Ecological consequences when organisms avoid a contaminated environment: A study evaluating the toxicity of fipronil.

The ability of aquatic organisms to sense the surrounding environment chemically and interpret these signals correctly is crucial to their survival and ecological niche. This study applied the Heterogenous Multi-Habitat Assay System - HeMHAS to evaluate the avoidance potential of Daphnia magna to detect fipronil-contaminated habitats in a connected landscape after a short (48 h), previous, forced exposure to an environmentally relevant concentration of the same insecticide. The swimming of daphnids was also analyzed by recording the total distance covered. D. magna preferred areas with less contamination, although the effect of fipronil on their swimming ability (a decrease) was observed for all the concentrations tested. The application of non-forced multi-compartment exposure methodologies is a recent trend and is ecologically relevant as it is based on how contamination can really produce changes in an organism's habitat selection. Finally, we consider the importance of more non-forced exposure approaches where Stress Ecology can be aggregated to improve systemic understanding of the risk that contaminants pose to aquatic ecosystems from a broader landscape perspective.

Personal care products: an emerging threat to the marine bivalve Ruditapes philippinarum.

In the last few decades, there has been a growing interest in understanding the behavior of personal care products (PCPs) in the aquatic environment. In this regard, the aim of this study is to estimate the accumulation and effects of four PCPs within the clam Ruditapes philippinarum. The PCPs selected were triclosan, OTNE, benzophenone-3, and octocrylene. A progressive uptake was observed and maximum concentrations in tissues were reached at the end of the exposure phase, up to levels of 0.68 µg g-1, 24 µg g-1, 0.81 µg g-1, and 1.52 µg g-1 for OTNE, BP-3, OC, and TCS, respectively. After the PCP post-exposure period, the removal percentages were higher than 65%. The estimated logarithm bioconcentration factor ranged from 3.34 to 2.93, in concordance with the lipophobicity of each substance. No lethal effects were found although significant changes were observed for ethoxyresorufin O-demethylase activity, glutathione S-transferase activity, lipid peroxidation, and DNA damage.

Evaluation of short-term copper toxicity in a co-culture of Synechococcus sp., Chaetoceros gracilis and Pleurochrisys cf. roscoffensis exposed to changes in temperature and salinity levels.

Metals such as copper (Cu) enter marine environments from natural and anthropogenic sources, causing changes in the biodiversity of marine microalgae and cyanobacteria. Cu plays a dual role as either a micronutrient or toxicant depending on the environmental concentration. Many studies have summarized the potential of Cu to become more toxic to microalgae under environmental stress (for instance climate change). Most of the data available on Cu toxicity concerning microalgae and cyanobacteria have been produced using single-species laboratory tests, and there is still a significant gap in the information concerning the behavior of a group of algae exposed to environmental stressors. Thus, the objective of this study was to evaluate the toxicity of Cu at two concentrations (C1 = 2 µg L-1 and C2 = 5 µg L-1) in multispecies bioassays using three phytoplankton species (one cyanobacteria, Synechococcus sp., and two microalgae, Chaetoceros gracilis and Pleurochrisys cf. roscoffensis). Combinations of two temperatures (20 and 23 °C) and two salinities (33 and 36 PSU), were applied in a 96 h study using flow cytometry analysis (FCM). Algal growth and reactive oxygen species (ROS) production by 2'7'-dichlorofluorescein (DCFH) were monitored by FCM. The results indicated that Synechococcus sp. was more sensitive than C. gracilis and P. roscoffensis to Cu stress at a temperature 23 °C and salinity of 36 PSU under both concentrations of Cu. Chlorophyll a fluorescence showed a significant decrease (p < 0.05) in Synechococcus sp. under 5 µg L-1 of Cu in the combined treatment of 20 °C and 33 PSU; however, there was a significant increase in P. roscoffensis in all combinations at C2 = 5 µg L-1 compared to the control with no Cu, indicating a potential hormetic response to Cu for P. roscoffensis. ROS levels were triggered in a combination of 23 °C and 33 PSU and 5 µg L-1 of Cu, which was higher than all the other combinations studied. Our study resulted in data concerning the potential impacts caused by possible future climate change scenarios in aquatic habitats chronically exposed to metals.

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).

Multi-level biological responses of Daphnia magna exposed to settleable atmospheric particulate matter from metallurgical industries.

Metallurgical industries are a continuous source of air pollution due to the amount of settleable particulate matter (SePM) they release. This SePM is a complex mixture formed by metallic nanoparticles and metals, which reach terrestrial and aquatic ecosystems and can be a significant source of contamination. The aim of this study was to evaluate the adverse effects of SePM at different levels of biological organization in order to estimate its ecological impacts on aquatic ecosystems. For this purpose, the crustacean Daphnia magna was exposed to different concentrations of SePM (0.01, 0.1, 1, 5, 10 g/L) using a multi-level response approach. The endpoints studied were: avoidance throughout 24 h in a non-forced exposure system, reproduction (total number of neonates per female after 21 days of exposure), acetylcholinesterase activity (AChE) after 48 h, and finally, the feeding rates during a short-term exposure (48 h) and a long-term exposure (21 day + 48 h). There was a negative effect of SePM on all responses measured at high concentrations. The avoidance was concentration-dependent and represented 88 % and 100 % at the two highest concentrations. The AChE activity was significantly inhibited at 5 and 10 g/L. The total number of neonates increased from 1 g/L of SePM and the first brood occurred earlier as of 5 g/L compared to control. The post-exposure feeding rates were lower during long-term exposure at the highest concentration. Chemical analyses were performed to characterize the metals present in this SePM, but this study did not report any direct relationship with toxicity, due to the chemical heterogeneity of the particles. The emission of compounds caused by anthropogenic activity may have significant ecological consequences, so it is important to consider these possible effects on aquatic biota generated by the mixture of metals present in SePM originated from metallurgical activities. Environmental and sectorial regulations are needed to prevent contamination and ecological disturbances.

Single and multispecies microalgae toxicological tests assessing the impact of several BPA analogues used by industry.

BPA is a hazard for human and environmental health and recently BPA was added to the Candidate List of substances of very high concern by European Chemical Agency (ECHA). In accordance with this proposal, the authorities have encouraged the replacement of BPA by BPA analogues; however, little is known about the impact of these compounds on the environment. Due to this situation five BPA analogues (BPS, BPAP, BPAF, BPFL and BPC) were chosen in order to study their effects on marine primary producers. Three marine microalgae species (Phaeodactylum tricornutum, Tetraselmis suecica and Nannochloropsis gaditana) were selected for single and multispecies tests concerning the ecotoxicological effects of these BPA analogues. Microalgae were exposed to BPs over 72 h at different dosages (5, 20, 40, 80, 150 and 300 µM). Responses such as: growth, ROS production, cell complexity, cell size, autofluorescence of chlorophyll a, effective quantum yield of PSII and pigment concentrations were assessed at 24, 48 and 72 h. The results revealed that BPS and BPA showed lower toxicity to microalgae in comparison with BPFL > BPAF > BPAP and >BPC for the endpoints studied. N. gaditana was the least sensitive microalgae in comparison to P. tricornutum and T. suecica. However, a different trend was found in the multispecies tests where T. suecica dominated the microalgae community in relation to N. gaditana and P. tricornutum. The results of this work revealed for first time that present day BPA analogues are a threat and not a safe substitute for BPA in terms of the marine phytoplanktonic community. Therefore, the results of their impact on aquatic organisms should be shared.

Short and long-term exposure to the pesticides fipronil and 2,4-D: Effects on behavior and life history of Daphnia magna.

The high levels of contamination in aquatic ecosystems caused by pesticides and the organisms' consequent continuous exposure to it has made them vulnerable to damage. However, mobile organisms can avoid this continued exposure to contaminants by moving to less disturbed habitats. Therefore, through the use of the Heterogenous Multi-Habitat Assay System (HeMHAS), our objective was to evaluate the ability of Daphnia magna to detect and avoid habitats contaminated by fipronil and 2,4-D, in a spatially connected landscape. Further, the role of contamination by these pesticides, isolated and in mixtures, concerning the colonization of habitats by daphnids was also evaluated. Given that not all organisms successfully escape contamination, the chronic toxicity of the same pesticides using different parameters for D. magna (maternal survival, fecundity and maternal body length) was also evaluated. When evaluating the avoidance response by D. magna exposed to pesticides, there was no preference for the less contaminated areas for both compounds. However, organisms did not move to contaminated zones in the colonization experiments, with no immigration of daphnids to the zones with intermediate and the highest levels of fipronil, nor to the highest concentration of 2,4-D. Finally, the colonization by daphnids was significantly prevented when exposed to a mixture of the pesticides, in which the areas with the highest combinations of pesticide concentrations were not colonized by D. magna. Regarding the long-term chronic effects, negative consequences were observed, particularly for maternal body length, fecundity and maternal survival, due to the exposure to fipronil. Considering that pesticides can limit the areas colonized by organisms by making them unattractive, the risk of local population extinction may be underestimated if only standard endpoints involving forced exposure are studied.

Single and joint effects of cadmium and selenium on bioaccumulation, oxidative stress and metabolomic responses in the clam Scrobicularia plana.

Selenium (Se) is a vital trace element for many living organisms inclusive of aquatic species. Although the antagonistic action of this element against other pollutants has been previously described for mammals and birds, limited information on the join effects in bivalves is available. To this end, bivalves of the species Scrobicularia plana were exposed to Se and Cd individually and jointly. Digestive glands were analysed to determine dose-dependent effects, the potential influence of Se on Cd bioaccumulationas well as the possible recover of the oxidative stress and metabolic alterations induced by Cd. Selenium co-exposure decreased the accumulation of Cd at low concentrations. Cd exposure significantly altered the metabolome of clams such as aminoacyltRNA biosynthesis, glycerophospholipid and amino acid metabolism, while Se co-exposure ameliorated several altered metabolites such asLysoPC (14:0), LysoPE (20:4), LysoPE (22:6), PE (14:0/18:0), PE (20:3/18:4) andpropionyl-l-carnitine.Additionally, Se seems to be able to regulate the redox status of the digestive gland of clams preventing the induction of oxidativedamage in this organ. This study shows the potential Se antagonism against Cd toxicity in S. plana and the importance to study join effects of pollutants to understand the mechanism underlined the effects.

Assessing the colonization by Daphnia magna of pesticide-disturbed habitats (chlorpyrifos, terbuthylazine and their mixtures) and the behavioral and neurotoxic effects.

The spread of pesticides in water bodies integrated into agricultural landscapes may prevent some areas from being colonized. In this study, the effects on the colonization responses of D. magna exerted by gradients of realistic environmental concentrations of the pesticides chlorpyrifos, terbuthylazine and their mixtures were tested in a novel multicompartment non-forced exposure system. Furthermore, the effects of both pesticides and their mixtures on the swimming behavior and the neurotransmission activity of D. magna were analyzed using a traditional forced exposure system. The synthesis and concentration of the main environmental metabolites of terbuthylazine were also analyzed. Results confirmed that D. magna exposed to mixture gradients were able to detect the pollutants and their colonization dynamics were drastically inhibited. The swimming behavior increased in D. magna exposed to the highest concentration of the mixture treatment. AChE activity was only significantly inhibited in the D. magna exposed to the highest concentration of chlorpyrifos. Changes in swimming behavior could not be directly related to the effects on AChE. Furthermore, the synthesis of the metabolite terbuthylazine 2-hydroxy during the course of the experiments was confirmed. These results demonstrate the importance of integrating pesticide mixtures in both non-forced and forced exposure systems during ecotoxicological assays.

Avoidance responses by Danio rerio reveal interactive effects of warming, pesticides and their mixtures.

Temperature variations and thermal extremes events caused by climate change can have profound implications for the toxicity of pesticides in aquatic organisms. Using an innovative system (Heterogeneous Multi-Habitat Test System - HeMHAS) that allows the simulation of different scenarios within a spatially heterogeneous landscape, the effects on the habitat selection of Danio rerio fish caused by the pesticides fipronil and 2,4-D were studied as single compounds and in mixture and integrated with air temperature variation (20, 24 and 28 °C). As a result, D. rerio detected and avoided both pesticides at air temperatures of 20 and 24 °C; however, at 28 °C no significant difference was observed in habitat choice by fish. Additionally, when pesticides were mixed in a heterogeneously contaminated landscape, it was observed that D. rerio detected contamination and preferred the clean zone at 20 and 24 °C; however, at 28 °C the potential to escape from the most contaminated areas was impaired. Thus, contamination by both pesticides made the habitat selection behavior of fish at 20 and 24 °C more noticeable. In addition, the association between pesticides and temperature showed negative effects on the response of fish to detect and escape from contaminated environments, suggesting the influence of temperature in altering the ability of the organism to provide an efficient response to stress.

The antibacterials ciprofloxacin, trimethoprim and sulfadiazine modulate gene expression, biomarkers and metabolites associated with stress and growth in gilthead sea bream (Sparus aurata).

The high consumption and subsequent input of antibacterial compounds in marine ecosystems has become a worldwide problem. Their continuous presence in these ecosystems allows a direct interaction with aquatic organisms and can cause negative effects over time. The objective of the present study was to evaluate the effects of exposure to three antibacterial compounds of high consumption and presence in marine ecosystems (Ciprofloxacin CIP, Sulfadiazine SULF and Trimethoprim TRIM) on the physiology of the gilthead sea bream, Sparus aurata. Plasma parameters, enzymatic biomarkers of oxidative stress and damage and expression of genes related to stress and growth were assessed in exposed S. aurata specimens. For this purpose, sea bream specimens were exposed to individual compounds at concentrations of 5.2 ± 2.1 µg L-1 for CIP, 3.8 ± 2.7 µg L-1 for SULF and 25.7 ± 10.8 µg L-1 for TRIM during 21 days. Exposure to CIP up-regulated transcription of genes associated with the hypothalamic-pituitary-thyroid (HPT) (thyrotropin-releasing hormone, trh) and hypothalamic-pituitary-interrenal (HPI) axes (corticotropin-releasing hormone-binding protein, crhbp) in the brain, as well as altering several hepatic stress biomarkers (catalase, CAT; glutathione reductase, GR; and lipid peroxidation, LPO). Similar alterations at the hepatic level were observed after exposure to TRIM. Overall, our study indicates that S. aurata is vulnerable to environmentally relevant concentrations of CIP and TRIM and that their exposure could lead to a stress situation, altering the activity of antioxidant defense mechanisms as well as the activity of HPT and HPI axes.

Bioaccumulation and biochemical responses in the peppery furrow shell Scrobicularia plana exposed to a pharmaceutical cocktail at sub-lethal concentrations.

Pharmaceutical drugs in the aquatic medium may pose significant risk to non-target organisms. In this study, the potential toxicity of a mixture of three compounds commonly detected in marine waters (ibuprofen, ciprofloxacin and flumequine) was assessed, by studying bioaccumulation, oxidative stress and neurotoxicity parameters (catalase CAT, superoxide dismutase SOD, glutathione reductase GR, glutathione S-transferase GST, lipid peroxidation LPO, glutathione peroxidase GPX, metallothionein MT and acetylcholinesterase AChE) in the clam Scrobicularia plana. Temporal evolution of selected endpoints was evaluated throughout an exposure period (1, 7 and 21 days) followed by a depuration phase. The accumulation of all drugs was fast, however clams showed the ability to control the internal content of drugs, keeping their concentration constant throughout the exposure and reducing their content after 7 days of depuration. The induction of biochemical alterations (SOD, CAT, LPO, MT, AChE) was observed in gills and digestive gland probably related to an imbalance in the redox state of clams as a consequence of the exposure to the drug mixture. These alterations were also maintained at the end of the depuration week when the high levels of SOD, CAT, GST and LPO indicated the persistence of oxidative stress and damage to lipids despite the fact that clams were no longer exposed to the mixture.

Products released from surgical face masks can provoke cytotoxicity in the marine diatom Phaeodactylum tricornutum.

Surgical face masks are more present than ever as personal protective equipment due to the COVID-19 pandemic. In this work, we show that the contents of regular surgical masks: i) polypropylene microfibres and ii) some added metals such as: Al, Fe, Cu, Mn, Zn and Ba, may be toxic to some marine life. This work has got two objectives: i) to study the release rate of the products from face masks in marine water and ii) to assess the toxicity in Phaeodactylum tricornutum of these by-products. To achieve these two objectives, we performed release kinetic experiments by adding masks in different stages of fragmentation to marine water (i.e. whole face masks and fragments of them 1.52 ± 0.86 mm). Released microfibres were found after one month in shaking marine water; 0.33 ± 0.24 and 21.13 ± 13.19 fibres·mL-1 were collected from the whole and fragmented face masks, respectively. Significant amounts of dissolved metals such as Mn, Zn and Ni, as well as functional groups only in the water containing the face mask fragments were detected. Water from both treatments was employed to study its toxicity on the marine diatom. Only the water from the face mask fragments showed a significant, dose-dependent, decrease in cell density in P. tricornutum; 53.09 % lower than in the controls. Although the water from the face mask fragments showed greater effects on the microalgae population than the water from the whole face mask, the latter treatment did show significant changes in the photosynthetic apparatus and intrinsic properties of the cells. These results indicate that during fragmentation and degradation face masks a significant chemical print can be observed in the marine environment.

Can a mixture of agrochemicals (glyphosate, chlorpyrifos and chlorothalonil) mask the perception of an individual chemical? A hidden trap underlying ecological risk.

As aquatic environments associated with conventional agriculture are exposed to various pesticides, it is important to identify any possible interactions that modify their effects when in a mixture. We applied avoidance tests with Danio rerio, exposing juveniles to three relevant current use pesticides: chlorpyrifos (CPF), chlorothalonil (CTL) and glyphosate (Gly), individually and in binary mixtures (CPF-Gly and CTL-Gly). Our goal was to identify the potential of contaminants to trigger the avoidance response in fish and detect any changes to that response resulting from binary mixtures. Avoidance was assessed for three hours using an open gradient system with six levels of increasing concentrations. Fish avoided environmentally relevant concentrations of the three compounds. The avoidance of CPF [AC50 = 7.95 (3.3-36.3) µg/L] and CTL [AC50 = 3.41 (1.2-41.6) µg/L] was evident during the entire period of observation. In the case of Gly, the response changed throughout the experiment: initially (until 100 min) the fish tolerated higher concentrations of the herbicide [AC50 = 52.2 (12.1-2700) µg/L] while during the later period (after 100 min) a clearer avoidance [1.5 (0.8-4.2) µg/L] was observed. The avoidance recorded using CPF and CTL alone was attenuated by the presence of Gly. Applying an additive concentration model, Gly initially acted synergistically with the other two compounds, although this interaction was not observed during the later period. Avoidance gives us an idea of how the distribution of populations may be altered by contamination, our results suggest that in some mixtures this response may be inhibited, at least temporarily, thus masking the ecological risk of the exposure.

Experimental evidence of contamination driven shrimp population dynamics: Susceptibility of populations to spatial isolation.

Contamination is likely to affect the composition of an ecological landscape, leading to the rupture of ecological connectivity among habitats (ecological fragmentation), which may impact on the distribution, persistence and abundance of populations. In the current study, different scenarios within a spatially heterogeneous landscape were simulated in the Heterogeneous Multi-Habitat Assay System (HeMHAS) to evaluate the potential effect that contamination (copper at 0.5 and 25 µg/L) might have on habitat selection by the estuarine shrimp Palaemon varians in combination with two other ecological factors: predator presence and food availability. As a result, P. varians detected and avoided copper; however, in the presence of the predation signal, shrimps shifted their response by moving to previously avoided regions, even if this resulted in a higher exposure to contamination. When encouraged to move towards environments with a high availability of food, a lower connectivity among the shrimp populations isolated by both contamination and predation risk simultaneously was evidenced, when compared to populations isolated only by the risk of predation. These results indicate that contamination might: (i) trigger avoidance in shrimps, (ii) prevent colonization of attractive foraging areas, (iii) enhance populations' isolation and (iv), make populations more susceptible to local extinction.

Are habitable clean areas in heterogeneously contaminated landscapes functioning as escape zones for fish populations to alleviate stress?

Environmental contamination is a problem that reduces the quality of ecosystems and may make them unsuitable to accommodate life. As many ecosystems are connected, some organisms avoid the stress from continuous exposure to contaminants by moving towards less disturbed areas. However, the landscapes in which organisms move might vary regarding the concentrations of contaminants, in the form of gradients or patches of contamination. Therefore, although it is expected that organisms prefer clean areas, their sporadic contact with contamination should not be ignored, as the greater the probability of being in contact with contaminated areas, the higher the stress. The aim of this study was to assess how the stress (cortisol levels) of zebrafish (Danio rerio) varies as a consequence of heterogeneity in the chemical composition of the habitats and the presence of uncontaminated areas in this heterogeneous landscape. Zebrafish were exposed to heterogeneous contamination scenarios containing different concentrations of copper along a free-choice multi-compartmented system, in which they were able to flee from the most contaminated areas. Fish escaped from the most contaminated areas with an avoidance by 50% of population (AC50) at concentrations of 41 (copper gradient scenario), 25 (spatially limited contamination scenario) and 69 (highly contaminated scenario) µg/L. Higher cortisol levels were observed in the populations exposed to homogeneously contaminated and highly contaminated (by copper) scenarios (both with no acceptable clean area to flee to). In summary, the uncontaminated areas might be crucial for the spatial dynamics of fish populations in a chemically heterogeneous landscape due to their role as escape zones to alleviate stress.

Could Contamination Avoidance Be an Endpoint That Protects the Environment? An Overview on How Species Respond to Copper, Glyphosate, and Silver Nanoparticles.

The use of non-forced multi-compartmented exposure systems has gained importance in the assessment of the contamination-driven spatial avoidance response. This new paradigm of exposure makes it possible to assess how contaminants fragment habitats, interfering in the spatial distribution and species' habitat selection processes. In this approach, organisms are exposed to a chemically heterogeneous scenario (a gradient or patches of contamination) and the response is focused on identifying the contamination levels considered aversive for organisms. Despite the interesting results that have been recently published, the use of this approach in ecotoxicological risk studies is still incipient. The current review aims to show the sensitivity of spatial avoidance in non-forced exposure systems in comparison with the traditional endpoints used in ecotoxicology under forced exposure. To do this, we have used the sensitivity profile by biological groups (SPBG) to offer an overview of the highly sensitive biological groups and the species sensitive distribution (SSD) to estimate the hazard concentration for 5% of the species (HC5). Three chemically different compounds were selected for this review: copper, glyphosate, and Ag-NPs. The results show that contamination-driven spatial avoidance is a very sensitive endpoint that could be integrated as a complementary tool to ecotoxicological studies in order to provide an overview of the level of repellence of contaminants. This repellence is a clear example of how contamination might fragment ecosystems, prevent connectivity among populations and condition the distribution of biodiversity.