João Carrola

Histopathological changes in liver and gill epithelium of Nile tilapia, Oreochromis niloticus, exposed to waterborne copper

Nile tilapia, Oreochromis niloticus, of both sexes were reared in freshwater and exposed to 0.5, 1.0 and 2.5mg L-1 of waterborne copper for a period of 21 days. Liver and gill samples were collected after 21 days of exposure to copper and lesions were analyzed by light microscopy. The main histopathological changes observed in gills exposed to the highest concentration were edema, lifting of lamellar epithelia and an intense vasodilatation of the lamellar vascular axis. Although less frequent, lamellar fusion caused by the filamentar epithelium proliferation and some lamellar aneurisms were also found. The liver of control group exhibited a quite normal architecture, while the fish exposed to copper showed vacuolation and necrosis. These hepatic alterations were more evident in fish exposed to 1.0 and 2.5mg L-1 copper concentrations. The number of hepatocytes nucleus per mm2 of hepatic tissue decreased with the increase of copper concentration. In contrast, the hepatic somatic index was high in fish exposed at 2.5mg L-1 of copper. In short, this work advance new knowledge as influence of copper in the gill and liver histology of O. niloticus and demonstrated that their effects could be observed at different concentrations.

Liver Histopathology in Brown Trout ( Salmo trutta f. fario) from the Tinhela River, Subjected to Mine Drainage from the Abandoned Jales Mine (Portugal)

The objective of this study was to compare the occurrence of toxicopathic liver lesions in brown trout (Salmo trutta fario) from Tinhela River near the Jales Mine, both before implementation (2002) and after completion of the governmental mitigation program (2006). Fish were caught in April 2002 and May 2006, using an electrofishing system at four sites: S0, reference station; S1, S2 and S3 as contaminated stations. In 2002, the hepatosomatic index (HSI) was significantly higher for trout captured at the contaminated sites S2 and S3 than in S0. After the rehabilitation program, the HSI of fish sampled at the contaminated sites did not differ from the reference group. The liver of trout caught at S0 exhibited the normal parenchymal and stromal architecture described for the species and there were no pathological abnormalities. In contrast, fish sampled at S3 and S2 sites had diverse toxicopathic alterations. Specifically, livers from the two contaminated sites showed bile duct hyperplasia, often with mild epithelial dysplasia and fibrotic adventitial sleeve, foci of smaller and more basophilic hepatocytes and foci of hepatocellular necrosis; the latter conditions were frequently associated. Compared with the reference animals, increased hepatocellular vacuolization was found in livers from the polluted sites. Histopathological examination revealed differences among sampling sites in the severity and diversity of hepatic lesions clearly related to the proximity of the tailings. No pathological alterations were observed in the livers of brown trout caught in the same four areas of the Tinhela River after the mitigation program in 2006. In conclusion, our results supported that drainage from the abandoned Jales Mine had deleterious toxicological effects in brown trout. Our data suggested that the governmental mitigation program may have reduced the impact of Jales tailings.

Gill histopathological alterations in Nile tilapia, Oreochromis niloticus exposed to treated sewage Water

Adult Nile tilapia, Oreochromis niloticus, of both sexes were exposed in wastewater from a sewage treatment plant for a period of 4 days. Gill samples were collected after 24, 48, 72 and 96 h and histopathological changes were analyzed by light and scanning electronic microscopy. Gill epithelium of control O. niloticus (freshwater group) was similar to that of other teleosts, while histopathological lesions were observed in exposed fishes. The main histopathological changes were edema, lifting of lamellar and filamentar epithelia and lamellar fusion. Cell proliferation with consequent thickening of the filament epithelium was also found in fishes exposed to the treated sewage water. The severity of the lesions increased with the time of exposure, namely the hyperplasia of the epithelial cells with proliferation of filamentar epithelium and fusion of lamellae observed at 96 h. Additionally, several histopathological results obtained by light microscopy were confirmed through scanning microscopy.

Mullet and gudgeon liver histopathology and macroinvertebrate indexes and metrics upstream and downstream from a wastewater treatment plant (Febros River--Portugal).

The increased pollution in ecosystems reinforces the importance of both chemical monitoring and biological monitoring of streams and rivers, as an effective water quality-based approach to assess aquatic ecosystem health. In this study, gudgeon (Gobio gobio) and mullet (Mugilcephalus) liver histopathology (biomarker) and some macroinvertebrate community indexes and metrics (bioindicator) were used to evaluate the effect of the wastewater treatment plant (WWTP) of Febros (Avintes) in Febros River water quality and ecosystem health. Regarding macroinvertebrate communities, the Belgian Biotic Index (BBI) and Iberian Biological Monitoring Working Party (IBMWP) indexes suggested that Febros water was slightly polluted, even though the worst situation was found downstream the WWTP discharge. Concerning community metrics, upstream percent of individuals in five numerically dominant taxa (80%) was slightly more superior than the downstream (78%). The presence of intolerant or sensible individuals, determined by percent of Ephemeroptera, Plecoptera, and Trichoptera individuals and number of Ephemeroptera, Plecoptera, and Trichoptera families metrics, was higher upstream WWTP, reflecting a better water quality. The histopathology shows the presence of hepatic lesions in gudgeon and mullet. The statistical analysis of the lesion gradation showed that only necrosis was significantly higher in gudgeon captured downstream the WWTP, while differences were not observed for mullet. The multivariate analysis of data confirmed the existence of differences in hepatic lesions between gudgeon and mullet and between sampling sites. Regarding macroinvertebrate community, this analysis showed that the organic contamination reflected by the BBI and IBMWP indexes values was a determinant factor in the spatial distribution of macroinvertebrates. This work showed that the study of different biological organization levels can be used for a better assessment of ecosystem ecological integrity and can be used as a tool to reveal anthropogenic activity effects in macroinvertebrate diversity and in fish liver pathology from Febros River.

In vitro exposure of Nile tilapia (Oreochromis niloticus) testis to estrogenic endocrine disrupting chemicals: mRNA expression of genes encoding steroidogenic enzymes

Many environmental pollutants can exert adverse effects on exposed organisms, including fish, leading to disruption of the endocrine system. Enzymes involved in the sex steroid biosynthesis are potential targets for the toxic action of pollutants. In this context, we investigated the hypothesis that selected estrogenic chemicals—the pharmaceutical estrogen ethinylestradiol (EE2), the phytoestrogen genistein (GEN), and the industrial compound bisphenol A (BPA)—may cause endocrine disruption by directly disturbing steps of fish steroidogenic pathways. We studied the mRNA expression of eight selected genes encoding steroidogenic enzymes (11β-HSD2, 20β-HSD, 3β-HSD1, 17β-HSD1, 17β-HSD8, 17β-HSD12, CYP19a, CYP19b) by quantitative real-time PCR. Testis slices from adult specimens of the model fish Nile tilapia were exposed in vitro for 3 and 8 h either to individual or to mixture solutions of EE2 (100 ng/L), GEN (200 ng/L), and BPA (10 µg/L); all at the peak concentrations observed in the Douro River estuary (Portugal). Our data revealed that only the mixture of the tested chemicals directly induced the expression of 11β-HSD2, 17β-HSD1, and 17β-HSD12, after 8 h, whereas no effect was seen for chemicals tested individually. The gene expression pattern agrees with the concept of dose addition for environmental mixtures, and for the first time an interference of estrogenic EDCs is reported for 17β-HSD1 and 17β-HSD12.

A multiple index integrating different levels of organization

Many methods in freshwater biomonitoring tend to be restricted to a few levels of biological organization, limiting the potential spectrum of measurable of cause-effect responses to different anthropogenic impacts. We combined distinct organisational levels, covering biological biomarkers (histopathological and biochemical reactions in liver and fish gills), community based bioindicators (fish guilds, invertebrate metrics/traits and chironomid pupal exuviae) and ecosystem functional indicators (decomposition rates) to assess ecological status at designated Water Framework Directive monitoring sites, covering a gradient of human impact across several rivers in northern Portugal. We used Random Forest to rank the variables that contributed more significantly to successfully predict the different classes of ecological status and also to provide specific cut levels to discriminate each WFD class based on reference condition. A total of 59 Biological Quality Elements and functional indicators were determined using this procedure and subsequently applied to develop the integrated Multiple Ecological Level Index (MELI Index), a potentially powerful bioassessment tool.

Antimicrobial-resistant Escherichia coli and Enterococcus spp. isolated from Miranda donkey (Equus asinus): an old problem from a new source with a different approach.

Purpose. The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals. Methodology. In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic‐resistant and virulent factors was analysed by PCR. Results. A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3′)‐IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole‐resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %). Conclusion. To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichia coli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic‐resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.