Challenges towards sustainable aquafeeds-plant nutrients and contaminants interactions- Use of biological models and indicators-

The development of new sustainable feed, by replacing marine feed resources for Atlantic salmon with vegetable feed ingredients, introduces new challenges in fish health and food safety. The increased use of vegetable oils has been shown to substantially reduce the levels of persistent organic pollutants in farmed salmonids. However, the use of new feed ingredients introduces new contaminates, like PAH and pesticides, normally not associated with farming of salmonides. Nutrients (eg. tocopherols and fatty acids) are well known to interact and even protect against contaminants (PAH, benzo[a]pyrene (BaP) and phenanthrene (Phe) and the pesticides, chlopyrifos (CHl) and endosulfan (Endo) toxicity. The increased replacement of marine ingredients with plant has increased the need for knowledge of new nutrients and contaminants and especially on interactions, and common biological outcome. In the present project the interactions of four contaminants (BaP, Phe, chlopyrifos and endosulfan) and four nutrients (plant nutrients; alfa tocopherol and arachidonic acid, marine nutrients; gamma tocopherol and EPA) are studied on 1) intestinal uptake mechanisms, 2) biological synergistic or antagonistic interaction effects and common mode of actions and 3) cellular end points. The intestinal uptake studies started with a first identification on intestinal sites of uptake of PAHs by autoradiogrammes of Atlantic salmon previously fed marine plant oil based feed and force feed with C14 labelled Phe and BaP. The uptake of the contaminants occurred at all intestinal sections with difference in uptake depedning on feed matrix. The relative intestinal uptake of Phe is higher than that of BaP, due to a lower metabolisation of Phe compared to BaP in the intestinal cells. Fish fed on fish oil had a higher metabolisation of BaP than fish fed on plant oil. Secondly, the intestinal uptake was studied at the apical membrane of intestinal cells from the cecea, mid and hind gut by use of isolated inside out apical vesicles. Preliminary results show high apical membrane loads and minor membrane transport into the cell. This uptake is independent of temperature and hence not mediated by active transport. Further studies investigate interaction with fatty acids at apical membranes as well as trans-epithelial uptake using ussing chambers. The biological interactions and mode of actions of contaminants and nutrients were studied in an in vitro Atlantic salmon primary hepatocyte cell culture model system. Firstly, the interactions of the contaminant and nutrient in combinations or alone on cell viability was assessed by using an xCELLigence in vitro system. The contaminant mixture alone showed mostly additive effects of the contaminants on cell viability but also a synergistic interaction response between chlorpyrifos and endosulfan. Further contaminant-nutrient mixture assessment indicate that the marine nutrients are more beneficial than the plant nutrients in preventing adverse cell viability effects caused by contaminant mixtures. The modes of actions in contaminant mixture exposures were assessed by genomic and metabolomic marker screening. Most dominant effects of contaminant mixture exposures were on lipid as and endocrine disruption with the pesticides giving the strongest responses. Chlorpyrifos suppressed the synthesis of unsaturated fatty acids. Endosulfan affected the VTG steroid hormone synthesis, while benzo(a)pyrene had an weak effect on steroid vitamin D3 metabolism. The marine nutrients EPA and alfa-tocopherol reduced the negative effects of PAH and pesticide mixtures while the vegetable nutrients such as gamma- tocopherol (plant vitamin E) or arachidonic acid (found in vegetable oil fed fish) enhance the effect.

The limited access to fish meal and fish oil for the rapidly growing aquaculture has lead to the replacement of fish meal and oil with plant ingredients over the last decade. This has reduced the level of notorious contaminants (e.g. dioxins), however, ha s also introduced new chemical contaminants (e.g. PAHs and pesticides) that are normally not associated with salmon farming. The use of plant ingredients has also altered the nutrient profile (fatty acid antioxidants). Nutrients are well known to interact and even protect against contaminant toxicity. The increased replacement of marine ingredients with plant ingredients has increased the need for knowledge of new nutrients and contaminants and especially on their interaction and common biological outcome . Nutrient-contaminant interactions can occur at several levels of biological organization including intestinal uptake, peripheral distribution, and/or central cellular pathways. The multitude of possible nutrient-contaminant combinations as well as inter actions points at cellular pathways requires an initial cytotoxic screening and marker identification, before studying cellular mechanisms of interaction or in vivo target animal evaluation. The present project aims to elucidate on the interactions of pla nt ingredient related contaminants and nutrients on dietary uptake, metabolism, and neurodevelopment related to novel aquafeed, with emphasis on generating basic knowledge on the combined effect by use of biological indicators in model systems at differen t levels of biological organization.