Development of analytical methods for arsenic speciation and their application to novel marine feed resources

By 2050, the world’s population is expected to surpass nine billion. Aquaculture has a huge potential to augment the demand for safe and nutritious food by the growing public. However, this would entail an increase in seafood supply, which translates to an additional feed volume requirement. In Norway, while the current composition of Atlantic salmon feed is predominantly plant-based ingredients, many have realized that the agricultural sector has turned into one of the biggest contributors of greenhouse gases. Thus, current research is directed towards novel marine feed resources which can alleviate aquaculture’s carbon footprint. Much focus has been given recently to marine resources at low-trophic levels due to their abundance and suitable nutritional composition, e.g. high in proteins and essential fatty acids. Primary producers and consumers such as microalgae, blue mussels, and mesopelagic organisms are currently considered as future ingredients for salmon feed.

Before novel marine resources can be fully utilized, it is necessary to document the levels of undesirable substances. Within the European Union (EU), maximum limits (MLs) are established for undesirable substances in feed and feed ingredients (Directive 2002/32 EC and amendments), which include toxic elements such as mercury, cadmium, lead, and arsenic. Arsenic (As) has over 100 naturally occurring As species in the marine environment. It is mainly recognized for its toxic properties associated with its inorganic forms, i.e. arsenite (As(III)) and arsenate (As(V)). In contrast, the non-toxic arsenobetaine (AB) is the predominant As compound in most marine organisms. Macrolagae, commonly known as seaweeds, contain significant proportions of arsenosugars (AsSug). In fatty fish, lipid-soluble As species, i.e. arsenolipids (AsLipids), are abundant. Recent studies have reported AsSug and AsLipids as potentially toxic compounds. Considering the varying toxicities of As species, the European Food Safety Authority (EFSA) recognizes the need for more As speciation data, which can only be realized when analytical methods for As speciation have been established.

In this PhD project, analytical methods for determining water-soluble As species in marine matrices were developed. A 2^7-3 fractional factorial design was performed to optimize the extraction procedure. Extraction temperature and the type of extraction solution were identified as significant factors. Arsenic speciation analysis was carried out using ion-exchange high-performance liquid chromatography coupled to inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). The mobile phase composition was also optimized by investigating the effects of mobile phase buffer and pH on the retention of analytes. Furthermore, the response of ICP-MS was enhanced by the addition of organic solvent in the mobile phase. The methods underwent single-laboratory validation using several marine certified reference materials (CRMs). Overall, satisfactory method performance characteristics were achieved.

The developed methods were applied to novel marine feed resources, i.e. mesopelagic organisms, blue mussels, and microalgae. The overall conclusion was that primary producers such as micro- and macroalgae, which are at the base of the aquatic food pyramid, do not contain AB but only the precursors. These precursors are then metabolized by higher-trophic animals to form AB. The effects of feed processing on As speciation was also studied through a lab-scale set-up with mesopelagic biomass as the starting raw material. An overall dilution effect was noted for total As (tAs) and most As species in mesopelagic meal and oil. However, the study also demonstrated the transfer of potentially toxic AsLipids from the biomass to the resulting mesopelagic meal, and further up-concentrated in mesopelagic oil.

The uptake and biotransformation of As in low-trophic marine food chain was likewise investigated by conducting an exposure and feeding experiment involving microalgae and blue mussels. The exposure of D. lutheri to higher iAs concentrations resulted to increased levels of iAs, DMA, and MA. However, at 10 μg/L, iAs accumulated, suggesting that the methylation threshold has been breached, and that detoxification mechanisms are overwhelmed.

Overall, novel marine feed resources will likely comply with current MLs for As (and iAs) in feed materials. However, since low-trophic marine organisms contain significant proportions of AsSug and, presumably, AsLipids, they will likely cause variation in As speciation compared to traditional feed raw materials, e.g. forage fish where AB is predominant. Studies on bioavailability in fish and possible accumulation of these compounds in final fish products should be endeavored to gain solid basis for risk assessment in terms of feed and food safety.