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.     

Cytogenetic effects of methylmercury in embryos of the killifish,Fundulus heteroclitus

Fertilized eggs of the killifish,Fundulus heteroclitus, were exposed experimentally to methylmercury (MeHg) to evaluate whether this compound induced cytogenetic effects expressed in the embryos. An additional objective was to assess cytogenetically whetherFundulus embryos from groups that were determined to be more susceptible to the teratogenic effects of MeHg were also more susceptible to its mutagenic effects. Embryos exposed to 0.05 mg/L MeHg for 1 and 7 days post-fertilization were preserved in 10% formalin for cytological examination, while unfixed embryos from the same clutches were evaluated for teratogenic effects. Untreated embyros from each clutch served as controls. Cytogenetic-cytological analyses of fixed treated susceptible embryos that exhibited teratogenic effects revealed decreased mitotic counts (5.0-fold), and increased chromosomal aberrations (2.5-fold) when compared to their controls. There were also decreased mitotic counts (1.5-fold) and increased chromosomal aberrations (2-fold) in embryos from resistant groups when compared to their controls. Exposure to MeHg also retarded development. Forty to fifty percent of the susceptible and resistant control embryos reached the tail-bud stage, but only 28% of the treated resistant and none of the treated susceptible embryos reached this stage. There was also a significant correlation (P < 0.05) between teratogenic and mutagenic effects of MeHg in early stages ofFundulus.     

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.     

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.     

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.     

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.     

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.     

Fish (Fundulus heteroclitus) populations with different exposure histories differ in tolerance of creosote-contaminated sediments

Prior studies suggest that field-collected fish (Fundulus heteroclitus) from a creosote-contaminated Superfund site (Atlantic Wood Industries site, Elizabeth River, VA, USA) have enhanced tolerance to local, contaminated sediments. This study was designed to test whether other populations in the Elizabeth River at less contaminated sites also show similar tolerance and whether this tolerance is heritable. To test this, F. heteroclitus populations were sampled from four sites within the Elizabeth River with varying sediment polycyclic aromatic hydrocarbon (PAH) concentrations (3.9-264 ng PAH/g dry wt 10(3)) and one reference site in a nearby, uncontaminated estuary (York River, VA, USA; 0.27 ng PAH/g dry wt x 10(3)). Embryo assays were performed to quantify population differences in teratogenic effects during contaminated sediment exposure. Atlantic Wood sediment was mixed with reference sediment to achieve a range of sediment concentrations. Minimal differences were observed in teratogenic effects among fish taken from sites within the Elizabeth River; however, embryos of fish collected from a nearby, uncontaminated York River site and exposed to contaminated sediments had a significantly higher proportion of embryos with cardiac abnormalities than those from the Elizabeth River sites. Embryos from wild-caught and laboratory-reared Elizabeth River F. heteroclitus were simultaneously exposed to contaminated sediments, and no significant tolerance differences were found between embryos from fish taken directly from the field and those reared for a generation in the lab. Differences between fish populations from the two estuaries were larger than differences within the Elizabeth River, and these differences in tolerance were heritable.     

Toxicity of methylmercury,mercuric chloride,and lead in killifish (Fundulus heteroclitus) from Southampton,New York

We have previously found great differences in susceptibility to teratogenic effects of methylmercury (meHg) among batches of eggs produced by different females of Fundulus heteroclitus. In the present study we investigated the relationships of resistance to meHg by a batch of eggs with other parameters of resistance. There was a positive correlation between embryonic meHg resistance and HgCl₂ resistance, although HgCl₂ generally caused less severe defects at a given dose level. The batches that were more susceptible to meHg had a greater uptake of mercury than the more resistant batches. There was a negative correlation of meHg resistance and Pb resistance (as measured by a skeletal index hatching), however. There was a positive correlation of meHg resistance of embryos and the subsequent meHg resistance of larvae after hatching.     

Effects of dietary methylmercury on reproductive behavior of fathead minnows (Pimephales promelas)

We examined the effects of dietary methylmercury on the production of testosterone in and the reproductive behavior of male fish. Juvenile fathead minnows (Pimephales promelas) were fed one of three diets contaminated with methylmercury at concentrations of 0.06 (control), 0.87 (low), and 3.93 (medium) microg Hg/g dry weight. After attaining sexual maturity, fish were paired for mating, and male behaviors were recorded. Carcass mercury and plasma testosterone concentrations also were measured. No significant differences were found in the amount of time spent by male fish in nest preparation or courtship activities, but dietary methylmercury suppressed mating behavior. Fish that were fed control diets spent approximately 5% of their time spawning; fish that were fed methylmercury-contaminated diets spent approximately 0.5% of their time spawning. Total mercury concentration in the carcass was not correlated with any individual reproductive behavior but was correlated with hypoactivity. Fish that were fed the low- and medium-methylmercury diets and with mean carcass concentrations of 0.71 to 4.2 microg Hg/g dry weight spent an average of 19 to 26% of their time being inactive; control fish with an average of 0.07 microg Hg/g dry weight spent only 8% of their time being inactive. No significant difference was found in testosterone concentrations among dietary treatments; however, testosterone was positively correlated with individual nest preparatory, courtship, and spawning behaviors and negatively correlated with the total amount of time spent in all nonreproductive behaviors. The relation between testosterone, reproductive behavior, and spawning success suggests that altered behavior is, in part, responsible for suppression of reproduction in methylmercury-exposed fish. Moreover, reproductive behavior may be more sensitive than steroid hormones to alteration by dietary methylmercury.     

Effects of kraft pulp mill condensates on plasma testosterone levels in mummichog (Fundulus heteroclitus)

Past studies at a bleached kraft pulp mill located in Saint John, NB, Canada have shown that chemical recovery condensates significantly depress circulating and gonadal steroids in mummichog (Fundulus heteroclitus), an endemic fish species. In the present study, compounds were extracted from the condensates, and a toxicity identification evaluation (TIE) was conducted to characterize the hormonally active substances (HASs) in the condensates. Extracts were fractionated by high performance liquid chromatography (HPLC) and mummichog were exposed to the fractions in a 7-day bioassay. Plasma testosterone was measured for each sex following exposure. Responses in fish exposed to the whole extract at 1% v/v were not as pronounced as had been observed previously (female plasma testosterone was reduced by 16% in the current study compared to 75% previously). Dose-response experiments showed an exposure concentration of 4% v/v was required to elicit significant plasma steroid reductions. Despite these responses, individual condensate fractions actually increased steroid levels in mummichog, which suggests that multiple HASs may need to act synergistically or additively to elicit effects, and if separated, the compounds may have different hormonal activity. The HASs in question caused a reduction in male gonad size at 4% v/v, and have sex-dependent mechanisms of action (males were more responsive to exposure than females).     

Effects of methylmercury exposure on the immune function of juvenile common loons (Gavia immer)

We conducted a dose-response laboratory study to quantify the level of exposure to dietary Hg, delivered as methylmercury chloride (CH3HgCl), that is associated with suppressed immune function in captive-reared common loon (Gavia immer) chicks. We used the phytohemagglutinin (PHA) skin test to assess T-lymphocyte function and the sheep red blood cell (SRBC) hemagglutination test to measure antibody-mediated immunity. The PHA stimulation index among chicks receiving dietary Hg treatment did not differ significantly from those of chicks on the control diet (p = 0.15). Total antibody (immunoglobulin [Ig] M [primary antibody] + IgG [secondary response]) production to the SRBC antigen in chicks treated with dietary methylmercury (MeHg), however, was suppressed (p = 0.04) relative to chicks on control diets. Analysis indicated suppression of total Ig production (p = 0.025 with comparisonwise alpha level = 0.017) between control and 0.4 microg Hg/g wet food intake treatment groups. Furthermore, the control group exhibited a higher degree of variability in antibody response compared to the Hg groups, suggesting that in addition to reducing the mean response, Hg treatment reduced the normal variation attributable to other biological factors. We observed bursal lymphoid depletion in chicks receiving the 1.2 microg Hg/g treatment (p = 0.017) and a marginally significant effect (p = 0.025) in chicks receiving the 0.4 microg Hg/g diet. These findings suggest that common loon chick immune systems may be compromised at an ecologically relevant dietary exposure concentration (0.4 microg Hg/g wet wt food intake). We also found that chicks hatched from eggs collected from low-pH lakes exhibited higher levels of lymphoid depletion in bursa tissue relative to chicks hatched from eggs collected from neutral-pH lakes.