Screening of hydrophobic DNA adducts in flounder (Platichthys flesus) from the Baltic Sea

Neoplasia and other histopathological lesions in flounder (Platichthys flesus) liver have been investigated in several European sea areas, including the Baltic Sea. Several studies have been able to link neoplasm epizootics in fish with the exposure to genotoxins such as polycyclic aromatic hydrocarbons (PAHs). The level of hydrophobic DNA adducts in tissue DNA reflects the exposure of the organism to PAHs.Using hydrophobic DNA adduct levels as biomarkers, possible PAH exposure was assessed in flounder from 10 different sites in the Baltic Sea, collected during the years 1995–1997. The results show that the overall levels of hepatic DNA adducts were low and, in general, the chromatograms appeared clean. The highest levels of DNA adducts were found at two sites in the southern Baltic Sea. There were no statistically significant differences in adduct levels between the sites. Our results indicate that flounder from studied off shore sites of the Baltic Sea had not been exposed to a greater extent to large polycyclic hydrophobic hydrocarbons in their environment.     

Accumulation of bis(tributyltin) oxide by the mud crab, Rhithropanopeusharrisii

Accumulation of ¹⁴C labelled bis(tributyltin) oxide (TBTO) by the mud crab, $\text{Rhithropanopeus}$$\text{harrisii}$, was determined through short-term exposure to labelled water and food. The potential for trophic accumulation during chronic low level exposure is emphasized.     

Effects of bis(tributyltin)oxide on antipredator behavior in threespine stickleback Gasterosteus aculeatus L.

Antipredator behavior was used as a parameter to detect effects caused by exposure to the organotin compound bis(tributyltin)oxide (TBTO). Three groups of threespine sticklebacks (Gasterosteus aculeatus L.) were exposed to 3, 9 and 27 ppb TBTO, respectively. A fourth control group was given the same treatment as the other three groups, but no TBTO. Antipredator behavior of the fish was evoked using a dummy heron (Ardea cinerea) bill. TBTO exposure caused significant changes in the spatial position of the fish in the aquarium (P(Loc)), their response to predator attack (P(Res)), recovery time (P(Rec)) and latency time (P(Lat)). Some of the effects were, however, reversible after the exposure was terminated. We suggest that behavior as an indicator of pollution may be used as an ecologically relevant integrative biomarker.     

Toxicity of anti-fouling biocides to encysted metacercariae of Echinoparyphium recurvatum (Digenea: Echinostomatidae) and their snail hosts

The toxicity of the anti-fouling biocides tributyltin (TBTO), copper, and Irgarol 1051 (irgarol) at a nominal concentration of 10 microg/l over a 30 day period were investigated against the viability of metacercarial cysts of the digenean parasite Echinoparyphium recurvatum resident within the body of two common freshwater snails, Lymnaea peregra and Physa fontinalis. Reduced parasite viability was found under most exposures in both snail species. However a greater effect of toxicant exposure was found in cysts within P. fontinalis compared to those in L. peregra. This was associated with an increased mortality of the host snail. Among all tested biocides, TBTO exposures induced the highest mortality to both the parasite and their hosts. These results suggest that parasite viability is interlinked with survival of the host snail. The mechanisms of differing toxicity between host species and its relevance to successful parasite transmission to the next host are discussed.     

Uptake, tissue distribution and metabolism of the insecticide endosulfan in Jenynsia multidentata (Anablepidae, Cyprinodontiformes)

The study reports the accumulation, distribution and metabolism of technical endosulfan in Jenynsia multidentata. Adult females were exposed to acute sublethal concentrations (0.072, 0.288 and 1.4 μg L⁻¹). After 24 h, fish were sacrificed and gills, liver, brain, intestine and muscle were removed. Results show that both isomers of technical-grade endosulfan (α- and β-) are accumulated in fish tissues and biotransformation to endosulfan sulfate occurs at all concentrations tested. Significantly differences in endosulfan accumulation were only found at 1.4 μg L⁻¹ but not between the lowest concentrations. However a similar distribution pattern was observed at all exposure levels where liver, intestine and brain had the highest levels of α-, β-endosulfan and endosulfan sulfate. Moreover, liver and brain showed the highest endosulfan sulfate:α-endosulfan ratios due to high biotransfomation capacity. J. multidentata demonstrated to be a sensitive species under exposure to technical endosulfan and, therefore, could be used to assess aquatic pollution.     

Histopathological lesions and DNA adducts in the liver of European flounder (Platichthys flesus) collected in the Seine estuary versus two reference estuarine systems on the French Atlantic coast

An epidemiological survey was conducted in the Seine estuary and in two smaller and relatively preserved estuaries on the French Atlantic coast in order to estimate the occurrence of liver lesions in European flounder, Platichthys flesus, and also to seek putative risk factors for the recorded pathologies. Four hundred and seventy-eight fish of both sexes and of different size ranges were sampled in the three studied areas, 338 of which in the Seine estuary. All fish were examined for histopathological liver lesions, while DNA adducts and otoliths were analyzed on a subsample. Five categories of hepatic lesions were recorded with the following prevalence for the Seine estuary: 36.7 % inflammations, 8 % parasites (mainly encysted nematodes), 6.5 % foci of cellular alteration (FCA), 5.3 % foci of necrosis or regeneration (FNR), and 1.5 % tumors. Inflammation occurrence increased according to age, contrary to parasitic infestations and FCA which were more prevalent in young fish, notably those of <1 year old (group 0). Tumors were only observed in females of more than two winters. Females exhibited a higher prevalence of tumors (3.0 %) and FCA (6.5 %) than males (0 and 2.6 %, respectively). Parasitic and infectious lesions and FNR were equally distributed in males and females. The prevalence of FNR was also shown to vary according to sampling season, with significantly more occurrences of liver necrosis in the fish collected in summer than in spring. Spatial differences were observed with a higher occurrence of encysted parasites in flounders from the upper Seine estuary, while inflammations predominated in flounders living downstream. Temporal trends were also noted, with an increased prevalence of parasitic infestations, inflammations, and FCA in the 2002–2003 period in comparison to the 1996–1997 one. The three flounder populations from the Seine estuary (Normandy), Ster estuary (Brittany), and Bay of Veys (Normandy) showed different spectra of hepatic lesions. Flounders from the Bay of Veys had relatively few liver lesions as compared to flounders from the two other estuaries. Flounders from the Ster estuary exhibited the highest prevalence of parasites (37.2 %) and inflammations (51.1 %). Finally, FCA and liver tumors occurred at very similar levels in both flounder populations from the Seine and the Ster estuaries. Group 0 flounders inhabiting the upper Seine estuary were more prone to parasitic and pre-neoplastic hepatic lesions and had higher levels of liver DNA adducts than the older ones living downstream. It was postulated that group 0 European flounders may serve as valuable bioindicators for assessing the quality of estuarine waters and the health status of euryhaline fish populations.     

Butyltins in shellfish, finfish, water and sediment from the Coos Bay estuary (Oregon, USA)

Butyltin was measured in tissue, water and sediment samples collected throughout Coos Bay estuary between 1986 and 1997. Butyltin-induced shell deformation in commercial oysters prompted this assessment. Butyltins are released from tributyltin oxide (TBTO)-containing boat paint. Clams from three sites contained tributyltin (TBT) levels ranging from 168 to 457 microg/kg in 1992-1994. Consequently, Oregon Health Division (OHD) issued a shellfish consumption advisory in 1995; it was rescinded in 1997 after US Environmental Protection Agency (EPA) revised the TBTO oral reference dose (RfD). The revised RfD changed the tissue screening level from 112 to 1120 microg TBT/kg. Fifty percent of sediment samples collected during 1991-1997 and analyzed for total butyltins (TotBT) exceeded the Puget Sound Dredge Disposal Analysis Screening Level (PSDDA SL) of 73 microg TBT/kg dw. TotBT at three shipyards selected for site assessment by EPA ranged from 24 to 68,613 microg/kg dw. Sediment and tissue butyltins have been decreasing in all but one area of the estuary.     

Low hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity and minor alterations in retinoid and thyroid hormone levels in flounder (Platichthys flesus) exposed to the polychlorinated biphenyl (PCB) mixture, Clophen A50

The effect of the polychlorinated biphenyl (PCB) mixture Clophen A50 on hepatic cytochrome P4501A1 dependent EROD (7-ethoxyresorufin-O-deethylase) activity, plasma thyroid hormone levels and plasma, kidney and liver retinoid concentrations of the euryhaline flatfish flounder (Platichthys flesus) was determined 2 and 10 days after i.p. (intraperitoneal) injection with 20, 100 and 500 mg Clophen A50/kg body weight. No effect of Clophen A50 on total cytochrome P450 content in flounder liver was observed at both time points. A six-fold, dose-dependent, significant increase in EROD activity was found at exposure day 10 in flounder receiving 100 or 500 mg Clophen A50/kg body weight. Plasma retinol concentrations were not altered at both time points after Clophen A50 administration, whereas renal retinol levels showed a minor dose-related increase at day 2 and day 10 of exposure. Significant alterations in hepatic retinoid concentrations were observed, which were not dependent on the dose of PCB administered. In addition Clophen A50 administration did not result in a dose-related alteration of total T4 concentrations in plasma. Total T3 concentrations in plasma were only significantly increased at day 2 after exposure, whereas free T4 concentrations were increased at both time points after Clophen A50 administration. These data indicate that with regard to the parameters investigated and in contrast to other fish species studied, the flounder is not a sensitive species to PCB exposure.     

Biochemical responses in armored catfish (Pterygoplichthys anisitsi) after short-term exposure to diesel oil,pure biodiesel and biodiesel blends

Biodiesel fuel is gradually replacing petroleum-based diesel oil use. Despite the biodiesel being considered friendlier to the environment, little is known about its effects in aquatic organisms. In this work we evaluated whether biodiesel exposure can affect oxidative stress parameters and biotransformation enzymes in armored catfish (Pterygoplichthys anisitsi, Loricariidae), a South American endemic species. Thus, fish were exposed for 2 and 7 d to 0.01 mL L⁻¹ and 0.1 mL L⁻¹ of pure diesel, pure biodiesel (B100) and blends of diesel with 5% (B5) and 20% (B20) biodiesel. Lipid peroxidation (malondialdehyde) levels and the activities of the enzymes glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase were measured in liver and gills. Also, DNA damage (8-oxo-7, 8-dihydro-2′-deoxyguanosine) levels in gills and 7-ethoxyresorufin-O-deethylase activity in liver were assessed. Pure diesel, B5 and B20 blends changed most of the enzymes tested and in some cases, B5 and B20 induced a higher enzyme activity than pure diesel. Antioxidant system activation in P. anisitsi was effective to counteract reactive oxygen species effects, since DNA damage and lipid peroxidation levels were maintained at basal levels after all treatments. However, fish gills exposed to B20 and B100 presented increased lipid peroxidation. Despite biodiesel being more biodegradable fuel that emits less greenhouse gases, the increased lipid peroxidation showed that biofuel and its blends also represent hazards to aquatic biota.     

Seabird feathers as monitors of the levels and persistence of heavy metal pollution after the Prestige oil spill

We measured heavy metal concentrations in yellow-legged gulls (n = 196) and European shags (n = 189) in order to assess the temporal pattern of contaminant exposure following the Prestige oil spill in November 2002. We analysed Pb, Cu, Zn, Cr, Ni and V levels in chick feathers sampled at four colonies during seven post-spill years (2003-2009), and compared results with pre-spill levels obtained from feathers of juvenile shag corpses (grown in spring/summer 2002). Following the Prestige wreck, Cu (4.3-10 μg g⁻¹) and Pb concentrations (1.0-1.4 μg g⁻¹) were, respectively, between two and five times higher than pre-spill levels (1.5-3.6 and 0.1-0.4 μg g⁻¹), but returned to previous background concentrations after three years. Our study highlights the suitability of chick feathers of seabirds for assessing the impact of oil spills on heavy metal contamination, and provides the best evidence to date on the persistence of oil pollution after the Prestige incident.     

Frequency of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from the Douro River estuary,Portugal

Liver lesions in wild fish have been associated with xenobiotic exposure. Facing reports of pollution in the Douro River estuary (north of Portugal), we have been making field surveys using fishes and targeting histopathological biomarkers of exposure and effect. Herein, we intended to better characterize and report the rate of one poorly understood lesion—hepatocellular fibrillar inclusions (HFI)—found in European flounder (Platichthys flesus). With this report, we aimed to establish sound baseline data that could be viewed as a starting point for future biomonitoring, while offering the world's second only pool of field data on such a liver toxicopathic lesion, which could be compared with data available from the UK estuaries. Sampling was done in the Douro River estuary over 1 year. A total of 72 animals were fished with nets, in spring–summer (SS) and autumn–winter (AW) campaigns. Livers were processed for histopathology and both routine and special staining procedures (alcian blue, periodic acid Schiff (PAS), tetrazonium coupling reaction). Immunohistochemistry targeted AE1/AE3 (pan cytokeratins). The severity of the HFI extent was graded using a system with four levels, varying from 0 (absence of HFI) to 3 (high relative density of cells with HFI). Cells (isolated/groups) with HFI appeared in 35 % or more of the fish, in the total samples of each season, and over 40 % in more homogeneous sub-samples. There were no significant differences when comparing samples versus sub-samples or SS versus AW. When merging the data sets from the two seasons, the frequency of fish with HFI was ≈36 % for the total sample and ≈49 % for the sub-sample. The extreme group (biggest and smallest fish) revealed a HFI frequency of only 16 %, which differed significantly from the total and sub-sampled groups. Immunostaining and PAS were negative for the HFI, and alcian blue could, at times, faintly stain the inclusions. These were positive with the tetrazonium reaction. We showed the presence of HFI in European flounder from the Douro River estuary, proving that they are essentially protein in nature, that no seasonal changes existed in the HFI frequency, and that it was rarer in the smallest and biggest fish groups. Within the ranges of weight/size of our total sample, we estimate that the frequency of HFI in the local flounder is ≈35 %. That rate stands as a baseline value for future assessments, namely for biomonitoring purposes targeting correlations with the estuary pollution status.     

Decontamination trends in the aquacultured fish gilthead seabream (Sparus aurata) after feeding long-term a PCDD/F spiked feed

Aquacultured fish gilthead seabream (Sparusaurata), previously exposed to low levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) trough the diet for a 13 months period, were fed on a clean feed for another 22 months. Gilthead seabream is a protandrous hermaphrodite species and this “decontamination” period coincided with the stages of sex differentiation, maturity and reproduction of the fish. PCDD/F levels in the fish tissues (i.e. muscle, liver, perivisceral fat and gonads), expressed in pg WHO-TEQ g⁻¹ fresh weight, showed a general decreasing trend during the “decontamination” period. However, this general trend varied among tissues and was also dependent on sex and lipid contents. Toxicological effects affecting fish behaviour and hepatic marker responses were also evaluated. The results pointed out that exposure to PCDD/Fs did not have an impact on fish development and reproduction, since the proportion of sexes found after the sex reversal process was within the normal range described for this species. In addition, long-term exposure to low PCDD/F levels did not significantly affect the response of most of the biochemical markers considered. On the contrary, some of them (e.g. EROD activity) showed variations in their responses during the sex differentiation process and onwards. Finally, the hepatic AhR mRNA levels increased during dioxin exposure but they returned to values typical for non-exposed fish after the “decontamination” period.     

Organotins in North Sea brown shrimp (Crangon crangon L.) after implementation of the TBT ban

The organotin (OT) compounds tributyltin (TBT) and triphenyltin (TPhT) are potent biocides that have been used ubiquitously in antifouling paints and pesticides since the mid-1970s. These biocides are extremely toxic to marine life, particularly marine gastropod populations. The European Union therefore took measures to reduce the use of TBT-based antifouling paints on ships and ultimately banned these paints in 2003. Despite sufficient data on OT concentrations in marine gastropods, data are scarce for other species such as the North Sea brown shrimp (Crangon crangon), a dominant crustacean species in North Sea inshore benthic communities. The present study provides the first spatial overview of OT concentrations in North Sea brown shrimp. We have compared these data with historical concentrations in shrimp as well as with sediment concentrations. We have also addressed the effect on the shrimp stock and any human health risks associated with the OT concentrations found. TBT and TPhT in shrimp tail muscle ranged from 4 to 124 and from 1 to 24 μg kg⁻¹ DW, respectively. High levels are accumulated in estuarine areas and are clearly related with sediment concentrations (biota-sediment accumulation factor ∼10). Levels have decreased approximately 10-fold since the ban took effect, coinciding with a recovery of the shrimp stock after 30 years of gradual regression. Furthermore, the OT levels found in brown shrimp no longer present a human health risk.     

Biological response of high-back crucian carp (Carassius auratus) during different life stages to wastewater treatment plant effluent

This study presents the adverse effects of endocrine disrupting chemicals (EDCs) in effluent of wastewater treatment plants (WwTPs) on fish health. A study of chronic exposure to WwTPs effluent for 10 months was undertaken in high-back crucian carp (Carassius auratus) during different life stages, covering early-life-stage (ELS), prespawning period, and postspawning period. Condition factor (CF), gonadosomatic index (GSI), hepatosomatic index (HSI), and plasma vitellogenin (VTG) levels were employed as indicators to assess biological effects of effluent on this gynogenesis species. Meanwhile, some high-back crucian carp were caged in Demonstration Base of Biological Purification for Filter-feeding Fish (hereinafter, Demonstration Base), as WwTPs effluent exposure controls. In the meantime, a depuration study was carried out to determine whether or not the estrogenic effects caused by effluent exposure could be reduced after moving fish into EDCs-free water. CF, HSI, GSI, and plasma Vtg levels of high-back crucian carp caged in Demonstration Base were generally in accordance with seasonal change. Effluent exposure inhibited gonadal growth, reducing GSI in ELS while increasing it around spawning, sharpened liver burdens, increasing HSI, and induced abnormal Vtg expression in juvenile high-back crucian carp, augmenting Vtg concentrations in plasma. Around spawning period, Vtg in high-back crucian carp were mainly induced by endogenous estrogens, and EDCs in effluent had less influence on them. Staying in EDCs-free water for 30 days made high-back crucian carp recover from effects of previous effluent exposure, relieving inhibition of gonadal development and hypertrophy of liver as well as reducing Vtg induced by EDCs in effluent. The results revealed that high-back crucian carp in ELS are more sensitive to WwTPs effluent exposure. Additionally, the depuration study showed a clearance of the estrogenic effects caused by effluent.     

Uptake of 17β-estradiol and biomarker responses in brown trout (Salmo trutta) exposed to pulses

In streams, chemicals such as 17β-estradiol (E2) are likely to occur in pulses. We investigated uptake and biomarker responses in juvenile brown trout (Salmo trutta) of 3- or 6-h pulses of concentrations up to 370 ng E2 L⁻¹. Uptake by the fish was estimated from disappearance of E2 from tank water. A single 6-h pulse of 370 ng E2 L⁻¹ increased the plasma vitellogenin concentration, liver Erα- and vitellogenin-mRNA. Exposure to 150-160 ng E2 L⁻¹ for 6 h increased vitellogenin in one experiment but not in another. Two 6-h pulses had a larger effect one pulse. Brown trout in the size range 24-74 g took up E2 linearly with time and exposure concentration with a concentration ratio rate of 20.2 h⁻¹. In conclusion, the threshold for induction of estrogenic effects in juvenile brown trout at short term pulse exposure appears to be in the range 150-200 ng E2 L⁻¹.     

Metallothioneins failed to reflect mercury external levels of exposure and bioaccumulation in marine fish--considerations on tissue and species specific responses

The suitability of metallothioneins (MT) in fish as biomarker of exposure to mercury has been questioned. Therefore, this study aimed at investigating the relationship between external levels of exposure, mercury accumulation and MT content, assessing species and tissue specificities. Two ecologically different fish species - Dicentrarchus labrax and Liza aurata - were surveyed in an estuary historically affected by mercury discharges. Total mercury (T-Hg) and MT content were determined in gills, blood, liver, kidney, muscle and brain. All tissues reflected differences in T-Hg accumulation in both species, although D. labrax accumulated higher levels. Regarding MT, D. labrax revealed a depletion in brain MT content and an incapacity to induce MT synthesis in all the other tissues, whereas L. aurata showed the ability to increase MT in liver and muscle. Tissue-specificities were exhibited in the MT inducing potential and in the susceptibility to MT decrease. L. aurata results presented muscle as the most responsive tissue. None of the investigated tissues displayed significant correlations between T-Hg and MT levels. Overall, the applicability of MT content in fish tissues as biomarker of exposure to mercury was uncertain, reporting limitations in reflecting the metal exposure levels and the subsequent accumulation extent.     

Bioaccumulation of butyltins and liver damage in the demersal fish Cathorops spixii (Siluriformes,Ariidae)

The toxicity of butyltin compounds (BTs), mainly tributyltin (TBT), has been reported in different organisms. However, such an analysis in fish after field exposure with reference to the related biomarkers has not been commonly observed in the literature. This study presents the uptake of BTs in the liver of a neotropical marine catfish Cathorops spixii in Paranagua Bay, an important estuarine system located in southern Brazil. Two different areas, close to and distant from the harbor, were used for chemical analysis evaluation of hepatotoxicity through genetic, enzymatic, and histopathological biomarkers. The presence of polycyclic aromatic hydrocarbons in bile was also considered as a biomarker. The results showed a significant relationship between TBT levels and the inhibition of biotransformation enzymes and high occurrence of melanomacrophages in fish collected close to the harbor site. These effects were linked to the absence of TBT metabolites in the liver. In the second site, the presence of DBT was associated with an increase in EROD and GST activity. The larger amount of DNA damage as well as the highest oxidative stress was noted in fish from the less TBT-polluted area, where DBT and bile PAHs occurred. These findings showed different impact levels due to or increased by the chronic exposure of biota to BTs.     

Mercury concentrations in wild mink (Mustela vison) and river otters (Lontra canadensis) collected from eastern and Atlantic Canada: relationship to age and parasitism

Total mercury (Hg) concentrations were measured in the fur, brain and liver of wild mink (Mustela vison) and river otters (Lontra canadensis) collected from eastern and Atlantic Canada. Total Hg concentrations in fur were strongly correlated with levels in the brain and liver. There was no difference in tissue concentrations between male and female mink; however, female otters had significantly higher fur, brain and liver Hg levels than males. Similarly, there was not a significant relationship between Hg concentration and age of mink, whereas in otters, Hg concentrations in all three tissues decreased significantly with age. In both species, only a very small percentage of the variability in Hg concentration was explained by age. After adjusting the data for site-to-site differences in Hg levels, Hg concentrations in the fur of mink infected by the parasite, Dioctophyma renale, were found to be significantly higher than Hg levels in uninfected mink.