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Cohort profile: The Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH) MeKiTamara Cohort Study: Influences of complex environmental exposures on maternal and child health in For peer Surinamereview only Journal: BMJ Open

Manuscript ID bmjopen-2019-034702

Article Type: Cohort profile

Date Submitted by the 02-Oct-2019 Author:

Complete List of Authors: Zijlmans, Wilco; Academic Hospital Paramaribo, Scientific Research Center Suriname ; Wickliffe, Jeffrey; Tulane University, Global Environmental Health Sciences Hindori-Mohangoo, Ashna; Netherlands Organization for Applied Scientific Research MacDonald-Ottevanger, Sigrid; Academic Medical Center, Amsterdam, The Netherlands Ouboter, Paul; Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname

Center Suriname on September 25, 2021 by guest. Protected copyright. Abdoel Wahid, Firoz; Tulane University Fortes Soares, Lissa; Tulane University Alcala, Cecilia; Tulane University Boedhoe, Esther; Academic Hospital Paramaribo Grünberg, Antoon; Ministry of Health, Paramaribo, Suriname, Department of Public Health Hawkins, William; Tulane University Shankar, Arti; Tulane University Harville, Emily; Tulane University School of Public Health, Epidemiology Drury, SS; Department of Psychiatry and Behavioral Sciences, Tulane University Covert, Hannah; Tulane University Lichtveld, Maureen; Tulane University School of Public Health and Tropical Medicine, Global environmental health sciences

Community child health < PAEDIATRICS, Developmental neurology & Keywords: neurodisability < PAEDIATRICS, PERINATOLOGY, PUBLIC HEALTH,

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1 2 3 Epidemiology < TROPICAL MEDICINE, Nutrition < TROPICAL MEDICINE 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 35

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1 2 3 Title: Cohort profile: The Caribbean Consortium for Research in Environmental and 4 5 6 Occupational Health (CCREOH) MeKiTamara Cohort Study: Influences of complex 7 8 environmental exposures on maternal and child health in Suriname 9 10 11 12 13 14 15 Author list 16 For peer review only 17 18 Wilco C.W.R. Zijlmans*1,2,3, Jeffrey K. Wickliffe2, Ashna D. Hindori-Mohangoo2,4,5, M. Sigrid 19 20 MacDonald-Ottevanger1,6, Paul E. Ouboter2,7,8, Gwendolyn A. Landburg7, John Codrington9, 21 22 9 3,10 3,11 1,3 23 Jimmy Roosblad , Gaitree K. Baldewsingh , Radha Ramjatan , Anisma Gokoel , Firoz 24 25 Abdoel Wahid1,2, Lissa Fortes Soares2, Cecilia S. Alcala2, Esther Boedhoe1, Antoon W.E. 26 27 Grünberg12, William B. Hawkins2,13, Arti Shankar2, Emily W. Harville14, Stacy S. Drury15, Hannah 28 29 H. Covert2, Maureen Y. Lichtveld2 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 Affiliations 37 38 39 1 Scientific Research Center Suriname, Academic Hospital Paramaribo, Paramaribo, Suriname 40

41 on September 25, 2021 by guest. Protected copyright. 42 2 Department of Global Environmental Health Sciences, School of Public Health and Tropical 43 44 Medicine, Tulane University, New Orleans, LA 70112, United States of America 45 46 47 3 Faculty of Medical Sciences, Anton de Kom University of Suriname, Paramaribo, Suriname 48 49 50 4 Perisur (Perinatal Interventions Suriname) Foundation, Paramaribo, Suriname 51 52 53 5 Netherlands Organization for Applied Scientific Research, TNO Healthy Living, Leiden, The 54 55 56 Netherlands 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 6 Department of Clinical Chemistry Medical Microbiology, Academic Medical Center, 4 5 6 Amsterdam, The Netherlands 7 8 7 9 National Zoological Collection of Suriname/Center for Environmental Research, Anton de Kom 10 11 University of Suriname, Paramaribo, Suriname 12 13 14 8 Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname 15 16 9 For peer review only 17 Department of Clinical Chemistry, Academic Hospital Paramaribo, Paramaribo, Suriname 18 19 10 20 Medical Mission Primary Health Care Suriname, Paramaribo, Suriname 21 22 11 23 Regional Health Service Nickerie, Suriname 24 25 12 26 Department of Public Health, Ministry of Health, Paramaribo, Suriname 27 28 13 29 Department of Health Policy, Vanderbilt University Medical Center, Nashville, TN, United 30 31 States of America 32

33 http://bmjopen.bmj.com/ 34 14 Department of Epidemiology, Tulane University School of Public Health and Tropical 35 36 Medicine, New Orleans, LA, United States of America 37 38 39 15 Tulane University School of Medicine, New Orleans, LA, United States of America 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 *Corresponding author. Scientific Research Center Suriname, Academic Hospital Paramaribo, 52 53 Picornistreet 11, Paramaribo, Suriname. Email: [email protected] 54 55 56 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Abstract 4 5 6 7 Purpose: The Caribbean Consortium for Research in Environmental and Occupational Health 8 9 10 (CCREOH) MeKiTamara population-based prospective longitudinal cohort study addresses the 11 12 13 potential adverse impact of complex chemical and non-chemical environmental exposures in 14 15 mother/child dyads in Suriname. Associations between levels of environmental elements and 16 For peer review only 17 18 toxicants in pregnant women and birth outcomes are determined and longitudinal 19 20 21 neurodevelopmental outcomes in infants 0-4 years are measured. Also, levels of environmental 22 23 24 elements and toxicants in fish, produce, and rice are assessed. 25 26 27 28 Participants: From December 2016 to date, 1067 pregnant women and 827 babies are enrolled. 29 30 31 Exposures to mixtures of toxicants including mercury, lead, and selected pesticides are monitored 32

33 and dietary, non-chemical stressors and environmental source characterization assessments take http://bmjopen.bmj.com/ 34 35 36 place during seven study time points: twice during pregnancy, birth, 12, 24, 36, and 48 months. 37 38 39 40 Findings to date: One out of four women, and predominantly those living in Suriname’s interior,

41 on September 25, 2021 by guest. Protected copyright. 42 43 have hair mercury levels exceeding values considered safe by international standards. Almost 25% 44 45 46 of women may suffer from depression, and 3 out of 10 have high stress levels. There was no 47 48 statistically significant association found between hair mercury levels and adverse birth outcomes. 49 50 51 52 53 54 55 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Future plans: Total mercury concentrations in blood are elevated in women from the interior of 4 5 6 Suriname where the primary source of exposure to mercury is through consumption of fish. Fish 7 8 9 consumption advisories may reduce exposure to these harmful substances. New research has 10 11 12 recently been funded to examine effects of potentially beneficial neuroprotective factors in fish 13 14 15 that may counter the neurotoxic effects of mercury. Long term effects of exposures to toxicant 16 For peer review only 17 18 mixtures in infants are being evaluated through pediatric neurodevelopmental assessments up to 19 20 21 four years of age. 22 23 24 25 26 Keywords environmental exposures, mercury, metal mixtures, pregnant women, pediatric 27 28 29 neurodevelopment, Suriname 30 31 32

33 http://bmjopen.bmj.com/ 34 35 Wordcount 3995 (excl. references, acknowledgements, tables & figures) 36 37 38 39 40

41 Strengths and limitations of this study on September 25, 2021 by guest. Protected copyright. 42 43 44 45  The study addresses two high priority public health threats in Suriname as well as for 46 47 neighboring countries in the Guiana shield with similar practices: the impact of mercury 48 49 exposure from artisanal gold mining and pesticide exposure associated with agricultural 50 51 52 practices in two vulnerable subpopulations: pregnant women and children aged <5 years 53 54 of age. 55 56 57 58 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3  The longitudinal follow up of children up to 48 months provides several timepoints to 4 5 6 assess neurodevelopmental outcomes. 7 8  The study has a biospecimen bank of approximately 12,000 samples, providing the 9 10 opportunity for future analyses within the cohort. 11 12 13  A linked research training grant facilitates the training of nine Surinamese PhD candidates 14 15 with dissertation research embedded in the study, thereby building critical in-country 16 For peer review only 17 environmental health research capacity. 18 19 20  In terms of initial challenges, the interior subcohort (N=200) is logistically difficult to 21 22 reach, resulting in delayed recruitment, data collection and transport of cord blood 23 24 25 specimens, this was corrected with additional team support onsite that facilitated 26 27 recruitment of the full subcohort, and additional heelprick sampling at birth. 28 29 30 31 32 Introduction

33 http://bmjopen.bmj.com/ 34 35 36 The Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH) 37 38 addresses the goal of high-priority environmental and occupational health risks in Suriname and 39 40 those common to the vulnerable Caribbean region, while preserving the unique assets pertaining

41 on September 25, 2021 by guest. Protected copyright. 42 43 to the health and cultural traditions of indigenous and other health disparate populations [1]. 44 45 Environmental contaminants may affect pregnancy, and pre- and postnatal health in multiple ways: 46 47 miscarriage, preterm delivery, intra-uterine growth retardation, and congenital malformations as 48 49 50 well as other behavioral and physical health problems that may not manifest until later in 51 52 development [2, 3, 4]. Prenatal exposure to multiple heavy metals is associated with adverse 53 54 pediatric health outcomes [5]. Pesticide exposure has been linked to fetal growth decrements and 55 56 57 58 5 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 preterm birth, and there is mounting evidence that exposure to pesticides during pre- and postnatal 4 5 6 development is associated with neurodevelopmental deficits in young children [6-10]. The 7 8 physiological effects of contaminants may differ in combination compared to individually; for 9 10 instance, lead, arsenic, and mercury can potentiate each other’s toxicity, even at individual levels 11 12 below concentration expected to result in adverse effects [11]. Despite this knowledge, no 13 14 15 cumulative risk assessments have been conducted to date addressing exposures to contaminant 16 For peer review only 17 mixtures in Caribbean Low- and Middle-Income Countries [12]. Beyond this, there remains a 18 19 paucity of studies that have examined the interactive impact of psychosocial exposures with 20 21 22 environmental exposures. 23 24 25 The CCREOH-MeKiTamara (MeKiTamara which means “making a mother and child's 26 27 tomorrow’’ in Suriname’s local language) environmental epidemiology cohort study fills this gap 28 29 by exploring the impact of exposures to organic and inorganic neurotoxicants, including mercury, 30 31 32 lead and multiple organophosphate pesticides, on Surinamese pregnant women and their offspring

33 http://bmjopen.bmj.com/ 34 (up to 48 months of age) using a cumulative risk approach. Specifically, the study assesses the 35 36 impact of exposures to chemical and non-chemical psychosocial stressors by examining the 37 38 interaction between exposure to environmental chemicals and social and psychological 39 40 determinants of early neurodevelopment. Few prospective studies have measured prenatal

41 on September 25, 2021 by guest. Protected copyright. 42 43 exposure through 48 months, and those that have showed contrasting results when evaluating 44 45 executive function, a key neurodevelopmental outcome [13-16]. Our hypothesis is that 46 47 48 environmental exposures to mixed neurodevelopmental toxicants and non-chemical stressors will 49 50 result in increased adverse birth outcomes and poorer child neurodevelopmental trajectories. Our 51 52 research aims are to: 53 54 55 56 57 58 6 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3  Identify exposures to a complex mixture of environmental elements and toxicants, 4 5 6 including mercury, lead, cadmium, aluminum, iron, manganese, tin, and selenium and 7 8 selected pesticides through comprehensive dietary, environmental, and occupational risk 9 10 assessments, and biomarker monitoring in Surinamese pregnant women and their 11 12 13 offspring; 14 15  Assess the levels of environmental elements and toxicants in fish, produce, rice, and 16 For peer review only 17 nutraceutical compounds; 18 19 20  Determine the association between birth outcomes and levels of environmental elements 21 22 and toxicants in pregnant women; 23 24  Assess the impact of mobile health technology-enabled community health workers on birth 25 26 27 outcomes and their associations with environmental contaminants; 28 29  Determine the association between prenatal, dietary, and environmental levels of elements 30 31 and toxicants and possible neuroprotective nutraceuticals on neurodevelopment. 32

33 http://bmjopen.bmj.com/ 34 CCREOH is funded by the Fogarty International Center at the US National Institutes of Health 35 36 37 (NIH). 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Cohort description 44 45 46 Study area 47 48 49 The Republic of Suriname is located on the northeastern coast of South America, bordered 50 51 by Brazil, Guyana, French Guiana, and the Atlantic Ocean. 90% of the population of 590,549 52 53 54 people live in the capital Paramaribo and the coastal area. The remainder live in the tropical 55 56 rainforest interior (90% of the landmass). Suriname’s multi-ethnic population consists of five main 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 groups: Hindustanis (27%), Tribal (22%), Creoles (16%), Javanese (14%), and Indigenous (4%) 4 5 6 [17]. 7 8 9 10 11 12 Recruitment 13 14 The MeKiTamara study enrolled mother/child dyads potentially exposed to complex 15 16 For peer review only 17 environmental chemical- and non-chemical stressors. Main maternal and infant/child determinants 18 19 included biological determinants: maternal anthropometrics, blood pressure, hemoglobin, liver and 20 21 kidney function, fetal and postnatal growth characteristics and health status, heavy metals (blood 22 23 24 and hair), pesticides (urine), telomere length (buccal swab) and medication use; environmental 25 26 determinants: maternal and child diet and exposure history; social determinants: ethnicity, social 27 28 support, trauma, prenatal life events, maternal education, household income, employment status, 29 30 31 and marital status. Main maternal and infant/child outcomes were growth and physical 32

33 development: pregnancy complications, fetal and postnatal growth patterns, risk factors for http://bmjopen.bmj.com/ 34 35 maternal liver and renal impairment; behavior and cognitive development: infant neuromotor 36 37 development, autism spectrum disorder and neuropsychological development; childhood diseases: 38 39 40 infectious diseases, respiratory and neurological disorders; health and healthcare: impact of mobile

41 on September 25, 2021 by guest. Protected copyright. 42 health technology-enabled Community Health Workers, quality of life and health care system 43 44 utilization and comparison. 45 46 47 Pregnant women were recruited from three regions of Suriname: (1) Paramaribo, where pesticides 48 49 are primarily used for residential purposes (2) Nickerie, the major rice producing region in western 50 51 Suriname where pesticide use is abundant; and (3) the tropical rainforest interior, where mercury 52 53 54 is used in artisanal gold mining and the population is highly dependent on consumption of 55 56 contaminated fish (Figure 1) [18]. Recruitment sites included all hospitals and clinics of the 57 58 8 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 11 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Regional Health Department in Paramaribo and Nickerie and the Medical Mission Primary Health 4 5 6 Care Suriname in the interior. Women who met the inclusion criteria were identified by their 7 8 treating physician, midwife, or health assistant during regular prenatal appointments and invited 9 10 to participate. Inclusion criteria were: pregnant women 16 years or older; speaking Dutch, Sranan 11 12 Tongo, Sarnami, Saramaccan or Trio; singleton gestation; planning to deliver at one of the study 13 14 15 hospitals, prenatal clinics or midwife facilities associated with those hospitals or clinics; and 16 For peer review only 17 signed informed consent. Assent was obtained from participants 16 and 17 years of age. 18 19 20 Children were excluded for follow-up if they had: gestational age <33 weeks and/or birthweight 21 22 <2000 grams, significant medical or neurological condition, Down syndrome, hydrocephalus, 23 24 25 cerebral palsy, or significant visual or hearing impairment inconsistent with neurocognitive testing. 26 27 28 29 30 31 Follow up 32

33 http://bmjopen.bmj.com/ 34 This study was IRB approved by both the Government of Suriname and the Tulane University to 35 36 consent 1200 pregnant women and their singleton birth children. Data were collected from the 37 38 women at two timepoints (trimester 1 or 2, and 3) and 24, 36, and 48 months, while the children 39 40 are followed at birth, 12, 24, 36 and 48 months (Table 1).

41 on September 25, 2021 by guest. Protected copyright. 42 43 From December 2016 until December 2018, 1067 pregnant women were enrolled; 76 (7.1%) were 44 45 46 ineligible (Figure 2). In comparison to participants, non-participants were more often of Creole 47 48 and tribal ethnicity, lived in Paramaribo, and had lower household income. Of the 898 babies born, 49 50 827 were enrolled (92%). Initial neurodevelopmental assessments at 12 months in 491 infants are 51 52 53 being analyzed. 54 55 56 57 58 9 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 12 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Data collection during pregnancy 4 5 6 Questionnaires 7 8 9 The main categories of data collected from the women are health status, demographics, 10 11 12 reproductive health history, social support, trauma history, exposure history, depression, perceived 13 14 stress, prenatal life events, health behavior, access to prenatal care, social status and diet (Table 15 16 1). Questions on healthFor behavior peer were adapted review from the Pregnancy only Risk Assessment Monitoring 17 18 System (PRAMS), a surveillance project by state health departments and the Centers for Disease 19 20 21 Control and Prevention (CDC). Information on maternal diet was obtained by a CDC’s National 22 23 Health and Nutrition Examination Survey (NHANES) based culturally tailored dietary survey 24 25 focusing on fish and produce consumption, including frequency of intake and portion sizes. Table 26 27 28 2 presents baseline maternal characteristics. 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Measurements of heavy metals in blood and hair 35 36 37 In 400 maternal whole blood samples (200 Paramaribo, 100 Nickerie, and 100 interior 38 39 communities), we analyzed lead, mercury, cadmium, aluminum, manganese, tin, selenium, and 40

41 iron using inductively coupled plasma-mass spectrometry (ICP-MS) or cold vapor atomic on September 25, 2021 by guest. Protected copyright. 42 43 fluorescence spectrometry (CVAFS). Total mercury and methylmercury using CVAFS was 44 45 46 assessed in a small subset of women (20-Paramaribo, 20-Nickerie, and 35-interior) to specify 47 48 exposure sources. 49 50 51 Samples collected from participants in remote areas were stored at 4°C for no more than 48 hours 52 53 prior to delivery and processing at the Academic Hospital Paramaribo clinical laboratory. Blood, 54 55 56 serum, plasma, and cord blood samples were aliquoted into 2 ml plastic freezer tubes and stored 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 at -80°C. Frozen samples were shipped for analysis to the Wisconsin State Laboratory of Hygiene 4 5 6 Trace Element Research Laboratory conform formal chain-of-custody. All internal and external 7 8 QA/QC were acceptable. Maternal hair samples were taken according to protocol, stored at room 9 10 temperature in a climate-controlled room and sent to the National Zoological Collection of 11 12 Suriname/Center for Environmental Research lab at the Anton de Kom University of Suriname 13 14 15 (NZCS/CMO) for total mercury analysis using Cold Vapour Atomic Absorption Spectrometry 16 For peer review only 17 (CVAAS). 18 19 20 To date, the total number of biospecimens collected from 1067 pregnant women include whole 21 22 blood for trace elements (N=1994), whole blood collected in K EDTA anticoagulant (N=1994), 23 2 24 25 serum collected in serum separator tubes (N=1994), plasma (N=1994), urine (N=1980), buccal 26 27 swabs (N=941), and hair (N=876); cord blood (N=441), blood from heelpricks (N=269), and 28 29 buccal swabs (N=363) are collected from 710 infants. All remaining samples not (yet) analyzed 30 31 32 are archived for future targeted and untargeted analyses.

33 http://bmjopen.bmj.com/ 34 35 36 37 Measurements of pesticides in urine 38 39 40 Urine samples were analyzed (subcohort N=220) from all three study sites. Urine samples were

41 on September 25, 2021 by guest. Protected copyright. 42 43 aliquoted into 10 ml plastic tubes, stored at -20°C and shipped for analysis by the U.S. Centers for 44 45 Disease Control and Prevention’s environmental health laboratory. The analysis panel consisted 46 47 of dialkylphosphate metabolites of organophosphate pesticides (DAP’s) including 48 49 50 dimethylphosphate, diethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, 51 52 diethylthiophosphate, and diethyldithiophosphate, and a universal panel (UP) including 1 53 54 herbicide (2,4D) and its metabolite, 2,4-dichlorophenoxyacetic acid; 4 OP insecticide metabolites 55 56 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 (3,5,6-trichloro-2-pyridinol, 2-isopropyl-4-methyl-6-hydroxypyrimidine, para-nitrophenol, 4 5 6 malathion dicarboxylic acid) and 3 pyrethroid metabolites (4-fluoro-3-phenoxybenzoic acid, 3- 7 8 phenoxybenzoic acid, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid). 9 10 11 12 13 14 Data collection from birth through 48 months 15 16 For peer review only 17 At birth, a cord blood or heelprick sample is taken from infants for measurements of heavy metals. 18 19 Baseline characteristics of all live births on e.g. gender, weight, gestational age, and Apgar score 20 21 are collected (Table 2). At each subsequent data collection timepoint (Table 1), the child’s growth 22 23 24 is measured and a blood sample is taken for heavy metal measurement. In addition, the child’s 25 26 health status is obtained through questionnaires on diet and history of infectious diseases, and 27 28 respiratory and neurological disorders. 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Telomere measurements 35 36 37 Consistent evidence demonstrates that telomere length (TL) is a marker of cellular aging and links 38 39 TL to negative health outcomes including diabetes, cardiovascular disease and cognitive decline 40

41 on September 25, 2021 by guest. Protected copyright. 42 with aging. Oxidative stress and DNA methylation, among other factors, influence TL. Arsenic 43 44 and pesticide exposure have both been linked to altered DNA methylation, while evidence also 45 46 suggests a link between mercury and elevated oxidative stress [19-23]. TL may reflect multiple 47 48 49 biological pathways through which environmental toxins influence child health and development 50 51 and potentially may be an earlier indicator of exposure. DNA in infants is collected at 12, 24, 36, 52 53 and 48 months of age using buccal swabs. The average relative TL, as represented by the telomere 54 55 56 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 repeat copy number to single gene (albumin) copy number (T/S) ratio, will be determined with 4 5 6 monochrome multiplex quantitative real-time PCR using a BioRad CFX96 [24]. 7 8 9 10 11 12 Behavior and cognitive development 13 14 Neurodevelopmental assessments include infant mental and motor development and behavior at 15 16 For peer review only 17 12 months using the BAYLEY scale of infant development as well as assessment of cognitive and 18 19 social-emotional development at 36 months, and measurement of executive function at 48 months 20 21 (Table 1). 22 23 24 25 26 27 Data management & statistical plan 28 29 30 Informed consent was required for successful recruitment and where indicated assent was 31 32 obtained. The CCREOH field team was trained in administering all questionnaires. Data from

33 http://bmjopen.bmj.com/ 34 35 administered questionnaires are recorded on secure, encrypted iPads, uploaded and managed using 36 37 REDCap electronic data management system [25]. As outlined earlier, biospecimens (blood, cord 38 39 blood, urine, buccal swabs) are collected at all study areas and then subsequently transported to 40

41 on September 25, 2021 by guest. Protected copyright. 42 the study’s laboratory at the Academic Hospital in Paramaribo. Hair samples were analyzed for 43 44 the presence of mercury at the NZCS/CMO Environmental laboratory. Fish samples were collected 45 46 in areas downstream and upstream of selected villages, including gold mining areas, and analyzed 47 48 49 for mercury. Biomarker assessments not possible in Suriname are conducted at US specialty 50 51 laboratories. 52 53 54 55 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 REDCap serves as the study’s integrated data management system. Data collected through iPads 4 5 6 by the field team are uploaded in the training site of REDCap for data cleaning prior to integrating 7 8 questionnaire and biospecimen data. Data records are maintained to see trends on source of errors; 9 10 duplicate records are scrubbed, and accuracy is validated through cross-checks with original data 11 12 files and medical records. A comprehensive biospecimen tracking system is nearing completion to 13 14 15 track analyzed samples and the overall study biospecimen repository. The data management team 16 For peer review only 17 develops de-identified data files upon request of CCREOH investigators as specified by a 18 19 standardized data request form. Statistical analyses are tailored to the specific research question. 20 21 22 CCREOH data have been presented at multiple local, regional, and international conferences, 23 24 including the Caribbean Public Health Agency, the International Society of Environmental 25 26 Epidemiology, the Consortium for Universities in Global Health, the American Public Health 27 28 29 Association and at scientific meetings convened by the US NIH. 30 31 32

33 http://bmjopen.bmj.com/ 34 35 Patient and public involvement 36 37 The CCREOH/ MeKiTamara cohort study was preceded by a pilot fish assessment study as well 38 39 40 as initial hair mercury assessment among communities in the interior which required approval

41 on September 25, 2021 by guest. Protected copyright. 42 from the village Chiefs or Tribal captain. This community engagement component provided us a 43 44 rich foundation to build on as we developed the research questions. The initial study design was 45 46 47 revised based on guidance from an External Scientific Advisory Board (EAB) and multi- 48 49 stakeholder Community Advisory Board (CAB; consisting of respected persons in society 50 51 representing organizations such as from the interior tribal and indigenous peoples, midwives, 52 53 54 general practitioners and green wildlife), all participating hospitals and clinicians, and 55 56 representatives from the Ministry of Health, the US Embassy, the Pan American Health 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Organization and the Caribbean Public Health Agency (CARPHA). For example, the CAB was 4 5 6 instrumental in urging the study team to lower the inclusion age to 16 years since many women in 7 8 the interior conceive at an earlier age. All stakeholders are convened annually to inform and give 9 10 updates about the study progress and results, and to seek advice on future directions and 11 12 dissemination of results. The study team is actively working with the EAB and the CAB on a 13 14 15 collaborative translation and dissemination strategy. However, a participant with neurotoxicant 16 For peer review only 17 levels of imminent public health concern is contacted by the study coordinator and asked to repeat 18 19 the test for confirmation. If the level remains of concern the participant is referred to her attending 20 21 22 physician according to the study’s formal health care provider referral protocol. Children that are 23 24 found to have a neurodevelopmental delay are referred to the child psychologist for further 25 26 evaluation. Participants and the general public were invited to multiple health fairs, meetings and 27 28 29 conferences, where the researchers discussed the study in a broader context, provided updates on 30 31 the latest results and answered questions. Additionally, a Facebook account was created for study 32

33 participants and others interested in this study, with general information on exposure to heavy http://bmjopen.bmj.com/ 34 35 metals and pesticides as well as the opportunity to ask questions of the research team and provide 36 37 38 feedback about the study. 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 Findings to date 45 46 47 Available results include total mercury concentrations in hair from pregnant women and urinary 48 49 50 pesticide metabolites that were measured in a subset of 220 women. Results on heavy metals in 51 52 blood, telomeres and neurodevelopmental assessments are pending. 53 54 55 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Biological specimens 4 5 6 Results for median total mercury concentrations in hair from pregnant women from Paramaribo 7 8 9 (N=522) were 0.64 µg/g hair (interquartile ranges (IQR) 0.36-1.09; minimum 0.00 – maximum 10 11 7.12), from Nickerie (N=176) 0.73 µg/g (IQR 0.45-1.05; 0.00-5.79) and the interior (N=178) 1.92 12 13 µg/g (IQR 1.92-7.39; 0.38-18.20). Pregnant women from the interior are exposed to high levels of 14 15 mercury compared to women in the coastal area and thus fetal exposures are expected to be high. 16 For peer review only 17 18 Median concentrations are well above health action levels of 1.1 µg/g hair for mercury [26]. Most 19 20 of these women are primarily exposed to methylmercury from consuming fish that are 21 22 contaminated by the artisanal gold-mining activities in those areas [18,27]. Other possible sources 23 24 25 of exposure to mercury may include active participation in or living very close to gold mining 26 27 activities. 28 29 30 Urinary pesticide metabolites measured in a subset of 220 women are presented in Table 3. 31 32 Concentrations varied geographically among participants: in Nickerie, participants’ pyrethroid and

33 http://bmjopen.bmj.com/ 34 35 herbicide metabolite concentrations were higher compared to Paramaribo and the interior, 36 37 probably because of widespread agricultural use. Organophosphate metabolite concentrations 38 39 were higher in Paramaribo compared to Nickerie and the interior, which could be attributed to 40

41 residential use and more intense mosquito control. on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Prenatal depression and perceived stress 48 49 50 Depression and prenatal stress in 722 participants were assessed using the standardized Edinburgh 51 52 53 Postnatal Depression Scale (EPDS) (cut off ≥12 for probable depression) and Cohen Perceived 54 55 Stress Scale (cut off ≥20 for high stress) respectively. One in 4 (24.9%) pregnant study participants 56 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 potentially exposed to mercury had EPDS scores indicative of probable depression. Three in ten 4 5 6 participants had high stress levels and nearly half of these women had probable depression. 7 8 9 10 11 Dietary exposure to pesticides 12 13 An assessment of Surinamese agricultural produce done by our group found pesticide residues 14 15 exceeding European Union maximum residue limits, including prohibited- worldwide endosulfan 16 For peer review only 17 18 and lindane in the leafy vegetable tannia, Xanthosoma brasiliense [27]. Tannia is commonly used 19 20 in baby food preparation in Suriname. 21 22 23 24 25 Dietary exposures to mercury 26 27 28 One in four pregnant women in our cohort, and predominantly those living in the interior who rely 29 30 31 heavily on local freshwater fish, have hair mercury levels exceeding those considered safe by the 32

33 US and the WHO. The dietary questionnaire (n=990) showed overall fish consumption of 96.1%. http://bmjopen.bmj.com/ 34 35 Previous environmental assessments of most frequently consumed carnivorous fish by interior 36 37 participants, Hoplias aimara, Serrasalmus rhombeus, and Cichla ocellaris, showed high levels of 38 39 40 mercury [28]. Respective consumption of these three carnivorous species was 44.4%, 19.3%, and

41 on September 25, 2021 by guest. Protected copyright. 42 26.3%. Carnivorous fish consumption was greater among the subcohort (N=123) living in the 43 44 interior: 89.4%, 67.5%, and 74.8%, respectively. 45 46 47 Polyunsaturated omega-3 fatty acids (PUFA-3) docosahexaenoic acid and eicosapentaenoic acid 48 49 50 from fish consumption offer neuroprotective benefits during prenatal and pediatric development 51 52 which could potentially counteract mercury neurotoxicity. To better inform fish consumption risk, 53 54 fatty acids in fish muscle tissue were measured in samples (N=5 per species) of five freshwater 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 and three marine fish species: freshwater species had higher levels of linoleic acid (2.0 vs. 0.2 4 5 6 mg/g), alpha-linoleic acid (0.4 vs. 0.1 mg/g), arachidonic acid (3.0 vs. 1.5 mg/g), omega-6 fatty 7 8 acids (6.5 vs. 2.3 mg/g) and lower levels of eicosapentaenoic acid (0.8 vs. 1.9 mg/g) compared to 9 10 marine species. Frequent consumption of Hoplias aimara, a freshwater fish with high mercury and 11 12 low PUFA-3 content, is likely to increase mercury exposure whereas consumption of Plagioscion 13 14 15 squamosissimus, a freshwater fish with high mercury and high PUFA-3, may have nutritional 16 For peer review only 17 benefits that outweigh mercury neurotoxicity. While freshwater species are a good source of these 18 19 nutrients, corresponding mercury content must be assessed and considered as well. 20 21 22 23 24 25 Mercury exposure and birth outcomes 26 27 28 Data from 763 pregnant women were analyzed for total hair mercury and associated with birth 29 30 31 outcomes. Adverse birth outcomes included preterm birth (PTB<37 weeks), low birthweight 32

33 (LBW<2500g) and low Apgar score (AS<7 at 5 minutes). 27.3% of the women had elevated total http://bmjopen.bmj.com/ 34 35 mercury hair levels that exceeded the USEPA action level, 23.8% had adverse birth outcomes, 36 37 18.4% PTB, 14.7% LBW and 5.1% had low AS. There was no statistically significant association 38 39 40 between hair mercury level and adverse birth outcomes. Neurodevelopmental assessment of the

41 on September 25, 2021 by guest. Protected copyright. 42 children is ongoing. 43 44 45 46 47 48 Strengths and limitations 49 50 51 The CCREOH-MeKiTamara environmental epidemiologic study is the first to examine the 52 53 potential impact of complex environmental exposures on maternal and child health in Suriname 54 55 56 by a multi-disciplinary team of scientists The study’s strengths include: 1) addressing two high 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 priority public health threats: the impact of mercury exposure from artisanal gold mining, and 4 5 6 pesticide exposure associated with agricultural practices; 2) inclusion in the cohort of two 7 8 vulnerable subpopulations: pregnant women and children aged <5 years; 3) the longitudinal follow 9 10 up with children up to 48 months with several built-in timepoints to assess neurodevelopmental 11 12 outcomes; and 4) a biospecimen bank of approximately 12,000 samples, providing the opportunity 13 14 15 for future analyses within the cohort. Moreover, a linked research training grant facilitates the 16 For peer review only 17 training of nine Surinamese PhD candidates with dissertation research embedded in the study, 18 19 thereby building critical in-country environmental health research capacity. Research 20 21 22 dissemination throughout the Caribbean region is completed in collaboration with the Caribbean 23 24 Public Health Agency. 25 26 27 In terms of initial challenges, the interior subcohort is logistically difficult to reach, resulting in 28 29 delayed recruitment and data collection. Additional team support onsite facilitated recruitment of 30 31 32 the full subcohort (N=200). Initial delays in transporting interior biospecimens rendered some

33 http://bmjopen.bmj.com/ 34 early cord blood specimens not analyzable. This has been corrected by collecting an additional 35 36 heelprick sample at birth. 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Future plans 44 45 46 Since the most likely source of exposure to mercury in pregnant women in Suriname is through 47 48 consumption of locally harvested fish, fish consumption advisories may aid in reducing exposure 49 50 to environmental contaminants. Recent additional funding allows us to examine potentially 51 52 53 beneficial neuroprotective factors such as selenium and dietary polyunsaturated fatty acids in fish 54 55 that may counter the neurotoxic effects of mercury. To address the large scale and poorly regulated 56 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 use of pesticides, we are developing a pesticide literacy scale through a recently funded fellowship. 4 5 6 Cohort-wide, we will undertake a comprehensive assessment of the neurodevelopmental outcomes 7 8 of the children, and create a robust, sharable data management system of biospecimen and non- 9 10 biospecimen data building on the current REDCap database. 11 12 13 14 15 16 For peer review only 17 Acknowledgements 18 19 20 We would like to thank our recruiters Jeetendra Jitan, Nishaira Doerga, Gimradj Dwarka, Sue-Ann 21 22 Sitaram, Devika Kalika, the Community Health Workers from Nickerie Lilian Singotiko, Henna 23 24 Mawo, Karien Jodiwongso, Karishma Bhondoe, Jay Kalicharan, Bhairam Goberdan, Azeez 25 26 27 Khodabaks, the midwives and laboratory personnel of the Academic Hospital Paramaribo, 28 29 Diakonessenhuis Hospital, St. Vincentius Hospital, ‘s-Lands Hospital, Mungra Medical Centre, 30 31 Regional Health Service clinics and Medical Mission Primary Health Care Suriname, the National 32

33 http://bmjopen.bmj.com/ 34 Zoological Collection of Suriname/Center for Environmental Research staff, Drs. Sila 35 36 Kisoensingh, Richenel Ellecom and Melissa Middleton for their help during the pediatric 37 38 neurodevelopment assessments, Arioene Vreedzaam for providing the study site map, and the 39 40 members of the External Advisory Board and Community Advisory Board for their guidance and

41 on September 25, 2021 by guest. Protected copyright. 42 43 valuable advice. 44 45 46 47 48 49 Collaborators 50 51 52 53 We welcome collaboration with fellow researchers working on similar projects. Specific research 54 55 proposals can be sent directly to the Principal Investigators Drs Wilco Zijlmans 56 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 ([email protected]) and Maureen Lichtveld ([email protected]). 4 5 6 Areas of collaboration include further environmental and biomarkers assessment of arsenic 7 8 exposure, occupational health risk assessments associated with mercury and pesticide exposure, 9 10 and exposomic analyses including metabolomic assessments. 11 12 13 14 15 16 Author ContributionsFor peer review only 17 18 19 MYL, JKW, HHC, EWH, AS, SSD and WCWRZ designed and established the cohort. MSMO, 20 21 ADHM, EWH and AG participated in the design of the questionnaires. WCWRZ, MSMO, ED, 22 23 WBH, GKB, RR and MYL are responsible for the continued management of the cohort. AG, GKB, 24 25 RR, AG and FAW are actively involved with recruitment of participants and collection of non- 26 27 28 biospecimen data and biospecimens. JC, JR, PEO and GAL store, archive and analyze the 29 30 biomarker samples. ADHM, AS, MSMO, AG, MYL and WCWRZ are responsible for data 31 32 management. SSD, AWEG, RR and EB manage neurodevelopmental testing of babies. JKW, LFS,

33 http://bmjopen.bmj.com/ 34 35 CSA, SSD interpret all results. WCWRZ, JKW, HHC and MYL drafted and edited the manuscript. 36 37 All authors critically reviewed and approved the final manuscript. 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Funding 44 45 46 Research reported in this publication was supported by the Fogarty International Center of the 47 48 National Institutes of Health under Award Numbers: U01TW010087 and U2RTW010104. The 49 50 51 content is solely the responsibility of the authors and does not necessarily represent the official 52 53 views of the National Institutes of Health. 54 55 56 57 58 21 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Competing interests 4 5 6 7 None declared 8 9 10 11 12 13 Patient consent for publication 14 15 16 Obtained For peer review only 17 18 19 20 21 22 23 Ethical approval 24 25 26 This study was IRB approved by both the Government of Suriname and the Tulane University, 27 28 New Orleans, Louisiana, USA, to consent 1200 pregnant women and their singleton birth 29 30 31 children. 32

33 http://bmjopen.bmj.com/ 34 35 36 37 Provenance and peer review 38 39 40 Not commissioned; externally peer reviewed.

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Data sharing statement 48 49 50 MekiTamara cohort data will be made publicly available upon publication of the results in 51 52 scientific articles. Questionnaire, registry and biospecimen data could be made accessible in de- 53 54 55 identified form after an application process that includes submission of a research plan that will 56 57 58 22 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 undergo evaluation by the CCREOH scientific board and relevant research ethics committees. 4 5 6 Currently, only part of the data is available since data collection is ongoing. 7 8 9 10 11 12 References 13 14 15 16 1. Lichtveld MY, For Hawkins peer WB, Ouboter review PE et al. A only One Health approach to interdict 17 18 environmental health threats in Suriname. Ann Glob Health 2016;82:444. 19 20 21 2. Silbergeld EK, Patrick TE. Environmental exposures, toxicologic mechanisms, and adverse 22 23 pregnancy outcomes. Am J Obstet Gynecol 2005;192:S11-21. 24 25 26 Ferguson KK, O'Neill MS, Meeker JD. Environmental contaminant exposures and preterm 27 3. 28 29 birth: a comprehensive review. J Toxicol Environ Health B Crit Rev 2013;16:69-113. 30 31 32 4. Wigle DT, Arbuckle TE, Turner MC et al. Epidemiologic evidence of relationships between

33 http://bmjopen.bmj.com/ 34 reproductive and child health outcomes and environmental chemical contaminants. J Toxicol 35 36 Environ Health B Crit Rev 2008;11:373-517. 37 38 39 Lewis M, Worobey J, Ramsay DS et al. Prenatal exposure to heavy metals: effect on childhood 40 5.

41 on September 25, 2021 by guest. Protected copyright. 42 cognitive skills and health status. Pediatrics 1992;89:1010-5. 43 44 45 6. Burns CJ, McIntosh LJ, Mink PJ, Jurek AM, Li AA. Pesticide exposure and 46 47 neurodevelopmental outcomes: Review of the epidemiologic and animal studies. J Toxicol 48 49 Environ Health B Crit Rev 2013;16:127-283. 50 51 52 53 54 55 56 57 58 23 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 26 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 7. Eskenazi B, Kogut K, Huen K et al. Organophosphate pesticide exposure, PON1, and 4 5 6 neurodevelopment in school-age children from the CHAMACOS study. Environ Res 7 8 2014;134C:149-57. 9 10 11 8. Furlong MA, Engel SM, Barr DB, Wolff MS. Prenatal exposure to organophosphate pesticides 12 13 and reciprocal social behavior in childhood. Environ Int 2014;70:125-31. 14 15 16 9. Quirós-Alcalá L,For Alkon AD,peer Boyce WT review et al. Maternal onlyprenatal and child organophosphate 17 18 19 pesticide exposures and children's autonomic function. Neurotoxicology 2011;32:646-55. 20 21 22 10. Rosas LG, Eskenazi B. Pesticides and child neurodevelopment. Curr Opin Pediatr 23 24 2008;20:191-7. 25 26 27 11. Belles M, Albina ML, Sanchez DJ et al. Interactions in developmental toxicology: effects of 28 29 concurrent exposure to lead, organic mercury, and arsenic in pregnant mice. Arch Environ 30 31 32 Contam Toxicol 2002;42:93-8.

33 http://bmjopen.bmj.com/ 34 35 12. Callahan MA, Sexton K. If cumulative risk assessment is the answer, what is the question? 36 37 Environ Health Perspect 2007;115:799-806. 38 39 40 13. Golding J, Rai D, Gregory S et al. Prenatal mercury exposure and features of autism: a

41 on September 25, 2021 by guest. Protected copyright. 42 prospective population study. Mol Autism 2018;9:30. 43 44 45 14. Valent F, Mariuz M, Bin M et al. Associations of prenatal mercury exposure from maternal 46 47 48 fish consumption and polyunsaturated fatty acids with child neurodevelopment: a prospective 49 50 cohort study in Italy. J Epidemiol 2013;23:360-70. 51 52 53 54 55 56 57 58 24 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 27 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 15. Sapbamrer R, Hongsibsong S. Effects of prenatal and postnatal exposure to organophosphate 4 5 6 pesticides on child neurodevelopment in different age groups: a systematic review. Environ 7 8 Sci Pollut Res Int 2019;26:18267-90. 9 10 11 16. Wang J, Wu W, Li H et al. Relation of prenatal low-level mercury exposure with early child 12 13 neurobehavioral development and exploration of the effects of sex and DHA on it. Environ Int 14 15 2019;126:14-23. 16 For peer review only 17 18 19 17. Results Eighth Population and Housing Census in Suriname (Volume 1) Demographic and 20 21 Social Characteristics and Migration. General Bureau of Statistic Studies in Suriname (ABS) 22 23 2013. 24 25 26 18. Ouboter PE, Landburg G, Satnarain GU et al. Mercury Levels in Women and Children from 27 28 29 Interior Villages in Suriname, South America. Int J Environ Res Public Health 2018;15:1007. 30 31 32 19. Frenck RW, Blackburn E, Shannon K. The rate of telomere sequence loss in human leukocytes

33 http://bmjopen.bmj.com/ 34 varies with age. Proc Natl Acad Sci 1998;95:5607-10. 35 36 37 20. Lung FW, Chen NC, Shu BC. Genetic pathway of major depressive disorder in shortening 38 39 telomeric length. Psychiatr Genet 2007;17:195-9. 40

41 on September 25, 2021 by guest. Protected copyright. 42 21. Von Zglinchi T. Oxidative Stress shortens telomeres. Trends Biochem Sci 2002;27:339-44. 43 44 45 22. Gonzalo S, Jaco I, Fraga MF et al. DNA methyltransferases control telomere length and 46 47 48 telomere recombination in mammalian cells. Nat Cell Biol 2006;8:416-24. 49 50 51 23. Damjanovic AK, Yang Y, Glaser R et al. Accelerated telomere erosion is associated with a 52 53 declining immune function of caregivers of Alzheimer's disease patients. J Immunol 54 55 2007;179:4249-54. 56 57 58 25 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 28 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 24. Cawthon RM. Telomere length measurement by a novel monochrome multiplex qualitative 4 5 6 PCR method. Nucleic Acids Res 2009;37:e21. 7 8 9 25. Harris PA, Taylor R, Minor BL et al. The REDCap consortium: Building an international 10 11 community of software partners, J Biomed Inform 2019;95:1-10. doi: 12 13 10.1016/j.jbi.2019.103208 14 15 16 26. Mercury Study For Report topeer Congress Volumereview V: Health only Effects of Mercury and Mercury 17 18 19 Compounds. Volume 5. United States Environmental Protection Agency; Washington, DC, 20 21 USA: 1997;1–348. 22 23 24 27. Abdoel Wahid F, Wickliffe J, Wilson M et al. Presence of pesticide residues on produce 25 26 cultivated in Suriname. Environ Monit Assess 2017;189:303. 27 28 29 28. Ouboter PE, Landburg GA, Quik JHM, Mol JHA, van der Lugt F. Mercury Levels in Pristine 30 31 32 and Gold Mining Impacted Aquatic Ecosystems of Suriname, South America. Ambio

33 http://bmjopen.bmj.com/ 34 2012;41:873-82. 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 26 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 29 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 Table 1. Assessments completed by the CCREOH-MeKiTamara Cohort with timeline 6 7 Assessments Trimester Birth 12 24 36 48 8 mos mos mos mos 9 1st/2nd 3rd 10 Mother 11 Obstetric history ● ● 12 Demographics ● 13 Residency ● ● 14 Anthropometrics ● ● 15 Marital status ● 16 For peer review only 17 Ethnicity ● 18 Occupation ● 19 Education ● 20 Income ● 21 Household composition ● 22 Maternity care ● ● 23 Questionnaires 24 SF 36 Health Survey ● ● 25 Social Support List ● 26 Brief Trauma Interview ● ● ● ● ● 27 Cohen Perceived Stress Scale ● ● ● ● 28 Edinburgh Depression Scale ● ● ● ● ● 29 ASSIST V3.0 ● ● ● ● ● 30 Exposure History ● 31 Prenatal Life Events Scale ● ● 32 Subjective Social Status ● ●

33 http://bmjopen.bmj.com/ Dietary Assessment ● ● 34 35 Family Environment Scale ● ● ● 36 Parenting Stress Index ● ● ● 37 Biological samples 38 Hair ● 39 Blood ● ● 40 Urine ● ● Buccal swab ● 41 on September 25, 2021 by guest. Protected copyright. 42 43 Infant at birth 44 Mode of delivery ● 45 Cord blood sample ● 46 Birth outcomes ● 47 48 Child 49 Physical examination ● ● ● ● 50 Questionnaires 51 General Health Questionnaire ● ● ● ● 52 M-CHAT ● 53 Child Behavior Checklist ● ● 54 55 Neurodevelopmental tests 56 Bayley ● 57 58 27 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 30 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 WIPPSI ● 4 CANTAB ● 5 Biological samples 6 Buccal swab ● ● ● ● 7 Blood ● ● ● 8 9 Notes: ASSIST V3.0 = Alcohol, Smoking and Substance Involvement Screening Test Version 3, Subjective 10 Social Status = MacArthur Scale of Subjective Social Status, M-CHAT = Modified Checklist for Autism 11 in Toddlers, Bayley = Bayley Scales of Infant Development, WIPPSI = Wechsler Preschool and Primary 12 Scale of Intelligence, CANTAB = Cambridge Neuropsychological Test Automated Battery. 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 28 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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1 2 3 Table 2. Maternal and infant characteristics 4 5 Maternal characteristics (N=1067) N % Infant characteristics (N=898) N % 6 Age at intake Gender 7 8 16-19 years 133 12.4 Male 461 52.3 9 20-24 years 239 22.4 Female 421 47.7 10 25-29 years 280 26.2 Missing 16 1.8 11 30-34 years 252 23.6 12 35-39 years For129 peer12.1 reviewBirth status only 13 40+ years 34 3.2 Live birth 863 96.1 14 Missing 0 0.0 Stillbirth 35 3.9 15 16 Missing 0 0.0 17 Parity

18 0 previous live births 344 33.0 Birthweight http://bmjopen.bmj.com/ 19 1 previous live birth 298 28.6 Low birthweight (<2500 g) 113 12.9 20 2 previous live births 174 16.7 Normal birthweight (≥2500g) 760 87.1 21 3 previous live births 102 9.8 Missing 25 2.8 22 23 4+ previous live births 124 11.9 24 Missing 25 2.3 Gestational age 25 Miscarriages 10 1.1

26 Ethnicity Very preterm births (22+0-32+6 weeks) on September 25, 2021 by guest. Protected copyright. 36 4.1 27 Creole 245 23.0 Moderately preterm births (33+0-36+6 weeks) 93 10.7 28 Hindustani 222 20.8 Term births (≥37+0 weeks) 733 84.1 29 Indigenous 149 14.0 Missing 26 2.9 30 31 Javanese 94 8.8 32 Tribal 223 20.9 Apgar score at 5 minutes 33 Mixed 128 12.0 0 - 6 46 5.2 34 Other 5 0.5 7 - 10 832 94.8 35 Missing 1 0.1 Missing 20 2.2 36 37 Educational level 38 39 No or primary 223 20.9 40 Lower vocational/secondary 375 35.2 41 Upper vocational/secondary 302 28.3 42 43 29 44 45 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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1 2 3 Tertiary 166 15.6 4 Missing 1 0.1 5 6 7 Household income SRD* 8 <1500 321 33.2 9 1500-2999 330 34.1 10 3000-4999 210 21.7 11 5000+ 106 11.0 12 Missing For100 peer 9.4 review only 13 14 15 Marital status 16 Married or living with partner 922 86.8 17 Not married/not living with 140 13.2

18 partner http://bmjopen.bmj.com/ 19 Missing 5 0.5 20 21 * SRD = Surinamese dollar, equivalent to 0.13 USD. 22 23 24 25

26 on September 25, 2021 by guest. Protected copyright. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 30 44 45 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Table 3: Urinary pesticide metabolites presented as arithmetic mean (± standard deviations) and median 4 * 5 concentrations. Ranges from minimum (LOD -corrected) to maximum are presented as well as the LODs and the 6 percentage of samples below the LOD. Total sample size is 218 7 8 Analyte Mean (Standard Deviation) Median Range LOD % Below 9 (ug/L) (ug/L) LOD 10 (ug/L) (ug/L) 11 12 13 Chemical Class: Phenoxy Acetic Acid Herbicide 14 15 2, 4- Dichlorophenoxyacetic acid 0.61 (0.92) 0.28 0.11-7.50 0.15 35.32 16 For peer review only 17 Chemical Class: Organophosphate Insecticide 18 19 Malathion dicarboxylic acid 0.82 (3.55) 0.35 0.35-51.68 0.50 76.61 20 3,5,6-Trichloro-2-pyridinol 1.45 (3.73) 0.56 0.07-46.70 0.10 22.02 21 22 2-isopropyl-4-methyl-6- 1.28 (4.32) 0.31 0.07-52.00 0.10 16.51 23 24 hydroxypyrimidine 25 26 para-Nitrophenol 1.28 (5.10) 0.59 0.07-73.30 0.10 40.92 27 Diethyldithiophosphate 0.07 (0.01) 0.07 0.07-0.14 0.10 98.62 28 29 Diethylphosphate 2.07 (5.12) 0.77 0.07-52.50 0.10 11.93 30 31 Dimethyldithiophosphate 0.36 (1.20) 0.07 0.07-11.70 0.10 72.48 32

33 Dimethylphosphate 1.90 (3.42) 1.06 0.07-42.50 0.10 3.67 http://bmjopen.bmj.com/ 34 35 Dimethylthiophosphate 2.64 (3.74) 1.22 0.07-25.00 0.10 5.05 36 37 Diethylthiophosphate 1.16 (4.54) 0.33 0.07-58.20 0.10 18.35 38 39 Chemical Class: Pyrethroid Insecticide 40 4-fluoro-3-phenoxybenzoic acid 0.07 (0.02) 0.07 0.07-0.29 0.10 98.17

41 on September 25, 2021 by guest. Protected copyright. 42 43 3-phenoxybenzoic acid 1.47 (3.88) 0.71 0.07-50.70 0.10 15.14 44 45 trans-3-(2,2-Dichlorovinyl)-2,2- 1.63 (3.32) 0.42 0.42-27.20 0.60 60.55 46 dimethylcyclopropane carboxylic 47 acid 48 49 *LOD = Limit of Detection. 50 51 52 53 54 55 56 57 58 31 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 Figure 1. CCREOH-MeKiTamara study area with recruitment sites from three regions of 51 Suriname: 1) the capital Paramaribo 2) Nickerie, the major rice-producing region in western 52 Suriname and 3) the tropical rainforest interior where mercury is used in artisanal gold mining. 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 36 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 Pregnant women recruited N=1067 7 Paramaribo N=724 8 Nickerie N=182 9 Interior N=161 10 Not eligible N=76 11 o Abortion/miscarriage N=37 12 o Multiple pregnancies N=2 13 o Other reasons (moved 14 abroad, unreachable, family 15 issues/interruption, consent 16 For peer review onlywithdrawn) N=37 17 Eligible for follow up N=993 18 19 20 21 22 23 24 25 26 Births to date N=898 27 Excluded N=71 28 29 o Stillbirths/neonatal deaths N=35 30 o Premature births (<33 weeks) N=36 31 32

33 http://bmjopen.bmj.com/ 34 Infants/children for postnatal follow up N=827 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 Neurodevelopmental assessment at 12 months to date N=491 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 37 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Figure 2. Flowchart with participant enrolment. 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

Manuscript ID bmjopen-2019-034702.R1

Article Type: Cohort profile

Date Submitted by the 19-Mar-2020 Author:

Complete List of Authors: Zijlmans, Wilco; Academic Hospital Paramaribo, Scientific Research Center Suriname ; Wickliffe, Jeffrey; Tulane University, Global Environmental Health Sciences Hindori-Mohangoo, Ashna; Perisur (Perinatal Interventions Suriname) Foundation MacDonald-Ottevanger, Sigrid; Academic Medical Center, Amsterdam, The Netherlands Ouboter, Paul; Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname

Primary Subject Occupational and environmental medicine Heading:

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1 2 3 Secondary Subject Heading: Epidemiology, Global health, Public health 4 5 Community child health < PAEDIATRICS, Developmental neurology & 6 Keywords: neurodisability < PAEDIATRICS, PERINATOLOGY, PUBLIC HEALTH, 7 Epidemiology < TROPICAL MEDICINE, Nutrition < TROPICAL MEDICINE 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

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1 2 3 Title: Cohort profile: The Caribbean Consortium for Research in Environmental and 4 5 6 Occupational Health (CCREOH) MeKiTamara Cohort Study: Influences of complex 7 8 environmental exposures on maternal and child health in Suriname 9 10 11 12 13 14 15 Author list 16 For peer review only 17 18 Wilco C.W.R. Zijlmans*1,2,3, Jeffrey K. Wickliffe2, Ashna D. Hindori-Mohangoo2,4, M. Sigrid 19 20 MacDonald-Ottevanger1,5, Paul E. Ouboter2,6,7, Gwendolyn A. Landburg6, John Codrington8, 21 22 8 3,9 3,10 1,3 23 Jimmy Roosblad , Gaitree K. Baldewsingh , Radha Ramjatan , Anisma Gokoel , Firoz 24 25 Abdoel Wahid1,2, Lissa Fortes Soares2, Cecilia S. Alcala2, Esther Boedhoe1, Antoon W.E. 26 27 Grünberg11, William B. Hawkins2,12, Arti Shankar2, Emily W. Harville13, Stacy S. Drury14, Hannah 28 29 H. Covert2, Maureen Y. Lichtveld2 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 Affiliations 37 38 39 1 Scientific Research Center Suriname, Academic Hospital Paramaribo, Paramaribo, Suriname 40

41 on September 25, 2021 by guest. Protected copyright. 42 2 Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, 43 44 Tulane University, New Orleans, LA 70112, United States of America 45 46 47 3 Faculty of Medical Sciences, Anton de Kom University of Suriname, Paramaribo, Suriname 48 49 50 4 Perisur (Perinatal Interventions Suriname) Foundation, Paramaribo, Suriname 51 52 53 5 Department of Clinical Chemistry Medical Microbiology, Academic Medical Center, 54 55 56 Amsterdam, The Netherlands 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 6 National Zoological Collection of Suriname/Center for Environmental Research, Anton de Kom 4 5 6 University of Suriname, Paramaribo, Suriname 7 8 7 9 Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname 10 11 8 12 Department of Clinical Chemistry, Academic Hospital Paramaribo, Paramaribo, Suriname 13 14 9 Medical Mission Primary Health Care Suriname, Paramaribo, Suriname 15 16 For peer review only 17 10 Regional Health Service Nickerie, Suriname 18 19 20 11 Department of Public Health, Ministry of Health, Paramaribo, Suriname 21 22 23 12 Department of Health Policy, Vanderbilt University Medical Center, Nashville, TN, United 24 25 26 States of America 27 28 13 29 Department of Epidemiology, Tulane University School of Public Health and Tropical 30 31 Medicine, New Orleans, LA, United States of America 32

33 http://bmjopen.bmj.com/ 34 14 Tulane University School of Medicine, New Orleans, LA, United States of America 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 *Corresponding author. Scientific Research Center Suriname, Academic Hospital Paramaribo, 47 48 Picornistreet 11, Paramaribo, Suriname. Email: [email protected] 49 50 51 52 53 54 55 56 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 7 Abstract 8 9 10 Purpose: The Caribbean Consortium for Research in Environmental and Occupational Health 11 12 13 (CCREOH) MeKiTamara prospective environmental epidemiologic cohort study addresses the 14 15 16 potential adverse impactFor of peer complex chemical review and non-chemical only environmental exposures in 17 18 19 mother/child dyads in Suriname. The study determines associations between levels of 20 21 22 environmental elements and toxicants in pregnant women and birth outcomes and 23 24 25 neurodevelopment in the child cohort. Environmental assessments include fish, produce, and rice. 26 27 28 Participants: From December 2016 to date, 1067 pregnant women and 827 babies were enrolled. 29 30 31 Data collection occurs at seven study time points: twice during pregnancy and at birth, 12, 24, 36, 32

33 http://bmjopen.bmj.com/ 34 and 48 months. 35 36 37 Findings to date: Four out of ten women, and predominantly those living in Suriname’s interior, 38 39 40 had hair mercury (Hg) levels exceeding values considered safe by international standards. Findings

41 on September 25, 2021 by guest. Protected copyright. 42 43 from a dietary survey indicated that 96.1% of women ate fish potentially contaminated with 44 45 46 mercury 3-5 times per week, while 89% frequently consumed a vegetable called tannia, samples 47 48 49 of which showed the presence of worldwide banned pesticides. One in four pregnant study 50 51 52 participants had high Edinburgh Postnatal Depression Scale scores indicative of probable 53 54 55 depression. 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 Future plans: Total blood mercury concentrations were elevated in interior women for whom fish 7 8 9 consumption is the primary source of mercury exposure. Culturally tailored fish consumption 10 11 12 advisories are in development. Research is ongoing to examine effects of potentially beneficial 13 14 15 neuroprotective factors in fish that may counter neurotoxic effects of mercury. A pesticide literacy 16 For peer review only 17 18 assessment in pregnant Surinamese women is in progress. Long-term effects of exposures to 19 20 21 toxicant mixtures in infants/toddlers are being evaluated through pediatric neurodevelopmental 22 23 assessments up to four years of age. Telomere length measurements of the mothers and children 24 25 26 as an indicator of prenatal exposure to environmental toxins are ongoing, as are the analyses of 27 28 29 biospecimen samples. 30 31 32

33 http://bmjopen.bmj.com/ 34 35 Keywords environmental exposures, mercury, metal mixtures, pregnant women, pediatric 36 37 38 neurodevelopment, Suriname 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 Word count 3997 (excl. references, acknowledgements, tables & figures) 45 46 47 48 49 50 Strengths and limitations of this study 51 52 53  The study addresses two high priority public health threats in Suriname and neighboring 54 55 56 countries in the Guiana Shield: the impact of mercury exposure from artisanal gold mining 57 58 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 and pesticide exposure associated with agricultural practices in two vulnerable 4 5 6 subpopulations: pregnant women and children younger than 5 years of age. 7 8  The longitudinal follow up of children to 48 months provides several timepoints to assess 9 10 neurodevelopmental outcomes. 11 12 13  The study has a biospecimen bank of approximately 12,000 samples, providing the 14 15 opportunity for future biomarker analyses. 16 For peer review only 17 A linked research training grant facilitates the training of nine Surinamese PhD candidates 18  19 20 with dissertation research embedded in the study, thereby building critical in-country 21 22 environmental health research capacity. 23 24 25  The interior subcohort (N=200) was logistically difficult to reach, resulting in delayed 26 27 recruitment, data collection and transport of cord blood specimens. Additional team 28 29 support onsite resulted in 100% recruitment of the subcohort, and heel prick sampling 30 31 32 replaced cord blood collection at birth.

33 http://bmjopen.bmj.com/ 34 35 36 37 Introduction 38 39 40 The Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH)

41 on September 25, 2021 by guest. Protected copyright. 42 43 addresses high-priority environmental and occupational health risks in Suriname and those 44 45 common to the vulnerable Caribbean region, while preserving unique cultural traditions of 46 47 indigenous people and other health disparate populations [1]. Exposure to environmental 48 49 50 contaminants at levels of public health concern may adversely affect pregnancy, and pre- and 51 52 postnatal health in multiple ways: miscarriage, preterm delivery, intra-uterine growth retardation, 53 54 congenital anomalies, and behavioral and physical consequences in later developmental stages [2, 55 56 57 58 5 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 3, 4]. Prenatal exposure to multiple heavy metals is associated with adverse pediatric health 4 5 6 outcomes [5]. Pesticide exposure has been linked to fetal growth decrements and preterm birth, 7 8 and there is mounting evidence that exposure to pesticides during pre- and postnatal development 9 10 is associated with neurodevelopmental deficits in young children [6-10]. Exposure to contaminant 11 12 mixtures can exacerbate adverse health effects: e.g. lead (Pb), arsenic (As), and mercury (Hg) can 13 14 15 potentiate each other’s toxicity, even at individual levels below concentration expected to result in 16 For peer review only 17 adverse effects [11]. Despite this knowledge, no cumulative risk assessments have been conducted 18 19 to date addressing exposures to contaminant mixtures in Caribbean Low- and Middle-Income 20 21 22 Countries [12]. Furthermore, a paucity of studies examined the interactive impact of exposures to 23 24 chemical and non-chemical (psychosocial) stressors. 25 26 27 The CCREOH-MeKiTamara (MeKiTamara means “making a mother and child's tomorrow’’ in 28 29 Suriname’s local language) environmental epidemiology cohort study fills this gap by exploring 30 31 32 the impact of exposures to organic and inorganic neurotoxicants, including Hg, Pb and multiple

33 http://bmjopen.bmj.com/ 34 organophosphate pesticides, on Surinamese pregnant women and their offspring using a 35 36 cumulative risk approach. Specifically, the study assesses the impact of exposures to chemical and 37 38 non-chemical stressors by examining the interaction between exposure to environmental chemicals 39 40 and social and psychological determinants of early neurodevelopment. Few prospective studies

41 on September 25, 2021 by guest. Protected copyright. 42 43 have measured exposure prenatally through 48 months in mother/child dyads, and those that have 44 45 showed contrasting results when evaluating executive function, a key neurodevelopmental 46 47 48 outcome [13-16]. Our hypothesis is that environmental exposures to mixtures of 49 50 neurodevelopmental toxicants and non-chemical stressors will result in increased adverse birth 51 52 outcomes and poorer child neurodevelopmental trajectories. Our research aims are to: 53 54 55 56 57 58 6 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3  Identify exposures to a complex mixture of environmental elements and toxicants, 4 5 6 including Hg, Pb, cadmium (Cd), aluminum (Al), iron (Fe), manganese (Mn), tin (Tn), 7 8 selenium (Se) and selected pesticides through comprehensive dietary and environmental 9 10 risk assessments, and biomarker monitoring in Surinamese pregnant women and their 11 12 13 offspring; 14 15  Assess the levels of environmental elements and toxicants in fish, produce, rice, and 16 For peer review only 17 nutraceutical compounds; 18 19 20  Determine the association between levels of environmental elements and toxicants in 21 22 pregnant women and birth outcomes; 23 24  Assess the impact of mobile health technology-enabled community health workers on birth 25 26 27 outcomes and their associations with environmental contaminants; 28 29  Determine the association between prenatal, dietary, and environmental levels of elements 30 31 and toxicants and possible neuroprotective nutraceuticals on neurodevelopment. 32

33 http://bmjopen.bmj.com/ 34 CCREOH is funded by the Fogarty International Center at the US National Institutes of Health 35 36 37 (NIH). 38 39 40

1 2 3 groups: Hindustani (27%), Tribal (22%), Creole (16%), Javanese (14%), and Indigenous (4%) 4 5 6 [17]. 7 8 9 10 11 12 Recruitment 13 14 The MeKiTamara study enrolled mother/child dyads potentially exposed to complex 15 16 For peer review only 17 environmental chemical- and non-chemical stressors. This study was approved by the Institutional 18 19 Review Boards of both the Government of Suriname and Tulane University to consent 1200 20 21 pregnant women and their singleton birth children. Pregnant women were recruited from three 22 23 24 regions of Suriname: (1) Paramaribo, where pesticides are primarily used for residential purposes; 25 26 (2) Nickerie, the major rice producing region in western Suriname where pesticide use is abundant; 27 28 and (3) the tropical rainforest interior, where Hg is used in artisanal gold mining and the population 29 30 31 is highly dependent on consumption of contaminated fish (Figure 1) [18]. 32

33 http://bmjopen.bmj.com/ 34 Recruitment sites included all hospitals and clinics of the Regional Health Department in 35 36 Paramaribo and Nickerie and the Medical Mission Primary Health Care Suriname in the interior. 37 38 In Nickerie, trained community health workers (CHWs) were integrated in the research team and 39 40 played a key role in every aspect of the study, from recruitment to assessments at every study time

41 on September 25, 2021 by guest. Protected copyright. 42 43 point prenatally and during the neurodevelopmental assessments of the child cohort. Women who 44 45 met the inclusion criteria were identified by their physician, midwife, CHW or health assistant 46 47 during regular prenatal appointments and invited to participate. Inclusion criteria were: pregnant 48 49 50 women 16 years or older; speaking Dutch, Sranan Tongo, Sarnami, Saramaccan or Trio; singleton 51 52 gestation; planning to deliver at one of the study hospitals, prenatal clinics or midwife facilities 53 54 55 56 57 58 8 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 11 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 associated with those hospitals or clinics; and signed informed consent. Assent was obtained from 4 5 6 participants 16 and 17 years of age. 7 8 9 Infants were not eligible for follow-up if they were born <33 completed weeks of gestation and/or 10 11 had a birthweight <2000 grams, significant medical or neurological condition, Down syndrome, 12 13 hydrocephalus, cerebral palsy, or significant visual or hearing impairment inconsistent with 14 15 neurocognitive testing. 16 For peer review only 17 18 Main maternal and infant/child determinants included biological determinants: maternal 19 20 21 anthropometrics, blood pressure, hemoglobin, liver and kidney function, fetal and postnatal growth 22 23 characteristics and health status, heavy metals (blood and hair), pesticides (urine), telomere length 24 25 (buccal swab) and medication use; environmental determinants: maternal and child diet and 26 27 28 exposure history; social determinants: ethnicity, social support, trauma, prenatal life events, 29 30 maternal education, household income, employment status, and marital status. Main maternal and 31 32 infant/child outcomes were growth and physical development: pregnancy complications, fetal and

33 http://bmjopen.bmj.com/ 34 35 postnatal growth patterns, risk factors for maternal liver and renal impairment; behavior and 36 37 cognitive development: infant neuromotor development, autism spectrum disorder and 38 39 neuropsychological development; childhood diseases: infectious diseases, respiratory and 40

41 neurological disorders; health and healthcare: impact of mobile health technology-enabled CHWs on September 25, 2021 by guest. Protected copyright. 42 43 44 on prenatal health and birth outcomes, quality of life assessments and the impact of health care 45 46 system utilization on prenatal health and birth outcomes. 47 48 49 50 51 52 Data management & statistical plan 53 54 55 56 57 58 9 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 12 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 The CCREOH field team was trained in administering all questionnaires. Data from administered 4 5 6 questionnaires are recorded on secure, encrypted iPads, uploaded and managed using REDCap, 7 8 which serves as the study electronic, integrated data management system [19]. Data collected 9 10 through iPads by the field team are uploaded in REDCap’s training site for data cleaning prior to 11 12 integrating questionnaire and biospecimen data. Data records are maintained to examine trends on 13 14 15 source of errors, duplicate records are scrubbed, and accuracy is validated through cross-checks 16 For peer review only 17 with original data files and medical records. A comprehensive biospecimen tracking system tracks 18 19 analyzed samples and the study’s overall biospecimen repository. Communication of data updates 20 21 22 and changes in the process are delivered through emails as well as during bimonthly meetings of 23 24 the data management team. The data management platform allows for the interrogation of 25 26 environmental, non-biospecimen and biospecimen data in an integrated fashion to facilitate 27 28 29 scientific inquiries associated with the impact of exposures to complex mixtures of chemical and 30 31 non-chemical stressors on pregnancy, birth outcomes and neurodevelopment. 32

33 http://bmjopen.bmj.com/ 34 The data management team develops de-identifies data files upon request of CCREOH 35 36 investigators as specified by a standardized data request form. Statistical analyses are tailored to 37 38 the specific research question. Frequency analyses are used for descriptive statistics and presented 39 40 as means with standard deviations or median with interquartile range for continuous variables and

41 on September 25, 2021 by guest. Protected copyright. 42 43 proportions for categorical variables. Associations between categorical variables are studied using 44 45 crosstabs; bivariate linear and logistic regression models are computed to study crude and 46 47 48 independent associations, respectively. Comparison between study sites will primarily be analyzed 49 50 by comparing participants, but can also be clustered depending on the research question. 51 52 53 54 55 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Data Collection 4 5 6 Maternal data were collected during pregnancy at two timepoints (trimester 1 or 2, and 3). Post- 7 8 9 partum assessments are ongoing and target both mothers and children: biomarker and 10 11 questionnaire data are collected from mothers during the child’s neurodevelopmental testing at 12, 12 13 24, 36, and 48 months. (Table 1). 14 15 16 For peer review only 17 18 19 Data collection during pregnancy 20 21 22 Questionnaires 23 24 25 Table 1 provides a list of standardized validated questionnaires to assess both physiological and 26 27 psychosocial prenatal health. All questionnaires were translated into Dutch and other local 28 29 30 languages to address the multiple language needs of our interior subcohort. The main categories 31 32 of data collected from maternal participants were health status, demographics, reproductive health

33 http://bmjopen.bmj.com/ 34 history, social support, trauma history, exposure history, depression, perceived stress, prenatal life 35 36 37 events, health behavior, access to prenatal care, social status and diet. Questions on health behavior 38 39 were adapted from the Alcohol, Smoking and Substance Involvement Screening Test version 3 40

41 (ASSIST V3.0) that was developed for the World Health Organization (WHO). Information on on September 25, 2021 by guest. Protected copyright. 42 43 maternal diet was obtained by adapting the CDC’s National Health and Nutrition Examination 44 45 46 Survey (NHANES) into a culturally tailored dietary survey focusing on fish and produce 47 48 consumption, including frequency of intake and portion sizes. 49 50 51 52 53 54 Biospecimen collection 55 56 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 The study’s biospecimen repository, housed at the Academic Hospital Paramaribo’s (AZP) clinical 4 5 6 laboratory, contains 12,000 samples. All biospecimens are collected at study site hospitals and 7 8 shipped to the AZP laboratory using established chain-of-custody procedures. Samples collected 9 10 from participants in remote areas were stored at 4°C for no more than 48 hours prior to delivery 11 12 and processing at the Academic Hospital Paramaribo clinical laboratory. Blood, serum, plasma, 13 14 15 and cord blood samples were aliquoted into 2 ml plastic freezer tubes and stored at -80°C. 16 For peer review only 17 18 400 frozen whole blood samples were shipped for analysis to the Wisconsin State Laboratory of 19 20 Hygiene Trace Element Research Laboratory. A chain-of-custody approach was used to ensure 21 22 samples were collected, maintained, processed, stored, shipped, and received according to 23 24 25 acceptable standards. Quality assurance/quality control (QA/QC) procedures for elemental 26 27 analyses and methylmercury analyses included reagent blanks, blank spikes (lab fortified blanks), 28 29 sample matrix spikes, ongoing precision and recovery spikes, second source spikes, sample matrix 30 31 32 duplicates, and external standard reference materials. All sample and QA/QC results were within

33 http://bmjopen.bmj.com/ 34 the acceptable recovery limits. Duplicates for all runs were within the acceptable relative percent 35 36 difference (%RPD) limits. 37 38 39 In these 400 maternal whole blood samples (200 Paramaribo, 100 Nickerie, and 100 interior 40

41 on September 25, 2021 by guest. Protected copyright. communities), we analyzed Pb, Hg, Cd, Al, Mn, Tn, Se, and Fe using inductively coupled plasma- 42 43 44 mass spectrometry (ICP-MS) or cold vapor atomic fluorescence spectrometry (CVAFS). Total Hg 45 46 and methylmercury using CVAFS was assessed in a small subset of women (20-Paramaribo, 20- 47 48 Nickerie, and 35-interior) to specify exposure sources. 49 50 51 Maternal hair samples were taken according to the following protocol: 1.5 gram of hair was cut as 52 53 54 close as possible to the hair roots with disinfected scissors and placed in patient coded ziplock 55 56 bags, stored at room temperature in a climate-controlled room and sent to the National Zoological 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Collection of Suriname/Center for Environmental Research lab at the Anton de Kom University 4 5 6 of Suriname (NZCS/CMO) for total mercury analysis using Cold Vapour Atomic Absorption 7 8 Spectrometry (CVAAS). 9 10 11 12 13 14 Urine collection for pesticides analyses 15 16 For peer review only 17 Urine samples from pregnant women were analyzed (subcohort N=218) from all three study sites. 18 19 Urine samples were aliquoted into 10 ml plastic tubes, stored at -20°C and shipped for analysis by 20 21 CDC’s environmental health laboratory using well-established protocols. The six dialkylphosphate 22 23 24 metabolites were measured using a modified method of Jayatilaka et al. [20] by solid phase 25 26 extraction high performance liquid chromatography-tandem mass spectrometry1. The eight 27 28 specific urinary metabolites were analyzed using a semi-automated solid phase extraction mass 29 30 31 spectrometry method and reversed-phase high performance liquid chromatography [21]. The 32

33 analysis panel consisted of dialkylphosphate metabolites of organophosphate pesticides (DAP’s) http://bmjopen.bmj.com/ 34 35 including dimethylphosphate, diethylphosphate, dimethylthiophosphate, 36 37 dimethyldithiophosphate, diethylthiophosphate, and diethyldithiophosphate, and a universal panel 38 39 40 (UP) including 1 herbicide (2,4D) and its metabolite, 2,4-dichlorophenoxyacetic acid; 4 OP

41 on September 25, 2021 by guest. Protected copyright. 42 insecticide metabolites (3,5,6-trichloro-2-pyridinol,2-isopropyl-4-methyl-6- 43 44 hydroxypyrimidine,para-nitrophenol, malathion dicarboxylic acid) and 3 pyrethroid metabolites 45 46 47 (4-fluoro-3-phenoxybenzoic acid, 3-phenoxybenzoic acid, trans-3-(2,2-dichlorovinyl)-2,2- 48 49 dimethylcyclopropane carboxylic acid). 50 51 52 53 54 55 Data collection from birth through 48 months 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 At birth, a cord blood sample was taken from infants for measurements of heavy metals. In case a 4 5 6 cord blood sample was not available, a blood sample was taken by heel prick shortly after birth. 7 8 Baseline characteristics of all live births (e.g. gender, weight, gestational age, and Apgar score) 9 10 were collected (Table 2). At each subsequent data collection timepoint (Table 1), the child’s 11 12 growth is measured and a blood sample is taken for heavy metal measurement. In addition, the 13 14 15 child’s health status is obtained through questionnaires on diet and history of infectious diseases, 16 For peer review only 17 and respiratory and neurological disorders. 18 19 20 21 22 23 Buccal swab collection for telomere assessments 24 25 26 Evidence exists that telomere length (TL) is a marker of cellular aging and links TL to negative 27 28 health outcomes including diabetes, cardiovascular disease and cognitive decline with aging. 29 30 31 Oxidative stress and DNA methylation, among other factors, influence TL. As and pesticide 32

33 exposure have both been linked to altered DNA methylation, while evidence also suggests a link http://bmjopen.bmj.com/ 34 35 between Hg and elevated oxidative stress [22-26]. TL may reflect multiple biological pathways 36 37 through which environmental toxins influence child health and development and potentially may 38 39 40 be an earlier indicator of exposure. Buccal swabs are collected to extract DNA and analyze TL

41 on September 25, 2021 by guest. Protected copyright. 42 prenatally in mothers and in children at 12, 24, 36, and 48 months of age. The average relative TL, 43 44 represented by the telomere repeat copy number to single gene (albumin) copy number (T/S) ratio, 45 46 47 is determined with monochrome multiplex quantitative real-time PCR using a BioRad CFX96 48 49 [27]. 50 51 52 53 54 55 Behavior and cognitive development 56 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Neurodevelopmental assessments include infant cognitive and motor development and behavior 4 5 6 at 12- 27 months using the Bayley Scales of Infant Development as well as an assessment of 7 8 cognitive and social-emotional development at 36 months, and measurement of executive function 9 10 at 48 months (Table 1). The Bayley assessment was administered by a team of trained 11 12 psychologists, psychiatrists, and members of the study team specifically trained to administer the 13 14 15 test. Data checking following data entry of each Bayley sub-scale is being conducted by other team 16 For peer review only 17 members. Administration of Bayley assessments will be completed by May 2020. Assessments at 18 19 36 and 48 months will take place as the cohort ages. 20 21 22 23 24 25 26 Patient and public involvement 27 28 29 The CCREOH/ MeKiTamara cohort study was preceded by a planning grant which included a 30 31 pilot fish assessment study as well as an initial hair mercury assessment among communities in 32

33 http://bmjopen.bmj.com/ 34 the interior which required approval from the village Chiefs or Tribal captains. This community 35 36 engagement component provided us a rich foundation to build on as we developed the research 37 38 questions. The initial study design was revised based on guidance from an External Scientific 39 40 Advisory Board (EAB) and a multi-stakeholder Community Advisory Board (CAB). For example,

41 on September 25, 2021 by guest. Protected copyright. 42 43 the CAB was instrumental in urging the study team to lower the inclusion age to 16 years since 44 45 many women in the interior conceive at an earlier age. The CAB consists of respected leaders 46 47 representing organizations from the interior tribal and indigenous peoples, midwives, general 48 49 50 practitioners and environmentalists. The EAB members represent medical directors and other 51 52 clinicians of all participating hospitals, representatives from the Ministry of Health, the US 53 54 Embassy, the Pan American Health Organization and the Caribbean Public Health Agency 55 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 (CARPHA). Both advisory boards are convened annually to inform and give updates about the 4 5 6 study progress and results, and to seek advice on future directions and dissemination of results. 7 8 9 10 11 12 Dissemination of study results 13 14 The study team is actively working with the EAB and the CAB on a collaborative translation and 15 16 For peer review only 17 dissemination strategy of overall study results. However, every participant with neurotoxicant 18 19 levels of imminent public health concern is contacted by the study coordinator and asked to repeat 20 21 the test for confirmation. If the level remains of concern, the participant is referred to her attending 22 23 24 physician according to the study’s formal health care provider referral protocol. Children that are 25 26 found to have a neurodevelopmental delay are referred to a child psychologist for further 27 28 evaluation. Participants and the general public were invited to multiple health fairs, meetings and 29 30 31 conferences, where the researchers discussed the study in a broader context, provided updates on 32

33 the latest results and answered questions. Additionally, a Facebook page was created for study http://bmjopen.bmj.com/ 34 35 participants and others interested in this study, with general information on exposure to heavy 36 37 metals and pesticides as well as the opportunity to ask questions of the research team and provide 38 39 40 feedback about the study. Periodic briefings were also provided to officials of the Suriname’s

41 on September 25, 2021 by guest. Protected copyright. 42 Ministry of Health. CCREOH data have been presented at multiple local, regional, and 43 44 international conferences, including CARPHA, the International Society of Environmental 45 46 47 Epidemiology, the Consortium for Universities in Global Health, the American Public Health 48 49 Association and at scientific meetings convened by the US NIH. 50 51 52 53 54 55 56 Findings to date 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 From December 2016 until December 2018, 1067 pregnant women were enrolled; 76 (7.1%) were 4 5 6 ineligible (Figure 2). In comparison to participants, non-participants were more often of Creole 7 8 and Tribal ethnicity, lived in Paramaribo, and had lower household income. Table 2 presents 9 10 baseline maternal characteristics: one in eight women was 16-19 years of age, ethnic distribution 11 12 was representative of the Surinamese population, and more than half had no or lower 13 14 15 vocational/secondary education level. 16 For peer review only 17 18 Of the 898 babies born, 827 were enrolled (92%). Initial neurodevelopmental assessments using 19 20 the Bayley instrument at 12-27 months in 824 infants are being analyzed. To date, the total number 21 22 of biospecimens collected from 1067 pregnant women include whole blood for trace elements 23 24 25 (N=1994), whole blood collected in K2EDTA anticoagulant (N=1994), serum collected in serum 26 27 separator tubes (N=1994), plasma (N=1994), urine (N=1980), buccal swabs (N=941), and hair 28 29 (N=876). Cord blood (N=441), blood from heel prick (N=269), and buccal swabs (N=363) have 30 31 32 been collected from 710 infants. All samples not yet analyzed are archived for future targeted and

33 http://bmjopen.bmj.com/ 34 untargeted analyses. Available results include total mercury concentrations in hair from pregnant 35 36 women, prenatal maternal depression and perceived stress, level of pesticides in produce, and 37 38 dietary exposure to Hg in fish. Results of heavy metals in blood, urinary pecticide metabolites, 39 40 telomere analysis, and neurodevelopmental assessments are pending.

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Biological specimens 48 49 50 Total hair Hg was measured in 876 pregnant women from all three study sites. Overall, 39.1% of 51 52 the women had elevated total mercury hair levels that exceeded the U.S. Environmental Protection 53 54 Agency’s (US EPA) action level [28]. For women from the interior, Nickerie and Paramaribo these 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 percentages were 95.8%, 26.3%, and 25.5% respectively. Results for median total Hg 4 5 6 concentrations in hair from pregnant women from Paramaribo (N=522) were 0.64 µg/g hair 7 8 (interquartile ranges (IQR) 0.36-1.09; range 0.00-7.12), from Nickerie (N=176) 0.73 µg/g (IQR 9 10 0.45-1.05; 0.00-5.79) and the interior (N=178) 1.92 µg/g (IQR 1.92-7.39; 0.38-18.20). Pregnant 11 12 women from the interior were exposed to high levels of Hg compared to women from the coastal 13 14 15 area and thus fetal exposures are expected to be high. Most of these women are primarily exposed 16 For peer review only 17 to methylmercury from consuming contaminated fish from local artisanal gold-mining activities 18 19 [18,29]. Other possible sources of exposure may include active participation in or living very close 20 21 22 to gold mining areas. 23 24 25 26 27 28 Prenatal depression and perceived stress 29 30 31 Depression and prenatal stress in 722 participants were assessed using the standardized Edinburgh 32

33 Postnatal Depression Scale (EPDS) (cut off ≥12 for probable depression) and Cohen Perceived http://bmjopen.bmj.com/ 34 35 Stress Scale (cut off ≥20 for high stress) respectively. One in four (24.9%) pregnant study 36 37 participants had EPDS scores indicative of probable depression. Three in ten participants had high 38 39 40 stress levels and nearly half of these women had probable depression.

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 Dietary exposure to pesticides 46 47 An assessment of Surinamese agricultural produce conducted by members of our study team found 48 49 50 pesticide residues exceeding European Union maximum residue limits, including prohibited- 51 52 worldwide endosulfan and lindane in the leafy vegetable tannia, Xanthosoma brasiliense [30]. An 53 54 interviewer-assisted NHANES-based dietary survey of 522 participants showed that 98.2% of 55 56 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 women reported consumption of leafy vegetables. Tannia was the most frequently consumed 4 5 6 (89.3%); 36.5% of surveyed women had high intake rates of tannia (≥36 gram/day) [31]. Tannia 7 8 is also a commonly used vegetable in baby food preparation in Suriname. 9 10 11 12 13 Dietary exposure to Hg in fish 14 15 16 One in four pregnantFor women inpeer our cohort, reviewand predominantly only those living in the interior who rely 17 18 heavily on local freshwater fish, have hair mercury levels exceeding those considered safe by the 19 20 21 US EPA and the World Health Organization (WHO). The dietary questionnaire (n=990) showed 22 23 overall fish consumption of 96.1%. Previous environmental assessments of most frequently 24 25 consumed carnivorous fish by interior participants, specifically Hoplias aimara, Serrasalmus 26 27 28 rhombeus, and Cichla ocellaris, showed high levels of Hg [29]. Respective consumption of these 29 30 three carnivorous species was 44.4%, 19.3%, and 26.3%. Carnivorous fish consumption was 31 32 greater among the subcohort (N=123) living in the interior: 89.4%, 67.5%, and 74.8%,

33 http://bmjopen.bmj.com/ 34 35 respectively. 36 37 Polyunsaturated omega-3 fatty acids (PUFA-3) docosahexaenoic acid and eicosapentaenoic acid 38 39 40 from fish consumption offer neuroprotective benefits during prenatal and pediatric development

41 on September 25, 2021 by guest. Protected copyright. 42 which could potentially counteract mercury neurotoxicity. To better inform fish consumption risk, 43 44 fatty acids in fish muscle tissue were measured in samples (N=5 per species) of five freshwater 45 46 47 and three marine fish species: freshwater species had higher levels of linoleic acid (2.0 vs. 0.2 48 49 mg/g), alpha-linoleic acid (0.4 vs. 0.1 mg/g), arachidonic acid (3.0 vs. 1.5 mg/g), omega-6 fatty 50 51 acids (6.5 vs. 2.3 mg/g) and lower levels of eicosapentaenoic acid (0.8 vs. 1.9 mg/g) compared to 52 53 54 marine species. Frequent consumption of Hoplias aimara, a freshwater fish with high Hg and low 55 56 PUFA-3 content, is likely to increase mercury exposure whereas consumption of Plagioscion 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 squamosissimus, a freshwater fish with high mercury and high PUFA-3, may have nutritional 4 5 6 benefits that outweigh mercury neurotoxicity. While freshwater species are a good source of these 7 8 nutrients, corresponding mercury content must be assessed and considered as well. 9 10 11 12 13 14 Strengths and limitations 15 16 For peer review only 17 The CCREOH-MeKiTamara environmental epidemiologic study is the first to examine the 18 19 potential impact of complex environmental exposures on maternal and child health in Suriname 20 21 22 by a multi-disciplinary team of research scientists. The study’s strengths include: 1) addressing 23 24 two high priority public health threats: the impact of Hg exposure from artisanal gold mining, and 25 26 pesticide exposure associated with agricultural practices; 2) inclusion in the cohort of two 27 28 29 vulnerable subpopulations: pregnant women and children; 3) the longitudinal follow up with 30 31 children until 48 months with built-in timepoints to assess neurodevelopmental outcomes; and 4) 32

33 a biospecimen bank of approximately 12,000 samples, providing the opportunity for future http://bmjopen.bmj.com/ 34 35 36 analyses within the cohort. Moreover, a linked research training grant facilitates the training of 37 38 nine Surinamese PhD candidates with dissertation research embedded in the study, thereby 39 40 building critical in-country environmental health research capacity. Research dissemination

41 on September 25, 2021 by guest. Protected copyright. 42 throughout the Caribbean region is implemented in collaboration with CARPHA. 43 44 45 The study team overcame two initial logistical challenges: 1) delayed recruitment and data 46 47 48 collection of the logistically difficult to reach subcohort in the interior region (N=200) was 49 50 ameliorated by additional team support onsite; and 2) delays in transporting interior biospecimens 51 52 rendered some early cord blood specimens not analyzable was corrected by collecting an additional 53 54 55 heelprick sample at birth. 56 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 7 Future plans 8 9 10 Culturally tailored fish consumption advisories are in development to reduce Hg exposure. Recent 11 12 additional funding allows us to examine potentially beneficial neuroprotective factors such as 13 14 15 selenium and dietary polyunsaturated fatty acids in fish that may counter the neurotoxic effects of 16 For peer review only 17 mercury. To address the large scale and poorly regulated use of pesticides, we are also developing 18 19 a pesticide literacy scale. Cohort-wide, we will undertake a comprehensive assessment of the 20 21 neurodevelopmental outcomes of the children, and create a robust, sharable data management 22 23 24 system of biospecimen and non-biospecimen data building on the current REDCap database. 25 26 27 28 29 30 Acknowledgements 31 32

33 We would like to thank our recruiters Jeetendra Jitan, Nishaira Doerga, Gimradj Dwarka, Sue-Ann http://bmjopen.bmj.com/ 34 35 36 Sitaram, Devika Kalika, the Community Health Workers from Nickerie Lilian Singotiko, Henna 37 38 Mawo, Karien Jodiwongso, Karishma Bhondoe, Jay Kalicharan, Bhairam Goberdan, Azeez 39 40 Khodabaks, the midwives and laboratory personnel of the Academic Hospital Paramaribo,

41 on September 25, 2021 by guest. Protected copyright. 42 43 Diakonessenhuis Hospital, St. Vincentius Hospital, ‘s-Lands Hospital, Mungra Medical Centre, 44 45 Regional Health Service clinics and Medical Mission Primary Health Care Suriname, the National 46 47 Zoological Collection of Suriname/Center for Environmental Research staff, Sila Kisoensingh 48 49 50 MSc, Dr. Richenel Ellecom and Dr. Melissa Middleton for their help during the pediatric 51 52 neurodevelopment assessments, Arioene Vreedzaam for providing the study site map, and the 53 54 55 56 57 58 21 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 members of the External Advisory Board and Community Advisory Board for their guidance and 4 5 6 valuable advice. 7 8 9 10 11 12 Collaborators 13 14 15 We welcome collaboration with fellow researchers working on similar projects. Specific research 16 For peer review only 17 18 proposals can be sent directly to the Principal Investigators Drs. Wilco Zijlmans 19 20 ([email protected]) and Maureen Lichtveld ([email protected]). 21 22 Areas of collaboration include further environmental and biomarkers assessment of arsenic 23 24 exposure, occupational health risk assessments associated with mercury and pesticide exposure, 25 26 27 and exposomic analyses including metabolomic assessments. 28 29 30 31 32 Author Contributions

33 http://bmjopen.bmj.com/ 34 35 MYL, JKW, HHC, EWH, AS, SSD and WCWRZ designed and established the cohort. MSMO, 36 37 ADHM, EWH and AG participated in the design of the questionnaires. WCWRZ, MSMO, EB, 38 39 40 WBH, GKB, RR and MYL are responsible for the continued management of the cohort. AG, GKB,

41 on September 25, 2021 by guest. Protected copyright. 42 RR, AG and FAW are actively involved with recruitment of participants and collection of non- 43 44 biospecimen data and biospecimens. JC, JR, PEO and GAL store, archive and analyze the 45 46 47 biomarker samples. ADHM, AS, MSMO, AG, MYL and WCWRZ are responsible for data 48 49 management. SSD, AWEG, RR and EB manage neurodevelopmental testing of babies. JKW, LFS, 50 51 CSA, SSD interpret all results. WCWRZ, JKW, HHC and MYL drafted and edited the manuscript. 52 53 All authors critically reviewed and approved the final manuscript. 54 55 56 57 58 22 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Funding 4 5 6 7 Research reported in this publication was supported by the Fogarty International Center of the 8 9 National Institutes of Health under Award Numbers: U01TW010087 and U2RTW010104. The 10 11 content is solely the responsibility of the authors and does not necessarily represent the official 12 13 14 views of the National Institutes of Health. 15 16 For peer review only 17 18 19 20 Competing interests 21 22 23 None declared 24 25 26 27 28 29 30 Patient consent for publication 31 32

33 Obtained http://bmjopen.bmj.com/ 34 35 36 37 38 39 Ethical approval 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 This study was IRB approved by both the Government of Suriname and the Tulane University, 44 45 New Orleans, Louisiana, USA, to consent 1200 pregnant women and their singleton birth 46 47 children. 48 49 50 51 52 53 54 Provenance and peer review 55 56 57 58 23 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 26 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Not commissioned; externally peer reviewed. 4 5 6 7 8 9 10 Data sharing statement 11 12 13 MeKiTamara cohort data will be made publicly available upon publication of the results in 14 15 scientific articles. Questionnaire, registry and biospecimen data could be made accessible in de- 16 For peer review only 17 18 identified form after an application process that includes submission of a research plan that will 19 20 undergo evaluation by the CCREOH scientific board and relevant research ethics committees. 21 22 Currently, data is partially available since data collection is ongoing. 23 24 25 26 27 28 29 References 30 31 32 1. Lichtveld MY, Hawkins WB, Ouboter PE et al. A One Health approach to interdict

33 http://bmjopen.bmj.com/ 34 35 environmental health threats in Suriname. Ann Glob Health 2016;82:444. 36 37 38 2. Silbergeld EK, Patrick TE. Environmental exposures, toxicologic mechanisms, and adverse 39 40 pregnancy outcomes. Am J Obstet Gynecol 2005;192:S11-21.

41 on September 25, 2021 by guest. Protected copyright. 42 43 3. Ferguson KK, O'Neill MS, Meeker JD. Environmental contaminant exposures and preterm 44 45 birth: a comprehensive review. J Toxicol Environ Health B Crit Rev 2013;16:69-113. 46 47 48 4. Wigle DT, Arbuckle TE, Turner MC et al. Epidemiologic evidence of relationships between 49 50 51 reproductive and child health outcomes and environmental chemical contaminants. J Toxicol 52 53 Environ Health B Crit Rev 2008;11:373-517. 54 55 56 57 58 24 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 27 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 5. Lewis M, Worobey J, Ramsay DS et al. Prenatal exposure to heavy metals: effect on childhood 4 5 6 cognitive skills and health status. Pediatrics 1992;89:1010-5. 7 8 9 6. Burns CJ, McIntosh LJ, Mink PJ, Jurek AM, Li AA. Pesticide exposure and 10 11 neurodevelopmental outcomes: Review of the epidemiologic and animal studies. J Toxicol 12 13 Environ Health B Crit Rev 2013;16:127-283. 14 15 16 7. Eskenazi B, KogutFor K, Huenpeer K et al.review Organophosphate only pesticide exposure, PON1, and 17 18 19 neurodevelopment in school-age children from the CHAMACOS study. Environ Res 20 21 2014;134C:149-57. 22 23 24 8. Furlong MA, Engel SM, Barr DB, Wolff MS. Prenatal exposure to organophosphate pesticides 25 26 and reciprocal social behavior in childhood. Environ Int 2014;70:125-31. 27 28 29 9. Quirós-Alcalá L, Alkon AD, Boyce WT et al. Maternal prenatal and child organophosphate 30 31 32 pesticide exposures and children's autonomic function. Neurotoxicology 2011;32:646-55.

33 http://bmjopen.bmj.com/ 34 35 10. Rosas LG, Eskenazi B. Pesticides and child neurodevelopment. Curr Opin Pediatr 36 37 2008;20:191-7. 38 39 40 11. Belles M, Albina ML, Sanchez DJ et al. Interactions in developmental toxicology: effects of

41 on September 25, 2021 by guest. Protected copyright. 42 concurrent exposure to lead, organic mercury, and arsenic in pregnant mice. Arch Environ 43 44 45 Contam Toxicol 2002;42:93-8. 46 47 48 12. Callahan MA, Sexton K. If cumulative risk assessment is the answer, what is the question? 49 50 Environ Health Perspect 2007;115:799-806. 51 52 53 13. Golding J, Rai D, Gregory S et al. Prenatal mercury exposure and features of autism: a 54 55 prospective population study. Mol Autism 2018;9:30. 56 57 58 25 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 28 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 14. Valent F, Mariuz M, Bin M et al. Associations of prenatal mercury exposure from maternal 4 5 6 fish consumption and polyunsaturated fatty acids with child neurodevelopment: a prospective 7 8 cohort study in Italy. J Epidemiol 2013;23:360-70. 9 10 11 15. Sapbamrer R, Hongsibsong S. Effects of prenatal and postnatal exposure to organophosphate 12 13 pesticides on child neurodevelopment in different age groups: a systematic review. Environ 14 15 Sci Pollut Res Int 2019;26:18267-90. 16 For peer review only 17 18 19 16. Wang J, Wu W, Li H et al. Relation of prenatal low-level mercury exposure with early child 20 21 neurobehavioral development and exploration of the effects of sex and DHA on it. Environ Int 22 23 2019;126:14-23. 24 25 26 17. Results Eighth Population and Housing Census in Suriname (Volume 1) Demographic and 27 28 29 Social Characteristics and Migration. General Bureau of Statistic Studies in Suriname (ABS) 30 31 2013. 32

33 http://bmjopen.bmj.com/ 34 18. Ouboter PE, Landburg G, Satnarain GU et al. Mercury Levels in Women and Children from 35 36 Interior Villages in Suriname, South America. Int J Environ Res Public Health 2018;15:1007. 37 38 39 19. Harris PA, Taylor R, Minor BL et al. The REDCap consortium: Building an international 40

41 on September 25, 2021 by guest. Protected copyright. 42 community of software partners, J Biomed Inform 2019;95:1-10. doi: 43 44 10.1016/j.jbi.2019.103208 45 46 47 20. Jayatilaka NK, Restrepo P, Williams L, et al. Quantification of three chlorinated dialkyl 48 49 phosphates, diphenyl phosphate, 2,3,4,5-tetrabromobenzoic acid, and four other 50 51 organophosphates in human urine by solid phase extraction-high performance liquid 52 53 54 chromatography-tandem mass spectrometry. Anal Bioanal Chem 2017;409:1323-32. 55 56 57 58 26 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 29 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 21. Davis MD, Wade EL, Restrepo PR, et al. Semi-automated solid phase extraction method for 4 5 6 the mass spectrometric quantification of 12 specific metabolites of organophosphorus 7 8 pesticides, synthetic pyrethroids, and select herbicides in human urine. Journal of 9 10 Chromatography B. 2013;929:18-26 11 12 13 22. Frenck RW, Blackburn E, Shannon K. The rate of telomere sequence loss in human leukocytes 14 15 varies with age. Proc Natl Acad Sci 1998;95:5607-10. 16 For peer review only 17 18 19 23. Lung FW, Chen NC, Shu BC. Genetic pathway of major depressive disorder in shortening 20 21 telomeric length. Psychiatr Genet 2007;17:195-9. 22 23 24 24. Von Zglinchi T. Oxidative Stress shortens telomeres. Trends Biochem Sci 2002;27:339-44. 25 26 27 25. Gonzalo S, Jaco I, Fraga MF et al. DNA methyltransferases control telomere length and 28 29 telomere recombination in mammalian cells. Nat Cell Biol 2006;8:416-24. 30 31 32 26. Damjanovic AK, Yang Y, Glaser R et al. Accelerated telomere erosion is associated with a

33 http://bmjopen.bmj.com/ 34 35 declining immune function of caregivers of Alzheimer's disease patients. J Immunol 36 37 2007;179:4249-54. 38 39 40 27. Cawthon RM. Telomere length measurement by a novel monochrome multiplex qualitative

41 on September 25, 2021 by guest. Protected copyright. 42 PCR method. Nucleic Acids Res 2009;37:e21. 43 44 45 28. Mercury Study Report to Congress Volume V: Health Effects of Mercury and Mercury 46 47 48 Compounds. Volume 5. United States Environmental Protection Agency; Washington, DC, 49 50 USA: 1997;1–348. 51 52 53 54 55 56 57 58 27 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 30 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 29. Ouboter PE, Landburg GA, Quik JHM, Mol JHA, van der Lugt F. Mercury Levels in Pristine 4 5 6 and Gold Mining Impacted Aquatic Ecosystems of Suriname, South America. Ambio 7 8 2012;41:873-82. 9 10 11 30. Abdoel Wahid F, Wickliffe J, Wilson M et al. Presence of pesticide residues on produce 12 13 cultivated in Suriname. Environ Monit Assess 2017;189:303. 14 15 16 31. F. Abdoel Wahid,For A. D. Hindori-Mohangoo,peer review M. Wilson etonly al. Dietary Exposure to Pesticides 17 18 19 in Pregnant Surinamese Women. Environ Health Perspect 2018; 20 21 https://doi.org/10.1289/isesisee.2018.P01.2380 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 28 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 31 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 Table 1. Assessments completed by the CCREOH-MeKiTamara Cohort with timeline 6 7 Assessments Trimester Birth 12 24 36 48 8 mos mos mos mos 9 1st/2nd 3rd 10 Mother 11 Obstetric history ● ● 12 Demographics ● 13 Residency ● ● 14 Anthropometrics ● ● 15 Marital status ● 16 For peer review only 17 Ethnicity ● 18 Occupation ● 19 Education ● 20 Income ● 21 Household composition ● 22 Maternity care ● ● 23 Questionnaires 24 SF 36 Health Survey ● ● 25 Social Support List ● 26 Brief Trauma Interview ● ● ● ● ● 27 Cohen’s Perceived Stress Scale ● ● ● ● 28 Edinburgh Depression Scale ● ● ● ● ● 29 ASSIST V3.0 ● ● ● ● ● 30 Exposure History ● 31 Prenatal Life Events Scale ● ● 32 Subjective Social Status ● ●

33 http://bmjopen.bmj.com/ Dietary Assessment ● ● 34 35 Family Environment Scale ● ● ● 36 Parenting Stress Index ● ● ● 37 Biological samples 38 Hair ● 39 Blood ● ● 40 Urine ● ● Buccal swab ● 41 on September 25, 2021 by guest. Protected copyright. 42 43 At birth 44 Mode of delivery ● 45 Cord or heelprick blood sample ● 46 Birth outcomes ● 47 48 Child development 49 Physical examination ● ● ● ● 50 Questionnaires 51 General Health Questionnaire ● ● ● ● 52 M-CHAT ● 53 Child Behavior Checklist ● ● 54 55 Bayley SEQ ● 56 Ages and Stages Questionnaire ● 57 58 29 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 32 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Neurodevelopmental tests 4 BSID-III ● 5 CANTAB ● 6 Biological samples 7 Buccal swab ● ● ● ● 8 Blood ● ● ● 9 10 Notes: ASSIST V3.0 = Alcohol, Smoking and Substance Involvement Screening Test Version 3, Subjective 11 Social Status = MacArthur Scale of Subjective Social Status, M-CHAT = Modified Checklist for Autism 12 in Toddlers, Bayley SEQ = Bayley Social Emotional Questionnaire, BSID-III = Bayley Scales of Infant 13 Development Third Edition, CANTAB = Cambridge Neuropsychological Test Automated Battery. 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 30 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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1 2 3 Table 2. Distribution of maternal and infant characteristics 4 5 Maternal characteristics (N=1067) N % Infant characteristics (N=898) N % 6 Age at intake Gender 7 8 16-19 years 133 12.4 Male 461 52.3 9 20-24 years 239 22.4 Female 421 47.7 10 25-29 years 280 26.2 Missing 16 1.8 11 30-34 years 252 23.6 12 35-39 years For129 peer12.1 reviewBirth status only 13 40+ years 34 3.2 Live birth 863 96.1 14 Missing 0 0.0 Stillbirth 35 3.9 15 16 Missing 0 0.0 17 Parity

26 Ethnicity Very preterm births (22+0-32+6 weeks) on September 25, 2021 by guest. Protected copyright. 36 4.1 27 Creole 245 23.0 Moderately preterm births (33+0-36+6 weeks) 93 10.7 28 Hindustani 222 20.8 Term births (≥37+0 weeks) 733 84.1 29 Indigenous 149 14.0 Missing 26 2.9 30 31 Javanese 94 8.8 32 Tribal 223 20.9 Apgar score at 5 minutes 33 Mixed 128 12.0 0 - 6 46 5.2 34 Other 5 0.5 7 - 10 832 94.8 35 Missing 1 0.1 Missing 20 2.2 36 37 Educational level 38 39 No or primary 223 20.9 40 Lower vocational/secondary 375 35.2 41 Upper vocational/secondary 302 28.3 42 43 31 44 45 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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18 partner http://bmjopen.bmj.com/ 19 Missing 5 0.5 20 21 * SRD = Surinamese dollar, equivalent to 0.13 USD. 22 23 24 25

1 2 3 Figure legends 4 5 6 7 Figure 1. CCREOH-MeKiTamara study area with recruitment sites from three regions of 8 Suriname: 1) the capital Paramaribo 2) Nickerie, the major rice-producing region in western 9 10 Suriname and 3) the tropical rainforest interior where mercury is used in artisanal gold mining. 11 12 13 Figure 2. Flowchart with participant enrolment. 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 50x61mm (300 x 300 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 37 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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Cohort profile: The Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH) Cohort Study: Influences of complex environmental exposures on maternal and child health in Suriname

ForJournal: peerBMJ Open review only Manuscript ID bmjopen-2019-034702.R2

Article Type: Cohort profile

Date Submitted by the 08-Jun-2020 Author:

Complete List of Authors: Zijlmans, Wilco; Anton de Kom University of Suriname Faculty of Medical Sciences, Pediatrics; Academic Hospital Paramaribo, Scientific Research Center Suriname Wickliffe, Jeffrey; Tulane University, Global Environmental Health Sciences Hindori-Mohangoo, Ashna; Perisur (Perinatal Interventions Suriname) Foundation MacDonald-Ottevanger, Sigrid; Academic Medical Center, Amsterdam, The Netherlands Ouboter, Paul; Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname

Landburg, Gwendolyn; Anton de Kom University of Suriname, http://bmjopen.bmj.com/ Paramaribo, Suriname, National Zoological Collection of Suriname/Center for Environmental Research Codrington, John; Academic Hospital Paramaribo, Department of Clinical Chemistry Roosblad, Jimmy; Academic Hospital Paramaribo Baldewsingh, Gaitree; Medical Mission Primary Health Care Suriname Ramjatan, Radha ; Regional Health Service Gokoel, Anisma; Academic Hospital Paramaribo, Scientific Research Center Suriname on September 25, 2021 by guest. Protected copyright. Abdoel Wahid, Firoz; Tulane University Fortes Soares, Lissa; Tulane University Alcala, Cecilia; Tulane University Boedhoe, Esther; Academic Hospital Paramaribo Grünberg, Antoon; Ministry of Health, Paramaribo, Suriname, Department of Public Health Hawkins, William; Tulane University Shankar, Arti; Tulane University Harville, Emily; Tulane University School of Public Health, Epidemiology Drury, SS; Department of Psychiatry and Behavioral Sciences, Tulane University Covert, Hannah; Tulane University Lichtveld, Maureen; Tulane University School of Public Health and Tropical Medicine, Global environmental health sciences

Primary Subject Occupational and environmental medicine Heading:

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1 2 3 Title: Cohort profile: The Caribbean Consortium for Research in Environmental and 4 5 6 Occupational Health (CCREOH) Cohort Study: Influences of complex environmental exposures 7 8 on maternal and child health in Suriname 9 10 11 12 13 14 15 Author list 16 For peer review only 17 18 Wilco C.W.R. Zijlmans*1,2,3, Jeffrey K. Wickliffe2, Ashna D. Hindori-Mohangoo2,4, M. Sigrid 19 20 MacDonald-Ottevanger1,5, Paul E. Ouboter2,6,7, Gwendolyn A. Landburg6, John Codrington8, 21 22 8 3,9 3,10 1,3 23 Jimmy Roosblad , Gaitree K. Baldewsingh , Radha Ramjatan , Anisma Gokoel , Firoz 24 25 Abdoel Wahid1,2, Lissa Fortes Soares2, Cecilia S. Alcala2, Esther Boedhoe1, Antoon W.E. 26 27 Grünberg4,11, William B. Hawkins2,12, Arti Shankar2, Emily W. Harville13, Stacy S. Drury14, 28 29 Hannah H. Covert2, Maureen Y. Lichtveld2 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 Affiliations 37 38 39 1 Scientific Research Center Suriname, Academic Hospital Paramaribo, Paramaribo, Suriname 40

41 on September 25, 2021 by guest. Protected copyright. 42 2 Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, 43 44 Tulane University, New Orleans, LA 70112, United States of America 45 46 47 3 Faculty of Medical Sciences, Anton de Kom University of Suriname, Paramaribo, Suriname 48 49 50 4 Foundation for Perinatal Interventions and Research in Suriname (Perisur), Paramaribo, 51 52 53 Suriname 54 55 56 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 5 Department of Clinical Chemistry Medical Microbiology, Academic Medical Center, 4 5 6 Amsterdam, The Netherlands 7 8 6 9 National Zoological Collection of Suriname/Center for Environmental Research, Anton de Kom 10 11 University of Suriname, Paramaribo, Suriname 12 13 14 7 Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname 15 16 8 For peer review only 17 Department of Clinical Chemistry, Academic Hospital Paramaribo, Paramaribo, Suriname 18 19 9 20 Medical Mission Primary Health Care Suriname, Paramaribo, Suriname 21 22 10 23 Regional Health Service Nickerie, Suriname 24 25 11 26 Department of Public Health, Ministry of Health, Paramaribo, Suriname 27 28 12 29 Department of Health Policy, Vanderbilt University Medical Center, Nashville, TN, United 30 31 States of America 32

33 http://bmjopen.bmj.com/ 34 13 Department of Epidemiology, Tulane University School of Public Health and Tropical 35 36 Medicine, New Orleans, LA, United States of America 37 38 39 14 Tulane University School of Medicine, New Orleans, LA, United States of America 40

1 2 3 Abstract 4 5 6 7 Purpose: The Caribbean Consortium for Research in Environmental and Occupational Health 8 9 10 (CCREOH) prospective environmental epidemiologic cohort study addresses the impact of 11 12 13 complex chemical and non-chemical environmental exposures in mother/child dyads in Suriname. 14 15 The study determines associations between levels of environmental elements and toxicants in 16 For peer review only 17 18 pregnant women and birth outcomes and neurodevelopment in the child cohort. 19 20 21 22 Participants: From December 2016 until July 2019, biomarker data on hair, blood, urine, buccal 23 24 25 swab and questionnaire data on physiological and psychosocial prenatal health were collected from 26 27 28 1143 enrolled pregnant women, neurodevelopmental assessments were completed in 832 of 992 29 30 31 eligible infants. 32

33 http://bmjopen.bmj.com/ 34 Findings to date: 39.1% of participants had hair mercury (Hg) levels exceeding values considered 35 36 37 safe by international standards. Median Hg concentrations in women from Paramaribo (N=522) 38 39 40 were 0.64µg/g hair (interquartile ranges (IQR) 0.36-1.09; range 0.00-7.12), from Nickerie (N=176)

41 on September 25, 2021 by guest. Protected copyright. 42 43 0.73µg/g (IQR 0.45-1.05;0.00-5.79) and the interior (N=178) 3.48µg/g (IQR 1.92-7.39;0.38- 44 45 46 18.20). 96.1% participants ate fish, respective consumption of the three most consumed 47 48 carnivorous species Hoplias aimara, Serrasalmus rhombeus, and Cichla ocellaris was 44.4%, 49 50 51 19.3%, and 26.3%, and was greater among the interior subcohort: 89.4%, 67.5%, and 74.8% 52 53 54 respectively; 89% frequently consumed the vegetable tannia, samples of which showed presence 55 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 of worldwide banned pesticides; 24.9% had high Edinburgh Postnatal Depression Scale scores 4 5 6 indicative of probable depression. 7 8 9 Future plans: Culturally tailored fish consumption advisories are in development, especially 10 11 12 relevant to interior women for whom fish consumption is the primary source of mercury exposure. 13 14 15 Research is ongoing to examine effects of potentially beneficial neuroprotective factors in fish that 16 For peer review only 17 18 may counter neurotoxic effects of mercury. A pesticide literacy assessment in pregnant Surinamese 19 20 21 women is in progress. Long-term effects of exposures to toxicant mixtures in infants/toddlers are 22 23 being evaluated through neurodevelopmental assessments. Telomere length measurements of the 24 25 26 mothers and children as an indicator of prenatal exposure to environmental toxins are ongoing. 27 28 29 30 31 32 Keywords environmental exposures, mercury, metal mixtures, pregnant women, pediatric

33 http://bmjopen.bmj.com/ 34 35 neurodevelopment, Suriname 36 37 38 39 40 Word count 3999 (excl. references, acknowledgements, tables & figures) 41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Strengths and limitations of this study 48 49 50  The study addresses two high priority public health threats in Suriname and neighboring 51 52 53 countries in the Guiana Shield: the impact of mercury exposure from artisanal gold mining 54 55 56 57 58 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 and pesticide exposure associated with agricultural practices in two vulnerable 4 5 6 subpopulations: pregnant women and children younger than 5 years of age. 7 8  The longitudinal follow up of children to 48 months provides several timepoints to assess 9 10 neurodevelopmental outcomes. 11 12 13  The study has a biospecimen bank of approximately 13,000 samples, providing the 14 15 opportunity for future biomarker analyses. 16 For peer review only 17 A linked research training grant facilitates the training of nine Surinamese PhD candidates 18  19 20 with dissertation research embedded in the study, thereby building critical in-country 21 22 environmental health research capacity. 23 24 25  The interior sub-cohort (N=200) was logistically difficult to reach, resulting in delayed 26 27 recruitment of some women in the second or early third trimester of pregnancy, data 28 29 collection and transport of cord blood specimens. This may limit our ability to understand 30 31 32 differential effects of exposure across gestation for the interior cohort; heel prick sampling

33 http://bmjopen.bmj.com/ 34 replaced cord blood collection at birth. 35 36 37 38 39 40 Introduction

41 on September 25, 2021 by guest. Protected copyright. 42 43 The Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH) 44 45 46 addresses high-priority environmental and occupational health risks in Suriname and those 47 48 common to the vulnerable Caribbean region, while preserving unique cultural traditions of 49 50 indigenous people and other health disparate populations [1]. Exposure to environmental 51 52 53 contaminants at levels of public health concern may adversely affect pregnancy, and pre- and 54 55 postnatal health in multiple ways: miscarriage, preterm delivery, intra-uterine growth retardation, 56 57 58 5 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 congenital anomalies, and behavioral and physical consequences in later developmental stages [2, 4 5 6 3, 4]. Prenatal exposure to multiple heavy metals is associated with adverse pediatric health 7 8 outcomes [5]. Pesticide exposure has been linked to fetal growth decrements and preterm birth, 9 10 and there is mounting evidence that exposure to pesticides during pre- and postnatal development 11 12 is associated with neurodevelopmental deficits in young children [6-10]. Exposure to contaminant 13 14 15 mixtures can exacerbate adverse health effects: e.g. lead, arsenic, and mercury (Hg) can potentiate 16 For peer review only 17 each other’s toxicity, even at individual levels below concentration expected to result in adverse 18 19 effects [11]. Despite this knowledge, no cumulative risk assessments have been conducted to date 20 21 22 addressing exposures to contaminant mixtures in Caribbean Low- and Middle-Income Countries 23 24 [12]. 25 26 27 The CCREOH- environmental epidemiology cohort study fills this gap by exploring the impact of 28 29 exposures to organic and inorganic neurotoxicants, including Hg, lead and multiple 30 31 32 organophosphate pesticides, on Surinamese pregnant women and their offspring using a

33 http://bmjopen.bmj.com/ 34 cumulative risk approach. Specifically, the study assesses the impact of exposures to chemical and 35 36 non-chemical stressors by examining the interaction between exposure to environmental chemicals 37 38 and social and psychological determinants of early neurodevelopment. Few prospective studies 39 40 have measured exposure prenatally through 48 months in mother/child dyads, and those that have

41 on September 25, 2021 by guest. Protected copyright. 42 43 showed contrasting results when evaluating executive function, a key neurodevelopmental 44 45 outcome [13-16]. Our hypothesis is that environmental exposures to mixtures of 46 47 48 neurodevelopmental toxicants and non-chemical stressors will result in increased adverse birth 49 50 outcomes and poorer child neurodevelopmental trajectories. Our research aims are to: 51 52 53  Identify exposures to a complex mixture of environmental elements and toxicants, 54 55 including Hg, lead, cadmium, aluminum, iron, manganese, tin, selenium and selected 56 57 58 6 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 pesticides through comprehensive dietary and environmental risk assessments, and 4 5 6 biomarker monitoring in Surinamese pregnant women and their offspring; 7 8  Assess levels of environmental elements and toxicants in fish, produce, rice, and 9 10 nutraceutical compounds; 11 12 13  Determine the association between levels of environmental elements and toxicants in 14 15 pregnant women and birth outcomes; 16 For peer review only 17  Assess the impact of mobile health technology-enabled community health workers 18 19 20 (CHWs) on birth outcomes and their associations with environmental contaminants; 21 22  Determine associations between prenatal, dietary, and environmental levels of elements 23 24 and toxicants and potential neuroprotective nutraceuticals on neurodevelopment. 25 26 27 CCREOH is funded by the Fogarty International Center at the US National Institutes of Health 28 29 30 (NIH). 31 32

33 http://bmjopen.bmj.com/ 34 35 36 Cohort description 37 38 39 Study area 40

41 on September 25, 2021 by guest. Protected copyright. 42 The Republic of Suriname is located on the northeastern coast of South America, bordered 43 44 by Brazil, Guyana, French Guiana, and the Atlantic Ocean. 90% of the population of 590,549 45 46 47 people live in the capital Paramaribo and the coastal area. The remainder live in the tropical 48 49 rainforest interior (90% of the landmass). Suriname’s multi-ethnic population consists of five main 50 51 groups: Hindustani (27%), Tribal (22%), Creole (16%), Javanese (14%), and Indigenous (4%) 52 53 54 [17]. 55 56 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 Recruitment 7 8 9 Pregnant women potentially exposed to complex environmental chemical- and non-chemical 10 11 12 stressors were recruited from three regions of Suriname: (1) Paramaribo, where pesticides are 13 14 primarily used for residential purposes; (2) Nickerie, the major rice producing region in western 15 16 Suriname where pesticideFor use peer is abundant; andreview (3) the tropical only rainforest interior, where Hg is used 17 18 in artisanal gold mining and the population is highly dependent on consumption of contaminated 19 20 21 fish [18]. Taking into account a potential 20% lost to follow up, we requested and were approved 22 23 to consent 1200 pregnant women and their singleton birth children by the Institutional Review 24 25 Boards of both the Government of Suriname and Tulane University. 26 27 28 85% of all deliveries in Suriname are hospital-based, with the remainder (14%) taking place in 29 30 31 primary health care clinics under the supervision of a general practitioner or midwife at the 32

33 Regional Health Department or a skilled healthcare worker at the Medical Mission Primary Health http://bmjopen.bmj.com/ 34 35 Care Suriname; a small proportion (1%) occurs at home. Recruitment sites included all hospitals 36 37 and clinics of the Regional Health Department in Paramaribo and Nickerie and the Medical 38 39 40 Mission Primary Health Care Suriname in the interior. In Nickerie, trained CHWs were integrated

41 on September 25, 2021 by guest. Protected copyright. 42 in the research team and played a key role in every aspect of the study, from recruitment to 43 44 assessments at every study time point prenatally and during the neurodevelopmental assessments 45 46 47 of the child cohort. Women who met the inclusion criteria were identified by their physician, 48 49 midwife, CHW or health assistant during regular prenatal appointments and invited to participate. 50 51 Inclusion criteria were: pregnant women 16 years or older; speaking Dutch, Sranan Tongo, 52 53 54 Sarnami, Saramaccan or Trio; singleton gestation; planning to deliver at one of the study hospitals, 55 56 prenatal clinics or midwife facilities associated with those hospitals or clinics; and signed informed 57 58 8 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 11 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 consent. Assent was obtained from participants aged 16 and 17 years. Informed consent forms and 4 5 6 questionnaires were translated into the local languages Sranan Tongo, Sarnami, Saramaccan or 7 8 Trio. If a participant was unable to read, the recruiter read the questions in the local language. 9 10 11 Infants were not eligible for follow-up if they were born before 33 completed weeks of gestation 12 13 and/or had a birthweight less than 2000 grams, significant medical or neurological condition, 14 15 Down syndrome, hydrocephalus, cerebral palsy, or significant visual or hearing impairment 16 For peer review only 17 18 inconsistent with neurocognitive testing. 19 20 21 Main maternal and infant/child determinants included biological determinants: maternal 22 23 anthropometrics, blood pressure, hemoglobin, liver and kidney function, fetal and postnatal growth 24 25 characteristics and health status, heavy metals (blood and hair), pesticides (urine), telomere length 26 27 28 (buccal swab) and medication use; environmental determinants: maternal and child diet and 29 30 exposure history; social determinants: ethnicity, social support, trauma, prenatal life events, 31 32 maternal education, household income, household size, employment status, and marital status.

33 http://bmjopen.bmj.com/ 34 35 Main maternal and infant/child outcomes were growth and physical development: pregnancy 36 37 complications, fetal and postnatal growth patterns, risk factors for maternal liver and renal 38 39 impairment; behavior and cognitive development: infant neuromotor development, autism 40

41 spectrum disorder and neuropsychological development; childhood diseases: infectious diseases, on September 25, 2021 by guest. Protected copyright. 42 43 44 respiratory and neurological disorders; health and healthcare: impact of mobile health technology- 45 46 enabled CHWs on prenatal health and birth outcomes, quality of life assessments and the impact 47 48 of health care system utilization on prenatal health and birth outcomes. 49 50 51 52 53 54 Data management & statistical plan 55 56 57 58 9 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 12 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 The CCREOH field team was trained in administering all questionnaires. Data from administered 4 5 6 questionnaires are recorded on secure, encrypted iPads, uploaded and managed using REDCap, 7 8 which serves as the study electronic, integrated data management system [19]. Data are uploaded 9 10 in REDCap’s training site for data cleaning prior to integrating questionnaire and biospecimen 11 12 data. Data records are maintained to examine trends on source of errors, duplicate records are 13 14 15 scrubbed, and accuracy is validated through cross-checks with original data files and medical 16 For peer review only 17 records obtained through the general practitioner, midwife, health assistant or hospital 18 19 administration. A comprehensive biospecimen tracking system tracks analyzed samples and the 20 21 22 study’s overall biospecimen repository. Communication of data updates and changes in the process 23 24 are delivered through emails and during bimonthly data management team meetings. The data 25 26 management platform allows for the interrogation of environmental, non-biospecimen and 27 28 29 biospecimen data in an integrated fashion to facilitate scientific inquiries associated with the 30 31 impact of exposures to complex mixtures of chemical and non-chemical stressors on pregnancy, 32

33 birth outcomes and neurodevelopment. http://bmjopen.bmj.com/ 34 35 36 De-identified data files are developed upon request of CCREOH investigators through 37 38 standardized data request forms; statistical analyses are tailored to the specific research question. 39 40 Frequency analyses are used for descriptive statistics and presented as means with standard

41 on September 25, 2021 by guest. Protected copyright. 42 43 deviations or median with interquartile range for continuous variables and proportions for 44 45 categorical variables. Associations between categorical variables are studied using crosstabs; 46 47 48 bivariate linear and logistic regression models are computed to study crude and independent 49 50 associations, respectively. Comparison between study sites will be analyzed by comparing 51 52 participants, or clustered depending the research question. 53 54 55 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Multiple logistic regression analysis will be used to develop predictive models for binomial and 4 5 6 multinomial variables, multiple linear regression to develop predictive models for continuous 7 8 outcome variables, and nonparametric methods if assumptions are not met. Sample size was 9 10 calculated based on a multiple linear regression model using a coefficient of determination of 0.10 11 12 and a R-square differential of 0.02. Using these parameters, a number of 495 was needed to have 13 14 15 an 80% power at a 0.05 level of significance. Multiple approaches to develop and compare models 16 For peer review only 17 for multiple exposures to toxic metals will be used. In the first approach, we will use a Bayesian 18 19 kernel machine regression, a nonparametric Bayesian variable selection framework that will allow 20 21 22 us to explore joint (or combined) effects of the multiple chemicals. In the second approach, 23 24 principal component analysis will be used to group correlated exposure variables using the 25 26 principal component scores as the main exposure measures. Finally, we will use structural equation 27 28 29 models to create latent constructs of similar exposure variables and then finding associations 30 31 between these latent constructs and the outcome variable. 32

33 http://bmjopen.bmj.com/ 34 35 36 37 Data Collection 38 39 40 Maternal data were collected during pregnancy at two timepoints (first/second and third trimester).

41 on September 25, 2021 by guest. Protected copyright. 42 Post- partum assessments are ongoing and target both mothers and children: biomarker and 43 44 questionnaire data are collected from mothers during the child’s neurodevelopmental testing at 12, 45 46 47 36, and 48 months. (Table 1). 48 49 50 51 52 53 Data collection during pregnancy 54 55 56 Questionnaires 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Table 1 provides a list of standardized validated questionnaires to assess both physiological and 4 5 6 psychosocial prenatal health. All questionnaires were translated into Dutch and other local 7 8 languages to address the multiple language needs of our interior sub-cohort. The main categories 9 10 of data collected from maternal participants were health status, demographics, reproductive health 11 12 history, social support, trauma history, exposure history, depression, perceived stress, prenatal life 13 14 15 events, health behavior, access to prenatal care, social status and diet. Questions on health behavior 16 For peer review only 17 were adapted from the Alcohol, Smoking and Substance Involvement Screening Test version 3 18 19 (ASSIST V3.0) that was developed for the World Health Organization (WHO). Information on 20 21 22 maternal diet was obtained by adapting the CDC’s National Health and Nutrition Examination 23 24 Survey (NHANES) into a culturally tailored dietary survey focusing on fish and produce 25 26 consumption, including frequency of intake and portion sizes. 27 28 29 30 31 32 Biospecimen collection

33 http://bmjopen.bmj.com/ 34 35 The study’s biospecimen repository, housed at the Academic Hospital Paramaribo’s (AZP) clinical 36 37 laboratory, contains 13,000 samples. All biospecimens are collected at study site hospitals and 38 39 40 shipped to the AZP laboratory using established chain-of-custody procedures. Samples collected

41 on September 25, 2021 by guest. Protected copyright. 42 from participants in remote areas were stored at 4°C for no more than 48 hours prior to delivery 43 44 and processing at the Academic Hospital Paramaribo clinical laboratory. Blood, serum, plasma, 45 46 47 and cord blood samples were aliquoted into 2 ml plastic freezer tubes and stored at -80°C. 48 49 50 400 frozen whole blood samples were shipped for analysis to the Wisconsin State Laboratory of 51 52 Hygiene Trace Element Research Laboratory. A chain-of-custody approach was used to ensure 53 54 samples were collected, maintained, processed, stored, shipped, and received according to 55 56 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 acceptable standards. Quality assurance/quality control (QA/QC) procedures for elemental 4 5 6 analyses and methylmercury analyses included reagent blanks, blank spikes (lab fortified blanks), 7 8 sample matrix spikes, ongoing precision and recovery spikes, second source spikes, sample matrix 9 10 duplicates, and external standard reference materials. All sample and QA/QC results were within 11 12 the acceptable recovery limits. Duplicates for all runs were within the acceptable relative percent 13 14 15 difference (%RPD) limits. 16 For peer review only 17 18 In these 400 maternal whole blood samples (200 Paramaribo, 100 Nickerie, and 100 interior 19 20 communities), we analyzed Hg, lead, cadmium, aluminum, manganese, tin, selenium and iron 21 22 using inductively coupled plasma-mass spectrometry (ICP-MS) or cold vapor atomic fluorescence 23 24 25 spectrometry (CVAFS). Total Hg and methylmercury using CVAFS were assessed in a small 26 27 subset of women (20-Paramaribo, 20-Nickerie, and 35-interior) to specify exposure sources. 28 29 30 Maternal hair samples were taken according to the following protocol: 1.5 gram of hair was cut as 31 32 close as possible to the hair roots with disinfected scissors and placed in patient coded ziplock

33 http://bmjopen.bmj.com/ 34 35 bags, stored at room temperature in a climate-controlled room and sent to the National Zoological 36 37 Collection of Suriname/Center for Environmental Research lab at the Anton de Kom University 38 39 of Suriname (NZCS/CMO) for total mercury analysis using Cold Vapour Atomic Absorption 40

41 Spectrometry (CVAAS). on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Urine collection for pesticides analyses 48 49 50 Urine samples from pregnant women were analyzed (sub-cohort N=218) from all three study sites. 51 52 53 Urine samples were aliquoted into 10 ml plastic tubes, stored at -20°C and shipped for analysis by 54 55 CDC’s environmental health laboratory using well-established protocols. The six dialkylphosphate 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 metabolites were measured using a modified method of Jayatilaka et al. [20] by solid phase 4 5 6 extraction high performance liquid chromatography-tandem mass spectrometry1. The eight 7 8 specific urinary metabolites were analyzed using a semi-automated solid phase extraction mass 9 10 spectrometry method and reversed-phase high performance liquid chromatography [21]. The 11 12 analysis panel consisted of dialkylphosphate metabolites of organophosphate pesticides (DAP’s) 13 14 15 including dimethylphosphate, diethylphosphate, dimethylthiophosphate, 16 For peer review only 17 dimethyldithiophosphate, diethylthiophosphate, and diethyldithiophosphate, and a universal panel 18 19 (UP) including 1 herbicide (2,4D) and its metabolite, 2,4-dichlorophenoxyacetic acid; 4 OP 20 21 22 insecticide metabolites (3,5,6-trichloro-2-pyridinol,2-isopropyl-4-methyl-6-hydroxypyrimidine, 23 24 para-nitrophenol, malathion dicarboxylic acid) and 3 pyrethroid metabolites (4-fluoro-3- 25 26 phenoxybenzoic acid, 3-phenoxybenzoic acid, trans-3-(2,2-dichlorovinyl)-2,2- 27 28 29 dimethylcyclopropane carboxylic acid). 30 31 32

33 http://bmjopen.bmj.com/ 34 35 Data collection from birth through 48 months 36 37 At birth, a cord blood sample was taken from infants for measurements of heavy metals. In case a 38 39 40 cord blood sample was not available, a blood sample was taken by heel prick shortly after birth.

41 on September 25, 2021 by guest. Protected copyright. 42 Baseline characteristics of all births (e.g. gender, weight, gestational age, and Apgar score) were 43 44 collected (Table 2). At each subsequent data collection timepoint (Table 1), the child’s growth is 45 46 47 measured and a blood sample is taken for heavy metal measurement. In addition, the child’s health 48 49 status is obtained through questionnaires on diet and history of infectious diseases, and respiratory 50 51 and neurological disorders. 52 53 54 55 56 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Buccal swab collection for telomere assessments 4 5 6 Isohelix SK1 buccal swabs (Cell Projects, Kent, United Kingdom) were used for the collection of 7 8 9 maternal and infant DNA for telomere length (TL) analyses. Swabs were air dried and then stored 10 11 with a dessicator pellet at 4 degrees until DNA is extracted. DNA is extracted using the QIAamp 12 13 DNA mini kit protocol (Qiagen, Valencia, CA) and stored at −80°C until assayed. Concentration 14 15 of dsDNA is quantified with a Qubit dsDNA BR assay kit (Invitrogen, Carlsbad, CA), purity of 16 For peer review only 17 18 the DNA is determined by using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, 19 20 Waltham, MA), and DNA integrity is confirmed by gel electrophoresis to ensure high molecular 21 22 weight DNA [22]. The average relative TL, represented by the telomere repeat copy number to 23 24 25 single gene (albumin) copy number (T/S) ratio, is determined with monochrome multiplex 26 27 quantitative real-time PCR using a BioRad CFX96 [23]. All samples are run in triplicate on 28 29 duplicate plates with a standard curve and known controls. 30 31 32

33 http://bmjopen.bmj.com/ 34 35 Behavior and cognitive development 36 37 38 Neurodevelopmental assessments include infant cognitive and motor development and behavior 39 40 at 12-27 months using the Bayley Scales of Infant Development as well as an assessment of

41 on September 25, 2021 by guest. Protected copyright. 42 43 cognitive and social-emotional development at 36 months, and measurement of executive function 44 45 at 48 months (Table 1). The Bayley assessment was administered by a team of trained 46 47 psychologists, psychiatrists, and members of the study team specifically trained to administer the 48 49 50 test. Data checking following data entry of each Bayley sub-scale is being conducted by other team 51 52 members. Administration of Bayley assessments will be completed by July 2020. Assessments at 53 54 36 and 48 months will take place as the cohort ages. 55 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 Data collection from fish 7 8 9 Previous assessments of carnivorous fish from different regions in the interior of Suriname showed 10 11 12 high levels of Hg, specifically the frequently consumed species Hoplias aimara (0.43-0.66ug/g) 13 14 and Serrasalmus rhombeus (0.23-1.38ug/g). These levels are well above international accepted 15 16 standards and in certainFor regions, peer up toreview 7 times the norm only for human consumption [24]. 17 18 Polyunsaturated omega-3 fatty acids (PUFA-3) docosahexaenoic acid and eicosapentaenoic acid 19 20 21 from fish consumption offer neuroprotective benefits during prenatal and pediatric development 22 23 which could potentially counteract mercury neurotoxicity [25-27]. To better inform fish 24 25 consumption risk, fatty acids in fish muscle tissue were measured in samples (N=5 per species) of 26 27 28 five freshwater and three marine fish species. 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Patient and public involvement 35 36 37 The CCREOH cohort study was preceded by a planning grant which included a pilot fish 38 39 40 assessment study as well as an initial hair mercury assessment among communities in the interior

41 on September 25, 2021 by guest. Protected copyright. 42 which required approval from the village Chiefs or Tribal captains. This community engagement 43 44 component provided us a rich foundation to build on as we developed the research questions. The 45 46 initial study design was revised based on guidance from an External Scientific Advisory Board 47 48 49 (EAB) and a multi-stakeholder Community Advisory Board (CAB). The CAB consists of 50 51 respected leaders representing organizations from the interior tribal and indigenous peoples, 52 53 midwives, general practitioners and environmentalists. The EAB members represent medical 54 55 56 directors and other clinicians of all participating hospitals, representatives from the Ministry of 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Health, the US Embassy, the Pan American Health Organization and the Caribbean Public Health 4 5 6 Agency (CARPHA). Both advisory boards are convened annually to inform and give updates 7 8 about the study progress and results, and to seek advice on future directions and dissemination of 9 10 results. 11 12 13 14 15 16 Dissemination of studyFor results peer review only 17 18 19 The study team is actively working with the EAB and CAB on a collaborative translation and 20 21 dissemination strategy of overall study results. Every participant with neurotoxicant levels of 22 23 24 imminent health concern is contacted and asked to repeat the test for confirmation. If the level 25 26 remains of concern, the participant is referred to her attending physician. Children with 27 28 neurodevelopmental delay are referred to a child psychologist. Participants and the general public 29 30 31 were invited to health fairs, meetings and conferences, where study results were presented and 32

33 discussed. CCREOH data have been presented at regional and international conferences, including http://bmjopen.bmj.com/ 34 35 CARPHA, the International Society of Environmental Epidemiology, the Consortium for 36 37 Universities in Global Health, the American Public Health Association and at scientific meetings 38 39 40 convened by the US NIH.

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 Findings to date 47 48 49 From December 2016 until July 2019, 1143 pregnant women were enrolled; 74 (6.5%) were 50 51 52 ineligible (Figure 1). Geographically, 738 women were enrolled in Paramaribo, 204 in Nickerie, 53 54 and 201 in the interior. In comparison to participants, non-participants were more often of Creole 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 and Tribal ethnicity, lived in Paramaribo, and had lower household income. Table 2 presents 4 5 6 baseline maternal characteristics: one in eight women was 16-19 years of age, ethnic distribution 7 8 was representative of the Surinamese population, and more than half had no or lower 9 10 vocational/secondary education level. 11 12 13 Of the 1069 babies born, 992 were enrolled (93%) (Figure 1). Initial neurodevelopmental 14 15 assessments using the Bayley instrument at 12-27 months in 832 infants are being analyzed. To 16 For peer review only 17 18 date, the total number of biospecimens (N=13,379) collected from 1143 pregnant women include 19 20 whole blood for trace elements (N=1994), whole blood collected in K2EDTA anticoagulant 21 22 (N=1994), serum collected in serum separator tubes (N=1994), plasma (N=1994), urine (N=1980), 23 24 25 buccal swabs (N=941), and hair (N=876); from 992 infants cord blood (N=441), blood from heel 26 27 prick (N=323), and buccal swabs (N=842). All samples not yet analyzed are archived for future 28 29 targeted and untargeted analyses. Available results include total mercury concentrations in hair 30 31 32 from pregnant women, prenatal maternal depression and perceived stress, level of pesticides in

33 http://bmjopen.bmj.com/ 34 produce, and dietary exposure to Hg in fish. Results of heavy metals in blood, urinary pesticide 35 36 metabolites, telomere analysis, and neurodevelopmental assessments are pending. 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 Biological specimens 43 44 45 Total hair Hg was measured in 876 participants from all three study sites. Overall, 39.1% had 46 47 elevated total mercury hair levels that exceeded the U.S. Environmental Protection Agency’s 48 49 50 (USEPA) action level (1.1µg/g hair) [28]. For women from the interior, Nickerie and Paramaribo 51 52 these percentages were 95.8%, 26.3%, and 25.5% respectively. Results for median total Hg 53 54 concentrations in hair from pregnant women from Paramaribo (N=522) were 0.64µg/g hair 55 56 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 (interquartile ranges (IQR) 0.36-1.09;range 0.00-7.12), from Nickerie (N=176) 0.73µg/g (IQR 4 5 6 0.45-1.05;0.00-5.79) and the interior (N=178) 3.48µg/g (IQR 1.92-7.39;0.38-18.20). Pregnant 7 8 women from the interior were exposed to high levels of Hg compared to women from the coastal 9 10 area and thus fetal exposures are expected to be high. Most of these women are primarily exposed 11 12 to methylmercury from consuming contaminated fish from local artisanal gold-mining activities 13 14 15 [18,24]. Other possible sources of exposure may include active participation in or living very close 16 For peer review only 17 to gold mining areas. Mercury concentrations from women residing in Paramaribo and Nickerie 18 19 are similar to those found in other studies [29,30]. 20 21 22 23 24 25 Prenatal depression and perceived stress 26 27 28 Depression and prenatal stress in 722 participants were assessed using the standardized Edinburgh 29 30 31 Postnatal Depression Scale (EPDS) (cut-off ≥12 for probable depression) and Cohen Perceived 32

33 Stress Scale (cut-off ≥20 for high perceived stress) respectively. One in four (24.9%) participants http://bmjopen.bmj.com/ 34 35 had EPDS scores indicative of probable depression, three in ten (30.2%) had high stress levels and 36 37 nearly half (49.1%) of these women had probable depression. 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Dietary exposure to pesticides 44 45 An assessment of Surinamese agricultural produce conducted by members of our study team found 46 47 pesticide residues exceeding European Union maximum residue limits, including prohibited- 48 49 50 worldwide endosulfan and lindane in the leafy vegetable tannia, Xanthosoma brasiliense [31]. An 51 52 interviewer-assisted NHANES-based dietary survey of 522 participants showed that 98.2% 53 54 reported consumption of leafy vegetables. Tannia was the most frequently consumed (89.3%); 55 56 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 36.5% participants had high intake rates of tannia (≥36 gram/day) [32]. Tannia is also a commonly 4 5 6 used vegetable in baby food preparation in Suriname. 7 8 9 10 11 Dietary exposure to Hg in fish 12 13 14 Dietary questionnaire analyses (n=990) showed an overall fish consumption of 96.1%. Respective 15 16 consumption in the Fortotal cohort peer of the three review most consumed carnivorousonly species Hoplias aimara, 17 18 Serrasalmus rhombeus and Cichla ocellaris, that are known to have high Hg levels, was 44.4%, 19 20 21 19.3%, and 26.3%, and was greater among the interior subcohort (N=123): 89.4%, 67.5%, and 22 23 74.8%, respectively. These dietary assessments support our hypothesis that fish consumption is 24 25 consistent with the Hg exposures. In addition, we have speciated Hg in blood and hair and found 26 27 28 that the majority of Hg in these human samples is methylmercury which is consistent with fish 29 30 consumption being the primary source of Hg exposure in our participants. 31 32

33 Measurements of fatty acids showed that freshwater species had higher levels of linoleic acid (2.0 http://bmjopen.bmj.com/ 34 35 vs. 0.2mg/g), alpha-linoleic acid (0.4 vs. 0.1mg/g), arachidonic acid (3.0 vs. 1.5mg/g), omega-6 36 37 fatty acids (6.5 vs. 2.3mg/g) and lower levels of eicosapentaenoic acid (0.8 vs. 1.9mg/g) compared 38 39 40 to marine species.

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 Strengths and limitations 47 48 49 This CCREOH study is the first to examine the potential impact of complex environmental 50 51 exposures on maternal and child health in Suriname by a multi-disciplinary team of research 52 53 54 scientists. Study’s strengths include: 1) addressing two high priority public health threats: the 55 56 impact of Hg exposure from artisanal gold mining, and pesticide exposure associated with 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 agricultural practices; 2) inclusion in the cohort of two vulnerable subpopulations: pregnant 4 5 6 women and children; 3) the longitudinal follow up with children until 48 months with built-in 7 8 timepoints to assess neurodevelopmental outcomes; and 4) a biospecimen bank of approximately 9 10 13,000 samples, providing the opportunity for future analyses within the cohort. Moreover, a 11 12 linked research training grant facilitates the training of nine Surinamese PhD candidates with 13 14 15 dissertation research embedded in the study, thereby building critical in-country environmental 16 For peer review only 17 health research capacity. 18 19 20 Delayed recruitment and data collection of the logistically difficult to reach sub-cohort in the 21 22 interior region (N=200) was ameliorated by additional team support onsite; delays in transporting 23 24 25 interior biospecimens rendered some early cord blood specimens not analyzable and was corrected 26 27 by collecting an additional heelprick sample. Other limitations include differences in educational 28 29 level between women from the three recruitment sites that could confound or impact literacy. 30 31 32 Participants may have moved between regions with different sources of exposure. Some

33 http://bmjopen.bmj.com/ 34 participants from the interior sub-cohort were recruited in the second or early third trimester 35 36 because of the distance to prenatal clinics. This may limit our ability to understand differential 37 38 effects of exposure across gestation for this sub-cohort. 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 Future plans 46 47 48 Culturally tailored fish consumption advisories are in development to reduce Hg exposure. Recent 49 50 additional funding allows us to examine potentially beneficial neuroprotective factors such as 51 52 53 selenium and dietary polyunsaturated fatty acids in fish that may counter the neurotoxic effects of 54 55 mercury. To address the large scale and poorly regulated use of pesticides, we are also developing 56 57 58 21 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 a pesticide literacy scale. Cohort-wide, we will undertake a comprehensive assessment of the 4 5 6 neurodevelopmental outcomes of the children, and create a robust, sharable data management 7 8 system of biospecimen and non-biospecimen data building on the current REDCap database. 9 10 11 12 13 14 Acknowledgements 15 16 For peer review only 17 18 We would like to thank our recruiters Jeetendra Jitan, Nishaira Doerga, Gimradj Dwarka, Sue-Ann 19 20 Sitaram, Devika Kalika, the Community Health Workers from Nickerie Lilian Singotiko, Henna 21 22 Mawo, Karien Jodiwongso, Karishma Bhondoe, Jay Kalicharan, Bhairam Goberdan, Azeez 23 24 Khodabaks, the midwives and laboratory personnel of the Academic Hospital Paramaribo, 25 26 27 Diakonessenhuis Hospital, St. Vincentius Hospital, ‘s-Lands Hospital, Mungra Medical Centre, 28 29 Regional Health Service clinics and Medical Mission Primary Health Care Suriname, the National 30 31 Zoological Collection of Suriname/Center for Environmental Research staff, Sila Kisoensingh 32

33 http://bmjopen.bmj.com/ 34 MSc, Dr. Richenel Ellecom and Dr. Melissa Middleton for their help during the pediatric 35 36 neurodevelopment assessments, and the members of the External Advisory Board and Community 37 38 Advisory Board for their guidance and valuable advice. 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 Collaborators 46 47 48 We welcome collaboration with fellow researchers working on similar projects. Specific research 49 50 proposals can be sent directly to the Principal Investigators Drs. Wilco Zijlmans 51 52 53 ([email protected]) and Maureen Lichtveld ([email protected]). 54 55 Areas of collaboration include further environmental and biomarkers assessment of arsenic 56 57 58 22 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 exposure, occupational health risk assessments associated with mercury and pesticide exposure, 4 5 6 and exposomic analyses including metabolomic assessments. 7 8 9 10 11 Author Contributions 12 13 14 MYL, JKW, HHC, EWH, AS, SSD and WCWRZ designed and established the cohort. MSMO, 15 16 ADHM, EWH, FAWFor and AG peer participated reviewin the design of the only questionnaires. WCWRZ, MSMO, 17 18 19 EB, WBH, GKB, RR and MYL are responsible for the continued management of the cohort. GKB, 20 21 RR, AG and FAW are actively involved with recruitment of participants and collection of non- 22 23 biospecimen data and biospecimens. JC, JR, PEO and GAL store, archive and analyze the 24 25 biomarker samples. ADHM, AS, MSMO, AG, MYL and WCWRZ are responsible for data 26 27 28 management. SSD, AWEG, RR and EB manage neurodevelopmental testing of babies. JKW, LFS, 29 30 CSA, SSD interpret all results. WCWRZ, JKW, HHC, ADHM and MYL drafted and edited the 31 32 manuscript. All authors critically reviewed and approved the final manuscript.

33 http://bmjopen.bmj.com/ 34 35 36 37 38 Funding 39 40

41 Research reported in this publication was supported by the Fogarty International Center of the on September 25, 2021 by guest. Protected copyright. 42 43 44 National Institutes of Health under Award Numbers: U01TW010087 and U2RTW010104. The 45 46 content is solely the responsibility of the authors and does not necessarily represent the official 47 48 views of the National Institutes of Health. 49 50 51 52 53 54 55 Competing interests 56 57 58 23 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 26 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 None declared 4 5 6 7 8 9 10 Patient consent for publication 11 12 13 Obtained 14 15 16 For peer review only 17 18 19 Ethical approval 20 21 22 23 This study was IRB approved by both the Government of Suriname and the Tulane University, 24 25 New Orleans, Louisiana, USA, to consent 1200 pregnant women and their singleton birth 26 27 children. 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Provenance and peer review 35 36 37 Not commissioned; externally peer reviewed. 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Data sharing statement 44 45 46 47 Our cohort study data will be made publicly available upon publication of the results in scientific 48 49 articles. Questionnaire, registry and biospecimen data could be made accessible in de-identified 50 51 form after an application process that includes submission of a research plan. We are currently 52 53 collaborating with Research Triangle Institute International (RTI), a global data management 54 55 56 enterprise, to develop an integrated database of biospecimen and non-biospecimen data. Once 57 58 24 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 27 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 that is fully developed, data can be made available based on a reasonable request to the PIs. Such 4 5 6 requests will be discussed with the full investigator Committee, the Data Management 7 8 Committee, and the Administrative Oversight Committee. 9 10 11 12 13 14 References 15 16 For peer review only 17 18 1. Lichtveld MY, Hawkins WB, Ouboter PE et al. A One Health approach to interdict 19 20 environmental health threats in Suriname. Ann Glob Health 2016;82:444. 21 22 23 Silbergeld EK, Patrick TE. Environmental exposures, toxicologic mechanisms, and adverse 24 2. 25 26 pregnancy outcomes. Am J Obstet Gynecol 2005;192:S11-21. 27 28 29 3. Ferguson KK, O'Neill MS, Meeker JD. Environmental contaminant exposures and preterm 30 31 birth: a comprehensive review. J Toxicol Environ Health B Crit Rev 2013;16:69-113. 32

33 http://bmjopen.bmj.com/ 34 4. Wigle DT, Arbuckle TE, Turner MC et al. Epidemiologic evidence of relationships between 35 36 reproductive and child health outcomes and environmental chemical contaminants. J Toxicol 37 38 39 Environ Health B Crit Rev 2008;11:373-517. 40

41 on September 25, 2021 by guest. Protected copyright. 42 5. Lewis M, Worobey J, Ramsay DS et al. Prenatal exposure to heavy metals: effect on childhood 43 44 cognitive skills and health status. Pediatrics 1992;89:1010-5. 45 46 47 6. Burns CJ, McIntosh LJ, Mink PJ, Jurek AM, Li AA. Pesticide exposure and 48 49 neurodevelopmental outcomes: Review of the epidemiologic and animal studies. J Toxicol 50 51 52 Environ Health B Crit Rev 2013;16:127-283. 53 54 55 56 57 58 25 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 28 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

41 on September 25, 2021 by guest. Protected copyright. 42 prospective population study. Mol Autism 2018;9:30. 43 44 45 14. Valent F, Mariuz M, Bin M et al. Associations of prenatal mercury exposure from maternal 46 47 48 fish consumption and polyunsaturated fatty acids with child neurodevelopment: a prospective 49 50 cohort study in Italy. J Epidemiol 2013;23:360-70. 51 52 53 54 55 56 57 58 26 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 29 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 15. Sapbamrer R, Hongsibsong S. Effects of prenatal and postnatal exposure to organophosphate 4 5 6 pesticides on child neurodevelopment in different age groups: a systematic review. Environ 7 8 Sci Pollut Res Int 2019;26:18267-90. 9 10 11 16. Wang J, Wu W, Li H et al. Relation of prenatal low-level mercury exposure with early child 12 13 neurobehavioral development and exploration of the effects of sex and DHA on it. Environ Int 14 15 2019;126:14-23. 16 For peer review only 17 18 19 17. Results Eighth Population and Housing Census in Suriname (Volume 1) Demographic and 20 21 Social Characteristics and Migration. General Bureau of Statistic Studies in Suriname (ABS) 22 23 2013. 24 25 26 18. Ouboter PE, Landburg G, Satnarain GU et al. Mercury Levels in Women and Children from 27 28 29 Interior Villages in Suriname, South America. Int J Environ Res Public Health 2018;15:1007. 30 31 32 19. Harris PA, Taylor R, Minor BL et al. The REDCap consortium: Building an international

33 http://bmjopen.bmj.com/ 34 community of software partners, J Biomed Inform 2019;95:1-10. doi: 35 36 10.1016/j.jbi.2019.103208 37 38 39 20. Jayatilaka NK, Restrepo P, Williams L, et al. Quantification of three chlorinated dialkyl 40

41 on September 25, 2021 by guest. Protected copyright. 42 phosphates, diphenyl phosphate, 2,3,4,5-tetrabromobenzoic acid, and four other 43 44 organophosphates in human urine by solid phase extraction-high performance liquid 45 46 chromatography-tandem mass spectrometry. Anal Bioanal Chem 2017;409:1323-32. 47 48 49 21. Davis MD, Wade EL, Restrepo PR, et al. Semi-automated solid phase extraction method for 50 51 the mass spectrometric quantification of 12 specific metabolites of organophosphorus 52 53 54 pesticides, synthetic pyrethroids, and select herbicides in human urine. Journal of 55 56 Chromatography B. 2013;929:18-26 57 58 27 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 30 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 22. Stacy S. Drury, Emily Mabile, Zoë H. Brett et al. The Association of Telomere Length With 4 5 6 Family Violence and Disruption. Pediatrics 2014;134:e128–e137. 7 8 9 23. Cawthon RM. Telomere length measurement by a novel monochrome multiplex qualitative 10 11 PCR method. Nucleic Acids Res 2009;37:e21. 12 13 14 24. Ouboter PE, Landburg GA, Quik JHM, Mol JHA, van der Lugt F. Mercury Levels in Pristine 15 16 and Gold MiningFor Impacted peer Aquatic review Ecosystems of Suriname,only South America. Ambio 17 18 19 2012;41:873-82. 20 21 22 25. Dellinger MJ, Olson JT, Holub BJ, Ripley MP. Mercury, polychlorinated biphenyls, selenium, 23 24 and fatty acids in tribal fish harvests of the upper great lakes. Risk Anal 2018;38:2029-2040. 25 26 27 26. Ginsberg GL, Toal BF, McCann PJ. Updated risk/benefit analysis of fish consumption effects 28 29 on neurodevelopment: Implications for setting advisories. Hum. Ecol. Risk Assess 30 31 32 2015;21:1810-1839.

33 http://bmjopen.bmj.com/ 34 35 27. Clarkson TW, Strain JJ. Nutritional factors may modify the toxic action of methyl mercury in 36 37 fish-eating populations. J. Nutr 2003;133:1539S-1543S. 38 39 40 28. Mercury Study Report to Congress Volume V: Health Effects of Mercury and Mercury

41 on September 25, 2021 by guest. Protected copyright. 42 Compounds. Volume 5. United States Environmental Protection Agency; Washington, DC, 43 44 45 USA: 1997;1–348. 46 47 48 29. Zilversmit L, Wickliffe J, Shankar A, Taylor R, Harville E. Correlations of biomarkers and 49 50 self-reported seafood consumption among pregnant and non-pregnant women in southeastern 51 52 Louisiana after the Gulf oil spill: the GROWH study. Int J Environ Res Public Health 2017;14: 53 54 55 784. 56 57 58 28 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 31 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 30. Castaño A, Cutanda F, Esteban M, et al. Fish consumption patterns and hair mercury levels in 4 5 6 children and their mothers in 17 EU countries. Environ Res 2015;141:58-68. 7 8 9 31. Abdoel Wahid F, Wickliffe J, Wilson M et al. Presence of pesticide residues on produce 10 11 cultivated in Suriname. Environ Monit Assess 2017;189:303. 12 13 14 32. F. Abdoel Wahid, A. D. Hindori-Mohangoo, M. Wilson et al. Dietary Exposure to Pesticides 15 16 in Pregnant For Surinamese peer Women. review Environ only Health Perspect 2018; 17 18 19 https://doi.org/10.1289/isesisee.2018.P01.2380 20 21 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 29 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 32 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 Table 1. Assessments completed by the CCREOH-MeKiTamara Cohort with timeline 6 7 Assessments Trimester Birth 12 36 48 8 mos mos mos 9 1st/2nd 3rd 10 Mother 11 Obstetric history ● ● 12 Demographics ● 13 Residency ● ● 14 Anthropometrics ● ● 15 Marital status ● 16 For peer review only 17 Ethnicity ● 18 Occupation ● 19 Education ● 20 Household income ● 21 Household composition ● 22 Maternity care ● ● 23 Medication ● 24 Questionnaires 25 SF 36 Health Survey ● ● 26 Social Support List ● 27 Brief Trauma Interview ● ● ● ● 28 Cohen’s Perceived Stress Scale ● ● ● ● 29 Edinburgh Depression Scale ● ● ● ● 30 ASSIST V3.0 ● ● ● ● 31 Exposure History ● 32 Prenatal Life Events Scale ● ●

33 http://bmjopen.bmj.com/ Subjective Social Status ● ● 34 35 Dietary Assessment ● ● 36 Family Environment Scale ● ● 37 Parenting Stress Index ● ● 38 Biological samples 39 Hair ● 40 Blood ● ● Urine ● ● 41 on September 25, 2021 by guest. Protected copyright. 42 Buccal swab ● 43 44 At birth 45 Mode of delivery ● 46 Cord or heelprick blood sample ● 47 Birth outcomes ● 48 49 Child development 50 Physical examination ● ● ● 51 Questionnaires 52 Generation R ● ● ● 53 M-CHAT ● 54 55 Child Behavior Checklist ● ● 56 Bayley SEQ ● 57 58 30 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 33 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Ages and Stages Questionnaire ● 4 Neurodevelopmental tests 5 BSID-III ● 6 CANTAB ● 7 Biological samples 8 Buccal swab ● ● ● 9 10 Blood ● ● 11 Notes: ASSIST V3.0 = Alcohol, Smoking and Substance Involvement Screening Test Version 3, Subjective 12 Social Status = MacArthur Scale of Subjective Social Status, M-CHAT = Modified Checklist for Autism 13 in Toddlers, Bayley SEQ = Bayley Social Emotional Questionnaire, BSID-III = Bayley Scales of Infant 14 Development Third Edition, CANTAB = Cambridge Neuropsychological Test Automated Battery. 15 Generation R = questionnaires on pediatric health and diet history (with permission from The Generation 16 R Study Group) For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 31 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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1 2 3 Table 2. Distribution of maternal and infant characteristics 4 5 Maternal characteristics N % Infant characteristics (N=971) N % 6 (N=1143) 7 8 Age at intake Gender 9 16-19 years 142 12.6 Male 510 52.5 10 20-24 years 260 22.8 Female 457 47.1 11 25-29 years 291 25.5 Missing 4 0.4 12 30-34 years For268 peer23.5 review only 13 35-39 years 140 12.3 Birth status 14 40+ years 39 3.4 Live birth 947 97.5 15 16 Missing 1 0.1 Stillbirth 23 2.4 17 Missing 1 0.1

18 Parity http://bmjopen.bmj.com/ 19 0 previous live births 384 33.7 Birthweight (in grams) 20 1 previous live birth 312 27.3 Low birthweight (<2500) 127 13.1 21 2 previous live births 187 16.4 Normal birthweight (≥2500) 835 86.0 22 23 3 previous live births 112 9.8 Missing 9 0.9 24 4+ previous live births 146 12.8 25 Missing 2 0.2 Gestational age (in weeks)

26 Very preterm births (22+0-32+6) on September 25, 2021 by guest. Protected copyright. 39 4.0 27 Ethnicity Moderately preterm births (33+0-36+6) 107 11.0 28 Creole 249 21.8 Term births (≥37+0) 817 84.1 29 Hindustani 233 20.4 Missing 8 0.8 30 31 Indigenous 155 13.6 32 Javanese 101 8.8 Apgar score at 5 minutes 33 Tribal 271 23.7 0 - 6 31 3.2 34 Mixed 127 11.1 7 - 10 918 94.5 35 Other 7 0.6 Missing 22 2.3 36 Missing 0 0.0 37 38 39 Educational level 40 No or primary 276 24.1 41 Lower vocational/secondary 382 33.4 42 43 32 44 45 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

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1 2 3 Upper vocational/secondary 317 27.7 4 Tertiary 168 14.7 5 6 Missing 0 0.0 7 8 Household income SRD* 9 <1500 401 35.1 10 1500-2999 362 33.0 11 3000-4999 221 20.2 12 5000+ For112 peer9.8 review only 13 14 Missing 47 4.1 15 16 Marital status 17 Married or living with partner 1000 87.6

18 Not married/not living with 141 12.4 http://bmjopen.bmj.com/ 19 partner 20 21 Missing 2 0.2 22 * SRD = Surinamese dollar, equivalent to 0.13 USD. 23 24 25

26 on September 25, 2021 by guest. Protected copyright. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 33 44 45 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 BMJ Open Page 36 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Figure legends 4 5 6 7 Figure 1. Flowchart with participant enrolment. 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 34 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 37 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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ForJournal: peerBMJ Open review only Manuscript ID bmjopen-2019-034702.R3

Article Type: Cohort profile

Date Submitted by the 28-Jul-2020 Author:

Primary Subject Occupational and environmental medicine Heading:

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33 http://bmjopen.bmj.com/ 34 35 36 Affiliations 37 38 39 1 Scientific Research Center Suriname, Academic Hospital Paramaribo, Paramaribo, Suriname 40

41 on September 25, 2021 by guest. Protected copyright. 42 2 Department of Environmental Health Sciences, Tulane University School of Public Health and 43 44 Tropical Medicine, New Orleans, LA, United States of America 45 46 47 3 Faculty of Medical Sciences, Anton de Kom University of Suriname, Paramaribo, Suriname 48 49 50 4 Foundation for Perinatal Interventions and Research in Suriname (Perisur), Paramaribo, 51 52 53 Suriname 54 55 56 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 5 Department of Clinical Chemistry Medical Microbiology, Academic Medical Center, 4 5 6 Amsterdam, The Netherlands 7 8 6 9 National Zoological Collection of Suriname/Center for Environmental Research, Anton de Kom 10 11 University of Suriname, Paramaribo, Suriname 12 13 14 7 Institute for Neotropical Wildlife & Environmental Studies, Paramaribo, Suriname 15 16 8 For peer review only 17 Department of Clinical Chemistry, Academic Hospital Paramaribo, Paramaribo, Suriname 18 19 9 20 Medical Mission Primary Health Care Suriname, Paramaribo, Suriname 21 22 10 23 Regional Health Service, Nickerie, Suriname 24 25 11 26 Department of Public Health, Ministry of Health, Paramaribo, Suriname 27 28 12 29 Department of Health Policy, Vanderbilt University Medical Center, Nashville, TN, United 30 31 States of America 32

1 2 3 Abstract 4 5 6 7 Purpose: The Caribbean Consortium for Research in Environmental and Occupational Health 8 9 10 (CCREOH) prospective environmental epidemiologic cohort study addresses the impact of 11 12 13 chemical and non-chemical environmental exposures in mother/child dyads in Suriname. The 14 15 study determines associations between levels of environmental elements and toxicants in pregnant 16 For peer review only 17 18 women, and birth outcomes and neurodevelopment in their children. 19 20 21 22 Participants: Pregnant women (N=1143) were enrolled from December 2016 to July 2019 from 23 24 25 three regions of Suriname: Paramaribo (N=738), Nickerie (N=204), and the tropical rainforest 26 27 28 interior (N=201). Infants (N=992) were enrolled at birth. Follow-up will take place until children 29 30 31 are 48 months old. 32

33 http://bmjopen.bmj.com/ 34 Findings to date: Biospecimens and questionnaire data on physiological and psychosocial health 35 36 37 in pregnant women have been analyzed. 39.1% had hair mercury (Hg) levels exceeding values 38 39 40 considered safe by international standards. Median hair-Hg concentrations in women from

41 on September 25, 2021 by guest. Protected copyright. 42 43 Paramaribo (N=522) were 0.64µg/g hair (interquartile ranges (IQR) 0.36-1.09; range 0.00-7.12), 44 45 46 from Nickerie (N=176) 0.73µg/g (IQR 0.45-1.05;0.00-5.79) and the interior (N=178) 3.48µg/g 47 48 (IQR 1.92-7.39;0.38-18.20). 96.1% of women ate fish, respective consumption of the three most 49 50 51 consumed carnivorous species, Hoplias aimara, Serrasalmus rhombeus, and Cichla ocellaris, 52 53 54 known to have high Hg levels, was 44.4%, 19.3%, and 26.3%, and was greater among the interior 55 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 subcohort. 89% frequently consumed the vegetable tannia, samples of which showed presence of 4 5 6 worldwide banned pesticides. 24.9% of pregnant women had Edinburgh Depression Scale scores 7 8 9 indicative of probable depression. 10 11 12 Future plans: Fish consumption advisories are in development, especially relevant to interior 13 14 15 women for whom fish consumption is likely to be the primary source of Hg exposure. Effects of 16 For peer review only 17 18 potentially beneficial neuroprotective factors in fish that may counter neurotoxic effects of Hg are 19 20 21 being examined. A pesticide literacy assessment in pregnant women is in progress. 22 23 Neurodevelopmental assessments and telomere length measurements of the children to evaluate 24 25 26 long-term effects of prenatal exposures to toxicant mixtures are ongoing. 27 28 29 30 31 32 Keywords environmental exposures, mercury, metal mixtures, pregnant women, pediatric

33 http://bmjopen.bmj.com/ 34 35 neurodevelopment, Suriname 36 37 38 39 40 Word count 3997 (excl. references, acknowledgements, tables & figures) 41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Strengths and limitations of this study 48 49 50  The study addresses two high priority public health threats in Suriname and neighboring 51 52 53 countries in the Guiana Shield: the impact of mercury exposure from artisanal gold mining 54 55 56 57 58 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 and pesticide exposure associated with agricultural practices in two vulnerable 4 5 6 subpopulations: pregnant women and children younger than 5 years of age. 7 8  The longitudinal follow up of children to 48 months provides several timepoints to assess 9 10 neurodevelopmental outcomes. 11 12 13  The study has a biospecimen bank of approximately 13,000 samples, providing the 14 15 opportunity for future biomarker analyses. 16 For peer review only 17 A linked research training grant facilitates the training of nine Surinamese PhD candidates 18  19 20 with dissertation research embedded in the study, thereby building critical in-country 21 22 environmental health research capacity. 23 24 25  The interior sub-cohort (N=201) was logistically difficult to reach, resulting in delayed 26 27 recruitment of some participants until the second or early third trimester. This may limit 28 29 our ability to understand differential effects of exposure across gestation. Transportation 30 31 32 challenges resulted in heel prick sampling replacing cord blood collection.

33 http://bmjopen.bmj.com/ 34 35 36 37 38 Introduction 39 40

41 The Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH) on September 25, 2021 by guest. Protected copyright. 42 43 addresses high-priority environmental and occupational health risks in Suriname and those 44 45 46 common to the vulnerable Caribbean region, while preserving unique cultural traditions of 47 48 indigenous people and other health disparate populations [1]. Exposure to environmental 49 50 contaminants at levels of public health concern may adversely affect pregnancy, and pre- and 51 52 53 postnatal health in multiple ways: miscarriage, preterm delivery, intra-uterine growth retardation, 54 55 congenital anomalies, and behavioral and physical consequences in later developmental stages [2, 56 57 58 5 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 3, 4]. Prenatal exposure to multiple heavy metals is associated with adverse pediatric health 4 5 6 outcomes [5]. Pesticide exposure has been linked to fetal growth decrements and preterm birth, 7 8 and there is mounting evidence that exposure to pesticides during pre- and postnatal development 9 10 is associated with neurodevelopmental deficits in young children [6-10]. Exposure to contaminant 11 12 mixtures can exacerbate adverse health effects: e.g. lead, arsenic, and mercury (Hg) can potentiate 13 14 15 each other’s toxicity, even at individual levels below concentration expected to result in adverse 16 For peer review only 17 effects [11]. Despite this knowledge, no cumulative risk assessments have been conducted to date 18 19 addressing exposures to contaminant mixtures in Caribbean Low- and Middle-Income Countries 20 21 22 [12]. 23 24 25 The CCREOH environmental epidemiology cohort study fills this gap by exploring the impact of 26 27 exposures to organic and inorganic neurotoxicants, including Hg, lead and multiple 28 29 organophosphate pesticides, on Surinamese pregnant women and their offspring using a 30 31 32 cumulative risk approach. Specifically, the study assesses the impact of exposures to chemical and

33 http://bmjopen.bmj.com/ 34 non-chemical stressors by examining the interaction between exposure to environmental chemicals 35 36 and social and psychological determinants of early neurodevelopment. Few prospective studies 37 38 have measured exposure prenatally through 48 months in mother/child dyads, and those that have 39 40 showed contrasting results when evaluating executive function, a key neurodevelopmental

41 on September 25, 2021 by guest. Protected copyright. 42 43 outcome [13-16]. Our hypothesis is that environmental exposures to mixtures of 44 45 neurodevelopmental toxicants and non-chemical stressors will result in increased adverse birth 46 47 48 outcomes and poorer child neurodevelopmental trajectories. Our research aims are to: 49 50 51  Identify exposures to a complex mixture of environmental elements and toxicants, 52 53 including Hg, lead, cadmium, aluminum, iron, manganese, tin, selenium and selected 54 55 56 57 58 6 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

33 http://bmjopen.bmj.com/ 34 35 36 Cohort description 37 38 39 Study area 40

41 on September 25, 2021 by guest. Protected copyright. 42 The Republic of Suriname is located on the northeastern coast of South America, bordered 43 44 by Brazil, Guyana, French Guiana, and the Atlantic Ocean. 90% of the 590,549 population lives 45 46 47 in the capital Paramaribo and the coastal area. The remainder live in the tropical rainforest interior 48 49 (90% of the landmass). Suriname’s multi-ethnic population consists of five main groups: 50 51 Hindustani (27%), Tribal (22%), Creole (16%), Javanese (14%), and Indigenous (4%) [17]. 52 53 54 55 56 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Recruitment 4 5 6 Pregnant women potentially exposed to complex environmental chemical- and non-chemical 7 8 9 stressors were recruited from three regions of Suriname: (1) Paramaribo, where pesticides are 10 11 primarily used for residential purposes; (2) Nickerie, the major rice producing region in western 12 13 Suriname where pesticide use is abundant; and (3) the tropical rainforest interior, where Hg is used 14 15 in artisanal gold mining and the population is highly dependent on consumption of contaminated 16 For peer review only 17 18 fish [18]. Taking into account a potential 20% lost to follow up, we requested and were approved 19 20 to consent 1200 pregnant women and their singleton birth children by the Institutional Review 21 22 Boards of both the Government of Suriname and Tulane University. 23 24 25 85% of all deliveries in Suriname are hospital-based, with the remainder (14%) taking place in 26 27 28 primary health care clinics under the supervision of a general practitioner or midwife at the 29 30 Regional Health Department or a skilled healthcare worker at the Medical Mission Primary Health 31 32 Care Suriname; a small proportion (1%) occurs at home. Recruitment sites included all hospitals

33 http://bmjopen.bmj.com/ 34 35 and clinics of the Regional Health Department in Paramaribo and Nickerie and the Medical 36 37 Mission Primary Health Care Suriname in the interior. In Nickerie, trained CHWs were integrated 38 39 in the research team and played a key role in every aspect of the study, from recruitment to 40

41 assessments at every study time point prenatally and during the neurodevelopmental assessments on September 25, 2021 by guest. Protected copyright. 42 43 44 of the child cohort. Women who met the inclusion criteria were identified by their physician, 45 46 midwife, CHW or health assistant during regular prenatal appointments and invited to participate. 47 48 Inclusion criteria were: pregnant women 16 years or older; speaking Dutch, Sranan Tongo, 49 50 51 Sarnami, Saramaccan or Trio; singleton gestation; planning to deliver at one of the study hospitals, 52 53 prenatal clinics or midwife facilities associated with those hospitals or clinics; and signed informed 54 55 consent. Assent was obtained from participants aged 16 and 17 years. Informed consent forms and 56 57 58 8 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 11 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 questionnaires were translated into the local languages Sranan Tongo, Sarnami, Saramaccan or 4 5 6 Trio. If a participant was unable to read, the recruiter read the questions in the local language. 7 8 9 Infants were not eligible for follow-up if they were born before 33 completed weeks of gestation 10 11 and/or had a birthweight less than 2000 grams, significant medical or neurological condition, 12 13 Down syndrome, hydrocephalus, cerebral palsy, or significant visual or hearing impairment 14 15 inconsistent with neurocognitive testing. 16 For peer review only 17 18 Main maternal and infant/child determinants included biological determinants: maternal 19 20 21 anthropometrics, blood pressure, hemoglobin, liver and kidney function, fetal and postnatal growth 22 23 characteristics and health status, heavy metals (blood and hair), pesticides (urine), telomere length 24 25 (buccal swab) and medication use; environmental determinants: maternal and child diet and 26 27 28 exposure history; social determinants: ethnicity, social support, trauma, prenatal life events, 29 30 maternal education, household income, household size, employment status, and marital status. 31 32 Main maternal and infant/child outcomes were growth and physical development: pregnancy

33 http://bmjopen.bmj.com/ 34 35 complications, fetal and postnatal growth patterns, risk factors for maternal liver and renal 36 37 impairment; behavior and cognitive development: infant neuromotor development, autism 38 39 spectrum disorder and neuropsychological development; childhood diseases: infectious diseases, 40

41 respiratory and neurological disorders; health and healthcare: impact of mobile health technology- on September 25, 2021 by guest. Protected copyright. 42 43 44 enabled CHWs on prenatal health and birth outcomes, quality of life assessments and the impact 45 46 of health care system utilization on prenatal health and birth outcomes. 47 48 49 50 51 52 Data management & statistical plan 53 54 55 56 57 58 9 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 12 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 The CCREOH field team was trained in administering all questionnaires. Data from administered 4 5 6 questionnaires are recorded on secure, encrypted iPads, uploaded and managed using REDCap, 7 8 which serves as the study electronic, integrated data management system [19]. Data are uploaded 9 10 in REDCap’s training site for data cleaning prior to integrating questionnaire and biospecimen 11 12 data. Data records are maintained to examine trends on source of errors, duplicate records are 13 14 15 scrubbed, and accuracy is validated through cross-checks with original data files and medical 16 For peer review only 17 records obtained through the general practitioner, midwife, health assistant or hospital 18 19 administration. A comprehensive biospecimen tracking system tracks analyzed samples and the 20 21 22 study’s overall biospecimen repository. Communication of data updates and changes in the process 23 24 are delivered through emails and during bimonthly data management team meetings. The data 25 26 management platform allows for the interrogation of environmental, non-biospecimen and 27 28 29 biospecimen data in an integrated fashion. 30 31 32 De-identified data files are developed upon request of CCREOH investigators through

33 http://bmjopen.bmj.com/ 34 standardized data request forms. Statistical analysis plans are tailored to specific research 35 36 questions and include descriptive statistics, bivariate linear and logistic regression models, 37 38 multiple logistic regression analysis to develop predictive models, and nonparametric methods if 39 40 assumptions are not met. Comparison between study sites will be analyzed by comparing

41 on September 25, 2021 by guest. Protected copyright. 42 43 participants, or clustered depending the research question. 44 45 46 Multiple approaches to develop and compare models for multiple exposures to toxic metals will 47 48 be used including a Bayesian kernel machine regression and a nonparametric Bayesian variable 49 50 51 selection framework to explore joint (or combined) effects of the multiple chemicals. Structural 52 53 equation models will be employed to create latent constructs of similar exposure variables and 54 55 then finding associations between these latent constructs and the outcome variable. Sample size 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 was calculated based on a multiple linear regression model using a coefficient of determination of 4 5 6 0.10 and an R-square differential ranging between 0.01 and 0.02. Using these parameters, a number 7 8 between 495 and 986 was needed to have an 80% power at a 0.05 level of significance. Using the 9 10 more conservative R square differential of 0.02 and to account for attrition we planned on 11 12 recruiting around 1200 participants. 13 14 15 16 For peer review only 17 18 Data Collection 19 20 21 Maternal data were collected during pregnancy at two timepoints (first/second and third trimester). 22 23 24 Post-partum assessments are ongoing and target both mothers and children: questionnaire data 25 26 from mothers and biospecimens and neurodevelopmental assessments data from children are 27 28 collected at 12, 36, and 48 months. (Table 1). 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Data collection during pregnancy 35 36 37 Questionnaires 38 39 40 Table 1 lists standardized validated questionnaires used to assess both physiological and

41 on September 25, 2021 by guest. Protected copyright. 42 psychosocial prenatal health. All questionnaires were translated into Dutch and other local 43 44 45 languages to address the multiple language needs of our interior sub-cohort. The main categories 46 47 of data collected from maternal participants were health status, demographics, reproductive health 48 49 history, social support, trauma history, exposure history, depression, perceived stress, prenatal life 50 51 52 events, health behavior, access to prenatal care, social status and diet. Questions on health behavior 53 54 were adapted from the Alcohol, Smoking and Substance Involvement Screening Test version 3 55 56 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 (ASSIST V3.0) that was developed for the World Health Organization (WHO). Information on 4 5 6 maternal diet was obtained by adapting the CDC’s National Health and Nutrition Examination 7 8 Survey (NHANES) into a culturally tailored dietary survey focusing on fish and produce 9 10 consumption, including frequency of intake and portion sizes. 11 12 13 Biospecimen collection 14 15 16 The study’s biospecimenFor repository, peer housed review at the Academic Hospitalonly Paramaribo’s (AZP) clinical 17 18 laboratory, contains 13,000 samples. Table 1 lists timepoints at which biospecimens are collected. 19 20 21 All biospecimens are collected at study site hospitals and shipped to the AZP laboratory using 22 23 established chain-of-custody procedures. Samples collected from participants in remote areas are 24 25 stored at 4°C for no more than 48 hours prior to delivery and processing at the Academic Hospital 26 27 28 Paramaribo clinical laboratory. Blood, serum, plasma, and cord blood samples are aliquoted into 29 30 2 ml plastic freezer tubes and stored at -80°C. Urine samples (60ml) are collected in sterile bar- 31 32 coded urine collection cups, aliquoted into 10 ml plastic tubes, stored in ziplock bags at -20°C and

33 http://bmjopen.bmj.com/ 34 35 shipped for analysis by CDC’s environmental health laboratory using well-established protocols. 36 37 Maternal hair samples, collected once during pregnancy, were taken according to the following 38 39 40 protocol: 1.5 gram of hair was cut as close as possible to the hair roots with disinfected scissors

41 on September 25, 2021 by guest. Protected copyright. 42 and placed in patient coded ziplock bags, stored at room temperature in a climate-controlled room 43 44 and sent to the National Zoological Collection of Suriname/Center for Environmental Research 45 46 47 lab at the Anton de Kom University of Suriname (NZCS/CMO) for total Hg analysis using Cold 48 49 Vapour Atomic Absorption Spectrometry (CVAAS). 50 51 52 Buccal swab collection for telomere assessments 53 54 55 56 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Isohelix SK1 buccal swabs (Cell Projects, Kent, United Kingdom) are used for the collection of 4 5 6 maternal DNA for telomere length (TL) analyses. Swabs are air dried and then stored with a 7 8 dessicator pellet at 4 degrees until DNA is extracted. DNA is extracted using the QIAamp DNA 9 10 mini kit protocol (Qiagen, Valencia, CA) and stored at −80°C until assayed. Concentration of 11 12 dsDNA is quantified with a Qubit dsDNA BR assay kit (Invitrogen, Carlsbad, CA), purity of the 13 14 15 DNA is determined by using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, 16 For peer review only 17 Waltham, MA), and DNA integrity is confirmed by gel electrophoresis to ensure high molecular 18 19 weight DNA [20]. The average relative TL, represented by the telomere repeat copy number to 20 21 22 single gene (albumin) copy number (T/S) ratio, is determined with monochrome multiplex 23 24 quantitative real-time PCR using a BioRad CFX96 [21]. All samples are run in triplicate on 25 26 duplicate plates with a standard curve and known controls. 27 28 29 Trace element analyses in blood 30 31 32 400 frozen maternal whole blood samples were shipped for analysis to the Wisconsin State

33 http://bmjopen.bmj.com/ 34 35 Laboratory of Hygiene Trace Element Research Laboratory. A chain-of-custody approach was 36 37 used to ensure samples were collected, maintained, processed, stored, shipped, and received 38 39 according to acceptable standards. Quality assurance/quality control (QA/QC) procedures for 40

41 elemental analyses and methylmercury analyses included reagent blanks, blank spikes (lab on September 25, 2021 by guest. Protected copyright. 42 43 44 fortified blanks), sample matrix spikes, ongoing precision and recovery spikes, second source 45 46 spikes, sample matrix duplicates, and external standard reference materials. All sample and 47 48 QA/QC results were within the acceptable recovery limits. Duplicates for all runs were within the 49 50 51 acceptable relative percent difference (%RPD) limits. In these 400 blood samples (200 52 53 Paramaribo, 100 Nickerie, and 100 interior communities), we analyzed Hg, lead, cadmium, 54 55 aluminum, manganese, tin, selenium and iron using inductively coupled plasma-mass spectrometry 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 (ICP-MS) or cold vapor atomic fluorescence spectrometry (CVAFS). Total Hg and methylmercury 4 5 6 using CVAFS were assessed in a small subset of women (20-Paramaribo, 20-Nickerie, and 35- 7 8 interior) to specify exposure sources. 9 10 11 Pesticide analyses in urine 12 13 14 Urine samples from pregnant women were analyzed (sub-cohort N=218) from all three study sites. 15 16 The six dialkylphosphateFor metabolites peer were review measured using a onlymodified method of Jayatilaka et al. 17 18 [22] by solid phase extraction high performance liquid chromatography-tandem mass 19 20 21 spectrometry1. The eight specific urinary metabolites were analyzed using a semi-automated solid 22 23 phase extraction mass spectrometry method and reversed-phase high performance liquid 24 25 chromatography [23]. The analysis panel consisted of dialkylphosphate metabolites of 26 27 28 organophosphate pesticides (DAP’s) including dimethylphosphate, diethylphosphate, 29 30 dimethylthiophosphate, dimethyldithiophosphate, diethylthiophosphate, and 31 32 diethyldithiophosphate, and a universal panel (UP) including 1 herbicide (2,4D) and its metabolite,

33 http://bmjopen.bmj.com/ 34 35 2,4-dichlorophenoxyacetic acid; 4 OP insecticide metabolites (3,5,6-trichloro-2-pyridinol,2- 36 37 isopropyl-4-methyl-6-hydroxypyrimidine, para-nitrophenol, malathion dicarboxylic acid) and 3 38 39 pyrethroid metabolites (4-fluoro-3-phenoxybenzoic acid, 3-phenoxybenzoic acid, trans-3-(2,2- 40

41 dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid). on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Data collection from birth through 48 months 48 49 50 At birth, a cord blood sample was taken from infants for measurements of heavy metals. When a 51 52 53 cord blood sample was not available, a blood sample was obtained by heel prick. Baseline 54 55 characteristics of all births (e.g. gender, weight, gestational age, and Apgar score) were collected 56 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 (Table 2). At each subsequent data collection timepoint (Table 1), the child’s growth is measured 4 5 6 and a buccal swab taken for TL analyses. In addition, the child’s health status is obtained through 7 8 questionnaires on diet, history of infectious diseases, and respiratory and neurological disorders. 9 10 To date, we have completed the first follow-up with 85% of children. A blood sample will be taken 11 12 for heavy metal measurement at 36 and 48 months. Blood samples and buccal swabs are collected 13 14 15 and stored in the same manner as described above for mothers. 16 For peer review only 17 18 Behavior and cognitive development 19 20 21 Neurodevelopmental assessments over the lifetime of the study include infant cognitive and motor 22 23 development and behavior at 12-27 months using the Bayley Scales of Infant Development as well 24 25 as assessments of cognitive and social-emotional development at 36 months, and executive 26 27 28 function at 48 months (Table 1). The Bayley Scales are currently being administered to enrolled 29 30 children by trained psychologists, psychiatrists, and study team members. Data checking following 31 32 data entry of each Bayley sub-scale is conducted by other team members. To date, 832 of 992

33 http://bmjopen.bmj.com/ 34 35 children have been tested; remaining assessments will be completed by September 2020. 36 37 Assessments at 36 and 48 months will take place as the cohort ages. 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Data collection from fish 44 45 46 Carnivorous fish from different regions in the interior of Suriname have shown high levels of Hg, 47 48 specifically the frequently consumed species Hoplias aimara (0.43-0.66ug/g) and Serrasalmus 49 50 rhombeus (0.23-1.38ug/g). These levels are well above international accepted standards and in 51 52 53 certain regions, up to 7 times the norm for human consumption [24]. Polyunsaturated omega-3 54 55 fatty acids (PUFA-3) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from fish 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 consumption may offer neuroprotective benefits during prenatal and pediatric development which 4 5 6 could potentially counteract mercury neurotoxicity [25-27]. Some studies examining 7 8 developmental trajectories of children born to women exposed to Hg in utero fail to link that 9 10 exposure to adverse neurodevelopmental outcomes, suggesting that fish nutrients may ameliorate 11 12 Hg toxicity and offer neuroprotective benefits [28-31]. 13 14 15 To better inform fish consumption risk, fatty acids in fish muscle tissue were measured in samples 16 For peer review only 17 18 (N=5 per species) of five freshwater and three marine fish species. Levels of 27 fatty acids, 19 20 including DHA and EPA, in fish muscle tissue were measured (expressed as mg fatty acid g-1 dry 21 22 weight) by the University of Texas Marine Science Institute. 50mg dry weight samples were 23 24 25 homogenized in a chloroform-methanol solution (2:1 v/v) for lipid cold extraction. Lipids were 26 27 then saponified in potassium hydroxide to yield fatty acid methyl esters, and then methylated 28 29 in a 14% boron trifluoride solution. Individual fatty acid concentrations were determined by 30 31 32 gas chromatography (Shimadzu GC-2014 gas chromatograph) set with a Suplecowax 10 fused

33 http://bmjopen.bmj.com/ 34 silica capillary column (Milipore Sigma). Freshwater species had higher levels of linoleic acid (2.0 35 36 vs. 0.2mg/g), alpha-linoleic acid (0.4 vs. 0.1mg/g), arachidonic acid (3.0 vs. 1.5mg/g), omega-6 37 38 fatty acids (6.5 vs. 2.3mg/g) and lower levels of eicosapentaenoic acid (0.8 vs. 1.9mg/g) compared 39 40 to marine species.

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 Patient and public involvement 48 49 50 The CCREOH cohort study was preceded by a planning grant which included pilot fish and hair 51 52 Hg assessments among interior communities approved by the village Chiefs or Tribal captains. 53 54 55 Building on this community partnership, the initial study design was revised based on guidance 56 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 from a multi-stakeholder Community Advisory Board (CAB) and an External Scientific Advisory 4 5 6 Board (EAB). The CAB consists of respected leaders representing organizations from the interior 7 8 tribal and indigenous peoples, midwives, general practitioners and environmentalists. The EAB 9 10 members represent medical directors and other clinicians of all participating hospitals, 11 12 representatives from the Ministry of Health, the US Embassy, the Pan American Health 13 14 15 Organization and the Caribbean Public Health Agency (CARPHA). Both advisory boards are 16 For peer review only 17 convened annually to inform and give updates about study progress and results, and to seek advice 18 19 on future directions and dissemination of results. 20 21 22 23 24 25 Dissemination of study results 26 27 28 Every participant with neurotoxicant levels of imminent health concern is contacted and asked to 29 30 31 repeat the test for confirmation. If the level remains of concern, the participant is referred to her 32

33 attending physician. Children with neurodevelopmental delay are referred to a child psychologist. http://bmjopen.bmj.com/ 34 35 Participants and the general public were invited to health fairs, meetings and conferences, where 36 37 preliminary study results were presented and discussed. CCREOH data have been presented at 38 39 40 regional and international conferences, including CARPHA, the International Society of

41 on September 25, 2021 by guest. Protected copyright. 42 Environmental Epidemiology, the Consortium for Universities in Global Health, the American 43 44 Public Health Association and at scientific meetings convened by the US NIH. The study team is 45 46 47 working with the EAB and CAB to develop a collaborative translation and dissemination strategy 48 49 for overall, final study results when those become available. 50 51 52 53 54 55 56 Findings to date 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 From December 2016 until July 2019, 1143 pregnant women were enrolled; 74 (6.5%) were 4 5 6 ineligible (Figure 1). Geographically, 738 women were enrolled in Paramaribo, 204 in Nickerie, 7 8 and 201 in the interior. In comparison to participants, non-participants were more often of Creole 9 10 and Tribal ethnicity, lived in Paramaribo, and had lower household income. Table 2 presents 11 12 baseline maternal characteristics: one in eight women was 16-19 years old, ethnic distribution was 13 14 15 representative of the Surinamese population, and >50% had no or lower vocational/secondary 16 For peer review only 17 education level. 18 19 20 Of the 1069 babies born, 992 were enrolled (93%) (Figure 1). To date, the total number of 21 22 biospecimens (N=13,379) collected from 1143 pregnant women include whole blood for trace 23 24 25 elements (N=1994), whole blood collected in K2EDTA anticoagulant (N=1994), serum collected 26 27 in serum separator tubes (N=1994), plasma (N=1994), urine (N=1980), buccal swabs (N=941), 28 29 and hair (N=876). From 992 infants, either cord blood (N=441) or blood from heel prick (N=323) 30 31 32 was collected at birth, and buccal swabs (N=842) during neurodevelopmental assessment at 12

33 http://bmjopen.bmj.com/ 34 months. All samples not yet analyzed are archived for future targeted and untargeted analyses. 35 36 Available results include total Hg concentrations in hair, prenatal maternal depression and 37 38 perceived stress, level of pesticides in produce, and dietary exposure to Hg in fish. Results of heavy 39 40 metals in blood, urinary pesticide metabolites, telomere analysis, and neurodevelopmental

41 on September 25, 2021 by guest. Protected copyright. 42 43 assessments are pending. 44 45 46 47 48 49 Biological specimens 50 51 52 Total hair Hg was measured in 876 participants from all three study sites. Overall, 39.1% had 53 54 elevated total Hg hair levels that exceeded the U.S. Environmental Protection Agency’s (USEPA) 55 56 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 action level (1.1µg/g hair) [32]. Elevated Hg levels were found in 95.8%, 26.3%, and 25.5% of 4 5 6 participants from the interior, Nickerie and Paramaribo, respectively. Results for median total Hg 7 8 concentrations in hair from pregnant women from Paramaribo (N=522) were 0.64µg/g hair 9 10 (interquartile ranges (IQR) 0.36-1.09; range 0.00-7.12), from Nickerie (N=176) 0.73µg/g (IQR 11 12 0.45-1.05;0.00-5.79) and the interior (N=178) 3.48µg/g (IQR 1.92-7.39;0.38-18.20). Pregnant 13 14 15 women from the interior were exposed to high levels of Hg compared to women from the coastal 16 For peer review only 17 area and thus fetal exposures are expected to be high. Most of these women are primarily exposed 18 19 to methylmercury from consuming contaminated fish from local artisanal gold-mining activities 20 21 22 [18,24]. Other possible sources of exposure may include active participation in or living very close 23 24 to gold mining areas. Hg concentrations from the Nickerie and Paramaribo sub-cohorts were 25 26 similar to other studies [33,34]. 27 28 29 30 31 32 Prenatal depression and perceived stress

33 http://bmjopen.bmj.com/ 34 35 Depression and prenatal stress in 722 participants were assessed using the standardized Edinburgh 36 37 Depression Scale (EDS) (cut-off ≥12 for probable depression) and the Cohen Perceived Stress 38 39 40 Scale (cut-off ≥20 for high perceived stress). One in four (24.9%) participants had EDS scores

41 on September 25, 2021 by guest. Protected copyright. 42 indicative of probable depression, three in ten (30.2%) had high stress levels and nearly half 43 44 (49.1%) of these women had probable depression. 45 46 47 48 49 50 Dietary exposure to pesticides 51 52 An assessment of Surinamese agricultural produce showed pesticide residues exceeding European 53 54 Union maximum residue limits, including prohibited-worldwide endosulfan and lindane in the 55 56 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 leafy vegetable tannia, Xanthosoma brasiliense [35]. An interviewer-assisted NHANES-based 4 5 6 dietary survey of 522 participants showed that 98.2% reported consumption of leafy vegetables. 7 8 Tannia was the most frequently consumed (89.3%); 36.5% participants had high intake rates of 9 10 tannia (≥36 gram/day) [36]. Tannia is also a commonly used vegetable in baby food preparation 11 12 in Suriname. 13 14 15 16 For peer review only 17 18 Dietary exposure to Hg in fish 19 20 21 Dietary questionnaire analyses (N=990) showed an overall fish consumption of 96.1%. 22 23 Consumption of the three most consumed carnivorous species- Hoplias aimara, Serrasalmus 24 25 rhombeus and Cichla ocellaris-, known to have high Hg levels, was 44.4%, 19.3%, and 26.3%, 26 27 28 and was greater among the interior subcohort (N=123): 89.4%, 67.5%, and 74.8%, respectively. 29 30 Intake rates (based on reported meal frequency and portion sizes) for these three carnivorous 31 32 species ranged between 0.01-2.5 kilograms per week. Hg speciation indicated that methylmercury

33 http://bmjopen.bmj.com/ 34 35 was predominantly found in the biospecimens. This is consistent with fish consumption likely 36 37 being the primary source of Hg exposure in our participants. 38 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 Strengths and limitations 44 45 46 This CCREOH study is the first to examine the potential impact of complex environmental 47 48 exposures on maternal and child health in Suriname by a multi-disciplinary team of scientists. 49 50 51 Study’s strengths include: 1) addressing two high priority public health threats: the impact of Hg 52 53 exposure from artisanal gold mining, and pesticide exposure associated with agricultural practices; 54 55 2) inclusion in the cohort of two vulnerable subpopulations: pregnant women and children; 3) the 56 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 longitudinal follow-up of children until 48 months with built-in timepoints to assess 4 5 6 neurodevelopmental outcomes; and 4) a biospecimen bank of approximately 13,000 samples, 7 8 providing the opportunity for future analyses. Moreover, a linked research training grant facilitates 9 10 the training of nine Surinamese PhD candidates with dissertation research embedded in the study, 11 12 thereby building critical in-country environmental health research capacity. 13 14 15 Delayed recruitment and data collection of the logistically difficult to reach sub-cohort in the 16 For peer review only 17 18 interior region (N=200) was ameliorated by additional team support onsite; delays in transporting 19 20 interior biospecimens rendered some early cord blood specimens not analyzable and was corrected 21 22 by collecting an additional heelprick sample. Other limitations include differences in educational 23 24 25 level between women from the three recruitment sites that could confound or impact literacy. 26 27 Participants may have moved between regions with different sources of exposure. Delayed 28 29 recruitment of some interior participants may limit our ability to examine differential effects of 30 31 32 exposure across gestation for this sub-cohort.

33 http://bmjopen.bmj.com/ 34 35 36 37 38 Future plans 39 40

41 Culturally-tailored fish consumption advisories are in development to reduce Hg exposure. An on September 25, 2021 by guest. Protected copyright. 42 43 44 expanded assessment of potentially neuroprotective fish nutrients is under way. A pesticide 45 46 literacy scale will assist in countering pesticide exposure. Repeated telomere length measurements 47 48 of children as an indicator of exposure to environmental toxins will be taken. Neurodevelopmental 49 50 assessments will take place at 36 and 48 months. An integrated, sharable data management system 51 52 53 is in development. 54 55 56 57 58 21 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Acknowledgements 4 5 6 7 We would like to thank our recruiters Jeetendra Jitan, Nishaira Doerga, Gimradj Dwarka, Sue-Ann 8 9 Sitaram, Devika Kalika, the Community Health Workers from Nickerie Lilian Singotiko, Henna 10 11 Mawo, Karien Jodiwongso, Karishma Bhondoe, Jay Kalicharan, Bhairam Goberdan, Azeez 12 13 14 Khodabaks, the midwives and laboratory personnel of the Academic Hospital Paramaribo, 15 16 Diakonessenhuis Hospital,For St. peer Vincentius reviewHospital, ‘s-Lands only Hospital, Mungra Medical Centre, 17 18 Regional Health Service clinics and Medical Mission Primary Health Care Suriname, the National 19 20 Zoological Collection of Suriname/Center for Environmental Research staff, Sila Kisoensingh 21 22 23 MSc, Dr. Richenel Ellecom and Dr. Melissa Middleton for their help during the pediatric 24 25 neurodevelopment assessments, and the members of the External Advisory Board and Community 26 27 Advisory Board for their guidance and valuable advice. 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Collaborators 35 36 37 We welcome collaboration with fellow researchers working on similar projects. Specific research 38 39 proposals can be sent directly to the Principal Investigators Drs. Wilco Zijlmans 40

41 on September 25, 2021 by guest. Protected copyright. 42 ([email protected]) and Maureen Lichtveld ([email protected]). Areas of collaboration 43 44 include further environmental and biomarkers assessment of arsenic exposure, occupational health 45 46 risk assessments associated with mercury and pesticide exposure, and exposomic analyses 47 48 49 including metabolomic assessments. 50 51 52 53 54 Author Contributions 55 56 57 58 22 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 MYL, JKW, HHC, EWH, AS, SSD and WCWRZ designed and established the cohort. MSMO, 4 5 6 ADHM, EWH, FAW and AG participated in the design of the questionnaires. WCWRZ, MSMO, 7 8 EB, WBH, GKB, RR and MYL are responsible for the continued management of the cohort. GKB, 9 10 RR, AG and FAW are actively involved with recruitment of participants and collection of non- 11 12 biospecimen data and biospecimens. JC, JR, PEO and GAL store, archive and analyze the 13 14 15 biomarker samples. ADHM, AS, MSMO, AG, MYL and WCWRZ are responsible for data 16 For peer review only 17 management. SSD, AWEG, RR and EB manage neurodevelopmental testing of babies. JKW, LFS, 18 19 CSA, SSD interpret all results. WCWRZ, JKW, HHC, ADHM and MYL drafted and edited the 20 21 22 manuscript. All authors critically reviewed and approved the final manuscript. 23 24 25 26 27 28 Funding 29 30 31 Research reported in this publication was supported by the Fogarty International Center of the 32

33 National Institutes of Health under Award Numbers: U01TW010087 and U2RTW010104. The http://bmjopen.bmj.com/ 34 35 content is solely the responsibility of the authors and does not necessarily represent the official 36 37 38 views of the National Institutes of Health. 39 40

41 on September 25, 2021 by guest. Protected copyright. 42 43 44 Competing interests 45 46 47 None declared 48 49 50 51 52 53 54 Patient consent for publication 55 56 57 58 23 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 26 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Obtained 4 5 6 7 8 9 10 Ethical approval 11 12 13 This study was IRB approved by both the Government of Suriname and the Tulane University, 14 15 New Orleans, Louisiana, USA, to consent 1200 pregnant women and their singleton birth 16 For peer review only 17 18 children. 19 20 21 22 23 24 Provenance and peer review 25 26 27 Not commissioned; externally peer reviewed. 28 29 30 31 32

33 http://bmjopen.bmj.com/ 34 Data sharing statement 35 36 37 Our cohort study data will be made publicly available upon publication of the results in scientific 38 39 articles. Questionnaire, registry and biospecimen data could be made accessible in de-identified 40

41 form after an application process that includes submission of a research plan. We are currently on September 25, 2021 by guest. Protected copyright. 42 43 44 collaborating with Research Triangle Institute International (RTI), a global data management 45 46 enterprise, to develop an integrated database of biospecimen and non-biospecimen data. Once 47 48 that is fully developed, data can be made available based on a reasonable request to the PIs. Such 49 50 51 requests will be discussed with the full investigator Committee, the Data Management 52 53 Committee, and the Administrative Oversight Committee. 54 55 56 57 58 24 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 27 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 4 5 6 7 References 8 9 10 Lichtveld MY, Hawkins WB, Ouboter PE et al. A One Health approach to interdict 11 1. 12 13 environmental health threats in Suriname. Ann Glob Health 2016;82:444. 14 15 16 2. Silbergeld EK, ForPatrick TE. peer Environmental review exposures, toxicologic only mechanisms, and adverse 17 18 pregnancy outcomes. Am J Obstet Gynecol 2005;192:S11-21. 19 20 21 3. Ferguson KK, O'Neill MS, Meeker JD. Environmental contaminant exposures and preterm 22 23 birth: a comprehensive review. J Toxicol Environ Health B Crit Rev 2013;16:69-113. 24 25 26 27 4. Wigle DT, Arbuckle TE, Turner MC et al. Epidemiologic evidence of relationships between 28 29 reproductive and child health outcomes and environmental chemical contaminants. J Toxicol 30 31 Environ Health B Crit Rev 2008;11:373-517. 32

33 http://bmjopen.bmj.com/ 34 5. Lewis M, Worobey J, Ramsay DS et al. Prenatal exposure to heavy metals: effect on childhood 35 36 cognitive skills and health status. Pediatrics 1992;89:1010-5. 37 38 39 40 6. Burns CJ, McIntosh LJ, Mink PJ, Jurek AM, Li AA. Pesticide exposure and

41 on September 25, 2021 by guest. Protected copyright. 42 neurodevelopmental outcomes: Review of the epidemiologic and animal studies. J Toxicol 43 44 Environ Health B Crit Rev 2013;16:127-283. 45 46 47 7. Eskenazi B, Kogut K, Huen K et al. Organophosphate pesticide exposure, PON1, and 48 49 neurodevelopment in school-age children from the CHAMACOS study. Environ Res 50 51 52 2014;134C:149-57. 53 54 55 56 57 58 25 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 28 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 8. Furlong MA, Engel SM, Barr DB, Wolff MS. Prenatal exposure to organophosphate pesticides 4 5 6 and reciprocal social behavior in childhood. Environ Int 2014;70:125-31. 7 8 9 9. Quirós-Alcalá L, Alkon AD, Boyce WT et al. Maternal prenatal and child organophosphate 10 11 pesticide exposures and children's autonomic function. Neurotoxicology 2011;32:646-55. 12 13 14 10. Rosas LG, Eskenazi B. Pesticides and child neurodevelopment. Curr Opin Pediatr 15 16 2008;20:191-7. For peer review only 17 18 19 11. Belles M, Albina ML, Sanchez DJ et al. Interactions in developmental toxicology: effects of 20 21 22 concurrent exposure to lead, organic mercury, and arsenic in pregnant mice. Arch Environ 23 24 Contam Toxicol 2002;42:93-8. 25 26 27 12. Callahan MA, Sexton K. If cumulative risk assessment is the answer, what is the question? 28 29 Environ Health Perspect 2007;115:799-806. 30 31 32 13. Golding J, Rai D, Gregory S et al. Prenatal mercury exposure and features of autism: a

33 http://bmjopen.bmj.com/ 34 35 prospective population study. Mol Autism 2018;9:30. 36 37 38 14. Valent F, Mariuz M, Bin M et al. Associations of prenatal mercury exposure from maternal 39 40 fish consumption and polyunsaturated fatty acids with child neurodevelopment: a prospective

41 on September 25, 2021 by guest. Protected copyright. 42 cohort study in Italy. J Epidemiol 2013;23:360-70. 43 44 45 15. Sapbamrer R, Hongsibsong S. Effects of prenatal and postnatal exposure to organophosphate 46 47 48 pesticides on child neurodevelopment in different age groups: a systematic review. Environ 49 50 Sci Pollut Res Int 2019;26:18267-90. 51 52 53 54 55 56 57 58 26 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 29 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 16. Wang J, Wu W, Li H et al. Relation of prenatal low-level mercury exposure with early child 4 5 6 neurobehavioral development and exploration of the effects of sex and DHA on it. Environ Int 7 8 2019;126:14-23. 9 10 11 17. Results Eighth Population and Housing Census in Suriname (Volume 1) Demographic and 12 13 Social Characteristics and Migration. General Bureau of Statistic Studies in Suriname (ABS) 14 15 2013. 16 For peer review only 17 18 19 18. Ouboter PE, Landburg G, Satnarain GU et al. Mercury Levels in Women and Children from 20 21 Interior Villages in Suriname, South America. Int J Environ Res Public Health 2018;15:1007. 22 23 24 19. Harris PA, Taylor R, Minor BL et al. The REDCap consortium: Building an international 25 26 community of software partners, J Biomed Inform 2019;95:1-10. 27 28 29 20. Stacy S. Drury, Emily Mabile, Zoë H. Brett et al. The Association of Telomere Length With 30 31 32 Family Violence and Disruption. Pediatrics 2014;134:e128–e137.

33 http://bmjopen.bmj.com/ 34 35 21. Cawthon RM. Telomere length measurement by a novel monochrome multiplex qualitative 36 37 PCR method. Nucleic Acids Res 2009;37:e21. 38 39 40 22. Jayatilaka NK, Restrepo P, Williams L, et al. Quantification of three chlorinated dialkyl

41 on September 25, 2021 by guest. Protected copyright. 42 phosphates, diphenyl phosphate, 2,3,4,5-tetrabromobenzoic acid, and four other 43 44 45 organophosphates in human urine by solid phase extraction-high performance liquid 46 47 chromatography-tandem mass spectrometry. Anal Bioanal Chem 2017;409:1323-32. 48 49 50 23. Davis MD, Wade EL, Restrepo PR, et al. Semi-automated solid phase extraction method for 51 52 the mass spectrometric quantification of 12 specific metabolites of organophosphorus 53 54 55 56 57 58 27 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 30 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 pesticides, synthetic pyrethroids, and select herbicides in human urine. Journal of 4 5 6 Chromatography B. 2013;929:18-26 7 8 9 24. Ouboter PE, Landburg GA, Quik JHM, Mol JHA, van der Lugt F. Mercury Levels in Pristine 10 11 and Gold Mining Impacted Aquatic Ecosystems of Suriname, South America. Ambio 12 13 2012;41:873-82. 14 15 16 25. Dellinger MJ, OlsonFor JT, Holubpeer BJ, Ripley review MP. Mercury, polychlorinatedonly biphenyls, selenium, 17 18 19 and fatty acids in tribal fish harvests of the upper great lakes. Risk Anal 2018;38:2029-2040. 20 21 22 26. Ginsberg GL, Toal BF, McCann PJ. Updated risk/benefit analysis of fish consumption effects 23 24 on neurodevelopment: Implications for setting advisories. Hum. Ecol. Risk Assess 25 26 2015;21:1810-1839. 27 28 29 27. Clarkson TW, Strain JJ. Nutritional factors may modify the toxic action of methyl mercury in 30 31 32 fish-eating populations. J. Nutr 2003;133:1539S-1543S.

33 http://bmjopen.bmj.com/ 34 35 28. Davidson PW, Strain JJ, Myers GJ et al. Neurodevelopmental effects of maternal nutritional 36 37 status and exposure to methylmercury from eating fish during pregnancy. Neurotoxicology 38 39 2008;29:767–75. 40

41 on September 25, 2021 by guest. Protected copyright. 42 29. Strain JJ, Yeates AJ, van Wijngaarden E et al. Prenatal exposure to methyl mercury from fish 43 44 45 consumption and polyunsaturated fatty acids: associations with child development at 20 mo of 46 47 age in an observational study in the Republic of Seychelles. Am J Clin Nutr 2015;101:530–7. 48 49 50 30. Taylor CM, Golding J, Emond AM. Blood mercury levels and fish consumption in pregnancy: 51 52 Risks and benefits for birth outcomes in a prospective observational birth cohort. Int J Hyg 53 54 55 Environ Health 2016;219:513-520. 56 57 58 28 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 31 of 35 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 31. Golding J, Gregory S, Emond A et al. Prenatal mercury exposure and offspring behaviour in 4 5 6 childhood and adolescence. Neurotoxicology 2016;57:87-94. 7 8 9 32. Mercury Study Report to Congress Volume V: Health Effects of Mercury and Mercury 10 11 Compounds. Volume 5. United States Environmental Protection Agency; Washington, DC, 12 13 USA: 1997;1–348. 14 15 16 33. Zilversmit L, WickliffeFor J,peer Shankar A, reviewTaylor R, Harville only E. Correlations of biomarkers and 17 18 19 self-reported seafood consumption among pregnant and non-pregnant women in southeastern 20 21 Louisiana after the Gulf oil spill: the GROWH study. Int J Environ Res Public Health 2017;14: 22 23 784. 24 25 26 34. Castaño A, Cutanda F, Esteban M, et al. Fish consumption patterns and hair mercury levels in 27 28 29 children and their mothers in 17 EU countries. Environ Res 2015;141:58-68. 30 31 32 35. Abdoel Wahid F, Wickliffe J, Wilson M et al. Presence of pesticide residues on produce

33 http://bmjopen.bmj.com/ 34 cultivated in Suriname. Environ Monit Assess 2017;189:303. 35 36 37 36. F. Abdoel Wahid, A. D. Hindori-Mohangoo, M. Wilson et al. Dietary Exposure to Pesticides 38 39 in Pregnant Surinamese Women. Environ Health Perspect 2018; 40

41 on September 25, 2021 by guest. Protected copyright. 42 https://doi.org/10.1289/isesisee.2018.P01.2380 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 29 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 32 of 35 BMJ Open: first published as 10.1136/bmjopen-2019-034702 on 13 September 2020. Downloaded from

1 2 3 Ages and Stages Questionnaire ● 4 Neurodevelopmental tests 5 BSID-III ● 6 CANTAB ● 7 Biological samples 8 Buccal swab ● ● ● 9 10 Blood ● ● 11 Urine ● ● 12 Notes: ASSIST V3.0 = Alcohol, Smoking and Substance Involvement Screening Test Version 3, Subjective 13 Social Status = MacArthur Scale of Subjective Social Status, M-CHAT = Modified Checklist for Autism 14 in Toddlers, Bayley SEQ = Bayley Social Emotional Questionnaire, BSID-III = Bayley Scales of Infant 15 Development Third Edition, CANTAB = Cambridge Neuropsychological Test Automated Battery. 16 Generation R = questionnairesFor onpeer pediatric health review and diet history only(with permission from The Generation 17 R Study Group) 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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18 Not married/not living with 141 12.4 http://bmjopen.bmj.com/ 19 partner 20 21 Missing 2 0.2 22 * SRD = Surinamese dollar, equivalent to 0.13 USD. 23 24 25

1 2 3 Figure legends 4 5 6 7 Figure 1. Flowchart with participant enrolment. 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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41 on September 25, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml