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Evidence from both laboratory and field studies has shown that currently used synthetic and naturally occurring chemical substances may potentially disrupt invertebrate endocrine systems, although the extent of this in field populations remains unclear. Translating concerns about potential endocrine disrupting chemicals (EDCs) into practical and effective regulatory action is currently hampered by the breadth of invertebrate endocrinology when compared to the better understood vertebrate systems, a lack of fundamental knowledge about the endocrinology of many invertebrate groups, and the resulting uncertainty when making regulatory decisions. This commentary (i) outlines the breadth of invertebrate endocrine pathways for which European Union regulation of potential EDCs may be relevant; (ii) reviews the extent to which current knowledge meets regulatory requirements for invertebrates, including an assessment of the suitability of current invertebrate test guidelines for detecting endocrine modes of action; and (iii) proposes a roadmap towards the regulation of potential EDCs with greater confidence, based on the Adverse Outcome Pathway (AOP) concept and a focus on identifying Molecular Initiating Events (MIEs) within AOPs. We conclude there are no validated tools to determine any invertebrate endocrine mode of action in vitro or in vivo. However, there are commonly used invertebrate toxicity tests which might capture adverse effects that could potentially result from an endocrine mode of action but would not identify the causal mechanisms. Therefore, EU regulatory requirements for the identification of EDCs cannot currently be satisfied for invertebrates, either in general or for the specific invertebrates used in standard ecotoxicological studies. We propose that the most important research need is compilation of a comprehensive list of endocrine-related MIEs across invertebrate taxa via use of high-throughput ‘omics in combination with bioinformatics reverse engineered analyses. Although tractable, such an approach would require significant resource investment for development and implementation.

Evidence from both laboratory and field studies has shown that currently used synthetic and naturally occurring chemical substances may potentially disrupt invertebrate endocrine systems, although the extent of this in field populations remains unclear. Translating concerns about potential endocrine disrupting chemicals (EDCs) into practical and effective regulatory action is currently hampered by the breadth of invertebrate endocrinology when compared to the better understood vertebrate systems, a lack of fundamental knowledge about the endocrinology of many invertebrate groups, and the resulting uncertainty when making regulatory decisions. This paper i) outlines the breadth of invertebrate endocrine pathways for which European Union regulation of potential EDCs may be relevant, ii) reviews the extent to which current knowledge meets regulatory requirements for invertebrates, including an assessment of the suitability of current invertebrate test guidelines for detecting endocrine modes of action; and iii) proposes a roadmap towards the regulation of potential EDCs with greater confidence, based on the Adverse Outcome Pathway (AOP) concept and a focus on identifying Molecular Initiating Events (MIEs) within AOPs. We propose that the most important research need is compilation of a comprehensive list of endocrine-related MIEs across invertebrate taxa via use of high throughput ‘omics in combination with bioinformatics reverse engineered analyses. Although tractable, such an approach would require significant resource investment for development and implementation.

The study assessed the environmental toxicity of three Norwegian mine tailings from Omya Hustadmarmor, Sydvaranger, and Sibelco, which are all released into a seawater recipient. Ecotoxicity assessments were performed on the overlying water extracted from the mine tailings, the transformation/dissolution waters obtained from the mine tailings, and whole sediment assessment using a suite of marine organisms including algae, Crustacea, and Mollusca. Overall, based on the toxicity evaluation of the transformation/dissolution data, Sibelco tailings resulted in the highest toxicity albeit at relatively high concentrations, followed by Sydvaranger and Hustadmarmor. Sibelco was the only mine where process chemicals were not used. In contrast, the Corophium sediment contact assay revealed a significantly higher toxicity exerted by Hustadmarmor tailings, which may indicate a physical impact of the fine tailings. The effects observed were discussed with respect to both the measured chemical concentrations of the tailings and the potential physical impact of the tailing particles on organism health.

Pharmaceuticals are produced to inflict a specific physiological response in organisms. However, as only partially metabolized after administration, these types of compounds can also originate harmful side effects to non-target organisms. Additionally, there is still a lack of knowledge on the toxicological effects of legacy pharmaceuticals such as the antibiotic azithromycin. This macrolide occurs at high concentrations in the aquatic environment and can constitute a threat to aquatic organisms that are at the basis of the aquatic food chain, namely microalgae. This study established a high‐throughput methodology to study the toxicity of azithromycin to the freshwater microalga Raphidocelis subcapitata. Flow cytometry and pulse amplitude modulated (PAM) fluorometry were used as screening tools. General toxicity was shown by effects in growth rate, cell size, cell complexity, cell viability and cell cycle. More specific outcomes were indicated by the analysis of mitochondrial and cytoplasmatic membrane potentials, DNA content, formation of ROS and LPO, natural pigments content and photosystem II performance. The specific mode of action (MoA) of azithromycin to crucial components of microalgae cells was revealed. Azithromycin had a negative impact on the regulation of energy dissipation at the PSII centers, along with an insufficient protection by the regulatory mechanisms leading to photodamage. The blockage of photosynthetic electrons led to ROS formation and consequent oxidative damage, affecting membranes and DNA. Overall, the used methodology exhibited its high potential for detecting the toxic MoA of compounds in microalgae and should be considered for future risk assessment of pharmaceuticals.

The recommended test for assessing if a chemical can be biodegraded in the marine environment is performed according to the Organisation for Economic Cooperation and Development Marine biodegradation test guideline (OECD 306). However, this test is known to generate highly variable test results when comparing interlaboratory test results for the same compound. One reason can be the relatively low bacterial content compared to the inoculum used for OECD readily biodegradation tests (OECD 301). Some of the variability in data obtained from OECD 306 tests can also be due to the flexibility on how to store the seawater inoculum before starting a test. Another variable in the seawater inoculum is the source of seawater used by different laboratories, i.e., geographical location and anthropogenic activities at the source. In this study, the effect of aging seawater and the source of seawater (sample time and depth) were investigated to determine differences in the biodegradation of the reference compound aniline. Aging the seawater before starting the test is recommended in OECD 306 to reduce the background levels of organic carbon in the water. However, it also functions to acclimatize the bacterial community from the environmental source temperature to the test temperature (normally 20 °C). Herein, the microbial community was monitored using flowcytometer during the aging process. As expected, the microbial community changed over time. In one experiment, aging significantly improved the biodegradation of aniline, while in two experiments, there was no significant difference in biodegradation. Interestingly however, there was significant variability in the biodegradation of aniline between sampling seasons and depths, even when all experiments were performed in the same lab, by the same operator and seawater obtained from the same source. This highlights the need for a more robust and consistent microbial inoculum source to reduce variability in seawater biodegradation tests.

Information about acute fish toxicity is routinely required in many jurisdictions for environmental risk assessment of chemicals. This information is typically obtained using a 96-hour juvenile fish test for lethality according to OECD test guideline (TG) 203 or equivalent regional guidelines. However, TG 203 has never been validated using the criteria currently required for new test methods including alternative methods. Characterization of the practicality and validity of TG 203 is important to provide a benchmark for alternative methods. This contribution systematically summarizes the available knowledge on limitations and uncertainties of TG 203, based on methodological, statistical, and biological considerations. Uncertainties stem from the historic flexibility (e.g., use of a broad range of species) and constraints of the basic test design (e.g., no replication). Other sources of uncertainty arise from environmental safety extrapolation based on TG 203 data. Environmental extrapolation models, combined with data from alternative methods, including mechanistic indicators of toxicity, may provide at least the same level of environmental protection. Yet, most importantly, the 3R advantages of alternative methods allow a better standardization, characterization, and an improved basic study design. This can enhance data reliability and thus facilitate the comparison of chemical toxicity, as well as the environmental classifications and prediction of no-effect concentrations of chemicals. Combined with the 3R gains and the potential for higher throughput, a reliable assessment of more chemicals can be achieved, leading to improved environmental protection., ALTEX, 38 (1), ISSN:1868-8551, ISSN:0946-7785, ISSN:1868-596X

Some of the largest extents of naturally occurring acidic waters are found across southern Australia. The origins of these systems remain poorly understood with many hypotheses for their genesis. Australian government agency groundwater datasets and mapping data (vegetation, geology, regolith and soils) for south-western Australia, unavailable to previous researchers, were statistically analysed to better understand the origins of acidic groundwater and guide additional fieldwork to study the origins of acidic saline groundwater. The groundwater data showed a distinct bimodal distribution in pH; the ‘acid’ population had a median pH of 3.5 and the larger ‘non-acid’ population had a median pH of 6.6. Acidic groundwater became progressively more common further from the coast towards the drier internally drained regions. Acidic groundwater was mostly confined to the lower slopes and valley floors with localised controls on distribution. Paradoxically, subsoil alkalinity within the internally drained inland regions had the strongest correlation with acidic groundwater (r2 = 0.85). Vegetation was also a strong predictor of acidic groundwater. Acidic groundwater had the highest occurrence under Eucalyptus woodlands and shrublands that grew on alkaline calcareous soils. Pre-clearing soil data in areas with acidic saline groundwater showed that the upper 1 m of the unsaturated zone had a pH around 8 while the pH at depths greater than 5 m decreased to 1.5 m. There, the iron is reduced around roots and the alkalinity generated by microbial iron reduction is removed by biogenic calcification processes. The iron moves in solution further down the profile following roots where it comes in contact with the oxygenated unsaturated zone matrix and is oxidised generating acid. The resulting acidic recharging solution acidifies the unsaturated zone matrix. Saline groundwater moving through the matrix becomes acidified due to ion exchange or direct recharge. The main chemical processes were modelled in PHREEQC to test the plausibility of the hypothesis and acidic solutions with a pH of 3.8 or lower were obtained.

The use of fish embryo toxicity (FET) data for hazard assessments of chemicals, in place of acute fish toxicity (AFT) data, has long been the goal for many environmental scientists. The FET test was first proposed as a replacement to the standardized AFT test nearly 15 y ago, but as of now, it has still not been accepted as a standalone replacement by regulatory authorities such as the European Chemicals Agency (ECHA). However, the ECHA has indicated that FET data can be used in a weight of evidence (WoE) approach, if enough information is available to support the conclusions related to the hazard assessment. To determine how such a WoE approach could be applied in practice has been challenging. To provide a conclusive WoE for FET data, we have developed a Bayesian network (BN) to incorporate multiple lines of evidence to predict AFT. There are 4 different lines of evidence in this BN model: 1) physicochemical properties, 2) AFT data from chemicals in a similar class or category, 3) ecotoxicity data from other trophic levels of organisms (e.g., daphnids and algae), and 4) measured FET data. The BN model was constructed from data obtained from a curated database and conditional probabilities assigned for the outcomes of each line of evidence. To evaluate the model, 20 data-rich chemicals, containing a minimum of 3 AFT and FET test data points, were selected to ensure a suitable comparison could be performed. The results of the AFT predictions indicated that the BN model could accurately predict the toxicity interval for 80% of the chemicals evaluated. For the remaining chemicals (20%), either daphnids or algae were the most sensitive test species, and for those chemicals, the daphnid or algal hazard data would have driven the environmental classification. Integr Environ Assess Manag 2020;16:452-460. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The formation of bacterial biofilms can have negative impacts on industrial processes and are typically difficult to control. The increase of antibiotic resistance, in combination with the requirement for more environmentally focused approaches, has placed pressure on industry and the scientific community to reassess biofilm control strategies. The discovery of bacterial quorum sensing, as an important mechanism in biofilm formation, has led to the development of new substances (such as halogenated thiophenones) to inhibit the quorum sensing process. However, there are currently limited data regarding the biodegradability or ecotoxicity of these substances. To assess the environmental fate and effects of thiophenones capable of quorum sensing inhibition, candidate substances were first identified that have potentially high biodegradability and low ecotoxicity using quantitative structure activity relationships. Subsequent confirmatory hazard assessment of these substances, using a marine alga and a marine crustacean, indicated that these estimates were significantly under predicted with acute toxicity values more than three orders of magnitude lower than anticipated combined with limited biodegradability. Therefore, although these quorum sensing inhibitors appear a promising approach to control biofilms, they may also have environmental impacts on certain aquatic organisms.

The aquatic bioconcentration of a chemical is typically determined using conventional fish tests. To foster the approach of alternatives to animal testing, a combination of computational models and in vitro substrate depletion bioassays (e.g., primary hepatocytes) can be used. One recently developed in vitro assay is the three-dimensional (3D) hepatic spheroid model from rainbow trout (Oncorhynchus mykiss). The aim of the present study was to evaluate the metabolic competence of the 3D spheroids from rainbow trout when exposed to pyrene, using 2 different sampling procedures (SP1 and SP2). The results were compared with previously published intrinsic clearance (CL) results from S9 fractions and primary hepatocyte assays. Extraction of pyrene using SP1 suggested that the spheroids had depleted 33% of the pyrene within 4 h of exposure, reducing to 91% after 30 h. However, when applying SP2 a substantial amount (36%) of the pyrene was bound to the exposure vial within 2 h, decreasing after 6 h of exposure. Formation of hydroxypyrene-glucuronide (OH-PYR-Glu) was obtained throughout the study, displaying the metabolic competence of the 3D spheroids. The 2 sampling procedures yielded different CLin vitro , where pyrene depletion using SP2 was very similar to published studies using primary hepatocytes. The 3D spheroids demonstrated reproducibile, log-linear biotransformation of pyrene and displayed formation of OH-PYR-Glu, indicating their metabolic competence for 30 h or more. Environ Toxicol Chem 2019;38:1738-1747. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Veterinary medicinal products (VMPs) are widely used within the fish farming industry to control sea lice infestations. There is concern that wild and farmed mussels in the vicinity to these fish farms may be exposed and subsequently bioaccumulate these chemicals, which could pose a threat to human health. To understand the fate of these chemicals in the environment, controlled laboratory exposures were performed to establish the uptake and depuration of selected VMPs in the blue mussel (Mytilus edulis). The VMPs included teflubenzuron, emamectin benzoate and deltamethrin. The effects of salinity on the bioaccumulation of teflubenzuron were also investigated to see whether mussels in brackish waters exhibit different bioaccumulation dynamics. Salinity had no significant effect on the uptake or depuration curves for teflubenzuron down to 15‰. The uptake rate constants (k1) for teflubenzuron, emamectin benzoate and deltamethrin in mussels were 192, 4.82 and 2003, with kinetic bioconcentration factors (BCFs) of 1304, 49 and 2516. Depuration rate constants (k2) were also found to differ between the three VMPs at 0.147, 0.048 and 0.796 for teflubenzuron, emamectin benzoate and deltamethrin, with calculated elimination half-lives (t1/2)of 4.7, 14 and 0.87 days. The longer elimination half-lives for teflubenzuron and emamectin benzoate, suggest that these chemicals accumulate in blue mussels and therefore have the potential to bioaccumulate in wild and farmed mussel populations in the environment.

Microalgal toxicity tests using integrative endpoints as algal growth are regularly required to analyse the toxicity of potentially hazardous substances in the aquatic environment. However, these do not provide mechanistic information on the toxic mode of action by which contaminants may affect algae. Bottled waters can be used as a substitute for culturing media and should not impose any stress to the cultured organisms. However, certain chemical components can interfere with specific cell targets which are not revealed by general toxicity assays. The present study investigated the sensitivity of flow cytometry (FCM) to analyse sub-lethal effects of different bottled waters to the freshwater microalgae Raphidocelis subcapitata. Several endpoints were analysed including growth rate, natural pigments content, cell size, complexity, viability and cycle, Reactive Oxygen Species (ROS) formation, mitochondrial membrane potential and Lipid Peroxidation (LPO). Additionally, photosystem II (PSII) performance was analysed by PAM fluorometry, to provide further information on the absorption, distribution and use of energy in photosynthesis. Results indicated that the most sensitive endpoints were the oxidative stress related endpoints ROS formation and LPO, pigment content, morphological endpoints as cell size, complexity and cycle, with growth rate being one of the least sensitive. Although being essential macronutrients for algal growth, the chemical elements Ca, Na, Mg, and NH4 were identified as being primarily responsible for the observed toxicological effects to exposed algae. The applied methodology proved to be of high throughput, simultaneously assembling information on morphological, biochemical, and physiological status of algal cells. FCM also showed potential to reveal mechanistic information on the toxic mode of action of the bottled waters before any effects on algal growth was observed. The used approach demonstrated its potential for being integrated into future microalgal toxicity bioassays for testing chemicals to improve the hazard information obtained from currently approved internationally accepted test guidelines.

We investigated the microbial community compositions in two sediment samples from the acidic (pH ∼3) and hypersaline (>4.5% NaCl) surface waters, which are widespread in Western Australia. In West Dalyup River, large amounts of NaCl, Fe(II) and sulfate are brought by the groundwater into the surface run-off. The presence of K-jarosite and schwertmannite minerals in the river sediments suggested the occurrence of microbial Fe(II) oxidation because chemical oxidation is greatly reduced at low pH. 16S rRNA gene diversity analyses revealed that sequences affiliated with an uncultured archaeal lineage named Aplasma, which has the genomic potential for Fe(II) oxidation, were dominant in both sediment samples. The acidophilic heterotrophs Acidiphilium and Acidocella were identified as the dominant bacterial groups. Acidiphilium strain AusYE3-1 obtained from the river sediment tolerated up to 6% NaCl at pH 3 under oxic conditions and cells of strain AusYE3-1 reduced the effects of high salt content by forming filamentous structure clumping as aggregates. Neither growth nor Fe(III) reduction by strain AusYE3-1 was observed in anoxic salt-containing medium. The detection of Aplasma group as potential Fe(II) oxidizers and the inhibited Fe(III)-reducing capacity of Acidiphilium contributes to our understanding of the microbial ecology of acidic hypersaline environments.

Diflubenzuron and teflubenzuron are benzoylureas that are used in aquaculture to control sea lice. Flubenzurons have low toxicity to many marine species such as fish and algae but by their nature are likely to have significant adverse effects on nontarget species such as crustaceans and amphipods. Although the exact mechanism of toxicity is not known, these compounds are thought to inhibit the production of the enzyme chitin synthase during molting of immature stages of arthropods. These chitin synthesis inhibitors are effective against the larval and pre-adult life stages of sea lice. Due to their low solubility and results of recent monitoring studies conducted in Norway, the sediment compartment is considered the most likely reservoir for these compounds and possible remobilization from the sediment to benthic crustaceans could be of importance. For this reason, the epibenthic copepod Tisbe battagliai was selected for investigations into the acute and developmental effects of these compounds. For comparative purposes, azamethiphos was investigated to identify differences in sensitivity and act as a negative control for developmental effects at environmentally relevant concentrations. Standard acute studies with adult copepods showed little or no acute toxicity at milligrams per liter levels with the flubenzurons, whereas a naupliar developmental test demonstrated that environmentally relevant concentrations (e.g., nanograms per liter) caused a complete cessation of molting and finally death in the exposed copepods.

The aim to reduce the number of animals in experiments has highlighted the need to develop and validate nonanimal methods as alternatives to bioaccumulation studies using fish. The present study details a novel 3-tier approach to develop a list of reference compounds to aid this process. The approach was based on 1) the inclusion of relevant chemical classes supported by high-quality in vivo data for the bioconcentration factor (BCF), whole-body biotransformation rates (Kmet), and metabolism characterization for rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) (tiers I and II); and 2) the refinement to ensure a broad coverage of hydrophobicity, bioconcentration potential, molecular weight, maximum molecular diameter, whole-body biotransformation half-lives, and metabolic pathways (tier III). In silico techniques were employed to predict maximal log BCF and molecular and metabolic properties. Of the 157 compounds considered as reference compounds, 144 were supported by high-quality BCF data, 8 were supported by Kmet data, and 5 were supported by in vivo metabolism data. Additional criteria for refinement of the list of reference compounds were suggested to aid practical implementation in experimental efforts. The present list of reference compounds is anticipated to facilitate the development of alternative approaches, enhance understanding of in vivo and in vitro bioaccumulation relationships, and refine in silico BCF and metabolism predictions. Environ Toxicol Chem 2014;33:2740–2752. © 2014 SETAC

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings. ISSN:1096-6080 ISSN:1096-0929

Performing ecotoxicity tests on poorly water soluble substances and in particular metals, metalloids, and metal oxides such as silica fume, can be problematic. Such substances may not be directly toxic to aquatic organisms but often have high concentrations of impurities present, due to production processes, which may result in ecotoxicological effects. This combined with possibly testing above the limit of solubility further exacerbates the interpretation of ecotoxicity test results. One approach to overcome this is to perform a transformation/dissolution (T/D) test to determine the quantities of elemental impurities which will consequently be in solution. These data can subsequently be compared to existing data to determine if there is likely to be an effect on aquatic organisms. This paper highlights research into determining the T/D potential of 2 different grades of amorphous silica fume (low and high grade purity) with complementary chronic ecotoxicity tests of the 2 substances to validate this approach. The low grade silica fume test substance was identified in the T/D assessments as being of concern for the potential to cause acute toxicity to aquatic organisms and had levels of impurities (e.g. Pb and Zn) in the solutions which exceeded the effect limits identified in the open literature. Consequently, silica fume would be hazard classified as acute 2 according to regulatory classification schemes. However, the results of the ecotoxicity hazard validation assessments in a Daphnia magna reproduction test and the sediment dwelling organism Chironomus riparius indicated that low and high grade silica fumes are not acutely or chronically toxic up to and including an initial loading concentration of 100 mg/L and 1000 mg/kg respectively. Hence, using the T/D test data alone may have resulted in a false hazard classification of silica fume (low grade).

Očuvanje bioraznolikosti primarna je zadaća planova upravljanja i mjera zaštite vlažnih staništa, no industrijski razvoj civilizacije omogućio je zadiranje čovjeka i u nasitnije pore ovih krhkih ekosustava. Smanjenje bioraznolikosti primani je pokazatelj u kojoj mjeri čovjek svojim djelatnostima zadire u određeni slatkovodni ekosustav. Slatkovodni ekosustavi pokrivaju svega 0, 01% površine Zemlje, no procjenjuje se da približno 126000 vrsta životinja obitava ili ovisi o njima, uslijed čega su dio biosfere s najvećim brojem vrsta po jedinici površine. Narušavanje životnih uvjeta svakoga slatkovodnoga ekosustava neprocjenjiv je gubitak, kako po prirodu, tako i po čovjeka. Kako bi se pravovremeno spoznao rizik pojave i prisutnosti određene tvari u vlažnom staništu te kako bi se na vrijeme poduzele određene mjere zaštite vlažnih staništa, pri utvrđivanju prisutnosti Daphnia magna slatkovodni je planktonski račić koji prirodno obitava u jezerima i plitkim ribnjacima. Ekotoksikološkim testovima na Daphnia magna moguće je odrediti akutnu i kroničnu toksičnost određene kemijske tvari, a prvi rezultati testova u slučaju ispitivanja akutne toksičnosti dostupni su roku od 24 sata, dok se testovi određivanja kronične toksičnosti provode tijekom 21 dana. određenog kontaminanta poželjno bi bilo utvrditi njegovu toksičnost na vodeni ekosustav. Utvrđivanje toksičnosti određene kemijske tvari provodi se ekotoksikološkim testovima kojima je moguće na prikladan i relativno brz način utvrditi biološku toksičnost tvari. Prednost je ekotoksikoloških testova što njihova provedba ne zahtijeva kompleksne i skupe postupke. Jedan od često u praksi primjenjivanih ekotoksikoloških testova za površinske slatkovodne sustave testovi su osjetljivosti organizma. Daphnia magna slatkovodni je planktonski račić koji prirodno obitava u jezerima i plitkim ribnjacima. Ekotoksikološkim testovima na Daphnia magna moguće je odrediti akutnu i kroničnu toksičnost određene kemijske tvari, a prvi rezultati testova u slučaju ispitivanja akutne toksičnosti dostupni su roku od 24 sata, dok se testovi određivanja kronične toksičnosti provode tijekom 21 dana.

Hazard assessments of Irgarol 1051, diuron, 2-(thiocyanomethylthio)benzothiazole (TCMTB), dichloro-octylisothiazolin (DCOIT), chlorothalonil, dichlofluanid, thiram, zinc pyrithione, copper pyrithione, triphenylborane pyridine (TPBP), capsaicin, nonivamide, tralopyril and medetomidine were performed to establish robust environmental quality standards (EQS), based on predicted no effect concentrations (PNECs). Microalgae, zooplankton, fish and amphibians were the most sensitive ecological groups to all the antifoulants evaluated, especially in the early life stages. No differences were identified between freshwater and seawater species. The use of toxicity tests with non-standard species is encouraged because they increase the datasets, allowing EQS to be derived from probabilistic-based PNECs whilst reducing uncertainties. The global ban of tributyltin (TBT) has been heralded as a major environmental success; however, substitute antifoulants may also pose risks to aquatic ecosystems. Environmental risk assessments (ERAs) have driven decision-makings for regulating antifouling products, but in many countries there is still a lack of regulation of antifouling biocides which should be addressed.

[1] Groundwater-dependent ecosystems (GDEs) in arid and semiarid environments play significant ecological roles, and, yet in many parts of the world, these ecosystems have been drained for agricultural use. In wetlands containing acid sulfate soils, the altered hydrology may trigger acidification and subsequent trace metal release. Quantifying shifts in hydrological regime and connectivity dynamics across wetlands is critical for understanding the resilience of these GDEs to anthropogenic impacts. Seasonal water balances for a wetland severely impacted by drainage and acidification were combined with laboratory geochemical data and field observations to develop a conceptual model describing hydrological connectivity across the wetland. The data indicated that, with the onset of the dry season, the superficial aquifer was lowered, exposing sulfides that oxidized to form sulfuric acid and dissolving metal salts. The following dry season enhanced capillary action causing upwelling of oxidized products to the surface where evaporative precipitation created acidity scalds. Subsequent winter rainfall and infiltration caused groundwater levels to rise, intersect with the ground surface, and form disconnected acidic pools. As the wet season progressed, connectivity was established between the pools, resulting in metal-rich acid discharge from the wetland. The degree of acid fluxes and metal release was controlled by the physicochemical characteristics of the soils, its exposure to the seasonally variable wetland hydrology, antecedent hydrological conditions, hydrological connectivity (both vertical and horizontal), and the resulting biogeochemical conditions.

The aims of the current study were to test the validity of the concentration addition (CA) and independent action (IA) calculation methods for mixture toxicity as well as the REACH guidelines for mixture exposure scenarios for paint products. Based on ecotoxicity tests of nine anticorrosive paint mixtures and the nine substances that contributed to classification of the mixtures as hazardous for the aquatic environment, neither CA nor IA was found to be appropriate calculation methods, although CA calculations were conservative for half of the mixtures tested. The REACH mixture approach, based on "lead" substances, resulted in conservative predictions for three out of five mixtures and the "lead" substance corresponded to the substance contributing the most in the CA mixture calculations for four out of six mixtures. The use of paints as a matrix for toxicity testing required adaptation of test procedures, development of sample handling and chemical analysis methods.

Animal alternative tests are gaining serious consideration in an array of environmental sciences, particularly as they relate to sound management of chemicals and wastewater discharges. The ILSI Health and Environmental Sciences Institute and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test in March, 2008. This relatively young discipline is following advances in animal alternatives for human safety sciences, and it is advisable to develop a broad comparison of how animal alternative tests involving fish are viewed in a regulatory context over a wide array of authorities or advising bodies. These include OECD, Western Europe, North America, and Japan. This paper summarizes representative practices from these regions. Presently, the global regulatory environment has varying stances regarding the protection of fish for use as an experimental animal. Such differences have a long-term potential to lead to a lack of harmony in approaches to fish toxicity testing, especially for chemicals in commerce across multiple geographic regions. Implementation of alternative methods and approaches will be most successful if accepted globally, including methods of fish toxicity testing. An important area for harmonization would be in the interpretation of protected and nonprotected life stages of fish. Use of fish embryos represent a promising alternative and allow bridging to more technically challenging alternatives with longer prospective timelines, including cell-based assays, ecotoxicogenomics, and QSARs.

In large areas of Western Australia, acidic groundwaters occur with pH values distinctly lower than 3, generation of which has been attributed to the oxidation of Fe(II). Incubation experiments performed with sediments from playas receiving acid groundwater demonstrated occurrence of reductive dissolution of ferric iron minerals at rates [670 nmol (g reactive iron)(-1) h(-1)] similar to those observed in sediments of acidic mining lakes (AML), indicating thatthe pH was established through an acidity-driven iron cycle in analogy to processes occurring in AML systems. The low pH values observed in acidic groundwaters and AML, however, can only be achieved if the anion corresponding to Fe(III) is that of a strong acid. In AML, sulfate is derived from pyrite oxidation. Because this process is reported not to occur in the acidic groundwater systems of Western Australia, we have derived a conceptual model according to which sulfate is generated upon reaction of weathering-derived alkalinity with gypsum to form calcite, which is abundant in these areas. The model proposes that part of the alkalinity generated during weathering is stared as calcite in the landscape, which leads to spatial separation of acidity and alkalinity.

There is currently limited guidance available for regulators and risk assessors on how to use data from non-guideline methods when assessing the bioaccumulation potential of a chemical. Furthermore, bioaccumulation assessments can be more subjective than they need to be due to the lack of a guidance framework on how to use/include the range of information that may be available for a substance. Under some circumstances, in silico, in vitro and/or in vivo non-test guideline data may be sufficient to classify whether a substance is bioaccumulative without the need for further animal testing. Classifying the bioaccumulative potential of a substance is especially difficult when the bioconcentration factor (BCF) is close to the threshold for defining it as bioaccumulative/very bioaccumulative (B/vB), and a more structured process is required to reduce uncertainty in the BCF estimates. In these situations, in silico and in vitro data can, and should, be used to provide greater confidence in classifying these substances. To aid future evaluations of bioaccumulation data, a proposed tiered assessment strategy is presented incorporating all available data on the bioaccumulative properties of a substance. In addition, a revised scheme is recommended for improving the classification of the bioaccumulative potential of a substance.

Activated carbon (AC) has long been associated with the capacity to effectively remove organic substances from aquatic and sediment matrices; however, its use in remediation purposes has drawn some concern due to possible impacts on benthic communities. Within the inner Oslofjord, the use of AC has been well documented for reducing the risks associated with dioxins or dioxin-like compounds from contaminated areas. However, benthic surveys performed on areas treated with AC have revealed that the abundance of organisms inhabiting these areas can be reduced significantly in the subsequent years following treatment. The reason for the reduction in the benthic communities is currently unknown, and therefore, an integrated approach to assess the effects of 2 different forms of AC (biogenic and petrogenic) on benthic organisms has been performed. A battery of 3 different benthic organisms with different feeding and life-cycle processes has been used encompassing sediment surface feeders, sediment ingestors, and sediment reworkers. Results of the tests indicated that although AC is not acutely toxic at concentrations up to 1000 mg/L, there may be physical effects of the substance on benthic dwelling organisms at environmentally relevant concentrations of AC at remediated sites.

We investigated the microbial community compositions in two sediment samples from the acidic (pH ∼3) and hypersaline (4.5% NaCl) surface waters, which are widespread in Western Australia. In West Dalyup River, large amounts of NaCl, Fe(II) and sulfate are brought by the groundwater into the surface run-off. The presence of K-jarosite and schwertmannite minerals in the river sediments suggested the occurrence of microbial Fe(II) oxidation because chemical oxidation is greatly reduced at low pH. 16S rRNA gene diversity analyses revealed that sequences affiliated with an uncultured archaeal lineage named Aplasma, which has the genomic potential for Fe(II) oxidation, were dominant in both sediment samples. The acidophilic heterotrophs Acidiphilium and Acidocella were identified as the dominant bacterial groups. Acidiphilium strain AusYE3-1 obtained from the river sediment tolerated up to 6% NaCl at pH 3 under oxic conditions and cells of strain AusYE3-1 reduced the effects of high salt content by forming filamentous structure clumping as aggregates. Neither growth nor Fe(III) reduction by strain AusYE3-1 was observed in anoxic salt-containing medium. The detection of Aplasma group as potential Fe(II) oxidizers and the inhibited Fe(III)-reducing capacity of Acidiphilium contributes to our understanding of the microbial ecology of acidic hypersaline environments.

Diflubenzuron and teflubenzuron are benzoylureas that are used in aquaculture to control sea lice. Flubenzurons have low toxicity to many marine species such as fish and algae but by their nature are likely to have significant adverse effects on nontarget species such as crustaceans and amphipods. Although the exact mechanism of toxicity is not known, these compounds are thought to inhibit the production of the enzyme chitin synthase during molting of immature stages of arthropods. These chitin synthesis inhibitors are effective against the larval and pre-adult life stages of sea lice. Due to their low solubility and results of recent monitoring studies conducted in Norway, the sediment compartment is considered the most likely reservoir for these compounds and possible remobilization from the sediment to benthic crustaceans could be of importance. For this reason, the epibenthic copepod Tisbe battagliai was selected for investigations into the acute and developmental effects of these compounds. For comparative purposes, azamethiphos was investigated to identify differences in sensitivity and act as a negative control for developmental effects at environmentally relevant concentrations. Standard acute studies with adult copepods showed little or no acute toxicity at milligrams per liter levels with the flubenzurons, whereas a naupliar developmental test demonstrated that environmentally relevant concentrations (e.g., nanograms per liter) caused a complete cessation of molting and finally death in the exposed copepods.

A The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96. h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96. h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV. 30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236.Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes. © 2014 The Authors.

The aim to reduce the number of animals in experiments has highlighted the need to develop and validate nonanimal methods as alternatives to bioaccumulation studies using fish. The present study details a novel 3-tier approach to develop a list of reference compounds to aid this process. The approach was based on 1) the inclusion of relevant chemical classes supported by high-quality in vivo data for the bioconcentration factor (BCF), whole-body biotransformation rates (K(met)), and metabolism characterization for rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) (tiers I and II); and 2) the refinement to ensure a broad coverage of hydrophobicity, bioconcentration potential, molecular weight, maximum molecular diameter, whole-body biotransformation half-lives, and metabolic pathways (tier III). In silico techniques were employed to predict maximal log BCF and molecular and metabolic properties. Of the 157 compounds considered as reference compounds, 144 were supported by high-quality BCF data, 8 were supported by K(met) data, and 5 were supported by in vivo metabolism data. Additional criteria for refinement of the list of reference compounds were suggested to aid practical implementation in experimental efforts. The present list of reference compounds is anticipated to facilitate the development of alternative approaches, enhance understanding of in vivo and in vitro bioaccumulation relationships, and refine in silico BCF and metabolism predictions.

Extremely acidic and saline groundwater occurs naturally in south-western Australia. Discharge of this water to surface waters has increased following extensive clearing of native vegetation for agriculture and is likely to have negative environmental impacts. The use of passive treatment systems to manage the acidic discharge and its impacts is complicated by the region's semi-arid climate with hot dry summers and resulting periods of no flow. This study evaluates the performance of a pilot-scale compost bioreactor treating extremely acidic and saline drainage under semi-arid climatic conditions over a period of 2.5 years. The bioreactor's substrate consisted of municipal waste organics (MWO) mixed with 10 wt% recycled limestone. After the start-up phase the compost bioreactor raised the pH from ≤3.7 to ≥7 and produced net alkaline outflow for 126 days. The bioreactor removed up to 28 g/m(2)/d CaCO3 equivalent of acidity and acidity removal was found to be load dependent during the first and third year. Extended drying over summer combined with high salinity caused the formation of a salt-clay surface layer on top of the substrate, which was both beneficial and detrimental for bioreactor performance. The surface layer prevented the dehydration of the substrate and ensured it remained waterlogged when the water level in the bioreactor fell below the substrate surface in summer. However, when flow resumed the salt-clay layer acted as a barrier between the water and substrate decreasing performance efficiency. Performance increased again when the surface layer was broken up indicating that the negative climatic impacts can be managed. Based on substrate analysis after 1.5 years of operation, limestone dissolution was found to be the dominant acidity removal process contributing up to 78-91% of alkalinity generation, while bacterial sulfate reduction produced at least 9-22% of the total alkalinity. The substrate might last up to five years before the limestone is exhausted and would need to be replenished. The MWO substrate was found to release metals (Zn, Cu, Pb, Ni and Cr) and cannot be recommended for use in passive treatment systems unless the risk of metal release is addressed.

Equilibrium passive sampling through in vivo implantation can help circumvent complex extractions of biological tissues, provide more accurate information on chemical contaminant burden based on the fugacity of a chemical in an organism rather than conventional normalization to lipid content, and improve the assessment of contaminant bioaccumulation potential. Here, we explored the feasibility of in vivo implantation for the passive sampling of neutral hydrophobic contaminants through the insertion of a silicone tag into brown trout (Salmo trutta). Implanted fish from the upper reaches of the River Alna (Oslo, Norway) were relocated to a polluted section of the river for a 28 day caged exposure. "Whole fish" lipid-silicone distribution coefficients (Dlip-sil) were calculated for chlorinated compounds measured in whole fish and in silicone tags of 13 fish. Dlip-sil ranged from 13.6 to 40.0 g g(-1) for polychlorinated biphenyl congeners 28-156 (CB28 and CB156), respectively, and are in close agreement with literature in vitro lipid phase and tissue-based lipid-silicone partition coefficients. After dissection a further of eight fish, muscle and liver samples were analyzed separately. Muscle-based Dlip-sil values similar to the whole fish data were observed. However, lipid-normalized concentrations in the liver tended to be lower than in muscle for most compounds (by up to 50%). Values of whole fish Dlip-sil for brominated diphenyl ethers determined for three fish were in the range of 8.6-51 g g(-1) and in agreement with chlorinated substances. Finally, fugacity ratios calculated from equilibrium concentrations in fish-implanted and water-exposed silicone provided information on the bioaccumulation for chlorinated compounds as well as for some polycyclic aromatic hydrocarbons. Equilibrium passive sampling through in vivo implantation can allow the comparison of a chemical's activity or fugacity in biotic as well as abiotic environmental compartments and at different trophic levels up to humans.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance. (C) 2013 Elsevier Inc. All rights reserved.

Reports of the intense (artificial) sweetener sucralose (1,6-dichloro-1,6-dideoxy-β-D-fructo-furanosyl 4-chloro-4-deoxy-α-D-galactopyranoside) in various environmental compartments have led to speculations about biological effects in nontarget species living in areas receiving discharges from anthropogenic activities. We have, as the first step in the risk assessment of this compound, conducted bioaccumulation studies in the freshwater alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna, and zebrafish (Danio rerio). The freshwater algae and the daphnid tests were performed using a 48-h static exposure system, whereas the zebrafish test was performed using a 48-h semi static exposure system followed by 48 h flow-through of clean water for the depuration phase. All three studies were conducted with two exposure concentrations (10 and 100 mg/L), and the concentrations of sucralose in water and biota were verified by liquid chromatography/mass spectrometry. The studies showed that uptake of sucralose was assumed to achieve a steady state within the first 48 h, and the bioconcentration factor at the assumed steady state (BCFSS) was calculated to be less than 1 for algae and between 1.6 to 2.2 for the daphnids. The fish BCFSS, assumed to occur between 24 to 48 hours, were calculated to be less than 1 for both concentrations tested. A first-order one-compartment (uptake phase) and a first-order two-compartment (elimination phase) model characterized the uptake and depuration kinetics in zebrafish (k1 = 0.027–0.038/h and k2 = 0.206–0.222/h, t95 = 13.5 to 14.6 h, t50 = 3.1 to 3.3 h, and BCFkinetic = 0.4 to 0.9). The current study shows that sucralose does not bioaccumulate in aquatic organisms from different tiers of the food web, and that the BCF's obtained were considerably lower than the criteria set to identify persistent, bioaccumulative, and toxic substances (i.e., BCF ≥ 2,000). Environ. Toxicol. Chem. 2011; 30:673–681. © 2011 SETAC

Animal alternatives research has historically focused on human safety assessments and has only recently been extended to environmental testing. This is particularly for those assays that involve the use of fish. A number of alternatives are being pursued by the scientific community including the fish embryo toxicity (FET) test, a proposed replacement alternative to the acute fish test. Discussion of the FET methodology and its application in environmental assessments on a global level was needed. With this emerging issue in mind, the ILSI Health and Environmental Sciences Institute (HESI) and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research in March, 2008. The workshop included approximately 40 scientists and regulators representing government, industry, academia, and non-governmental organizations from North America, Europe, and Asia. The goal was to review the state of the science regarding the investigation of fish embryonic tests, pain and distress in fish, emerging approaches utilizing fish embryos, and the use of fish embryo toxicity test data in various types of environmental assessments (e.g., hazard, risk, effluent, and classification and labeling of chemicals). Some specific key outcomes included agreement that risk assessors need fish data for decision-making, that extending the FET to include eluethereombryos was desirable, that relevant endpoints are being used, and that additional endpoints could facilitate additional uses beyond acute toxicity testing. The FET was, however, not yet considered validated sensu OECD. An important action step will be to provide guidance on how all fish tests can be used to assess chemical hazard and to harmonize the diverse terminology used in test guidelines adopted over the past decades. Use of the FET in context of effluent assessments was considered and it is not known if fish embryos are sufficiently sensitive for consideration as a surrogate to the sub-chronic 7-day larval fish growth and survival test used in the United States, for example. Addressing these needs by via workshops, research, and additional data reviews were identified for future action by scientists and regulators.

When addressing the use of fish for the environmental safety of chemicals and effluents, there are many opportunities for applying the principles of the 3Rs: Reduce, Refine, and Replace. The current environmental regulatory testing strategy for bioconcentration and secondary poisoning has been reviewed, and alternative approaches that provide useful information are described. Several approaches can be used to reduce the number of fish used in the Organization for Economic Cooperation and Development (OECD) Test Guideline 305, including alternative in vivo test methods such as the dietary accumulation test and the static exposure approach. The best replacement approach would seem to use read-across, chemical grouping, and quantitative structure-activity relationships with an assessment of the key processes in bioconcentration: Adsorption, distribution, metabolism, and excretion. Biomimetic extraction has particular usefulness in addressing bioavailable chemicals and is in some circumstances capable of predicting uptake. Use of alternative organisms such as invertebrates should also be considered. A single cut-off value for molecular weight and size beyond which no absorption will take place cannot be identified. Recommendations for their use in bioaccumulative (B) categorization schemes are provided. Assessment of biotransformation with in vitro assays and in silico approaches holds significant promise. Further research is needed to identify their variability and confidence limits and the ways to use this as a basis to estimate bioconcentration factors. A tiered bioconcentration testing strategy has been developed taking account of the alternatives discussed.

Laboratory studies were conducted to investigate potential adverse effects on development, growth, reproduction and biomarker responses (vitellogenin [VTG] and gonad histology) in fathead minnows (Pimephales promelas) exposed to tamoxifen citrate. Based on the results of a partial life cycle study (nominal [mean measured] concentrations ranged from 0.18 [0.11] to 18 [15.74] microg/L), a 284-d fish full life-cycle (FFLC) flow-through study was conducted using newly fertilized embryos (24 h postfertilization) exposed to nominal (mean measured) concentrations of 14C-tamoxifen citrate that ranged from 0.01 (0.007) to 5.12 (4.08) microg/L. Triethylene glycol (2.0 microl/L) was used as a solvent carrier, with 17beta-estradiol (E2) as a positive control (nominal 0.1 microg/L). Among the biomarkers measured, significant effects on VTG and gonad histology were observed, although these results required care in their interpretation. Among important population-relevant endpoints, no effects on reproduction were observed at nominal concentrationsor = 5.12 microg/L. Effects on growth (length and weight) were observed in some treatments; however, some of these showed irregular concentration-response relationships, which made interpretation uncertain, or were deemed transient in nature (e.g., reduction in growth of F1 28-d posthatch larval fish at nominal concentrations of 0.08, 0.64, and 5.12 microg/L) and judged not to be biologically significant. Interpretation of results from fish chronic studies is challenging and frequently calls for scientific judgement about statistical and biological significance and what constitutes an adverse effect. Using the principles used in mammalian toxicology studies, data from partial and FFLC studies were evaluated from both statistical and biological perspectives in order to determine no-observed-adverse effect concentrations (expressed as (adverse)NOEC) for use in environmental risk assessment. Careful consideration of both biological and statistical outcomes from these studies suggested overall (adverse)NOEC concentration and lowest-observed-effect concentration ((adverse)LOEC) values for tamoxifen citrate of 5.12 microg/L and 5.6 microg/L, respectively.

The pharmaceutical industry gives high priority to animal welfare in the process of drug discovery and safety assessment. In the context of environmental assessments of active pharmaceutical ingredients (APIs), existing U.S. Food and Drug Administration and draft European regulations may require testing of APIs for acute ecotoxicity to algae, daphnids, and fish (base-set ecotoxicity data used to derive the predicted no-effect concentration [PNECwater] from the most sensitive of three species). Subject to regulatory approval, it is proposed that testing can be moved from fish median lethal concentration (LC50) testing (typically using > or = 42 fish/API) to acute threshold tests using fewer fish (typically 10 fish/API). To support this strategy, we have collated base-set ecotoxicity data from regulatory studies of 91 APIs (names coded for commercial reasons). For 73 of the 91 APIs, the algal median effect concentration (EC50) and daphnid EC50 values were lower than or equal to the fish LC50 data. Thus, for approximately 80% of these APIs, algal and daphnid acute EC50 data could have been used in the absence of fish LC50 data to derive PNECwater values. For the other 18 APIs, use of an acute threshold test with a step-down factor of 3.2 is predicted to give comparable PNECwater outcomes. Based on this preliminary scenario of 91 APIs, this approach is predicted to reduce the total number of fish used from 3,822 to 1,025 (approximately 73%). The present study, although preliminary, suggests that the current regulatory requirement for fish LC50 data regarding APIs should be succeeded by fish acute threshold (step-down) test data, thereby achieving significant animal welfare benefits with no loss of data for PNECwater estimates.

The need for alternative approaches to the use of vertebrate animals for hazard assessment of chemicals and pollutants has become of increasing importance. It is now the first consideration when initiating a vertebrate ecotoxicity test, to ensure that unnecessary use of vertebrate organisms is minimized wherever possible. For some regulatory purposes, the use of vertebrate organisms for environmental risk assessments has been banned; in other situations, the number of organisms tested has been dramatically reduced or the severity of the procedure refined. However, there is still a long way to go to achieve a complete replacement of vertebrate organisms to generate environmental hazard data. The development of animal alternatives is based not just on ethical considerations but also on reducing the cost of performing vertebrate ecotoxicity tests and in some cases on providing better information aimed at improving environmental risk assessments. The present Focus article provides an overview of the considerable advances that have been made toward alternative approaches for ecotoxicity assessments over the last few decades. Environ Toxicol Chem 2016;35:2637-2646. © 2016 SETAC.