Toxicity of perfluoroalkyl substances (PFAS) toward embryonic stages of mahi-mahi (Coryphaena hippurus)

Perfluoroalkyl substances (PFAS) are highly persistent organic pollutants that have been detected in a wide array of environmental matrices and, in turn, diverse biota including humans and wildlife wherein they have been associated with a multitude of toxic, and otherwise adverse effects, including ecosystem impacts. In the present study, we developed a toxicity assay for embryonic stages of mahi-mahi (Coryphaena hippurus), as an environmentally relevant pelagic fish species, and applied this assay to the evaluation of the toxicity of “legacy” and “next-generation” PFAS including, respectively, perfluorooctanoic acid (PFOA) and several perfluoroethercarboxylic acids (PFECA). Acute embryotoxicity, in the form of lethality, was measured for all five PFAS toward mahi-mahi embryos with median lethal concentrations (LC₅₀) in the micromolar range. Consistent with studies in other similar model systems, and specifically the zebrafish, embryotoxicity in mahi-mahi generally (1) correlated with fluoroalkyl/fluoroether chain length and hydrophobicity, i.e., log P, of PFAS, and thus, aligned with a role of uptake in the relative toxicity; and (2) increased with continuous exposure, suggesting a possible role of development stage specifically including a contribution of hatching (and loss of protective chorion) and/or differentiation of target systems (e.g., liver). Compared to prior studies in the zebrafish embryo model, mahi-mahi was significantly more sensitive to PFAS which may be related to differences in either exposure conditions (e.g., salinity) and uptake, or possibly differential susceptibility of relevant targets, for the two species. Moreover, when considered in the context of the previously reported concentration of PFAS within upper sea surface layers, and co-localization of buoyant eggs (i.e., embryos) and other early development stages (i.e., larvae, juveniles) of pelagic fish species to the sea surface, the observed toxicity potentially aligns with environmentally relevant concentrations in these marine systems. Thus, impacts on ecosystems including, in particular, population recruitment are a possibility. The present study is the first to demonstrate embryotoxicity of PFAS in a pelagic marine fish species, and suggests that mahi-mahi represents a potentially informative, and moreover, environmentally relevant, ecotoxicological model for PFAS in marine systems.

     

Copper toxicity across salinities from freshwater to seawater in the euryhaline fish Fundulus heteroclitus: is copper an ionoregulatory toxicant in high salinities?

Two waterborne Cu exposures were performed to investigate if Cu is an ionoregulatory toxicant at all salinities in the killifish, Fundulus heteroclitus. A 30-day flow through exposure in 0 (FW), 5, 11, 22, and 28 ppt (SW) and three [Cu]'s (nominal 0, 30, and 150 microg Cu L(-1)) revealed no apparent Cu induced mortality at the intermediate salinities and high mortality in FW and SW. Fish were sampled at 4, 12, and 30 days after the start of the exposure and both Na+/K+ adenosine triphosphatase (Na+/K+ ATPase) and carbonic anhydrase (CA) activity in the gill and intestine as well as whole body [Na+], and [Cl-] were measured. At the high [Cu] a reduction of whole body [Na+] after 4 days of exposure in FW was the only physiological parameter influenced. A second static 24h Cu exposure was performed in FW, 5, 13, and 29 ppt (SW) and two [Cu]'s (nominal 0 and 110 microg Cu L(-1)). In addition to the parameters listed above, ammonia flux was measured at all salinities and Na+ flux was measured in FW fish. Cu affected ionoregulation in FW where decreased Na+ uptake associated with inhibition of Na+/K+ ATPase led to decreased whole body [Na+] after 24h. The only affected parameter in SW was net ammonia excretion suggesting that Cu is not an ionoregulatory toxicant in SW at the concentrations employed. We propose that physiology rather than chemistry explain much of the variation in Cu toxicity seen across salinities.     

Effects of Copper, Cadmium, and Zinc on the Hatching Success of Brine Shrimp (Artemia franciscana)

Previous studies indicate that the hatching success of brine shrimp (Artemia franciscana) cysts is surprisingly sensitive to ambient metal concentrations. These studies estimated median effective concentrations (EC₅₀s) of 7, 5, and 28 μg l⁻¹ for Cd, Cu, and Zn, suggesting that the hatching end point for A. franciscana is the most sensitive tested to date for Cd and Zn in saline environments and comparable in sensitivity with the most sensitive tested to date for Cu. Furthermore, these data suggest that brine shrimp are at significant risk from Cu and Zn in Great Salt Lake (GSL), UT, where ambient concentrations as high as 10 and 14 μg l⁻¹, respectively, have been measured. Given that brine shrimp appear to be successfully reproducing in GSL, we hypothesized that these toxicity values were either biased low as a result of an artifact of the test method used or that site-specific water-quality conditions in the lake had decreased metal bioavailability such that brine shrimp could successfully reproduce. To test these hypotheses, we initiated a step-wise series of experiments. First we investigated the effects of pretreatment of brine shrimp cysts with antibiotics on brine shrimp sensitivity to metals because previous investigators as part of their test methods have used antibiotics. Next we considered the effect of ionic composition of the artificial test media on sensitivity. Finally, we evaluated the effects of the site-specific water quality of the GSL on metal bioavailability and toxicity. Results indicate that pretreatment of cysts with antibiotics had no effect on sensitivity. However, we were unable to repeat the previous values for Cd and Zn, obtaining EC₅₀s of 11,859 and 289 μg l⁻¹ for Cd and Zn, respectively. For Cu, however, we estimated an EC₅₀ of 12 μg l⁻¹, so we conducted further testing on the artificial media, adjusting the media composition to better reflect the Ca²⁺ and HCO ₃ ⁻ concentration of normal seawater. This increased the EC₅₀ to 28 μg l⁻¹. Finally we evaluated the toxicity of Cu in GSL water and obtained an EC₅₀ of 68 μg l⁻¹, suggesting that the increased dissolved organic carbon in GSL has a significant protective effect. Overall, the results of this study suggest that brine shrimp hatching success is not particularly sensitive to Cd and Zn, but it is sensitive to Cu. However, site-specific water-quality conditions ensure that brine shrimp cyst hatching success is not significantly affected by any of these metals at the normal background concentrations that occur in GSL (<15 μg l⁻¹).     

Physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates

The present paper presents original data and a review of the copper (Cu) toxicity literature for estuarine and marine environments. For the first time, acute Cu toxicity across the full salinity range was determined. Killifish, Fundulus heteroclitus, eggs were hatched in freshwater (FW), 2.5, 5, 10, 15, 22 and 35 ppt (seawater, SW) and juveniles were allowed to acclimate for 7 days prior to acute toxicity testing. Sensitivity was highest in FW (96 h LC50: 18 microg/l), followed by SW (96 h LC50: 294 microg/l) with fish at intermediate salinities being the most tolerant (96 h LC50 > 963 microg/l at 10 ppt). This approximately 50-fold, non-linear variation in sensitivity could not be accounted for by Cu speciation or competition among cations but can be explained by physiology. The relative Na(+) gradient from the blood plasma to the water is greatest in FW followed by SW and is smallest at 10 ppt. Regression of Cu toxicity versus the equilibrium potential for Na(+), which reflects the relative Na(+) gradient, revealed that 93% of the variation can be attributed to Na(+) gradients and thus osmoregulatory physiology. Examination of the existing literature on acute Cu toxicity in SW (defined as >25 ppt) confirmed that early life stages generally are most sensitive but this pattern may be attributable to size rather than developmental stage. Regardless of developmental stage and phylogeny, size clearly matters for Cu sensitivity. The existing literature on the influence of salinity on acute Cu toxicity as well as studies of mechanisms of Cu toxicity in fish and invertebrates are reviewed.     

Validation study of the acute biotic ligand model for silver

An important final step in development of an acute biotic ligand model for silver is to validate predictive capabilities of the biotic ligand model developed for fish and invertebrates. To accomplish this, eight natural waters, collected from across North America, were characterized with respect to ionic composition, pH, dissolved organic carbon, and sulfide. Tests were conducted with the cladoceran Ceriodaphnia dubia (48-h static) and the fish Pimephales promelas (96-h static renewal) to determine the concentrations causing lethality to 50% of the organisms (LC50s) for silver in each of these waters. Overall, the biotic ligand model adequately predicted silver toxicity to C. dubia; however, in some cases, predicted LC50 values exceeded measured values. The accuracy of the biotic ligand model predictions was less convincing for silver toxicity to P. promelas with pronounced problems in low-ionic strength waters. Another issue was the use of acclimated organisms in toxicity studies because the biotic ligand model has been developed with the use of a mix of studies with acclimated and nonacclimated test organisms of varying ages and sizes. To evaluate whether effects of acclimation to test waters influence biotic ligand model predictions, a subset of the natural waters were also tested with P. promelas that had been acclimated to the natural water for 7 d before testing. These experiments revealed no differences in toxicity between acclimated and nonacclimated P. promelas. To determine the influence of organism size, which has been previously correlated to Na(+) turnover and acute silver toxicity across multiple species, Na(+) and Cl(-) influx rates were measured in P. promelas of different sizes. Our results show that Na(+) and Cl(-) influx rates were inversely related to fish mass and positively correlated with silver sensitivity.     

Uptake and effects of nitrite in the marine teleost fish Platichthys flesus

The route of NO(2)(-) uptake and subsequent physiological effects were examined in the marine teleost, European flounder (Platichthys flesus), during exposure to 1 mM ambient NO(2)(-) for up to 11 days. Drinking of seawater resulted in a similar nitrite concentration in the anterior part of the intestine as in the ambient water. The NO(2)(-) concentration decreased along the gastro-intestinal tract, suggesting NO(2)(-) uptake across the intestinal epithelium. Comparison of NO(2)(-) uptake in fish that drank NO(2)(-)-contaminated seawater with fish that did not (i.e. had the intestine perfused with a NO(2)(-)-free saline during NO(2)(-) exposure) revealed that the intestinal route contributed some 66% of whole-body NO(2)(-) uptake. Plasma [NO(2)(-)] stayed below the ambient level. It reached a maximum of 0.35-0.4 mM on days 3-6 and then declined to 0.2 mM on day 11. The physiological effects of NO(2)(-) exposure were relatively minor compared with those reported in freshwater fish. Blood methemoglobin levels increased from approximately 4% in non-exposed fish to a maximum of 18% of total hemoglobin in exposed fish. An extracellular hyperkalemia was observed from day 3 of NO(2)(-) exposure, with a maximal increase in plasma K(+) concentrations of 38%. No mortality occurred during the 11 days of NO(2)(-) exposure. The lack of mortality can be related to the relatively low NO(2)(-) accumulation in the plasma and the relatively minor physiological disturbances.     

The effects of metals on embryo-larval and adult life stages of the sea urchin, Diadema antillarum

Since the massive population decline of the long-spined sea urchin, Diadema antillarum, in the early 1980s, the dynamics of coral reef ecosystems in the Caribbean have changed tremendously. The absence of D. antillarum, once a keystone herbivore, has led to macroalgal dominance in many of these reef communities. D. antillarum is not only important ecologically, but may also be a sensitive bioindicator species for toxicant exposure. Echinoderm larval development tests were conducted with D. antillarum exposed to elevated levels of aqueous copper (Cu), silver (Ag), nickel (Ni), or selenium (Se). All metals significantly affected larval development, based on normal development to the pluteus stage. The EC50s based on dissolved metal concentrations were 11 microg/L Cu, 6 microg/L Ag, 15 microg/L Ni, and 26 microg/L Se. Adult sea urchins were exposed to aqueous copper under flow through conditions for 96 h. The 96-h LC50 for this exposure was 25 microg/L dissolved Cu. Additionally, behavioral and physiological disturbance was observed. The physiological responses included both acid-base balance disturbance, as evidenced by reduced coelomic fluid pH and apparent ionoregulatory effects. In addition, behavioral effects included spatial orientation within the exposure tank, spine closure, and loss of spines. The high sensitivity of both adult and larval D. antillarum to these metals supports the use of this organism as an important biological indicator for metal exposure in marine environments.     

Physiological responses to acute silver exposure in the freshwater crayfish (Cambarus diogenes diogenes)--a model invertebrate?

Adult crayfish (Cambarus diogenes diogenes) exposed to 8.41 +/- 0.17 microg silver/L (19.4% as Ag+) in moderately hard freshwater under flow-through conditions for 96 h exhibited ionoregulatory disturbance, elevated metabolic ammonia (T(amm)) production and substantial silver accumulation in the gills, hemolymph, and hepatopancreas. The ionoregulatory disturbance included both a generally reduced unidirectional Na+ influx and an increased unidirectional Na+ efflux, leading to a substantial net loss of Na+ from the silver-exposed crayfish. The Na+ uptake in silver-exposed crayfish differed overall from controls, while the increased Na+ efflux recovered to control values 48 h into the 96 h of exposure. The general inhibition of Na+ uptake could be explained by a reduced sodium/potassium-adenosine triphosphatase (Na/K-ATPase) activity in terminally obtained gill samples from the silver-exposed crayfish. The silver-induced effect on Na+ uptake and loss translated to reduced hemolymph Na+ concentrations but not significantly reduced hemolymph Cl- concentrations. Hemolymph T(anim) and T(amm) efflux both increased in silver-exposed crayfish, indicating an increased metabolic T(amm) production. The present study demonstrates that the toxic mechanism of waterborne silver exposure in freshwater crayfish resembles that of freshwater teleost fish. The crayfish might therefore be a useful model system for extending current environmental regulatory strategies, currently based on teleost fish, to invertebrates.     

The Effects of Dietary Silver on Larval Growth in the Echinoderm Lytechinus variegatus

Previous studies have demonstrated that the euryhaline copepod Acartia tonsa is extremely sensitive to dietborne silver (Ag) exposure, with a 20 % inhibition (EC(20)) of survival occurring when copepods are fed algae with 1.6 μg g(-1) dry weight (dw) Ag, corresponding to a waterborne Ag concentration of 0.46 μg l(-1) Ag. In contrast, 43 μg l(-1) Ag is required to elicit similar effects in copepods exposed to Ag by way of water. In the current study, we investigated whether another planktonic marine organism might also be sensitive to dietary Ag. Specifically, we tested larvae of the echinoderm, Lytechinus variegatus in an 18-day study in which larvae were continuously exposed to Ag-laden algae (Isochrysis galbana). After 7 days of exposure, no significant effects were observed on larval growth up to the highest concentration tested (10.68 μg g(-1) dw Ag in algae after exposure to 3.88 μg l(-1) waterborne Ag). After 18 days, significant effects were observed in all Ag treatments resulting in a lowest-observable effect concentration of 0.68 μg g(-1) dw Ag in algae and corresponding waterborne Ag concentration of 0.05-0.07 μg l(-1) Ag (depending on background Ag [see Results]). However, the dose-response relationship was quite flat with a similar level of growth inhibition (approximately 15 %) in all Ag treatments, resulting in an EC(20) of >10.68 μg g(-1) dw Ag in algae (>3.88 μg l(-1) Ag in water). This flat dose-response relationship is characteristic of dietary metal (silver, copper, cadmium, nickel, and zinc) toxicity to copepods as well, although the effect is slightly more robust (approximately 20-30 % inhibition of survival or reproduction). We conclude that echinoderm larvae may be similar to copepods in their sensitivity to dietary Ag, although a better understanding of the mechanisms underlying the apparent flat dose-response relationships is clearly needed.     

The effects of silver on intestinal ion and acid-base regulation in the marine teleost fish, Parophrys vetulus

Exposure to elevated silver (as AgNO3) concentrations (6-9 microM) in seawater was associated with comparably high silver concentrations in the intestinal fluids of the lemon sole (Parophrys vetulus), and a tendency for reduced drinking rate. The effects of silver on intestinal ion and acid-base regulation were studied using in situ perfusion of the intestine. Intestinal net Cl- uptake was reduced from 0.4 to 0.1 and intestinal net Na+ uptake from 0.2 to 0 mmol kg(-1) x h(-1) during silver exposure (9 microM). At the same time, intestinal HCO3- net efflux was reduced from 0.2 to 0.1 mmol kg(-1) x h(-1). Both intestinal Na+ and Cl- uptake and Cl-/HCO3- exchange are thus sensitive to silver, but to different extents. None of the observed effects were reversible during 24 h of recovery. Intestinal water transport was highly variable in vivo in the perfused preparation, and no significant effect of silver exposure was observed. However, in vitro intestine preparations exhibited reduction of intestinal net water flux from 4 to 1 microl cm(-2) x h(-1) during silver exposure together with reduced unidirectional Cl- influx. Reduced water intake and transepithelial water transport in silver-exposed fish resulted in moderate hemoconcentration evident from higher hematocrit values, but not in increased plasma ion levels. The latter could reflect a compensatory response via increased branchial Na+/K+-ATPase levels, observed in silver-exposed fish, indicative of increased branchial ion transport capacity. Impairment of intestinal ion and water transport as a result of silver intake via drinking could be an important part of the fatal cascade of physiological effects observed in marine fish during acute silver exposure.