The current study is the first report of embryotoxicity of PFAS in a pelagic marine fish species. Dose-dependent embryotoxicity (Fig. 2), and specifically, lethality, was observed for mahi-mahi embryos exposed to all PFAS evaluated within 24 h of exposure in the micromolar range (Table 1). Measured toxicity in the mahi-mahi embryo generally aligns with previous studies in the zebrafish model which have reported quantitatively similar (i.e., micromolar) embryotoxicity for both legacy PFAS (i.e., PFOA and PFOS) and next-generation PFAS including PFECA (Gaballah et al. 2020; Gebreab et al. 2020; Godfrey et al. 2017; Hagenaars et al. 2011; Jantzen et al. 2016; Pecquet et al. 2020; Shi et al. 2008; Weiss-Errico et al. 2017; Ye et al. 2009; Zheng et al. 2012). Observations in both the present (i.e. mahi-mahi) and past (i.e., zebrafish) studies are notable as PFECA (e.g., GenX) have been adopted as purportedly less toxic alternatives (Bowman 2015), and these results, thereby, agree with prior studies which have concluded that toxicity of these replacement compounds is, on the contrary, potentially comparable to legacy PFAS (Gaballah et al. 2020; Gebreab et al. 2020).
Relative embryotoxicity for mahi-mahi, based on calculated 24-h LC50 values, was found to be GenX < PFOA < PFO2DA < PFDMMOBA < PFO3TDA, and was generally correlated with PFAS chain length (inclusive of all fluorocarbon and ether groups) with the notable exception of the relatively high toxicity of PFDMMOB. Indeed, LC50 was significantly correlated (p < 0.001) when PFMMOBA was not included (Fig. 3). This trend - and, furthermore, the exception of the PFMMOBA - is noteworthy as a similar significant correlation between embryotoxicity and chain length of PFAS has been consistently demonstrated in numerous, previous studies (in the zebrafish embryo model; Buhrke et al. 2013; Gaballah et al. 2020; Menger et al. 2020; Ulhaq et al. 2013; Wasel et al. 2021) including a recent assessment of the same PFECA (Gebreab et al. 2020; see Table 1). These studies collectively demonstrate a highly reproducible, quantitative toxicity of PFAS in embryonic fish models. It has, likewise, been recently shown that bioconcentration factors (BCF) of PFAS (alongside relative toxicity) in zebrafish are, likewise, significantly correlated with chain length, and it has been concluded that toxic potential is likely correlated with relative uptake potential (Menger et al. 2020; Vogs et al. 2019). Moreover, a recent metabolomics analysis in the zebrafish embryo model (Gebreab et al. 2020) identified nearly identical patterns of altered metabolic profiles between embryos exposed to both PFOA, and long- and short-chain PFECA (i.e., PFO3TDA and GenX, respectively). This observation suggests shared mechanistic pathways of toxicity, despite observed quantitative differences, and correlations between embryotoxicity and chain-length, and thus, further support a role of differential uptake (rather than difference in mechanism) in the relative toxicity of perfluorocarboxylic acids. Relative uptake is presumably a function of hydrophobicity, and indeed, a similar correlation between toxicity and calculated log P values for PFAS (with similar exception of PFDMMOBA) was observed in the present study (Fig. 3). That said, while hydrophobicity and, in turn, uptake, of PFAS is expected to primarily increase with chain length, previous studies have suggested other structural features including, a role of terminal functional groups (e.g., carboxylic versus sulfonic acids) in the relative uptake and, thus, toxic potential (Menger et al. 2020; Vogs et al. 2019). Thus, correlations as observed here are likely to only apply to perfluorinated carboxylic acids.
Although the reason for the higher than expected toxicity of PFDMMOBA in the present study is unclear, and remains to be investigated further, it is worth noting that this congener is among the only two (with the other being the most lipophilic, long-chain PFO3TDA) for which LC50 and LOAEL/NOAEL (Table 1) did not significantly change following an additional 24 h of exposure (discussed further below). And, in fact, a similar lack of significant increase in toxicity during an equivalent developmental window (7 dpf, i.e., fully hatched, early larval stage) was, likewise, observed for PFDMMOBA in a previous study of zebrafish embryotoxicity (Gebreab et al. 2020). It is, therefore, proposed that more rapid uptake, or alternatively perhaps, different cellular or molecular targets during early development, may explain higher than expected toxicity observed for this congener. Such a role of toxicokinetics and/or other mechanistic differences in the exceptional toxicity of this congener, however, remains to be addressed.
With the exception of PFDMMOBA, as well as most toxic PFO3TDA, lethality significantly increased for all compounds tested over 48 h of static exposure, as evidenced by significantly (p < 0.05) lower LC50, and NOAEL/LOAEL (Table 1). Similarly increased toxicity, based on both lethality and other toxicological endpoints, with continuous exposure has been observed in previous studies of PFAS including PFOA and PFECA in zebrafish embryos (Gebreab et al. 2020; Hagenaars et al. 2011; Ye et al. 2009; Zheng et al. 2012). With respect to developing embryos, increased toxicity at 48 h is notable as mahi-mahi embryos typically hatch within approximately 36-48 hpf (Perrichon et al. 2019), and nearly all (96%) of the embryos in the current study were, in fact, fully hatched at 48 h. Increased toxicity, therefore, may be associated with loss of the protective chorion as a barrier for uptake of PFAS. A role of the chorion would, in turn, have ecotoxicological implications for environmental exposure of post-hatch eleutheroembryos and larvae of mahi-mahi. A similarly significant increase in toxicity coincident with hatching (~ 72 hpf) has previously been observed for the same compounds (except PFMDMMOBA) in zebrafish (Gebreab et al. 2020). A recent study (Vogs et al. 2019) has similarly documented a “biphasic” pattern of slower uptake prior to hatching, and accelerated uptake post-hatch, in the zebrafish embryo model generally concluding that the chorion, indeed, serves as a protective barrier. The current results are consistent with such a bi-phasic pattern, yet reflect differential effects of the chorion for PFAS congeners: specifically, rapid uptake (and little effect of chorion) of PFO3TDA and PFDMMOBA is seemingly reflected in the observation of a near maximum toxicity of these congeners within 24 h, whereas the chorion limits uptake of PFOA, GenX and PFO2DA until hatching. Notably, the correlation between toxicity and chain-length is not maintained after 48 h exposure (Supplementary Fig. 1), and the differential role of the chorion may, furthermore, explain this lack of correlation: in short, as hindrance of the chorion does not exist post-hatch, equivalent uptake of congeners leads to a convergence of lethal concentrations (in the low micromolar range) after hatching.
Alternatively, it is possible that increased toxicity with exposure time might relate, at least in part, to the development of relevant cellular and molecular targets of PFAS. As recognized hepatotoxins, for example, both liver (i.e., hepatocytes) and enzymes associated with phase I hepatic detoxification including, in particular, cytochrome P450 have been well documented as targets of PFAS(Bassler et al. 2019; Cheng and Klaassen 2008; Dale et al. 2020). Studies in the zebrafish have recently suggested a role of the differentiation of the liver and hepatic enzymes in the observed stage-dependent increase in embryotoxicity of both PFOA and PFECA (Gebreab et al. 2020), as well as other hepatotoxins (Zuberi et al. 2019). Expression of genes associated with differentiation of hepatocytes, and associated liver enzymes, in mahi-mahi has been similarly found (Xu et al. 2017) to occur over a time-frame (i.e., 36 to 48 hpf) coincident with increased embryotoxicity in the current study, suggesting a possibly similar contribution of hepatic developmental in this toxicity. Elevated mortality may, of course, be simply due to the cumulative exposure to the compounds during the continuous exposure period. Whether the observed increased toxicity is due to the loss of the protective chorion barrier, development of the liver (or possibly other targets), or simply, to the prolonged duration (and cumulative effects) of exposure remains to be clarified.
Although observed toxicity of PFOA and PFECA in the mahi-mahi embryo system was approximately comparable in scale (i.e., µM concentrations) to that previously observed in the zebrafish model, and significantly correlated, with respect to the relative toxicity (with the exception of PFDMMOBA), between the two species (Fig. 3), comparison of the current data to our previous assessments of the same compounds in the zebrafish embryo model (Gebreab et al. 2020) demonstrated consistently higher toxicity, as evidenced by LC50 and LOAEL values (Table 1). The consensus of previous studies (Gaballah et al. 2020; Godfrey et al. 2017; Hagenaars et al. 2011; Pecquet et al. 2020; Ye et al. 2009; Zheng et al. 2012) of PFAS including PFOA and PFECA, e.g., GenX, in the zebrafish embryo model have, likewise, generally observed lower toxicity, i.e., LC50, compared to that currently reported for mahi-mahi. Higher relative toxicity for mahi-mahi, compared to zebrafish, may be related to a number of factors including differences in toxicokinetics (i.e., uptake) and susceptibility of relevant biochemical, molecular or cellular targets between the two species, as well as respective assay parameters including, in particular, exposure media (e.g., seawater versus non-saline medium, pH, etc.). A positive correlation between salinity and BCF in fish has, for example, been previously demonstrated (Jeon et al. 2010), suggesting a possible role of both exposure medium (i.e., salinity), and consequent toxicokinetics, in the higher toxicity among mahi-mahi embryos. Alternatively, however, higher sensitivity of mahi-mahi may relate to the targeting of interrelated pathways of cellular energy metabolism by PFAS. Numerous previous studies have implicated metabolic dysfunction including lipid, amino acid and carbohydrate among the adverse effects of PFOA and PFECA (Alderete et al. 2019; Chen et al. 2020; Yu et al. 2016) including such effects in early life (i.e., embryo and larval) stages of zebrafish (Gebreab et al. 2020; Sant et al. 2021). At the same time, it has been shown that rapidly developing mahi-mahi embryos are among the most metabolically active of marine fish species (Pasparakis et al. 2016), and much higher than zebrafish embryos. Taken together, it is possible that targeting of energy metabolism by PFAS may accentuate toxicity in mahi-mahi embryos, alongside any contributions of differential uptake and toxicokinetics.
Regardless of cause, the higher sensitivity of mahi-mahi may have implications for potential exposure of embryos to toxic concentrations of PFAS in ecologically relevant (i.e., marine) waters. Indeed, a lingering ecotoxicological question, in this regard, is whether environmentally relevant concentrations of PFAS in aquatic systems are sufficient for toxicity. With 48-h LOAEL (for lethality) in the low micromolar range for PFAS (Table 1), it is possible that sub-acute toxicity may, indeed, effectively extend into the nanomolar range. While typically in the sub-ppt range, PFAS concentrations approaching nanomolar concentrations (e.g., 200 ppt PFOA) have been measured, particularly in nearshore marine waters (Yamashita et al. 2004). Moreover, 100-fold concentration factors of PFAS within the SSML – and as much as 5000-fold in aerosols – have been reported (Casas et al. 2020; Ju et al. 2008), suggesting potentially nanomolar concentrations within ecologically relevant upper-layer surface waters where buoyant eggs (i.e., embryos) and larval stages of mahi-mahi, and many other marine fish species, are distributed. Whether these early life stages of marine fish are, in fact, exposed to effectively toxic concentrations, however, remains to be investigated.
In conclusion, embryos of mahi-mahi, as a representative pelagic marine fish species, were found to be a quantitative model of the toxicity of PFAS, largely comparable in this regard to the zebrafish embryo as an established laboratory model. Alongside quantitative potential of this model with respect to structure-activity (i.e., chain-length and relative toxicity), these studies point to interactive effects of uptake and developmental stage (e.g., hatching/loss of chorion, development of relevant target organs). Moreover, these studies identified toxicity at exposure concentrations sufficiently low to approach environmentally relevant concentrations, particularly in marine waters, and especially the sea surface where both PFAS and buoyant eggs of many marine fish species are generally concentrated. This finding opens the door to future studies to evaluate ecotoxicological impacts of PFAS on marine fish populations.