Micronucleus test in freshwater fish species: an evaluation of its sensitivity for application in field surveys

Brown trout, Salmo trutta, European eel, Anguilla anguilla, and European minnow, Phoxinus phoxinus, three fish species inhabiting European freshwater ecosystems, were evaluated for their use as in situ pollution biomarkers using the micronucleus test in renal erythrocytes. Experimental exposure (by immersion) to different concentrations of cyclophosphamide, colchicine, and cadmium showed that brown trout are more sensitive to the three compounds than minnows and eels. In situ surveys of wild freshwater ecosystems with different levels of pollution showed that minnows and eels living in polluted sites do not present higher micronuclei averages than those caught in clean rivers systems, whereas micronuclei are induced in brown trout inhabiting polluted sites. Our results demonstrated the suitability of brown trout for in situ biomonitoring of freshwater ecosystems as well as for laboratory tests using the micronucleus test.     

Metallothionein and heavy metals in brown trout (Salmo trutta) and European eel (Anguilla anguilla): a comparative study

The levels and the cellular distribution of heavy metals, and the extent by which the metals binds to metallothionein (MT) in brown trout (Salmo trutta) and European eel (Anguilla anguilla), were analyzed in order to assess the natural conditions of MT and heavy metals in these two fish species. There were no differences in heavy metals and MT concentrations between males and females of brown trout in a nonreproductive status and between adult brown trout individuals. Brown trout presented higher Cu content than European eel. The cellular distribution of Cu was also different between the two fish species; while in brown trout most of the Cu was in the noncytosolic fraction, Cu was mainly located in the cytosol in European eel. However, the cellular distribution of Zn, Cd, and Pb was similar in the two fish species. There was also an important difference in the metal content of MT between both species. Whereas, in brown trout, Cu-binding MT represented 75% of total metal-binding MT, this value was 25% in European eel. The between-species differences found in this study are intrinsic characteristics not associated with environmental factors. These results establish the basis to use MT as a bioindicator.     

The further development of ionoregulatory measures as biomarkers of sensitivity and effect in fish species

Extensive season-by-season sampling was used to establish the normal range of whole-body Na+ and Cl- and Na+ uptake in healthy populations of two fish species, rainbow trout, Oncorhynchus mykiss, and fathead minnow, Pimephales promelas, of known differences in sensitivity to ionoregulatory toxicants (low pH, trace metals). These data together with responses of both species to six different ionoregulatory challenge tests of increasing severity (mild handling, exposure to low Ca2+ water, epinephrine injection, net-confinement stress, exposure to copper, and osmotic shock) were evaluated for their potential as biomarkers of sensitivity and of effect of ionoregulatory toxicants. There were no obvious biomarkers of sensitivity in the ion measures themselves, but four of the six challenges (exposure to low Ca2+ water, epinephrine injection, exposure to copper, and osmotic shock) produced a significantly greater effect in the more sensitive of the two species, fathead minnow. Based on the responses of both species, this article makes a number of recommendations for the application of ion measures alone and in combination with challenge tests to the assessment of chronic effects in populations experiencing sublethal ionoregulatory stress.     

Induction of micronuclei in eel (Anguilla anguilla L.) by heavy metals

Micronucleus test was performed in situ on eels (Anguilla anguilla) from river sites with different levels of heavy metal pollution (cadmium and mercury). Cadmium content in eel liver but not micronuclei averages in kidney were associated with cadmium content in sediments. Mercury content in liver was not significantly associated with mercury content in sediments. Both cadmium and mercury induced micronuclei expression in eels when injected, the concentration tested being 1.7 mg metal/kg body weight and the micronuclei induction being 2.64 and 2.35 micronuclei per 1000 cells for cadmium and mercury respectively. It was concluded that these heavy metals are genotoxic for European eel, that eel liver metal content is a sensitive indicator for environmental monitoring of cadmium pollution, and that the micronuclei scores in eels are not a sensitive method to detect heavy metals pollution in freshwater ecosytems.     

Oil shale processing as a source of aquatic pollution: monitoring of the biologic effects in caged and feral freshwater fish.

The biologic effects of the oil shale industry on caged rainbow trout (Oncorhynchus mykiss) as well as on feral perch (Perca fluviatilis) and roach (Rutilus rutilus) were studied in the River Narva in northeast Estonia. The River Narva passes the oil shale mining and processing area and thus receives elevated amounts of polycyclic aromatic hydrocarbons (PAHs), heavy metals, and sulfates. The effects of the chemical load were monitored by measuring cytochrome P4501A (CYP1A)-dependent monooxygenase (MO) activities [7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase (AHH)] as well as conjugation enzyme activities [glutathione S-transferase (GST) and UDP-glucuronosyltransferase] in the liver of fish. CYP1A induction was further studied by detecting the amount and occurrence of the CYP1A protein. Histopathology of tissues (liver, kidney, spleen, and intestine) and the percentage of micronuclei in fish erythrocytes were also determined. Selected PAHs and heavy metals (Cd, Cu, Hg, and Pb) were measured from fish muscle and liver. In spite of the significant accumulation of PAHs, there was no induction of MO activities in any studied fish species. When compared to reference samples, AHH activities were even decreased in feral fish at some of the exposed sites. Detection of CYP1A protein content and the distribution of the CYP1A enzyme by immunohistochemistry also did not show extensive CYP1A induction. Instead, GST activities were significantly increased at exposed sites. Detection of histopathology did not reveal major changes in the morphology of tissues. The micronucleus test also did not show any evidence of genotoxicity. Thus, from the parameters studied, GST activity was most affected. The lack of catalytic CYP1A induction in spite of the heavy loading of PAHs was not studied but has been attributed to the elevated content of other compounds such as heavy metals, some of which can act as inhibitors for MOs. Another possible explanation of this lack of induction is that through adaptation processes the fish could have lost some of their sensitivity to PAHs. Either complex pollution caused by oil shale processing masked part of the harmful effects measured in this study, or oil shale industry did not have any severe effects on fish in the River Narva. Our study illustrates the difficulties in estimating risk in cases where there are numerous various contaminants affecting the biota.     

Relative sensitivity of bull trout (Salvelinus confluentus) and rainbow trout (Oncorhynchus mykiss) to acute copper toxicity

Bull trout (Salvelinus confluentus) were recently listed as threatened in the United States under the federal Endangered Species Act. Past and present habitat for this species includes waterways contaminated with heavy metals released from mining activities. Because the sensitivity of this species to copper was previously unknown, we conducted acute copper toxicity tests with bull and rainbow trout (Oncorhynchus mykiss) in side-by-side comparison tests. Bioassays were conducted using water at two temperatures (8 degrees C and 16 degrees C) and two hardness levels (100 and 220 mg/L as CaCO3). At a water hardness of 100 mg/L, both species were less sensitive to copper when tested at 16 degrees C compared to 8 degrees C. The two species had similar sensitivity to copper in 100-mg/ L hardness water, but bull trout were 2.5 to 4 times less sensitive than rainbow trout in 220-mg/L hardness water. However, when our results were viewed in the context of the broader literature on rainbow trout sensitivity to copper, the sensitivities of the two species appeared similar. This suggests that adoption of toxicity thresholds that are protective of rainbow trout would be protective of bull trout; however, an additional safety factor may be warranted because of the additional level of protection necessary for this federally threatened species.     

Assessment of Genotoxic Induction and Deterioration of Fish Quality in Commercial Species Due to Heavy-Metal Exposure in an Urban Reservoir

The aim of the study was to evaluate the effect of heavy-metal contamination on two fish species (Channa striatus and Heteropneustes fossilis) inhabiting a small freshwater body of northern India. After being captured, each specimen was weighed, measured, and analyzed for heavy metals (chromium [Cr], nickel [Ni], and lead [Pb]). Accumulation of heavy metals was found to be significantly greater (p < 0.05) in different tissues (gill, liver, kidney, and muscle) of fishes captured from the reservoir than from the reference site. Levels of heavy-metal contamination in Shah jamal water was Cr (1.51 mg/l) > Ni (1.22 mg/l) > Pb (0.38 mg/l), which is significantly greater than World Health Organization standards. Bioaccumulation factor was calculated, and it was observed that Pb was most detrimental heavy metal. Condition factor was also influenced. Micronucleus test of fish erythrocytes and comet assay of liver cells confirmed genotoxicity induced by heavy-metal contamination in fishes. Heavy metals (Cr, Ni, and Pb) were increased in both fish species as determined using recommended values of Federal Environmental Protection Agency for edible fishes. This raises a serious concern because these fishes are consumed by the local populations and hence would ultimately affect human health.     

Metallothionein as bioindicator of freshwater metal pollution: European eel and brown trout

The aim of this work was to evaluate the potential of metallothionein (MT) as a bioindicator of heavy metal pollution in brown trout and European eel in the field situation. River Ferrerias (North Spain) provided a good gradient of metal contamination: concentrations of heavy metals were elevated both in water and in sediments at the downstream (polluted) site and were low at the upstream (unpolluted) site. MT levels of brown trout exhibited statistically significant differences between sites. Although European eel at the polluted site had a higher MT content, differences were not significant. It is concluded that MT is a good bioindicator of heavy metal pollution in brown trout.     

Eel (Anguilla anguilla) and brown trout (Salmo trutta) target species to assess the biological impact of trace metal pollution in freshwater ecosystems

Copper, lead, and cadmium pollution were measured in water and sediments of two Northern Spanish rivers: Piles and Pigüeña. Liver contents of these heavy metals were analyzed in two fish species (eel, Anguilla anguilla, and brown trout, Salmo trutta) collected from the same locations. Significant levels of heavy metal pollution were found in a 38% of fish that are potential catches for sport fishermen. The results indicate that adult eel could be a good metal bioindicator if sampled at a homogenous age. In contrast, brown trout could be considered as a bioindicator only during the first year of life.     

Mutagenicity, Genotoxicity, and Estrogenic Activity of River Porewaters

We investigated mutagenicity, genotoxicity, and estrogenic activity in the porewaters of two river basins in southern Italy that had different features. Three samples from each site were collected in different seasons from 7 sites for a total of 21 samples. Mutagenicity was measured with the Ames test with and without metabolic activation (S9) using Salmonella typhimurium TA98 and TA100 strains. Genotoxicity was measured with two tests: one involved a chromophore that detected DNA damage in Escherichia coli PQ37 (SOS chromotest), and the other measured micronuclei formation in the root cells of Vicia faba. Estrogenic activity was measured with a yeast-based estrogen receptor assay and an MCF-7 cell-based, estrogen-sensitive proliferation assay. We also applied chemical analyses to detect alkylphenols, pesticides, natural and synthetic hormones, and heavy metals. The porewaters of both river sediments showed mutagenic/genotoxic activity on V. faba test and Ames test, the latter both with and without S9 liver fraction. The SOS chromotest without metabolic activation was not sufficiently sensitive to detect genotoxicity of the porewaters, but the SOS DNA repair system in E. coli PQ37 was activated in the presence of S9 mix. Good correlations were found between mutagenicity/genotoxicity and the concentration of cadmium and between estrogenic activity and the presence of copper. This study assessed the chemical concentrations of some bioavailable pollutants in porewater and detected the overall effects of multiple pollutants that contributed to mutagenicity, genotoxicity, and estrogenic activity of these two basin porewaters, thus increasing our understanding of the environmental consequences of polluted aquatic ecosystems.     

Assessing Contaminant Sensitivity of Endangered and Threatened Aquatic Species: Part I. Acute Toxicity of Five Chemicals

Assessment of contaminant impacts to federally identified endangered, threatened and candidate, and state-identified endangered species (collectively referred to as listed species) requires understanding of a species sensitivities to particular chemicals. The most direct approach would be to determine the sensitivity of a listed species to a particular contaminant or perturbation. An indirect approach for aquatic species would be application of toxicity data obtained from standard test procedures and species commonly used in laboratory toxicity tests. Common test species (fathead minnow, Pimephales promelas; sheepshead minnow, Cyprinodon variegatus; and rainbow trout, Oncorhynchus mykiss) and 17 listed or closely related species were tested in acute 96-hour water exposures with five chemicals (carbaryl, copper, 4-nonylphenol, pentachlorophenol, and permethrin) representing a broad range of toxic modes of action. No single species was the most sensitive to all chemicals. For the three standard test species evaluated, the rainbow trout was more sensitive than either the fathead minnow or sheepshead minnow and was equal to or more sensitive than listed and related species 81% of the time. To estimate an LC50 for a listed species, a factor of 0.63 can be applied to the geometric mean LC50 of rainbow trout toxicity data, and more conservative factors can be determined using variance estimates (0.46 based on 1 SD of the mean and 0.33 based on 2 SD of the mean). Additionally, a low- or no-acute effect concentration can be estimated by multiplying the respective LC50 by a factor of approximately 0.56, which supports the United States Environmental Protection Agency approach of multiplying the final acute value by 0.5 (division by 2). When captive or locally abundant populations of listed fish are available, consideration should be given to direct testing. When direct toxicity testing cannot be performed, approaches for developing protective measures using common test species toxicity data are available.     

Heavy Metals Alter the Survival, Growth, Metamorphosis, and Antipredatory Behavior of Columbia Spotted Frog (Rana luteiventris) Tadpoles

Amphibian populations appear to be declining around the world. Although there is no single cause, one factor may be pollution from heavy metals. As a result of mining in the Silver Valley of Idaho, heavy metals have been released into habitats containing many species of sensitive organisms, including spotted frogs (Rana luteiventris). While the gross extent of pollution has been well documented, the more subtle behavioral effects of heavy metals such as lead, zinc, and cadmium are less well studied. We tested the effects of heavy metals on the short-term survival (LC₅₀) of spotted frog tadpoles. Compared to single metals, metals presented together were toxic at lower doses. We also raised the tadpoles in outdoor mini-ecosystems containing either a single heavy metal or soil from an EPA Superfund site in the Silver Valley known to be composed of numerous heavy metals. Exposure to Silver Valley soil resulted in delayed metamorphosis. We tested the ability of metal-exposed tadpoles to detect and respond to chemical cues emanating from predacious rainbow trout. We found that high levels of Silver Valley soil, medium levels of zinc, and medium and high levels of lead resulted in a decreased fright response. Low levels of cadmium, zinc, and lead did not cause a significant effect, but low levels of soil did result in a decreased fright response. Heavy metals may alter interactions between tadpoles and their predators. Received: 9 October 1997/Accepted: 25 March 1998     

Acute toxicity of selenium dioxide to freshwater fishes.

Acute toxicity tests of selenium dioxide were conducted for 96 to 336 hr in intermittent-flow bioassay systems using six species of freshwater fish. The decreasing order of species sensitivity was: fathead minnow, flagfish, brook trout, channel catfish, goldfish, and bluegil. Curves relating median lethal concentration to exposure time for each species exposed for more than 168 hr were sigmoid in shape and were characterized by a change in slope indicating a more rapid mortality rate after 96 to 168 hr toxicant exposure. The 96-hr LC50 estimates ranged from 2.9 mg/L SeO2 for fathead minnow fry to 40.0 mg/L for bluegill juveniles. Effects of brief toxicant exposure (24 hr) on fathead minnow and flagfish juveniles included limited delayed mortality and no effects on growth over a 28-day period.     

Acute toxicity of selenium dioxide to freshwater fishes

Acute toxicity tests of selenium dioxide were conducted for 96 to 336 hr in intermittent-flow bioassay systems using six species of freshwater fish. The decreasing order of species sensitivity was: fathead minnow, flagfish, brook trout, channel catfish, goldfish, and bluegill. Curves relating median lethal concentration to exposure time for each species exposed for more than 168 hr were sigmoid in shape and were characterized by a change in slope indicating a more rapid mortality rate after 96 to 168 hr toxicant exposure. The 96-hr LC50 estimates ranged from 2.9 mg/L SeO₂ for fathead minnow fry to 40.0 mg/L for bluegill juveniles.Effects of brief toxicant exposure (24 hr) on fathead minnow and flagfish juveniles included limited delayed mortality and no effects on growth over a 28-day period.Research supported by the U. S. Environmental Protection Agency National Water Quality Laboratory, Duluth, Minnesota. (Contract No. 68-01-0748).     

Toxicity of selected priority pollutants to various aquatic organisms

Toxicity tests were conducted with selected compounds listed by the United States Environmental Protection Agency (EPA) as priority pollutants. Acute toxicity information was determined for acenaphthene, arsenic trioxide, cadmium chloride, mercury(II) chloride, silver nitrate, chlordane, endosulfan, and heptachlor. Acute tests were conducted using one or more of the following species: fathead minnows (Pimephales promelas), channel catfish (Ictalurus punctatus), rainbow trout (Salmo gairdneri), brown trout (Salmo trutta), brook trout (Salvelinus fontinalis), bluegills (Lepomis macrochirus), snails (Aplexa hypnorum), or chironomids (Tanytarsus dissimilis). Acute values from these tests ranged from a silver nitrate 96-hr LC50 of 6.7 micrograms/liter for fathead minnows to an arsenic trioxide 48-hr LC50 of 97,000 micrograms/liter for chironomids. In addition to acute tests, a fathead minnow embryo-larval exposure was conducted with silver nitrate to estimate chronic toxicity. The estimated maximum acceptable toxicant concentration for silver nitrate, based on fathead minnow survival, lies between 0.37 and 0.65 micrograms/liter.     

Indirect Effects of Heavy Metals on Parasites May Cause Shifts in Snail Species Compositions

We studied the direct and indirect effects of pollution on the distributions and abundances of two closely related species of pulmonate freshwater snails. Physella columbiana is more numerous at heavy metal–polluted lakes, and Lymnaea palustris is more numerous at reference lakes. Both species are present at all sites, as are predatory bluegill sunfish (Lepomis macrochirus). The direct effects examined included the snails' growth and reproduction in both the presence and absence of heavy metals and their short-term survival when exposed to large concentrations of heavy metals. The indirect effects were the species' ability to elude capture by sunfish and the diversity and abundance of parasites within the snails. We found that heavy metals had little direct effect on growth and reproduction and that both species acquired similar levels of metals in their tissues. Interestingly, P. columbiana (the more abundant species in polluted lakes) actually exhibited higher recruitment in the absence of metals than did L. palustris (reference lakes). L. palustris has life history characteristics that favor increased growth and reduced reproduction. These characteristics resulted in decreased predation of adults by gape-limited predators and a greater ability to cope with heavy parasite burdens. P. columbiana exhibited slower growth, which resulted in increased predation although higher reproduction rates may compensate. The major effect of heavy metals on species distributions was indirect on the snails' parasites. Parasites appeared to be very susceptible to metals, and this resulted in lower parasite diversity and intensities at polluted sites for both species of snails. P. columbiana may only be able to outcompete L. palustris at polluted sites due to the indirect effects of heavy metals; the negative effect of heavy metals on parasites, and a proposed negative effect of metals on the foraging ability of sunfish that favors the faster-reproducing P. columbiana. Received: 13 August 2001/Accepted: 19 January 2002     

Relative sensitivity of bull trout (Salvelinus confluentus) and rainbow trout (Oncorhynchus mykiss) to acute exposures of cadmium and zinc

Bull trout (Salvelinus confluentus) were recently listed as threatened in the United States under the federal Endangered Species Act. Present and historical habitat of this species includes waterways that have been impacted by metals released from mining and mineral processing activities. We conducted paired bioassays with bull trout and rainbow trout (Oncorhynchus mykiss) to examine the relative sensitivity of each species to Cd and Zn independently and as a mixture. A total of 15 pairs of acute toxicity bioassays were completed to evaluate the effects of different water hardness (30 or 90 mg/L as CaCO3), pH (6.5 or 7.5), and temperature (8 or 12 degrees C) on Cd and Zn toxicity. For both species, the acute toxicity of both Cd and Zn was greater than previously observed in laboratory studies. Bull trout were about twice as tolerant of Cd and about 50% more tolerant of Zn than were rainbow trout. Higher hardness and lower pH water produced lower toxicity and slower rates of toxicity in both species. Elevated temperature significantly increased the sensitivity of bull trout to Zn but decreased the sensitivity (not significantly) of rainbow trout to Zn. At a hardness of 30 mg/L, the toxicity values (i.e., median lethal concentration; 120-h LC50) for both species were lower than the current U.S. national water quality criteria for protection of aquatic life, indicating that current national criteria may not be protective of sensitive salmonids--including the threatened bull trout--in low calcium waters.     

Acute and embryo-larval toxicity of phenolic compounds to aquatic biota

Because of the prevalence of phenolic compounds in various types of effluents, both acute and embryo-larval bioassays were performed on eight phenolic compounds with rainbow trout, fathead minnows andDaphnia pulicaria. In flow-through bioassays, the 96-hr LC₅₀ values for rainbow trout and fathead minnows ranged from <0.1 mg/L for hydroquinone to >100 mg/L for resorcinol.Daphnia pulicaria was consistently the least sensitive species tested as measured in 48-hr bioassays, while fathead minnows and rainbow trout varied in their relative sensitivity to phenolics as measured in 96-hr tests. Fathead minnows were more sensitive to phenol at 25°C than at 14°C.In embryo-larval bioassays with phenol, fathead minnow growth was significantly reduced by 2.5 mg/L phenol, while rainbow trout growth was significantly reduced by 0.20 mg/L phenol. For both species the embryolarval effects concentration was 1.1% of the 96-hr LC₅₀. Another embryolarval bioassay was attempted withp-benzoquinone, a highly toxic phenolic compound found in fossil fuel processing wastewaters, which was discontinued because the compound was rapidly degraded chemically or biologically in the headtank and aquaria.Work funded under an Interagency Agreement between the U.S. Department of Energy and the U.S. Environmental Protection Agency under Contract No. DE-AS20-79 LC 01761 to the Rocky Mountain Institute of Energy and Environment, University of Wyoming.     

Comparison of relative binding affinities of endocrine active compounds to fathead minnow and rainbow trout estrogen receptors

Twelve chemicals were tested for binding affinity to rainbow trout liver estrogen receptor (rbtER) and fathead minnow liver ER (fhmER). The chemicals included estradiol (E2), diethylstilbestrol (DES), ethinylestradiol (EE2), estrone (El), estriol, tamoxifen (TAM), genistein (GEN), p-nonylphenol (PNP), p-tert-octylphenol (PTOP), methoxychlor (MXC), testosterone, and methyltestosterone (MT). Relative binding affinity (RBA) was calculated for each chemical as a function of E2 binding to the receptor. The estrogens DES, EE2, and E1 bound with high affinity to both receptors, with respective RBAs of 583, 166, and 28% (fathead minnow) and 179, 89, and 5% (rainbow trout). Relative binding affinity of E3, TAM, and GEN for both fhmER and rbtER were moderate, with values between 0.3 and 5%. The alkylphenols had weak affinity for the ERs with RBAs for the fhmER of 0.1 and 0.01 for PNP and PTOP, respectively. Corresponding values for the rbtER are 0.027 and 0.009. Estradiol ([3H]E2) only partially was displaced from both the fhmER and the rbtER by MXC, T, and MT. Comparison of RBAs of the chemicals tested for fhmER and rbtER indicates that the rank order of RBAs essentially are the same for both species.