Socio-economic factors affecting the distribution of marine litter: The Portuguese case study.

Marine litter is a growing global problem with serious environmental, economic, social, and health threats. Understanding the socio-economic factors that influence the types and amounts of litter is of utmost importance. In this study, an integrative analysis of the socio-economic factors that characterize the beach litter distribution in continental Portugal and the Azores archipelago was conducted via a cluster analysis, implementing a novel technique to support the difficult task of marine litter characterization. The results highlighted that the most abundant beach litter material is plastic (92.9 %), followed by paper (2.2 %), wood (1.5 %), and metal (1.3 %). The majority of the items could not be attributed to a specific source (46.5 %). The remaining were attributed to public litter (34.5 % of total aggregated items), fishing (9.8 %), sewage-related debris (6.4 %) and shipping (2.2 %). The top-three beach litter categories were small plastic pieces (0-2.5 cm, 43.5 %), cigarette butts (30.1 %), and medium plastic pieces (2.5-50 cm, 26.4 %). A positive relation between both municipality environment expenditures and population density and the quantity and typology of litter was found. Beach litter quantity and categories were also associated with specific economic sectors, as well as with geographical/hydrodynamic conditions, demonstrating the utility of the technique and its applicability to other regions.

Are biopesticides safe for the environment? Effects of pyrethrum extract on the non-target species Daphnia magna.

Biopesticides are natural compounds considered more safe and sustainable for the environment. However, it is also important to evaluate the potential risk in non-target organisms. Pyrethrum extract (PE) is a biopesticide, widely used for agriculture, veterinary, and aquaculture. This work aimed to evaluate acute (0.6 - 40.0 µg/L; 96 h; E(L)C50 toxicity) and sub-chronic (0.7 - 1.1 µg/L; 10 d; life-history parameters) effects of PE on Daphnia magna. Moreover, a biomarkers approach using antioxidant and biotransformation capacity, lipid peroxidation (LPO), neurotoxicity, and energy reserves content were evaluated. Acute effects (mortality, changes in swimming behavior, oxidative stress, lipid peroxidation, neurotoxicity) were recorded with the increase in PE concentration. Sub-chronic assay showed an increase in energy reserves content, antioxidant parameters, and LPO demonstrating that PE unbalances oxidative metabolism. This study can conclude that PE potentiates toxic effects in D. magna and demonstrates the vulnerability of a non-target organism to PE that is considered environmentally safe.

Can biochemical endpoints improve the sensitivity of the biomonitoring strategy using bioassays with standard species, for water quality evaluation?

The Water Framework Directive (WFD) was adopted in 2000 and is a common framework for water policy, management and protection in Europe. The WFD assesses specific parameters; however, it ignores indicators of ecosystem functioning and sub-individual performance. Reservoirs are strongly influenced by anthropogenic activities that promote their imbalance. Bioassays and biomarkers are useful tools to link the chemical, ecological and toxicological assessments in water quality assessments. These approaches can be complementary to WFD methodologies, allowing the detection of impacts on the ecosystem. This study evaluated if the biochemical parameters can improve the sensitivity of the biomonitoring strategy using bioassays with the standard species Daphnia magna, in the assessment of the ecological quality of water reservoirs. To this end, water samples of Portuguese reservoirs were analysed in three sampling periods (Autumn 2018 and Spring, Autumn 2019). In parallel, a physicochemical characterization of waters was performed. D. magna feeding rate assays were performed for 24 h. After exposure, metabolism, oxidative stress and lipid peroxidation biomarkers were evaluated. Feeding rate assays showed sensitivity to different reservoirs. Biomarkers showed a higher sensitivity and can therefore improve the sensitivity of the biomonitoring strategy using bioassays. Bioassays and biomarkers approach allowed to highlight potential sources of stress, more related to the quality of the seston than to chemical contamination. This work highlights the complementarity between bioassays and biomarkers to identify ecotoxicological effects of surface waters, and can be extremely useful, especially in cases where the biotic indices are difficult to establish, such as reservoirs.

Assessment of water quality in Aguieira reservoir: Ecotoxicological tools in addition to the Water Framework Directive.

To assess the ecological potential (EP), the Water Framework Directive (WFD) uses specific parameters but ignores ecosystem functioning and individual/subindividual biological responses. The heavily modified water bodies (reservoirs) are strongly influenced by human activities that promote their imbalance, with a loss or compromise the biodiversity and the functioning of these ecosystems. This work aims to determinate the EP according physical, chemical and biological parameters of WFD and evaluate the functionality and sensitivity of ecotoxicological tools in the evaluation of water quality of Aguieira reservoir. Four sampling sites were defined in Aguieira reservoir and sampling was conducted in autumn 2018, spring and autumn 2019. WFD showed overall that Aguieira reservoir presented moderate EP. The autumn samples and the two reservoir upstream sites had the worst classification. These results were complemented by a set of ecotoxicological assays (growth inhibition of Raphidocelis subcapitata and Spirodela polyrhiza, and feeding rate of Daphnia longispina) using three natural water treatments. R. subcapitata was the most sensitive species, indicating the presence of a risk that is not detected by chemical analysis, nor by organisms of different trophic levels. D. longispina only showed sensitivity in A3 and A4 in autumn, however this results was difficult to interpret since no reason can be observed to respond the F1 lower feeding rate observed in almost sites. S. polyrhiza, as well as the total chlorophyll, lipid peroxidation, and proline content did not show sensitivity, since no discrimination among the water treatments were recorded. The relevance of bioassays is associated with the fact that an integrated response to a complex mixture can be obtained, which can contain more substances than the measured ones. Thus, we may conclude that WFD are insufficient to draw conclusions about water quality, and the growth inhibition of Raphidocelis subcapitata assay is an important parameter to complement the WFD. In addition, species of different trophic levels are recommended for ecotoxicological analyses due to differences in species sensitivities.

Sensitivity to salinization and acclimation potential of amphibian (Pelophylax perezi) and fish (Lepomis gibbosus) models.

Predictions of the International Panel for Climate Changes on sea level rise foresee that the number of coastal regions impacted with salinization will increase in a near future. The present work intended to evaluate the sensitivity to salinization of two freshwater vertebrate species (the frog Pelophylax perezi and the fish Lepomis gibbosus) and their ability to acclimate to this stressor. For this, three specific objectives were targeted: (i) to assess if NaCl may be used as a safe surrogate for risk assessment of seawater (SW) intrusion for freshwater vertebrates; (ii) to evaluate the sensitivity of two freshwater vertebrate models to increased salinity (both due to NaCl or SW); (iii) to determine the capacity of the studied species to acclimate to low levels of salinization. To assess specific objectives (i) and (ii), organisms were exposed to serial concentrations of NaCl or SW dilutions. To assess the capacity of acclimation of both species to salinization, organisms were exposed to low serial concentrations of NaCl during the embryonic development or for a period of two months, respectively, and their sensitivity to NaCl was re-evaluated after this period. Results showed that fish juveniles were more tolerant (96-h LC50 of 21.3 mS cm-1 for NaCl and 23.6 mS cm-1 for SW) than frog embryos (96-h LC50 of 10.7 mS cm-1 for NaCl and 10.7 mS cm-1 for SW) and tadpoles (96-h LC50 of 19.4 mS cm-1 for NaCl and 8.72 mS cm-1 for SW). The fish was able to cope with conductivities of almost one third of SW conductivity, while effect conductivities computed for the amphibian were much lower than SW conductivity (≈ 52 mS cm-1). The two-fold difference between the sensitivity of the two tested species reinforces the idea that ecological risk assessment for amphibians based on fish toxicity data may underestimate the risk to the former. Acclimation to low levels of salinity caused an increase in tolerance to salinization in P. perezi tadpoles but not in fish.

Sensitivity of freshwater species under single and multigenerational exposure to seawater intrusion.

Salinization of coastal freshwater ecosystems is already occurring in some regions of the world. This phenomenon raises serious concerns on the protection of coastal freshwater ecosystems, since many of them support and shelter a large number of species and are considered hotspots of biodiversity. This work intended to assess the adverse effects that salinization, caused by the intrusion of seawater (SW), may pose to freshwater organisms. In this study, three specific goals were addressed: (i) to assess if sodium chloride (NaCl) may be used as a surrogate of natural SW at early-stages of risk assessment; (ii) to identify the most sensitive freshwater species to salinity NaCl; and (iii) to determine if increased tolerance to salinity may be acquired after multigenerational exposure to low levels of salinization (induced with NaCl). A total of 12 standard monospecific bioassays were carried out by exposing organisms from different taxonomic groups (Cyanobacteria: one species, Tracheophyta: two species, Rotifera: one species, Arthropoda: two species and Mollusca: one species) to a series of concentrations of NaCl (ranging from 0.95 to 22.8 mS cm-1) or dilutions of SW (ranging from 1.70 to 52.3 mS cm-1). In general, NaCl exerted similar or higher toxicity than SW, both at lethal and sublethal levels, suggesting that it may be proposed as a protective surrogate of SW for first tiers of salinization risk assessment. Among all tested species, the cyanobacterium Cylindrospermopsis raciborskii, the daphnid Daphnia longispina and the rotifer Brachionus plicatilis were the most sensitive taxa to salinization (EC50 ≤ 4.38 mS cm-1). Given their position at the basis of the food web, it is suggested that small increments of salinity may be enough to induce structural changes in freshwater communities or induce changes in trophic relations. No clear evidences of increased tolerance after multigenerational exposure to low levels of salinity were found.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

Ecotoxicological evaluation of gilthead seabream (Sparus aurata) exposed to the antibiotic oxytetracycline using a multibiomarker approach.

Oxytetracycline (OTC) is an antibiotic widely used in human and veterinary medicines. Since the primary toxicity occurs mainly at molecular/biochemical levels, the study of different biological responses corresponds to a sensitive and crucial approach. The aim of the present study was to assess the toxic effects of OTC in gilthead seabream (Sparus aurata) through the use of multibiomarkers and elucidate about the possible toxicological mechanisms involved. S. aurata were acutely (96 h: 0.04-400 µg/L) and chronically (28 days: 0.0004-4 µg/L) exposed to OTC. Detoxification, antioxidant defense, lipid peroxidation, genotoxicity, neurotransmission and energy metabolism biomarkers were evaluated. OTC impaired the detoxification pathways and caused peroxidative damage and genotoxicity. The relevance of the here-obtained data is high, since significant effects were recorded for levels already reported to occur in the wild, meaning that environmentally-exposed marine organisms (including those cultured at fish farms) are not completely exempt of risks posed by OTC.

Oxytetracycline effects in specific biochemical pathways of detoxification, neurotransmission and energy production in Oncorhynchus mykiss.

Oxytetracycline (OTC) is a tetracycline antibiotic, widely used in human and veterinary medicines, including in aquaculture. Given this use, OTC has been detected in different aquatic environments. Some recent works have demonstrated unintentional biological activity of OTC in non-target aquatic organisms. This study investigated the acute and chronic effects of OTC on the physiology of the fish species Oncorhynchus mykiss (rainbow trout), namely through the quantification of the activity of enzymes involved in different biochemical pathways, such as detoxification (phase II - glutathione S-transferases - GSTs, uridine-diphosphate-glucuronosyltransferases - UGTs), neurotransmission (acetylcholinesterase - AChE) and energy production (lactate dehydrogenase - LDH). The here-obtained data demonstrated the induction of GSTs activity in gills, and inhibition of AChE activity in eyes tissue, in chronically exposed organisms, as well as alterations in LDH activity following both exposures. Considering this set of results, we can infer that OTC exposure may have induced the glutathione pathway of detoxification in gills with the involvement of GSTs, or indirectly due to the metabolites that may have been produced. In turn, these metabolites may have interfered with the mechanism of neurotransmission, also causing physiological and biochemical disturbances in rainbow trout after OTC exposure, namely disturbances in energetic metabolism. In addition, it is important to stress that such occurrences took place at low, environmentally realistic levels of OTC, suggesting that organisms exposed in the wild may be putative targets of toxic effects by commonly used drugs such as antibiotics.

Assessment of ecotoxicological effects of ciprofloxacin in Daphnia magna: life-history traits, biochemical and genotoxic effects.

Antibiotics (e.g. ciprofloxacin) have been detected in surface water and groundwater for several decades. In order to understand the potential impact of the continuous exposure of aquatic organisms to ciprofloxacin, a chronic assay was carried out with Daphnia magna. This approach allowed evaluation of the effects of ciprofloxacin on life-history and sub-individual parameters (antioxidant status and metabolic response: activities of catalase and glutathione S-transferases - GSTs; peroxidative damage; thiobarbituric acid reactive substances and genotoxic effects (genetic damage index, measured by the comet assay). Life-history parameters of D. magna showed no significant effects after ciprofloxacin exposure. Concerning oxidative stress and metabolism parameters, no significant alterations were reported for catalase and GSTs activities. However, a dual response was observed, with a significant decrease in lipid peroxidation levels at low ciprofloxacin concentrations (<0.013 mg/L), while a significant increase was verified at high ciprofloxacin concentrations (0.078 mg/L). The genotoxicity assay detected a significant increase in genetic damage index up to 0.013 mg/L of ciprofloxacin. The here-tested ciprofloxacin concentrations, which are ecologically relevant, did not cause significant impacts concerning the life-history parameters of D. magna; however, at the same levels of ciprofloxacin an oxidative stress and genotoxic damage scenarios were recorded.

Transgenerational Effects of Toxicants: An Extension of the Daphnia 21-day Chronic Assay?

The assessment of transgenerational effects should be incorporated in standard chronic toxicity protocols for the sake of a realistic extrapolation of contaminant effects to the population level. We propose a simple add-on to the standard 21-day chronic Daphnia magna assay, allowing the assessment of the reproductive performance of the offspring (F1 generation) born from the first clutch of the parental (F0) generation. The extended generational assay was performed simultaneously with the standard reproduction assay. With this design, we evaluated the lethal, reproductive, and transgenerational effects of four widespread and extensively used substances: a biocide/anti-fouling (copper sulphate), an industrial oxidizing agent (potassium dichromate), a pharmaceutical (paracetamol), and a quaternary ammonium compound (benzalkonium chloride). Benzalkonium chloride was the most toxic in terms of lethality, whereas paracetamol, copper sulphate, and potassium dichromate caused deleterious effects in the reproductive performance of exposed D. magna. Adverse effects in the fitness of the daughter (F1) generation were observed in the case of maternal exposure to paracetamol and copper sulphate, although they were not very pronounced. These findings highlight the usefulness of our approach and reinforce the view-shared by other authors-of the need for a generalised formal assessment of the transgenerational effects of pollutants.

Adequacy of planctomycetes as supplementary food source for Daphnia magna.

The nutritional quality of daphnids diet can influence their growth, reproduction and survival. In aquatic ecosystems, bacteria can contribute significantly to Daphnia diet by supporting, for instances, their high needs for phosphorus. The laboratory feeding of the model organisms Daphnia spp. is algal based, but should be improved to allow their better performance. The aim of this study was to evaluate the potential of two planctomycetes, Gemmata obscuriglobus and Rhodopirellula rubra, from exponential and stationary growth phases as alternative or supplementary food source for Daphnia magna. The actinobacterium Arthrobacter sp. was used for comparison. The feeding with only bacteria showed the inefficacy of both planctomycetes and actinobacteria as the only food source. However, when used in supplement to Raphidocelis subcapitata, a decrease in the age of first reproduction, a significant increase in reproductive output, in somatic growth and in rate of population increase was found for the highest cell densities of bacteria tested. The typical pink coloration of these bacteria present in daphnids body and eggs confirmed bacterial absorption and metabolization of their pigment. Planctomycetes yielded better results than the actinobacteria Arthrobacter but G. obscuriglobus that possesses sterols did not induce a better performance comparatively to R. rubra. No relation could be established between the feeding treatments that allowed improvement of Daphnia performance and the different kind of Daphnia' Fatty Acid Methyl Esters. The use of sonication to separate planctomycetal cells before feeding the daphnids proved to be efficient. We confirmed that R. subcapitata supplemented by bacteria allows a better growth performance of D. magna.

Histological alterations in gills and liver of rainbow trout (Oncorhynchus mykiss) after exposure to the antibiotic oxytetracycline.

The aim of this study was to investigate the histopathological effects of oxytetracycline (OTC) on the gill and liver tissues of rainbow trouts (Oncorhynchus mykiss) following acute (96h: 0.005-50mg/L) and chronic (28days: 0.3125-5µg/L) exposures. Results suggest the existence of a cause-and-effect relationship between the exposure to OTC and tissue damage. Most predominant disorders observed in gills were progressive (e.g. hypertrophy of mucous cells and hyperplasia of epithelial cells) in acute exposure and regressive (e.g. lamellar fusion, epithelial lifting of lamellae and some changes in tissue architecture) in chronic exposure. However, only the acute exposure was responsible for a significant increase of the total gill pathological index. PAGE index, reflecting the extent of gill tissue available for gas exchanges in fish, remained unchanged for both exposures. In liver, circulatory (e.g. hemorrhage and increase of sinusoidal space), regressive (e.g. pyknotic nucleus, vacuolization and hepatocellular degenerations) and progressive (e.g. hypertrophy of hepatocytes) changes were observed, but just after acute exposure. After chronic exposure, only inflammatory changes (e.g. leucocytes infiltration) were observed. Following both exposures, a significant increase of the total liver pathological index was recorded. Despite the increase of the histological damage in individuals exposed to OTC, lesions observed were of minimal or moderate pathological importance, non-specific and reversible. The data gathered following acute and chronic exposures also suggest the onset of adaptive mechanisms of fish, namely for longer exposure periods. Furthermore the observed histological alterations appear to be result of several physio-metabolic disorders consequence of the biochemical and molecular modes of action of OTC.

Whole-organism and biomarker endpoints in Daphnia magna show uncoupling of oxidative stress and endocrine disruption in phenolic derivatives.

During the past century, the amount of chemicals released into water bodies has increased, with particular emphasis being attributed to xenobiotics with endocrine disruption properties and/or pro-oxidant effects. Among these, it is possible to identify a specific chemical class, alkylphenols, which are of widespread use, and include a variety of chemicals with multiple uses. Bisphenol A is an important chemical used in industrial production of plastics, and has been extensively described as an endocrine disruptor. Paracetamol is a pharmaceutical compound used in human medicine, known for its therapeutic action but also for its evident pro-oxidant features. Additionally, previous studies have suggested that paracetamol may also exert endocrine disruption. The main goal of this study was to assess the effects of both paracetamol and bisphenol A as endocrine disruptors, and as promoters of oxidative stress and damage, on the freshwater microcrustacean Daphnia magna. The obtained results showed that bisphenol A was capable of altering population traits of exposed organisms, by impairing molting. On the contrary, paracetamol was not causative of any significant change in this parameter, despite having caused extensive oxidative stress.

Effects of chronic exposure to benzalkonium chloride in Oncorhynchus mykiss: cholinergic neurotoxicity, oxidative stress, peroxidative damage and genotoxicity.

Benzalkonium chloride (BAC) is one of the most used conservatives in pharmaceutical preparations. However, its use is limited to a small set of external use formulations, due to its high toxicity. Benzalkonium chloride effects are related to the potential exertion of deleterious effects, mediated via oxidative stress and through interaction with membrane enzymes, leading to cellular damage. To address the ecotoxicity of this specific compound rainbow trouts were chronically exposed to BAC at environmental relevant concentrations (ranging from 0.100 to 1.050mg/L), and the biological response of cholinergic neurotoxicity, modulation of the antioxidant defense, phase II metabolism, lipid peroxidation and genotoxicity was studied. The obtained results showed a dual pattern of antioxidant response, with significant alterations in catalase activity (starting at 0.180mg/L), and lipid peroxidation, for intermediate (0.180 and 0.324mg/L) concentrations. No significant alterations occurred for glutathione-S-transferases activity. An unexpected increased of the acetylcholinesterase activity was also recorded for the individuals exposed to higher concentrations of BAC (starting at 0.180mg/L). Furthermore, exposure to BAC resulted in the establishment of genotoxic alterations, observable (for the specific case of the comet assay results) for all tested BAC concentrations. However, and considering that the oxidative response was not devisable, other mechanisms may be involved in the genotoxic effects reported here.

Multibiomarker toxicity characterization of uranium mine drainages to the fish Carassius auratus.

The release of acidic effluents, naturally enriched in metals and radionuclides, is the main legacy of uranium mines. Generally, metals dissolved by these acidic effluents can cause significant alterations in exposed organisms, with distinct toxicological outcomes. In this study, 72 individuals of the freshwater fish species Carassius auratus were exposed in situ for different periods (8, 16, 24, and 48 h) to water from a pond (treatment pond (TP)) with a chemically treated effluent and a reference pond (PRP), in the vicinity of the Cunha Baixa uranium mine (Portugal). Comparing the water of the two ponds, the PRP pond was characterized by higher pH and oxygen values and lower conductivity and hardness values. Regarding total metal concentrations, among others, magnesium (56,000 µg/L), sodium (17,400 µg/L), zinc (86 µg/L), manganese (6340 µg/L), and uranium (1380 µg/L) concentrations in the TP pond were above the values obtained for the PRP pond. The values of manganese and uranium exceeded the values of quality criteria established for surface waters for cyprinids and for irrigation purposes. After exposure to pond water, significant differences were recorded for several biomarkers: (i) between ponds for acetylcholinesterase (AChE) with higher activities for animals from the PRP and glutathione-S-transferase (GST) activities that were particularly enhanced in animals from the TP pond; (ii) between ponds and exposure periods for lactate dehydrogenase (LDH) activity, since organisms from PRP pond presented always higher values than those from the TP pond, and among these, organisms exposed for the longer period presented a further depression in LDH activity; and (iii) between exposure periods for erythrocyte micronucleus. GSTs and LDH were the most sensitive biomarkers within the timeframe of the in situ assay performed. Despite the alleged efficacy of the chemical treatment (evidenced by a significantly lower pH), some metals persisted in the treated effluent (TP pond), potentially contributing to the induction of oxidative stress or increased conjugation metabolic activity in fish.

Acute and chronic effects of erythromycin exposure on oxidative stress and genotoxicity parameters of Oncorhynchus mykiss.

Erythromycin (ERY) is a macrolide antibiotic used in human and veterinary medicine, and has been detected in various aquatic compartments. Recent studies have indicated that this compound can exert biological activity on non-target organisms environmentally exposed. The present study aimed to assess the toxic effects of ERY in Oncorhynchus mykiss after acute and chronic exposures. The here adopted strategy involved exposure to three levels of ERY, the first being similar to concentrations reported to occur in the wild, thus ecologically relevant. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by performing analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and of erythrocytic nuclear abnormalities (ENAs). The results suggest the occurrence of a mild, but significant, oxidative stress scenario in gills. For acutely exposed organisms, significant alterations were observed in CAT and GRed activities, and also in TBARS levels, which however are modifications with uncertain biological interpretation, despite indicating involvement of an oxidative effect and response. After chronic exposure, a significant decrease of CAT activity, increase of GPx activity and TBARS levels in gills was noticed. In liver, significant decrease in TBARS levels were observed in both exposures. Comet and ENAs assays indicated significant increases on genotoxic damage of O. mykiss, after erythromycin exposures. This set of data (acute and chronic) suggests that erythromycin has the potential to induce DNA strand breaks in blood cells, and demonstrate the induction of chromosome breakage and/or segregational abnormalities. Overall results indicate that both DNA damaging effects induced by erythromycin may be related to the oxidative damage observed, shown to occur at environmentally relevant concentrations of erythromycin.

Biomonitoring of environmental stress in Pollicipes pollicipes from the northern coast of Portugal: a non-destructive approach using haemolymph.

In the intertidal area, the interactions between anthropogenic contaminants and natural variations (biotic and abiotic factors) are poorly understood. Consequently, there is a great need for new assessment procedures to characterize the biological responses occurring in organisms from this extreme environment. Considering the intrinsic inter-individual variations among organisms from a single population, it is important to propose new methods that address this variability, by validating a sampling strategy in target groups of organisms, encompassing seasonal fluctuations. This strategy must however be less invasive than traditional methods, avoiding the mandatory sacrifice of the sampled organisms. By doing so, it is also possible to increase the ecological relevance of obtainable data, and contribute to minimize damage to endangered species. The main purpose of the present study was to assess the influence of seasonal variations in the responses elicited by anthropogenic compounds on a marine crustacean species, by using a biomarker-based approach. According to this purpose, the seasonal variations in key physiological responses (biomarkers) were investigated in the crustacean Pollicipes pollicipes from the Northern coast of Portugal. Biomarkers used for this purpose were the activity of the phase II biotransformation isoenzymes glutathione-S-transferases (GSTs), the activity of cholinesterases (ChEs), and the levels of lipid peroxidation (TBARS). All biomarkers were quantified in distinct tissues (such as cirri, and peduncle) and haemolymph (a non-destructive source of biological samples). The glycogen content in peduncle tissue, and the variation in haemocyte number in haemolymph were also analyzed. Samples were collected monthly, during a year, in Lavadores, located in the proximity of an estuarine area (Douro River). The results showed a seasonal pattern in all tested biomarkers. The results also showed a significant increase in GSTs activities, and in peroxidative damage, especially during warmer months. The lowest ChEs values were recorded during the rainy season. The results also showed a similar pattern among all tested tissues, validating the proposed use of the haemolymph as a source tissue for non-lethal sampling strategy for biomarker determinations. Glycogen content was apparently related to the reproductive cycle, with lower values being observed in spring and summer. Total haemocyte count (THC) increased during summer months. The results pointed to an influence of natural variations in the levels of biomarkers in P. pollicipes, highly dependent upon abiotic factors, a factor that must be considered when interpreting biological responses elicited by anthropogenic contaminants from the marine coastal environment. The validation of haemolymph as a non-lethal source tissue for the quantification of biomarkers was successfully attained, opening the possibility of less invasive and almost unlimited sampling in a small number of organisms.

Ecotoxicological effect of zinc pyrithione in the freshwater fish Gambusia holbrooki.

Currently diverse biocidal agents can be used for distinct applications, such as personal hygiene, disinfection, antiparasitic activity, and antifouling effects. Zinc pyrithione is an organometallic biocide, with bactericidal, algicidal and fungicidal activities. It has been recently incorporated in antifouling formulas, such as paints, which prevent the establishment of a biofilm on surfaces exposed to the aquatic environment. It has also been used in cosmetics, such as anti-dandruff shampoos and soaps. Previously reported data has shown the presence of this substance in the aquatic compartment, a factor contributing to the potential exertion of toxic effects, and there is also evidence that photodegradation products of zinc pyrithione were involved in neurotoxic effects, namely by inhibiting cholinesterases in fish species. Additional evidence points to the involvement of zinc pyrithione in alterations of metal homeostasis and oxidative stress, in both aquatic organisms and human cell models. The present work assesses the potential ecotoxicity elicited by zinc pyrithione in the freshwater fish Gambusia holbrooki after an acute (96 h) exposure. The oxidative stress was assessed by the quantification of the activities of specific enzymes from the antioxidant defense system, such as catalase, and glutathione-S-transferases; and the extent of peroxidative damage was quantified by measuring the thiobarbituric acid reactive substances levels. Neurotoxicity was assessed through measurement of acetylcholinesterase activity; and a standardized method for the description and assessment of histological changes in liver and gills of was also used. Zinc pyrithione caused non-specific and reversible tissue alterations, both in liver and gills of exposed organisms. However, histopathological indices were not significantly different from the control group. In terms of oxidative stress biomarkers, none of the tested biomarkers indicated the occurrence of pro-oxidative effects, suggesting that the oxidative pathway is not the major toxicological outcome of exposure to zinc pyrithione.

Acute effects of tetracycline exposure in the freshwater fish Gambusia holbrooki: antioxidant effects, neurotoxicity and histological alterations.

A large body of evidence was compiled in the recent decades showing a noteworthy increase in the detection of pharmaceutical drugs in aquatic ecosystems. Due to its ubiquitous presence, chemical nature, and practical purpose, this type of contaminant can exert toxic effects in nontarget organisms. Exposure to pharmaceutical drugs can result in adaptive alterations, such as changes in tissues, or in key homeostatic mechanisms, such as antioxidant mechanisms, biochemical/physiological pathways, and cellular damage. These alterations can be monitored to determine the impact of these compounds on exposed aquatic organisms. Among pharmaceutical drugs in the environment, antibiotics are particularly important because they include a variety of substances widely used in medical and veterinary practice, livestock production, and aquaculture. This wide use constitutes a decisive factor contributing for their frequent detection in the aquatic environment. Tetracyclines are the individual antibiotic subclass with the second highest frequency of detection in environmental matrices. The characterization of the potential ecotoxicological effects of tetracycline is a much-required task; to attain this objective, the present study assessed the acute toxic effects of tetracycline in the freshwater fish species Gambusia holbrooki by the determination of histological changes in the gills and liver, changes in antioxidant defense [glutathione S-transferase (GST), catalase (CAT), and lipoperoxidative damage] as well as potential neurotoxicity (acetylcholinesterase activity). The obtained results suggest the existence of a cause-and-effect relationship between the exposure to tetracycline and histological alterations (more specifically in gills) and enzymatic activity (particularly the enzyme CAT in liver and GST in gills) indicating that this compound can exert a pro-oxidative activity.

Ecotoxicological effects of salicylic acid in the freshwater fish Salmo trutta fario: antioxidant mechanisms and histological alterations.

The presence of pharmaceutical drugs in aquatic ecosystems has been widely reported during the past years. Salicylic acid (SA) is mainly used in human medicine as an analgesic and antipyretic drug, being also active in preventing platelet aggregation. To study the ecotoxicological effects potentially elicited by SA in freshwater fish, brown trout individuals (Salmo trutta fario) were chronically exposed (28 days) to this drug, in order to evaluate the enzymatic and histological effects, in both gills and liver. A qualitative and semi-qualitative evaluation of the gills and liver was performed, and also a quantitative evaluation of various lamellar structures. Oxidative stress was quantified trough the determination of glutathione S-transferases (GSTs), glutathione reductase (GRed), total and selenium-dependent glutathione peroxidase (GPx) and Catalase (Cat) activities. Lipid peroxidative damage was also assessed by the quantification of thiobarbituric acid reactive substances (TBARS) in the liver. The here-obtained data showed the occurrence of oxidative stress, reflected by an increased activity of GPx and GRed in the liver; additionally, it was possible to observe non-specific histological changes in gills. The global significance of the entire set of results is discussed, giving emphasis to the ecological relevance of the responses.