Resumen de Risk assessment of exposure to organic chemicals in children and mothers using human biomonitoring
Today, global chemical manufacturing is increasing each year, making environmental pollution and exposure to chemicals a serious public health problem linked to the development of many non-genetic diseases. Approximately, 70 % of chemicals consumed in Europe in 2019 were hazardous for human health. These harmful substances can enter to the food chain and remain in the environment, making humans exposed to them mainly by ingestion, inhalation or dermal exposure. Consequently, exposure to chemicals should be continuously monitored for assuring health protection of citizens by two ways: i) studying their presence in environmental compartments, such as water, food, soil or air, by environmental monitoring, and ii) determining their presence in biological samples, such as urine, blood, hair, etc., by human biomonitoring (HBM). In contrast to environmental monitoring studies, HBM data reflect the total body burden integrating all potential pathways of exposure to chemicals.
The overall aim of this doctoral thesis was to develop different target analytical tools for their application to HBM projects, and to design and carry out some specific HBM studies on urine samples of children (5 – 11 years old) and lactating mothers of the BIOVAL and BETTERMILK projects, respectively, focusing on non-persistent pesticides, polycyclic aromatic hydrocarbons (PAHs), and acrylamide (AA), which are priority substances in public health and food safety.
In Chapter I, a method based on QuEChERS and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous analysis of six non-specific metabolites of organophosphate pesticides (OPs), known as dialkyl phosphates (DAPs), in urine (Article I.1). Urinary levels of DAPs, together with other 20 metabolites of OPs, pyrethroids and herbicides, were analyzed in 116 mothers (Article I.2) and 568 children (Article I.3) living in Spain. The highest detection frequencies (DFs) were observed for diethyl phosphate (non-specific metabolite of OPs), 3,5,6-trichloro-2-pyridinol (a specific metabolite of chlorpyrifos), p-nitrophenol (specific metabolite of parathion), and 3-phenoxybenzoic acid (PBA, a non-specific metabolite of pyrethroids), with geometric means (GMs) ranging from 0.8 and 1.9 ng·ml-1 in mothers, and from 1.03 to 1.53 ng·ml-1 in children. In a global context, the Spanish population under study seemed to be at higher exposure to pyrethroids, since they presented higher urinary levels of PBA compared to other countries. The statistical analysis showed that closeness to farming activities, place of residence and presence of garden/plants at home might be important contributors to pesticide exposure in the mothers under study. In children, the consumption of vegetables, legumes and cereals seemed to be significant predictors of exposure to these substances. Mean estimated daily intakes (EDIs) were also calculated, ranging from 0.08 (chlorpyrifos) to 1.62 μg·kg-1·day-1 (λ-cyhalothrin) in children, and from 0.09 (dimethoate) to 0.40 μg·kg-1·day-1 (chlorpyrifos) in mothers. Risk assessment revealed a relatively low health risk of exposure to the analyzed pesticides in both target population groups. However, due to the extensive use of pesticides in Spain, it is highly advisable to continuously monitor time trends in the Spanish population’s exposure.
In Chapter II, the development and validation of a liquid-liquid extraction method followed by LC-MS/MS detection of 11 urinary PAH metabolites (OHPAHs) was performed and applied to urine samples of Spanish mothers (n = 110) and children (n = 566) (Article II.1 and Article II.2, respectively). In total, 9 hydroxy-metabolites of naphthalene, fluorene, phenanthrene and pyrene were detected in more than 78 % of the analyzed samples. The most abundant biomarker was 2-hydroxynaphthalene (2OHNAP), with a GM of 7 and 10 ng·ml-1 for mothers and children, respectively. In general, Spanish children and mothers under study were at greater exposure to naphthalene than those populations from other countries based on their urinary levels of 2OHNAP. According to the multiple regression analysis, smoker mothers and young children were observed to be at higher exposure to PAHs. In addition, some dietary habits, such as intake of oils and fats, smoked fish and coffee, as well as, potato fried products and plastic- packed foods, were statistically associated with a higher PAH exposure in lactating women and children, respectively. Exposure to PAHs was evaluated by calculating EDIs, which were between 9 (fluorene) and 446 ng·kg-1·day-1 (naphthalene) in mothers, and between 5 (fluorene) and 204 ng·kg-1·day-1 (naphthalene). In a risk context, no potential non-cancer risk associated to cumulative PAH exposure was observed. However, more attention should be paid to exposure to naphthalene, considered as a Group 2B (Possibly carcinogenic to humans), since it was by far the highest contributor to cumulative PAH exposure in this study.
In Chapter III, N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl- 2-hydroxyethyl)-L-cysteine (GAMA3) and N-Acetyl-3-[(3-amino-3-oxopropyl)sulfinyl]-L- alanine (AAMA-Sul), which are exposure biomarkers of AA in urine, were analyzed by a fast and validated methodology based on ‘dilute-and-shoot’ and LC-MS/MS determination. This method was applied to urine samples of 114 lactating mothers (Article III.1) and 612 children (Article III.2), resulting in DFs of 100 % with GMs in the range of 18 (GAMA3) – 79 ng·ml-1 (AAMA) in children, and 15 (GAMA3) – 70 ng·ml-1 (AAMA) in women. Comparing with previous studies, similar exposure levels were found in other countries, showing the widespread AA exposure in many populations around the world. Statistical analysis suggested that intake of fried potato products and biscuits, as well as coffee, bread and precooked food products, could be associated with higher levels of urinary AA in children and mothers, respectively. Mean EDIs for AA in the target populations were between 1.2 – 1.9 μg·kg-1·day-1, and risk assessment estimations showed that exposure levels were above current guidance values, revealing that a health concern with respect to non-neoplastic toxicity of AA could not be discarded in the Spanish population. Therefore, there is a need of further regulation and risk management activities, such as promotion of mitigation measures of AA exposure at households and a risk assessment follow-up, to minimize its exposure in Spain and other countries.
In conclusion, three LC-MS/MS methods validated under a high-quality control/quality assurance system with low limits of quantification were developed to determine urinary biomarkers of exposure to pesticides, PAHs and AA in HBM studies. Their applicability has been successfully tested by the analysis of urines of lactating mothers and children of the BETTERMILK and BIOVAL studies, and it allowed to potentially identify determinants of exposure and estimate the associated risks. Therefore, HBM studies are shown to be powerful tools to guide and evaluate strategies for prevention, intervention, and reduction of chemical exposure in general and the most vulnerable population.
