Wastewater-based epidemiology for tracking human exposure to mycotoxins
Graphical abstract
Introduction
Mycotoxins are toxic compounds naturally produced by certain types of moulds (fungi) that grow on numerous foodstuffs such as cereals, dried fruits, nuts and spices under warm, humid conditions (WHO, 2011). Over the last decades, concern about mycotoxins in food and their adverse effects for human and animal health has increased. Mycotoxins occur variably in agricultural products, but it is generally estimated that 25% of the world’s crops are contaminated to some extent; nevertheless this proportion is expected to increase in the near future, with growing global food demand and global climatic and environmental changes (Bennett et al., 2003).
Most mycotoxins are chemically stable, they survive storage and processing, and could even remain in cooked food (Rodríguez-Carrasco et al., 2014), so humans are mainly exposed through direct ingestion of contaminated foods or of animals fed contaminated feeds. Secondary routes of exposure are inhalation of moulds containing mycotoxins, or skin and mucosa contact (Fromme et al., 2016). Around 400 compounds are classified as mycotoxins but only 10–15 are priority food contaminants on account of their toxic effects (Bennett et al., 2003; Fromme et al., 2016). Thus, maximum acceptable levels have been established for some mycotoxins in foodstuffs (Commission Regulation 1881/2006, 2006, Commission Regulation 1881/2006, 2006Commission Regulation 1881/2006, 2006Commission Regulation 1881/2006, 2006), and Health-Based Guidance values (i.e. tolerable daily intakes) have been set by the European Food Safety Authority (EFSA) and by the Joint Food and Agriculture Organization of the United Nations /World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA).
So far, estimates of human mycotoxins intake have been based on the analysis of a variety of foods such as cereals, nuts, dried fruit, coffee, cocoa, alcoholic beverages, milk, eggs or baby food (Turner et al., 2015; Beltrán et al., 2013), and on dietary habit surveys (indirect methods). Additionally, human biomonitoring studies (HBM) consisting in the analysis of food and environmental contaminants in human specimens such as tissues and fluids (e.g. urine) are valuable additional method to assess human intake to mycotoxins. However, so far only two HBM studies have looked at mycotoxins in Europe and only aflatoxin and deoxynivalenol (DON) were studied, so there is a lack of information on human exposure to these substances (Choi et al., 2015). All these studies have some limitations, related mainly to the subjective nature of data collected through food frequency questionnaires, high costs of analysis of food and human specimens, long duration or difficulty in extrapolating results from a few individuals to the general population.
Wastewater-based epidemiology (WBE) is a novel biomonitoring approach with the potential to provide direct information on human intake/exposure to food and environmental chemicals. It is based on the analysis of specific human metabolic excretion products (biomarkers) in urban wastewater that directly reflect the intake of contaminants in an entire community. It was implemented for the first time in Italy for evaluating illicit drug consumption (Zuccato et al., 2005, 2008) and was later employed to assess other lifestyle-related factors (Gracia-Lor et al., 2017). Recently, WBE was successfully applied for the first time to assess human exposure to pesticides by measuring some of the urinary metabolites commonly included in HBM studies (Rousis et al., 2017a, b). Biomarker levels in wastewater reflected those in HBM studies (Rousis et al., 2016) and WBE results in different European countries were in line with several national statistics (Rousis et al., 2017b). Additional WBE applications have been developed to examine human exposure to environmental contaminants such as phthalate plasticizers (González-Mariño et al., 2017) and phosphorus flame retardants (Been et al., 2017).
The aim of this study was to extend the WBE approach to other priority food toxicants, mycotoxins, whose proven toxicity for humans has led to regulations for admissible intake levels by different agencies and organisations. However, information on the real human exposure to mycotoxins is still scant and very few extensive HBM studies are available. WBE for mycotoxins can rapidly assess human exposure at the population level, with contained costs, so it can be very useful to improve this knowledge and foster the implementation of future policies.
Preliminary data indicated the presence of mycotoxins in wastewater (Wettstein and Bucheli, 2010; Schenzel et al., 2010, 2012; Kolpin et al., 2014; Laganà et al., 2004), but none of the above studies employed WBE (Gracia-Lor et al., 2017). In the present study, a set of mycotoxins and metabolites (biomarkers) was selected by a systematic review of HBM information in urine, and an analytical method was developed to measure these biomarkers in wastewater. The selected substances were screened as biomarkers of intake/exposure for mycotoxins by studying their specificity and stability with the aim of finding suitable WBE biomarkers. A WBE approach was developed and tested to estimate DON intake at some locations in Italy and Spain.
Section snippets
Chemical and reagents
All reference standards were purchased from Sigma–Aldrich (St Louis, MO, USA), except deepoxy-deoxynivalenol (DOM-1) and β-zearalenol (β-ZEN) that were supplied by Romer Labs Diagnostic GmbH-Europe (Tulln, Austria) and Santa Cruz Biotechnology (Santa Cruz, California, USA), respectively. Isotopically labelled 3-acetyl-deoxynivalenol-d3 (3AcDON-d3) was obtained from Santa Cruz Biotechnology, and α-zeranol-d2 (α-ZEL-d2) was synthetized in our laboratory; both compounds were used as internal
Stability in wastewater
Results of the stability tests for mycotoxins are reported in Table S7. Most of the substances were stable under the two conditions assayed (room temperature and 4 °C). Differences in the concentrations after 24 h at room temperature (-3, -37%) were larger than at 4 °C (-1, -26%), and a few substances decreased by approximately 30% (3AcDON, T2-toxin, B-ZEN). Nevertheless, 24 h is a worst case situation, because in the cities considered the residence time in the sewer system is less than 8 h.
Conclusions
To the best of our knowledge, this is the first study where WBE has been used to estimate the collective intake of a population to mycotoxins. Only four mycotoxins (DON and fumonisins) were found in wastewater and DON was prevalent. These results validate the application of WBE for assessing human exposure to mycotoxins, because it reflected the urinary levels reported in HBM studies and also in national foodstuff analyses.
Despite being the first application of WBE in this field, it
Acknowledgments
Emma Gracia-Lor is very grateful for the financial support provided by the Atracción de Talento Program of the Comunidad de Madrid (Ref: 2017-T2/AMB-5466). The COST Action ES1307 “SCORE - Sewage biomarker analysis for community health assessment”, supported part of this work. We thank Franceso Riva, Carlo Pacciani and Eddie Fonseca for help with samples analyses. We are grateful to the personnel of all the WWTPs for their assistance in providing samples.
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