Method development for extraction and determination of the presence and quantity of endocrine disrupting chemicals in selected food items sold in South African open markets

Abstract

Exposure to Endocrine Disrupting Chemicals (EDCs) has potential carcinogenic and mutagenic effects to human health. Endocrine Disrupting Chemicals are contaminants that have the potential to disrupt the processes of natural body hormones that enables the organism to respond normally to its environment. The primary source for EDC exposure is through the consumption of foodstuff like contaminated fruits, vegetables, fish, and beef. In fruits and vegetable farms, most EDCs originate from the fertilizers applied to the soil. The EDCs are constantly being leached from the soil environment and released into rivers and other aquatic ecosystems. Eventually, they make their way into the lipid membranes and further into the muscles of fish via the process of bioaccumulation. For beef, some of the EDCs bioaccumulates within their muscles when the cattle (cow or bull) drink the contaminated river water when grazing. When humans consume the foodstuff contaminated with the EDCs, the EDCs are passed onto their body muscles via the process of biomagnification. Due to the EDC toxicity in humans from from diet, it is essential to investigate their levels in foodstuff like fruits, vegetables, fish and beef muscle. On the other hand, bioeconomy is an interesting research field which is gaining a lot of attention. Currently, there is considerable need to maximize the use of plant based biomass in analytical chemical processes. One such source is the use of Moringa Oleifera (M.O) seed protein bio sorbent which can be extracted, purified, and applied for a greener and low-cost sample clean-up. The aim of this study was to optimize the QuEChERS method using Primary secondary amine (PSA) sorbent and then comparing it with Moringa Oleifera protein biosorbent sorbents and other sorbents like Hemp cellulose, activated carbon sorbent for the extraction of selected organic EDCs in foodstuff. The optimization was done using chemometrics called Response Surface Methodology (RSM). A Design of Experiment (DoE) was used to investigate the effect of the sample mass (0.5-3 g), centrifuge speed (3400- 4000 rpm) , time (5-20 mins), mass of 𝑁𝑎𝐶𝑙 and 𝑀𝑔𝑆𝑂4(1 − 3 𝑔), and solvent extraction volume (5-10 mL). Firstly, a screening on the aforesaid parameters was done using an L8 (3 levels) linear model. Thereafter, a Central Composite Orthogonal (CCO) approach was used to v optimize the QuEChERS technique for maximum extraction of the selected EDCs. The analysis was done using Gas chromatography – Electron capture detection (GC-ECD) and 1 dimensional gas chromatography mass spectroscopy time of flight (1D GCxGC MSTOF). The PSA method presented optimal values of 3 g of sample, 150 mg PSA, 4000 rpm for 6 mins centrifuge conditions, 2 g NaCl, 2 g 𝑀𝑔𝑆𝑂4 extracted in 10 mL methanol, respectively. The PSA method presented recoveries between 74.1 ± 1.12% and 107.4 ± 0.88%. Furthermore, the Moringa Oleifera method presented recoveries between 76.2 ± 0.85% and 105.1 ± 2.24%. Limits of detection and quantification ranged between 0.16 to 1.77 𝜇𝑔 𝑘𝑔−1 and 6.1 to 17.7 𝜇𝑔 𝑘𝑔−1with RSD values ≤ 13.32 ,respectively. From this investigation, the Moringa Oleifera seed protein bio sorbent is a promising alternative to the synthetic PSA and other sorbents like cellulose, C18 and activated carbon in clean-up of food samples using QuEChERS approach as it was more selective towards target compounds The optimal Moringa Oleifera method was then applied to thirty-eight different samples from Johannesburg (CBD Open markets, City deep main market and a farm along Hennops river) and Pretoria (Open markets and Tshwane main market) including fruits and vegetables, fish and beef samples. The maximum detected concentrations of six organic Endocrine Disrupting Chemicals extracted (Dimethyl phthalate, Diethyl phthalate, 4-n-Nonylphenol, 4,4’-DDT, 4,4’-DDD and 4,4’-DDE) were 65.93 ± 9.80, 111.61 ± 11.27, 115.15 ±12.18, 101.17 ± 9.65, 114.7 ±20.76 and 105.45 ± 18.33 𝜇𝑔 𝑘𝑔 −1 , respectively. WHO regulation comparison of the determined concentrations revealed that consumers staying in the two cities are not at risk of cancer from ingestion of the studied organic EDCs in the detected levels. Secondly, the study aimed at extracting and determining the levels of inorganic Endocrine Disrupting Chemicals (selected metals) which are toxic at certain doses. The same thirty-eight samples collected for organic analysis were digested using Anton Paar microwave digestion system using 10% nitric acid and hydrogen peroxide. Thereafter, the total metal concentration analysis was done with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The selected metals of interest from the totals were Aluminum vi (Al), Arsenic (As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Arsenic (As), Lead (Pb), Manganese (Mn), Selenium (Se) and Zinc (Zn). The results show that the mean metal concentrations for Johannesburg foodstuff is not significantly higher (𝑃 > 0.05) than the mean concentration for Pretoria foodstuff. However, the two samples from the Hennops river farm (Rape and Spinach), exhibited the highest metal concentrations among all samples. Most of the metal concentrations were generally higher than the limits set by the World Health Organization (WHO), which alerts critical endocrine disrupting issues associated with the two cities under study. However, to critically conclude this regard, future work for thorough carcinogenic risk assessments must be established in terms of Daily limit Intake of Metals (DIM) and Hazard index (HI).