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.

Electrical and electronic equipment reaching the end of its useful life is currently being disposed of at such an alarmingly high pace that raises environmental concerns. Together with other potentially dangerous compounds, electronic waste contains the rare-earth element gadolinium (Gd), which has already been reported in aquatic systems. Additionally, the vulnerability of aquatic species to this element may also be modified when climate change related factors, like increase in temperature, are taken into consideration. Thus, the present study aimed to evaluate the toxicity of Gd under a scenario of increased temperature in Mytilus galloprovincialis mussels. A multi-biomarker approach and Gd bioaccumulation were assessed in mussels exposed for 28 days to 0 and 10 μg/L of Gd at two temperatures (control - 17 °C; increased - 22 °C). Results confirmed that temperature had a strong influence on the bioaccumulation of Gd. Moreover, mussels exposed to Gd alone reduced their metabolism, possibly to prevent further accumulation, and despite catalase and glutathione S-transferases were activated, cellular damage seen as increased lipid peroxidation was not avoided. Under enhanced temperature, cellular damage in Gd-exposed mussels was even greater, as defense mechanisms were not activated, possibly due to heat stress. In fact, with increased temperature alone, organisms experienced a general metabolic depression, particularly evidenced in defense enzymes, similar to the results obtained under Gd-exposure. Overall, this study underlines the importance of conducting environmental risk assessment taking into consideration anticipated climate change scenarios and exposures to emerging contaminants at relevant environmental concentrations.

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.

Climate change is expected to alter the dynamics of water masses, with consequent changes in water quality parameters such as dissolved organic carbon (DOC) concentration. DOC levels play a critical role in the fate of organic chemicals, influencing their bioavailability and toxicity to aquatic organisms. This study aimed to evaluate the effects of DOC, particularly humic acids (HA), in the toxicity of gemfibrozil (GEM) - a human pharmaceutical frequently detected in surface waters. Lethal and sublethal effects (genotoxic, biochemical and behavioural alterations) were evaluated in zebrafish embryos exposed to several concentrations of GEM and three HA levels, in a full factorial design. HA significantly increased GEM LC50 values, mainly in the first 72 h of exposure, showing a protective effect. At sublethal levels, however, such protection was not observed since HA per se elicited adverse effects. At a biochemical level, individual exposure to HA (20 mg/L) elicited significant decreases in cholinesterase and glutathione S-transferase activities. Regarding behaviour, effects of individual exposure to HA appear to surpass the GEM effects, reducing the total distance moved by larvae. Both GEM and HA significantly increased DNA damage. Hence, this study demonstrated that abiotic factors, namely HA, should be considered in the assessment of pharmaceuticals toxicity. Moreover, it showed that lethality may not be enough to characterize combined effects since different patterns of response may occur at different levels of biological organization. Testing sublethal relevant endpoints is thus recommended to achieve a robust risk assessment in realistic scenarios.

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds.