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).

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.

An abiotic typology and reference conditions for nutrients and chlorophyll-a in subtropical reservoirs (São Paulo State, Brazil).

The establishment of reference conditions and typology are two important steps in water resources management. The reference conditions enable the determination of how impacted an ecosystem is, while the typology facilitates the implementation of management procedures. A study of subtropical reservoirs in São Paulo State (Brazil) was performed to obtain (1) an abiotic typology, using principal component analysis (PCA) and cluster analysis (Ward's method with Euclidean distances), and (2) reference conditions for total phosphorus (TP), total nitrogen (TN), and chlorophyll-a (Chl-a) in the dry and rainy seasons for one of the established typologies. Two main statistical techniques were used: the lake population distribution approach and the trisection method. PCA identified an environmental gradient in the east-west direction, with reservoirs at higher latitudes and with larger dimensions in the west, and smaller reservoirs in eastern areas with higher altitude and higher average annual rainfall. The PCA and cluster analysis indicated that there were four main types of reservoirs. The nutrient criteria techniques, obtained for 13 type I rainy-east reservoirs, were not significantly different (t test, p < 0.05). Although the methods resulted in similar reference conditions, one-way ANOVA indicated significant differences between the seasons for Chl-a, which levels were slightly higher in the rainy season. As far as we know, this is the first study to simultaneously provide an abiotic typology and reference conditions for chlorophyll-a and nutrients in different seasons, considering subtropical reservoirs in South America. This investigation makes an important contribution to the monitoring and management of subtropical reservoirs, and the promotion of dialog between the scientific community and managers, aiming at ensuring the sustainability of water bodies.

Not Only Toxic but Repellent: What Can Organisms' Responses Tell Us about Contamination and What Are the Ecological Consequences When They Flee from an Environment?

The ability of aquatic organisms to sense the surrounding environment chemically and interpret such signals correctly is crucial for their ecological niche and survival. Although it is an oversimplification of the ecological interactions, we could consider that a significant part of the decisions taken by organisms are, to some extent, chemically driven. Accordingly, chemical contamination might interfere in the way organisms behave and interact with the environment. Just as any environmental factor, contamination can make a habitat less attractive or even unsuitable to accommodate life, conditioning to some degree the decision of organisms to stay in, or move from, an ecosystem. If we consider that contamination is not always spatially homogeneous and that many organisms can avoid it, the ability of contaminants to repel organisms should also be of concern. Thus, in this critical review, we have discussed the dual role of contamination: toxicity (disruption of the physiological and behavioral homeostasis) vs. repellency (contamination-driven changes in spatial distribution/habitat selection). The discussion is centered on methodologies (forced exposure against non-forced multi-compartmented exposure systems) and conceptual improvements (individual stress due to the toxic effects caused by a continuous exposure against contamination-driven spatial distribution). Finally, we propose an approach in which Stress and Landscape Ecology could be integrated with each other to improve our understanding of the threat contaminants represent to aquatic ecosystems.

Predicting zebrafish spatial avoidance triggered by discharges of dairy wastewater: An experimental approach based on self-purification in a model river.

Wastewater discharges from dairy industries can cause a range of harmful effects in aquatic ecosystems, including a decline in biodiversity due to species evasion. Therefore, it is important to know the purification potential of rivers for the removal of pollutants released in dairy wastewater (DWW). The hypothesis adopted in this work was that the release of DWW into stretches of the Ribeirão dos Pombos River (São Paulo State, Brazil) might trigger an avoidance response, resulting in fish migrating to other regions, with the response being greater when the self-cleaning potential of the river is smaller. Therefore, the goals of the present study were to: (i) investigate how land use and seasonality of the rainfall regime influence the quality of the water in different areas of the river (P1: river source; P2: urban region; P3: rural region); (ii) assess the potential of the river to purify DWW; and (iii) evaluate the potential toxicity and repellency of DWW to the freshwater fish Danio rerio, using acute toxicity (mortality) and non-forced avoidance tests, respectively. P1 was shown to be the most preserved area. The chemical composition of the river varied seasonally, with higher concentrations of Cl- and SO42- at P3 during the rainy period. The river purification potential for DWW was higher at P2, due to greater microbiological activity (associated with higher BOD). The DWW was more acutely toxic in water from P2. The avoidance response was strongly determined by the concentration of DWW, especially for water from P2. The high capacity for self-cleaning at P2 did not seem sufficient to maintain the stability of the ecosystem. Finally, the non-forced exposure system proved to be a suitable approach that can assist in predicting how contaminants may affect the spatial distributions of organisms.

Long-term performance and acute toxicity assessment of scaled-up air-cathode microbial fuel cell fed by dairy wastewater.

Long-term performance of a scaled-up air-cathode microbial fuel cell (MFC) and toxicity removal were studied with dairy wastewater (DW) used as the substrate. The MFC in a semi-continuous flow was strategically inoculated with consortium of Shewanella oneidensis and Clostridium butyricum. The scaled-up approach delivered a maximum power density of 0.48 W/m3 (internal resistance of 73 Ω) removing 93% of total chemical oxygen demand and 95% of total biochemical oxygen demand at organic loading rate (OLR) of 0.9 kg COD/m3/d and hydraulic retention time (HRT) of 21 days. It also achieved high removal efficiency of nitrate (100%), organic nitrogen (57%), sulfate (90%) and organic phosphorus (90%). The power generation and DW degradation performance decreased with OLR of 1.8 kg COD/m3/d and HRT of 10.5 days. Furthermore, testing of acute toxicity with the microcrustacean, Daphnia similis, revealed high toxic effect of the raw DW, but no toxic effects of the MFC effluent during 95 days of operation. These outcomes demonstrated that scaled-up MFC fed with high-strength DW should be an effective system for pollutants removal and simultaneously energy recovery.

Risk of triclosan based on avoidance by the shrimp Palaemon varians in a heterogeneous contamination scenario: How sensitive is this approach?

As the exposure of organisms to contaminants can provoke harmful effects, some organisms try to avoid a continuous exposure by using different strategies. The aim of the current study was to assess the ability of the shrimp Palaemon varians to detect a triclosan gradient and escape to less contaminated areas. Two multi-compartmented exposure systems (the linear system and the HeMHAS-Heterogeneous Multi-Habitat Assay System) were used and then results were compared. Finally, it was aimed how sensitive the avoidance response is by comparing it with other endpoints through a sensitivity profile by biological groups and the species sensitive distribution. The distribution of the shrimps along the triclosan gradient was dependent on the concentrations, not exceeding 3% for 54 µg/L in the linear system and 7% for 81 µg/L in the HeMHAS; 25% of organisms preferred the compartment with the lowest concentrations in both systems. Half of the population seems to avoid concentrations around 40-50 µg/L. The triclosan concentration that might start (threshold) to trigger an important avoidance (around 20%) was estimated to be of 18 µg/L. The profile of sensitivity to triclosan showed that avoidance by shrimps was less sensitive than microalgae growth and avoidance by guppy; however, it might occur even at concentrations considered safe for more than 95% of the species. In summary, (i) the HeMHAS proved to be a suitable system to simulate heterogeneous contamination scenarios, (ii) triclosan triggered the avoidance response in P. varians, and (iii) the avoidance was very sensitive compared to other ecotoxicological responses.

Influence of interspecific interactions on avoidance response to contamination.

An increasing number of studies have shown the ability of organisms to escape from toxic effects due to contamination, by moving spatially towards less contaminated habitats. However, this issue has been investigated in monospecific scenarios, without considering possible interactions between species during the contamination avoidance process. It is widely known that the spatial distribution of one species can be affected by another one, in different ways. Therefore, the main question addressed in the present study was as follows: Might interspecific interaction between the freshwater fish Danio rerio (zebrafish) and Poecilia reticulata (guppy) change their behavior patterns in terms of avoidance in the presence of a copper gradient? Zebrafish and guppies exposed to a copper gradient were tested for avoidance responses in a free-choice, non-forced, static, multi-compartmented exposure system, using two distinct approaches: (1) monospecific tests, in which only one species was exposed to the copper gradient, at two different population densities; and (2) multispecific tests, in which both species were tested simultaneously. In the control (with no copper) monospecific tests, both species were randomly distributed; however, in the control multispecific test, P. reticulata tended to aggregate. In the monospecific tests with a copper gradient, both species avoided copper in a similar way, with AC50 (concentration triggering avoidance in 50% of the exposed population) values between 15 and 18 µg·L-1, irrespective of the population density. However, in the multispecific tests, P. reticulata displaced D. rerio to previously avoided copper levels, consequently increasing the AC50 of D. rerio to 75 µg·L-1. This study shows the importance of understanding the interactions among species in contaminated areas, and the way that one species can prevent the avoidance behavior of another.

Bisphenol risk in fish exposed to a contamination gradient: Triggering of spatial avoidance.

Bisphenol A (BPA) is an emerging contaminant widely used in various industrial products. Sublethal toxicity of BPA on aquatic organisms is expected to occur at a concentration of around 500 µg L-1, which is much higher than environmentally realistic concentrations found in water bodies (up to 0.41 µg L-1). However, there is no information concerning how a BPA contamination gradient could affect the spatial displacement of organisms. We hypothesized that fish might be able to detect an environmentally realistic BPA contamination gradient and avoid potential toxic effects due to continuous exposure. Therefore, the objectives of this work were: (i) to determine if BPA could trigger an avoidance response in the freshwater fish Poecilia reticulata; (ii) to assess whether BPA-driven avoidance occurs at environmentally relevant concentrations; and (iii) to estimate the population immediate decline (PID) at the local scale, considering avoidance and mortality as endpoints. Avoidance experiments were performed in a seven-compartment non-forced exposure system, in which a BPA contamination gradient was simulated. The results indicated that BPA triggered avoidance in P. reticulata. In a traditional forced acute toxicity test, lethal effects in 50% of the population occurred at a BPA concentration of 1660 µg L-1, while in the non-forced system with a BPA concentration gradient, avoidance of 50% of the population occurred at a concentration four orders of magnitude lower (0.20 µg L-1). At environmentally relevant BPA concentrations, PID was mainly determined by the avoidance response. Avoidance in P. reticulata populations is expected to occur at BPA concentrations below those that cause sublethal effects on fish and are considered safe by international agencies (≤1 µg L-1). The approach used in the present study represents a valuable tool for use in environmental risk assessment strategies, providing a novel and ecologically relevant response that is complementary to traditional ecotoxicological tests.

Habitat fragmentation caused by contaminants: Atrazine as a chemical barrier isolating fish populations.

Information on how atrazine can affect the spatial distribution of organisms is non-existent. As this effect has been observed for some other contaminants, we hypothesized that atrazine-containing leachates/discharges could trigger spatial avoidance by the fish Poecilia reticulata and form a chemical barrier isolating upstream and downstream populations. Firstly, guppies were exposed to an atrazine gradient in a non-forced exposure system, in which organisms moved freely among the concentrations, to assess their ability to avoid atrazine. Secondly, a chemical barrier formed by atrazine, separating two clean habitats (extremities of the non-forced system), was simulated to assess whether the presence of the contaminant could prevent guppies from migrating to the other side of the system. Fish were able to avoid atrazine contamination at environmentally relevant concentrations (0.02 µg L-1), below those described to cause sub-lethal effects. The AC50 (atrazine concentration causing avoidance to 50% of the population) was 0.065 µg L-1. The chemical barrier formed by atrazine at 150 µg L-1 (concentration that should produce an avoidance around 82%) caused a reduction in the migratory potential of the fish by 47%; while the chemical barrier at 1058 µg L-1 (concentration that produces torpidity) caused a reduction in the migratory potential of the fish by 91%. Contamination by atrazine, besides driving the spatial distribution of fish populations, has potential to act as a chemical barrier by isolating fish populations. This study includes a novel approach to be integrated in environmental risk assessment schemes to assess high-tier contamination effects such as habitat fragmentation and population displacement and isolation.

Potential effects of triclosan on spatial displacement and local population decline of the fish Poecilia reticulata using a non-forced system.

Triclosan (TCS) is an emerging contaminant of concern in environmental studies due to its potential adverse effects on fish behavior. Since avoidance has been shown to be a relevant behavioral endpoint, our aims were: (i) to determine if TCS is able to trigger an avoidance response in Poecilia reticulata; (ii) to predict the population immediate decline (PID) caused by TCS exposure, by integrating lethality and avoidance responses; and (iii) to verify the overestimation of risk when mortality is assessed under forced exposure. Fish were exposed to TCS in a forced exposure system, to assess mortality, and to a TCS gradient in a non-forced exposure (NFE) system. Two NFE scenarios were simulated: (#1) a spatially permanent gradient, including low and high concentrations; and (#2) a scenario with high concentrations, simulating a local discharge. The fish avoided TCS concentrations as low as 0.2 µg L-1 (avoidance of 22%). The AC50 obtained from scenario #1 (8.04 µg L-1) was about 15 times more sensitive than that from scenario #2 (118.4 µg L-1). In general, up to the highest concentration tested (2000 µg L-1), the PID was determined by the avoidance. Mortality from the forced exposure was overestimated (48 h-LC50 of 1650 mg L-1), relative to the NFE. The reduced mortality in a non-forced environment does not imply a lower effect, because part of the population is expected to disappear by moving towards favorable environments. TCS is a potential environmental disturber, since at environmentally relevant concentrations (<2 µg L-1) it could cause a decline in the fish population.