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Every day, people use personal care products containing UV filters. Although their use initially showed a protective role, toxicity is a concern for human health as several UV filters are endocrine-disrupting chemicals (EDCs). Exposure to EDCs may induce cardiovascular diseases and can affect the health of sensitive people, such as pregnant women. Currently, the world’s most widely used UV-B filter is octylmethoxycinnamate (OMC), an EDC. However, the disruptive effects on pregnant women are little known. The present work proposed to understand how long-term exposure to OMC affects vascular homeostasis. Endothelium-denuded human umbilical artery (HUA) rings were incubated in an organ bath system. Long-term effects of exposure to OMC (0.001–50 μmol/L) were evaluated on the contractile responses of HUA to the application of the contractile agents, serotonin (5-HT) and histamine (Hist). To investigate in more detail the vascular mode of action of OMC, through which it impairs the vascular homeostasis of HUA, the activity and expression of different 5-HT and Hist-receptors involved in contractility processes were studied. Our findings pointed out an increase in the reactivity of HUA to 5-HT and Hist due to OMC exposure. These alterations in reactivity may be precursors of preeclampsia development and/or gestational hypertension.

Ultraviolet (UV) filters are widely used in personal care products and due to their lipophilicity these chemicals tend to bioaccumulate in the aquatic biota. 4-Methylbenzylidene camphor (4-MBC) is one of the most used UV-filters, and it is commonly detected in freshwater fish tissues. This substance is suspected to be an endocrine disruptor due to its interaction with Hypothalamus-Pituitary-Gonadal (HPG) and HP-Thyroid (HPT)-axis. The main objective of this study was to evaluate the effects of 4-MBC on apical endpoints, biochemical markers and on genes involved in endocrine pathways in Danio rerio. Zebrafish embryos were exposed to 4-MBC (0.083–0.77 mg/l) from 0 to 96 h post-fertilization (hpf). Hatching, heart rate and malformations were the apical endpoints assessed. Alterations on neurotransmission and oxidative stress were evaluated through acetylcholinesterase (AChE), catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. Endocrine effects were analysed by the expression of genes involved in HPG and HPT-axis of embryos exposed 96 h to the EC10 of 4-MBC (0.19 mg/l). Exposure to 4-MBC induced morphological abnormalities during embryonic development, including notochord curvature, delayed absorption of yolk sac and pericardial oedema. Concentration of 0.77 mg/l 4-MBC decreased embryo heart rate at 48h. At neurotransmission level, an induction of AChE at concentrations above 0.15 mg/l was observed. Malformations and decreased heart rate along with alterations observed at neurotransmission level might have compromised zebrafish larvae equilibrium. Glutathione S-transferase induction above 0.15 mg/l 4-MBC suggests activation of detoxification processes. Furthermore, observed brain aromatase gene down-regulation by 4-MBC suggests impairment of normal functioning of HPG axis in zebrafish.

Polychlorinated biphenyls (PCBs) are a class of industrial chemicals that cause endocrine changes, since they are able to bind to estrogen receptors and interfere with estrogen-regulated processes, such as fish vitellogenesis. Therefore, the present work aimed to assess potential endocrine effects of PCB-77 exposure during zebrafish (Danio rerio) gonadal differentiation. To achieve that, zebrafish juveniles were exposed to increasing concentrations of PCB-77 for 14 days during a critical window of gonad differentiation (30-44 days post-fertilization). Vitellogenin (Vtg) levels and several endpoints such as survival, growth, gonadosomatic index (GSI) and hepatosomatic index were recorded at the end of exposure and/or after 3 months in clean medium. The results obtained showed a reduction of Vtg-like proteins in juveniles, just after exposure to PCB-77 accompanied, after 3 months, by a decrease in gonadal Vtg levels and GSI of females. These results suggest that exposure to PCB-77 during the critical window of gonadal differentiation decreased vitellogenesis in juvenile zebrafish which lasted until adulthood affecting the normal development of female gonad, which might have further implications in reproduction success.

Increasing evidence relating the exposure and/or bioaccumulation of endocrine-disrupting compounds (EDCs) with cardiovascular system are arising. Octylmethoxycinnamate (OMC) is the most widely used UV-B filter and as EDC interacts with TH receptors. However, their effects on thyroid diseases during pregnancy remain unknown. The purpose of this work was to assess the short- and long-term effects of OMC on arterial tonus of pregnant women with hypothyroidism. To elucidate this, human umbilical artery (HUA) rings without endothelium were used to explore the vascular effects of OMC by arterial and cellular experiments. The binding energy and the modes of interaction of the OMC into the active center of the TSHR and THR&alpha, were analyzed by molecular docking studies. Our results indicated that OMC altered the contractility patterns of HUA contracted with serotonin, histamine and KCl, possibly due to an interference with serotonin and histamine receptors or an involvement of the Ca2+ channels. The molecular docking analysis show that OMC compete with T3 for the binding center of THR&alpha, Taken together, these findings pointed out to alterations in HUA reactivity as result of OMC-exposure, which may be involved in the development and increased risk of cardiovascular diseases.

Gold nanorods (AuNR) have been explored for many applications, including innovative nanomedicines, which also might contribute to its increase in the environment, namely due to inadequate disposable of wastes into aquatic environments. Early-life stages of amphibians are usually aquatic and sensitive to chemical contamination. Accordingly, this study aimed to determine the sublethal effects of CTAB functionalized AuNR on Xenopus laevis tadpoles. As such, tadpoles were exposed to serial concentrations of AuNR for 72 h. A reduction in the rate of feeding (EC50 = 4 μg.L−1), snout to vent growth (EC50 = 5 μg.L−1) and weight gain (EC50 = 6 μg.L−1), was observed for AuNR-exposed tadpoles. Also, tadpoles actively avoided concentrations ≥ 4 μg.L-1 of AuNR, after 12 h of exposure. At the biochemical level, AuNR caused impairments in antioxidant and nervous system related enzymes. Exposure to CTAB alone caused a high mortality. Results indicated that CTAB functionalized AuNR may induce several sublethal effects that may compromise the organism’s fitness. Avoidance behavior (which corresponds to the disappearance of organisms, thus, similar to their death), observed at concentrations matching those inducing sublethal effects, suggest that it should be considered in the risk assessment for amphibians.

Personal care products (PCPs) are a group of diverse substances widely used daily for health, beauty, and cleanliness. More than 90% of all PCPs contain the UV-B filter octylmethoxycinnamate (OMC) as a protective function, however, their safety has recently been questioned. The purpose of the present work was to understand how the long-term exposure of UV-filter OMC, used daily by pregnant women, disrupts their vascular homeostasis, altering vascular responses of proteins and channels involved in contractile processes. The long-term effects of 24 h of exposure to OMC (1, 10, and 50 μmol/L) were evaluated on contractile responses of human umbilical arteries (HUA) to serotonin and potassium chloride. Since OMC altered vascular homeostasis of arteries, its vascular mode of action was explored in more detail through the analysis of the activity of cGMP and Ca2+-channels, two pathways involved in their relaxation and contraction, respectively. Our findings showed that long-term exposure of UV-filter OMC impaired the main vasorelaxant mechanism of HUA, once OMC altered the vasorelaxant response pattern of sodium nitroprusside and nifedipine. Results also showed that long-term exposure to OMC induced a decreased vasorelaxation response on HUA due to an interference with the NO/sGC/cGMP/PKG pathway. Moreover, OMC seems to modulate the L-type Ca2+ channels, the BKCa 1.1 α-subunit channels, and the PKG. Overall, since OMC compromises the vascular homeostasis of pregnant women it can be an inductor of pregnancy hypertensive disorders.

Pyrethroid insecticides have been used for decades and their worldwide market continues to increase, despite their high toxicity to non-target insects. Recent studies reveal that it is essential to investigate the secondary mechanisms of action of type II pyrethroids to understand their cellular effects on invertebrates. The aim of this study was to evaluate the lethality, behaviour and physiological alterations and energetic costs in caddisfly larvae exposed to environmentally relevant concentrations of esfenvalerate (ESF). ESF caused both mortality and feeding inhibition of exposed caddisfly larvae: nominal ESF 96 h LC50 was 2.29 μg/L; feeding activity was impaired at concentrations equal or above 0.25 μg/L. At the cellular level, glutathione-S-transferase (GST) activity was increased on caddisfly larvae exposed to 0.25 and 0.5 μg/L ESF, which might contribute to prevent oxidative damage since levels of lipid peroxidation (LPO) were not altered. The energy budget of exposed caddisfly larvae was impaired by exposure to 0.25 μg/L ESF since sugar and protein contents decreased, while a decline of energy consumption was observed. The analysis of feeding, energy reserves and consumption data through structural equation modelling (SEM) allowed to quantify the direct and indirect effects of ESF exposure on bioenergetics of caddisfly larvae. SEM analysis showed a strong negative direct influence of ESF onto feeding activity, sugars content and energy consumption, highlighting a significant positive relationship between sugars and protein contents. These results show that energy expenditure is related to oxidative defense mechanisms induced by ESF stress that may lead to deleterious effects on growth and development.

Despite the frequent detection of organic ultraviolet-filters (UV-filters) in freshwater sediments, there is a lack of ecotoxicological data undermining a correct risk assessment for these emerging contaminants. The present study assessed the effects of three of the most commonly used UV-filters (benzophenone-3 - BP3; 3-(4-methylbenzylidene)camphor - 4-MBC and octocrylene - OC) on Chironomus riparius life history and biochemical responses. Standard ecotoxicological assays confirmed that all compounds impaired growth of C. riparius larvae and induced developmental effects such as delayed emergence and a reduction of imagoes weight. Concerning the biochemical responses analysed no evidences of oxidative damage in lipids or neurotoxicity (tested assessing acetylcholinesterase activity) were observed for any of the tested compounds. However, 4-MBC exposure induced a decrease in catalase activity and an increase in glutathione-S-transferase activity at 14.13mg/Kg while OC exposure caused an increase in total glutathione levels at 0.23 and 18.23mg/Kg. Exposure to all UV-filters tested, increased energy consumption on C. riparius with significant differences above 1.00mg/Kg for BP3, 0.09mg/Kg for 4-MBC and 2.13mg/Kg for OC. These results suggest that environmental relevant concentrations of UV-filters can cause deleterious effects to aquatic benthic species, such as C. riparius, and call for further research concerning effects of organic UV-filters on natural invertebrate communities and ecosystem functioning.

Ultraviolet radiation (UVR) reaching the Earth surface is increasing and scarce information is available regarding effects of this stressor to early life stages of marine vertebrates. Therefore, this work aims to study the effects of UVR exposure during early development stages of the flatfish Solea senegalensis. Firstly, fish were exposed to UVR (six daily doses between 3.4 ± 0.08 and 8.6 ± 0.14 kJ m−2) at the following moments: gastrula stage (24 h post fertilization, hpf), 1 and 2 days after hatching (dah, 48 and 72 hpf, respectively). In a second bioassay, fish at the beginning of metamorphosis were exposed to UVR (one or two daily doses of 7.2 ± 0.39 or 11.1 ± 0.49 kJ m−2) and then maintained until the end of metamorphosis. Mortality and effects on development, growth and behaviour were evaluated at the end of both bioassays (3 dah and 18 dah, respectively). Biomarkers of neurotransmission (acetylcholinesterase, AChE), oxidative stress (catalase, CAT) and biotransformation (glutathione S-transferase, GST) were also determined at the end of the early larvae bioassay, and metamorphosis progression was evaluated during the second bioassay. UVR exposure caused distinct effects depending on life stage. Altered pigmentation, decreased growth, impaired fish behaviour and AChE and GST inhibition were observed at the earlier larval phase. Whereas, decrease in growth was the main effect observed at the metamorphosis stage. In summary, the exposure of S. senegalensis early stages to environmentally relevant UVR doses led to adverse responses at different levels of biological organization, which might lead to implications in later life stages.

Caddisflies have been included in ecotoxicological studies because of their sensitivity and ecological relevance. The present study aimed to assess the sublethal effects of an anthranilic diamide insecticide, chlorantraniliprole (CAP), to Sericostoma vittatum. Used worldwide, CAP is a persistent compound that has been found in surface waters at concentrations from 0.1 μg/L to 9.7 μg/L. It targets the ryanodine receptors, and the present ecotoxicological assessment focused on biomarkers related to neurotransmission, biotransformation, oxidative stress damage, and endpoints related to energy processing (feeding, energy reserves, and cellular metabolism). Six days of exposure trials revealed that feeding activity was significantly decreased in S. vittatum larvae exposed to 0.9 μg/L CAP. Concomitantly, a reduction in cellular metabolism and a significant decrease in protein content were also observed in caddisfly larvae exposed to CAP, suggesting metabolic depression. The results show that sublethal concentrations of CAP can cause detrimental sublethal effects on S. vittatum total glutathione content at concentrations as low as 0.2 μg/L. Bioenergetics can be used to assess physiological effects of contaminants, and the present results show that exposure to low, environmentally relevant, concentrations of CAP alter energy acquisition and metabolism in nontarget aquatic insects with potential population level effects. Environ Toxicol Chem 2017;36:1584-1591. © 2016 SETAC.

Stream ecosystems face ever-increasing pressures by the presence of emergent contaminants, such as, personal care products. N, N-diethyl-3-methylbenzamide (DEET) is a synthetic insect repellent that is being found in surface waters environments in concentrations up to 33.4 μg/L. Information concerning DEET's toxicity in the aquatic environment is still limited and focused only on its acute effects on model species. Our main objective was to assess the effects of DEET exposure to a caddisfly non-target species using sub-lethal endpoints. For that, we chose Sericostoma vittatum, an important shredder in Portuguese freshwaters that has been already used in different ecotoxicological assays. Besides acute tests, S. vittatum were exposed during 6 days to a gradient of DEET concentrations (8, 18 and 40.5 mg/L) to assess effects on feeding behaviour and biochemical responses, such as, lipid peroxidation levels (LPO), catalase and acetylcholinesterase (AChE) activities, and also assess effects on energy reserves and consumption. Acute tests revealed a 48 h-LC50 of 80.12 mg/L and DEET exposure caused feeding inhibition with a LOEC of 36.80 mg/L. Concerning the biochemical responses, DEET caused no effects in LPO nor on catalase activity. A non-significant decrease in AChE activity was observed. Regarding energetic reserves, exposure to DEET caused a significant reduction in S. vittatum carbohydrates levels. These results add important information for the risk assessment of insect repellents in the aquatic environment and suggest that reported environmental concentrations of DEET are not toxic to non-target freshwater insects.

Freshwater ecosystems are essential for humans; however, input of several types of contamination has led to the degradation of these ecosystems. Thus, it is urgent to assess their health to allow actions for prevention and remediation. The level of trace metals can be enhanced by natural or anthropogenic sources. Essential metals, such as copper and zinc, become toxic when present in the environment above threshold concentrations. To evaluate the physiological effects of these 2 essential metals for 2 freshwater detritivores, the shrimp Atyaephyra desmarestii and the amphipod Echinogammarus meridionalis, acute tests were performed. Forty-eight hour median lethal concentration (LC50) values were estimated for these species using static bioassays with copper and zinc. Sublethal assays for both metals with several phases were also done to evaluate the effects on feeding behavior. The LC50 values of copper for the shrimp A. desmarestii and amphipod E. meridionalis were 0.128 mg/L and 0.050 mg/L and those of zinc were 7.951 mg/L and 11.860 mg/L, respectively. The results indicated that copper is more toxic to both species. Only E. meridionalis showed deleterious effects of copper on feeding rate. Zinc showed some tendency for feeding inhibition in both species. Environ Toxicol Chem 2016;35:1442-1448. © 2015 SETAC.

Some sacoglossan sea slugs incorporate intracellular functional algal chloroplasts (kleptoplasty) for periods ranging from a few days to several months. Whether this association modulates the photobehaviour of solar-powered sea slugs is unknown. In this study, the long-term kleptoplast retention species

Presence of pesticides in the environment and their possible effects on aquatic organisms are of great concern worldwide. The extensive use of herbicides in agricultural areas are one of the factors contributing to the known decline of amphibian populations. Thus, as non-target species, amphibians can be exposed in early life stages to herbicides in aquatic systems. In this context, this study aims to evaluate effects of increasing concentrations of two maize herbicides, linuron and S-metolachlor on embryos of the Perez' frog (Pelophylax perezi) during 192 h. Apical endpoints were determined for each herbicide: mortality, hatching rate, malformations and length. Frog embryos presented a LC50 of 21 mg/l linuron and 37.5 mg/l S-metolachlor. Furthermore, sub-lethal concentrations of both herbicides affected normal embryonic development, delaying hatching, decreasing larvae length and causing several malformations. Length of larvae decreased with increasing concentrations of each herbicide, even at the lower concentrations tested. Malformations observed in larvae exposed to both herbicides were oedemas, spinal curvature and deformation, blistering and microphtalmia. Overall, these results highlight the need to assess adverse effects of xenobiotics to early life stages of amphibians regarding beside mortality the embryonic development, which could result in impairments at later stages. However, to unravel mechanisms involved in toxicity of these herbicides further studies regarding lower levels of biological organisation such as biochemical and genomic level should be performed.

The widespread and increasing use of personal care products (PCPs) have led to environmental contamination by substances included in these products. These substances have been detected in aquatic compartments and shown to cause adverse effects on non-target aquatic organisms. In this work toxicity of the antimicrobial triclosan (TCS) and of the UV-filter 3-(4-methylbenzylidene) camphor (4-MBC) was assessed in the embryos of Perez' frog Pelophylax perezi. Lethal and sub-lethal parameters were evaluated in embryos in Gosner stage 8-9 exposed to 0.00013-1.3 mg/l of 4-MBC and 0.25-2.50 mg/l of TCS during 144 h. Survival, malformations, length and hatching were evaluated as apical endpoints. Biomarkers of neurotransmission, oxidative stress, energy metabolism and estrogenicity were determined at the biochemical level through the activities of cholinesterase (ChE), catalase (CAT), glutathione S-transferase (GST), lactate dehydrogenase (LDH) and levels of lipid peroxidation (LPO) and vitellogenin (Vtg). Embryo exposure to 4-MBC led to few developmental malformations (up to 3%) and a GST induction at 0.013 mg/l. Triclosan exposure reduced survival, delayed hatching (at 72 h) and development and induced malformations. In addiction ChE was inhibited in the highest concentrations tested and GST and LDH were induced at 0.79 mg/l, the LOEC registered for TCS in Perez' frogs. Overall, our study showed that TCS might exert adverse effects on P. perezi early life stages, but only at four orders of magnitude above the concentrations found in environment. Furthermore, our results highlight the need to assess PCPs toxicity at different levels of biological organization.

Evaluation of the effects of linuron and S-metolachlor on apical, biochemical and transcriptional endpoints in zebrafish (Danio rerio) early life stages was the main purpose of this work. Embryos were exposed for 96h to a range of concentrations of each herbicide to determine lethal and sub-lethal effects on apical (e.g. malformations, hatching) and biochemical parameters (cholinesterase, ChE; catalase, CAT; glutathione S-transferase, GST; lipid peroxidation, LPO and lactate dehydrogenase, LDH). To evaluate endocrine disruption effects, embryos were exposed during 96h to 0.88mg/L linuron and 9.66mg/L S-metolachlor, isolated or in binary mixture. Expression of a suite of genes involved in HPT, HPG and HPA-axis was then assessed. Highest concentration of linuron (5.0mg/L) decreased hatching rate to 5% and 70.0mg/L S-metolachlor completely inhibited hatching, about 100%. Both herbicides impaired development by inducing several malformations (100% in 5.0mg/L linuron and 70.0mg/L S-metolachlor). Linuron only affected GST and CAT at concentrations of 0.25 and 0.0025mg/L, respectively. S-metolachlor induced GST (to 256%), inhibited ChE (to 61%) and LDH (to 60%) and reduced LPO levels (to 63%). Linuron isolated treatment seems to have an estrogenic mode of action due to the observed induction of vtg1. Exposure to S-metolachlor seems to interfere with steroidogenesis and with HPT and HPA-axis, since it has inhibited cyp19a2, TSHβ and CRH gene expression. In addition to vtg1 induction and CRH inhibition, herbicide combination also induced sox9b that has a role in regulation of sexual development in zebrafish. This study pointed out adverse effects of linuron and S-metolachlor, namely impairment of neurotransmission and energy production, induction of steroidogenesis, and interference with HPT and HPA-axis. These results contributed to elucidate modes of action of linuron and S-metolachlor in zebrafish embryo model. Furthermore, gene expression patterns obtained are indicative of endocrine disruption action of these herbicides.

Caddisflies have been included in ecotoxicological studies because of their sensitivity and ecological relevance. The present study aimed to assess the sublethal effects of an anthranilic diamide insecticide, chlorantraniliprole (CAP), to Sericostoma vittatum. Used worldwide, CAP is a persistent compound that has been found in surface waters at concentrations from 0.1 μg/L to 9.7 μg/L. It targets the ryanodine receptors, and the present ecotoxicological assessment focused on biomarkers related to neurotransmission, biotransformation, oxidative stress damage, and endpoints related to energy processing (feeding, energy reserves, and cellular metabolism). Six days of exposure trials revealed that feeding activity was significantly decreased in S. vittatum larvae exposed to 0.9 μg/L CAP. Concomitantly, a reduction in cellular metabolism and a significant decrease in protein content were also observed in caddisfly larvae exposed to CAP, suggesting metabolic depression. The results show that sublethal concentrations of CAP can cause detrimental sublethal effects on S. vittatum total glutathione content at concentrations as low as 0.2 μg/L. Bioenergetics can be used to assess physiological effects of contaminants, and the present results show that exposure to low, environmentally relevant, concentrations of CAP alter energy acquisition and metabolism in nontarget aquatic insects with potential population level effects. Environ Toxicol Chem 2017;36:1584-1591. © 2016 SETAC.

Juveniles of the estuarine fish Pomatoschistus microps were collected from autumn 2001 to summer 2002 in five stations along the Portuguese Northwest coast with different types and/or levels of environmental contamination: two reference sites with low levels of contamination (R1 and R2) and three differently impacted areas with higher levels of contamination. UI is located in an estuary under the influence of urban and industrial effluents, AA in a channel that receives intensive agriculture run-off and IE in a highly impacted industrial area. The activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferases (GST) were used as environmental biomarkers on P. microps. A significant seasonality effect on all the enzymatic activities was found, lower levels being registered in winter and spring on AChE, in autumn on LDH, and in winter on GST and EROD. The battery of biomarkers used was capable of discriminating sites with different types and/or levels of contamination, R1 and UI being the highest discriminated (91.7% and 66.7%, respectively). At R1 significantly lower levels of AChE and LDH were found, and EROD was significantly inducted at UI. Furthermore, IE presented higher levels of GST, and R2 and AA an inhibition of AChE in winter and spring. The results indicated that the battery of biomarkers used in this study seems to be a useful tool to distinguish between different types of environmental contamination in estuarine systems, and that P. microps is a suitable species to be used as bioindicator.

N,N-diethyl-3-methylbenzamide (DEET) is the active ingredient of many commercial insect repellents. Despite being detected worldwide in effluents, surface water and groundwater, there is still limited information on DEET's toxicity toward non-target aquatic invertebrates. Thus, our main objective was to assess the effects of DEET in the life cycle of Chironomus riparius and assess its biochemical effects. Laboratory assays showed that DEET reduced developmental rates (reduced larval growth, delayed emergence) of C. riparius larvae and also caused a decrease in the size of adult midges. Concerning the biochemical responses, a short exposure to DEET caused no effects in lipid peroxidation, despite the significant inhibition of catalase and glutathione-S-transferase activities and of total glutathione contents. Moreover, inhibition of acetylcholinesterase activity was also observed showing neurotoxic effects. Environmental risk assessment of insect repellents is needed. Our results showed moderate toxicity of DEET toward C. riparius, however, due to their mode of action, indirect ecological effects of DEET and of other insect repellents cannot be excluded and should be evaluated.

Integrative studies focused on sub-organismal responses to pyrethroid exposure are important to understand life history responses. In this study, the ecotoxicological effects of esfenvalerate (ESF) on Chironomus riparius were assessed using five biochemical biomarkers related to neurophysiological function (acetylcholinesterase) and oxidative stress (catalase; glutathione-S-transferase; total glutathione and lipid peroxidation). In addition, effects on cellular energy allocation were assessed and all results were compared with organismal level responses (larval growth, emergence and sex ratio). Exposure to sublethal concentrations of ESF caused the failure of C. riparius antioxidant defenses (inhibition of catalase activity and decreased levels of total glutathione), which was reflected as oxidative damage. C. riparius energy budget was decreased by exposure to ESF due to an increased energy consumption. Life cycle tests showed that exposure to ESF impaired C. riparius developmental rates and increased male: female ratios, thereby confirming its toxicity and potential population level effects at environmentally relevant concentrations.

The input of metals into freshwater ecosystems from natural and anthropogenic sources impairs water quality and can lead to biological alterations in organisms and plants, compromising the structure and the function of these ecosystems. Biochemical biomarkers may provide early detection of exposure to contaminants and indicate potential effects at higher levels of biological organisation. The effects of 48h exposures to copper and zinc on Atyaephyra desmarestii and Echinogammarus meridionalis were evaluated with a battery of biomarkers of oxidative stress and the determination of ingestion rates. The results showed different responses of biomarkers between species and each metal. Copper inhibited the enzymatic defence system of both species without signs of oxidative damage. Zinc induced the defence system in E. meriodionalis with no evidence of oxidative damage. However, in A. desmarestii exposed to zinc was observed oxidative damage. In addition, only zinc had significantly reduced the ingestion rate and just for E. meridionalis. The value of the integrated biomarkers response increased with concentration of both metals, which indicates that might be a valuable tool to interpretation of data as a whole, as different parameters have different weight according to type of exposure.

The need to overcome pesticide resistance has led to the development of novel insecticides such as chlorantraniliprole (CAP), an anthranilic diamide. CAP disrupts calcium homeostasis in nerve and muscle cells and is used in several agricultural crops due to its potency and selectivity. However, chronic toxicity data for aquatic invertebrates are limited. Our objective was to evaluate the toxicity of CAP at different levels of biological organization using Chironomus riparius. Organismal endpoints (survival, larval growth and emergence), and 5 biomarkers associated with important physiological functions (acetylcholinesterase - AChE; catalase - CAT; glutathione-S-transferase - GST; total glutathione - TG; and lipid peroxidation - LPO) were investigated. Effects of CAP on cellular energy allocation (CEA) were also assessed. Acute tests revealed a 48 h LC50 for C. riparius of 77.5 μg/L and life-cycle tests revealed a chronic LOEC of 3.1 μg/L based on effects on C. riparius larval growth and emergence. C. riparius females exposed as larvae to low concentrations of CAP emerged at a smaller size which might also translate into effects on reproduction. Chironomid larvae were not under oxidative stress, since short exposures to CAP did not affect LPO levels, despite the significant inhibition of GST (0.6-9.6 μg/L) and CAT (9.6 μg/L). It seems that detoxification of reactive intermediates and ROS is still achieved due to glutathione consumption, since TG levels were significantly decreased in organisms exposed to CAP (0.6-9.6 μg/L). Moreover, it was observed that CEA was disturbed due to increased activity of the electron transport system (ETS), suggesting extra energy expenditure in larvae. These results show that environmental concentrations of CAP can impair the fitness of C. riparius natural populations and at the same time that chironomids, as for most insecticides, are suitable test organisms to evaluate the organismal and biochemical effects of anthranilic diamides.

The equilibrium of the structure and functioning of freshwater ecosystems is dependent of detritivores that link all the other functional groups. The preference for feeding leaves with different diameters (particle size) and leaves with metal contamination (several concentrations of the essential metals copper and zinc) were determined for two detritivores, the decapod Atyaephyra desmarestii and the amphipod Echinogammarus meridionalis. Several no-choice and multi-choice assays were done to determinate which leaf diameter the amphipod and the decapod species would eat when they had or not had alternatives available and include a set of dual-choice assays with contaminated and uncontaminated foods. No significant preference was shown by either species relative to the diameter of leaves, either on no-choice or multi-choice assays. The presence of essential metals on food did not had any influence on the feeding choice of these organisms over the concentration range studied. Both showed no preference on ingesting food spiked with these essential metals, except E. meridionalis which preferred ingesting leaves with 2.19 μg.l(-1) of copper instead of uncontaminated leaves. For further works, despite no preference for leaves with a certain diameter, the leaves with 0.70 cm (0.385 cm(2)of area) and with 0.50 cm (1.767 cm(2) of area) should be used for A. desmarestii and E. meridionalis, respectively. Furthermore, to maintain E. meridionalis, the diet should include some percentage of copper in order to accomplish metabolic needs.

Metals are released into freshwater ecosystems from natural and anthropogenic sources, compromising their structural and functional equilibrium. As early warning tools, cholinesterases (ChEs) are usually used to assess the effects of organophosphate and carbamate pesticides, but are also known to be inhibited by metals. The objectives of this work were to characterise the activity of ChE present in the amphipod Echinogammarus meridionalis and the shrimp Atyaephyra desmarestii and to evaluate the in vivo effects of the metals copper and zinc in their ChE activity. To achieve this, firstly the activity of ChE forms were characterised using different in vitro assays with substrates and selective inhibitors. Then, the in vivo effects of 48 h exposures to increasing concentrations of copper and zinc on ChE activity were determined. The ChE form present in both species was acetylcholinesterase (AChE) since both revealed preference for the acetylthiocholine iodide substrate, total inhibition with eserine, the inhibitor of ChEs, and with 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, the specific inhibitor of AChE, and presented insensitivity to iso-OMPA, a specific inhibitor of butyrylcholinesterase. The activity of ChEs was inhibited by zinc exposures in the amphipod species, but was not affected by copper. Exposure to copper and zinc did not affect ChEs activity in the shrimp at the concentrations tested. This work is a relevant contribution as foundation for the use of AChE in freshwater crustaceans in further studies including biomonitoring campaigns in different contamination scenarios.

In this study, the toxicities of four pesticides (the herbicides atrazine, terbuthylazine, metolachlor and the insecticide chlorpyrifos) previously detected in the Alqueva reservoir/dam (south of Portugal) were evaluated individually and in binary combinations of the herbicides and the insecticide using fourth-instar larvae of the aquatic midge Chironomus riparius. Chlorpyrifos induced toxicity to midges in all the 48 h toxicity bioassays performed. The swimming behaviour of the larvae was impaired, with EC50 values ranging from 0.15 to 0.17 μg/L. However, neither s-triazine (atrazine and terbuthylazine) herbicides nor metolachlor alone at concentrations up to 200 μg/L caused significant toxicity to C. riparius. When combined with both s-triazine herbicides, chlorpyrifos toxicity was enhanced by approximately 2-fold when tested in a binary mixture experimental setup, at the 50% effective concentration levels. To evaluate how chlorpyrifos toxicity was being increased, the cholinesterases (ChE) were characterized biochemically using different substrates and selective inhibitors. The results obtained suggested that the main enzyme present in this species is acetylcholinesterase (AChE) and therefore it was assayed upon C. riparius exposures to all pesticides individually and as binary mixtures. Although atrazine and terbuthylazine are not effective inhibitors of AChE, the potentiation of chlorpyrifos toxicity by the two s-triazine herbicides was associated with a potentiation in the inhibition of AChE in midges; both s-triazine herbicides at 200 μg/L increased the inhibition of the AChE activity by 7 and 8-fold, respectively. A strong correlation was observed between swimming behaviour disturbances of larvae and the inhibition of the AChE activity. In contrast, metolachlor did not affect chlorpyrifos toxicity at any of the concentrations tested. Therefore, the herbicides atrazine and terbuthylazine can act as synergists in the presence of chlorpyrifos, increasing the toxicity and consequently underestimating risk based on single chemical levels.

Acclimation of organisms for ecotoxicity testing is in general processed according to Organisation for Economic Co-operation and Development (OECD) and/or Environmental Protection Agency (EPA) guidelines, under controlled conditions. However, when organisms are collected in the field, their capture, transport and adaptation to laboratory conditions are factors of stress. In their natural habitat, estuarine fish are exposed to considerable fluctuations of environmental variables, while in laboratory they are acclimated to constant conditions and this can be per se a factor of stress that may influence biomarker responses. Therefore, it is important to investigate the effects of these procedures on estuarine fish performance before using them as test organisms in ecotoxicity bioassays. The goal of the present study was to investigate the effects of transporting the common goby, Pomatoschistus microps from the field (natural populations) to the laboratory and of its acclimation to laboratory conditions on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GSTs). Fish were collected in a reference site of the Minho River estuary (NW of Portugal) and the activities of the biomarkers were monitored before and after the transport of organisms to the laboratory and during the acclimation period (at 5, 10 and 15 days). The activities of all the enzymes indicated that capture and transport conditions had no effects on enzymatic activities. Furthermore, AChE, LDH and GST presented higher activities at the end of the acclimation period than at beginning, suggesting a physiological adaptation to laboratory conditions. This adaptation should be taken into consideration in the experimental design to avoid bias in the interpretation of effects of xenobiotics on biomarkers.

The degradation of estuaries is a result of human activities which overloads the environment with substances of both industrial and/or natural origins. Bioindicators have been consistently used to interpret effects of contaminants in the environment. In this study, the use of biomarkers (particular measurable characteristics of a bioindicator organism) was used to evaluate the contamination by xenobiotics of Crangon crangon natural populations. The central aim was to evaluate the capability of a battery of biomarkers to discriminate sites with different types of contamination. The activity of the enzymes cholinesterases (ChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GST) were used as biomarkers. In addition, the ChE form(s) present in the cephalotorax of C. crangon were characterised. Organisms were seasonally sampled from winter 2001/2002 to autumn of 2002, at "reference" sites and at sites that receive agricultural, industrial and/or urban effluents. Results obtained in the characterisation of ChE with different substrates and selective inhibitors demonstrate that the form of ChE present in the cephalotorax of C. crangon shows proprieties of vertebrates' AChE and therefore it may be classified as true AChE-like ChE. The battery of biomarkers exhibited seasonal and local variations, apparently related to agricultural, industrial or urban effluent contamination. The tested biomarkers proved to be able to discriminate sources of environmental contamination, and confirms C. crangon as a sensitive species suitable to be used as a bioindicator.

The zooplankton of the lower part of Canal de Mira (Ria de Aveiro) was sampled during one lunar month. The sampling programme consisted of nine 25 hour fixed-cycles, separated weekly. In each cycle, samples were collected every two hours at three depths (surface, mid-water and above the bottom) with a 500 µm mesh net. The overall effect of the tidal phase was analyzed, taking into account the day and depth of the vertical position of the organisms through a 3-way analysis of variance (ANOVA). The zooplankton densities were, in general, higher at the surface during the night and near the bottom during the day, mainly at spring tide. The variation in the number of species followed a similar pattern to that observed for abundance. Significant differences (p

The aromatic amine 3,4-dichloroaniline (DCA) is a model environmental contaminant, precursor for synthesis and degradation product of several herbicides, which is commonly found in European estuarine ecosystems. In this work, the possibility of using biochemical and histological markers to assess sub-lethal effects of DCA in natural populations of Pomatoschistus microps juveniles was investigated. Alterations on the activities of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferase (GST) and histological alterations on spleen were investigated after 96 h of exposure to sublethal concentrations of DCA (0.50-1.49 mg/l). At the concentrations tested, DCA had no effect on AChE activity. LDH and GST activities were significant altered in treated animals when compared to control groups. As already described for mammals, DCA induced splenic histological alterations in P. microps, including expansion of red pulp and deposition of hemosiderin granules in a concentration-dependent manner. This suggests that DCA is a xenobiotic of concern in estuaries receiving agricultural effluents.

In recent years biomarkers have been widely used for the assessment of effects and/or exposure to environmental contaminants. One of the most frequently used biomarkers is the inhibition of cholinesterases (ChE), which is a useful indicator of organophosphate and carbamate exposure and/or effects. Recent studies indicated that more than one ChE may be present in tissues of fish and that different forms may vary in their sensitivity to anticholinesterase agents. Cholinesterase activity of the juvenile of the common goby (Pomatoschistus microps), a widespread fish in estuaries of the Atlantic coast of northwestern Europe, was characterized using four substrates (acetylthiocholine iodide, acetyl-beta-metylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulfate, BW284C51, and iso-OMPA) in different tissues of the fish head. In addition, the range of ChE activity that may be considered as "normal" for non-exposed P. microps was determined. The results suggest the presence of two types of ChE in the whole-head homogenate. The present study underscores the relevance of ChE characterization before its use as a biomarker in biomonitoring studies.