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.     

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.     

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.     

Can Excrement and Feathers of Nestling Songbirds Be Used as Biomonitors for Heavy Metal Pollution?

Although birds have been frequently used as indicators of heavy metal pollution, few studies have examined pollutant levels in nestling passerines. In this paper we determined the levels of two essential (zinc and copper) and three nonessential heavy metals (lead, cadmium, and arsenic) in the excrement and feathers of great (Parus major) and blue tit (Parus caeruleus) nestlings at a polluted site (near a metallurgic factory) and a reference site (4 km farther east). The excrement of both great and blue tit nestlings contained significantly higher concentrations of arsenic, cadmium, copper, and lead at the polluted site. Zinc concentrations did not differ significantly between sites for both species. The feathers of great and blue tit nestlings accumulated significantly higher concentrations of lead at the polluted site than at the reference site. Zinc levels in the feathers of great tit nestlings were significantly higher at the reference site than at the polluted site. For all other elements considered, concentrations did not differ significantly between the two sites. There were no interspecific differences in metal levels between great and blue tits in both excrement and feathers. There was a significant positive correlation between the lead concentration in the excrement and feathers for both great and blue tits. We therefore conclude that excrement of great and blue tit nestlings can be used as a biomonitor for heavy metals (lead, cadmium, arsenic, and copper), whereas feathers appear only to be suitable as a biomonitor for lead pollution. Received: 21 March 2000/Accepted: 9 July 2000     

Effects of Heavy Metal Exposure on Aggressive Behavior in a Small Territorial Songbird

Several studies have shown that elevated levels of certain heavy metals may affect the behavior of birds and mammals. However, most of these studies were carried out under controlled laboratory conditions; results from free-living populations are scarce. In this study we examined the possible effects of exposure to high heavy metal concentrations on the aggressive behavior of resident great tit (Parus major) males by means of simulated territorial intrusion experiments during the egg-laying and incubation period at a highly polluted and a reference site. We also examined the effects of heavy metal exposure on the breeding performance of the tested couples, and we examined the plasma testosterone concentrations of great tit males in both study sites. We found no significant differences in the aggressive behavior between great tits at the polluted and the reference site during the egg-laying period. During the incubation period, responding male great tits at the polluted site performed significantly more aggressive flights around the decoy than did males at the reference site. During this period, there was also a tendency that more responding males were out of sight of the observer for some time during the experiment at the reference site than at the polluted site and this also for a longer period of time. Other behavioral parameters did not differ significantly between the two sites. Although plasma testosterone levels were higher at the polluted site, they did not differ significantly between the two sites. The tested couples at the polluted site interrupted their laying sequence significantly more and hatching success was significantly reduced compared to the tested birds at the reference site. We found no significant difference in clutch size, the length of the laying interruptions, fledging success, and fledgling age between the two sites. Although our results showed only minor alterations in the territorial aggressive behavior of male great tits exposed to heavy metal pollution, these small changes may have an important influence on the breeding and survival of these birds.Received: 22 June 2002/Accepted: 1 December 2002     

Antioxidant Enzymes and Heavy Metal Levels in Tissues of the Black Chocolate Clam <Emphasis Type="Italic">Megapitaria squalida</Emphasis> in Bahía de La Paz,Mexico

The black chocolate clam Megapitaria squalida is a commercially important bivalve along the Pacific coast of Mexico and is used as a bioindicator in environmental health studies. Previous studies of Bahía de La Paz indicated significant differences in heavy metal content among localities in this clam. To identify areas affected by heavy metal pollution, lipid peroxidation (TBARS) levels and activity of superoxide dismutase (SOD) and glutathione S-transferase (GST) were measured in muscle, gonad, and digestive gland tissue and correlated with heavy metal levels of clams from six localities along the shore of the bay. Significant differences in TBARS levels and antioxidant enzyme activity were found among these tissues at all localities. Such differences are ascribed to the physiology of each organ. Higher levels of TBARS were found in areas with higher cadmium content. SOD activity in muscle correlated positively with cadmium, lead, and iron in areas with a higher content of those elements. This study suggests that TBARS levels and SOD and GST activity are affected by biotic and abiotic factors and have potential as indicators of heavy metal contamination.     

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.     

Breeding performance of great tits (Parus major) along a gradient of heavy metal pollution

The possible effects of heavy metal pollution on various breeding parameters of the great tit (Parus major) were studied at four study sites in a pollution gradient near a large nonferrous smelter in Belgium during three consecutive breeding seasons. Significantly more females interrupted their laying period near the pollution source than did females farther away. At the two most polluted sites, hatching success was significantly reduced compared to two lesser-polluted study sites. Overall breeding success was also significantly lower toward the factory complex. This lowered breeding success was caused not only by the decreased hatching success toward the pollution source but also by the significant difference in fledging success among sites. Although a clear relationship was observed between the exposure to heavy metal pollution and an impaired reproduction, not all reproductive parameters were affected. We found no significant differences in the onset of laying, clutch size, length of the incubation period, and the proportion of nests with low-quality eggs among sites. Although nest box occupancy was every year slightly lower toward the smelter compared to farther away, differences among study sites were never significant.     

Effects of environmental pollution on microsatellite DNA diversity in wood mouse (Apodemus sylvaticus) populations

Ten microsatellite DNA loci were surveyed to investigate the effects of heavy metal pollution on the genetic diversity and population genetic structure of seven wood mouse (Apodemus sylvaticus) populations along a heavy metal pollution gradient away from a nonferrous smelter in the south of Antwerp (Flanders, Belgium). Analysis of soil heavy metal concentrations showed that soil Ag, As, Cd, Cu, and Pb decreased with increasing distance from the smelter. Genetic analyses revealed high levels of genetic variation in all populations, but populations from the most polluted sites in the gradient did not differ from those of less-polluted sites in terms of mean observed and expected heterozygosity level and mean allelic richness. No correlation was found between measures of genetic diversity and the degree of heavy metal pollution. However, an analysis of molecular variance and a neighbor-joining tree suggested a contamination-related pattern of genetic structuring between the most polluted and less polluted sites. Pairwise F(ST) values indicated that populations were significantly genetically differentiated, and assignment tests and direct estimates of recent migration rates suggested restricted gene flow among populations. Additionally, genetic differentiation increased significantly with geographical distance, which is consistent with an isolation-by-distance model. We conclude that, at least for our microsatellite DNA markers, genetic diversity in the studied wood mouse populations is not affected greatly by the heavy metal pollution.     

Associations between trace metals in sediment, water, and guppy, Poecilia reticulata (Peters), from urban streams of Semarang, Indonesia

The present study aims to answer the question whether the amount of metal in aquatic biota reflects the concentrations in the sediment and water, and whether the physico-chemical properties of the water and sediment have any influence on the suspected relationship. A study was made of 101 small streams in the city of Semarang, Central Java, Indonesia. Data on fish occurrence in 63 streams of the greater Semarang indicated that the guppies did not avoid the highly polluted sites. No significant difference in body weight between sites was found. Significant differences were found in metal body concentrations (Pb and Zn) between fish collected from sites with different degrees of pollution. A significant declining trend of Pb concentrations with increasing organism size was observed, whereas for two other metals, Zn and Cu, the concentrations did not depend on the body weight. Apparently, body concentrations of these two metals are regulated and maintained at a certain concentration. For the relationships between metal concentrations in water, sediment and fish, water and sediment parameters, and fish dry weight, the presence of significant multiple correlations and bivariate correlation (in 17 of 91 pairs of variables) indicated that, in general, abiotic parameters and body size had no influence on the metal flux from sediment to water, and into fish. Results of partial correlation analyses further suggested that metal concentrations in the sediments were the most important factor governing the metal body concentrations of fish. The present study indicates that the guppy Poecilia reticulata from urban streams is a potential bioindicator for urban metal pollution, especially with respect to their (1) spatial distribution over sites of all pollution regimes and (2) variation in metal accumulation levels reflecting the degree of pollution.     

Effects of heavy metals on riverine benthic macroinvertebrate assemblages with reference to potential food availability for drift-feeding fishes

We examined the influence of heavy metal pollution from an abandoned mine on benthic macroinvertebrates, at population and community levels, and the potential amount of food available for drift-feeding fish in northern Japanese streams. We studied multiple polluted and unpolluted sites with similar longitudinal positions to avoid problems caused by upstream-downstream comparisons. The ranges of zinc, copper, cadmium, and lead concentrations among the study sites were 5 to 812 microg/L, less than 0.12 to 5.2 microg/L, less than 0.0026 to 4.9 microg/L, and 0.1 to 18.6 microg/L, respectively. The abundance of several populations and community metrics showed a significant negative response to heavy metal pollution. Mayfly diversity and abundance was relatively sensitive to heavy metal pollution. In addition, the biomass of groups of macroinvertebrate taxa that are highly available for salmonids were significantly reduced at metal-polluted sites; this decrease in the most highly available group was noticeable (99% at the heavily polluted upper sites and 69% at the moderately polluted lower sites in spring). These results suggest that we should consider the indirect effect of pollution on food availability for the conservation of fish populations that depend on drifting macroinvertebrates.     

Pollution-induced community tolerance and functional redundancy in a decomposer food web in metal-stressed soil

Pollution may lead to the development of pollution-induced community tolerance (PICT) in a stressed community. We studied the presence of PICT in soil food webs using soil microcosms. Soil microcosms containing soil invertebrates and microbes were collected from polluted and unpolluted areas and exposed to a range of soil zinc concentrations. A pine seedling was planted in each microcosm to measure the effects of the origin of the community and Zn pollution on above-ground plant production. The effects of the treatments on nutrient content in the soil were also measured. The diversity of soil microarthropods and the soil's mineral nutrient content were low at the Zn-polluted site. We did not observe an increasing Zn tolerance among the soil organisms in the polluted soil. However, low population growth rates of soil invertebrates from the polluted site may indicate the deleterious effects on fitness of long-lasting pollution. In the soil from the nonpolluted site, Zn additions caused changes in the invertebrate food web structure. These changes were explained by the good physiological condition of the animals and their insensitivity to Zn. The fact that the food web structure in soil from the polluted site did not change can be used as a rough indicator of PICT. Structural stability is presumed by the lack of Zn-sensitive species at this site and the inability of populations to acclimate by altering their growth or reproduction patterns in response to changing soil conditions. Although microbial-based soil decomposer systems may have a high functional redundancy, our results indicate that metal stress at the polluted site exceeds the tolerance limits of the system. As a consequence, ecosystem function at this site is endangered. This study also shows that the evolution of metal tolerance by soil decomposer organisms may not be a common reaction to soil pollution, although changes of population and community structure indicated severe metal stress on organisms.     

Brown Trout and European Minnow as Target Species for Genotoxicity Tests: Differential Sensitivity to Heavy Metals

Brown trout (Salmo trutta) and European minnow (Phoxinus phoxinus) were evaluated as target species to carry out genotoxicity tests. Assessment was made of their relative abundance in wild; their distribution areas; and their sensitivity to heavy metals, intraperitoneally exposing individuals of both species to a low dose (1.7 mg/kg body weight) of different heavy metals. Micronuclei were scored in renal erythrocytes 24 h after treatment. Cadmium chloride significantly induced micronuclei in both species whereas mercury nitrate induced micronuclei increase only in brown trout. Brown trout is abundant, present in all studied freshwater ecosystems, and more sensitive to toxic heavy metals than minnow; therefore it is presented as a target species for studies on heavy metal genotoxicity.     

Studies of peripheral benzodiazepine receptors in mussels: comparison between a polluted and a nonpolluted site

The aim of this study was to investigate the peripheral benzodiazepine receptors in soft tissue membranes of the mussel Mytilus galloprovincialis from both polluted and nonpolluted seawater populations, using a radioligand specific for this receptor, [3H]PK11195. Mussels were dissected into four body parts--mantle, gills, digestive gland, and muscles-to determine the distribution of tissue-specific peripheral benzodiazepine receptors (PBRs). The specific binding was saturable and reversible. A statistically significant increase (muscle, 537% and mantle, 201%, as absolute percentages) in the maximal number of binding sites (B(max)) was found in mussels from the polluted site, compared with mussels from the nonpolluted site. By contrast, the value of the dissociation constant (K(d)) at equilibrium does not show a statistically significant variation between the two groups. In competitive experiments of the compounds clonazepam, flumazenil, flunitrazepam, Ro5-4864, PK11195, and protoporphyrin IX, only PK11195 and protoporphyrin IX displaced [3H]PK11195 specifically bound to soft tissue membranes, revealing that the binding sites of peripheral benzodiazepine receptors of mussels have pharmacological properties comparable to those of low vertebrates such as trout. M. galloprovincialis was also tested as an indicator of heavy metal exposure, and metal accumulation in the digestive gland was measured by atomic absorption spectrophotometry (AAS). The contents of Pb, Mn, and Zn in mussels collected off the polluted site were higher than those in mussels from the nonpolluted site. These data suggest that PBRs are present in the soft tissues of the mussel M. galloprovincialis. Here we report preliminary evidence of biochemical alterations in mussels from the polluted site.     

Do seasonal changes affect metallothionein induction by metals in mussels, Mytilus edulis?

Mussels have been proposed as biomonitors of metal pollution based on the determination of metallothionein (MT) concentrations as a biomarker, but a comprehensive study taking into account both intersite and long-term temporal variations in MT and metal concentrations in different organs is lacking. Thus, the present study was designed to examine the concentrations of cytosolic and insoluble Cd, Cu, Zn, and MT in gills and digestive gland of mussels (Mytilus edulis) of homogeneous size and age obtained from aquaculture and kept on a reference site or translocated to a metal-rich site throughout their reproductive season (March-October 1997). Relatively significant binding of metals to the insoluble fraction was observed in both tissues. In the digestive gland, monthly MT concentrations were strongly correlated to cytosolic metal levels. Moreover, despite significant temporal variations, the grand mean MT concentration based on all individual determinations in the digestive gland (reference, n=54; transplants, n=50) was significantly higher in mussels from the metal-rich site. On the other hand, gill MT concentrations did not reflect metal contamination as reliably.     

Function of metallothioneins in carp Cyprinus carpio from two field sites in Western Ukraine

The aim of the present study was to elucidate the seasonal and spatial regularity of the properties of metallothioneins (MT) from the liver and gills of carp Cyprinus carpio L. in rural (R) and industrial (I) sites in Western Ukraine. The MT is represented by two chromatographic forms, the features of which exhibit seasonal rather than spatial dependence. The pronounced differences between the sites were due to the lower levels of Zn in the liver and the higher levels of Zn in MT of carp from site I, providing evidence of the higher overall anthropogenic impact here that leads to the distortion of this essential metal accumulation and to the activation of metal-binding function of MT. In spring, higher levels of Cu and Cd in MT and in the tissues were reflected at site R probably as the result of the permitting pollution here. The principal component analysis demonstrated the correlation of MT-bound metal levels to their levels in water and the absence of such relation for general tissue metal levels for Zn.     

Altered exoskeleton composition and vitellogenesis in the crustacean Gammarus sp. collected at polluted sites in the Saguenay Fjord, Quebec, Canada

Gammarus sp. individuals were collected at four intertidal sites subjected to direct sources of pollution (marinas, ferry traffic, and harbors) and at one site with no direct source of pollution. Levels of vitellogenin-like proteins (Vtg), metallothioneins (MT), alkali-labile phosphates (ALPs) in proteins, and lipogenic enzyme activities (i.e., glucose-6-dehydrogenase, isocitrate dehydrogenase, and malate enzyme) were measured in whole soft tissues. In exoskeletons, levels of pH-dependent extractable protein and chitin were determined to assess the possible impacts of pollution on exoskeleton integrity and the molting process. Results show that males were consistently heavier than females regardless of site quality but that the whole-body weight of both sexes was significantly lower at polluted sites. Females displayed either induced or decreased Vtg-like proteins at polluted sites, indicating significant changes in gametogenesis activity. MT levels were not sex dependent and tended to be induced at all impacted sites. ALP levels in acetone-fractionated proteins indicate altered phosphate mobilization at some impacted sites, where females tended to display higher ALP levels. Lipogenic enzyme activities did not vary by sex but were readily increased at impacted sites, suggesting a delay in gonad maturation rates. Exoskeleton protein characteristics revealed that the proportion of chitin in exoskeletons was a lower at most impacted site, suggesting disruption of chitin and pH-dependent protein mobilization. Principal component analysis revealed that gammarids collected at affected sites displayed substantial changes in the proportion of chitin, arthropodin, sclerotin, MTs, and intermediary glucose metabolism (glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase in soft tissues) and thus suffered from disturbed gametogenesis and exoskeleton integrity.     

Great and blue tits as indicators of heavy metal contamination in terrestrial ecosystems.

Although there are abundant data on heavy metal contamination in the feathers of seabird species and birds of prey, relatively few studies have addressed the use of passerine birds as indicators of point-source contamination. Concentrations of three essential elements (Al, Zn, and Cu) and two toxic metals (Cd and Pb) were determined in the outer tail feathers of great and blue tits (Parus major and Parus caeruleus), two small insectivorous passerines, at a presumed polluted site (in an urban area close to household waste incinerators) and at a reference site (in a nature reserve). For the great tit, both Cd and Cu levels were significantly higher at the polluted site than in the nature reserve. For the blue tit, Cd, Pb, and Cu levels were significantly higher at the polluted site than at the reference site. There were striking differences in metal levels between blue and great tits inhabiting the same polluted site: lead, zinc, and copper were significantly higher in the smaller blue tit than in the great tit. We suggest that tit (Parus) species, which have been important models in behavioral and ecological research, may be very useful as biomonitors for local contamination.     

Burrowing Crab (Neohelice granulata) as a Potential Bioindicator of Heavy Metals in the Bahía Blanca Estuary, Argentina

Bioindicators can be used to determine spatial and/or temporal variations of heavy metals in a certain coastal or marine environments. This study investigated the potential use of the burrowing crab Neohelice granulata from two different locations in the Bahía Blanca estuary, a moderately polluted ecosystem. Concentrations of zinc (Zn), nickel (Ni), and lead (Pb) in soft tissues of male and female crabs were measured. In addition, concentrations of the three metals in eggs were compared with concentrations in female crabs. No geographical differences were found for any of the three metals, whereas sexual and seasonal differences were obtained for Zn and Ni, with the winter season posing lower concentrations. Moreover, the three metals were detectable in eggs and were lower than concentrations in female crabs (except for Zn). Finally, the usefulness of this species as a potential bioindicator of heavy-metal pollution within this estuarine ecosystem is discussed.