Effect of methylmercury on egg and juvenile viability in two populations of killifish Fundulus heteroclitus

Killifish (Fundulus heteroclitus) eggs from a polluted creek (Piles Creek (PC)) and a relatively pristine estuary in Long Island (LI) were exposed for 20 min to various concentrations of methylmercuric chloride (MeHg) prior to combination with untreated sperm. PC killifish eggs showed a higher LC50 value (1.7 mg/liter) than LI eggs (0.7 mg/liter). PC eggs that were fertilized by nontreated sperm after exposure to 1.0 or 2.5 mg/liter meHg and then placed in clean sea water (15 parts per thousand) for 1 week showed a 5 and 7% malformations of the embryos, respectively. However, exposure of LI eggs to 1.0 mg/liter prior to fertilization caused 32% malformations of the embryos, and at 2.5 mg/liter almost all the embryos died. The data indicate that LI killifish eggs are less tolerant to meHg than PC eggs. This is in keeping with previous data on embryonic tolerance to meHg in these two populations. However, 96-hr LC50 values of juvenile fish (25-45 mm standard length) did not differ between these two populations.     

Salinity effects on the bioavailability of aqueous metals for the estuarine killifish Fundulus heteroclitus

Estuarine organisms experience varying salinity conditions on a daily and seasonal basis, and these fluctuations could influence the amount of metal accumulated from the aqueous phase. The present study experimentally assessed the role of salinity (0, 2, 6, 12, and 25 ppt) on the uptake of As, Cd, Cr, inorganic Hg [Hg(II)], and methylmercury (MeHg) into the euryhaline killifish (Fundulus heteroclitus) from the aqueous phase using gamma-emitting radioisotopes. Patterns of metal uptake as a function of salinity varied by metal. Chromium showed no relationship with salinity; Cd, which was most affected by salinity, showed an inverse relationship; and As, Hg(II), and MeHg uptake increased as salinity increased from 0 ppt to 25 ppt. Arsenic (salinities ≤ 6 ppt) and Cr were regulated by the fish, whereas Cd, Hg(II), and MeHg were not. Cadmium, Hg(II), and MeHg are chloro-complexed, increasing bioavailability for Hg(II) and MeHg, and reducing bioavailability for Cd. Concentration factors (CFs) were >1 at all salinities for Cd, Hg(II), and MeHg, indicating that the fish were more enriched in the metal than the surrounding water, whereas As and Cr CFs were <1 at all salinities. Uptake rate constants (k(u)s) were highest for MeHg (0.79-2.29 L g(-1) d(-1)), followed by Hg(II), Cd, Cr, and lowest for As (0.0004-0.0008 L g(-1) d(-1)). Tissue distribution of each metal was determined by dissections. Data for Cd showed that as salinity increased, the concentration of this metal increased in the viscera, whereas it decreased in the head and gills, suggesting that drinking to osmoregulate may account for a portion of Cd uptake from the aqueous phase in marine fish.     

Reproductive and transgenerational effects of methylmercury or Aroclor 1268 on Fundulus heteroclitus

This research determined the potential for methylmercury or Aroclor 1268 to disrupt reproduction and sexual differentiation in Fundulus heteroclitus. The research determined whether fish that are exposed to mercury or Aroclor 1268 survive and successfully reproduce; whether offspring of exposed fish hatch, survive, produce eggs, and fertilize them; and whether the second-generation offspring of exposed fish hatch and survive. Fundulus heteroclitus were exposed to mercury or Aroclor 1268 via contaminated food. Endpoints evaluated included survival, growth, fecundity, fertilization success, hatch success, larval survival, sex ratios, and the prevalence of gonadal abnormalities. In general, polychlorinated biphenyls were highly bioavailable and accumulated well through feeding. The only statistically significant effect observed as a result of treatment with Aroclor 1268 was an increase in growth in the offspring of exposed fish. Mercury was accumulated in a dose-dependent fashion via food exposures. Exposure to mercury in food increased mortality in male F. heteroclitus, which possibly occurred as a result of behavioral alterations. Increased mortality was observed at body burdens of 0.2 to 0.47 microgram/g. Offspring of F. heteroclitus fed mercury-contaminated food were less able to successfully reproduce, with reduced fertilization success observed at egg concentrations of 0.01 to 0.63 microgram/g, which corresponds with parent whole-body concentrations of 1.1 to 1.2 micrograms/g. Offspring of exposed fish also had altered sex ratios, with treatment at moderate concentrations producing fewer females and treatment at the highest concentration producing more females than expected. Alterations in sex ratios were observed at concentrations of less than 0.01 microgram/g in eggs or between 0.44 and 1.1 micrograms/g in parents. Offspring of mercury-exposed fish also had increased growth in moderate treatments, when egg concentrations were less than 0.02 microgram/g, or when parent whole bodies contained 0.2 to 0.47 microgram/g. In summary, exposure to mercury reduced male survival, reduced the ability of offspring to successfully reproduce, and altered sex ratios in offspring. Both direct effects on exposed fish and transgenerational effects were observed.     

The influence of exposure history on arsenic accumulation and toxicity in the killifish, Fundulus heteroclitus

Exposure to arsenic is known to cause adverse effects in aquatic biota and wildlife and is of major concern to human health. Although numerous studies have investigated the toxicity of arsenic, little is known about the effects of acquired tolerance on arsenic accumulation and toxicity outside of cell culture models. Accordingly, studies were conducted on the estuarine fish, Fundulus heteroclitus, that were preexposed to nontoxic concentrations of arsenic (as sodium arsenite; 0.7 and 106 micromol As/L) for 96 h or na?ve to elevated arsenic to determine the effects of acclimation on arsenic toxicity and accumulation. Tolerance to arsenic was rapidly (96 h) acquired in killifish that were preexposed. In toxicity tests with arsenic-acclimated killifish, preexposure to 106 micromol As/L resulted in a reduction in toxicity when compared to na?ve animals. Toxicity in arsenic-acclimated fish also was distinguished by a delayed onset of mortality that manifested in dose-dependent fashion and was significant even for the lower acclimation concentration (0.7 micromol As/L). The increase tolerance acquired following preexposure to 106 micromol As/L for 96 h was associated with lower concentrations of arsenic in all monitored tissues (e.g., gill, liver, kidney) and the whole body when fish were exposed to 240 micromol As/L for an additional 96 h. In accordance with these observations, expression of the multidrug resistance- associated protein (MRP)-2 gene, which is responsible for transporting arsenic conjugated to glutathione out of cells, was increased in the liver of arsenic-acclimated fish.     

Effects of salinity on copper accumulation in the common killifish (Fundulus heteroclitus)

Results of laboratory and field studies have demonstrated that salinity influences the accumulation of copper. The present study is, to our knowledge, the first to examine the effect of salinity on copper accumulation in a teleost fish across a comprehensive range of salinity from freshwater to seawater. This was done in an effort to identify potential target tissues and differences in chemical interactions across salinities that will aid in the development of a seawater biotic ligand model (BLM) for copper. Killifish (Fundulus heteroclitus) were acclimated to five salinities (0, 5, 11, 22, and 28 ppt) and exposed to three copper concentrations (0 [nominal], 30, and 150 microg L(-1), yielding 15 treatment groups. Fish from each group were sampled for tissue copper analysis at 0, 4, 12, and 30 d postexposure. Whole-body and liver accumulations were highest at lower salinities. The liver accounted for 57 to 86% of the whole-body copper even though it accounted for less than 4% of the body mass. Similarly, the gill accumulated more copper at lower salinities, whereas the intestine generally accumulated more copper at higher salinities. Speciation calculations indicate that CuCO3 likely accounts for much of the accumulation, possibly with some contributions from CuOH+ and Cu(OH)2. The free ion, Cu2+, does not appear to be associated with copper accumulation. However, the differences in physiology and in the concentrations of competing cations across salinities suggest that speciation alone cannot explain accumulation. The present findings may have implications for future development of a BLM for saline environments by identifying potential target tissues.     

Melatonin effects on Fundulus heteroclitus reproduction

This study aimed to investigate the effects of two different doses (100nM (M1) and 1μM (M2)) of exogenous melatonin on the reproductive capacity of Fundulus heteroclitus. Eight days of melatonin exposure significantly increased the fecundity and embryo survival of F. heteroclitus only in the M2 group compared with the control; the hatching rate was unaffected. Moreover, increases in the local expression of the melatonin receptor (mtnr) gene during follicle maturation were found; however, there were no differences between the experimental groups. Furthermore, in vitro melatonin-treated follicles showed a significantly higher germinal vesicle break down percentage compared with the control, while SDS-PAGE showed no difference in the electrophoretic pattern of the major yolk proteins. Nevertheless, densitometry revealed a greater intensity of the 118-, 95- and 40-kDa components in groups treated with melatonin. Finally, Fourier transform infrared microspectroscopy was applied to classify the different stages of oocyte development (Stages I-II, III and IV) on the basis of their macromolecular composition. The effects induced by melatonin on oogenesis were investigated by comparing vibrational spectra of females exposed to melatonin with those of controls. Changes to the Amide I band, corresponding to an increase in β-structure, were found in oocytes of females exposed to the highest melatonin dose. These results highlight the positive role of melatonin, which is able to enhance the reproductive capacity of F. heteroclitus. Further studies are in progress to better explain the molecular mechanisms by which melatonin treatment affects reproduction in this marine species.     

Influence of humic acid on the uptake of aqueous metals by the killifish Fundulus heteroclitus

The role of humic acids, over a concentration range of 0 to 20?mg?L(-1) , was investigated in the uptake of three metals (Cd, Cr, and Hg-as both inorganic Hg [Hg(II)] and methylmercury [MeHg]) and a metalloid (As) from the aqueous phase by the killifish (Fundulus heteroclitus). Cadmium uptake showed no relationship with humic acid concentration, whereas Cr, Hg(II), and MeHg uptake showed an inverse relationship, and As uptake increased with increasing humic acid concentration. Concentration factors were >1 for Cd, Hg(II), and MeHg at all humic acid concentrations, indicating killifish were more enriched in the metal than the experimental media, whereas As and Cr generally had concentration factors <1 at the end of a 72-h exposure. The uptake of As and Cr reached steady state within the 72-h exposure, whereas uptake of Cd, Hg(II), and MeHg did not. Uptake rate constants (k(u) s;?ml?g(-1) d(-1) ) were highest for MeHg (91-3,936), followed by Hg(II), Cd, and Cr, and lowest for As (0.17-0.29). Dissection data revealed that the gills generally had the highest concentration of all metals under all humic acid treatments. The present study concludes that changes in humic acid concentration can influence the accumulation of aqueous metals in killifish and should be considered when modeling metal bioaccumulation. Environ. Toxicol. Chem. 2012; 31: 2225-2232. ? 2012 SETAC.     

Effects of methylmercury on sperm and egg viability of two populations of killifish (fundulus heteroclitus)

Exposure of sperm of killifish (Fundulus heteroclitus) from a relatively clean area in Long Island (LI) to 0.01 mg/L methylmercury (meHg) in 15%o sea water caused significant reduction of fertilization success. However, exposure of killifish sperm from polluted Piles Creek (PC) to either 0.01 or 0.05 mg/L meHg in 15%o sea water prior to insemination had no effect on fertilization success. Exposure of LI killifish sperm to 0.05 mg/L meHg caused significant reduction in motility. However, PC killifish sperm showed no significant difference in motility between 0 and 0.05 mg/L meHg exposure. Exposure for 5 min to 0.05 mg/L meHg caused significant reduction in motility. These data indicate that meHg is less toxic to PC killifish sperm than LI killifish sperm. Exposure of PC and LI killifish sperm to 0.05 mg/L meHg for 15 min had no effect on sperm morphology. PC killifish sperm also showed higher (20 min) motility in 15%o sea water than LI killifish sperm (10 min). Exposure of PC and LI killifish eggs up to 25 min to 0.05 mg/L meHg prior to fertilization had no effect on fertilization success.     

Teratogenic effects of injected methylmercury on avian embryos

Controlled laboratory studies with game farm mallards (Anas platyrhynchos) and chickens (Gallus gallus) have demonstrated that methylmercury can cause teratogenic effects in birds, but studies with wild species of birds are lacking. To address this need, doses of methylmercury chloride were injected into the eggs of 25 species of birds, and the dead embryos and hatched chicks were examined for external deformities. When data for controls were summed across all 25 species tested and across all types of deformities, 24 individuals out of a total of 1,533 (a rate of 1.57%) exhibited at least one deformity. In contrast, when data for all of the mercury treatments and all 25 species were summed, 188 deformed individuals out of a total of 2,292 (8.20%) were found. Some deformities, such as lordosis and scoliosis (twisting of the spine), misshapen heads, shortening or twisting of the neck, and deformities of the wings, were seldom observed in controls but occurred in much greater frequency in Hg-treated individuals. Only 0.59% of individual control dead embryos and hatchlings exhibited multiple deformities versus 3.18% for Hg-dosed dead embryos and hatchlings. Methylmercury seems to have a widespread teratogenic potential across many species of birds.     

Cadmium acclimation and hormesis in Fundulus heteroclitus during fin regeneration

We have previously demonstrated that exposure to cadmium at 0.1 and 0.5 mg/liter will retard the rate of fin regeneration in Fundulus heteroclitus. Since there is a body of literature indicating that preexposure to low levels of metals may increase tolerance during subsequent exposure, these experiments were designed to investigate the effects of preexposure to cadmium, using fin regeneration as the parameter of effect. Fish were exposed for 1 week to 0, 0.01, or 0.05 mg/liter Cd. Following this preexposure, the lower third of the caudal fin was amputated, and regrowth of the fins measured by ocular micrometer twice weekly for 2 weeks. During regeneration fish were maintained in either clean water (25% S, 23-25 degrees C) or in water with 0.1 mg/liter Cd. Fish newly placed in Cd (C----0.1 Cd) regenerated at the slowest rate, while the 0.01----0.1 Cd group approached the growth of control fish regenerating in clean water, indicating a protective effect or acclimation brought about by the preexposure. The 0.05----0.1 Cd group regenerated even more rapidly than controls, a phenomenon which has been termed "hormesis" and is believed to be due to an overcompensation by homeostatic regulatory mechanisms. In a second set of experiments in which regeneration took place in 0.5 mg/liter rather than 0.1 mg/liter, a protective effect of preexposure was again seen, but no hormesis was observed. Uptake data indicated that preexposed groups took up less Cd than the groups not preexposed.     

Trivalent chromium alters gene expression in the mummichog (Fundulus heteroclitus)

Mummichogs (Fundulus heteroclitus) were used as a model fish species to study the effects of trivalent chromium exposure. To ascertain chromium's effects, we examined altered gene expression by differential display between fish exposed in the laboratory and fish collected from a chromium-impacted estuarine site. Twenty differentially expressed genes were found from either laboratory-exposed fish or in fish collected from the field site. Database sequence searches indicated that several of these genes are highly homologous to known sequences, including a fatty acid-binding protein (FABP), cytochrome P4502N2 (CYP2N2), and a precursor to the translation initiation factor eIF2B. Verification of the differentially expressed genes by real-time polymerase chain reaction (PCR) revealed that the fatty acid-binding protein was repressed to a 3.6-times greater extent (3.6-fold) in the field-site animals as compared to a reference site, eIF2B was repressed 2-fold, and an expressed sequence tag (EST) termed A31 was induced 2.6-fold. In the laboratory-exposed animals, A31 was also induced between 2- and 4-fold. However, in contrast to the field-site fish, FABP was upregulated in the chromium-exposed animals. We hope to be able to use A31 as a biomarker for ascertaining the impacts of chromium exposure on fish.     

Combined use of biochemical indicators to assess sublethal pollution effects on the Gulf killifish (Fundulus grandis)

Sublethal biochemical markers were used to identify liver enzyme induction in fish from a bayou in Pensacola, Florida. Gulf killifish,Fundulus grandis, from, a nonpolluted site were used in the study which included laboratory-induced fish and their various controls as well as the fish captured in the bayou. The biochemical markers tested were total content of cytochrome P-450, aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase activities, and the specific induction of the LM_4bisozyme of the cytochrome P-450 system. The findings suggest that enzyme induction occurred at a sublethal level, indicating the presence of liver cytochrome P-450-inducing substances in the bayou.