Comparison of neutral lipid fatty acid composition in organisms from different trophic levels.

The profiles of total fatty acids (TFAs) and the neutral lipid fatty acids (NLFAs) were compared for the bacterium Rhodopirellula rubra and the alga Raphidocelis subcapitata (conventional food source for Daphnia magna). D. magna NLFAs were assessed when this crustacean was fed with bacterium and alga, individually or in combination. After NLFA extraction, the profiles of the various organisms were characterized by gas chromatography. Results evidenced the relevance of the different composition of the fatty acid (FAs) fractions in the different organisms, R. rubra and R. subcapitata. In these species, the NFLA analyses revealed high amounts of long chain FAs (C19). The FA profile of D. magna was influenced by the different diets provided although the preferred diet was the alga. D. magna showed the capacity to adapt to the available food resources as it defines its FA profile according to its needs, namely for the long chain FAs (C19).

Assessment of Rhodopirellula rubra as a supplementary and nutritional food source to the microcrustacean Daphnia magna.

The daily use of the planctomycete Rhodopirellula rubra as an alternative or supplementary food source for Daphnia magna and its feasibility in the nutrition of transgenerational populations were studied. The life history parameters, fatty acids (saturated, mono- and polyunsaturated; SFAs, MUFAs and PUFAs), glycogen and protein contents of organisms during feeding assays and of the first generation were analysed. An increase in the yields of D. magna with the increase of the cell concentration of R. rubra was evident, but overall, bacteria supplied as the only food source was nutritionally insufficient as observed for all the parameters analysed. However, when R. rubra was added as supplement to the microalgae Raphidocelis subcapitata a significant improvement in the life history parameters was observed namely in the reproductive output and the somatic growth rate. The identified SFAs, MUFAs and PUFAs were the fatty acids more abundant in daphniids, and the feed regimens influenced daphniids fatty acid profiles. Additionally, the mixed diet resulted in a larger number and size of offspring in the different F1 broods as also observed with the results of F0 generation. The pink colouration present in D. magna body and eggs confirmed that bacteria were absorbed, the pigment(s) retained and passed on to the next generation. Our results showed that R. rubra can play an essential role in D. magna diet as a nutritional supplement showing potential biotechnological applications.

Histopathological effects of the antibiotic erythromycin on the freshwater fish species Oncorhynchus mykiss.

Pharmaceuticals are found in the aquatic compartment due to their continuous release in wastewater effluents or direct dispersal in aquaculture practices raising serious threats to human and environmental health. Erythromycin (ERY) is a macrolide antibiotic widely prescribed in human and veterinary medicine to threat a number of bacterial infections, being consequently found in the aquatic environment. The present work intends to evaluate the sub-lethal effects of ERY on juveniles of rainbow trout (Oncorhynchus mykiss) in terms of tissue damage using histochemical staining procedures. Individuals were exposed for 96 h (acute exposure: 0.001-10 mg/L) and 28 days (chronic exposure: 0.05-0.8 µg/L) to environmentally realistic concentrations of ERY. Qualitative and quantitative approaches were used to assess O. mykiss gills and liver tissue alterations after exposure to ERY. For both exposures the most common gill changes recorded were progressive (e.g. hypertrophy of mucous cells and hyperplasia of the epithelial cells). However, circulatory (e.g. aneurysms and oedemas) and regressive (e.g. epithelial lifting of lamellae and lamellar fusion) changes were also observed in the acute assay. Gill morphometric analysis revealed to be a good indicator of subtle alterations in gill architecture in agreement with the qualitative scoring system. In liver, regressive (e.g. cytoplasmic vacuolization, pyknotic nucleus and hepatocellular degeneration) and circulatory disturbances (e.g. hemorrhage and increase of sinusoidal space) were the most frequently observed alterations, but only for the acute assay. Furthermore, all histological changes observed contributed to a significant increase in the pathological index for both organs. The current data demonstrate the existence of a direct dose-effect relationship between the exposure to this specific macrolide antibiotic and the histological disorders recorded in different tissues of the exposed fish. The histopathological findings observed in this study may have been the result of several physio-metabolic dysfunctions. However, the observed tissue lesions were of minimal or moderate pathological importance, non-specific and reversible. Further investigation into the cellular mechanism of action of ERY is needed.

Rainbow trout (Oncorhynchus mykiss) pro-oxidant and genotoxic responses following acute and chronic exposure to the antibiotic oxytetracycline.

Oxytetracycline (OTC), an antibacterial agent, is extensively used in aquaculture practices all over the world, but also in human and veterinary medicines. Because of its intensive use, low rates of absorption by treated animals, inadequate disposal, and low efficiency of removal in wastewater treatment plants, the potential harmful effects on aquatic organisms are of great concern. This work aimed to assess the effects of this antibiotic in rainbow trout, following both acute and chronic exposures. 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 analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and erythrocytic nuclear abnormalities (ENAs). The obtained results showed a mild pattern of antioxidant response, with modifications in CAT and GPx activities in gills, and lipid peroxidation in liver. These results suggest that despite the occurrence of oxidative effects, a full scenario of oxidative stress is not likely. However, exposure to OTC resulted in the establishment of genotoxic alterations with the induction of DNA strand breaks in blood cells (increase of GDI), and of chromosome breakage and/or segregational abnormalities (increase of ENAs). Considering that the oxidative response was not totally devisable, other mechanisms may be involved in the genotoxic effects reported.

Clams sensitivity towards As and Hg: A comprehensive assessment of native and exotic species.

To assess the environmental impact of As and Hg, bioindicator organisms such as bivalves have been used. Nevertheless, few studies have assessed the impacts of As and Hg in Ruditapes decussatus and Ruditapes philippinarum, which are native and exotic species in Europe, respectively. The main goal of the present study was to assess elements' partitioning and detoxification strategies of R. decussatus and R. philippinarum. Both clams showed a higher capacity to bioconcentrate Hg (BCF 2.29-7.49), when compared to As (0.59-1.09). Furthermore, As accumulation in both species was similar in the soluble and insoluble fractions, while in both species the majority of Hg was found in the insoluble fraction. Clams exposed to As showed different detoxification strategies, since R. decussatus had higher ability to enhance antioxidant enzymes and metallothioneins in order to reduce toxicity, and R.philippinarum increased glutathione S-transferase Ω activity, that catalyzes monomethyl arsenate reduction, the rate-limiting reaction in arsenic biotransformation. When exposed to Hg, R. decussatus presented, higher synthesis of antioxidant enzymes and lower LPO, being able to better tolerate Hg than the exotic species R. philippinarum. Thus under relevant levels of As and Hg contamination our work evidenced the higher ability of R. decussatus to survive and inhabit coastal environments not heavily contaminated by Hg and As.

Acute and chronic ecotoxicological effects of four pharmaceuticals drugs on cladoceran Daphnia magna.

The occurrence of pharmaceuticals in the aquatic environment has received increasing attention in recent years, as concerns have risen about their environmental persistence, biological activity and different effects toward nontarget organisms. Considering the magnitude of concentrations (ng L(-1) to mg L(-1)) and their often-specific modes of action, the assessment of physiological responses of exposed aquatic biota may provide significant information regarding the potential ecological consequences of exposure to these contaminants. The present study intended to assess the acute and chronic effects of four pharmaceuticals: acetaminophen, chlorpromazine, diclofenac sodium and propranolol in the cladoceran species Daphnia magna. Parameters such as immobility, total of offspring and rate of population increase were analyzed. Results of acute exposures showed a considerable variability of toxicity among pharmaceuticals, with the following ranking of toxicity: diclofenac (EC50 = 123.3 mg L(-1)) < propranolol (EC50 = 5.531 mg L(-1)) < acetaminophen (EC50 = 2.831 mg L(-1)) < chlorpromazine (EC50 = 1.805 mg L(-1)). The chronic toxicity data showed the exertion of reproductive adverse effects. The compounds chlorpromazine and propranolol caused a significant decrease in fecundity, and the rate of population increase parameter suffered a significant decrease from 0.33 mg L(-1) to 0.128 mg L(-1) onwards, respectively. The levels of exposure to which our test organism was acutely and chronically exposed were above those already reported in the wild. Nevertheless, the extensive production, prescription and release of pharmaceuticals drugs will continue to grow in the future, and consequently their loadings to the environment can result in potential long-term ecological risks to aquatic biota.

Evaluation of ecotoxicological effects of drugs on Daphnia magna using different enzymatic biomarkers.

The increasing occurrence of pharmaceutical drugs in the aquatic environment is cause of concern, due to the possibility of toxic phenomena in non-target species, including oxidative stress and neurotoxicity. The present study aimed to assess the acute effect of four widely used therapeutic agents: acetaminophen (analgesic), chlorpromazine (antipsychotic), diclofenac (anti-inflammatory) and propranolol (antihypertensive), in the cladoceran species Daphnia magna. Considering the involvement of the mentioned compounds in the impairment of cholinesterasic activity and modifications in cellular redox systems, the purpose of this study was to analyze their effects on biomarkers of neuronal regulation, such as total cholinesterases (ChEs), and enzymatic oxidative stress defense, including as catalase (CAT), glutathione-S-transferases (GSTs), and total and selenium-dependent glutathione-peroxidase (total GPx; Se-GPx) activities. Exposure to acetaminophen caused a significant inhibition of AChE and Se-GPx activities in D. magna relative to the control. Among the biomarkers of oxidative stress, only the activity of CAT was significantly altered in concentration of 0.001mg L(-1) of chlorpromazine, which was not always consistent with the literature. Diclofenac caused a significant inhibition of AChE and Se-dependent GPx, and also in total GPx activities. Propranolol was responsible for a significant decrease in the activity of the latter two enzymes, and also a slight increase of GSTs activity. The results indicated that the exposure to all the tested compounds induced alterations on the cellular redox status in the studied species. In addition, acetaminophen and diclofenac were shown to have the capability of interfering with D. magna neurotransmission, through the inhibition of ChEs. Our data enlighten the need for more research on the ecological consequences of pharmaceuticals in non-target organisms.

Alterations in gills of Lepomis gibbosus, after acute exposure to several xenobiotics (pesticide, detergent and pharmaceuticals): morphometric and biochemical evaluation.

In recent decades, scientific research about the effects of anthropogenic xenobiotics on non-target organisms has increased. Among the likely effects, some studies reported the evaluation of biochemical and morphological changes in specific tissues or organs of fishes, such as gills, which are key organs for the direct action of pollutants in the aquatic environment. This work intended to assess biochemical [oxidative stress/phase II conjugation isoenzymes glutathione S-transferase (GSTs)] and morphological [secondary lamellar length (SLL), secondary lamellar width (SLW), interlamellar distance (ID), basal epithelial thickness (BET) and proportion of the secondary lamellae available for gas exchange (PAGE)] changes in gills, after acute exposure to the pesticide chlorfenvinphos, the detergent sodium dodecylsulphate (SDS) and to the anticholinesterasic pharmaceuticals (neostigmine and pyridostigmine). Our results point to a significant, eventually hormetic, effect in the activity of GSTs following exposure to chlorfenvinphos that significantly increased the activity of GSTs at concentration of 0.2 mg/L. The activity of GSTs increased significantly after exposure to 100 mg/L of neostigmine. Considering the morphometric analysis of the gills, the data obtained showed that chlorfenvinphos exerted mainly minor architectural alterations in gills, with the exception of the highest tested concentration of chlorfenvinphos that produced also a slight decrease of the PAGE. The overall conclusions point to a null or negligible toxicity of the selected toxicants towards L. gibbosus, which may be reverted if exposure is withdrawn.

Toxic potential of paracetamol to freshwater organisms: a headache to environmental regulators?

Paracetamol is one of the most prescribed drugs globally, due to its antipyretic and analgesic properties. However, it is highly toxic at elevated doses, with involvement of an already described oxidative stress pathway. Despite this, the number of ecotoxicological studies on potential effects of paracetamol in wild organisms is still scarce. The present article presents a comprehensive series of standardized assays for the assessment of paracetamol effects in freshwater organisms. The results show that paracetamol toxicity is widely variable among species, even when these species are phylogenetically related. Furthermore, comparisons between data from the literature and our results reinforce this conclusion, providing evidence of the inadequacy of standardized toxicity testing guidelines for pharmaceutical compounds in wild organisms. Paracetamol toxicity can be modulated by unpredictable physiological conditions that might compromise extrapolations and comparisons of responsiveness among species. The ecological relevance of data obtained from classical tests for this compound is further discussed.

From bacteria to fish: ecotoxicological insights into sulfamethoxazole and trimethoprim.

Sulfamethoxazole (SMX) and trimethoprim (TRIM) are two of the most used antibiotics in the last 50 years, to prevent and treat bacterial infections; however, the available literature about toxicity to non-target organisms is quite discrepant and incomplete. This study aims to assess the SMX and TRIM ecotoxicological effects in standard species: Aliivibrio fischeri (bioluminescence inhibition), Escherichia coli ATCC 25922 (growth inhibition), Lemna minor (growth inhibition and biochemical biomarkers), Daphnia magna (immobilization/mortality, life history traits, and biochemical biomarkers), and Danio rerio (survival, hatching, abnormalities, and biochemical biomarkers). The species tested showed different acute sensitivities to SMX (A. fischeri < D. magna < E. coli < L. minor) and TRIM (L. minor < A. fischeri < D. magna < E. coli). Overall, TRIM reveals less toxicity than SMX, except for E. coli (Ecotoxicological approach based on Antimicrobial Susceptibility Testing - EcoAST procedure). Both antibiotics affect individually (e.g., growth and survival) and sub-individually (e.g., antioxidant defenses) L. minor, D. magna, and D. rerio. This study allowed us to generate relevant data and fill gaps in the literature regarding the effects of SMX and TRIM in aquatic organisms. The here-obtained results can be used to (i) complete and re-evaluate the Safety Data Sheet to improve the assessment of environmental safety and management of national and international entities; (ii) clarify the environmental risks of these antibiotics in aquatic ecosystems reinforcing the inclusion in the 4th Watch List of priority substances to be monitored in whole inland waters by the Water Framework Directive; and (iii) combat the development of antimicrobial resistance, as well as supporting the definition of environmental measurements in the context of European One Health Action Plan. However, it is essential to continue studying these antibiotics to better understand their toxicity at ecologically relevant concentrations and their long-term effects under different climatic change scenarios.

Biopesticide spinosad: Unraveling ecotoxicological effects on zebrafish, Danio rerio.

Biopesticides are natural compounds considered more safe and sustainable for the environment. Spinosad (SPI) is a bioinsecticide used in marketed worldwide, to eradicate a variety of pests. This study aimed to assess the impacts of the SPI on the non-target organism zebrafish (Danio rerio). Several concentrations of SPI were tested to evaluate the acute (0.07-1.0 mg/L) and chronic (0.006-0.100 mg/L) ecotoxicological effects. To evaluate sub-individual effects, antioxidant defense, lipid peroxidation, energy sources, and cholinergic biomarkers were quantified. In both exposures, SPI induced significant effects on antioxidant defense indicating oxidative stress, disrupting energy pathways, and exhibiting neurotoxic effects, under environmentally relevant conditions. Integrated Biomarker Response (IBRv2) showed that with increasing SPI concentrations, an increase in impacts on organisms was recorded. This study demonstrates the vulnerability of a non-target organism to SPI, a bioinsecticide considered environmentally safe. Further research is essential to fully understand the implications of spinosad to aquatic biota.

Assessment of ecotoxicological effects of Fojo coal mine waste elutriate in aquatic species (Douro Coalfield, North Portugal).

Introduction: The exploitation of anthracite A in the Pejão mining complex (Douro Coalfield, North Portugal) resulted in the formation of several coal waste piles without proper environmental control. In 2017, a new pedological zonation emerged in the Fojo area, after the ignition and self-burning of some of the coal waste piles, namely: unburned coal waste (UW); burned coal waste, and a cover layer (BW and CL, respectively); uphill soil (US); mixed burned coal waste (MBW); downhill soil (DS). This study aimed to evaluate the toxic effects of 25 soil elutriates from different pedological materials. Methods: Allivibrio fischeri bioluminescence inhibition assay, Lemna minor growth inhibition assay, and Daphnia magna acute assay were used to assess the toxicity effects. Additionally, total chlorophyll and malondialdehyde (MDA) content and catalase (CAT) activity were also evaluated in L. minor. Results and Discussion: The results obtained from each endpoint demonstrated the extremely heterogeneous nature of soil properties, and the species showed different sensibilities to soil elutriates, however, in general, the species showed the same sensitivity trend (A. fischeri > L. minor > D. magna). The potentially toxic elements (PTE) present in the soil elutriates (e.g., Al, Pb, Cd, Ni, Zn) affected significantly the species understudy. All elutriates revealed toxicity for A. fischeri, while US1 and UW5 were the most toxic for L. minor (growth inhibition and significant alterations in CAT activity) and D. magna (100% mortality). This study highlights the importance of studying soil aqueous phase toxicity since the mobilization and percolation of bioavailable PTE can cause environmental impacts on aquatic ecosystems and biota.

Community-level effects in edaphic fauna from an abandoned mining area: integration with chemical and toxicological lines of evidence.

As a part of the Ecological Risk Assessment of a deactivated uranium mining area (Cunha Baixa), the aim of this study was to assess the drivers of litter arthropod community (ecological line of evidence) inhabiting soils with different degrees of contamination. Litter arthropods were collected in the mining area using a total of 70 pitfall traps, in the spring and autumn of 2004. Unlike information previously collected in the chemical and ecotoxicological lines of evidence, we found no clear evidence of impacts of soil contamination on the edaphic arthropod assemblage. Multivariate analyses were unable to extract relevant environmental gradients related to contamination, as most of the sites shared the same taxa overall. Given the consistency of the chemical and ecotoxicological lines of evidence, we must conclude that the litter arthropod assemblage underestimated the impacts of contamination in this abandoned mining area. In part, this could be due to the uncertainty caused by confounding factors that affect the litter arthropod community in the area. Nevertheless, despite the overall lack of responsiveness of the epigeic arthropod community data, a few taxa were negatively correlated with metal concentrations (Clubionidae and Staphylinidae), while Pseudoscorpionida were associated with the toxicological profile of the sites. These evidences suggest that community-level approaches with other animal and plant assemblages are necessary to reduce uncertainty relatively to the assessment of risks in higher evaluation tiers in the Cunha Baixa mine area.

The Silent Threat: Exploring the Ecological and Ecotoxicological Impacts of Chlorinated Aniline Derivatives and the Metabolites on the Aquatic Ecosystem.

The growing concern over the environmental impacts of industrial chemicals on aquatic ecosystems has prompted increased attention and regulation. Aromatic amines have drawn scrutiny due to their potential to disturb aquatic ecosystems. 4-chloroaniline and 3,4-dichloroaniline are chlorinated derivatives of aniline used as intermediates in the synthesis of pharmaceuticals, dyes, pesticides, cosmetics, and laboratory chemicals. While industrial applications are crucial, these compounds represent significant risks to aquatic environments. This article aims to shed light on aromatic amines' ecological and ecotoxicological impacts on aquatic ecosystems, given as examples 4-chloroaniline and 3,4-dichloroaniline, highlighting the need for stringent regulation and management to safeguard water resources. Moreover, these compounds are not included in the current Watch List of the Water Framework Directive, though there is already some information about aquatic ecotoxicity, which raises some concerns. This paper primarily focuses on the inherent environmental problem related to the proliferation and persistence of aromatic amines, particularly 4-chloroaniline and 3,4-dichloroaniline, in aquatic ecosystems. Although significant research underscores the hazardous effects of these compounds, the urgency of addressing this issue appears to be underestimated. As such, we underscore the necessity of advancing detection and mitigation efforts and implementing improved regulatory measures to safeguard the water bodies against these potential threats.

Phytotoxicity of coal waste elutriates (Douro Coalfield, North Portugal) in Lactuca sativa.

One of the most important mining areas in the Douro Carboniferous Basin is the Pejão Coalfield. In the summer of 2017, a wildfire promoted the ignition and self-burning some of the coal waste piles in the area and caused important environmental changes, promoting a new heterogenic pedological zonation. This study aims to assess the ecotoxicological effects of 25 soil elutriates from these different soil types in seed germination and individual (emergence, growth, and morphologic alterations) and subindividual parameters in Lactuca sativa. The different evaluated endpoints were differently affected regarding the soil elutriate revealing the high heterogeneity of soil characteristics. The presence of different potentially toxic elements (e.g., Cd, Cr, Pb, Zn) in soil elutriates, even in low concentrations, caused effects on L. sativa development. Unburned coal wastes and downhill soil elutriates were able to inhibit the germination of L. sativa and affect them individually and sub-individually (decrease in size, biomass, and presence of morphological alterations). Additionally, it was observed that all soil elutriates induce a decrease in root size. The results highlight the importance of using elutriate samples in phytotoxicity studies of coal mining waste, since the tailings lixiviate may reduce plant establishment and growth, affecting the terrestrial ecosystems. The integrated use of seed germination assays with the analysis of morphological and biochemical alterations in plants proved to be sensitive parameters to evaluate the phytotoxicity of coal mining wastes.

A forecast effects of climate change and anthropogenic compounds in Gambusia holbrooki: ecotoxicological effects of salinity and metformin.

Due to global warming and extreme weather events, estuarine and coastal ecosystems are facing sudden fluctuations in salinity. These ecosystems are also threatened by organic and inorganic compounds that increase water pollution. Metformin is an antidiabetic drug commonly used by patients with type-2 diabetes, and an increase in environmental concentration has been recorded. To better understand the impacts of these two stressors on aquatic organisms, this study assessed: 1) the acute (96 h) ecotoxicological effects (antioxidant and biotransformation capacity, oxidative damage, energetic reserves, and protein content, neurotoxicity) induced by a range of metformin concentrations in Gambusia holbrooki under different salinities (17, 24, 31 expressed as Practical Salinity Units - PSU); and 2) the same endpoints after chronic exposure (28 d) under a range of metformin concentrations at a salinity of 17. The results obtained from the acute exposure showed interactions between salinity and metformin in G. holbrooki superoxide dismutase (SOD) activity, body protein, and glycogen (GLY) contents. The results revealed that an increase in salinity can modulate the response of G. holbrooki to metformin. Chronically exposed organisms showed that metformin led to a significant decrease in SOD activity at most of the tested concentrations (0.5, 1.0, and 10 µg/L). In addition, glutathione S-transferases increased and glutathione peroxidase activity decreased significantly at concentrations of metformin of 5 and 10 at the µg/L, respectively. Therefore, overall, metformin can lead to potential oxidative stress in G. holbrooki the highest metformin concentrations tested and the GLY content in G. holbrooki increased after exposure to metformin concentrations of 0.5, 1.0 and 5.0 µg/L. Published studies have already shown that metformin alone can lead to oxidative damage in aquatic species, endangering the biodiversity of aquatic ecosystems. Therefore, additional ecotoxicological studies should be performed to characterize if other metformin concentrations combined with salinity, or other climate change-related factors, might impact non-target species. Standard toxicity bioassays may not be predictive of actual pollutants (e.g. metformin) toxicity under variable environmental conditions, and the investigation of a wider range of exposure conditions could improve the accuracy of chemical risk assessments.

Insights into environmental caffeine contamination in ecotoxicological biomarkers and potential health effects of Danio rerio.

Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 µg/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (superoxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 µg/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 µg/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments.

Are metallothioneins equally good biomarkers of metal and oxidative stress?

Several researchers investigated the induction of metallothioneins (MTs) in the presence of metals, namely Cadmium (Cd). Fewer studies observed the induction of MTs due to oxidizing agents, and literature comparing the sensitivity of MTs to different stressors is even more scarce or even nonexistent. The role of MTs in metal and oxidative stress and thus their use as a stress biomarker, remains to be clearly elucidated. To better understand the role of MTs as a biomarker in Cerastoderma edule, a bivalve widely used as bioindicator, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. Results showed that induction of MTs occurred at very different levels in metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress was very high, and mortality rates were higher than 50 percent. On the contrary, C. edule survived to all Cd concentrations used and significant MTs increases, compared to the control, were observed in all Cd exposures. The present work also revealed that the number of ions and the metal bound to MTs varied with the exposure conditions. In the absence of disturbance, MTs bound most (60-70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind to Cu and bound less to Zn. When organisms were exposed to Cd, the total number of ions bound per MT molecule did not change, compared to control. However the sort of ions bound per MT molecule differed; part of the Zn and all Cu ions where displaced by Cd ions. For organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to control and Cd conditions, which indicates a partial oxidation of thiol groups in the cysteine residues through ROS scavenging. The present results suggest that MTs are excellent markers of metal stress, but not of oxidative stress.

Effects of anticholinesterase drugs on biomarkers and behavior of pumpkinseed, Lepomis gibbosus (Linnaeus, 1758).

The presence of pharmaceutical residues in the aquatic environment has recently received great attention, as potential adverse effects may arise from their presence. Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. However, other widespread anthropogenic contaminants - including pharmaceutical drugs - can exert toxic effects through ChE inhibition. Studies with aquatic species have shown that inhibition of ChE is associated with behavioral changes. Bearing this in mind, this work aimed to study the effects on individual behavior and acetylcholinesterase (AChE) activity of selected tissues of Lepomis gibbosus, after exposure to the anticholinesterasic drugs neostigmine and pyridostigmine. Results revealed that neostigmine significantly decreased the activity of AChE in the head (LOEC = 100 mg L(-1)), but not in dorsal muscle. On the other hand, pyridostigmine significantly decreased the activity of AChE in both the head (LOEC = 0.001 mg L(-1)) and dorsal muscle homogenates (LOEC = 100 mg L(-1)). The impairment of this enzymatic form was attained at ecologically relevant concentrations (in the case of pyridostigmine, for head AChE), which is an interesting finding, specially considering that these toxic effects occurred for a pharmaceutical compound. Contrarily, there were no significant differences in the behavior of L. gibbosus in any parameter, for neither drug. These results suggest that the behavioral parameters analyzed (scototaxis and lethargy) in L. gibbosus could not be regarded as suitable markers to assess the effects of drugs such as neostigmine and pyridostigmine. In contrast, the pattern of response elicited by cholinesterasic inhibition showed the usefulness of this toxicological parameter for the assessment of pharmaceuticals in the environment, namely anticholinesterasics.

Cholinesterase characterization in Corbicula fluminea and effects of relevant environmental contaminants: a pesticide (chlorfenvinphos) and a detergent (SDS).

This study characterizes the cholinesterasic forms present in the bivalve Corbicula fluminea, through the analysis of differential affinity towards various substrates and inhibitors. This approach also permitted the validation of cholinesterase inhibition in the mentioned species for monitoring purposes by evaluating the inhibitory activity of a detergent (SDS) and a pesticide (chlorfenvinphos), both in vitro and in vivo. The cholinesterasic form present in the tissues of C. fluminea showed intermediate properties in terms of its relation with substrates and inhibitors. Furthermore, it was not inhibited by the detergent SDS under any of the test conditions, but was clearly inhibited by the selected organophosphate. The data show clearly that the main cholinesterasic form in C. fluminea is responsive to the organophosphate chlorfenvinphos, thus supporting the use of this biomarker for the environmental monitoring of specific contaminants such as anticholinesterasic pesticides. Despite not being commonly attained in the wild, the levels of chlorfenvinphos required to elicit a significant ChE inhibition were only one order of magnitude above actual values of aquatic contamination by this pesticide.