Effects of wastewater-spiked nanoparticles of silver and titanium dioxide on survival, growth, reproduction and biochemical markers of Daphnia magna.

Silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) are released into aquatic environments through wastewater treatment plants (WWTPs). Even though these NPs are mostly retained in WWTPs, a small fraction can be found in released effluents and may exert toxic effects on aquatic biota. Currently, the available information about the sublethal effects of wastewater-borne NPs on aquatic organisms is inconclusive and the importance of exposure media remains poorly understood. Previously, we demonstrated that rainbow trout juveniles chronically exposed to wastewater-borne AgNPs or TiO2NPs caused no effects on growth, but antioxidative stress mechanisms were triggered in fish organs. Accordingly, this study aimed to: (i) assess the chronic (21-d) effects of wastewater-borne AgNPs (0.3-23.5 µg L-1 Ag) and TiO2NPs (2.7-3.9 µg L-1 Ti) on survival, growth and reproduction of Daphnia magna; (ii) determine the short-term (96-h) effects of wastewater-borne AgNPs (30.3 µg L-1 Ag) and TiO2NPs (6.3 µg L-1 Ti) at the subcellular level (biochemical markers of neurotoxicity, anaerobic metabolism and oxidative stress); and (iii) compare the effects obtained in (i) and (ii) with the corresponding ones induced by effluent-supplemented and water-dispersed NPs. Total Ag and Ti levels were analytically quantified in all treatments. It was demonstrated that both wastewater-borne NPs are considered non-toxic to daphnids at tested concentrations, considering the endpoints at the individual (survival, growth, reproduction) and subcellular (biochemical markers) levels. Contrarily, when pristine forms of NPs were supplemented to effluents or water, concentration-dependent effects were noticed, particularly on cumulative offspring of daphnids. The significant effects on anaerobic metabolism and detoxification pathways caused by the effluent indicate background toxicity. Bearing in mind the achievement of a suitable risk assessment of NPs in aquatic environments, this combined approach looking at both the individual and subcellular levels responses come up with relevant information about the ecotoxicological harmlessness of wastewater-borne NPs in complex environmental matrices like WWTP effluents.

Effects of nanostructure antifouling biocides towards a coral species in the context of global changes.

Biofouling prevention is one of the biggest challenges faced by the maritime industry, but antifouling agents commonly impact marine ecosystems. Advances in antifouling technology include the use of nanomaterials. Herein we test an antifouling nano-additive based on the encapsulation of the biocide 4,5-dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) in engineered silica nanocontainers (SiNC). The work aims to assess the biochemical and physiological effects on the symbiotic coral Sarcophyton cf. glaucum caused by (1) thermal stress and (2) DCOIT exposure (free or nanoencapsulated forms), in a climate change scenario. Accordingly, the following hypotheses were addressed: (H1) ocean warming can cause toxicity on S. cf. glaucum; (H2) the nanoencapsulation process decreases DCOIT toxicity towards this species; (H3) the biocide toxicity, free or encapsulated forms, can be affected by ocean warming. Coral fragments were exposed for seven days to DCOIT in both free and encapsulated forms, SiNC and negative controls, under two water temperature regimes (26 °C and 30.5 °C). Coral polyp behavior and photosynthetic efficiency were determined in the holobiont, while biochemical markers were assessed individually in the endosymbiont and coral host. Results showed transient coral polyp retraction and diminished photosynthetic efficiency in the presence of heat stress or free DCOIT, with effects being magnified in the presence of both stressors. The activity of catalase and glutathione-S-transferase were modulated by temperature in each partner of the symbiosis. The shifts in enzymatic activity were more pronounced in the presence of free DCOIT, but to a lower extent for encapsulated DCOIT. Increased levels of oxidative damage were detected under heat conditions. The findings highlight the physiological constrains elicited by the increase of seawater temperature to symbiotic corals and demonstrate that DCOIT toxicity can be minimized through encapsulation in SiNC. The presence of both stressors magnifies toxicity and confirm that ocean warming enhances the vulnerability of tropical photosynthetic corals to local stressors.

Physiological and Biochemical Effects of Cd Stress in Thlaspi Arvense L-A Non-Accumulator of Metals.

The field pennycress (Thlaspi arvense L.) is a sensitive non-accumulator of metals. The main objective of the present work was to evaluate the effects of Cd stress in T. arvense using a physiological approach. Four-week-old plants were exposed to increasing concentrations of Cd (0, 10, 50 and 100 µM Cd(NO3)2) for 14 days. Plants were harvested at days 0, 3, 7 and 14 to assess the following parameters: shoot and root length and fresh weight; osmolality and conductivity in leaves; chlorophyll a and b contents and photosynthetic efficiency in leaves (young and expanded); water content, Cd accumulation and nutrient imbalances in leaves (young and expanded) and roots. Thlaspi arvense presented a reduction of 27% in the length of roots exposed to 100 µM Cd, and in plants exposed to 50 and 100 µM Cd, a reduction in the fresh weight of both shoots (53 and 58%, respectively) and roots (58 and 63%, respectively) was observed. In general, in plants exposed to the highest concentrations of Cd, a decrease in leaves osmolality and membrane permeability and in water content of both roots and leaves were observed, as well as several nutrient imbalances. As for the photosynthetic parameters, young and expanded leaves responded differently to Cd stress. Exposed young leaves presented no alterations in photosynthetic efficiency or chlorophyll contents, while expanded leaves of 100 µM Cd-treated plants showed a reduction of up to 67% in chlorophyll contents and a concomitant reduction of 30% in photosynthetic efficiency. Overall, Cd-induced senescence in the non-accumulator plant T. arvense exposed to high concentrations of Cd, which was measured as a decrease in several physiological and biochemical parameters and nutrient imbalances. These Cd-induced alterations resulted in lower plant growth, which might have further implications on plant performance at the population level.

Effects of exposure to the UV-filter 4-MBC during Solea senegalensis metamorphosis.

Many personal care products integrate UV-filters, such as 4-methylbenzylidene camphor (4-MBC), a compound frequently detected in aquatic habitats, including coastal areas. However, the potential effects of 4-MBC to saltwater species have been poorly studied. Therefore, the main objective of this work is to study the effects of 4-MBC exposure on Solea senegalensis during metamorphosis, a sensitive life stage of this flatfish. To achieve this, fish were exposed to 4-MBC (0.2-2.0 mg L-1) for 48 h at the beginning of metamorphosis (13 days after hatching, dah). After this period, the fish were transferred to a clean medium. They were fed and maintained until more than 80% of individuals in the control group completed the metamorphosis (24 dah). Mortality, malformations, and metamorphic progression were studied daily. Growth, behavior, and biochemical markers of neurotransmission (acetylcholinesterase, AChE), oxidative stress (catalase, CAT; lipid peroxidation, LPO), detoxification (glutathione S-transferase, GST), and anaerobic metabolism (lactate dehydrogenase, LDH) were also determined at the end of the experiment. An acceleration of metamorphosis progression was observed during and 2 days after the 4-MBC exposure in all concentrations tested. In addition, reduced length, inhibition of CAT activity, and induction of oxidative damage were observed (lowest observed effect concentration, LOEC = 0.928 mg L-1 4-MBC for length, CAT, and LPO). Short-term exposure to 4-MBC at the onset of metamorphosis affected S. senegalensis at several levels of organization, even after 9 days in a clean medium, including growth and metamorphic progression, suggesting possible long-term adverse effects in this species.

Effects of ultraviolet radiation to Solea senegalensis during early development.

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 ±â€¯0.08 and 8.6 ±â€¯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 ±â€¯0.39 or 11.1 ±â€¯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.

Mercury Uptake Affects the Development of Larus fuscus Chicks.

Current emission and mobilization rates of mercury (Hg) in the environment pose extensive threats to both wildlife and human health. Assessing the exposure risk and effects of Hg contamination in model species such as seabirds is essential to understand Hg risks at the population and ecosystem levels. The lesser black-backed gull (Larus fuscus), a generalist seabird species, is an excellent model species because it forages in both marine and terrestrial habitats, which in turn differ in their Hg exposure risk. To identify possible deleterious effects of Hg exposure on developing L. fuscus chicks, a dietary experiment was carried out and chicks were provided a marine, terrestrial, or mixed diet. The effects of embryonic and dietary Hg exposure on chick body condition and physiological state were assessed at different developmental stages until fledging age (30 d). Overall physiological condition was lower in chicks fed a predominantly marine diet, which coincided with higher Hg loads in blood and primary feathers. However, no effect of dietary uptake of Hg was observed on body condition or in terms of genotoxic damage. Body condition and genotoxic damage correlated instead with Hg exposure during embryonic development, which seems to indicate that embryonic exposure to Hg may result in carry-over effects on later chick development. Environ Toxicol Chem 2020;39:2008-2017. © 2020 SETAC.

Chronic effects of wastewater-borne silver and titanium dioxide nanoparticles on the rainbow trout (Oncorhynchus mykiss).

Even though nanoparticles (NPs) are mostly removed by wastewater treatment plants, wastewater-borne NPs may show an altered toxicity to aquatic organisms. The main objectives of this work were: i) to assess the chronic (28 days) effects of wastewater-borne NPs of silver (AgNPs, 1.4-36.2 µg L-1) and titanium dioxide (TiO2NPs, 3.1-50.2 µg L-1) at the individual (growth) and biochemical (biomarkers of neurotoxicity, oxidative stress and energy metabolism) levels in rainbow trout Oncorhynchus mykiss; and ii) to compare them with their effluent-supplemented and water-dispersed counterparts. The total Ag and Ti levels were determined in several fish organs. The growth of O. mykiss was not affected by the NPs in any treatment, except a 29% increase at 5.5 µg L-1 of total Ag supplemented to effluents. The Ag level in organs of O. mykiss was significantly higher after exposure to water-dispersed AgNPs than their wastewater-borne or effluent-supplemented counterparts. No significant Ti uptake could be observed. Effluent-supplemented TiO2NPs (50.1 µg L-1 Ti) potentially induced neurotoxic effects, indicated by a 24% increase in acetylcholinesterase activity comparatively to controls. Energy reserves were unaffected by TiO2 treatments, while nearly all AgNP-containing treatments caused a depletion of total lipids, proteins and carbohydrates in the muscle, suggesting an increased energy demand for detoxification processes to cope with AgNPs. Besides NPs, the effluent matrix and dispersing agent (for AgNPs) induced significant effects on energetic reserves and oxidative stress, indicating background toxicity of both treatments at the biochemical level. Our study is the first to assess chronic effects of wastewater-borne NPs on rainbow trout. While no effects were found at the individual level, several biochemical markers were changed by the NPs exposure. Our results highlight the importance of using complex matrices for a reliable risk assessment of NPs in the aquatic environment.

Impact of wastewater-borne nanoparticles of silver and titanium dioxide on the swimming behaviour and biochemical markers of Daphnia magna: An integrated approach.

Due to their widespread use, silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) are commonly discharged into aquatic environments via wastewater treatment plants. The study was aimed to assess the effects of wastewater-borne AgNPs (NM-300 K; 15.5 ±â€¯2.4 nm; 25-125 µg L-1) and TiO2NPs (NM-105; 23.1 ±â€¯6.2 nm; 12.5-100 µg L-1), from a laboratory-scale wastewater treatment plant, on Daphnia magna, at individual and subcellular level. For effect comparison, animals were also exposed to ASTM-dispersed NPs at the same nominal concentrations. The behaviour of D. magna was evaluated through monitoring of swimming height and allocation time for preferred zones after 0 h and 96 h of exposure. Biochemical markers of neurotransmission, anaerobic metabolism, biotransformation, and oxidative stress were subsequently determined. No 96-h EC50 (immobilization ≤ 4 %) could be obtained with wastewater-borne NPs and ASTM-dispersed TiO2NPs, whereas the ASTM-dispersed AgNPs resulted in an immobilization 96-h EC50 of 113.8 µg L-1. However, both wastewater-borne and ASTM-dispersed TiO2NPs, at 12.5 µg L-1, caused immediate (0 h) alterations on the swimming height. Allocation time analyses showed that animals exposed to ASTM-dispersed AgNPs spent more time on the surface and bottom at 0 h, and in the middle and bottom at 96 h. This pattern was not observed with ASTM-dispersed TiO2NPs nor with wastewater-borne AgNPs and wastewater-borne TiO2NPs. At the biochemical level, the more pronounced effects were observed with wastewater-borne AgNPs (e.g. induction of lactate dehydrogenase and glutathione S-transferase activities, and inhibition of catalase activity). This integrative approach showed that: (i) the behavioural and biochemical response-patterns were distinct in D. magna exposed to environmentally relevant concentrations of wastewater-borne and ASTM-dispersed NPs; (ii) the most pronounced effects on allocation time were induced by ASTM-dispersed AgNPs; and (iii) at the subcellular level, wastewater-borne AgNPs were more toxic than wastewater-borne TiO2NPs. This study highlights the need for the assessment of the effects of wastewater-borne NPs under realistic exposure scenarios, since processes in wastewater treatment plants may influence their toxicity.

Assay optimisation and age-related baseline variation in biochemical markers in Lesser Black-backed gulls.

Free-ranging animals are often used as bioindicators of both short- and long-term changes in ecosystem health, mainly to detect the presence and effects of contaminants. Birds, and gulls in particular, have been used as bioindicators over a broad range of marine and terrestrial ecosystems. In this study, we standardise the conditions for the use of a suite of biochemical markers in non-destructive matrices of Lesser Black-backed Gull (Larus fuscus) to facilitate future biomonitoring of marine and terrestrial contaminants. We characterized cholinesterase (ChE) in plasma and optimized assay conditions for ChE activity as a marker of neurotoxic damage. Moreover, we quantified variation in activity of ChE, lactate dehydrogenase (LDH), glutathione-S-transferase (GST) and catalase (CAT) as well as variation ranges of lipid peroxidation (LPO), in free-ranging adults and captive chicks. The main ChE form present in plasma of both adults and chicks was butyrylcholinesterase (BChE) followed by acetylcholinesterase (AChE), whose relative proportion in plasma tended to decrease with increased chick age. LPO levels and GST activity in blood cells (BCs) decreased significantly with increasing chick age, while BChE and LDH activity in plasma were not age-dependent. CAT in BCs tended to decline non-significantly in older chicks. Results of this study underscore the importance of standardising assay conditions and assessing intrinsic baseline variation in biochemical markers, before biochemical quantification. Data presented here provide a foundation for future use of BChE and LDH activity in plasma, as well as oxidative stress markers (LPO, CAT and GST) in BCs, to monitor environmental stress effects in Lesser Black-backed gulls.

Effects of PCB-77 in adult zebrafish after exposure during early life stages.

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.

Toxicity effects of the organic UV-filter 4-Methylbenzylidene camphor in zebrafish embryos.

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.

Effects of the herbicides linuron and S-metolachlor on Perez's frog embryos.

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.

Offspring Hg exposure relates to parental feeding strategies in a generalist bird with strong individual foraging specialization.

Generalist species can potentially exploit a wide variety of resources, but at the individual level they often show a certain degree of foraging specialization. Specific foraging strategies, however, may increase exposure to environmental contaminants that can alter the cost-benefit balance of consuming particular food items. The Lesser Black-backed Gull (Larus fuscus) is known to opportunistically feed on a wide range of marine and terrestrial prey that differ in contaminant load, such as mercury (Hg) that strongly biomagnifies through the aquatic food web. The hypothesis tested in this study were: i) a predominant use of marine prey by females during egg-formation and by both parents during chick rearing increases the exposure to Hg during embryonic development and chick growth, and ii) this affects parental investment in clutch volume, chick growth and body condition. Total Hg burden and isotopic signatures of carbon (δ13C) and nitrogen (δ15N) were determined for eggs, down feathers, and primary feathers of L. fuscus chicks collected at a coastal colony in Belgium. As expected, eggs and feathers of chicks from parents with a stable isotope signature that suggested a predominantly marine diet had higher levels of Hg. The use of marine resources by females during the egg-formation period positively correlated to maternal investment in egg size, though entailing the cost of increased Hg-concentrations which in turn negatively affected clutch volume. Furthermore, it is shown that the use of chick down feathers is a suitable matrix to non-lethally estimate Hg concentrations in eggs. Contrary to our expectations, no relationship between Hg exposure and chick growth or chick body condition was found, which may be due the low concentrations found. We conclude that currently Hg contamination does not constitute a risk for development and condition of L. fuscus offspring at the levels currently observed at the Belgian coast.

Mercury levels in parturient and newborns from Aveiro region, Portugal.

Since the outbreak of methylmercury (MeHg) poisoning in Japan and Iraq, mercury (Hg) is classified as well-established teratogen. The Portuguese region of Aveiro faced some decades ago an environmental Hg contamination due to activities from a chlor-alkali plant. Until now, no apparent evaluation was conducted regarding prenatal exposure to Hg in this area. The main objectives of this study were to: i) assess maternal and fetal exposure to Hg in the Aveiro region using noninvasive biological matrices; ii) examine the influence of variables that may contribute to Hg exposure during pregnancy; and iii) improve knowledge regarding metal accumulation and distribution over the maternal-fetal-placental unit. This study was performed in 50 mother-newborn pairs from the Aveiro district. Total Hg (THg) was quantified in maternal scalp hair, placenta, amniotic membrane, and umbilical cord. Maternal hair presented THg levels with a mean value of 900 ng/g, which is lower than the USEPA and WHO acceptable threshold. Regarding THg levels in placenta and umbilical cord, mean values were similar (decidua basalis: 32.84 ng/g; chorionic plate: 30.18 ng/g; umbilical cord: 30.67 ng/g). The amniotic membrane presented the highest THg levels with a mean concentration of 42.35 ng/g, reaching a maximum of 134.1 ng/g. Further, a significant positive correlation was noted between THg levels found in hair, and all matrices analyzed reinforcing the use of hair in biomonitoring studies with respect to maternal exposure to Hg. In general, levels of THg found in our study were lower than those in previous studies performed in Europe. Consumption of fish rich in selenium and bottled water was negatively correlated with THg levels. Finally, data demonstrated that Hg is capable of crossing the placental barrier and accumulate in placental tissues. Amniotic membrane seemed to play a role in metal detoxification, but further investigations are necessary to examine whether this catabolic process affects Hg accumulation.

Effects of 4-MBC and triclosan in embryos of the frog Pelophylax perezi.

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.

Assessment of DNA damage in Ardea cinerea and Ciconia ciconia: A 5-year study in Portuguese birds retrieved for rehabilitation.

Over the past decades, the presence of micronucleated blood cells has been used to detect genotoxic effects of xenobiotics in fish, amphibians and birds. This study assessed the frequency of micronuclei (MN) and other nuclear abnormalities in erythrocytes of individuals of Ardea cinerea and Ciconia ciconia retrieved for rehabilitation in order to evaluate the influence of age, temporal and spatial factors on the occurrence of DNA damage in Portuguese wild birds. Blood smears from 65 birds with different life-history backgrounds (e.g. geographic origin, age) were collected between 2007 and 2011 and the frequency of erythrocyte nuclear abnormalities (ENAs) was analysed. Differences in DNA damage between ages were observed to occur in C. ciconia, with chicks displaying significantly higher frequencies of ENAs (both when looking at total ENAs or only MN frequency) than juveniles and adults. Additionally, significant differences in ENAs frequencies were observed between different years and geographic origins, whereas MN frequency alone did not show significant alterations concerning spatial and temporal variations. These results suggest that the assessment of ENAs rather than MN frequency alone may be a useful and valuable tool to complement the evaluation of DNA damage in populations of birds, as prompted by individual life-history traits and environmental factors.

Multilevel assessment of ivermectin effects using different zebrafish life stages.

Several studies have shown high toxicity of the veterinary pharmaceutical ivermectin (a semisynthetic avermectin) for aquatic invertebrates however, few data is found for fish species. The present study evaluated the toxicity of ivermectin, to embryos, juveniles, and adults of zebrafish at different levels of biological organization including developmental, behavioural and biochemical. Toxicity tests were performed based on OECD protocols and mortality and behavioural changes were assed for all stages. Biochemical responses were assessed in adults and embryos and included cholinesterases (ChEs), catalase (CAT) (only in embryos), glutathione-S-Transferase (GST), lactate dehydrogenase (LDH) and vitellogenin (VTG) like proteins (only in embryos). Genotoxicity was evaluated in adults. Results showed a higher sensitivity of juvenile and adults of zebrafish (96h-LC10 values of 14.0 and 55.4µg/L, respectively). For embryos a 96h-LC10 of 147.1µg/L was calculated, moreover developmental anomalies and hatching inhibition were observed only at high concentrations (>400µg/L), whereas biochemical and behavioural responses occurred at lower concentrations (<60µg/L). Behavioural responses (lethargy) occurred in all life stages. Biochemical responses were observed including the inhibition of GST in adults and changes in ChE, CAT, LDH activities and VTG levels in embryos. Ivermectin did not show to be genotoxic for adult fish. The species sensitivity distribution analysis, based on fish and invertebrate species, indicated a Hazardous Concentration for 5% of the population (HC5) value of 0.057µg/L; suggesting high sensitivity of both groups to ivermectin and a high risk of this compound to aquatic ecosystems.

Brain cholinesterase reactivation as a marker of exposure to anticholinesterase pesticides: a case study in a population of yellow-legged gull Larus michahellis (Naumann, 1840) along the northern coast of Portugal.

Between late 2010 to early 2011, an increased mortality in gulls was observed along the northern coast of Portugal, with individuals exhibiting neurologic disorders consistent with an eventual anticholinesterase pesticide poisoning event. To clarify if this mortality was related to organophosphate (OP) and/or carbamate (CB) poisoning, chemical and spontaneous cholinesterase (ChE) reactivation was tested in the brain of the yellow-legged gull (Larus michahellis). Initial brain ChE activity in L. michahellis was 40.92 ± 5.23 U/mg of protein (average ± SE). Following chemical and spontaneous reactivation, ChE activity increased in average 70.38 ± 48.59% and 131.95 ± 92.64%, respectively. ChE reactivation was found to decrease at increasing concentrations of the oxime pyridine-2-aldoxime methochloride and dilution factor, underscoring the importance of first optimizing the assay conditions prior to its use on bird species. These results suggest that birds analysed could have been exposed to OP and CB pesticide compounds and that in most cases CB exposure appeared to be the main cause of birds poisoning. These results are an important contribution to environmental monitoring as it demonstrates the suitability of L. michaellis as sentinel species of OP and CB pesticides within an urban environment.

Biomarkers of endocrine disruption in juveniles and females of the estuarine fish Pomatoschistus microps.

The presence of endocrine disruptor chemicals (EDCs) in aquatic compartments, including estuaries, has been object of major concern. However, the effects of EDCs on autochthonous estuarine fish species are far less known than in freshwater fish. Therefore, the response of biomarkers in the estuarine fish Pomatoschistus microps was evaluated after 21-days of exposure to different EDCs: 17ß-estradiol (E2); PCB-77 and p,p'DDE. In juveniles, Vtg (vitellogenin)-like proteins were significantly induced by E2. PCB-77 led to a decrease of Vtg-like proteins in juveniles, while in female liver they increased at the highest concentrations tested, suggesting a mode of action of PCB-77 that depends on the fish life stage. p,p'-DDE did not induce significant changes in Vtg-like proteins. Overall, P. microps juveniles seemed to respond to environmental relevant concentrations of EDCs. Thus, the evaluation of Vtg-like proteins in this life stage seems to be a promising tool to track EDC contamination in biomonitoring studies.

Characterization of cholinesterases in Chironomus riparius and the effects of three herbicides on chlorpyrifos toxicity.

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.