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.

The 2,4-dichlorophenoxyacetic acid (2,4-D) is an auxinic herbicide widely used in agriculture that is effective in controlling weeds. It is directly applied to the soil, to ponds or sprayed onto crops; thus, it can progressively accumulate in environmental compartments and affect non-target organisms. The aim of the present meta-analytic review is to investigate the toxic effects of 2,4-D, based on a compilation of results from different studies, which were synthesized to form a statistically reliable conclusion about the lethal effect of potentially ecological concentrations of 2,4-D in several animal species. The search was carried out in the Web of Science and Scopus databases. After the selection process was over, 87 datasets were generated and analyzed. The overall effect has indicated significant increase in the mortality rate recorded for animals exposed to environmental concentrations of 2,4-D compared to the control in the experiment (unexposed animals). The segregation of animals into taxonomic categories has shown that fish and birds presented higher mortality rates after exposure to the investigated substance. The present meta-analysis indicated larval and adult animals were susceptible among the ontogenetic development stages. Juvenile individuals exposed to different 2,4-D concentrations did not show significant difference in comparison to the control. Organisms exposed to 2,4-D immersion were the most impacted compared to those exposed by oral, spray and contact. Animals subjected to commercial formulation presented higher mortality rate than the analytical standard. Thus, 2,4-D can, in fact, increase mortality rate in animals, but it depends on species sensitivity, life stage and exposure route. This is the first meta-analytical study evaluating the mortality rate after 2,4-D exposure in several animal species.

Glyphosate (GLY) is a broad-spectrum herbicide that is the most commonly applied pesticide in terrestrial ecosystems in the U.S. and, potentially, worldwide. However, the combined effects of warming associated with climate change and exposure to GLY and GLY-based formulations (GBFs) on terrestrial animals are poorly understood. Animals progress through several life stages (e.g., embryonic, larval, and juvenile stages) that may exhibit different sensitivities to stressors. Therefore, we factorially manipulated temperature and GLY/GBF exposure in the variable field cricket (Gryllus lineaticeps) during two life stages—nymphal development and adulthood—and examined key animal traits, such as developmental rate, body size, food consumption, reproductive investment, and lifespan. A thermal environment simulating future climate warming obligated several costs to fitness-related traits. For example, warming experienced during nymphal development reduced survival, adult body mass and size, and investment into flight capacity and reproduction. Warming experienced by adults reduced lifespan and growth rate. Alternatively, the effects of GBF exposure were more subtle, often context-dependent (e.g., effects were only detected in one sex or temperature regime), and were stronger during adult exposure relative to exposure during development. There was evidence of additive costs of warming and GBF exposure to rates of feeding and growth in adults. Yet, the negative effect of GBF exposure to adult lifespan did not occur in warming conditions, suggesting that ongoing climate change may obscure some of the costs of GBFs to non-target organisms. The effects of GLY alone (i.e., in the absence of proprietary surfactants found in commercial formulations) were non-existent. Animals will be increasingly exposed to warming and GBFs, and our results indicate that GBF exposure and warming can entail additive costs for an animal taxon (insects) that plays critical roles in terrestrial ecosystems.

Morphological tools can assist in the evaluation of effects of insecticides on non-target insects. Pyriproxyfen, a juvenile hormone analog, is known to interfere with growth and metamorphosis of insects. However, there are studies showing indirect effects on natural enemies, including green lacewings. Few prior studies describe morphological effects of pyriproxyfen on target insect organs, especially on natural enemies. Through morphological tools, this study aimed to characterize the midgut and fat body, both important organs of digestion and great metabolic activity respectively, of the predator Ceraeochrysa claveri after chronic exposure to pyriproxyfen. Larvae of C. claveri were fed Diatraea saccharalis egg clusters treated with pyriproxyfen in solution of 50 or 100 mg a.i. L⁻¹ throughout the larval stage. The biological data revealed significant increases in development time, especially in the third instar, and in cumulative mortality from the prepupal into the pupal stage. Morphological analysis of adult midgut (≤24 h old) showed damage including formation of epithelial folds, intercellular spaces, emission of cytoplasmic protrusions. Both fat body regions presented decrease of lipid droplets, vacuolization of trophocytes and mitochondrial injury featuring a multisystemic action. In both organs, pyriproxyfen exposure induced significant oxidative stress by mitochondrial superoxide production. Cytoprotective responses were induced in midgut and fat body cells by augmenting the number of cytoplasmic granules containing calcium and expression of HSP 90. Both organs proved to be efficient in presenting histopathological alterations, showing the sensitivity and applicability of this morphological tool for evaluating other insecticides in non-target organisms.

Both amphibian metamorphosis assay (AMA) and larval amphibian growth and development assay (LAGDA) can detect thyroid-mediated modality and adversity on the basis of morphological changes during the thyroid hormone-dependent metamorphosis. They are used for identification of thyroid hormone system disrupting chemicals (TDCs) for non-target organisms or the environment. The EU Guidance recommends that the AMA and the LAGDA should be used to address sufficient investigation of the thyroid-mediated modality and adversity, respectively. In the EU discussions over identification of TDCs, the necessity of using LAGDA as a follow-up of positive results of the AMA has been questioned because of the overlap between the endpoints and the exposure of both tests. This study analyzed similarities, differences, and sensitivity of these two assays in detection of TDCs. For agonists and most of antagonists of the hypothalamic-pituitary-thyroid (HPT) axis, both AMA and LAGDA can detect the thyroid-mediated modality and adversity. The LAGDA, as a follow-up of the positive results of the AMA, may not be needed because the results of AMA are considered enough for identification of TDCs. For chemicals like inhibitors of iodotyrosine deiodinase, the LAGDA is considered necessary for identification of TDCs because the thyroid-mediated adversity cannot be detected until Nieuwkoop and Faber (NF) stage 62. Incorporation of mechanistic endpoints into existing test guidelines and the use of Xenopus Eleutheroembryo Thyroid Assay (XETA), extended amphibian metamorphosis assay (EAMA) and adverse outcome pathways (AOPs) for testing and identification of TDCs are further discussed.

Across the planet, winter de-icing practices have caused secondary salinization of freshwater habitats. Many amphibians are vulnerable because of permeable skin and reliance on small ponds, where salinity can be high. Early developmental stages of amphibians are especially sensitive to salt, and larvae developing in salt-polluted environments must osmoregulate through ion exchange in gills. Though ionoregulation in amphibian gills is generally understood, the role of gill morphology remains poorly described. Yet gill structure should affect ionoregulatory capacity, for instance in terms of available surface area. As larval amphibian gills also play critical roles in gas exchange and foraging, changes in gill morphology from salt pollution potentially affect not only osmoregulation, but also respiration and feeding. Here, we used an exposure experiment to quantify salinity effects on larval gill morphology in wood frogs (Rana sylvatica). We measured a suite of morphological traits on gill tufts—where ionoregulation and gas exchange occur—and on gill filters used in feeding. Larvae raised in elevated salinity developed larger gill tufts but with lower surface area to volume ratio. Epithelial cells on these tufts were less circular but occurred at higher densities. Gill filters showed increased spacing, likely reducing feeding efficiency. Many morphological gill traits responded quadratically, suggesting that salinity might induce plasticity in gills at intermediate concentrations until energetic demands exceed plasticity. Together, these changes likely diminish ionoregulatory and respiratory functionality of gill tufts, and compromise feeding functionality of gill filters. Thus, a singular change in aquatic environment from a widespread pollutant appears to generate a suite of consequences via changes in gill morphology. Critically, these changes in traits likely compound the severity of fitness impacts in populations dwelling in salinized environments, whereby ionoregulatory energetic demands should increase respiratory and foraging demands, but in individuals who possess structures poorly adapted for these functions.

Plastic additives (PAs) are chemical compounds incorporated into the plastic during the manufacturing process. Phthalate acid esters, bisphenols, and nonylphenols are all PAs found in marine environments and associated with endocrine-disrupting processes. However, our knowledge regarding the impact of endocrine-disrupting PAs on coral-reef organisms is limited. As reef population structure is directly linked to reproduction and larval settlement processes, interference with hormonal systems can impact coral-reef community structure, particularly if the effects of PAs differ among species. In the current study we exposed the reproductive products of four tropical coral-reef invertebrates to environmentally-relevant concentrations of four prevalent PAs in seawater: dibutyl phthalate (DBP), dimethyl phthalate, (DMP), 4-nonylphenol (4-NP), and bisphenol A (BPA), as well as to 10³ higher laboratory concentrations of these PAs. Our results revealed that apart from the significant negative effect of the 1 μg/L of 4-NP on the settlement of the soft coral Rhytisma fulvum, none of the other tested materials demonstrated a significant effect on the exposed organisms at environmentally-relevant concentrations in seawater. The 4-NP high laboratory concentration (1000 μg/L), however, had significant negative effects on all the examined species. The high laboratory BPA concentration (1000 μg/L) significantly reduced fertilization success in the solitary ascidian Herdmania momus, up to its complete failure to reproduce. Moreover, the high laboratory DMP concentration (100 μg/L) had a significant negative effect on planulae settlement of the stony coral Stylophora pistillata. Our findings demonstrate the negative and selective effects of PAs on the development and reproduction of coral-reef organisms; and, specifically, the significant effect found following exposure to 4-NP. Consequently, if we aim to fully understand the impact of these contaminants on this endangered ecosystem, we suggest that the actual concentrations within the living organism tissues should be tested in order to produce relevant risk assessments for brooding-coral species.

Insufficient evidence exists regarding the effects of microplastics (MPs) on the neuronal toxicity of heavy metals in the early stages of organisms. Herein, the effects of micro-polystyrene (μ-PS; 157 μm) and nano-polystyrene (n-PS; 100 nm) particles on the neurodevelopmental toxicity of mercury (Hg) in zebrafish embryos were compared. Zebrafish embryos exposed to Hg at the concentration of 0.1 mg L⁻¹ revealed blood disorders, delayed hatching, and malformations such as pericardial oedema and tail deformity. The length of the larval head was significantly reduced (P < 0.01) and in vivo expression of atoh1a in the cerebellum of neuron-specific transgenic zebrafish Tg(atoh1a:dTomato) larvae was inhibited by 29.46% under the Hg treatment. Most of the toxic effects were inhibited by the combined exposure to μ-PS or n-PS with Hg, and n-PS decreased the neurodevelopmental toxicity of Hg more significantly than μ-PS. Metabolomic analysis revealed that in addition to inhibiting the amino acid metabolism pathway as in the μ-PS+Hg treatment, the n-PS+Hg treatment inhibited unsaturated fatty acid metabolism in zebrafish larvae, likely because of a greater reduction in Hg bioavailability, thus reducing the oxidative damage caused by Hg in the larvae. The combined effects of MPs and heavy metals differ greatly among different species and their targeted effects. We conclude that the combined toxicity mechanisms of MPs and heavy metals require further clarification.

Plastic additives are utilized during the production of plastic to modify the attributes and stability of the polymer. As oceanic plastic waste degrades, these additives can leach, and are harmful to global marine ecosystems. Despite the high abundance of additives leached into the marine environment, little is known about their direct impact on marine zooplankton. Here we test for impacts of four plastic additives, UV-327, Irganox 1010, DEHP, and methylparaben, all commonly used in plastic manufacturing, on purple sea urchin (Strongylocentrotus purpuratus) larval growth and survival in a serial dose response for 4 days. Methylparaben, UV-327, and Irganox 1010 significantly reduced larval body length by about 5% for at least one dose. In contrast, all compounds reduced larval survival by 20–70% with strongest effects at intermediate rather than high doses. Our results highlight that plastic additives should be tested for their effects on marine organisms.

There is an urgent need to understand how organisms respond to multiple, potentially interacting drivers in today's world. The effects of the pollutants anthropogenic sound (pile driving sound playbacks) and waterborne cadmium were investigated across multiple levels of biology in larval and juvenile Norway lobster, Nephrops norvegicus under controlled laboratory conditions. The combination of pile driving playbacks (170 dBₚₖ₋ₚₖ re 1 μPa) and cadmium combined synergistically at concentrations >9.62 μg[Cd] L⁻¹ resulting in increased larval mortality, with sound playbacks otherwise being antagonistic to cadmium toxicity. Exposure to 63.52 μg[Cd] L⁻¹ caused significant delays in larval development, dropping to 6.48 μg[Cd] L⁻¹ in the presence of piling playbacks. Pre-exposure to the combination of piling playbacks and 6.48 μg[Cd] L⁻¹ led to significant differences in the swimming behaviour of the first juvenile stage. Biomarker analysis suggested oxidative stress as the mechanism resultant deleterious effects, with cellular metallothionein (MT) being the predominant protective mechanism.