The toxicity of phycotoxins has received increasing attention with the increase of frequency, scale and magnitude of toxic harmful algal blooms (HABs) in recent years. Many studies have been mostly focused on the impacts of phycotoxins on mammals (such as mice, dogs, human cell lines and etc) to meet the demand of seafood safety control and pollution monitoring (EFSA CONTAM, 2010). However, the toxicological data in aquatic organisms is really limited (Table 2), making it difficult to fully evaluate the ecological risk of phycotoxins and toxic HABs. In this study, the short-term toxicity of 14 common phycotoxins (OA, DTX1, PTX2, YTX, hYTX, GYM, SPX1, AZA1, AZA2, AZA3, STX, dcSTX, PbTx2 and PbTx3) in A. salina was investigated. Among the 14 tested phycotoxins, AZA3 (with a LC50 of 0.0203 µg/ml) was the most toxic phycotoxin in artemia, followed by AZA2 (with a LC50 of 0.0273 µg/ml). AZAs (including AZA-1, AZA-2, AZA-3, AZA-4, AZA-5 and etc) are a group of phycotoxins produced by Azadinium spinosum (Ferreiro et al., 2016). Although the mode of action of the AZAs has not been fully elucidated, AZAs are found to inhibit endocytosis (Sala et al., 2013) and to induce cytoskeleton disorganization (Twiner et al., 2005). In this study, AZA3 (with a LC50 of 0.0203 µg/ml) and AZA2 (with a LC50 of 0.0273 µg/ml) showed higher toxicity than AZA1 (with a LC50 of 0.106 µg/ml). Similarly, a study in mice shows that after intraperitoneal administration, AZA2 (with a minimum lethal dose of 110 µg/kg) and AZA3 (140 µg/kg) are more toxic than AZA1 (150 µg/kg) (Hajime,2006; Twiner et al., 2008). These results reinforce the concept that the toxicity of analogues might can vary significantly.
To prevent human intoxications, the European Union (EU) has set regulatory limits of phycotoxins in shellfish mainly based on the toxicity on mice (Alarcan et al., 2018). The limits of OA, AZA, PTX, STX and YTX in 1 kg shellfish meat are 160 µg, 160 µg, 160 µg, 800 µg and 1 mg, respectively. This suggest that YTX might be the least toxic phycotoxin among the five phycotoxins, followed by STX. In the present study, YTX (with a LC50 of 0.172 µg/ml) is found to be more toxic than STX (with a LC50 of 1.042 µg/ml) and OA (with a LC50 of 0.728 µg/ml) in artemia. This suggest that the toxic effects of phycotoxins in mammals (like mice) and in zooplanktons (like artemia) might be distinct. Therefore, besides of the human-health concerns, the investigation of deleterious effects of phycotoxins on marine food web also requires attention.
As phycotoxins do not only occur singly (Ferron et al., 2016), it is of importance to clarify the combined effects of two phycotoxins. In this study, additive effects were observed in OA + DTX1. OA and DTX1 belong to the polyether fatty acid toxins (Farabegoli et al., 2018). They share similar mode of action, that attacking the serine/threonine phosphoprotein phosphatases (PPs), in particular PP2A, and as secondary targets, PP1 and PP2B (Farabegoli et al., 2018). Therefore, the observed additive effects of OA and DTX1 in artemia are probably due to the “dose addition”. On the other hand, the combination of OA and PTX2 exhibited antagonistic effects. Similarly, a recent study in human intestinal Caco-2 cells shows that the combination of OA with PTX2 results in reduced toxicity (including, ROS production, IL-8 release and γ-H2AX phosphorylation) at low concentrations (Alarcan et al., 2019). It is reported that OA can interact with regulatory nuclear receptors such as PXR (Fidler et al., 2012; Ferron et al., 2016), which regulate the expression of some cytochrome P450 enzymes (Wang et al., 2012). PTX-2 is believed to interact with the AhR and induce P450 1A protein in hepatic cells (Alarcan et al., 2017; Alarcan et al., 2019). Therefore, one possible explanation is that the mixture of OA and PTX2 might induce cytochrome P450 activity and efflux transporter expression, resulting in higher detoxification/excretion of toxins and thus decreased toxic effects (Alarcan et al., 2019).
In this study, the binary exposure to DTX1 + STX, DTX1 + YTX or DTX1 + PTX2 dramatically elevated the mortality in artemia, compared to the individual exposure, suggesting that DTX1 can interact with STX, YTX and PTX2, and then induce greater effects than additive. The synergetic effects of two phycotoxins have been documented. For instance, the mixture of AZA-1 and YTX shows synergism in human intestinal cell models (Caco-2 cells and the human intestinal epithelial crypt-like (Ferron et al., 2016). The combination of YTX and OA with a ratio of 1:26.5 exhibits synergistic effects in the human intestinal epithelial crypt-like cells. Our results further highlight the hazard potency of the mixtures of DTX1 and other phycotoxins (like STX, YTX and PTX2) with regard to the ecological risk.
In summary, this study demonstrates the individual toxicity of 14 phycotoxins in artemia. On the basis of 48 h LC50, the order of toxicity in artemia is AZA3>AZA2>PTX2>DTX1>AZA1> SPX1>YTX>dcSTX>OA>STX>GYM>PbTx3>hYTX>PbTx2. Furthermore, the combination of two phycotoxins exhibits additive (OA + DTX1; OA + DTX1), antagonistic (OA + PTX2; OA + STK) or synergetic (DTX1 + STX; DTX1 + YTX; DTX1 + PTX2; PTX2 + hYTX) effects with regard to the mortality of artemia. The findings enrich the toxicological data of HABs and phycotoxins in zooplanktons and marine ecosystems, and also help better understand the ecological risk of toxic HABs and phycotoxins.