Recommended rates of azoxystrobin and tebuconazole seem to be environmentally safe but ineffective against target fungi.

The use of fungicides in agriculture has been playing a role in the enhancement of agricultural yields through the control of pathogens causing serious diseases in crops. Still, adverse environmental and human health effects resulting from its application have been reported. In this study, the possibility of readjusting the formulation of a commercial product combining azoxystrobin and tebuconazole (active ingredients - AIs; Custodia®) towards environmentally safer alternative(s) was investigated. Specifically, the sensitivity of non-target aquatic communities to each AI was first evaluated by applying the Species Sensitivity Distributions (SSDs) approach. Then, mixtures of these AIs were tested in a non-target organism (Raphidocelis subcapitata) denoting sensitivity to both AIs as assessed from SSDs. The resulting data supported the design of the last stage of this study, where mixtures of those AIs at equivalent vs. alternative ratios and rates as in the commercial formulation were tested against two target fungal species: Pyrenophora teres CBS 123929 and Rhynchosporium secalis CBS 110524. The comparison between the sensitivity of non-target aquatic species and the corresponding efficacy towards target fungi revealed that currently applied mixture and rates of these AIs are generally environmentally safe (antagonistic interaction; concentrations below the EC1 for R. subcapitata and generally below the HC5 for aquatic non-target communities), but ineffective against target organisms (maximum levels of inhibition of 70 and 50% in P. teres CBS 123929 and R. secalis CBS 110524, respectively). Results additionally suggest a potentiation of the effects of the AIs by the other formulants added to the commercial product at tested rates. Overall, this study corroborates that commercial products can be optimized during design stages based on a systematic ecotoxicological testing for ingredient interactions and actual efficacy against targets. This could be a valuable pathway to reduce environmental contamination during transition to a more sustainable agricultural production.

A review on aquatic toxins - Do we really know it all regarding the environmental risk posed by phytoplankton neurotoxins?

Aquatic toxins are potent natural toxins produced by certain cyanobacteria and marine algae species during harmful cyanobacterial and algal blooms (CyanoHABs and HABs, respectively). These harmful bloom events and the toxins produced during these events are a human and environmental health concern worldwide, with occurrence, frequency and severity of CyanoHABs and HABs being predicted to keep increasing due to ongoing climate change scenarios. These contexts, as well as human health consequences of some toxins produced during bloom events have been thoroughly reviewed before. Conversely, the wider picture that includes the non-human biota in the assessment of noxious effects of toxins is much less covered in the literature and barely covered by review works. Despite direct human exposure to aquatic toxins and related deleterious effects being responsible for the majority of the public attention to the blooms' problematic, it constitutes a very limited fraction of the real environmental risk posed by these toxins. The disruption of ecological and trophic interactions caused by these toxins in the aquatic biota building on deleterious effects they may induce in different species is paramount as a modulator of the overall magnitude of the environmental risk potentially involved, thus necessarily constraining the quality and efficiency of the management strategies that should be placed. In this way, this review aims at updating and consolidating current knowledge regarding the adverse effects of aquatic toxins, attempting to going beyond their main toxicity pathways in human and related models' health, i.e., also focusing on ecologically relevant model organisms. For conciseness and considering the severity in terms of documented human health risks as a reference, we restricted the detailed revision work to neurotoxic cyanotoxins and marine toxins. This comprehensive revision of the systemic effects of aquatic neurotoxins provides a broad overview of the exposure and the hazard that these compounds pose to human and environmental health. Regulatory approaches they are given worldwide, as well as (eco)toxicity data available were hence thoroughly reviewed. Critical research gaps were identified particularly regarding (i) the toxic effects other than those typical of the recognized disease/disorder each toxin causes following acute exposure in humans and also in other biota; and (ii) alternative detection tools capable of being early-warning signals for aquatic toxins occurrence and therefore provide better human and environmental safety insurance. Future directions on aquatic toxins research are discussed in face of the existent knowledge, with particular emphasis on the much-needed development and implementation of effective alternative (eco)toxicological biomarkers for these toxins. The wide-spanning approach followed herein will hopefully stimulate future research more broadly addressing the environmental hazardous potential of aquatic toxins.

Transgenerational Inheritance of DNA Hypomethylation in Daphnia magna in Response to Salinity Stress.

Epigenetic mechanisms have been found to play important roles in environmental stress response and regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we focus on the aquatic microcrustacean Daphnia, a parthenogenetic model species, and its response to salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of environmental perturbation affecting freshwater ecosystems. We exposed one generation of D. magna to high levels of salinity (F0) and found that the exposure provoked specific methylation patterns that were transferred to the three consequent nonexposed generations (F1, F2, and F3). This was the case for the hypomethylation of six protein-coding genes with important roles in the organisms' response to environmental change: DNA damage repair, cytoskeleton organization, and protein synthesis. This suggests that epigenetic changes in Daphnia are particularly targeted to genes involved in coping with general cellular stress responses. Our results highlight that epigenetic marks are affected by environmental stressors and can be transferred to subsequent unexposed generations. Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this parthenogenetic model system. Furthermore, no life history costs seem to be associated with the maintenance of hypomethylation across unexposed generations in Daphnia following a single stress exposure.

The role of contamination history and gender on the genotoxic responses of the crayfish Procambarus clarkii to a penoxsulam-based herbicide.

The responses of non-target organisms to pesticide exposure are still poorly explored in what concerns the development of adjustments favouring population success. Owing to the vital role of DNA integrity, it is important to identify genome-maintenance skills and their determinant factors. Thus, the major aims of the present study were: (i) to assess the genotoxicity of the penoxsulam-based herbicide (Viper®) to the crayfish Procambarus clarkii; (ii) to understand the influence of gender and contamination history in the genotoxic responses following exposure to this herbicide; (iii) to investigate the damage mechanisms involved in putative adjustments shown by P. clarkii. Two populations were tested, one from a reference site and the other from a historically contaminated site. Specimens from both populations were exposed to Viper®, considering environmentally relevant penoxsulam concentrations (20 and 40 µg L-1) and to a model genotoxicant (EMS). Comet assay was adopted to assess the genetic damage in gills. The results disclosed the genotoxicity of the herbicide to crayfish (a non-target organism). Additionally, organisms exposed to the highest concentration of penoxsulam signalized the influence of factor "population" towards the genotoxic pressure (measured as effective DNA breaks): P2 males from the historically impacted population displayed a significantly higher susceptibly (by up to 53.98%) when compared to control, while the homologous group from the reference population presented levels similar to its respective control. When DNA lesion-repair enzymes were considered, DNA oxidation patterns suggested an increased ability of this gender (39.75% lower than negative control) to deal with this particular type of damage, namely considering pyrimidines oxidation. It is worth remarking that the influence of the exposure history on the protection/vulnerability to the penoxsulam-based herbicide was only evident in males, despite depending on the type of DNA damage: when the non-specific damage was considered, organisms from the impacted population seemed to be more vulnerable while regarding to the oxidative damage, males from the impacted population appeared to be more protected than organisms that have never been exposed to penoxsulam. Overall, the influence of factors "gender" and "contamination history" was demonstrated as well as its dependence on DNA damage type was evident. EMS groups did not present the differences between populations, reinforcing the agent-specific adjustment hypothesis.These findings highlighted the importance of considering differential physiological backgrounds in ecogenotoxicological analysis, hence favouring the elaboration of more plausible and holistic approaches integrating the environmental risk assessment of pesticides.

Ecotoxicological assessment of the herbicide Winner Top and its active substances-are the other formulants truly inert?

Formulants used in Plant Protection Products (PPPs) to promote their efficiency are normally undisclosed in the PPP documentation, unless they bear a human health or environmental hazardous potential per se. PPP regulation also demands the assessment of putative interactions among formulants within each product recipe and consequent effects, but these results are often unavailable. Such a case is that of the herbicide Winner Top (Selectis®, Portugal), which we selected as a model commercial formulation in the present study specifically aiming at (i) characterising its aquatic toxicity towards sensitive eco-receptors (Raphidocelis subcapitata, Chlorella vulgaris, Lemna minor and Lemna gibba), as well as that of its active substances (a.s.) nicosulfuron and terbuthylazine; (ii) comparing the ecotoxicity among the commercial formulation, the corresponding mixture of its a.s. and this a.s.'s mixture increasingly enriched with the formulants. Single chemical testing revealed that terbuthylazine was the strongest microalgae growth inhibitor and nicosulfuron was the strongest macrophyte growth inhibitor. On the other hand, the commercial formulation was consistently less toxic than the corresponding mixture of the a.s., suggesting that Winner Top formulants (72.9% of the commercial formulation) interact with the a.s., promoting less than additive effects in the selected non-target species. Importantly, this environmentally protective effect of the formulation can be apparent. Because macrophytes share most physiological features with the weeds targeted by the studied herbicide, it is likely that increased application doses are required to reach desired efficacy levels with the consequent detrimental increase of PPP residues load in edge-of-field freshwater ecosystems.

Bioremediation of metal-rich effluents: could the invasive bivalve work as a biofilter?

Industrial effluents are important sources of contamination of water and sediments, frequently causing serious damage at different levels of biological organization. Management and treatment of harmful industrial wastes is thus a major concern. Metal-bearing effluents, such as acid mine drainage (AMD), are particularly problematic because metals can easily bioaccumulate in organisms and biomagnify across the trophic chain. Several solutions have been proposed to treat AMD, including active methods involving the addition of neutralizing agents and passive techniques that use natural energy sources for remediation. However, increasing environmental and economic requirements lead the constant search for more sustainable solutions. The present study explores the possibility of using , an invasive freshwater bivalve, as a bioremediation tool using AMD as a model, metal-bearing effluent. The study compares untreated and biotreated effluents at two dilution levels (4 and 10% v/v) following two distinct approaches: (i) chemical characterization of the metal concentrations in water complemented by determination of the accumulation in the clams' soft tissues and shells; and (ii) ecotoxicity assessment using standard organisms (the bacterium , the microalgae , and the cladoceran ). Significant removal of metals from water was recorded for both effluent dilutions, with higher purification levels found for the 4% effluent. The environmental toxicity of the effluents generally decreased after the treatment with the clams. Thus, this study provides evidence for the suitability of as a bioremediator for metal-bearing effluents, especially if the treatment can be materialized in a multistage configuration system.

The impact of paracetamol on selected biomarkers of the mollusc species Corbicula fluminea.

The Asian clam Corbicula fluminea is an invasive bivalve that has recently spread in Europe and currently represents a large portion of the aquatic biomass in specific areas. Because of the impacts that the species may have in invaded ecosystems, increased knowledge on the physiologic features of the species life-cycle under different environmental scenarios (e.g., contamination events) is critical to understand the dynamics of the invasion and resulting ecosystem imbalance. The presence of pharmaceutical residues in the aquatic environment has recently received great attention since high levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. The present article reports toxicological biochemical effects of paracetamol to Corbicula fluminea following short- and long-term exposures. Oxidative stress parameters were specially focused namely catalase (CAT), glutathione S-transferases (GSTs), and glutathione reductase (GRed). The effect of tested substances on lipid peroxidation was also investigated. Paracetamol did not induce alterations on CAT activity, caused a significant decrease of GSTs activity following short- and long-term exposure (LOEC values of 532.78 mg L(-1) and 30.98 µg L(-1) , respectively), and was responsible for a significant and dose-dependent decrease of GRed activity in short- and long-term exposures. These results indicate that exposure to paracetamol can provoke significant alterations on the cellular redox status of C. fluminea.

Toxicity testing with the benthic diatom Navicula libonensis (Schoeman 1970): procedure optimisation and assessment of the species sensitivity to reference chemicals.

Periphytic communities are good indicators of river quality due to their general sensitivity to several pollutants. The primary objective of this study was to develop and optimize an ecotoxicological testing methodology using the freshwater benthic diatom Navicula libonensis. This species was selected due to its ubiquity and suitability for use under laboratory conditions. In the most suitable test medium (Chu10) the diatom demonstrated comparable sensitivity to potassium dichromate and 3,5-dichlorophenol using growth rate as the reference parameter, with median effect concentrations (ErC50) in the same order of magnitude (0.119 and 0.799 mg L(-1)) respectively. Yield-based estimates did not confirm this pattern and potassium dichromate was one order of magnitude more toxic than 3,5-dichlorophenol. The sensitivity of N. libonensis to the reference chemicals was higher than that published in the literature for several standard planktonic microalgae. This advantage, as well as the ability to grow the species in the laboratory, supports further efforts towards the standardisation of a toxicity testing protocol. In addition, the functional role of benthic diatoms in lotic ecosystems justifies their inclusion in risk assessment test batteries to better cover an environmental compartment that has so far been neglected.

Potential effects of the discharge of wastewater treatment plant (WWTP) effluents in benthic communities: evidence from three distinct WWTP systems.

Wastewater treatment plant (WWTP) effluents can be sources of environmental contamination. In this study, we aimed to understand whether effluents of three different WWTPs may have ecological effects in riverine recipient ecosystems. To achieve this, we assessed benthic phytobenthos and macroinvertebrate communities at three different locations relative to the effluent discharge: immediately upstream, immediately downstream and 500-m downstream the effluent discharge. Two approaches were employed: the ecological status classification as defined in the Water Framework Directive (WFD) based on biological indicators; constrained multivariate analysis to disentangle the environmental drivers (physicochemical variables and contaminants, namely metals, polycyclic aromatic hydrocarbons, pharmaceuticals, and personal care products) of ecological changes across the study sites. The results showed inconsistencies between the WFD approach and the multivariate approach, as well as between the responses of macroinvertebrates and diatoms. The WWTP effluents impacted benthic communities in a single case: macroinvertebrates were negatively affected by one of the WWTP effluents, likely by the transported pharmaceuticals (other stressors are essentially homogeneous among sites). Given the findings and the scarcity of consistent evidence on ecological impacts that WWTP effluents may have in recipient ecosystems, further research is needed towards more sustainable regulation and linked environmental protection measures.

Redox Homeostasis Disclosed in the Saltmarsh Plant Halimione portulacoides upon Short Waterborne Exposure to Inorganic Mercury.

The saltmarsh plant Halimione portulacoides was shortly exposed to realistic levels of inorganic mercury (iHg) with the aim of investigating the adaptative processes of the roots and leaves regarding redox homeostasis, physiology, and Hg accumulation. Plants were collected at a contaminated (CONT) and a reference (REF) site to address the interference of contamination backgrounds. The influence of major abiotic variables (i.e., temperature and light) was also examined. Total Hg levels, antioxidant enzymes, lipid peroxidation (LPO), and photosynthetic activity were analyzed after 2 and 4 h of exposure. A poor accumulation of Hg in the roots was noticed, and no translocation to the stems and leaves was found, but plants from the CONT site seemed more prone to iHg uptake (in winter). Despite this, antioxidant modulation in the roots and leaves was found, disclosing, in winter, higher thresholds for the induction of enzymatic antioxidants in CONT leaves compared to REF plants, denoting that the former are better prepared to cope with iHg redox pressure. Consistently, CONT leaves exposed to iHg had remarkably lower LPO levels. Exposure did not impair photosynthetic activity, pinpointing H. portulacoides' ability to cope with iHg toxicity under very-short-term exposure. Biochemical changes were noticed before enhancements in accumulation, reinforcing the relevance of these responses in precociously signaling iHg toxicity.

A review of the order mysida in marine ecosystems: What we know what is yet to be known.

Mysids have a high ecological importance, particularly by their role in marine food chains as a link between the benthic and pelagic realms. Here we describe the relevant taxonomy, ecological aspects such as distribution and production, and their potential as ideal test organisms for environmental research. We also highlight their importance in estuarine communities, trophic webs, and their life history, while demonstrating their potential in addressing emergent problems. This review emphasizes the importance of mysids in understanding the impacts of climate change and their role in the ecology of estuarine communities. Although there is a dearth of research in genomic studies, this review emphasizes the relevance of mysids and their potential as a model organism in environmental assessment studies of prospective or retrospective nature and highlights the need for further research to enhance our understanding of this group's ecological significance.

Behavioural, developmental and biochemical effects in zebrafish caused by ibuprofen, irgarol and terbuthylazine.

The increasing use of chemicals and their release into aquatic ecosystems are harming aquatic biota. Despite extensive ecotoxicological research, many environmental pollutants' ecological effects are still unknown. This study examined the spatial avoidance, behavioural and biochemical impacts of ibuprofen, irgarol, and terbuthylazine on the early life stages of zebrafish (Danio rerio) under a range of ecologically relevant concentrations (0-500 µg/L). Embryos were exposed following the OECD guideline "fish embryo toxicity test" complemented with biochemical assessment of AChE activity and behavioural analyses (swimming activity) using the video tracking system Zebrabox. Moreover, spatial avoidance was assessed by exposing 120 hpf-old larvae of D. rerio to a gradient of each chemical, by using the heterogeneous multi-habitat assay system (HeMHAS). The results obtained revealed that the 3 compounds delayed hatching at concentrations of 50 and 500 µg/L for both ibuprofen and irgarol and 500 µg/L for terbuthylazine. Moreover, all chemicals elicited a dose-dependent depression of movement (swimming distance) with LOEC values of 5, 500 and 50 µg/L for ibuprofen, irgarol and terbuthylazine, respectively. Zebrafish larvae avoided the three chemicals studied, with 4 h-AC50 values for ibuprofen, irgarol, and terbuthylazine of 64.32, 79.86, and 131.04 µg/L, respectively. The results of the HeMHAS assay suggest that larvae may early on avoid (just after 4 h of exposure) concentrations of the three chemicals that may later induce, apical and biochemical effects. Findings from this study make clear some advantages of using HeMHAS in ecotoxicology as it is: ecologically relevant (by simulating a chemically heterogeneous environmental scenario), sensitive (the perception of chemicals and the avoidance can occur at concentrations lower than those producing lethal or sublethal effects) and more humane and refined approach (organisms are not mandatorily exposed to concentrations that can produce individual toxicity).

Do Freshwater and Marine Bivalves Differ in Their Response to Wildfire Ash? Effects on the Antioxidant Defense System and Metal Body Burden.

Wildfires constitute a source of contamination to both freshwater and marine ecosystems. This study aimed to compare the antioxidant defense response of the freshwater clam Corbicula fluminea and the marine cockle (Cerastoderma edule) to wildfire ash exposure and the concomitant metal body burden. Organisms were exposed to different concentrations (0%, 12.5%, 25%, 50%, and 100%) of aqueous extracts of Eucalypt ash (AEAs) from a moderate-to-high severity wildfire. The activity of various enzymes, as well as lipid peroxidation, protein content, and metal body burden, were determined after 96 h of exposure. A significant increase in the protein content of soft tissues was observed for C. edule at AEA concentrations ≥ 25%, unlike for C. fluminea. Similarly, significant effects on lipid peroxidation were observed for cockles, but not for clams. For both species, a significant effect in the total glutathione peroxidase activity was observed at AEA concentrations ≥ 25%. Relative to the control, AEAs-exposed clams showed higher Cd content, whereas AEAs-exposed cockles showed higher Cu content, thus exhibiting different responses to the exposure to wildfire ash. The susceptibility of bivalves to ashes, at environmentally relevant concentrations, raises concern about the effects of post-fire runoff to bivalve species.

Multigenerational DNA methylation responses to copper exposure in Daphnia: Potential targets for epigenetic biomarkers?

Epigenetic mechanisms are moving to the forefront of environmental sciences, as environmentally induced epigenetic changes shape biological responses to chemical contamination. This work focused on Daphnia as a representative of potentially threatened freshwater biota, aiming to gain an insight into the involvement of epigenetic mechanisms in their response and eventual adaptation to metal contamination. Copper-induced DNA methylation changes, their potential transgenerational inheritance, and life-history traits were assessed. Organisms with different histories of past exposure to copper were exposed to toxic levels of the element for one generation (F0) and then monitored for three subsequent unexposed generations (F1, F2, and F3). Overall, methylation changes targeted important genes for counteracting the effects of metals and oxidative stress, including dynein light chain, ribosomal kinase and nuclear fragile X mental retardation-interacting protein. Also, contrasting overall and gene-specific methylation responses were observed in organisms differing in their history of exposure to copper, with different transgenerational methylation responses being also identified among the two groups, without apparent life-history costs. Taken together, these results demonstrate the capacity of copper to promote epigenetic transgenerational inheritance in a manner related explicitly to history of exposure, thereby supporting the development and incorporation of epigenetic biomarkers in risk assessment frameworks.

Chemical characterization of riverine sediments affected by wastewater treatment plant effluent discharge.

This study was aimed at assessing the contribution of wastewater treatment effluents to the contamination profile of the sediments of receiving waterways. Three wastewater treatment plants (WWTP) were addressed, encompassing different population equivalent sizes, urbanization degrees and treatment methods translating differences in expected contamination patterns. Within each WWTP system, the assessment targeted the effluent and sediment samples collected upstream and downstream the effluent discharge point; contaminants belonging to several concerning chemical classes (metals and metalloids; pesticides; pharmaceuticals and personal care products, PPCPs; and polycyclic aromatic hydrocarbons, PAHs) were quantified both in effluent and sediment samples. Clear associations between contaminants present in the effluent and corresponding sediment samples were not always verified. In fact, a noticeable difference between the number or abundance of contaminants detected in effluents and in sediments, suggesting that effluents are not always the most likely source (e.g. PAHs). However, sediment contaminants that were likely sourced by the effluents were also identified (e.g. PPCPs). Sediment analysis offers an important historical view of contamination, especially in flowing recipient ecosystems where any characterization over the water matrix is ephemeral and linking exclusively to the moment of sampling. Hence, sediments should be considered for the establishment of WWTP operational benchmarks regulating the emission of contaminants, which is currently focused mostly on effluent composition thus potentially over/underestimating the longer-term impact of effluent discharge in the recipient waterways.

Performance of standard media in toxicological assessments with Daphnia magna: chelators and ionic composition versus metal toxicity.

Fully artificial test media can increase reproducibility and standardization in ecotoxicological assessments, but there is still a lack of convergence among ecotoxicology laboratories in aquatic test media with respect to ionic composition, chelators, and organic supplements. We compared the performance of Daphnia magna in three widely-used reconstituted media. The tested media differed in composition: (a) ADaM, an artificial medium based in a synthetic sea salt, with no a priori known chelating properties; (b) ASTM hard water supplemented with algal extract, a semi-artificial medium with unknown chelating properties; and (c) M7, a complex artificial medium containing EDTA as a chelator. All three media were suitable for rearing D. magna (although performance in M7 was suboptimal) and acute EC(50) values for reference substances (3,4-DCA, K(2)Cr(2)O(7)) were similar between media. In acute exposures to Cu and Cd, daphniids were least sensitive when reared in M7, as expected due to metal chelation by EDTA. Daphnia sensitivity to Cd was low in ADaM. Thus, these two media were suboptimal for assessing the toxicity of some metals to D. magna in acute tests. We suggest that both the ionic composition of the medium and the presence of chelators should be taken into account when metal toxicity is concerned. Chronic toxicity profiles for Cu suggested a mild chelating effect of the algal extract in ASTM medium. Still, ASTM hard water persists as one of the most suitable media for acute toxicity assessments of metals and metal-contaminated samples.

Sensitivity of a widespread groundwater copepod to different contaminants.

Groundwater is an indispensable resource for humankind and sustainable biomes functioning. Anthropogenic disturbance threatens groundwater ecosystems globally, but to which extent groundwater organisms respond to stressors remains poorly understood. Groundwater animals are rare, with small populations, difficult to find and to breed in the lab, which poses a main challenge to the assessment of their responses to pollutants. Despite the difficulties, assessing the toxicity of a large spectrum of stressors to groundwater organisms is a priority to inform towards appropriate environmental protection of these ecosystems. We tested the sensitivity to CuSO4, diclofenac, and NaCl of a groundwater population of the copepod Diacyclops crassicaudis crassicaudis and compared its sensitivity with the model organism Daphnia magna. We ranked its sensitivity using a species sensitivity distribution (SSD) approach using the feasible data available for groundwater and surface crustaceans. Our results show that the most toxic compound was CuSO4 for which higher amount of data was recorded and wider variability in response was observed. It was followed by diclofenac, largely lacking data for groundwater-adapted organisms, and the least toxic compound was NaCl. The differential sensitivity between D. crassicaudis and D. magna was contaminant-dependent. As a general trend D. crassicaudis was always distributed in the upper part of the SSD curves together with other groundwater-adapted organisms. Our results highlight that the widespread groundwater populations of the D. crassicaudis species complex, which can be successfully breed in the lab, may provide a reasonable approach to assess the ecological effects of anthropogenic stressors in groundwater ecosystems.

Impacts of wildfires in aquatic organisms: biomarker responses and erythrocyte nuclear abnormalities in Gambusia holbrooki exposed in situ.

Wildfires are an environmental concern due to the loss of forest area and biodiversity, but also because their role as drivers of freshwater systems contamination by metals. In this context, the fish Gambusia holbrooki was used as a model, deployed for in situ exposure in watercourses standing within a recently burnt area and further assessment of toxic effects. The fish were exposed during 4 days at four different sites: one upstream and another downstream the burnt area and two within the burnt area. Biochemical biomarkers for oxidative stress and damage were assessed. The extent of lipoperoxidative damage was monitored by quantifying malondialdehyde and DNA damage evaluated through erythrocyte nuclear abnormalities observation. Chemical analysis revealed higher metal levels within the burnt area, and exposed fish consistently showed pro-oxidative responses therein, particularly an increase of gill glutathione peroxidase and glutathione reductase activity, the records doubling compared to samples from sites in the unburnt area; also the activity of glutathione-S-transferases comparatively increased (by 2-fold in the liver) in samples from the burnt area, and malondialdehyde was produced twice as much therein and in samples downstream the burnt area reflecting oxidative damage. Consistently, the frequency of erythrocyte nuclear abnormalities was higher at sites within and downstream the burnt area. This study supports the use of sensitive oxidative stress and genotoxicity biomarkers for an early detection of potentially noxious ecological effects of wildfires runoff.

Cytotoxic effects of wildfire ashes: In-vitro responses of skin cells.

Wildfires are a complex environmental problem worldwide. The ashes produced during the fire bear metals and PAHs with high toxicity and environmental persistence. These are mobilized into downhill waterbodies, where they can impair water quality and human health. In this context, the present study aimed at assessing the toxicity of mimicked wildfire runoff to human skin cells, providing a first view on the human health hazardous potential of such matrices. Human keratinocytes (HaCaT) were exposed to aqueous extracts of ashes (AEA) prepared from ash deposited in the soil after wildfires burned a pine or a eucalypt forest stand. Cytotoxicity (MTT assay) and changes in cell cycle dynamics (flow cytometry) were assessed. Cell viability decreased with increasing concentrations of AEA, regardless of the ash source, the extracts preparation method (filtered or unfiltered to address the dissolved or the total fractions of contaminants, respectively) or the exposure period (24 and 48 h). The cells growth was also negatively affected by the tested AEA matrices, as evidenced by a deceleration of the progress through the cell cycle, namely from phase G0/G1 to G2. The cytotoxicity of AEA could be related to particulate and dissolved metal content, but the particles themselves may directly affect the cell membrane. Eucalypt ash was apparently more cytotoxic than pine ash due to differential ash metal burden and mobility to the water phase. The deceleration of the cell cycle can be explained by the attempt of cells to repair metal-induced DNA damage, while if this checkpoint and repair pathways are not well coordinated by metal interference, genomic instability may occur. Globally, our results trigger public health concerns since the burnt areas frequently stand in slopes of watershed that serve as recreation sites and sources of drinking water, thus promoting human exposure to wildfire-driven contamination.

Responses of benthic diatoms to waters affected by post-fire contamination.

Wildfire effects go beyond direct impact in terrestrial ecosystems. Specifically, the periphytic communities of aquatic ecosystems standing within and downstream the burnt areas are relevant ecological receptors of post-fire runoff contamination. Nevertheless, the off-site impacts of wildfires in these communities are limitedly studied so far. The present study aimed to assess the effects of river water contaminated with ash-loaded runoff in the growth benthic diatom Navicula libonensis (Schoeman 1970). Four surface water samples were collected approximately one year after the wildfire for laboratory testing with the diatom: one was collected from a site upstream the burnt area, within the Unhais river (UU); three were collected from sites standing within the burnt area, one in the Unhais river (UB) and two in the Zêzere river (Z1 and Z2), reflecting different hydrological regimes. N. libonensis was proven able to discriminate among river sites affected and unaffected by wildfire runoff, reflecting, in general, the expected trends considering the physico-chemical characterization of the water samples. The water samples from the sites standing within the burnt area inhibited the biomass yield and growth rate of the tested diatom, ranking the samples regarding toxicity as follows: Z1 > UB > Z2 > UU. However, UB rather than Z1 presented the highest contaminant burden, namely metal elements, and some were found above widely accepted safety benchmarks (polycyclic aromatic hydrocarbons were not detected). This inconsistency can be linked to unknown interactions among metals within each water sample, to differential nutrient enrichment of samples, as well as hydrological factors. Overall, our results suggest that monospecific laboratory assays with sensitive diatoms can be valuable as cost-effective screening tools to prioritize sites affected by wildfires runoff requiring in-depth monitoring of negative effects in benthic producer communities.