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.

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.

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.

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.

Microplastic fibers influence Ag toxicity and bioaccumulation in Eisenia andrei but not in Enchytraeus crypticus.

Microplastic fibers (MF) are released from synthetic textiles during washing and end up in the wastewater. Similarly, silver nanoparticles (AgNP), incorporated in textiles as antimicrobial agents, are released in washing machines, also reaching the wastewater treatment plants. Therefore, both MF and AgNP co-exist in the environment and enter the soil compartment mainly via the application of biosolids. Yet, the combined effect of MF and AgNP has not been studied. Here, we assessed the effects of polyester MF on the toxicity of AgNP and AgNO3 to the earthworm Eisenia andrei and the enchytraeid Enchytraeus crypticus. The organisms were exposed to a range of concentration of AgNP (32, 100, 320, 1000, 3200 mg Ag/kg) and AgNO3 (12.8, 32, 80, 200, 500 mg Ag/kg) in LUFA 2.2 soil in the absence or presence of MF (0.01% DW). Reproduction tests were conducted and the toxicity outcomes compared between soils with and without MF. The exposure to MF caused a decrease in the number of juveniles and changed the biochemical composition of earthworms. Moreover, the presence of MF increased the toxicity of AgNP to earthworm reproduction (EC50 = 165 mg Ag/kg) when compared to AgNP exposure alone (EC50 = 450 mg Ag/kg), but did not alter the toxicity of AgNO3 (EC50 = 40 mg Ag/kg). For enchytraeids, no significant difference in Ag toxicity could be detected when MF was added to the soil for both AgNP and AgNO3. Overall, Ag bioaccumulation was not affected by MF, except for a decrease in earthworm body concentration at the highest Ag soil concentration (3200 mg Ag/kg). Our results suggest that the presence of MF in the soil compartment may be a cause of concern, and that the joint exposure to Ag may be deleterious depending on the Ag form, organism, and endpoint. The present work provides the first evidence that a realistic MF concentration in soil lowers AgNP concentration necessary to provoke reproductive impairment in earthworms. The influence of MF on the risk assessment of AgNP should be considered.

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.

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.

Modulations of immune parameters caused by bacterial and viral infections in the terrestrial crustacean Porcellio scaber: Implications for potential markers in environmental research.

The terrestrial crustacean Porcellio scaber (Crustacea: Isopoda) is an established invertebrate model in environmental research. Preceding research using isopods did not widely use immune markers. In order to advance their use in research, knowledge of the reference values in control animals as well as variations during infections is of importance. This study presents, for the first time, the morphology, and ultrastructure of the three main haemocyte types of Porcellio scaber as semigranulocytes (SGCs), granulocytes (GCs), and hyalinocytes (HCs), with the latter having two subtypes, using various light and electron microscopy approaches. The modulation of selected immune cellular and humoral parameters of P. scaber in symptomatic phases of Rhabdochlamydia porcellionis and Iridovirus IIV-31 infections is presented. A clear difference in the immune responses of bacterial and viral infections was shown. Remarkable changes in total haemocyte count (THC) values and the proportions of three different haemocyte types were found in animals with a viral infection, which were not as significant in bacterially infected animals. Modified NO levels and SOD activity were more pronounced in cases of bacterial infection. Knowledge of the morphological and ultrastructural features of distinct haemocyte types, understanding the baseline values of immune parameters in control animals without evident symptoms of infection, and the influence that infections can have on these parameters can serve as a basis for the further use of P. scaber immune markers in environmental research.

Exploring the impacts of plastics in soil - The effects of polyester textile fibers on soil invertebrates.

Polyester fiber is one of the most abundant types of microplastics in the environment. A major proportion of the fibers entering wastewater treatment plants end up in sewage sludge, which is used as a soil fertilizer in many countries. As their impacts in the terrestrial environment are still poorly understood, we studied the effects of polyester fibers on enchytraeids (Enchytraeus crypticus), springtails (Folsomia candida), isopods (Porcellio scaber) and oribatid mites (Oppia nitens), all playing an important role in soil decomposer food webs. We exposed these invertebrates in the laboratory to short (12 µm-2.87 mm) and long (4-24 mm) polyester fibers, spiked in soil or in food at five concentrations ranging from 0.02% to 1.5% (w/w) and using five replicates. Overall the effects of polyester fibers on the soil invertebrates were slight. Energy reserves of the isopods were slightly affected by both fiber types, and enchytraeid reproduction decreased up to 30% with increasing fiber concentration, but only for long fibers in soil. The low ingestion of long fibers by the enchytraeids suggests that this negative impact arose from a physical harm outside the organism, or from indirect effects resulting from changes in environmental conditions. The short fibers were clearly ingested by enchytraeids and isopods, with the rate of ingestion positively related to fiber concentration in the soil. This study shows that polyester fibers are not very harmful to soil invertebrates upon short-term exposure. However, longer lasting, multigeneration studies with functional endpoints are needed to reveal the possible long-term effects on soil invertebrates and their role in the decomposition process. This study also shows that polyester fibers can enter terrestrial food web via ingestion of fibers by soil invertebrates.