Quantification of the role of reactive oxygen species, phycotoxins and fatty acids in fish toxicity by harmful marine microalgae remains inconclusive. An in vitro fish gill (from rainbow trout Oncorhynchus mykiss) assay was used to simultaneously assess the effect in superoxide dismutase, catalase and lactate dehydrogenase enzymatic activities caused by seven species of ichthyotoxic microalgae (Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, Alexandrium catenella, Karlodinium veneficum, Prymnesium parvum). Quantification of superoxide production by these algae was also performed. The effect of purified phycotoxins and crude extracts was compared, and the effect of fatty acids is discussed. The raphidophyte Chattonella was the most ichthyotoxic (gill cell viability down to 35%) and also the major producer of superoxide radicals (14 pmol cell^-1 hr^-1) especially after cell lysis. The raphidophyte Heterosigma and dinoflagellate Alexandrium were the least toxic and had low superoxide production, except when A. catenella was lysed (5.6 pmol cell^-1 hr^-1). Catalase showed no changes in activity in all the treatments. Superoxide dismutase (SOD) and lactate dehydrogenase exhibited significant activity increases of ≤23% and 51.2% TCC (total cellular content), respectively, after exposure to C. marina, but SOD showed insignificant changes with remaining algal species. A strong relationship between gill cell viability and superoxide production or superoxide dismutase was not observed. Purified brevetoxins PbTx-2 and -3 (from Karenia brevis, LC₅₀ of 22.1 versus 35.2 μg mL^-1) and karlotoxin KmTx-2 (from Karlodinium; LC₅₀ = 380 ng mL^-1) could almost entirely account for the fish killing activity by those two dinoflagellates. However, the paralytic shellfish toxins (PST) GTX1&4, C1&C2, and STX did not account for Alexandrium ichthyotoxicity. Only aqueous extracts of Alexandrium were cytotoxic (≤65% decrease of viability), whereas crude methanol and acetone extracts of Chattonella, Fibrocapsa, Heterosigma, Karlodinium and Prymnesium decreased cell viability down to 0%. These and our previous findings involving the role of fatty acids confirm that superoxide radicals are only partially involved in ichthyotoxicity and point to a highly variable contribution by other compounds such as lipid peroxidation products (e.g. aldehydes).

The large phagotrophic dinoflagellate Noctiluca has become a prominent red tide organism in southeast Australian waters since the 2000s, raising concerns for beach tourism, grazing impacts as well as ichthyotoxicity for finfish aquaculture. Satisfactory culture growth rates (0.23–0.56 per day) were obtained by feeding with small Thalassiosira diatom and Tetraselmis flagellate diets, while optimal growth rates sustained for up to 8 months (0.69 per day) were achieved by feeding in a plankton wheel with the large chain-forming dinoflagellate Gymnodinium catenatum. Noctiluca was highly tolerant towards salinities from 20 to 35 and growth was stimulated by temperatures increasing from 10 to 23°C, which in combination with the key factor of prey abundance explains the incidence in southeast Australia of predominantly summer and spring but occasionally also winter blooms. Fatty acid biomarkers suggest that Tasmanian field populations indiscriminately feed on available diatom and dinoflagellate mixtures. Noctiluca exhibited very limited ichthyotoxicity, and only at the highest cell concentrations of 2 000 000/L (50% reduction in RTgill W1 cell viability). Only the densest red tide surface slicks contained acutely toxic levels of unionized ammonia of 242 to 510 μg/L while inshore slicks generated oxygen concentrations as low as 0–1.5 ppm. Lipid phycotoxins (eicosapentaenoic acid, docosahexaenoic acid) did not appear to contribute to Noctiluca ichthyotoxicity. The fatty acid 20:0 eicosanoic acid may serve as a potential Noctiluca biomarker in marine food webs and sediments.

Clay minerals have previously been used to mitigate algal blooms because of their ability to flocculate algal cells or remove nutrients, but also offer considerable potential to remove ichthyotoxins. When a barramundi farm in tropical Australia suffered substantial fish mortalities due to a bloom of the ichthyotoxic haptophyte Prymnesium parvum, the farm manager decided to manipulate pond water N:P ratios through removal of phosphorus by the addition of lanthanum-modified bentonite clay (Phoslock™) to successfully mitigate ichthyotoxic effects. We conducted Prymnesium culture experiments under a range of N:P ratios, screening 14 different clays (two zeolites, four kaolins, six bentonites and two types of Korean loess) at pH 7 and 9 for cell flocculation and removal of ichthyotoxicity assessed with the RTgill-W1 cell line assay. Application of Phoslock™ to cultures grown at different N:P effectively removed 60%–100% of water-soluble toxicity of live Prymnesium (dependent on nutritional status). While most clays efficiently flocculated Prymnesium cells (≥80% removal), cell removal proved a poor predictor of ichthyotoxin adsorption. Extensive clay screening revealed that at elevated pH, as commonly associated with dense algal blooms, most clays either exacerbated ichthyotoxicity or exhibited significantly reduced toxin adsorption. Interpretation of changes in clay zeta potential at pH 7 and 9 provided valuable insight into clay/ichthyotoxin interactions, yet further research is required to completely understand the adsorption mechanisms. Bentonite-type clays proved best suited for ichthyotoxin removal purposes (100% removal at ecologically relevant pH 9) and offer great potential for on-farm emergency response.