Toward High-Throughput Fish Embryo Toxicity Tests in Aquatic Toxicology.

Addressing the shift from classical animal testing to high-throughput in vitro and/or simplified in vivo proxy models has been defined as one of the upcoming challenges in aquatic toxicology. In this regard, the fish embryo toxicity test (FET) has gained significant popularity and wide standardization as one of the sensitive alternative approaches to acute fish toxicity tests in chemical risk assessment and water quality evaluation. Nevertheless, despite the growing regulatory acceptance, the actual manipulation, dispensing, and analysis of living fish embryos remains very labor intensive. Moreover, the FET is commonly performed in plastic multiwell plates under static or semistatic conditions, potentially inadequate for toxicity assessment of some organic, easily degradable or highly adsorptive toxicants. Recent technological advances in the field of mechatronics, fluidics and digital vision systems demonstrate promising future opportunities for automation of many analytical stages in embryo toxicity testing. In this review, we highlight emerging advances in fluidic and laboratory automation systems that can prospectively enable high-throughput FET testing (HT-FET) akin to pipelines commonly found in in vitro drug discovery pipelines. We also outline the existing challenges, barriers to future development and provide an outlook of ground-breaking fluidic technologies in embryo toxicity testing.

"Nanosize effect" in the metal-handling strategy of the bivalve Scrobicularia plana exposed to CuO nanoparticles and copper ions in whole-sediment toxicity tests.

To date, the occurrence, fate and toxicity of metal-based NPs in the environment is under investigated. Their unique physicochemical, biological and optical properties, responsible for their advantageous application, make them intrinsically different from their bulk counterpart, raising the issue of their potential toxic specificity or "nanosize effect". The aim of this study was to investigate copper bioaccumulation, subcellular distribution and toxic effect in the marine benthic species Scrobicularia plana exposed to two forms of sediment-associated copper, as nanoparticles (CuO NPs) and as soluble ions (CuCl2). Results showed that the exposure to different copper forms activated specific organism's metal handling strategies. Clams bioaccumulated soluble copper at higher concentrations than those exposed to sediment spiked with CuO NPs. Moreover, CuO NPs exposure elicited a stronger detoxification response mediated by a prompt mobilization of CuO NPs to metal-containing granules as well as a delayed induction of MT-like proteins, which conversely, sequestered soluble copper since the beginning of the exposure at levels significantly different from the control. Eventually, exposure to high concentrations of either copper form led to the same acute toxic effect (100% mortality) but the outcome was delayed in bivalves exposed to CuO NPs suggesting that the mechanisms underlying toxicity were copper form-specific. Indeed, while most of soluble copper was associated to the mitochondrial fraction suggesting an impairment of the ATP synthesis capacity at mitochondrial level, CuO NPs toxicity was most likely caused by the oxidative stress mediated by their bioaccumulation in the enzymatic and mitochondrial metabolically available fractions.

Emerging prospects of integrated bioanalytical systems in neuro-behavioral toxicology.

Neurotoxicity effects of industrial contaminants are currently significantly under investigated and require innovative analytical approaches to assess health and environmental risks at individual, population and ecosystem levels. Behavioral changes assessed using small aquatic invertebrates as standard biological indicators of the aggregate toxic effects, have been broadly postulated as highly integrative indicators of neurotoxicity with physiological and ecological relevance. Despite recent increase in understanding of the emerging value of behavioral biotests, their wider implementation especially in high-throughput environmental risk assessment assays, is largely limited by the lack of advances in analytical technologies. To date, most of the behavioral biotests have only been performed with larger-volumes and lacked dynamic flow-through conditions. They also lack features necessary for development of higher throughput neuro-behavioral ecotoxicity assays such as miniaturization and integration of automated components. We postulate that some contemporary analytical limitations can be effectively addressed by innovative Lab-on-a-Chip (LOC) technologies, an emerging and multidisciplinary field poised to bring significant miniaturization to aquatic ecotoxicity testing. Recent developments in this emerging field demonstrate particular opportunities to study a plethora of behavioral responses of small model organisms in a high-throughput fashion. In this review, we highlight recent advances in this budding new interdisciplinary field of research. We also outline the existing challenges, barriers to development and provide a future outlook in the evolving field of neurobehavioral ecotoxicology.

Unsuitable use of DMSO for assessing behavioral endpoints in aquatic model species.

Dimethyl sulfoxide (DMSO) is a universally used aprotic solvent with the ability to permeate biological membranes and thus is commonly used to achieve appropriate biological availability of hydrophobic toxicants. While DMSO as a carrier medium has a reportedly low toxicity and is routinely employed in ecotoxicology, very little is known about its effect on dynamic behavioral parameters. This study presents a comparative analysis of the lethal and behavioral effects of exposures to DMSO concentrations of 0.1-10% on several test species such as: neonates of the freshwater crustacean Daphnia magna, nauplii of the marine crustacean Artemia franciscana, the marine crustacean Allorchestes compressa, embryos and larvae of the freshwater fish Danio rerio. The results demonstrated that DMSO did not cause statistically significant mortality even at concentrations close to 1% but induced clear and significant behavioral abnormalities in response to sublethal concentrations on all test species. These included hypoactivity syndrome in A. franciscana, A. compressa, D. magna and zebrafish larvae while a slight time-dependent hyperactivity response was observed in zebrafish embryos. For the majority of test species, behavioral changes such as moving distance, acceleration and burst movement were often observed during the first hours of exposure. These results indicate that caution should be exercised when using DMSO as a carrier solvent in experiments assessing behavioral endpoints.

Ecotoxicology Goes on a Chip: Embracing Miniaturized Bioanalysis in Aquatic Risk Assessment.

Biological and environmental sciences are, more than ever, becoming highly dependent on technological and multidisciplinary approaches that warrant advanced analytical capabilities. Microfluidic lab-on-a-chip technologies are perhaps one the most groundbreaking offshoots of bioengineering, enabling design of an entirely new generation of bioanalytical instrumentation. They represent a unique approach to combine microscale engineering and physics with specific biological questions, providing technological advances that allow for fundamentally new capabilities in the spatiotemporal analysis of molecules, cells, tissues, and even small metazoan organisms. While these miniaturized analytical technologies experience an explosive growth worldwide, with a substantial promise of a direct impact on biosciences, it seems that lab-on-a-chip systems have so far escaped the attention of aquatic ecotoxicologists. In this Critical Review, potential applications of the currently existing and emerging chip-based technologies for aquatic ecotoxicology and water quality monitoring are highlighted. We also offer suggestions on how aquatic ecotoxicology can benefit from adoption of microfluidic lab-on-a-chip devices for accelerated bioanalysis.

A Millifluidic System for Analysis of Daphnia magna Locomotory Responses to Water-born Toxicants.

Aquatic toxicity testing in environmental monitoring and chemical risk assessment is critical to assess water quality for human use as well as predict impact of pollutants on ecosystems. In recent years, studies have increasingly focused on the relevance of sub-lethal effects of environmental contaminants. Sub-lethal toxicity endpoints such as behavioural responses are highly integrative and have distinct benefits for assessing water quality because they occur rapidly and thus can be used to sense the presence of toxicants. Our work describes a Lab-on-a-Chip system for the automated analysis of freshwater cladoceran Daphnia magna locomotory responses to water-born toxicants. The design combines a Lab-on-a-Chip system for Daphnia sp. culture under perfusion with time-resolved videomicroscopy and software tracking locomotory activity of multiple specimens. The application of the system to analyse the swimming behaviour of water fleas exposed to different concentrations of water-born toxicants demonstrated that Lab-on-a-Chip devices can become important research tools for behavioural ecotoxicology and water quality biomonitoring.

Bioaccumulation kinetics of copper in Ruditapes philippinarum exposed to increasing, continuous and pulsed exposure: Implications for growth.

Metal bioaccumulation and toxicity to aquatic organisms depends on factors such as magnitude, duration and frequency of the exposure. The type of the exposure affects the toxicokinetic processes in the organisms. In this study, we carried out 30-day toxicity tests on juveniles of Ruditapes philippinarum exposed to increasing, continuous and pulsed exposure. Organisms were exposed to copper-spiked sediments followed by a 10-day recovery period. We assessed the interaction between the kinetics of subcellular copper partitioning and the growth response. Results showed that the growth rate of the bivalve was inversely correlated to the bioaccumulation rate and that sublethal copper concentrations stimulated the detoxification mechanisms inside the organism regardless the type of the exposure. However, a large stimulatory effect on growth was observed during the recovery period, associated with significant negative accumulation rate values and dependent on the type of antecedent exposure. This suggested that on individual and short-term basis pulsed exposures have a more adverse effect compared to increasing or continuous exposure scenarios.

Importance of subcellular metal partitioning and kinetics to predicting sublethal effects of copper in two deposit-feeding organisms.

The role of subcellular partitioning of copper on the sublethal effects to two deposit-feeding organisms (41-day growth in the bivalve Tellina deltoidalis and 11-day reproduction in the amphipod Melita plumulosa) was assessed for copper-spiked sediments with different geochemical properties. Large differences in bioaccumulation and detoxification strategies were observed. The bivalve accumulated copper faster than the amphipod, and can be considered a relatively strong net bioaccumulator. The bivalve, however, appears to regulate the metabolically available fraction (MAF) of the total metal pool by increasing the net accumulation rate of copper in the biologically detoxified metal pool (BDM), where most of the copper is stored. In the amphipod, BDM concentration remained constant with increasing copper exposures and it can be considered a very weak net bioaccumulator of copper. This regulation of copper, with relatively little stored in detoxified forms, appears to best describe the strategy applied by the amphipod to minimize the potential toxic effects of copper. When the EC50 values for growth and reproduction are expressed based on the MAF of copper, the sensitivity of the two species appears similar, however when expressed based on the net accumulation rate of copper in the metabolically available fraction (MAFrate), the bivalve appears more sensitive to copper. These results indicate that describing the causality of metal effects in terms of kinetics of uptake, detoxification, and excretion rather than threshold metal body concentrations is more effective in predicting the toxic effects of copper. Although the expression of metal toxicity in terms of the rate at which the metal is bioaccumulated into metabolically available forms may not be feasible for routine assessments, a deeper understanding of uptake rates from all exposure routes may improve our ability to assess the risk posed by metal-contaminated sediments.

Demonstrating the appropriateness of developing sediment quality guidelines based on sediment geochemical properties.

The pool of bioavailable metals in sediments is typically much smaller than the total metal concentration and is strongly influenced by metal-binding with acid-volatile sulfide (AVS), particulate organic carbon (OC), and iron and manganese oxide solid phases. We have investigated how the properties of relatively oxidized sediments influence the exposure and effects of copper on the survival and growth rate of the deposit-feeding benthic bivalve Tellina deltoidalis. Growth rate was a much more sensitive end point than survival. Toxic effects to growth were consistently observed in sediment where both pore water and overlying water copper concentrations were below the effect threshold for dissolved copper. Decreases in growth of the bivalve were largely attributable to dietary exposure to sediment-bound copper, as the organism was observed to actively feed on fine materials from the sediment surface. For sediments with the same total copper concentrations, effects were less for sediments with greater concentrations of fine particles (<63 µm sediment) or particulate organic carbon (OC). Based on the concentration-response relationship, a no-effect value of 5.5 mg <63 µm Cu g(-1) OC for growth of T. deltoidalis was calculated. The results confirm the appropriateness of using OC-normalized copper concentration in the <63 µm sediment fraction to develop sediment quality guidelines (SQGs) that vary with sediment properties. For sediments where the amount of AVS is not sufficient to bind metals in non bioavailable forms, the metal-binding capacity provided by OC and iron and manganese oxyhydroxides associated with the fine sediments considerably reduced metal bioavailability. These sediment properties should be considered when assessing the risks posed by metal-contaminated sediments.

Evaluating the suitability of Hydrobia ulvae as a test species for sediment metal toxicity testing applying a tissue residue approach to metal mixtures in laboratory and field exposures.

A major weakness in evaluating the suitability of a biomonitor organism is the poor ability to predict the variability of the bioavailability of metals from measured environmental concentrations. In this study, the intertidal gastropod Hydrobia ulvae was used to evaluate its suitability as a test organism for assessing sediment metal toxicity. Toxicity tests were run with sediments spiked with copper, cadmium and zinc applied both as single metal and as a mixture to investigate toxicological interactions evaluating different lethal and sublethal effects. Dose-response relationships were constructed based both on tissue residue approach and particulate metal concentrations. Because metal-spiked sediments used in routine toxicity tests often do not exhibit the same adsorption/desorption kinetics as the natural sediments, the laboratory results were compared to 10-d bioassays conducted with natural field sediments collected from the Guadalete estuary (SW Spain). Highly significant correlations between tissue residue concentrations and particulate metal concentrations were found for all metal-spiked or field-collected and demonstrated that: (i) H. ulvae readily accumulated copper and cadmium in response to contamination and (ii) dietary uptake was determined to be the most significant route of metal exposure. The comparison of the modeled tissue residue-response curve developed from the mixture tests was in good agreement with the results from the bioassay conducted with field sediments and strongly demonstrated that H. ulvae is also a suitable test organism for assessing copper sediment toxicity. In contrast, the dose-response curve expressed as a function of total particulate metal concentrations would fail in predicting effect, erroneously assessing higher metal toxicity.

Sub-lethal effects of copper to benthic invertebrates explained by sediment properties and dietary exposure.

The next generation of sediment quality guidelines (SQGs) requires better established causal links between the chronic exposure and effects of metals from both dissolved and dietary sources. The potential for dietary exposure from sediment metals to cause toxic effects to benthic invertebrates is strongly influenced by the metal-binding properties of the sediments. For relatively oxidized sediments, sublethal effects of copper to the epibenthic deposit-feeding amphipod, Melita plumulosa, and the benthic harpacticoid copepod, Nitocra spinipes, were investigated. Effects on reproduction were strongly influenced by the properties of the sediments and sediment-bound copper was found to be the major contribution to the toxicity. For sediments with the same total copper concentrations, effects were less for sediments with greater concentrations of fine particles (<63 µm sediment) or particulate organic carbon (OC). The OC-normalized copper concentration in the <63 µm sediment fraction provided a single effects threshold for all sediment types. For M. plumulosa and N. spinipes, the 10% effect concentrations (EC10s) were 5.2 and 4.8 mg <63 µm Cu g(-1) OC. These chronic EC10s indicate that a SQG of 3.5 mg <63 µm Cu g(-1) OC, that was previously proposed based on a species sensitivity distribution of acute no effects thresholds data for 12 benthic organisms, will be protective for these species. The study confirms the appropriateness of using SQGs that vary with sediment properties and that SQGs of this form provide adequate protection for metal exposure via both dissolved and dietary exposure pathways.

Effect of lead on ALA-D activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus.

The effects of lead (Pb) on ALA-D activity, metallothionein (MT) levels, and lipid peroxidation in liver, kidney, and blood of the toadfish Halobatrachus didactylus were investigated. A time-course experiment was performed with sampling on days 0, 2, 5, and 7 following intraperitoneal Pb injection. This indicated a rank order for lead concentration of kidney > liver > blood in fish exposed to Pb. No significant variation of ALA-D activity was observed in liver and kidney while in blood, a slight decrease of ALA-D activity was found but this was not attributed to acute metal stress. Hepatic and renal MT levels were both affected in different ways by metal uptake. The progressive decrease of MDA concentration in the liver and the lack of a clear induction in kidney suggested the hypothesis that Pb is not a good inductor of lipid peroxidation. The histological and histochemical results demonstrated degenerative effects of lead accumulation on the tissues and the activation of lysosomal responses to induced stress.