Responses of European eel (Anguilla anguilla L.) in two polluted environments: in situ experiments

European eels (Anguilla anguilla L.) were caged for 8 and 48 h in two polluted areas to assess the contamination cleanup process in the Vouga River 2 years after the official closing of a bleached kraft pulp mill effluent (BKPME; experiment 1), and to monitor the effects induced by contaminated offward fishing harbor waters (experiment 2). Plasma cortisol, glucose, and lactate were evaluated as stress responses. In experiment 1, plasma cortisol, glucose, and lactate increased after 8 h of exposure in site 3, which is located farthest from the deactivated sewage outlet. However, A. anguilla seemed to adapt after 48 h of exposure in site 3, because all three parameters returned to control levels. Plasma glucose also significantly increased after 8 h of exposure at sites 1, 2, and 3, returning to control levels after 48 h. Plasma lactate levels increased after 8 h of exposure at site 3 and after 48 h of exposure at site 1. In experiment 2, A. anguilla exposed to contaminated harbor waters increased their plasma lactate after 8 and 48 h of exposure, whereas their cortisol and glucose plasma were elevated only after 48 h of exposure. The results demonstrate that even 2 years after the official closing of the BKPME sewage outlet, the river Vouga water remains contaminated by the sediment associated chemicals. Because the fishing harbor induced in A. anguilla the same type of stress responses, it is also an area of concern. The adopted stress parameters allied to a caging strategy are recommended for future environmental monitoring assessments.     

Biotransformation,genotoxic, and histopathological effects of environmental contaminants in European eel (Anguilla anguilla L.)

A prolonged toxicity study was carried out in young European eel (Anguilla anguilla L.) to evaluate the effects of environmental contaminants, namely, two individual standard compounds, benzo[a]pyrene (BaP) and dehydroabietic acid (DHAA), and a complex mixture, bleached kraft pulp mill effluent (BKPME). Fish were exposed to BaP (0.22, 0.45, and 0.9 microM) and BKPME (3.12%, 6.25%, and 12.5% (v/v)) for 3, 7, and 30 days and to DHAA (0.07, 0.15, and 0.30 microM) for 3, 7, 30, 90, and 180 days. The biomarkers include biotransformation and genotoxicity indicators, such as total ethoxyresorufin O-deethylase (EROD) activity and frequency of erythrocytic nuclear abnormalities (ENAs), respectively. Hematological dynamics was assessed as frequency of immature erythrocytes (IEs). Histopathological examinations were carried out for the highest concentrations and for 30 days and longer exposures. Total EROD increases significantly only after 180 days of DHAA exposure. However, significant ENA induction was generally observed during exposure to all contaminants tested. Nevertheless, some of the ENA results suggest an altered genotoxic response, which may arise either from short-term exposures to the highest contaminant levels or long-term exposures to the lowest contaminant levels. IE frequency decreased significantly after 30 days of exposure to 0.45 microM BaP and 180 days of exposure to the entire DHAA concentration range. Increased density of pigmented macrophage aggregates in 30-day BaP- and BKPME-exposed fish as well as in 90- and 180-day DHAA-exposed fish confirmed histopathological liver alterations. Bile accumulation in hepatocytes after BaP treatment, cytoplasmic vacuolization and cell atrophy following DHAA exposure, as well as liver loss of parenchymal cells in BKPME-exposed fish, were also detected. Dispersed necrosis and focal inflammation were observed in the livers of all treated groups. Fish exposed to DHAA and BKPME showed skin and gill disruption as well as kidney Malpighian corpuscle alterations. All 30-day-treated groups revealed intense spleen hemosiderosis, indicating increased erythrophagia. This splenic effect may be strongly correlated with the observed disappearance of ENAs. Neoplastic lesions were not found. A multibiomarker strategy, which includes EROD, ENA, and IE assays as well as histopathological studies, contributed to a better understanding of the global toxic process.     

European eel (Anguilla anguilla L.) metallothionein, endocrine, metabolic and genotoxic responses to copper exposure

This study investigated Anguilla anguilla (European eel) physiological and genotoxic responses to copper (Cu) and their relation with metallothionein (MT) protection. Eels were exposed during 7 days to Cu 0.2 micromol/L. MT induction was assessed in gill and liver, revealing significant response only in liver. Endocrine responses displayed a plasma free triiodothyronine (T3) and cortisol significant decrease, though the thyroid-stimulating hormone (TSH) and free thyroxine (T4) concentrations were unaltered. A significant plasma glucose increase was observed whereas lactate was significantly decreased. Despite the absence of DNA integrity decrease in blood, gill, liver and kidney, erythrocytic nuclear abnormalities (ENA) frequency significantly increased in Cu exposed group. MT induction was insufficient to prevent endocrine and metabolic alterations as well as genotoxicity/clastogenicity in blood. However, MT protection was evident in liver by preventing DNA integrity loss. Globally, it was demonstrated that Cu environmentally realistic levels may pose a serious ecological risk to fish.     

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.     

Benzo[a]pyrene and beta-naphthoflavone mutagenic activation by European eel (Anguilla anguilla L.) S9 liver fraction

Is Anguilla anguilla L. (eel) liver ethoxyresorufin O-deethylase (EROD) induction absolutely necessary in order to convert promutagens as benzo[a]pyrene (BaP) into a mutagenic compound? Eels were exposed for 8 h to clean (control) and 0.3 microM beta-naphthoflavone (BNF)-contaminated water. The 8-h exposure to 0.3 microM BNF brought about a very high EROD induction (10 pmol/min/mg protein) compared to control animals (1 pmol/min/mg protein). The Ames test (Maron and Ames, 1983) was carried out with Salmonella typhimurium TA 98 strain (TA98 His-) and eel isolated S9 liver fraction was used as a metabolic BaP activator. The BaP and BNF dose range concentrations tested were 0 (blank), 0.015, 0.08, 0.15, 0.38, 0.75, 1.5, 3.8, and 7.5 microM/plate and 0 (blank), 0.412, 1.235, 3.704, 11.1, 33.0, and 100 nM BNF, respectively. A dose-response relationship between BaP concentration and mutagenic activity was observed in the presence of S9 fractions in control and 0.3 microM BNF-exposed eels. Significant positive results, as TA98 His+ revertants, were observed at 0.38, 0.75, 1.5, 3.8, and 7.5 microM BaP/plate induced by BNF S9 fractions. Significant BaP mutagenic activation by liver control S9 was detected only at 1.5, 3.8, and 7.5 microM/plate. The BaP 1.5, 3.8, and 7.5 microM/plate mutagenic activation by BNF S9 and control S9 were not significantly different. Relative to BNF activation, it was only possible to detect His+ reversion at 11.1 nM BNF concentration with 0.3 microM BNF-induced S9. The above results demonstrate that the eel S9 liver fraction has the capacity to biotransform high BaP concentrations and convert it into a mutagenic compound with or without previous liver BNF biotransformation induction. The same does not apply to low BaP concentrations, where liver S9 induction by BNF is necessary to promote mutagenesis.     

Metabolic effects of kraft mill effluents on the eel Anguilla anguilla L

Yellow eels (Anguilla anguilla L.) with an average weight of 60 g were used in this experiment. The fish were caught in June/July at the Aveiro Lagoon on the Portuguese West Coast, transported to the Department of Biology, Aveiro University, and kept in aerated aquaria for 1 week before the experiment started. The eels were then exposed for 1 and 3 weeks to 75 and 50% of the kraft pulp mill effluent. The eels exposed to the kraft pulp mill effluent developed an increase in red blood cell number per cubic millimeter and several biochemical changes, such as an increase in plasma lactate and sodium and a decrease in plasma pyruvate and potassium. Histological examination of the experimental eels exposed to the 50% kraft pulp mill effluent revealed deep alteration of the tissue structure, such as disruption of the skin and edematous hypertrophy of covering epithelial cells in secondary gill lamellae. The kidney had damage of the renal tubules. The liver developed necrosis supported by a significant decrease in GOT and GPT activity. The spleen had an increase in blood content as well as in pigment centers. Previous results indicated the kraft pulp mill effluent causes tissue damage and consequent metabolic changes in the eel Anguilla anguilla L.     

Induction of biotransformation in the liver of Eel (Anguilla anguilla L.) by sublethal exposure to dinitro-o-cresol: an ultrastructural and biochemical study

Structural and functional alterations in hepatocytes of the European eel, Anguilla anguilla, following a 4-week-exposure to 5, 50, and 250 micrograms/liter dinitro-o-cresol (DNOC) were investigated by means of electron microscopy and biochemistry and compared to liver pathology in eels exposed to the chemical spill into the Rhine river at Basle in November 1986. Whereas phenological parameters (growth, condition factor) are unaffected, ultrastructural and biochemical alterations are detectable at greater than or equal to 50 and 5 micrograms/liter DNOC, respectively. Structural modifications include: rounding-up of the nuclei; fractionation and reduction of the rough endoplasmic reticulum; proliferation of the smooth endoplasmic reticulum (SER), mitochondria, peroxisomes, and lysosomes; bundles of rod-shaped SER profiles; annulate lamellae; membrane whorls within mitochondria; crystallization of the peroxisomal matrix and glycogen bodies; glycogen depletion and lipid augmentation. Structural changes can be correlated to an increase in hepatic lipid and protein contents as well as stimulation of mitochondrial (cytochrome c oxidase), peroxisomal (catalase, allantoinase, uricase), lysosomal (arylsulfatase), and microsomal (esterase) enzymes. An increase in NADPH-cytochrome c reductase and cytochrome P450 as well as UDP-glucuronyltransferase and arylsulfotransferase activities in the microsomal fraction document an induction of hepatic biotransformation as a functional correlate to SER proliferation. Maximum inducibility of biotransformation enzymes at 50 micrograms/liter indicates a biphasic, concentration-dependent reaction of eel liver. Comparison of DNOC-induced effects with liver pathology in eel exposed to the chemical spill in 1986 reveals striking similarities so that DNOC may not be excluded as a possible factor in the fish kill in the Rhine river.     

Role of glutathione in Thiobencarb resistance in the European eel Anguilla anguilla

Glutathione-dependent defense against xenobiotic toxicity is a multifaceted phenomenon that has been well characterized in mammals. In the present study, eels of species Anguilla anguilla were exposed to 15 ppm of the herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate) for 96 h. Eels exposed to the pesticide were grouped in 24-h intervals according to their time of death, while surviving intoxicated eels constituted another group (live animals). Glutathione content (GSx, GSH, GSSG) was determined in liver and muscle tissues of the dead and live (intoxicated) animals and compared to control values (nonexposed eels). The fish that died before 96 h of exposure were considered susceptible to thiobencarb, while those dead after 96 h and the surviving ones were called resistant. Hepatic glutathione (GSH) content in susceptible eels was lower than that in the control fish, while resistant eels presented GSH levels threefold higher than those of controls. Muscle glutathione levels in dead eels were practically unaffected, but there was a significant decrease in GSH levels in the surviving intoxicated eels. These results indicate that the eels that were able to induce glutathione synthesis in the liver due to the presence of thiobencarb in the medium demonstrated a greater longevity than those who lost glutathione homeostasis.     

Ammonia sensitivity of the glass eel (Anguilla anguilla L.): salinity dependence and the role of branchial sodium/potassium adenosine triphosphatase

Eel aquaculture is capture based and thus dependent on the fishery for juvenile glass eels. This fishery typically takes place in estuaries where salinity varies and ammonia levels can be elevated. Also, during capture and transport glass eels are kept at high densities and ammonia from endogenous production can increase to toxic levels. Ammonia is known to have detrimental effects on fish growth and survival. In the present study, the salinity dependence of ammonia sensitivity in glass eels acclimated to either seawater or freshwater was determined, and the possible role of branchial sodium/potassium adenosine triphosphatase (Na+/K+ -ATPase) was investigated. Freshwater-acclimated glass eels were found to be more sensitive to ammonia with a lethal concentration to 50% of the test organisms (LC50) value for 96 h of 3.30 mM for total ammonia nitrogen (TAN) and 117 microM for un-ionized ammonia (UIA), versus values of 4.95 mM and 138 microM, respectively, for seawater-acclimated animals. Freshwater glass eels also had significantly lower body TAN levels than seawater-acclimated glass eels, although body accumulation profiles during exposure were similar. The higher branchial Na+/K+ -ATPase activities in seawater glass eels might explain this difference in sensitivity; however, activities decreased significantly with increasing ammonia levels. There was also no salinity dependence of net ammonia flux rates (0.388 micromol of TAN/g/h). Holding glass eels at high densities characteristic of transport conditions resulted in elevated ammonia concentrations to approximately 3 mM, which coincidently approaches to the freshwater LC50 value and may therefore contribute to mortality.     

Influence of inorganic lead on the biochemical blood composition of the eel, Anguilla anguilla L

Yellow eels (Anguilla anguilla L.) with an average weight of 50 g were caught in September in the Aveiro Lagoon on the Portuguese West Coast and kept in aerated aquaria for 1 week before the experiment started. The eels were exposed to an inorganic lead concentration of 300 micrograms Pb/liter for 30 days. Differential white blood cell count showed an increase in the number of lymphocytes in exposed fish. Blood hemoglobin and number of red blood cells per cubic millimeter showed no differences between the control and the exposed fish. Biochemical analyses of blood composition, such as plasma glucose, total plasma protein, total plasma cholesterol, and sodium and potassium plasma, showed no differences between treated and control animals. Plasma lactate levels increased in treated animals compared with those in controls.     

Capacity of the European eel (Anguilla anguilla) to elongate and desaturate dietary linoleic acid

The ability of eels (Anguilla anguilla) to further desaturate and chain elongate linoleic acid, 18:2n-6, was studied by feeding diets containing either corn oil or a fish oil to groups of elvers for 12 wk and analyzing proportions of fatty acids in tissue lipids. Over the 12-wk period elvers given both dietary treatments increased in weight by fourfold. The proportion of arachidonic acid, 20:4n-6, present in polar lipids of elvers fed the diet containing corn oil increased from an initial value of 5% by weight to 12% by weight; the corresponding value for elvers given a diet containing fish oil, with little linoleic acid in it, was less than 4% by weight. The capacity of this fish to modify dietary linoleic acid metabolically was confirmed by examining the metabolic fate of radioactive carbon after giving [1-14C]linoleic acid orally to eels. Seven days after administration of the linoleic acid approximately 10% of the radioactivity recovered in liver fatty acids was present in trienes and tetraenes with about 4% occurring in arachidonic acid. Compositional analyses from the feeding experiment also indicated that dietary docosaenoic acid, 22:1n-11, is preferentially oxidized by eels.     

Induction of mixed function oxidases by petroleum in the American eel,Anguilla rostrata

American eels,Anguilla rostrata, were exposed to crude oil by ingestion of a 10, 100, or 500 l/kg fish dose per day for five days. Depuration was followed for an additional twelve days. All oil doses caused an induction of hepatic microsomal enzymes, maximally by three days of exposure. Benzo(a)pyrene hydroxylase (BaPH) showed a dose related response, with greater induction at 100 l/kg than at the other doses. The highest dose was hepatotoxic. Cytochrome P-450 induction was dose independent, and remained induced maximally for the entire experimental period, in contrast to BaPH which declined in activity. Reaction optimum for BaPH was at pH 7.5 and 27°C. A study of tissue distribution showed the liver to account for nearly all BaPH activity. A significant increase in the protein content of the hepatic postmitochondrial fraction of oil-exposed fish was also observed.     

Interactions of 2,4,6-trinitrotoluene (TNT) with xenobiotic biotransformation system in European eel Anguilla anguilla (Linnaeus, 1758)

The aim of the present study was to investigate the interaction of 2,4,6-trinitrotoluene (TNT) with liver biotransformation enzymes in European eel Anguilla anguilla (Linnaeus, 1758). Eels were exposed to 0.5, 1 and 2.5 mg/l nominal concentrations of TNT for 6 and 24 h. Modulation of CYP1A1, UDPGT and GST genes was investigated by real-time PCR. Total CYP450 content, NADPH cytochrome c reductase activity, CYP1A and CYP2B-like activities, such as EROD, MROD and BROD, as well as GST and UDPGT activities, were measured by biochemical assays. An in vitro study was performed on EROD in order to evaluate catalytic modulation by TNT. No modulation of the CYP1A1 gene or protein was observed in TNT-exposed eels. On the other hand, a significant decline of EROD and MROD activities was observed in vivo. An increase in NADPH cyt c reductase, and phase II enzymes (UDPGT and GST) were observed at both gene expression and activity levels. The overall results indicated that TNT is a potential competitive inhibitor of CYP1A activities. A TNT metabolic pathway involving NADPH cyt c reductase and phase II enzymes is also suggested.     

Toxicity of heavy metals and salts to eurasian watermilfoil (Myriophyllum spicatum L.)

The toxicity of various heavy metals and salts to Eurasian watermilfoil (Myriophyllum spicatum L.) was determined under controlled growth conditions. Toxicants were added to water or to soil in systems with and without woods earth in the substrate.Fifty precent inhibition of root weight occurred with concentrations of 0.25 ppm Cu⁺², 1.9 ppm Cr₂O₇ ^–2, 3.4 ppm Hg⁺², 2.9 ppm AsO₂ ^–1, 7.4 ppm Cd⁺², 2.5 ppm Al⁺³, 9.9 ppm Cr⁺³, 41.2 ppm Ba⁺², 21.6 ppm Zn⁺², 13.3 ppm NH₄ ⁺¹, 22.4 ppm CN^–1, 143 ppm B₄O₇ ^–2, 363 ppm Pb⁺², 10,228 ppm Na₂SO₄, and 8,183 ppm NaCl. Soil increased toxicity of Cr⁺² and Ba⁺² but decreased toxicity of Cr₂O₇ ^–2, Cu⁺², Cd⁺², Al⁺³, and Hg⁺². In distilled water, CaCl₂ increased toxicity of Cr⁺³ but not Cr₂O₇ ^–2.For most toxicants there was a consistent relationship between inhibition of length and inhibition of weight and between inhibition of roots and inhibition of shoots. However, Cr₂O₇ ^–2 disproportionately decreased dry weight, and Hg⁺² and Na₂SO₄ disproportionately decreased stem length growth. With Cd⁺² and Cu⁺² stem length was greater relative to other measures of growth. Toxicity of Na₂SO₄ and NaCl was the same when concentrations were calculated as osmotic pressure but not when calculated as Na atoms or as total molecules/L.     

Effects of propanil on the European eel Anguilla anguilla and post-exposure recovery using selected biomarkers as effect criteria

The aim of this study was to assess the physiological response of Anguilla anguilla to propanil and the degree of recovery after being moved to clean water. Preliminary acute toxicity test was carried out in the laboratory and the median lethal concentration (LC50) at 96 h was calculated as 31.33 mg/L (29.60-33.59 mg/L). NOEC and LOEC values (at 96 h) were also calculated as 20 and 25mg/L, respectively. The fish were exposed to 0.63 and 3.16 mg/L of propanil for 72 h and allowed to recover for 144 h. Total proteins (TPs), gamma-glutamil transpeptidase (gamma-GT), alanin aminotransferase (AlAT), alkaline phosphatase (AP), lactate dehydrogenase (LDH) and water content (WC) were assayed in muscle and liver tissues, liver somatic index (LSI) was also determined. Liver TPs and gamma-GT activity decreased after propanil exposure while AlAT and LDH increased. Muscular AP, AlAT and proteins decreased in intoxicated eels while LDH and gamma-GT activities increased. WC increased in both tissues after herbicide exposure as well as LSI. These results revealed that propanil affects the intermediary metabolism of A. anguilla and that the assayed enzymes can be used as good biomarkers of herbicide contamination. However a longer recovery period should be necessary to re-establish eel physiology. The parameters measured in the present study can be used as herbicide toxicity indicators and are recommended for environmental monitoring assessments.     

Anguilla anguilla L. genotoxic and liver biotransformation responses to abietic acid exposure

Adult eels (Anguilla anguilla L.) were exposed for 8, 16, 24, and 72 h to 0, 0.1, 0.3, 0.9, and 2.7 microM abietic acid (AA). Genotoxicity was measured as erythrocytic nuclear abnormalities (ENA), as well as DNA strand breaks in blood and liver. Liver cytochrome P450 (P450) content, liver ethoxyresorufin O-deethylase (EROD), and glutathione S-transferase (GST) activities were determined as biotransformation biomarkers. Liver alanine transaminase (ALT) activity was also measured as an indication of tissue damage. Low AA concentrations, such as 0.1 and 0.3 microM, result in a delayed induction of A. anguilla L. liver EROD activity, whereas the higher AA concentration (2.7 microM AA) also has a delayed effect probably as a consequence of liver tissue high inhibitory concentration. The current eel liver GST activity results demonstrate that only low AA concentrations promote liver increases in GST, whereas high AA concentrations, such as 0.9 and 2.7 microM, do not alter it. The results concerning eel liver ALT activity indicate that significant liver damage is induced by high AA concentrations, such as 2.7 and 0.9 microM. The eel ENA result analysis reveals that AA is a weak ENA inducer in A. anguilla L. Blood DNA integrity results suggest that low AA concentrations promote late decreases in blood DNA integrity; nevertheless, high AA concentrations are early blood genotoxic inducers compared with low AA doses. According to the present research results with respect to eel liver DNA damage, all of the AA exposure concentrations decreased liver DNA integrity.     

Anguilla anguilla L. liver EROD induction and genotoxic responses after retene exposure

Anguilla anguilla L. (European eel) were exposed for 8, 16, 24, and 72 h to 0 (control), 0.1, 0.3, 0.9, and 2.7 microM retene. A. anguilla L. liver ethoxyresorufin-O-deethylase EROD activity significantly increased during the whole exposure period to all retene concentrations, when compared to their controls. The liver cytochrome P450 content only increased after exposure to high retene concentrations (0.9 and 2.7 microM) from 8 to 24 and 72 h, respectively. Generally, liver DNA integrity decreased with increased retene concentrations. Thus, a low retene concentration (0.1 microM) was only effective at 16 h, 0.3 and 0.9 microM had an early and prolonged effect up to 24h, and 2.7 microM decreased liver DNA integrity during the whole exposure period. However, blood DNA integrity decrease was observed in eels after 24h exposure to 0.1 microM retene, and at 16 h to 0.3 and 0.9 microM retene, despite an early blood DNA integrity decrease at 8 and 16 h exposure to 2.7 microM retene. An early genotoxic response to retene was also observed as erythrocyte nuclear abnormalities plus Notched (ENA+Not) frequency increase at 8, 16, and 24h exposure to 0.1 and 0.3 microM retene as well as at 8, 16, 24, and 72 h to 0.9 microM retene. Though, the highest retene concentration (2.7 microM) only induced ENA+Not and erythrocyte nuclear abnormalities minus Notched (ENA-Not) at 16 h exposure. The eel ENA+Not increase was more sensitive than the ENA-Not increase as a measure of retene genotoxicity. Eel liver alanine amino transferase (ALT) increased activity reveals its enhanced transamination capacity after short-term exposure to retene. The A. anguilla L. ratio between hemoglobin concentration and red blood cells count (Hb/RBC) increased at 8h exposure to 0.1, 0.3, and 0.9 microM retene, suggesting an initial homeostatic process.