Ecotoxicological Tests Research Articles

Natural daily patterns in fish behaviour may confound results of ecotoxicological testing.

Low doses of neuroactive chemicals end up in the environment and disrupt behaviour of non-target organisms. Although a whole range of studies have documented pollutant-induced changes in behaviour, natural daily variability in behaviour is rarely taken into account. This is surprising because biological rhythms may affect the outcome of experiments, are adaptive and are expected to be sensitive to neurochemical exposure. Here, we exploit daily behavioural variation in the fish model Nothobranchius furzeri to examine if behavioural effects of chronic exposure (74 days) to an environmentally relevant level (28ng/L) of the neurochemical fluoxetine depend on the time of day. Fluoxetine exposure induced an increase in anxiety-related behaviour that was slightly more pronounced in the evening compared to the morning. Moreover, open-field locomotor activity was disrupted and daily patterns in activity lifted upon exposure to the compound. These results imply that short-term behavioural variability should be considered both to standardise ecological risk assessment of neuroactive chemicals as well as to better understand the environmental impact of such compounds in aquatic ecosystems.

Aquatic contaminants in Solomon Islands and Vanuatu: Evidence from passive samplers and Microtox toxicity assessment

Water Quality issues in many Pacific countries are rising, with the increase in coastal populations and associated urban runoff but management requires contamination issues in the aquatic environment to be identified and prioritised. In Vanuatu and Solomon Islands there are few laboratories and resources to assess for the presence or impact of complex chemical contaminants. The extent and impact of chemical contamination of the marine and coastal environment is poorly described.Passive chemical samplers were used to measure a range of aquatic pollutants around the capital cities, Honiara (Solomon Islands) and Port Vila (Vanuatu). We detected a range of chemicals indicative of agricultural and industrial contamination and a few sites had concerning concentrations of specific hydrocarbons and pesticides. The rapid ecotoxicology test, Microtox, indicated toxic impacts in rivers, coastal sites and urban drains This work provides new data on chemical contamination and possible impacts of that contamination for both countries. The techniques could be applied widely across the region to generate critical data for environmental management, guide monitoring efforts and measure the impact of policy or land-use changes.

Twists and turns of an oyster's life: effects of different depuration periods on physiological biochemical functions of oysters.

Aquaculture activities are often established in the vicinity of highly populated, potentially contaminated areas. Animals cultured at such locations, namely bivalves, are frequently used as test organisms in ecotoxicological testing. In this case, a period of depuration is required to allow the normalization of physiological processes, which are likely to be altered after exposure to a multiplicity of waterborne contaminants occurring in the wild. One of the most important species in modern marine aquaculture is the oyster species Crassostrea gigas. The aim of this study was to assess if the current depuration time frame of 24 h (adopted by most aquaculture facilities), is long enough to permit oysters to revert potential toxic effects exerted by environmental contaminants, allowing their use in laboratory-based ecotoxicological studies. The selected approach involved the monitoring of biochemical (antioxidant defence, oxidative damage, phase II metabolism, and neurological homeostasis) and physiological (condition index) parameters, along a period of 42 days. The obtained results showed that a period of 24 h does not revert any of the potential toxic effects caused by environmental contaminants to which animals may have been previously subjected; even a period of 42 days was not long enough for the oysters to completely normalize the levels of their antioxidant defences, namely total GPx activity, which increased over time. Lipid peroxidation was also increased during the depuration period, and the activity of the metabolic isoenzymes GSTs was significantly decreased. Furthermore, AChE activity measured in the adductor muscle of oysters was increased over time. These assumptions suggest that a period of depuration longer than 24 h is mandatory to obtain adequate test organisms of this oyster species, to be used for ecotoxicological testing purposes.

Evaluation of an in vitro assay to screen for the immunotoxic potential of chemicals to fish

A wide variety of environmental contaminants has been shown to disrupt immune functions of fish and may compromise their defense capability against pathogens. Immunotoxic effects, however, are rarely considered in ecotoxicological testing strategies. The aim of this study was to systematically evaluate the suitability of an in vitro immuno-assay using selected fish immune parameters to screen for chemicals with known immunotoxic potential and to differentiate them from non-immunotoxicants. Non-stimulated and lipopolysaccharide-stimulated head kidney leukocytes of rainbow trout (Oncorhynchus mykiss) were exposed for 3 h or 19 h to chemicals with different modes of action. As immune parameters, phagocytosis activity, oxidative burst activity and cytokine transcription (IL-1β, TNFα, IL-10) were examined, accompanied by in silico modelling. The immunotoxicants dexamethasone, benzo(a)pyrene, ethinylestradiol and bisphenol A significantly altered the immune parameters at non-cytotoxic concentrations whereas diclofenac had only weak effects. However, the two baseline chemicals with no known immunotoxic potential, butanol and ethylene glycol, caused significant effects, too. From our results it appears that the in vitro fish leukocyte assay as performed in the present study has only a limited capacity for discriminating between immunotoxicants and non-immunotoxicants.

A novel technique for Cd removal from soil based on alginate-derived floatable spheres

This paper proposes a fast, facile and eco-friendly strategy for removing Cd from soil. By combining two types of floatable alginate-derived multifunctional spheres: magnetic hollow carbon composite (MHCC) and calcium alginate aerogel (CAA) spheres, the fixed state Cd in soil is activated and removed sequentially. MHCC spheres activate and desorb the solid phase anchored Cd into mobile Cd2+ ions, and CAA spheres effectively absorb the free Cd2+ ions from the liquid phase via the massive absorption sites of carboxy and hydroxy groups. The remediation results of 5-round successive MHCC-CAA treatments (1.5 g MHCC-0.45 g CAA in 60 g soil) show that 44.02% of Cd is removed from the soil. MHCC spheres demonstrate excellent recyclability of 88.87 and 94.45% in flooding and non-flooding soil, respectively. The texture, cation exchange capacity (CEC) and pH value of the remediated soil suggest that the MHCC-CAA system has little interference on the soil properties. No remarkable hindrance of microbial growth by MHCC or CAA spheres is observed in the eco-toxicological test, indicating the ecological friendliness of MHCC-CAA remediation system.

Effective processing and evaluation of chemical imaging data with respect to morphological features of the zebrafish embryo

A workflow was developed and implemented in a software tool for the automated combination of spatially resolved laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) data and data on the morphology of the biological tissue. Making use of a recently published biological annotation software, FishImager automatically assigns the biological feature as regions of interest (ROIs) and overlays them with the quantitative LA-ICP-MS data. Furthermore, statistical tools including cluster algorithms can be applied to the elemental intensity data and directly compared with the ROIs. This is effectively visualized in heatmaps. This allows gaining statistical significance on distribution and co-localization patterns. Finally, the biological functions of the assigned ROIs can then be easily linked with elemental distributions. We demonstrate the versatility of FishImager with quantitative LA-ICP-MS data of the zebrafish embryo tissue. The distribution of natural elements and xenobiotics is analyzed and discussed. With the help of FishImager, it was possible to identify compartments affected by toxicity effects or biological mechanisms to eliminate the xenobiotic. The presented workflow can be used for clinical and ecotoxicological testing, for example. Ultimately, it is a tool to simplify and reproduce interpretations of imaging LA-ICP-MS data in many applications.Graphical abstract

Germination, root elongation, and photosynthetic performance of plants exposed to sodium lauryl ether sulfate (SLES): an emerging contaminant

The anionic surfactant SLES (sodium lauryl ether sulfate) is an emerging contaminant, being the main component of foaming agents that are increasingly used by the tunnel construction industry. To fill the gap of knowledge about the potential SLES toxicity on plants, acute and chronic effects were assessed under controlled conditions. The acute ecotoxicological test was performed on Lepidum sativum L. (cress) and Zea mays L. (maize). Germination of both species was not affected by SLES in soil, even at concentrations (1200 mg kg−1) more than twice higher than the maximum realistic values found in contaminated debris, thus confirming the low acute SLES toxicity on terrestrial plants. The root elongation of the more sensitive species (cress) was instead reduced at the highest SLES concentration. In the chronic phytotoxicity experiment, photosynthesis of maize was downregulated, and the photosynthetic performance (PITOT) significantly reduced already under realistic exposures (360 mg kg−1), owing to the SLES ability to interfere with water and/or nutrients uptake by roots. However, such reduction was transient, likely due to the rapid biodegradation of the surfactant by the soil microbial community. Indeed, SLES amount decreased in soil more than 90% of the initial concentration in only 11 days. A significant reduction of the maximum photosynthetic capacity (Pnmax) was still evident at the end of the experiment, suggesting the persistence of negative SLES effects on plant growth and productivity. Overall results, although confirming the low phytotoxicity and high biodegradability of SLES in natural soils, highlight the importance of considering both acute and nonlethal stress effects to evaluate the environmental compatibility of soil containing SLES residues.

Ecotoxicological risk assessment of pentachlorophenol, an emerging DBP to plants: evaluation of oxidative stress and antioxidant responses

Chlorophenols are not only noticed in an effluvium of industries but also can emerge from the water treatment plants for domestic supply which poses a high threat for crop production and human health. Therefore, research on their risks to ecosystem and human health via ecotoxicological tests to derivate permissible environmental contaminant concentrations is necessary. The chlorophenols produced in the course of chlorination of potable water is an outcome of natural carboxylic acids/organic material and those chlorophenols occurred as emerging disinfection byproducts (EDBPs). Among chlorophenols, pentachlorophenol (PCP) has been recently identified as one of the important EDBPs. The main objective was to evaluate the PCP-induced genotoxicity and the oxidative damage in two plant species, i.e., Allium cepa and Vigna radiata. Genotoxicity of PCP was examined at three selected concentrations based on EC50 (half-maximal effective concentrations) values in both the plants along with the defense mechanism. EC50 value for A. cepa and V. radiata was 0.7mg/L and 35mg/L. Root length inhibition, DNA laddering, lipid peroxidation, H2O2 content, and antioxidant enzymatic assays evaluated revealed a dose-dependent response. PCP influenced defense enzyme glutathione peroxidase (GPX) and ascorbate peroxidase (APX) action in both plants and showed deprivement of catalase (CAT) with the increase of PCP concentrations. PCP-invaded toxicity management by these plants implied that A. cepa is more sensitive than V. radiata regarding PCP-induced toxicity.

Environmental Impact of Geosynthetics in Coastal Protection.

Geosynthetic materials are applied in measures for coastal protection. Weathering or any damage of constructions, as shown by a field study in Kaliningrad Oblast (Russia), could lead to the littering of the beach or the sea (marine littering) and the discharge of possibly harmful additives into the marine environment. The ageing behavior of a widely used geotextile made of polypropylene was studied by artificial accelerated ageing in water-filled autoclaves at temperatures of 30 to 80 °C and pressures of 10 to 50 bar. Tensile strength tests were used to evaluate the progress of ageing, concluding that temperature rather than pressure was the main factor influencing the ageing of geotextiles. Using a modified Arrhenius equation, it was possible to calculate the half-life for the loss of 50% of the strain, which corresponds to approximately 330 years. Dynamic surface leaching and ecotoxicological tests were performed to determine the possible release of contaminants. No harmful effects on the test organisms were observed.

Bioassays for the evaluation of reclaimed opencast coal mining areas.

This study aimed to use bioassays (single and multispecies) with organisms from different trophic levels to assess soil quality in reclaimed coal mining areas. Soil samples were collected from four sites: two sites with recent reclaim processes (one using topsoil and other using clayey soil), an natural attenuation site, and a control soil. The evaluated parameters were divided into (1) ecotoxicological tests (avoidance test with Eisenia andrei (earthworms) and Armadillidium vulgare (isopods); germination test with Sinapis alba seeds (mustard); reproduction tests with Folsomia candida (collembolans); bacterial toxicity test); (2) population and community assessments (a fungal count; microbial community analysis using Biolog EcoPlatesTM); (3) microcosms scale evaluation (the MS-3 multispecies system); and (4) chemical analysis (soil parameters, soil metal, and cations and anions in soil leachate). Results pointed to toxicity in the natural attenuation site that compromised of habitat function, probably due to low pH and low nutrient levels. The most recent reclaim process, using topsoil and clay soil, improved soil quality and fertility, with a further increase in habitat quality and heterogeneity. This study shows that some techniques used to reclaim degraded mining areas are effective in rebuilding habitats, sustaining soil biota, and reestablishing ecosystem services.

Performance of Chlorella Vulgaris Exposed to Heavy Metal Mixtures: Linking Measured Endpoints and Mechanisms.

Microalgae growth inhibition assays are candidates for referent ecotoxicology as a fundamental part of the strategy to reduce the use of fish and other animal models in aquatic toxicology. In the present work, the performance of Chlorella vulgaris exposed to heavy metals following standardized growth and photosynthesis inhibition assays was assessed in two different scenarios: (1) dilutions of single heavy metals and (2) an artificial mixture of heavy metals at similar levels as those found in natural rivers. Chemical speciation of heavy metals was estimated with Visual MINTEQ software; free heavy metal ion concentrations were used as input data, together with microalgae growth and photosynthesis inhibition, to compare different effects and explain possible toxicity mechanisms. The final goal was to assess the suitability of the ecotoxicological test based on the growth and photosynthesis inhibition of microalgae cultures, supported by mathematic models for regulatory and decision-making purposes. The C. vulgaris algae growth inhibition test was more sensitive for As, Zn, and Pb exposure whereas the photosynthesis inhibition test was more sensitive for Cu and Ni exposure. The effects on growth and photosynthesis were not related. C. vulgaris evidenced the formation of mucilaginous aggregations at lower copper concentrations. We found that the toxicity of a given heavy metal is not only determined by its chemical speciation; other chemical compounds (as nutrient loads) and biological interactions play an important role in the final toxicity. Predictive mixture effect models tend to overestimate the effects of metal mixtures in C. vulgaris for both growth and photosynthesis inhibition tests. Growth and photosynthesis inhibition tests give complementary information, and both are a fast, cheap, and sensitive alternative to animal testing. More research is needed to solve the challenge of complex pollutant mixtures as they are present in natural environments, where microalgae-based assays can be suitable monitoring tools for pollution management and regulatory purposes.

Mesocosm Experiments at a Tunnelling Construction Site for Assessing Re-Use of Spoil Material as a By-Product

Mechanized excavation of tunnels with Earth Pressure Balance-Tunnel Boring Machines requires the use of foaming agents. The latter contain the anionic surfactant sodium lauryl ether sulphate (SLES) as the main compound. The re-use as a by-product of excavated soil containing foaming agents (spoil material) can pose a risk for soil and particularly for aquatic ecosystems if they are close to the spoil material final destination site. This work reports the chemical results (SLES residual concentrations) and ecotoxicological effects (battery of five tests) of 28 day-mesocosm studies performed at a tunnelling construction site. The soil mesocosms were set up with two different lithologies, which contained four different foaming agent products at the highest amounts used for excavation. The decrease in SLES concentrations and the ecotoxicological tests were performed in soil and its water extract (elutriate) at different times (0, 7, 14, 28 d). Elutriates were prepared in order to simulate a possible SLES leaching from soil to water. The results showed a decrease in SLES over time and different ecotoxicological responses depending not only on the initial amount of each product, but also on the soil lithology and organism tested (aquatic or terrestrial). This study showed how only site-specific ecotoxicological evaluations can ensure a safe management of the spoil material, making possible the re-use of soil and avoiding production of waste.

A multivariate approach to chlorophyll a fluorescence data for trace element ecotoxicological trials using a model marine diatom

The increasing uncontrolled development of human activities and consequent increase in the production and release into the marine realm of potentially harmful substances highlights the need to develop efficient and high-throughput screening (HTS) tools. Bio-optical tools, such as laser-induced fluorescence (LIF) and pulse amplitude modulated (PAM) fluorometry, emerge as efficient non-invasive techniques for toxicophenomic evaluation in ecotoxicological trials. Both techniques generate large datasets that can be applied in multivariate analysis to evaluate canonical classification efficiency of the exposure types and levels to which photosynthetic organisms, such as diatoms, are subjected to. In the present work, marine diatom cultures were exposed to two trace elements known to have physiological roles and different toxicity ranges (Zn and Cu), and to two other trace elements without known metabolic functions and very different toxicological profiles (Cr and Hg). All the tested approaches were able to disentangle the control groups from the test groups. Moreover, the application of LIF raw-data showed that this technique had the higher classification efficiency, providing very good separation of the different doses applied of each trace element tested. Additionally, PAM chlorophyll fast induction kinetics raw data also produced good classification efficiencies and provided data that can be useful for interpreting the physiological shifts induced by trace element exposure. In sum, LIF and PAM techniques appear as completely non-invasive HTS techniques that, when applied together, produce a correct toxicophenomic classification and disentangle the physiological effects behind the observed phenomic changes. Therefore, they are worth to be included in future ecotoxicological assessment test protocols.

Ecotoxicological assessment of sewage sludge-derived biochars-amended soil.

The study aimed to evaluate the ecotoxicity of soil (S) amended with biochars (BCKN) produced by the thermal conversion of sewage sludge (SSL) at temperatures of 500°C, 600°C, or 700°C and SSL itself. The ecotoxicological tests were carried out on organisms representing various trophic levels (Lepidium sativum in plant, Folsomia candida in invertebrates, and Aliivibrio fischeri in bacteria). Moreover, the study evaluated the effects of three plants (Lolium perenne, Trifolium repens, and Arabidopsis thaliana) growing on BCKN700-amended soil on its ecotoxicological properties. The experiment was carried out for six months. In most tests, the conversion of sewage sludge into biochar caused a significant decrease in toxicity by adding it to the soil. The pyrolysis temperature directly determined this effect. The soil amended with the biochars produced at higher temperatures (600°C and 700°C) generally exhibited lower toxicity to the test organisms than the SSL. Because of aging, all the biochars lost their inhibition properties against the tested organisms in the solid-phase tests and had a stimulating influence on the reproductive ability of F.candida. With time, the fertilizing effect of the BCKN700 amended soil also increased. The aged biochars also did not have an inhibitory effect on A.fischeri luminescence in the leachate tests. The study has also demonstrated that the cultivation of an appropriate plant species may additionally reduce the toxicity of soil fertilized with biochar. The obtained results show that the conversion of sewage sludge to biochar carried out at an appropriate temperature can become a useful method in reducing the toxicity of the waste and while being safe for agricultural purposes.

Pharmaceuticals in farms and surrounding surface water bodies: Hazard and ecotoxicity in a swine production area in Costa Rica

The presence of pharmaceuticals in the environment is known to have multiple origins; livestock activities comprise one scarcely studied source, both globally and specially in Latin-America. This work aims to study the occurrence of pharmaceuticals in wastewater from swine farms and their surrounding surface waters, in a highland livestock production area of Costa Rica. The monitoring of 70 pharmaceutical active compounds resulted in the detection of 10 molecules in farm wastewater (influents and effluents of the on-farm treatment system), including compounds of animal and human use. A 57% of effluents showed high hazard (ΣHQ > 1), mainly due to the compounds risperidone, ketoprofen, ibuprofen and naproxen. Additionally, ecotoxicological tests with Daphnia magna and Microtox classified at least 21% of the effluents as very toxic (10 < TU ≤ 100); likewise, 86% of effluents exhibited germination index (GI) inhibition values over 90% for Lactuca sativa. Seven molecules were detected in surface water, six of them of human use (1,7-dimethylxanthine, caffeine, cephalexin, carbamazepine, gemfibrozil, ibuprofen) and one (acetaminophen) of dual (human and veterinary) use; nonetheless, most of the detections were found in sampling points closer to human settlements than animal farms. Considering the set of molecules and their distribution, the livestock influence on surface water seems minimal in comparison with the urban influence. Only 16% of surface water samples showed high risk, mainly due to ibuprofen, gemfibrozil and caffeine; similarly, 45% samples presented GI inhibition >20% (no toxicity was determined towards Daphnia magna or Microtox). These findings in surface water suggest an incipient environmental risk in the area.

Testing of Eluates from Waterproof Building Materials for Potential Environmental Effects Due to the Behavior of Enchytraeus albidus.

In order to determine the potential environmental impact of construction products, it is necessary to evaluate their influence on organisms exposed to them or their eluates under environmental conditions. The behavior of the white worm Enchytraeus albidus is a useful tool for assessing the potential environmental impact of construction products in contact with water and soil. This study investigates the environmental effects of eluates from two construction products, a reactive waterproofing product, and an injection resin, on the reproduction and avoidance behavior of E. albidus. The eluates were prepared according to existing guidelines. The soil used for the tests was moistened with the eluates of the construction products. The reproduction results of the worms were collected after six weeks of exposure. Offsprings were counted under the microscope and statistically analyzed. Results from the avoidance behavior were collected after 48 h of exposure, and results were compared with the reproduction results. The eluates from both construction products induced significant changes in the reproduction behavior of E. albidus. Undiluted or only slightly diluted eluates of the injection resin drastically reduced the reproduction of the worms, whereas the leaches of the reactive waterproofing product only had a minor effect. The avoidance results for the injection resin indicates that its presence in the habitat is clearly detrimental to the survival of E. albidus, while the avoidance results for the waterproofing resin showed an initial avoidance of the eluates, but no harmful effects were observed. The avoidance test is a way of rapid toxicity screening of environmental samples when time is a critical parameter to measure possible environmental effects. This study shows that ecotoxicological tests using Enchytraeids are a valuable and important tool for understanding the mode of action of eluates from construction products in the environment.

Ecotoxicological assessment of silicate rock fertilizers using soil invertebrates

Abstract: The objective of this work was to evaluate the possible adverse effects of rock dusts on the avoidance behavior of earthworms and collembolans, as well as on the survival and reproduction of enchytraeids, in ecotoxicological tests using artificial and natural soils. The tests were performed following international guidelines (ISO 17512-2/2011, ISO 11267/1999, and NBR ISO 16387/2012), with some adaptations, in order to evaluate the effect of dusts from two silicate rocks, biotite schist and phonolite, at the concentrations of 0, 0.1, 1.0, 10, and 100%, in artificial and natural (Arenosol and Ferralsol) soils. The avoidance behavior of the earthworm Eisenia andrei and the collembolan Folsomia candida was evaluated, as well as the survival and reproduction of the enchytraeid Enchytraeus sp. Biotite schist and phonolite affected negatively earthworms when applied to the soil at concentrations equal to or higher than 1.0%. The observed effect was associated to the greater presence of salts, especially in the phonolite, and to changes in soil texture when higher concentrations were used. Negative effects on collembolans and enchytraeids were verified at concentrations of 10% or using pure rock dusts. Agronomic rates smaller than 1.0% (approximately 20 Mg ha-1) rock dusts do not cause avoidance behavior or reduction in the survival and reproduction of invertebrates in the soil.

MÉTODO ANALÍTICO PARA DETERMINAÇÃO DOS CORANTES AURAMINA EM TESTES DE ECOTOXICIDADE E EM ÁGUAS SUPERFICIAIS

ANALYTICAL METHOD FOR DETERMINATION OF AURAMINE DYES IN ECOTOXICITY TESTS AND IN SURFACE WATERS. Dyes can be considered important environmental contaminants. Auramine dyes, known commercially as Solvent Yellow 34 and Basic Yellow 2, are used in several industrial products, including textile and paper. In this study, an analytical method was developed and validated using Liquid-Liquid Extraction and High-Performance Liquid Chromatography coupled to Diode Array Detector (LLE and HPLC-DAD) to determine the exposure concentrations in the ecotoxicological tests with auramine dyes, increasing the results reliability about the hazard of these compounds. The method was also used to monitor auramine dyes in surface water samples, in a region considered one of the most critical regions for dyes contamination in Brazil. The results showed no risk for the aquatic organisms considering the analyzed samples. The method using simple instrumentation showed the results of selectivity, linearity (R2 &gt; 0.99), precision (CV: 5.6-29%) and accuracy (recovery: 82-90%) considered satisfactory for the two purposes with Instrumental LOQ of the 12 µg L-1 and the Method LOQ of the 12 ng L-1. The results indicated that the proposed method was effective and practical for the auramine determination in environmental levels and will be an important tool to verify the potential risk of auramine dyes in future studies.

Calidad de agua del Arroyo Guazú del Departamento Central evaluada mediante indicadores biológicos, parámetros fisicoquímicos y ecotoxicológicos

The waters of urban streams in the Metropolitan Area of Greater Asuncion are under great pressure from anthropogenic pollution of urban and industrial origin. The Guazu Stream is a watercourse that crosses the cities of Nemby and San Antonio and is affected by pollution throughout its course. The quality of its water based on its physicochemical, biological and ecotoxicological characteristics was used in this study to compare it with current national legislation. Water samples were collected at five different points in the months of July and November 2017. The results of the physicochemical parameters indicate that its waters possess class II and class III characteristics as established by the Ministry of Environment and Sustainable Development of Paraguay. Some parameters such as nitrate, total nitrogen, total phosphorus, dissolved oxygen, BOD5 and fecal coliforms presented values outside the allowed limits. The Water Quality Index gave Fair and Poor ratings, preferably towards the lower basin in the area where it flows into the Paraguay River, while the trophic status index categorized the stream as mesotrophic to hypereutrophic. There was a decrease in zooplankton diversity towards the mouth of the river. Therefore, the results indicated an increase in aquatic pollution. Ecotoxicological tests on Daphnia magna and Lactuca sativa did not detect the presence of toxic pollutants in the waters of the Guazu Stream.

Early Life Stage Assays in Zebrafish.

Fish embryo toxicity (FET) test using zebrafish (Danio rerio) has been established as an alternative assay to animal experimentation. The FET assay enables the assessment of multiple morphological endpoints during the development of zebrafish early life stages, showing high impact to the field of ecotoxicology on risk assessment of chemicals and pollutants. Moreover, it is also applied to screening drug-induced toxicity and human diseases, due to the high genetic and physiological orthology between zebrafish and humans. Here, we describe FET test, with all steps and several adaptations involved in the methodological procedures. To demonstrate the efficiency of this method, results using the reference substance 3,4-dichloroaniline (DCA) were included to demonstrate sublethal and teratogenic malformations on zebrafish embryos. Thus, there is a strong tendency for using FET tests as a replacement strategy of traditional tests in toxicology and ecotoxicology.