- tailed p

- values and, nevertheless did not observe statistically significant differences between groups in their primary analyses. Confidence intervals were not reported.

The approach to confounding was not described in detail. Familiarity and use of computers, a potential confounder in this population, was assessed in only a subset of the population.

Although t he study was not sufficiently powered to examine associations among subsets of the study population, the authors stratified some of their results by gender and location. It was not clear from the report that effect modification by these factors was conside red a priori

.

It was not clear from the report that the authors’ assessment of improvement in completion of tests between sessions (observed in children from both agricultural and non

- agricultural areas, but greater in children from non

- agricultural comm unities) was conducted to test an a priori hypothesis.

No statistically significant differences for any of the tests of neurobehavioral performance were observed when comparing all children from agricultural communities to those from non

agricultural are as. The interpretation of the two statistically significant differences observed in male children is limited by issues of multiple testing of one

- sided hypotheses, and the specter of residual confounding. Issues concerning validity aside, the study is like ly generalizable to other immigrant communities in agricultural areas of the US. Overall, this study provides little evidence consistent with the hypothesis that OP pesticide exposure is associated with neurobehavioral function at age four to six years of age.References cited

McCauley LA, Michaels S, Rothlein J, Muniz J, Lasarev MR, Ebbert C. Pesticide exposure and self

- reported home hygiene practices in agricultural families. Am Assoc Occup Health Nurses J 2003;51:113

– 9.Study 28

. Samarawickrema et al. (2008)

Fetal effects of environmental exposure of pregnant women to organophosphorus compounds in a rural farming community in Sri Lanka. Nirma Samarawickrema, Arunasalam Pathmeswaran, Rajitha Wickremasinghe, Roshini Peiris

- John, Madawa Karunaratna, N ick Buckley, Andrew Dawson, and Janaka De Silva. Clinical Toxicology
46:489

– 495, 2008.

Study Summary

Samarawickrema et al. conducted a cross

- sectional study of pregnant women from a rural farming community in Southern Sri Lanka. The objective of the stu dy was to evaluate the effects of chronic, low level OP pesticide exposure on biomarkers of neonatal health including antioxidant status, oxidative stress, and fragmentation of fetal DNA.

Pregnant women age 20

- 30 years of age who lived in a farming area f or the duration of their pregnancy, and who had not directly handled or been knowingly exposed to OP pesticides were recruited for participation in the study when they presented for delivery at the hospital serving the rural farming community.

Forty

- one w omen enrolled during two spray seasons (July

- August, 2005, and January

- February 2006) constituted the OP

- exposed group. An additional 25 women recruited at the end of the “in

between spray season” were defined as the unexposed comparison group.

An intervi ew

- guided questionnaire was completed at the time of admission for delivery to determine eligibility for the study, and also to collect information on gestational age, distance from place of residence to closest OP pesticide spraying site, approximate freq uency of spraying during the month prior to the interview, and diet.

Maternal and cord blood samples were obtained at delivery, and a breast milk sample was collected within 24 hours of delivery. (Maternal blood samples were not obtained for exposed

women enrolled between July and August, 2005, i.e. during the first of the two spray seasons.)

Breast milk and plasma from maternal and cord blood samples were assayed for the presence of four OP pesticides chlorpyrifos, diazinon, dimethoate, and fenthion. Qua ntification of these OP pesticides was conducted using HPLC

- Tandem mass spectrometry, a method with a level ofdetection of 0.5 μg/L. In addition, serum chlorpyrifos and diazinon levels were quantified using gas chromatography with minimum levels of detect ion listed as 40 μg/L and 20 μg/L, respectively. The investigators also quantified oxidative butyrylcholiesterase (BChE) activity inhibition in blood and breast milk samples as a marker of OP pesticide exposure.

The antioxidant status of the fetus was cha racterized by superoxide dismutase (SOD) activity assayed in maternal and cord blood samples as well as in breast milk samples. Fetal oxidative stress was characterized by quantifying malondialdehyde (MDA) concentrations in cord blood. Fetal DNA fragmentat ion was assessed by electrophoresis of DNA extracted from erythrocytes from cord blood samples. The authors noted that the assessment of DNA damage was conducted by a researcher blinded to the exposure status of the samples. A similar statement was not mad e with respect to assessment of the other fetal outcomes.

The authors did not describe their statistical methods in the report. The investigators compared the exposed and non

- exposed groups with respect to unspecified demographic and clinical measures, le vels of the four OP pesticide biomarkers in blood and breast milk samples, as well as BChE, SOD, and MDA concentrations in the collected biological media using t

- tests and Chi

square tests.

Statistically significant differences in clinical and demographic characteristics were not observed
between the women characterized as OP

- exposed (delivered during a spray season) and not exposed to OP pesticide (delivered in between spray seasons). Similarly, the women
characterized as OP

- exposed and not exposed were s imilar with respect to the OP

- residues quantified in the collected biological media. In fact, OP residues were detected in only two participants

- chlorpyrifos in maternal and cord blood samples obtained from one exposed women, and dimethoate in breast mil k of one unexposed women. BChE activity in cord blood samples collected from women delivering during the spray seasons was significantly decreased, relative to that observed among the women who delivered in

- between spray seasons.

Slightly higher SOD observed in cord blood samples of the women classified as OP

- exposed were not statistically significant (p=0.5) On average, cord blood MDA concentration, a marker of lipid peroxidation and oxidative stress, was significantly higher among the women who deli vered during the spray seasons, relative to those who delivered in between spray seasons. The proportion of fetal DNA breakage was also significantly higher among the OP

- exposed group.Study Review

Limited inference can be garnered from this small cross

- sectional study. Although the use of OP

exposure and fetal effect biomarkers was reasonable, limitations of the study design, particularly the lack of true differences in OP pesticide exposures betwee n the two comparison groups, likely account for the largely null findings. Moreover, it is unclear whether the differences observed in
markers of fetal oxidative stress can be ascribed to differences in OP pesticide exposure, given the similarities in expo sure profiles between the two groups.

Inclusion and exclusion criteria were generally reasonable, though the decision to exclude women who directly handled or were knowingly exposed to pesticides (in order to focus on low

level exposures) limited the stat istical power of the study. By design, the exposed and non

exposed women were likely to have similar OP pesticide exposure profiles. Indeed, the two groups could not be distinguished based on of BCHE activity levels of OP residues in blood or
breast milk s amples. This is evidence that notable OP pesticide exposure episodes did not occur prior to delivery. The OP residue biomarker assessment methods were likely not sensitive enough to resolve the low

- level environmental (rather than occupational) exposures t hat were the subject of this investigation.

By design, OP pesticide exposures were low, if they occurred at all. Moreover, the exact types of pesticides used in spray activities were not identified. That said, the investigators' decision to quantify four specific OPs in biological media was guided by information from the Sri Lanka Department of Agriculture and the findings of a previous study that characterized types of pesticides used during different seasons in the study area. These sources identified ch lorpyrifos, diazinon, dimethoate, and fenthion.

Given the similarity in OP pesticide exposure profiles between the two groups, the sample size of this study was likely inadequate to detect small differences in the fetal outcomes between them.
This issue w as compounded by the failure to collect maternal blood samples for the exposed group recruited during the first of the two growing seasons assessed in this investigation.

The timing of the collection of biological media for exposure biomarker assessment ( during or just after delivery) in this cross

- sectional study may not resolve OP pesticide exposures at biologically relevant time windows for fetal exposure effects.The assessment of fetal outcomes were appropriately selected and substantiated by adequat e discussions of biological plausibility. Errors in their measurement are likely to have been non

differential with respect to exposure.

In their report, Samarawickrema et al. did not adequately detail the statistical analysis conducted in this investigat ion. The T

- tests used to evaluate mean differences in continuously distributed variables between the exposed and non

- exposed groups was likely reasonable, though evaluation of the distribution of the variables being compared were not described in the repor t. Chi

- square tests were used to evaluate categorical variables. This test relies on large sample sizes, and the use of the test to compare DNA damage in the two groups, was not appropriate, as only two
individuals in the non

- exposed group had observed DNA fragmentation.

The authors did not find statistical differences between the exposed and non

- exposed women with respect to demographic and clinical details, and did not conduct multivariate adjusted analyses. Though the lack of observed differences betwee n the two groups with respect to measured baseline characteristics provides some evidence of comparability of the two groups, there was no description in the report a detailed assessment of potential confounding factors. As such, residual confounding of th e associations between fetal outcomes and indicators of OP pesticide exposure is possible.

The findings of increased markers of fetal oxidative stress and DNA fragmentation among
mother

- infant pairs delivered during spray seasons, along with the finding o f lower BChE in this group, relative to those delivering during the in

- between spray periods, led the authors to conclude that these infants may be at “increased risk for health problems,” though they add that
the nature and extent of the risk is unknown.” The strength of the authors’ conclusion is diminished by the failure to observe direct evidence of differences in OP pesticide exposure between the two groups. In fact, no measurable maternal OP pesticide exposure or BChE inhibition was observed, indicati ng low levels of, or absence of, recent OP pesticide exposure. This study therefore provides weak evidence consistent with the hypothesis that chronic, low

level OP pesticide exposure is associated with fetal decreased antioxidant status, increased
oxidati ve stress, and fragmentation of fetal DNA.Study 29

. Savitz et al. (1997)

Male pesticide exposure and pregnancy outcome. David A. Savitz, Tye Arbuckle, Diane Kaczor, and Kathryn M. Curtis. American Journal of Epidemiology 146(12):1025

- 1036,
1997.

Study Summary

Savitz et al. reported on results of the Ontario Farm Family Health Study, one of the few studies specifically evaluating male pesticide exposure during the peri

- conception period and subsequent pregnancy outcomes in a large retrospective c ohort study. Set in Ontario, Canada, the study leveraged the 1986 Canadian Census of Agriculture to identify couples operating full

- time, family run farms. Couples identified who were subsequently provided a survey on which to report farm activities, chemi cal applications, and birth outcomes.

The objective of the study, conducted in 1991

- 1992, was to assess associations between male exposure to agricultural pesticides, as indicated by self

- reported farm activities (to application of OP pesticides) in the 3 months prior to conception through the month of conception, and adverse pregnancy outcomes (miscarriage, pre

- term delivery, and small

- for

- gestational

- age birth, and altered sex ratio). Although a large number of chemical applications (e.g. insecticides, fungicides, herbicides) and farm activities (e.g., mixing/use of chemicals for crops yard, buildings, and livestock, use of personal protective equipment) were reported, this summary will focus on the associations reported between birth outcomes (miscarria ge, pre

- term delivery, and
small

- for

- gestational

- age birth) and self

- reported farm activities specific to OP pesticides.

7,379 farms were sampled from the Canadian agriculture census, 2,693 of which met the eligibility criteria. Farm couples were eligible if they were living year

- round on or near the farm and the woman was younger than age 45 years old (at the time of the screening interview); 2,946 couples met these eligibility criteria, of whom 1,898 (64%) completed and provided survey
forms for the farm operator, husband, and wife. These couples reported on 5,853 pregnancies, of which 3,984 (68%) were included in the analysis. Among these, there were 1,548 live births reported (417 women with a single birth, 595 reporting two births, 354 with three, and 182 with more than three births).

Exposure was defined through self

- reporting of engagement in any activities associated with direct pesticide exposure for one or more months in the 3

- month period prior to conception bymale study participants. Frequency of adverse birth outcomes among pregnancies classified as exposed were compared to those of a reference group of couples for whom the man reported no exposure to any activities associated with direct pesticide exposure. This review focuses on OP
insecticid e use for crops, livestock, and buildings, though the study evaluated and reported on a
wider set of farm

- related chemical use and related activities. Objective measures or biomarkers of pesticide exposure were not obtained in this large, retrospective sur vey study.

Adverse birth outcomes were recalled and self

- reported by the female of each farm family. The recall interval was in the range of 5

- 15 years, for the post part (the actual range was not reported). Births were classified as a miscarriage if a r ecognized pregnancy was lost prior to 20 weeks of completed gestation and stillbirth if pregnancy loss occurred after 20 weeks of completed gestation. Pre

- term delivery (PTD) was defined as birth prior to completion of 37 weeks of gestation and small for g estational age (SGA) was defined as the newborn falling
below the 10 th percentile of birth weight for gestational age based on Canadian percentiles. Evaluation of PTD and SGA were restricted to the subset of singleton live births.

For their primary analys es, the authors built a multivariate adjusted logistic regression model for each of the pregnancy outcomes. The authors approached model building by first estimated crude (unadjusted) risk ratios for each outcome and each of the following potential confoun ders: Mother's age, parity, mother's education, father's education, per capita income, mother's off

- farm job, father's off

- farm job, mother's smoking during pregnancy, mother's alcohol use during pregnancy, conception

- to

- interview time interval, father's a ge, tobacco use, alcohol use, caffeine use, mother's language, ethnicity, religion, and child's sex. They then retained only those potential covariates, and collapsed categories of covariates, that yielded risk ratios less than 0.8 or greater than 1.2. Sub sequent elimination and re

- specification of covariates occurred “as appropriate,” but the rationale was not specified by the authors. All covariates except for the last 8 listed above were adjusted for in final models of the relationship between adverse bi rth
outcomes and self

- reported indicators of peri

- conception pesticide exposure.

The authors also built “several” models using generalized estimating equations (GEE) and a logit link to evaluate the degree to which including multiple pregnancies per woman (i.e., potential

non

- independence of observed outcomes) resulted in biased estimation of variances and corresponding 95% confidence intervals. They concluded that the overall impact of failure to
account for within

- family correlations among pregnancies wa s minimal and reported only the results of the conventional logistic regression models.The authors did not observe any statistically significant associations between peri

- conception pesticide exposure among male farm workers and risk of miscarriage, sti llbirth, PTD, or SGA. The highest elevation in relative risk was observed for PTD in association with reported exposure to livestock organophosphate pesticide exposure (OR=2.7; 95% CI: 0.7

- 11).

Study Review

The interpretation of the results of this cohort study of pesticide exposure and birth outcomes conducted among farm families living in Ontario, Canada is constrained by limitations of the retrospective study design, particularly the methods used to assess male pesticide exposure and birth outcomes. Some degree of both differential and non

- differential errors in recall of exposures and outcomes is probable. The potential for selection bias, and, to a lesser extent, an inappropriate choice of statistical met hods and potential for residual confounding further limit the study. Finally, the (fortunately) low prevalence of adverse birth outcomes in this study population results in relatively imprecise estimation of odds ratios, as reflected in the observed
confid ence intervals, despite the relatively large size of the study.

The inclusion/exclusion criteria employed in the study were appropriate and likely resulted in recruitment of a study population that was relatively homogeneous with respect to confounders

o f the male pesticide exposure

- pregnancy outcome relationship, and the potential for confounding was thus limited by design. The study was large, relative to other similar studies, and employed a detailed questionnaire to obtain detailed information on both farm

- related exposures and pregnancy outcomes.

The retrospective design and the reliance on survey

- derived indicators of pesticide exposure and birth outcomes are two of study features that, although logistically efficient, may have resulted in
errors i n classification. The authors referred to their assignment of exposure as “probabilistic.” That is, men who engaged in specific farm activities were more likely to be truly pesticide

exposed than men who did not engage; Men classified as unexposed may neve rtheless have been exposed to pesticides and men classified as exposed may nevertheless have not been exposed to pesticides. This exposure classification scheme likely resulted in ‘exposed’ and ‘not exposed’ groups being more similar to each other with res pect to their true pesticide exposures, relative to a 100% accurate and precise exposure measurement scheme. The effect of this design element on the association estimates (i.e., the estimated odds ratios) is attenuation toward the null

– no observed effec t. Moreover, quantitative inferences regarding dose are not attainable. Frank errors in recall of exposure are also a potential problem. For example, the authors note that the lengthyrecall interval between pregnancies and completion of the study survey m ay have adversely impacted the quality both pesticide exposure and birth outcome information; most of the pregnancies occurred greater than 5 years prior to the start of the study, and some occurred as much as 15 years prior to the completion of the questi onnaires by the study participants. On average, errors in recall of OP pesticide exposure that are independent of the occurrence of adverse pregnancy outcomes result in observed odds ratio estimates that are attenuated (i.e.
closer to the null value of 1.0 ), relative to odds ratios estimated in the absence of such errors. These errors may result in ‘false

- negative’ findings, and are a potential alternative explanation for the largely null findings of this study. Although perhaps less likely, similarly non

- d ifferential errors in the recall of pregnancy outcomes would also engender ‘false negative’ findings consistent with the observed odds ratios reported in the study. The authors cite a previous study, for example, that noted increasing errors in reporting o f miscarriage after approximately 10 years (Wilcox and Horney 1984). It is also possible that the probability that participants’ imperfect recall of pesticide exposure in this study is not independent of the occurrence of a birth outcome. If, for example, fathers who have experienced an adverse pregnancy outcome are more likely to incorrectly recall exposure to pesticides, relative to fathers who have not had a similar experience, the observed odds ratio would overestimate the true odds ratio. Finally, simi lar errors and subsequent classification of self

- recalled exposure to confounding factors may have resulted in some degree of residual confounding, despite efforts of the investigators to control for confounding in their statistical analyses.

The investi gators’ intent to ascertain information regarding exposure to specific pesticides, rather than just reporting on associations between birth outcomes and agricultural use of chemicals, generally, is laudable. However, the attempt at specificity likely impac ts both the validity and the statistical efficiency (power) of the study; the efficiency with which odds ratios were estimated is adversely impacted because the number of men exposed to specific pesticide classes is smaller than the aggregate indicators of exposure. The validity is adversely affected because, although men may accurately recall that they were exposed to a chemical, they may imperfectly recall the actual chemical to which they were exposed during the pre

- conception period.

Only 64% of eligib le couples completed the study surveys. It is not possible to determine the degree to which those who chose not to participate are represented by those who did chose to participate in the study. Bias due to differential errors in the selection of study par ticipants is likely to have occurred in this study, the magnitude of which is dependent on the degree to which participation in the study was dependent on both exposure and outcome. Although many scenarios are plausible, it is possible, for example, that c ouples are more likely to havecompleted the study surveys if the male is concerned about his past pesticide exposures and the female is motivated to participate due to her having experienced an adverse pregnancy outcome,
relative to those that have not ha d similar experiences.

Two aspects of the statistical methods employed in this investigation were not optimal. First, the investigators did not report having accounted for multiple testing, despite evaluating associations between five pregnancy outcomes and more than two dozen i ndicators of farm

- related activities associated with pesticide exposure. Second, the conventional variance estimator calculated by the standard logistic regression model and used by the authors was not appropriate. The investigators were aware of potential bias in estimation of variances due to their inclusion of multiple pregnancies per couple, and they explored the impact of their model by employing the correct statistical technique (i.e. GEE with empirical variance estimators) in an unspecified
subset of the analyses they conducted. The authors reported that the influence on the width of the confidence intervals was “quite modest” for both indicators of pesticide exposure and included covariates. They noted that all of the confidence intervals estimated u sing GEE were within 20% of the estimates observed using conventional logistic regression models. The authors also observed that around 40% percent of the confidence intervals estimated using the GEE models

were narrower than those estimated with conventio nal logistic regression. However, regardless of whether it generates a confidence interval that is wider or narrower than the corresponding empirical variance estimate, the conventional variance estimator is not appropriate in the presence of correlated ou tcomes.

The investigators’ approach to evaluating and adjusting for potential confounders was generally appropriate, although certain aspects of the final steps of the model building process were not reported in sufficient detail.

Assuming they are val id, the study results are largely generalizable to similar populations of families living on or near, and operating, family farms. However, overall, this study provides very little evidence consistent with the hypothesis that OP pesticide exposure among ma les causes an increase in the risk of miscarriage, stillbirth, pre

- term delivery or small

- for

- gestational age outcomes. Some, but not all of the observed odds ratio estimates of the association between OP pesticides and adverse birth outcomes were greater than one, but most were small in magnitude, and none were statistically significant. Selection bias, confounding, and information bias are potential explanations for the negative findings, as is the limited statistical power to
assess rare outcomes in this study. The information bias, in particular, is likely to have resulted in attenuation of the true (unobserved) associations, toward (observed) associations that arerelatively smaller, or null. Differential recall bias, and the other biases too, may have also resulted in observed associations that are greater in magnitude than the true association between male pre

- conception exposure to agricultural OP pesticides and subsequent adverse birth

outcomes.

References cited

Wilcox AJ, Horney LF. Accuracy of s pontaneous abortion recall. American Journal of Epidemiology. 1984;120:727

- 33.Study 30. Wickerham et al. (2012)

Reduced birth weight in relation to pesticide mixtures detected in cord blood of full

- term infants. Erin L. Wickerham, Betsy Lozoff, Jie Shao, Niko Kaciroti, Yankai Xia, John D.
Meeker. Environment International 47:80

– 85, 2012.

Study Summary

Wickerham et al. reported on results of a cross

- sectional pilot study evaluating differences in birth weight associated with prenatal OP and other n on

- persistent pesticides quantified in umbilical cord blood samples of children born to healthy mothers living in a rural area of China.

This pilot study was set in Zhejiang Province, using data collected from a subset of healthy pregnant women participating in a previously initiated study of iron deficiency and neurodevelopment. Pregnant women at 36 weeks gestation with an apparently healt hy, uncomplicated, singleton pregnancy were recruited from a Maternal and Children's hospital and invited to participate in the study. Exclusion criteria included prevalent chronic diseases, complicated pregnancies, and hereditary or metabolic diseases. Ov er 99% of eligible pregnant women agreed to participate in the original study. Consecutive participants in the original iron deficiency study with due dates between April and June, 2009 were included in this pilot study of pesticide exposure effects on bir th weight. The final study sample consisted of 112 of 116 pregnant women who gave birth to full

- term infants and for whom cord blood samples were analyzed for pesticides; Two women for whom demographic information was unavailable, one woman with missing ma ternal hemoglobin levels at delivery, and one women whose data were observed to be highly influential were excluded from the multivariate analyses.

The mother’s demographic information and medical history were gathered from medical records during two pre natal visits (~16 weeks and ~36 weeks gestation), and additional maternal and neonate characteristics, including birth weight, were obtained from medical records from the delivery.

A 30 ml umbilical cord blood sample was collected after delivery of the ch ild, processed, and shipped to Institute of Toxicology at Nanjing Medical University for pesticides residue quantification. The 20 pesticides evaluated were chosen based on pesticide usage data as well as availability of analytical standards, method compat ibility, and preliminary data. The OP pesticides included chlorpyrifos, diazinon, fonofos, malathion, parathion

- ethyl, parathion

-methyl, profenofos, and terbufos. Concentrations of the pesticides in cord serum were analyzed by GC and DSQ mass spectrometry. More than half of all OP pesticide concentrations in the cord blood samples were below the lower level of detection (LOD); For 5 of 8 OP pesticides analyzed, 75% of the concentrations were not detectable in the cord blood samples. For the analyses in whic h individual pesticide concentrations were included in a model as a continuous variable, levels below the lower LOD were assigned a value of the LOD/√2.

Three pesticide exposure metrics were defined for subsequent analysis: the number of pesticides
detect ed in the cord blood sample, presence or absence of individual pesticides (assessed only for pesticides detected in samples from at least four subjects), and a 3

- level ordinal variable (concentrations below the LOD, medium concentrations, and high concentr ations defined by the median among detectable pesticide levels).

Multivariate linear regression models constructed that adjusted a priori for gestational age, maternal age, maternal BMI at early pregnancy, and maternal hemoglobin at delivery. Newborn

se x and newborn serum ferritin were also considered as covariates but were not empirically associated with both pesticide exposure and birth weight in the study sample and where therefore not included in final models. Regression diagnostics were conducted, i ncluding assessment of influential observations; those with a studentized residual greater than 3 or less than −3 were considered influential. As mentioned earlier, one observation with a residual greater than 3 was
not included in final models.

No stati stically significant associations were observed between the number of OP pesticides detected in cord blood and birth weight. Similarly, no differences in mean birth weight were observed between those with OP pesticides classified as a dichotomous variable or as a three

level ordinal variable.

Study Review

The interpretation of the null results of this pilot study conducted among healthy Chinese women and their babies living in a rural area of China is hampered by the cross

- sectional design and especially by the large proportion of cord blood samples in which OP pesticide residues were not
detectable.

Selection of participants was reasonable, though based largely on convenience. The sample size was small but adequate for a pilot study.The cord blood pesticide biomarker assessment was well conducted, though the exposure classification based on the number of detectable pesticide residues present in the sample is not directly applicable to the assessment of OP pesticide exposure effects. T he authors reasoning for using the number of pesticides detected as their primary exposure metric was based on the observation that humans are exposed to multiple environmental toxicants which may exert synergistic effects. More likely, though, this decisi on was one of convenience, and guided by the observation of very few detectable levels of many of the pesticides in the cord blood samples.

The selection and consideration of covariates for inclusion in multivariate regression models was appropriate. The multivariate modeling process was also reasonable.

This study provides no evidence consistent with the hypothesis that prenatal OP pesticide exposures affect birth weight. The quantification of a large number of pesticide residues in cord blood specimens is a novel approach to evaluating prenatal exposure effects on birth outcomes. However, the sensitivity of the analytic methodology was largely insufficient to detect pestici de residues in this population.Study 31

. Naksen et al (2005 )

Naksen W, Prapamontol T, Mangklabruks A, Chantara S, Thavornyutikarn P, Srinual N, Panuwet P, Ryan PB, Riederer AM, Barr DB. Associations of maternal organophosphate pesticide exposure and PON1 activity with birth outcomes in SAWASDEE birth cohort,
Thailan d. Environ mental Research142: 288

– 296, 2015. Study Summary

Naksen W., et al., (2015) evaluated the environmental chemical exposures and their relation to birth outcomes and infant neurodevelopment in 52 pregnant farmworkers in Fang district, Chiang Mai province, T hailand. This cohort is named, the Study of Asian Women and Offspring's Development and Environmental Exposures (SAWASDEE) cohort. A large dataset was collected during pregnancy including urine samples, three blood samples, and by using a questionnaire. The authors investigated the paraoxonase1 (PON1) activities, changes in maternal acetylcholinesterase (AChE) and their relation to urinary dialkylphosphates (DAPs) metabolites of OP pesticides, during pregnancy. Maternal AChE, butyrylcholinesterase (BChE), and (PON1) activities were measured three times (at enrollment; 32 weeks of pregnancy and at delivery) during pregnancy, and six urinary DAP concentrations [including di

- methylphosphate (DMP), dimethylthiophosphate (DMTP), di

- methydithiophosphate (DMDTP ), diethylphosphate (DEP), diethylthiophosphate (DETP), and diethydithiophosphate (DEDTP)] were measured in urine samples on average 8 times during pregnancy (from enrollment until delivery). In this study, a direct correlation between maternal biomarkers of pesticide exposure (as measured by urinary DAPs) and negative birth outcomes (head circumference and birth weight) was only observed after stratification by PON1 phenotype. However, an association between maternal enzyme activities and negative birth outcomes was not observed. Among the individuals in the group with low maternal PON1 activity (n=23), new born head circumference was inversely correlated with log10 maternal ΣDEAP (molar sum of DEP, DETP and DETDP) and ΣDAP (molar sum of all six DAPs), a t enrollment (gestational age = 12 ± 3 weeks; β=

- 1.0 cm, p= 0.03 and β=

- 1.8 cm, p< 0.01, respectively), and at 32 weeks pregnancy (β =

- 1.1cm, p = 0.04 and β =

- 2.6 cm, p= 0.01, respectively). Furthermore, among these mothers, new born birthweight was also inversely associated with log 10 maternal ΣDEAP and ΣDAP at enrollment (β=

- 219.7g, p= 0.05 and β=

- 371.3 g, p= 0.02, respectively). Associations between maternal DAP levels and newborn outcomes (birth weight, length and head circumference) were not observed in the group of mothers wit h high PON1 activity. This study highlights the finding of the negative association of prenatal OP exposure and birth outcomes especially in low PON1 activity mothers.

Study ReviewIn reviewing this study, a few errors (for example, accuracy of statistic al analysis of association between urine cumulative DAPs and birth outcomes, stratified by maternal PON1 phenotype; and authors did not show adjustment for multiple comparisons) were identified in the data tables, raising concerns about the validity of the study results. Given these errors, the small sample size (N=59), and the lack of discussion of study weaknesses, this study was rated to be of low quality overall.