Response of the terrestrial isopod Porcellio scaber to lipopolysaccharide challenge after microplastic and insecticide exposure.

The exposure of organisms to microplastics could compromise their ability to cope with other environmental stressors, such as infections. In this context, we investigated the effects of a 14-day exposure of the terrestrial isopod Porcellio scaber to tire particles in soil (1.5 % w w-1 dry weight) on the organisms' response to a secondary exposure, i.e., injection of the bacterial endotoxin lipopolysaccharide. In addition, the insecticide chlorpyrifos (2 mg kg-1 dry weight) was tested as a positive control. The survival and immune response of P. scaber was assessed at the end of the 7- and 14-day primary exposure and two days after the secondary exposure, by analyzing selected haemolymph immune parameters (total haemocyte count, differential haemocyte count, and haemocyte viability). No change in survival was observed after primary exposure of P. scaber to tire particles or chlorpyrifos. However, primary exposure to chlorpyrifos triggered a strong activation of the immune response, which was not the case following exposure to the tire particles. Further injection of lipopolysaccharide into the body did not affect the survival of animals exposed to tire particles or chlorpyrifos, while a strong immunomodulatory change was observed, particularly with chlorpyrifos, and to some extent, tire particles. Based on these results, we conclude that exposure of P. scaber to tire particles or chlorpyrifos has no significant effect on the susceptibility of the organism to lipopolysaccharide in terms of their mortality, but primary exposure to an insecticide significantly modulates the immune response of the organisms to a second stressor. We discuss the "stress on stress" approach for testing low-toxic substances, such as microplastics, where an environmentally realistic exposure is followed by a secondary exposure.

Cryptic Species in Ecotoxicology.

The advent of genetic methods has led to the discovery of an increasing number of species that previously could not be distinguished from each other on the basis of morphological characteristics. Even though there has been an exponential growth of publications on cryptic species, such species are rarely considered in ecotoxicology. Thus, the particular question of ecological differentiation and the sensitivity of closely related cryptic species is rarely addressed. Tackling this question, however, is of key importance for evolutionary ecology, conservation biology, and, in particular, regulatory ecotoxicology. At the same time, the use of species with (known or unknown) cryptic diversity might be a reason for the lack of reproducibility of ecotoxicological experiments and implies a false extrapolation of the findings. Our critical review includes a database and literature search through which we investigated how many of the species most frequently used in ecotoxicological assessments show evidence of cryptic diversity. We found a high proportion of reports indicating overlooked species diversity, especially in invertebrates. In terrestrial and aquatic realms, at least 67% and 54% of commonly used species, respectively, were identified as cryptic species complexes. The issue is less prominent in vertebrates, in which we found evidence for cryptic species complexes in 27% of aquatic and 6.7% of terrestrial vertebrates. We further exemplified why different evolutionary histories may significantly determine cryptic species' ecology and sensitivity to pollutants. This in turn may have a major impact on the results of ecotoxicological tests and, consequently, the outcome of environmental risk assessments. Finally, we provide a brief guideline on how to deal practically with cryptic diversity in ecotoxicological studies in general and its implementation in risk assessment procedures in particular. Environ Toxicol Chem 2023;42:1889-1914. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Influence of Weathering on the Degradation of Cellulose Acetate Microplastics Obtained from Used Cigarette Butts.

Cellulose acetate is used in many applications, including for cigarette filters. Unfortunately, unlike cellulose, its (bio)degradability is under question, yet it often ends up uncontrolled in the natural environment. The main purpose of this study is to compare the effects of weathering on two types of cigarette filter (classic filters and newer filters that have more recently arrived on the market) following their use and disposal in nature. Microplastics were prepared from polymer parts of used (classic and heated tobacco products-HTP) cigarettes and artificially aged. TG/DTA, FTIR, and SEM analyses were performed both before and after the aging process. Newer tobacco products contain an additional film made of a poly(lactic acid) polymer which, like cellulose acetate, burdens the environment and poses a risk to the ecosystem. Numerous studies have been conducted on the disposal and recycling of cigarette butts and cigarette butt extracts, revealing alarming data that have also influenced the decisions of the EU, who addressed the disposal of tobacco products in the EU Directive (EU) 2019/904. Despite this, there is still no systematic analysis in the literature evaluating the impact of weathering (i.e., accelerated aging) on the degradation of cellulose acetate in classic cigarettes compared with that in newer tobacco products that have recently appeared on the market. This is of particular interest given that the latter have been promoted as being healthier and environmentally friendly. The results show that in cellulose acetate cigarette filters the particle size decreased after accelerated aging. Also, the thermal analysis revealed differences in the behavior of the aged samples, while the FTIR spectra showed no shifts in the position of the peaks. Organic substances break down under UV light, which can be seen by measuring the color change. The PLA film was found to be more stable than cellulose acetate under the influence of UV light.

Proteomics of the haemolymph of the terrestrial crustacean Porcellio scaber reveals components of its innate immunity under baseline conditions.

The terrestrial crustacean Porcellio scaber is an established test organism in environmental research. We analysed the haemolymph proteome of P. scaber using a classical proteomic approach based on one-dimensional gel electrophoresis and tandem mass spectrometry. Using a publicly available protein database and our P. scaber transcriptome data, we have identified 76 proteins involved in cytoskeleton formation, protein degradation, vesicular transport, genetic information processing, detoxification, carbohydrate and lipid metabolism reflecting haemocyte metabolic activity, active intracellular transport, and intercellular communication. Compared with the data reported for other crustaceans, 28 of these P. scaber proteins have been linked to its immunity, among them hemocyanin, α-2-macroglobulin, phenoloxidase 3, superoxide dismutase, glutathione S-transferase, haemolymph clottable protein, and histones H4 and H2B. Our results thus provide a firm base for studying the innate immune response of P. scaber at the level of the haemolymph proteome. This knowledge is of particular importance in ecotoxicity studies with various environmental stressors where understanding physiological changes is important to reveal possible modes of action.

Defining Quality Criteria for Nanoplastic Hazard Evaluation: The Case of Polystyrene Nanoplastics and Aquatic Invertebrate Daphnia spp.

Polystyrene nanoparticles are the most investigated type of nanoplastics in environmental hazard studies. It remains unclear whether nanoplastic particles pose a hazard towards aquatic organisms. Thus, it was our aim to investigate whether the existing studies and data provided therein are reliable in terms of data completeness. We used the example of Daphnia spp. studies for the purpose of polystyrene nanoplastic (nanoPS) hazard evaluation. First, a set of quality criteria recently proposed for nanoplastic ecotoxicity studies was applied. These rather general criteria for all types of nanoplastics and different test organisms were then, in the second step, tailored and refined specifically for Daphnia spp. and nanoPS. Finally, a scoring system was established by setting mandatory (high importance) as well as desirable (medium importance) criteria and defining a threshold to pass the evaluation. Among the existing studies on nanoPS ecotoxicity for Daphnia spp. (n = 38), only 18% passed the evaluation for usability in hazard evaluation. The few studies that passed the evaluation did not allow for conclusions on the hazard potential of nanoPS because there was no consensus among the studies. The greatest challenge we identified is in data reporting, as only a few studies presented complete data for hazard evaluation.

Tire microplastics exposure in soil induces changes in expression profile of immune-related genes in terrestrial crustacean Porcellio scaber.

Tire particles pose a potential threat to terrestrial organisms because they are deposited in large quantities in the soil by tire wear abrasion, and moreover their chemical complexity poses an additional risk. Microplastics can affect several physiological processes in organisms, including those related to immunity. Therefore, we investigated the expression profile of selected immune-related genes (MnSod, Manganese Superoxide dismutase; Cat, Catalase; CypG, Cyclophilin G; Nos, Nitric oxide synthase; Ppae2a, Prophenoloxidase-activating enzyme 2a; Dscam, Down syndrome cell adhesion molecule; Myd88, Myeloid-differentiation factor 88; Toll4, Toll-like receptor 4; Mas-like, Masquerade-like protein) in haemocytes and the digestive gland hepatopancreas of terrestrial crustacean Porcellio scaber after two different time exposures (4 and 14 days) to tire particles in soil. Our results reveal for the first time the response of P. scaber after microplastic exposure at the transcriptome level. We observed time- and tissue-dependent changes in the expression of the analysed genes, with more pronounced alterations in haemocytes after 14 days of exposure. Some minor changes were also observed in hepatopancreas after 4 days. Changes in the expression profile of the analysed genes are a direct indication of a modulated immune status of the test organism, which, however, does not represent an adverse effect on the test organism under the given conditions. Nevertheless, the question remains whether the observed change in immune status affects the immunocompetence of the test organism.

Effects of microplastics from disposable medical masks on terrestrial invertebrates.

This study investigated impacts of microplastics from disposable polypropylene medical masks on woodlice Porcellio scaber, mealworm larvae Tenebrio molitor and enchytraeids Enchytraeus crypticus. Effects of microplastics on survival, reproduction, immune parameters and energy-related traits were assessed after 21 days exposure in soil. Microplastics obtained from each medical mask layer separately differed in size and shape (inner frontal layer: 45.1 ± 21.5 µm, fibers; middle filtering layer: 55.6 ± 28.5 µm, fragments; outer layer: 42.0 ± 17.8 µm, fibers) and composition of additives. Overall, the concentrations of metals and organic chemicals were too low to cause effects on soil invertebrates. The microplastics from disposable medical masks at 0.06%, 0.5%, 1.5%, w/w did not induce severe adverse effects on survival or reproduction (for enchytraeids). A transient immune response of woodlice and a change in energy-related traits in mealworms were observed, which was most clearly seen for the microplastics from the outer layer. This was reflected in increased electron transfer system activity of mealworms and different immune response dynamics of woodlice. In conclusion, the tested soil invertebrates respond to microplastics from disposable medical masks, but it remains unclear what these changes mean for their fitness on the long term.

Time-Course of the Innate Immune Response of the Terrestrial Crustacean Porcellio scaber After Injection of a Single Dose of Lipopolysaccharide.

Invertebrates, including crustaceans, rely on cellular and humoral immune responses to protect against extrinsic and intrinsic factors that threaten their integrity. Recently, different immune parameters have been increasingly used as biomarkers of effects of pollutants and environmental change. Here, we describe the dynamics of the innate immune response of the terrestrial crustacean Porcellio scaber to injection of a single dose of lipopolysaccharide (LPS), an important molecular surface component of the outer membrane of Gram-negative bacteria. The aim was to provide a basis for interpretation of change in immune parameters as a result of different challenges, including microplastics and nanoplastics exposure. Changes in total and differential numbers of hemocytes, hemocyte viability, and humoral immune parameters (i.e., phenoloxidase-like activity, nitric oxide levels) were assessed at different times (3, 6, 12, 24, 48 h). An injection of 0.5 µg/µL LPS into the body of P. scaber resulted in a rapid decrease (3 h after LPS injection) in the total number of hemocytes and reduced viability of the hemocytes. This was accompanied by changed proportions of the different hemocyte types, as a decrease in the numbers of semigranulocytes and granulocytes, and a marked increase in the numbers of hyalinocytes. In addition, phenoloxidase-like activity and nitric oxide levels in the hemolymph were increased at 3 h and 6 h, respectively, after the LPS challenge. Forty-eight hours after LPS injection, the immune parameters in the hemolymph of P. scaber had returned to those before the LPS challenge. This suggests that the innate immune system successfully protected P. scaber from the deleterious effects of the LPS challenge. These data indicate the need to consider the dynamics of innate immune responses of P. scaber when effects of infections, pollutants, or environmental changes are studied. We also propose an approach to test the immunocompetence of organisms after different challenges in ecotoxicity studies, based on the dynamics of their immune responses.

Screening of NaCl salinity sensitivity across eight species of subterranean amphipod genus Niphargus.

Secondary salinization of freshwater is becoming a growing environmental problem. Currently, there is few data available on the effects of salinisation on subterranean crustaceans that are vital for the maintenance of groundwater ecosystem functioning. In this study, the sensitivity of subterranean Niphargus amphipods to NaCl was investigated. We expected that cave-dwelling species would be more sensitive as surface-subterranean boundary species. Eight ecologically different Niphargus species were tested: four live at the boundary between the surface and subterranean ecosystems (N. timavi, N. krameri, N. sphagnicolus, N. spinulifemur), three live in cave streams (N. stygius, N. scopicauda, N. podpecanus), and one species (N. hebereri) lives in anchialine caves and wells. The organisms were exposed to five concentrations of NaCl for 96 h and afterwards the immobility, mortality, and electron transfer system (ETS) activity (a measure for metabolic rate of animals) were evaluated. As expected, the most tolerant species was N. hebereri dwelling in naturally high-salinity habitat. However, contrary to our expectations, the species collected at the surface-subterranean boundary were more sensitive as cave stream species when their immobility and mortality were assessed. Interestingly, the majority of Niphargus tested were more NaCl tolerant as can be deduced from currently available data for subterranean and surface crustaceans. We could not observe a clear trend in ETS activity changes between groups of surface-subterranean boundary and cave streams species after exposure to NaCl stress, but it appears that osmotic stress-induced metabolic rate changes are species-specific. This study shows that amphipods Niphargus can be a valuable subterranean environmental research model and further ecotoxicity research is of interest.

Long-term interactions between microplastics and floating macrophyte Lemna minor: The potential for phytoremediation of microplastics in the aquatic environment.

The presence of microplastics (MPs) in the environment has raised many concerns, and therefore approaches and technologies to remove them in situ are of high interest. In this context, we investigated the interactions between polyethylene MPs (fragments with a mean size of 149 ± 75 µm) and an aquatic floating macrophyte Lemna minor in order to assess its potential use for in situ phytoremediation. We first investigated the long-term effects of a high (100 mg/L = 9600 MPs/L), but still environmentally relevant concentration of MPs on L. minor. Subsequently bioadhesion of MPs was studied and the number and strength of MPs adhering to plant biomass were assessed. MPs did not adversely affect various parameters of plants (e.g., specific growth rate, chlorophyll contents, total antioxidant capacity, electron transport system activity, and contents of energy-rich molecules) throughout the duration of the experiment (12 weeks), except for the first week of the experiment, when protein content and total antioxidant capacity were affected. On the other hand, MPs affected the root length of L. minor during the first eight weeks of the experiment, while further exposure resulted in a decrease in the effects, indicating the ability of L. minor to tolerate the presence of MPs for a long period of time. MPs adhered rapidly to the plant biomass and the average percentages of strongly and weakly adhered particles were 6.5% and 20.0%, respectively, of the total MPs applied. In summary, results of this study suggest that L. minor can tolerate hotspot concentrations of MPs and can collect MPs from the water surface. Therefore, phytoremediation using floating plants could be considered as a potential method for in situ removal of MPs from the aquatic environment.

Environmental hazard of polypropylene microplastics from disposable medical masks: acute toxicity towards Daphnia magna and current knowledge on other polypropylene microplastics.

The COVID-19 pandemic has increased the use of disposable plastics, including medical masks, which have become a necessity in our daily lives. As these are often improperly disposed of, they represent an important potential source of microplastics in the environment. We prepared microplastics from polypropylene medical masks and characterised their size, shape, organic chemical leaching, and acute toxicity to the planktonic crustacean Daphnia magna. The three layers of the masks were separately milled and characterised. Each of the inner frontal, middle filtering, and outer layers yielded different types of microplastics: fibres were obtained from the inner and outer layer, but irregular fragments from the middle layer. The shape of the obtained microplastics differed from the initial fibrous structure of the intact medical mask layers, which indicates that the material is deformed during cryo-milling. The chemical compositions of plastics-associated chemicals also varied between the different layers. Typically, the inner layer contained more chemicals related to antimicrobial function and flavouring. The other two layers also contained antioxidants and their degradation products, plasticisers, cross-linking agents, antistatic agents, lubricants, and non-ionic surfactants. An acute study with D. magna showed that these microplastics do not cause immobility but do physically interact with the daphnids. Further long-term studies with these microplastics are needed using a suite of test organisms. Indeed, studies with other polypropylene microplastics have shown numerous adverse effects on other organisms at concentrations that have already been reported in the environment. Further efforts should be made to investigate the environmental hazards of polypropylene microplastics from medical masks and how to handle this new source of environmental burden. PLEASE CHECK THE SI WORD DOCUMENT THE AUTHORS ARE NOT LISTED THERE I CANNOT EDIT THAT FILE PLEASE ADD THE AUTHORS SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s43591-021-00020-0.

Time-dependent immune response in Porcellio scaber following exposure to microplastics and natural particles.

Microplastics are very common contaminants in the environment. Despite increasing efforts to assess the effects of microplastics on soil organisms, there remains a lack of knowledge on how organisms respond to diverse types of microplastics after different exposure durations. In the present study, we investigated the immune response of the terrestrial crustacean Porcellio scaber exposed to the two most common microplastic particles in the environment: polyester fibres and tyre particles. We also tested two natural particles: wood dust and silica powder, with all treatments performed at 1.5% w/w. The response of P. scaber was evaluated at the level of the immune system, and also the biochemical, organism and population level, after different exposure durations (1, 2, 4, 7, 14, 21 days). These data reveal dynamic changes in the levels of some immune parameters shortly after exposure, with a gradual return to control values. The total number of haemocytes was significantly decreased after 4 days of exposure to tyre particles, while the proportion of different haemocyte types in the haemolymph was altered shortly after exposure to both polyester fibres and tyre particles. Moreover, 7 days of exposure to tyre particles resulted in increased superoxide dismutase activity in the haemolymph, while metabolic activity in whole woodlice (measured as electron transport system activity) was increased after exposure for 7, 14 and 21 days. In contrast, the natural particles did not elicit any significant changes in the measured parameters. Survival and feeding of P. scaber were not altered by exposure to the microplastics and natural particles in soil. Overall, this study defines a time-dependent transient immune response of P. scaber, which indicates that immune parameters represent sensitive biomarkers of exposure to microplastics. We discuss the importance of using natural particles in studies of microplastics exposure and their effects.

Effects of Bioinsecticidal Aegerolysin-Based Cytolytic Complexes on Non-Target Organisms.

Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus Pleurotus bind strongly to an invertebrate-specific membrane sphingolipid, and together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. This pore formation is the basis for the selective insecticidal activity of aegerolysin/PlyB complexes against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm. In this study, we evaluated the toxicities of these aegerolysin/PlyB complexes using feeding tests with two ecologically important non-target arthropod species: the woodlouse and the honey bee. The mammalian toxicity of the EryA/PlyB complex was also evaluated after intravenous administration to mice. None of the aegerolysin/PlyB complexes were toxic against woodlice, but OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees, with 48 h mean lethal concentrations (LC50) of 0.22 and 0.39 mg/mL, respectively, in their food. EryA/PlyB was also tested intravenously in mice up to 3 mg/kg body mass, without showing toxicity. With no toxicity seen for EryA/PlyB for environmentally beneficial arthropods and mammals at the tested concentrations, these EryA/PlyB complexes are of particular interest for development of new bioinsecticides for control of selected coleopteran pests.

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology.

The scopes related to the interplay between stem cells and the immune system are broad and range from the basic understanding of organism's physiology and ecology to translational studies, further contributing to (eco)toxicology, biotechnology, and medicine as well as regulatory and ethical aspects. Stem cells originate immune cells through hematopoiesis, and the interplay between the two cell types is required in processes like regeneration. In addition, stem and immune cell anomalies directly affect the organism's functions, its ability to cope with environmental changes and, indirectly, its role in ecosystem services. However, stem cells and immune cells continue to be considered parts of two branches of biological research with few interconnections between them. This review aims to bridge these two seemingly disparate disciplines towards much more integrative and transformative approaches with examples deriving mainly from aquatic invertebrates. We discuss the current understanding of cross-disciplinary collaborative and emerging issues, raising novel hypotheses and comments. We also discuss the problems and perspectives of the two disciplines and how to integrate their conceptual frameworks to address basic equations in biology in a new, innovative way.

Exploring the impacts of microplastics and associated chemicals in the terrestrial environment - Exposure of soil invertebrates to tire particles.

Abrasion of tire wear is one of the largest sources of microplastics to the environment. Although most tire particles settle into soils, studies on their ecotoxicological impacts on the terrestrial environment are scarce. Here, the effects of tire particles (<180 µm) on three ecologically relevant soil invertebrate species, the enchytraeid worm Enchytraeus crypticus, the springtail Folsomia candida and the woodlouse Porcellio scaber, were studied. These species were exposed to tire particles spiked in soil or in food at concentrations of 0.02%, 0.06%, 0.17%, 0.5% and 1.5% (w/w). Tire particles contained a variety of potentially harmful substances. Zinc (21 900 mg kg-1) was the dominant trace element, whilst the highest concentrations of the measured organic compounds were detected for benzothiazole (89.2 mg kg-1), pyrene (4.85 mg kg-1), chlorpyrifos (0.351 mg kg-1), HCB (0.134 mg kg-1), methoxychlor (0.116 mg kg-1) and BDE 28 (0.100 mg kg-1). At the highest test concentration in soil (1.5%), the tire particles decreased F. candida reproduction by 38% and survival by 24%, and acetylcholinesterase (AChE) activity of P. scaber by 65%, whilst the slight decrease in the reproduction of E. crypticus was not dose-dependent. In food, the highest test concentration of tire particles reduced F. candida survival by 38%. These results suggest that micro-sized tire particles can affect soil invertebrates at concentrations found at roadsides, whilst short-term impacts at concentrations found further from the roadsides are unlikely.

Stressor-Dependant Changes in Immune Parameters in the Terrestrial Isopod Crustacean, Porcellio scaber: A Focus on Nanomaterials.

We compared the changes of selected immune parameters of Porcellio scaber to different stressors. The animals were either fed for two weeks with Au nanoparticles (NPs), CeO2 NPs, or Au ions or body-injected with Au NPs, CeO2 NPs, or lipopolysaccharide endotoxin. Contrary to expectations, the feeding experiment showed that both NPs caused a significant increase in the total haemocyte count (THC). In contrast, the ion-positive control resulted in a significantly decreased THC. Additionally, changes in phenoloxidase (PO)-like activity, haemocyte viability, and nitric oxide (NO) levels seemed to depend on the stressor. Injection experiments also showed stressor-dependant changes in measured parameters, such as CeO2 NPs and lipopolysaccharide endotoxin (LPS), caused more significant responses than Au NPs. These results show that feeding and injection of NPs caused an immune response and that the response differed significantly, depending on the exposure route. We did not expect the response to ingested NPs, due to the low exposure concentrations (100 µg/g dry weight food) and a firm gut epithelia, along with a lack of phagocytosis in the digestive system, which would theoretically prevent NPs from crossing the biological barrier. It remains a challenge for future research to reveal what the physiological and ecological significance is for the organism to sense and respond, via the immune system, to ingested foreign material.

Long Term Exposure to Virgin and Recycled LDPE Microplastics Induced Minor Effects in the Freshwater and Terrestrial Crustaceans Daphnia magna and Porcellio scaber.

The effects of microplastics (MP) are extensively studied, yet hazard data from long-term exposure studies are scarce. Moreover, for sustainable circular use in the future, knowledge on the biological impact of recycled plastics is essential. The aim of this study was to provide long-term toxicity data of virgin vs recycled (mechanical recycling) low density polyethylene (LDPE) for two commonly used ecotoxicity models, the freshwater crustacean Daphnia magna and the terrestrial crustacean Porcellio scaber. LDPE MP was tested as fragments of 39.8 ± 8.82 µm (virgin) and 205 ± 144 µm (recycled) at chronic exposure levels of 1-100 mg LDPE/L (D. magna) and 0.2-15 g LDPE/kg soil (P. scaber). Mortality, reproduction, body length, total lipid content, feeding and immune response were evaluated. With the exception of very low inconsistent offspring mortality at 10 mg/L and 100 mg/L of recycled LDPE, no MP exposure-related adverse effects were recorded for D. magna. For P. scaber, increased feeding on non-contaminated leaves was observed for virgin LDPE at 5 g/kg and 15 g/kg. In addition, both LDPE induced a slight immune response at 5 g/kg and 15 g/kg with more parameters altered for virgin LDPE. Our results indicated different sublethal responses upon exposure to recycled compared to virgin LDPE MP.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Microplastics, chlorpyrifos and their mixtures modulate immune processes in the terrestrial crustacean Porcellio scaber.

Microplastics and agrochemicals are common pollutants in terrestrial ecosystems. Their interaction during coexistence in soils may influence their fate and adverse effects on terrestrial organisms. The aim of this study was to investigate how the exposure to two types of microplastics; polyester fibres, and crumb rubber; induce changes in immune parameters of Porcellio scaber and if the co-exposure of microplastics affects the response induced by the organophosphate pesticide chlorpyrifos. A number of immune parameters, such as total haemocyte count, differential haemocyte count, and phenoloxidase-like activity were assessed. In addition, the acetylcholinesterase (AChE) activity in the haemolymph was evaluated as a measure of the bioavailability of chlorpyrifos. After three weeks of exposure, the most noticeable changes in the measured immune parameters and also a significantly reduced AChE activity were seen in chlorpyrifos-exposed animals. Both types of microplastic at environmentally relevant concentrations caused only slight changes in immune parameters which were not dependent on the type of microplastic, although the two types differed significantly in terms of the chemical complexity of the additives. Mixtures of chlorpyrifos and microplastics induced changes that differed from individual exposures. For example, alterations in some measured parameters suggested a reduced bioavailability of chlorpyrifos (AChE activity, haemocyte viability) caused by both types of microplastics exposure, but the increase of haemocyte count was promoted by the presence of fibres implying their joint action. In conclusion, this study suggests that immune processes in P. scaber are slightly changed upon exposure to both types of microplastics and microplastics can significantly modulate the effects of other co-exposed chemicals. Further research is needed on the short-term and long-term joint effects of microplastics and agrochemicals on the immunity of soil invertebrates.

Comparison of sublethal effects of natural acaricides carvacrol and thymol on honeybees.

The parasitic mite Varroa destructor is a threat to beekeeping colonies. Among naturally derived acaricides, the monoterpenoid essential oil compound thymol is used in beekeeping for varroa mite control, but adverse impacts on honeybees has been already documented. Carvacrol, another monoterpenoid, also has a high acaricidal potential and could thus be promising for regular use in beekeeping, but information is scarce regarding the effects of prolonged systemic administration of carvacrol on honeybees. In this study, we evaluate and compared the sublethal effects of long term consumption of carvacrol and thymol on Carnolian honeybee workers (Apis mellifera carnica). Survival and feeding rate were determined preliminary to assess sublethal concentrations. The sublethal effects were analysed by the activity of the acetylcholinesterase (AChE), enzyme involved in the control of neurotransmission, and the activity of detoxifying enzyme glutathione S-transferase (GST) in heads and thoraces. We found that, thymol and carvacrol, caused mortality only at the highest concentrations tested, 1% and 5% respectively. As demonstrated by others, both substances could be effective against varroa at concentrations ten times lower than those causing significant honeybee mortality. However, we demonstrated sublethal effects at the 0.05% carvacrol and thymol exposure concentrations evidenced as increased activity of AChE and GST in the honeybee heads. In conclusion, prolonged treatment with thymol and carvacrol affects bee nervous system and induce detoxification processes possibly resulting in a limited use for acaricidal purposes. We postulate that under the same chronic exposure conditions carvacrol and thymol will have similar sublethal effects on honeybees.