Integrated Approach for Synthetic Cathinone Drug Prioritization and Risk Assessment: In Silico Approach and Sub-Chronic Studies in Daphnia magna and Tetrahymena thermophila.

Synthetic cathinones (SC) are drugs of abuse that have been reported in wastewaters and rivers raising concern about potential hazards to non-target organisms. In this work, 44 SC were selected for in silico studies, and a group of five emerging SC was prioritized for further in vivo ecotoxicity studies: buphedrone (BPD), 3,4-dimethylmethcathinone (3,4-DMMC), butylone (BTL), 3-methylmethcathinone (3-MMC), and 3,4-methylenedioxypyrovalerone (MDPV). In vivo short-term exposures were performed with the protozoan Tetrahymena thermophila (28 h growth inhibition assay) and the microcrustacean Daphnia magna by checking different indicators of toxicity across life stage (8 days sublethal assay at 10.00 µg L-1). The in silico approaches predicted a higher toxic potential of MDPV and lower toxicity of BTL to the model organisms (green algae, protozoan, daphnia, and fish), regarding the selected SC for the in vivo experiments. The in vivo assays showed protozoan growth inhibition with MDPV > BPD > 3,4-DMMC, whereas no effects were observed for BTL and stimulation of growth was observed for 3-MMC. For daphnia, the responses were dependent on the substance and life stage. Briefly, all five SC interfered with the morphophysiological parameters of juveniles and/or adults. Changes in swimming behavior were observed for BPD and 3,4-DMMC, and reproductive parameters were affected by MDPV. Oxidative stress and changes in enzymatic activities were noted except for 3-MMC. Overall, the in silico data agreed with the in vivo protozoan experiments except for 3-MMC, whereas daphnia in vivo experiments showed that at sublethal concentrations, all selected SC interfered with different endpoints. This study shows the importance to assess SC ecotoxicity as it can distress aquatic species and interfere with food web ecology and ecosystem balance.

Toxicity of the 3,4-Methylenedioxymethamphetamine and Its Enantiomers to Daphnia magna after Isolation by Semipreparative Chromatography.

MDMA (3,4-methylenedioxymethamphetamine) is a chiral psychoactive recreational drug sold in illicit markets as racemate. Studies on the impact of MDMA on aquatic organisms are scarce. While enantioselectivity in toxicity in animals and humans has been reported, none is reported on aquatic organisms. This study aimed to investigate the ecotoxicological effects of MDMA and its enantiomers in Daphnia magna. For that, enantiomers (enantiomeric purity > 97%) were separated by liquid chromatography using a homemade semipreparative chiral column. Daphnids were exposed to three concentrations of (R,S)-MDMA (0.1, 1.0 and 10.0 µg L-1) and two concentrations of (R)- and (S)-enantiomers (0.1 and 1.0 µg L-1) over the course of 8 days. Morphophysiological responses were dependent on the substance form and daphnia development stage, and they were overall not affected by the (R)-enantiomer. Changes in swimming behaviour were observed for both the racemate and its enantiomers, but enantioselective effects were not observed. Reproductive or biochemical changes were not observed for enantiomers whereas a significant decrease in acetylcholinesterase and catalase activity was noted at the highest concentration of (R,S)-MDMA (10 µg L-1). Overall, this study showed that sub-chronic exposure to MDMA racemate and its enantiomers can interfere with morphophysiological and swimming behaviour of D. magna. In general, the (R)-enantiomer demonstrated less toxicity than the (S)-enantiomer.

Acoustic barriers as an acoustic deterrent for native potamodromous migratory fish species.

This study focused on the use of sound playbacks as acoustic deterrents to direct native potamodromous migratory species away from all kind of traps. The effects of two acoustic treatments, a repeated sine sweep up to 2 kHz (sweep-up stimulus) and an intermittent 140 Hz tone, were tested in three fish species native to Iberia: Salmo trutta, Pseudochondrostoma duriense and Luciobarbus bocagei. In contrast with S. trutta, the endemic cyprinids P. duriense and L. bocagei exhibited a strong repulse reaction to the frequency sweep-up sound. The 140 Hz stimulus did not seem to alter significantly the behaviour of any of the studied species. These results highlight the potential of acoustic stimuli as fish behavioural barriers and their application to in situ conservation measures of native Iberian fish populations, to protect them from hydropower dams. In addition, this study shows that acoustic deterrents can be used selectively on target species.

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.

Alkaline Comet Assay to Assess Genotoxicity in Zebrafish Larvae.

The zebrafish (Danio rerio) is a model organism widely used in several research fields due to its characteristics and numerous advantages, such as optical embryo transparency, fully sequenced genome, orthologous genes to humans, small size, high reproductive rate, easy gene editing and relatively low costs. Thus, a number of protocols have been developed that allow the use of this vertebrate model for toxic effect evaluation at various biological levels, including genotoxicity, using the comet assay technique.The comet assay or single-cell gel electrophoresis is a popular and sensitive method to study DNA damage in cells, which is described in this chapter. Briefly, cells suspended in agarose on a microscope slide are lysed, denatured, electrophoresed, neutralized, and stained to study the migration of DNA strand breaks. As a result, cells with increased DNA damage present a high fluorescence intensity and an increase of comet tail length. For the visual score, comets are classified according to the head integrity, tail intensity, and tail length into five classes, namely, class 0 until class 4 (comets with high damage and with almost all the DNA in the tail). These data are used to calculate the Genetic Damage Index (GDI) expressed as Arbitrary Units (AU).

Unveil the toxicity induced on early life stages of zebrafish (Danio rerio) exposed to 3,4-methylenedioxymethamphetamine (MDMA) and its enantiomers.

The increased detection of the recreational drug 3,4-methylenedioxymethamphetamine (MDMA) in aquatic ecosystems, has raised concern worldwide about its possible negative impacts on wildlife. MDMA is produced as racemate but its enantioselective effects on non-target organisms are poorly understood. Therefore, this study aimed to provide a comprehensive study of the toxicity of MDMA and its enantiomers in the early life stages of zebrafish (Danio rerio). Zebrafish embryos (≈3 h post fertilization) were exposed to different concentrations (0.02, 0.2, 2, 20, and 200 µg/L) of (R,S)-MDMA and both pure enantiomers. Both enantiomers induced effects on embryonic development, DNA integrity, and behaviour and enantioselective effects were noted. (S)-MDMA exhibits higher toxic effects on embryonic development level with increased mortality and severity of teratogenic effects, and behavioural abnormalities in acoustic startle-habituation response. (R)-MDMA affected general activity and avoidance behaviour, showing greater inhibitory effects on behavioural activity. Additionally, (R,S)-MDMA induced higher genotoxic effects than the two isolated enantiomers. These results are of concern at populational levels since effects on mortality, development, and behaviour were observed even at environmentally relevant concentrations, which can cause a reduction of larval viability and in the number of individuals in each generation, and an increase in the risk of predation of the organisms. Yet, the bioaccumulation studies showed that MDMA is not accumulated in zebrafish. Therefore, this work demonstrated for the first time the occurrence of MDMA enantiotoxicity in the early life stages of zebrafish, which should be considered in further environmental risk assessments involving fish species or other non-target aquatic organisms.

Toxicity of butylone and its enantiomers to Daphnia magna and its degradation/toxicity potential using advanced oxidation technologies.

Butylone (BTL) is a chiral synthetic cathinone available as a racemate and reported as contaminant in wastewater effluents. However, there are no studies on its impact on ecosystems and possible enantioselectivity in ecotoxicity. This work aimed to evaluate: (i) the possible ecotoxicity of BTL as racemate or its isolated (R)- and (S)- enantiomers using Daphnia magna; and (ii) the efficiency of advanced oxidation technologies (AOTs) in the removal of BTL and reduction of toxic effects caused by wastewaters. Enantiomers of BTL were obtained by liquid chromatography (LC) using a chiral semi-preparative column. Enantiomeric purity of each enantiomer was > 97 %. For toxicity assessment, a 9-day sub-chronic assay was performed with the racemate (at 0.10, 1.0 or 10 µg L-1) or each enantiomer (at 0.10 or 1.0 µg L-1). Changes in morphophysiological, behavioural, biochemical and reproductive endpoints were observed, which were dependent on the form of the substance and life stage of the organism (juvenile or adult). Removal rates of BTL in spiked wastewater (10 µg L-1) treated with different AOTs (ultraviolet, UV; ozonation, O3; and UV/O3) were similar and lower than 29 %. The 48 h D. magna acute toxicity assays demonstrated a reduction in the toxicity of the treated spiked effluents, but no differences were found amongst AOTs treatments. These results warn for the contamination and negative impact of BTL on ecosystems and highlight the need for efficient removal processes.

Effects of acute metaphedrone exposure on the development, behaviour, and DNA integrity of zebrafish (Danio rerio).

The presence of new psychoactive substances (NPS), like metaphedrone (3-MMC), in aquatic environments raises concern about the potential negative effects on ichthyofauna. Therefore, the aim of this study was to evaluate the potential effects of 3-MMC on zebrafish embryonic development, behaviour, and DNA integrity. For that, embryos were exposed during 96 h post-fertilization to 3-MMC (0.1, 1, 10, and 100 µg/L). Overall, an increase in the eye area of zebrafish larvae was observed for the concentrations of 1 µg/L (increase of 24%) and 100 µg/L (increase of 25%) in comparison with the control group. Genetic damage was noted at the highest concentration (100 µg/L) with an increase of DNA damage (increase of 48%) and hyperactivity and disorganised swimming pattern characterised by an increase in speed (increase of 49%), total distance moved (increase of 53%), and absolute turn angle (increase of 48%) of zebrafish larvae. These findings pointed that, at environmental low levels, 3-MMC harmful effects are not expected to occur during critical development life stages of fish.

Enantioselectivity in the Ecotoxicity of Amphetamine Using Daphnia magna as the Aquatic Model Organism: Morphophysiological, Behavioral, Reproductive and Biochemical Parameters.

Amphetamine (AMP) is a chiral psychoactive substance that exhibits enantioselectivity in its pharmacological properties. It has been detected in wastewaters and surface waters and can occur as enantiomeric mixtures, but little is known about its environmental risk and potential enantioselective toxicity to aquatic organisms. Our study aimed to target enantioselectivity in AMP toxicity to the freshwater invertebrate Daphnia magna. Daphnids were subchronically exposed to the racemate (rac-AMP: 0.1, 1.0, and 10 µg/L) and pure enantiomers, (R)-AMP and (S)-AMP (0.1, and 1.0 µg/L, respectively), for 8 days. Morphophysiological, swimming behavior, reproductive and biochemical variables were evaluated during critical life stages (juveniles vs. adults). Some responses were context-dependent and often enantioselective, varying between racemate and enantiomers and across the life stage of the organisms. Overall, rac-AMP stimulated D. magna growth, decreased heart rate and area, affected behavior, and stimulated reproduction. The effect of enantiomers was totally or partially concordant with rac-AMP, except for swimming behavior and reproduction. Enantioselectivity was observed for body size, number of eggs/daphnia, and heart rate (steeper decrease caused by (R)-AMP on day 3). Changes in biochemical parameters were also observed: AMP caused a significant decrease in catalase activity as racemate or pure enantiomers, whereas a decrease in acetylcholinesterase activity was found only for rac-AMP. Evidence for oxidative stress was contradictory, although both enantiomers caused a significant decrease in reactive oxygen species (unlike rac-AMP). Overall, these results show that AMP can interfere in an enantioselective way with aquatic organisms at low concentrations (e.g., 0.1 µg/L), demonstrating the relevance of this kind of study to an accurate environmental risk assessment regarding medium- to long-term exposure to this psychoactive drug. Environ Toxicol Chem 2023;42:1743-1754. © 2023 SETAC.

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 (11ß-HSD2, 20ß-HSD, 3ß-HSD1, 17ß-HSD1, 17ß-HSD8, 17ß-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 11ß-HSD2, 17ß-HSD1, and 17ß-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 17ß-HSD1 and 17ß-HSD12.

Enantioselective Ecotoxicity of Venlafaxine in Aquatic Organisms: Daphnia and Zebrafish.

Venlafaxine is a chiral antidepressant detected in aquatic compartments. It was recently included in the 3rd Watch List from the European Union. The present study aimed to investigate venlafaxine toxicity effects, targeting possible enantioselective effects, using two aquatic organisms, daphnia (Daphnia magna) and zebrafish (Danio rerio). Specimens were exposed to both racemate, (R,S)-venlafaxine (VEN), and to pure enantiomers. Acute assays with daphnia showed that up to 50 000 µg/L of the (R,S)-VEN induced no toxicity. Organisms were also exposed to sublethal concentrations (25-400 µg/L) of (R,S)-, (R)- and (S)-VEN, for 21 days. No significant effects on mortality, age at first reproduction, and size of the first clutch were observed. However, a decrease in fecundity was observed for both enantiomers at the highest concentration. Regarding zebrafish, the effects of venlafaxine on mortality, embryo development, behavior, biochemistry, and melanin pigmentation were investigated after 96 h of exposure to the range of 0.3-3000 µg/L. (R)-VEN significantly increased the percentage of malformations in comparison with (S)-VEN. Behavior was also enantiomer dependent, with a decrease in the total distance moved and an increase in avoidance behavior observed in organisms exposed to (R)-VEN. Despite the biochemical variations, no changes in redox homeostasis were observed. (R)-VEN also led to an increase in zebrafish pigmentation. The different susceptibility to venlafaxine and enantioselective effects were observed in zebrafish. Our results suggest that at environmental levels (R,S)-VEN and pure enantiomers are not expected to induce harmful effects in both organisms, but (R)-VEN increased malformations in zebrafish larvae, even at reported environmental levels. These results highlight the importance of including enantioselective studies for an accurate risk assessment of chiral pollutants. Environ Toxicol Chem 2022;41:1851-1864. © 2022 SETAC.

Ketamine and Norketamine: Enantioresolution and Enantioselective Aquatic Ecotoxicity Studies.

Ketamine is a chiral drug used for various clinical purposes but often misused. It is metabolized to norketamine, an active chiral metabolite. Both substances have been detected in environmental matrices, but studies about their enantioselective toxic effects are scarce. In the present study, the enantiomers of ketamine and norketamine were separated by a semipreparative enantioselective liquid chromatography method, and their toxicity was investigated in different aquatic organisms. The enantioseparation was achieved using a homemade semipreparative chiral column. Optimized conditions allowed the recovery of compounds with enantiomeric purity higher than 99%, except for (R)-ketamine (97%). The absolute configuration of the enantiomers was achieved by experimental electronic circular dichroism (ECD). The ecotoxicity assays were performed with the microcrustacean Daphnia magna and the protozoan Tetrahymena thermophila using Toxkit MicroBioTests. Different concentrations were tested (0.1-10 000 µg/L) to include environmental levels (~0.5-~100 µg/L), for racemates (R,S) and the isolated enantiomers (R or S) of ketamine and norketamine. No toxicity was observed in either organism at environmental levels. However, at greater concentrations, (R,S)-ketamine presented higher mortality for D. magna compared with its metabolite (R,S)-norketamine (85 and 20%, respectively), and the (S)-ketamine enantiomer showed higher toxicity than the (R)-ketamine enantiomer. In addition, (S)-ketamine also presented higher growth inhibition than (R)-ketamine for T. thermophila at the highest concentrations (5000 and 10 000 µg/L). Contrary to D. magna, growth inhibition was observed for both enantiomers of norketamine and in the same magnitude order of the (S)-ketamine enantiomer. The results showed that the 2 organisms had different susceptibilities to norketamine and that the toxicity of ketamine at high concentrations is enantioselective for both organisms. Environ Toxicol Chem 2022;41:569-579. © 2020 SETAC.

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 (Mugil cephalus) 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.

Uncommon hepatic macrophagic foamy-cell nodules in Iberian barbel (Luciobarbus bocagei Steindachner, 1864) from the Vizela River (Portugal).

The main intent of this work (after the by chance finding, in archived histological slides) is to characterize one previously non-described liver lesion of the Iberian barbel from the Vizela River (Portugal). This ran through a textile and dyeing industrial region. The lesion type was made of groups of foamy cells (presumptive macrophages), which appear either as a "smaller non-nodular form," without a connective tissue capsule and displaying an irregular profile, or as a "bigger nodular form," presenting a thin capsule and a circular profile. The nodular forms could appear multi-layered, resembling "cross-sectioned onions". The lesions number, dimension, and structural complexity were greater in bigger fish, appearing only after a history of poor water quality. In extreme cases, the lesions slightly protruded the liver surface. Special histological staining proved the connective tissue nature of the capsule (and its eventual septa), the presence of proteins, glycoproteins, lipofuscin, melanin, iron (putative hemosiderin), and copper, in a variable number of foamy cells within the lesions. At times, degenerating hepatocytes appeared at the border of the lesions. It is proposed that this lesion type incorporates both macrophages and degenerating hepatocytes, looking as one (unpublished) form of a macrophage aggregate. The term "foamy-cell nodules" was advanced for this abnormality. The lesion could have been induced by pollution, because: no parasites were ever associated with the lesion; there was co-existence of the lesion with a water quality status scored as "bad"; such lesion neither existed in fish sampled after mitigation and remediation measures nor in reference fish.

The Impact of Land Use Transformations on Zooplankton Communities in a Small Mountain River (The Corgo River, Northern Portugal).

Transformation of the river catchment and the river bed cause significant changes in the functioning of river ecosystems. The main effects of anthropogenic transformations are hydrological changes, such as lower current velocity or an increase of nutrient content, and higher temperature. Zooplankton reacts rapidly to the new environmental conditions in rivers, increasing its richness and abundance. We tried to answer two questions: what type of catchment use has a greater influence on the zooplankton communities in a river and how do dam impoundments influence the zooplankton communities downstream? The study was conducted in the Corgo river (drainage of the Douro river, Northern Portugal) at 17 sampling sites in the lotic, free-flowing sections. Crustaceans present in the Corgo can attain relatively high densities in the rural section, which offers them better trophic conditions. The urban catchment use and the presence of dams have a greater impact on the rotifer density and the increase of zooplankton density downstream. The results of this study confirm that zooplankton properties allow for the evaluation of the degree of river-bed transformation.

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 Escherichiacoli 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.

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.

Frequency of micronuclei and of other nuclear abnormalities in erythrocytes of the grey mullet from the Mondego, Douro and Ave estuaries--Portugal.

Fish are bioindicators of water pollution, and an increased rate of their erythrocyte nuclear morphological abnormalities (ENMAs)-and particularly of erythrocyte micronuclei (EMN)-is used as a genotoxicity biomarker. Despite the potential value of ENMAs and MN, there is scarce information about fish captured in Iberian estuaries. This is the case of the Portuguese estuaries of the Mondego, Douro and Ave, suffering from different levels of environmental stress and where chemical surveys have been disclosing significant amounts of certain pollutants. So, the aim of this study was to evaluate genotoxicants impacts and infer about the exposure at those ecosystems, using the grey mullet (Mugil cephalus) as bioindicator and considering the type and frequency of nuclear abnormalities of erythrocytes as proxies of genotoxicity. Sampling of mullets was done throughout the year in the important Mondego, Douro and Ave River estuaries (centre and north-western Portugal). The fish (total n = 242) were caught in campaigns made in spring-summer and autumn-winter, using nets or fishing rods. The sampled mullets were comparable between locations in terms of the basic biometric parameters. Blood smears were stained with Diff-Quik to assess the frequencies of six types of ENMAs and MN (given per 1,000 erythrocytes). Some basic water physicochemical parameters were recorded to search for fluctuations matching the ENMAs. Overall, the most frequent nucleus abnormality was the polymorphic type, sequentially followed by the blebbed/lobed/notched, segmented, kidney shaped, vacuolated, MN and binucleated. The total average frequency of the ENMAs ranged from 73 ‰ in the Mondego to 108 ‰ in the Ave. The polymorphic type was typically ≥50 % of the total ENMAs, averaging about 51 ‰, when considering all three estuaries. The most serious lesion-the MN-in fish from Mondego and Douro had a similar frequency (≈0.38 ‰), which was significantly lower than that in the Ave (0.75 ‰). No significant seasonal differences existed as to the MN rates and seasonal differences existed almost only in the Douro, with the higher values in AW. In general, the pattern of ENMAs frequencies was unrelated with the water physicochemical parameters. Considering the data for both the total ENMAs and for each specific abnormality, and bearing in mind that values of MN in fish erythrocytes >0.3 ‰ usually reflect pollution by genotoxicants, it is suggested that mullets were likely being chronically exposed to such compounds, even in the allegedly less polluted ecosystem (Mondego). Moreover, data supported the following pollution exposure gradient: Mondego < Douro < Ave. The scenario and inferences nicely agree with the published data from chemical monitoring.

Effects of nebivolol on liver fibrosis induced by bile duct ligation in Wistar rats.

AIM: To study the effect of nebivolol on liver fibrosis induced by common bile duct ligation (BDL) in rats. MATERIALS AND METHODS: After BDL, Wistar rats were divided into three groups (n=24): SO, sham-operated animals; BDL, BDL rats without treatment; BDL+N, BDL rats treated with nebivolol for five weeks. Alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, total bilirubin and albumin levels were assessed. Liver samples collected were stained with hematoxylin-eosin, Masson's trichrom and reticulin. RESULTS: Mortality reached 37.5% in the BDL group, whereas no deaths were observed in the SO and BDL+N groups. The BDL group showed hepatic damage as evidenced by elevation in serum biochemical parameters and fibrosis scores. These pathophysiological changes were attenuated in the BDL+N group. However, there was no significant difference between these two groups. CONCLUSION: Nebivolol improved the survival rate of animals with BDL, but was unable to significantly improve liver function or reduce liver fibrosis.

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.

Tilápias adultas, Oreochromis niloticus, de ambos os sexos foram expostas em águas residuais de uma estação de tratamento de esgoto durante 4 dias. Amostras de brânquia foram recolhidas após 24, 48, 72 e 96 h e as alterações histopatológicas foram analisadas por microscopia óptica e eletrônica de varredura. O epitélio da brânquia do grupo controle apresentou uma morfologia similar à de outros peixes teleosteos, enquanto foram observadas lesões nos peixes expostos. As principais alterações histopatológicas foram edema, destacamento dos epitélios lamelar e filamentar e fusão lamelar. Os peixes expostos às águas residuais mostraram também proliferação celular com consequente aumento da espessura do filamento branquial. A severidade das lesões aumentou com o tempo de exposição, nomeadamente a hiperplasia das células epiteliais com proliferação do epitélio filamentar e fusão das lamelas observadas preferencialmente às 96 h. Adicionalmente, alguns dos resultados obtidos pela análise em microscopia de luz foram confirmados com o recurso da microscopia eletrônica de varredura.