Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.

In situ assays based on feeding depression can be powerful ecotoxicological tools that can link physiological organism-level responses to population and/or community-level effects. Amphipods are traditional target species for toxicity tests due to their high sensitivity to contaminants, availability in the field and ease of handling. However, cost-effective in situ assays based on feeding depression are not yet available for amphipods that inhabit estuarine ecosystems. The aim of this work was to assess a short-term in situ assay based on postexposure feeding rates on easily quantifiable food items with an estuarine amphipod.Experiments were carried out under laboratory conditions using juvenile Echinogammarus marinus as the target individual. When 60 Artemia franciscana nauplii (as prey) were provided per individual for a period of 30 min in dark conditions, feeding rates could be easily quantified. As an endpoint, postexposure feeding inhibition in E. marinus was more sensitive to cadmium contamination than mortality. Assay calibration under field conditions demonstrated the relevance of sediment particle size in explaining individual feeding rates in uncontaminated water bodies. An evaluation of the 48-h in situ bioassay based on postexposure feeding rates indicated that it is able to discriminate between unpolluted and polluted estuarine sites. Using the harmonized protocol described here, the in situ postexposure feeding assay with E. marinus was found to be a potentially useful, cost-effective tool for assessing estuarine sediment and water quality.

Brine shrimp Artemia sp. has been recognised as an important ecotoxicity and nanotoxicity test model organism for salt-rich aquatic environments, but currently there is still no harmonised testing protocol which would ensure the comparable results for hazard identification. In this paper we aimed to design the harmonised protocol for nanomaterial toxicity testing using Artemia franciscana and present a case study to validate the protocol with silver nanoparticles (AgNPs). We (i) revised the existing nanotoxicity test protocols with Artemia sp. (ii) optimised certain methodological steps based on the experiments with AgNPs and potassium dichromate (K2Cr2O7) as a soluble reference chemical and (iii) tested the optimised protocol in an international inter-laboratory exercise conducted within the EU FP7 NanoValid project. The intra- and inter-laboratory reproducibility of the proposed protocol with a soluble reference chemical K2Cr2O7 was good, which confirms the suitability of this assay for conventional chemicals. However, the variability of AgNPs toxicity results was very high showing again that nanomaterials are inherently challenging for toxicity studies, especially those which toxic effect is linked to shed metal ions. Among the identified sources for this variability were: the hatching conditions, the type of test plate incubation and the illumination regime. The latter induced variations assumingly due to the changes in bioavailable silver species concentrations. Up to our knowledge this is the first inter-laboratory comparison of the Artemia sp. toxicity study involving nanomaterials. Although the inter-laboratory exercise revealed poor repeatability of AgNPs toxicity results, this study provides valuable information regarding the importance of harmonisation of all steps in the test procedure. Also, the presented AgNPs toxicity case study may serve as a platform for further validation steps with other types of NMs.

This study investigated the trophic transfer of nanoplastics in marine food chains. We fed nanoplastic-exposed Artemia franciscana (brine shrimp) to Larimichthys polyactis (small yellow croaker) daily for eight days. Subsequently, the overall health condition, histopathological damage to the liver and digestive tract, and swimming ability of the fish were measured. After the sub-acute exposure to nanoplastics via trophic transfer, the fish showed inhibited growth, severe liver damage, as well as a poorer swimming ability compared to the control. The swimming ability was especially affected, in terms of the overall movement as well as thigmotaxis. The results thus clarified that even an indirect exposure to nanoplastics could induce neurotoxic effects and affect the swimming ability of the fish. As fish are well-known human food resources, the possibility of such trophic transfers affecting higher trophic level organisms, such as humans, cannot be ruled out.

We examined the synergistic effects of CO2 injection on electro-chlorination in disinfection of plankton and bacteria in simulated ballast water. Chlorination was performed at dosages of 4 and 6ppm with and without CO2 injection on electro-chlorination. Testing was performed in both seawater and brackish water quality as defined by IMO G8 guidelines. CO2 injection notably decreased from the control the number of Artemia franciscana, a brine shrimp, surviving during a 5-day post-treatment incubation (1.8 and 2.3 log10 reduction in seawater and brackish water, respectively at 6ppm TRO+CO2) compared with water electro-chlorinated only (1.2 and 1.3 log10 reduction in seawater and brackish water, respectively at 6ppm TRO). The phytoplankton Tetraselmis suecica, was completely disinfected with no live cell found at >4ppm TRO with and without CO2 addition. The effects of CO2 addition on heterotrophic bacterial growth was not different from electro-chlorination only. Total residual oxidant concentration (TRO) more rapidly declined in electro-chlorination of both marine and brackish waters compared to chlorine+CO2 treated waters, with significantly higher amount of TRO being left in waters treated with the CO2 addition. Total concentration of trihalomethanes (THMs) and haloacetic acids (HAAs) measured at day 0 in brackish water test were found to be 2- to 3-fold higher in 6ppm TRO+CO2-treated water than in 6ppm TRO treated water. The addition of CO2 to electro-chlorination may improve the efficiency of this sterilizing treatment of ballast water, yet the increased production of some disinfection byproducts needs further study.

Assessing oil spill toxicity in real time is challenging due to dynamic field exposures and lack of simple, rapid, and sensitive tests. We investigated the relative sensitivity of two commercially available marine toxicity tests to aromatic hydrocarbons using the target lipid model (TLM). State of the art passive dosing in sealed vials was used to assess the sensitivity of brine shrimp (Artemia franciscana) and rotifer (Brachionus plicatilis). Organisms were exposed to toluene, 1-methylnaphthalene and phenanthrene for 24 h. Toxicity results were analysed using the TLM to estimate the critical target lipid body burden and support comparison to empirical data for 79 other aquatic organisms. Our findings demonstrate the applicability of passive dosing to test small volumes and indicate that the two rapid cyst-based assays are insensitive in detecting hydrocarbon exposures compared to other aquatic species. Our results highlight the limitations of applying these tests for oil pollution monitoring and decision-making.

Coolia are marine benthic dinoflagellates which are globally distributed and potentially toxic. This study provides the first investigation of species diversity and toxicity assessment of Coolia in Hong Kong waters. Fifty-one strains of four Coolia species, including C. malayensis, C. canariensis, C. tropicalis, and C. palmyrensis, were isolated from twelve sub-tidal habitats, and identified phylogenetically using 28S rDNA sequences. Exposure experiments (48-hour) demonstrated that the algal lysates extracted from the four Coolia species exhibited different toxic effects on the lethality and abnormality of two invertebrate larvae, i.e., brine shrimp Artemia franciscana and sea urchin Heliocidaris crassispina. Heliocidaris crassispina was more sensitive to the toxic effects of Coolia species than A. franciscana. Toxicity tests from both larvae revealed that C. malayensis was generally more toxic, and caused higher mortality rates when compared with the other three species. The emerging threat of harmful benthic dinoflagellates to marine environments and sensitive biota is discussed.

Studies were conducted to assess the biodegradability and toxicity of the cationic surfactant dodecyl trimethyl ammonium chloride (DTMAC) in sea water samples collected from the Gulf of Cadiz (Spain). Ultimate biodegradation was studied following the guideline proposed by the United States Environmental Protection Agency (USEPA). Growth inhibition tests on five marine microalgae species and mortality tests on a marine crustacean (Artemia franciscana) were carried out. Biodegradation process was modelled according to a logistic kinetic model. Lag time and half-life were 15.17 and 26.95 days, respectively. Depending on the microalgae, 96-h EC50 values ranged from 0.69 to 6.34 mg L⁻¹ DTMAC, respectively. 48-h and 72-h LC50 to A. franciscana were 46.74 and 34.19 mg L⁻¹ DTMAC, respectively. The results indicate that DTMAC can be mineralised in sea water. Marine crustacean was more resistant than the microalgae. Surfactant tolerance on microalgae followed this order: T. chuii > N. gaditana > C. gracilis ≈ I. galbana ≈ D. salina, being the Green microalgae T. chuii the most tolerant.

Microplastics (MPs) are ubiquitously present in the world’s seas with unknown potential toxic effects on aquatic ecosystems. The aim of this study was to evaluate biochemical responses caused by 1–5 μm diameter plastic fluorescent red polymer microspheres (FRM), under short-term exposure of nauplii and juveniles of Artemia franciscana, using a set of biomarkers involved in important physiological processes such as biotransformation, neuronal transmission and oxidative stress. Two FRM concentrations (0.4 and 1.6 mg mL⁻¹) present in the water at ecologically relevant concentrations were used to study their toxicity. No significant differences were found in growth, survival and feeding behaviour of nauplii, after 2 days of exposure to both FRM concentrations. However, in juveniles, survival decreased after 5 days of exposure to FRM1.6; but no significant differences were found in either growth or feeding behaviour. It was observed that nauplii and juveniles, under short-term exposure, had the ability to ingest and egest FRM particles, although their accumulation was higher in nauplii than in juveniles, maybe related with the capacity of the latter to empty their gut content faster, in the presence of food. Regarding biomarkers responses in nauplii, all enzymatic activities increased significantly, after short-term exposure to the higher FRM concentration tested (FRM1.6), which could be related with detoxifying MPs-triggered oxidative stress. In juveniles, the inhibition of ChE and the decrease in the activity of antioxidant enzymes, after 5 days of exposure to FRM1.6, might indicate a neurotoxic effect and oxidative damage induced by FRM. This study provides further evidences that accumulation of MPs in the gut by nauplii and juveniles of A. franciscana can induce negative effects on important physiological processes with influence on their health, highlighting the general concern about the negative effects of MPs pollution on aquatic species, as well as the need to understand the mechanism of MPs toxicity and its possible impacts on environmental safety. Graphical abstract

A column experiment was conducted to evaluate the effectiveness of nanoscale zerovalent iron (nZVI) for the in situ immobilization of Pb and Zn in an acidic soil. The impact of nZVI on soil was evaluated by monitoring the physicochemical characteristics of the leachates and their ecotoxicological effects on three species, Vibrio fischeri, Artemia franciscana, and Caenorhabditis elegans. Treatment with nZVI resulted in more effective Pb immobilization in comparison to Zn and reduced the leachability by 98 and 72 %, respectively; the immobilization was stable throughout the experiment. Leachates from nZVI-treated soils showed lower toxicity than leachates from untreated ones. The highest toxicity in treated soils was observed in the first leachate, which presented high values of electrical conductivity due to the leachability of soil ions and those provided by the commercial nanoparticle suspension (Na and Fe). V. fischeri and C. elegans were more sensitive to leachates from nZVI-treated soils polluted with Zn than those from soils polluted with Pb; A. franciscana showed the opposite trend.