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Lead in childhood is well known to be associated with poor neurodevelopment. As part of a study on maternal anemia and offspring neurodevelopment, we analyzed blood lead level (BLL) with no prior knowledge of lead exposure in 225 mothers and 685 offspring 1 to 2 years old from Allada, a semi-rural area in Benin, sub-Saharan Africa, between May 2011 and May 2013. Blood samples were analyzed by inductively coupled plasma mass spectrometry. Environmental assessments in households and isotopic ratio measurements were performed for eight children with BLL > 100 mg/L. High lead levels (BLL > 50 mg/L) were found in 44% of mothers and 58% of children. The median BLL was 55.1 (interquartile range 39.2–85.0) and 46.6 (36.5–60.1) mg/L, respectively. Maternal BLL was associated with offspring’s consumption of piped water and animals killed by ammunition. Children’s BLL was associated with presence of paint chips in the house and consumption of animals killed by ammunition. In this population, with 98% of children still breastfed, children’s BLL was highly associated with maternal BLL on multivariate analyses. Environmental measures and isotopic ratios supported these findings. Offspring may be highly exposed to lead in utero and probably via breastfeeding in addition to lead paint exposure.
As the investigation of elevated BLL was not initially planned but due to incidental discovery, the methods section contains successive steps. Our study sample included singletons born to pregnant women enrolled in the “Malaria in Pregnancy Preventive Alternative Drugs” (MiPPAD) clinical trial ({“type”:”clinical-trial”,”attrs”:{“text”:”NCT00811421″,”term_id”:”NCT00811421″}}NCT00811421) comparing two intermittent preventive treatments of malaria in pregnancy [11]. The study was conducted in three health centers in the Allada district (Sekou, Allada and Attogon), South Benin. All surviving infants of recruited pregnant women were invited to undergo neurocognitive assessment in the TOVI study (Fon language: Tovi means child from the country) when the child was 12 months old [12]. From May 2011 to May 2013, blood samples were taken from 685 children 12 to 24 months old. Children with BLL > 250 µg/L were invited to be assessed for free a second time for BLL (9 of 14 reassessed). Mothers were not initially planned to be assessed for lead. After the first 50 BLL assessments, we decided to assess as many mothers as possible when offspring were 18 or 24 months through the TOLIMMUNPAL project, which resulted in 227 blood samples from mothers. Information on socioeconomic status and home environment were gathered during a home visit when the child was 12 months old. This questionnaire administered by a nurse or a psychologist included information on potential sources of lead including presence of paint and paint chips in the household, maternal and paternal occupation classified by risk of lead exposure [13], breastfeeding and sociodemographic characteristics. A family wealth scale involved a scoring instrument incorporating a checklist of material possessions (radio, television, bike, motorbike, and car), keeping cows, and access to electricity. Following the first results of BLL, we included a second questionnaire on potential sources of lead including sources of water for the child, pica behavior (ingestion of substances; here white and green clay) in children and in mothers during pregnancy, gasoline stored at the home, cooking and eating utensils [14], the child’s consumption of meat from animals killed by lead ammunition, maternal use of cosmetics, activities in the house or neighborhood, and number of hours the child played outside the house. This questionnaire was completed for 623 children: 53% when the child was > 12 months old because of the delay in implementing this second questionnaire (median 20.1 months, range 11.3–35.1). We collected 8 ml venous blood from each participant, 4 mL in a tube containing dipotassium EDTA and 4 mL in an iron-free dry tube. Blood samples were analyzed at the Centre de Toxicologie, Institut National de Santé Publique du Québec (Québec, Canada), by inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer Sciex Elan DRC II ICP-MS instrument) before 20-fold dilution in amonia 0.5% v/v and 0.1% v/v surfactant Triton-X. The limit of detection for blood analysis was 0.2 µg/L. Furthermore, to investigate exposure pathways, we visited eight households for environmental and food assessments. Households were selected if the child’s BLL was > 100 µg/L (very high BLL) and a sufficient amount of blood was available to perform lead isotope ratio (LIR) measurements. The LIR is the ratio of abundance of lead with different atomic weight because of a different neutron number. LIR can provide information from lead contamination origins because the isotopic signature varies by the age of the original ore. The concentration of lead in soil, water, gasoline, dishes, food and paint, when present, was determined in each household. Ammunition for animal hunting was purchased from a local market. All samples were analyzed by ICP-MS (Agilent technologies 7500ce ICP-MS instrument) at a school of public health (EHESP, Rennes, France). For water, the limit of quantification (LOQ) was 1 µg/L. Before ICP-MS analysis, food, soil and gasoline were mineralized with a mixture of nitric and hydrochloric acid (1/3 HNO3 and 2/3 HCl) by microwave (Multiprep 41, Milestone) and dust by a graphite block digestion system (Digiprep, SCP Science). The LOQ was 0.1 mg/kg for food, 0.1 mg/kg for soil, 0.5 mg/L for gasoline and 2 µg/m2 for dust. Dishes were soaked in a solution of acetic acid (4%) for 24 h before analysis, and the LOQ was 2 µg/L. The LIR in children’s blood and possible sources of lead was determined by the concentration of lead in each source for a given child. The LIR was determined in the most concentrated sample of each exposure media by using quadrupole ICP-MS (Agilent technologies 7500ce ICP-MS instrument) with environmental samples at EHESP. The mass bias was corrected with a certified reference material (Common Lead Isotopic Standard, SRM 981, NIST) with the standard bracketing technique [15]. Lead stable isotope ratios in blood were established with low-resolution quadropole ICP-MS (Perkin Elmer NexION 300S instrument) after proper dilution to an obtained final lead concentration of 5 µg/L in a 0.5% (v/v) ammonia solution with 0.1% v/v surfactant Triton-X. Instrumental isotopic ratio responses were calibrated with certified reference materiel NIST SRM 981 and controlled with SRM NIST 982. Intercalibrated LIR was calculated by the two laboratories and involved two aqueous leaded samples. Each of the two laboratories used its own method for determining mass correction with the standard SRM 981 and correction of blanks. The results were comparable, except for LIR containing 204. Interpretation was based on the proximity of graphical isotopic ratios between blood and potential sources, accounting for measurement accuracy (a source was considered compatible with blood with recovery between the error bars of the source and blood) [16] and assessed on isotopic ratios 208/207 vs. 207/206. In statistical analyses, BLL was log-transformed for normal distribution (natural logarithm). We first described BLL in children and mothers, potential sources of lead, and sociodemographic characteristics of the study population. Second, we performed univariate analysis to assess the crude association between the potential sources of lead in both children and mothers, sociodemographic characteristics, and BLL. Third, we conducted multivariate analyses in three steps for children with logBLL and BLL >50 µg/L as dependent variables: model 1 included potential sources of lead identified on univariate analyses with p < 0.05; model 2 included socioeconomic factors in addition to sources of lead; model 3 included maternal BLL in addition to the former variables. The first two models were also used to analyze maternal BLL. Student t test and chi-square test were used to compare means and proportions, respectively. Multiple linear regression and logistic regression were used for continuous and binary outcomes, respectively. Statistical significance was defined as p < 0.05. Statistical analyses involved use of SAS 9.3. The study was approved by the institutional review boards of the University of Abomey-Calavi in Benin and New York University in the United States (IRB#09-1253). At recruitment, we obtained informed consent from all pregnant women and guardians of children who participated in this study.
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