Terrestrial Isopod Porcellio Scaber Research Articles

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.

Microplastic ingestion affects hydrogen production and microbiomes in the gut of the terrestrial isopod Porcellio scaber.

Microplastic (MP) is an environmental burden and enters food webs via ingestion by macrofauna, including isopods (Porcellio scaber) in terrestrial ecosystems. Isopods represent ubiquitously abundant, ecologically important detritivores. However, MP-polymer specific effects on the host and its gut microbiota are unknown. We tested the hypothesis that biodegradable (polylactic acid [PLA]) and non-biodegradable (polyethylene terephthalate [PET]; polystyrene [PS]) MPs have contrasting effects on P. scaber mediated by changes of the gut microbiota. The isopod fitness after an 8-week MP-exposure was generally unaffected, although the isopods showed avoidance behaviour to PS-food. MP-polymer specific effects on gut microbes were detected, including a stimulation of microbial activity by PLA compared with MP-free controls. PLA stimulated hydrogen emission from isopod guts, while PET and PS were inhibitory. We roughly estimated 107 kg year-1 hydrogen emitted from the isopods globally and identified their guts as anoxic, significant mobile sources of reductant for soil microbes despite the absence of classical obligate anaerobes, likely due to Enterobacteriaceae-related fermentation activities that were stimulated by lactate generated during PLA-degradation. The findings suggest negative effects of PET and PS on gut fermentation, modulation of important isopod hydrogen emissions by MP pollution and the potential of MP to affect terrestrial food webs.

Avoidance behaviour toxicity tests should account for animal gregariousness: a case study on the terrestrial isopod Porcellioscaber.

Avoidance behaviour enables woodlice to escape suboptimal environmental conditions and to mitigate harmful effects of pollutants. However, several studies have shown that at least in some woodlice species the tendency to aggregate can lead to suboptimal responses as the between-conspecific attraction can outweigh the aversive stimuli. The present study evaluated the influence of gregariousness on the behaviour of Porcellioscaber in a heterogeneously polluted environment. The hypothesis was that the tendency for aggregation outweighs the tendency for exploratory activity, therefore animals in groups will be less active. Consequently, this will affect their avoidance of polluted environmental patches. To test this hypothesis, isolated individuals or pairs of individuals were monitored in free-choice arenas where animals could select between uncontaminated and pyrethrin-contaminated soils. Animals were video recorded for 3 h in darkness using infrared light and analysed for avoidance behaviour and locomotor activity. In general, isolated animals were more locomotory active and avoided the contaminated soil more than paired animals. It can be concluded that aggregation behaviour suppresses exploratory behaviour and consequently also the avoidance of polluted environments. This should be accounted for when interpreting results of avoidance tests with groups of gregarious animals, which may underestimate the effect of pollutants.

Invertebrate Species for the Bioavailability and Accumulation Assessment of Manufactured Polymer-Based Nano- and Microplastics.

Bioaccumulation tests with invertebrates have recently been discussed as a suitable alternative to bioaccumulation tests with metal- or metal oxide-based nanoparticles in fish for regulatory assessment. In the present study, as a first step, we investigated the suitability of three invertebrate species for bioaccumulation tests with nano- and microplastics. In a laboratory approach the freshwater bivalve Corbicula fluminea, the freshwater amphipod Hyalella azteca, and the terrestrial isopod Porcellio scaber were exposed to fluorescently labeled nano- and microplastics to evaluate their suitability to estimate the bioavailability and bioaccumulation of these test items. No bioaccumulation was observed in H. azteca or P. scaber. In contrast, the measurement of the relative fluorescence of the test items in the soft tissue and the feces of the filter-feeding bivalve allowed us to derive data that may be useful for the regulatory bioaccumulation assessment of manufactured nano- and microplastics. The developed measurement method using fluorescence represents a time-efficient and cost-effective analytical method for manufactured nano- and microplastics in laboratory studies for regulatory assessment. Environ Toxicol Chem 2022;41:961-974. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The epidermis cells of mandible teeth in the terrestrial isopod Porcellio scaber: Differentiations for mineralisation with calcium phosphate and carbonate

Generally, the mineralisation of the crustacean cuticle occurs when the cuticle has expanded after moulting. However, in the partes incisivae of Porcellio scaber, cuticle mineralisation with calcium phosphate already occurs before the moult. We investigated the ultrastructure and distribution of organelles within the epidermis cells and searched for calcium-containing organelles using EDX and EFTEM analysis. We found two different cell types. Calcium carbonate-secreting C-cells, which resemble the epithelial cells of the general integument, and the P-cells, which, as an unusual feature, have cell extensions up to 400 μm long. During secretion of the partes incisivae, these extensions end at the unmineralised tip and the phosphate-containing middle region. Their cell bodies contain most of the mitochondria located in basal folds and a high amount of endoplasmic reticulum. The cell extensions contain many microtubules, endoplasmic reticulum, large and small vesicles and densely stained rod-shaped cisternae. The rod-shaped cisternae and the endoplasmic reticulum contain calcium. During cuticle mineralisation, vesicles, which probably belong to the endo-lysosomal system, contain calcium and phosphorus. They occur at some distance and close to the cuticle. The mineral in these vesicles has a similar composition to that within the cuticle, suggesting that they play a role in cuticle mineralisation.

Effective Techniques for the Care, Reproduction & Utilization of the Terrestrial Isopod Porcellio scaber in Your Science Classroom

Terrestrial isopods (often referred to as sow bugs or pill bugs) are small, land-dwelling crustaceans. Because of their gentle nature, terrestrial isopods are excellent animals to have in a science classroom. There are many fun and effective classroom activities that utilize terrestrial isopods to teach science concepts, but there are multiple barriers to acquiring the large number of terrestrial isopods needed for classroom activities. Furthermore, when terrestrial isopods are brought into the classroom, the possibility exists that they will die inhumanely because finding good information on their proper care and reproduction can be difficult. Websites, books, and magazines frequently provide contradictory, inaccurate, incomplete, overly complex, and inhumane care information. To address these issues, this article explains how to set up a simple, low-maintenance, and inexpensive classroom enclosure for the terrestrial isopod species Porcellio scaber. These techniques, which I use in my classroom, produce large numbers of healthy captive-bred P. scaber. This article also presents examples of effective, hands-on classroom activities utilizing P. scaber.

Vanishing benefits - The loss of actinobacterial symbionts at elevated temperatures.

Only a few insect species are known to engage in symbiotic associations with antibiotic-producing Actinobacteria and profit from this kind of protection against pathogens. However, it still remains elusive how widespread the symbiotic interactions with Actinobacteria in other organisms are and how these partnerships benefit the hosts in terms of the growth and survival. We characterized a drastic temperature-induced change in the occurrence of Actinobacteria in the gut of the terrestrial isopod Porcellio scaber reared under two different temperature (15 °C and 22 °C) and oxygen conditions (10% and 22% O2) using 16S rRNA gene sequencing. We show that the relative abundance of actinobacterial gut symbionts correlates with increased host growth at lower temperature. Actinobacterial symbionts were almost completely absent at 22 °C under both high and low oxygen conditions. In addition, we identified members of nearly half of the known actinobacterial families in the isopod microbiome, and most of these include members that are known to produce antibiotics. Our study suggests that hosting diverse actinobacterial symbionts may provide conditions favorable for host growth. These findings show how a temperature-driven decline in microbiome diversity may cause a loss of beneficial functions with negative effects on ectotherms.

Silver nanoparticles in sewage sludge: Bioavailability of sulfidized silver to the terrestrial isopod Porcellio scaber

Silver nanoparticles (AgNPs) are efficiently converted during the wastewater-treatment process into sparingly soluble Ag sulfides (Ag2 S). In several countries, sewage sludge is used as a fertilizer in agriculture. The bioavailability of sulfidized Ag to the terrestrial isopod Porcellio scaber was investigated. Sewage sludge containing transformed AgNPs was obtained from a laboratory-scale sewage-treatment plant operated according to Organisation for Economic Co-operation and Development (OECD) guideline 303a. The results of transmission electron microscopy with energy dispersive X-ray of sludge samples suggest that AgNPs were completely transformed to Ag2 S. Adult isopods were exposed to OECD 207 soil substrate amended with the AgNP spiked sludge for 14 d (uptake phase) followed by an elimination phase in unspiked soil of equal duration. Most of the Ag measured in P. scaber at the end of the uptake phase was found in the hindgut (71%), indicating that only a minor part of the estimated Ag content was actually assimilated by the isopods with 16.3 and 12.7% found in the carcass and hepatopancreas, respectively. As a result of this, the Ag content of the animals dropped following transition to unspiked sludge within 2 d to one-third of the previously measured Ag concentration and remained stable at this level until the end of the elimination period. The present study shows that Ag2 S in sewage sludge is bioavailable to the terrestrial isopod P. scaber. Environ Toxicol Chem 2018;37:1606-1613. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

In field conditions, commercial pigment grade TiO2 was not harmful to terrestrial isopods but reduced leaf litter fragmentation

We investigated the effects of a commercial pigment grade rutile TiO2 on the terrestrial isopod Porcellio scaber in three locations that differed in terms of abiotic and biotic conditions: the laboratory, open air, and the closed barn. Mortality and isopod energy reserves (digestive gland total proteins, lipids and carbohydrates) were not affected following 14days exposure to up to 1000mg TiO2 per kg dry leaves (mg/kg) under any experimental scenario. However, in the field tests, isopods consumption of TiO2-coated leaves was reduced compared to that of uncoated leaves and the decrease was not dose-dependent. The highest reduction was in the closed barn (45–56%) rather than in the open-air (38–40%). In laboratory-based food choice tests, isopods neither preferred nor avoided leaves coated with TiO2, suggesting that rather than sensing the TiO2 on the leaves directly, the isopods under open-air and barn exposure responded to altered attractiveness and/or palatability of the TiO2 amended leaves. We propose that this could be due to altered microbial population on the leaves, a hypothesis that requires further investigation. Although short-term exposure to atmospheric deposition of up to 1000mg/kg commercial TiO2 is unlikely to pose an immediate threat to isopod mortality and energy balance, reduced leaf feeding may have implications for the decomposition of plant material.

Bioaccumulation of hexachlorobenzene in the terrestrial isopod Porcellio scaber.

A test system to investigate the biomagnification of organic chemicals in the terrestrial isopod Porcellio scaber was developed and validated. Adult isopods were fed on alder leaf powder (Alnus glutinosa) spiked with [14 C]hexachlorobenzene (HCB). Test animals, sampled regularly during the uptake (16 d) and depuration phases (16 d), were analyzed, and the kinetics of tissue concentrations were determined. Uptake (k1 ) and depuration rates (k2 ) were calculated to estimate kinetic biomagnification factors (BMFs). In addition, the effect of coprophagy on the uptake and accumulation of HCB as well as the tissue distribution of HCB in P. scaber was investigated. The test system was shown to be suitable for investigations into the terrestrial bioaccumulation of chemicals. Coprophagy had no effect on the bioaccumulation of HCB in P. scaber. The hepatopancreas was identified as the main target tissue for HCB accumulation. The low BMF of 0.057 resulted from an assimilation efficiency (α) of 31.42%, a low uptake rate k1 (0.009 d-1 ), and a high depuration rate k2 (0.164 d-1 ). The results indicate that the terrestrial bioaccumulation of organic chemicals in P. scaber might not represent a worst-case scenario for biomagnification, limiting the value of the test system for the regulatory assessment of organic chemicals. Environ Toxicol Chem 2016;35:2867-2873. © 2016 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

Gold nanoparticles do not induce adverse effects on terrestrial isopods Porcellio scaber after 14-day exposure

Despite the anticipated environmental release of anthropogenic gold nanoparticles (AuNPs), there is currently not enough data on their potential impact on terrestrial environment. In the current study, we investigated the effects of considerably low concentrations of AuNPs on terrestrial isopods (Porcellio scaber) after 14 days of exposure. The effects on mortality, weight change, feeding rate, avoidance/preference feeding behavior, and cell membrane destabilization of digestive gland cells were followed. In parallel, the accumulation of Au in the digestive glands was measured. Our results show that none of the tested parameters was affected in isopods under given exposure doses (10 and 60 µg Au/g dry leaf) and exposure duration. No Au was assimilated in the digestive glands. Also, the same doses of the reference chemical, AuCl3, showed no effect. We conclude that these concentrations of AuNPs are safe for terrestrial isopods. We encourage reporting the results showing no adverse effects of nanoparticles to balance the prevailing publication of their adverse effects. This will help to build a realistic public perception of the environmental risk of nanomaterials.

Effects of food salinization on terrestrial crustaceans Porcellio scaber

Salinization of soil as the main cause for soil degradation is a global issue. Despite this, the data concerning the effects of different salts on terrestrial organisms are still missing. Our aim was to address the effects of four different salts (NaCl, KCl, NaNO3, and KNO3) on terrestrial isopods Porcellio scaber. Two types of experiments were performed. In the first experiment, the isopods were exposed to 1, 2, and 5g salt/kg dry leaf for 14 days and afterwards animal mortality, feeding activity, moult and growth were monitored. In the second experiment, a 48-h soil selection tests was done to investigate their preference/avoidance to certain salts. The feeding rate and growth efficiency of isopods decreased in a dose-dependent manner after 14 days exposure to KNO3 and NaNO3. We suggest that nitrate (NO3−) may be the predominant reason for these effects, because (i) the effects were only observed in the case of NO3−-salts, and (ii) K+ and Na+ induced no effects in the case of Cl−-salts where the concentrations of these two cations were even higher as in the case of NO3− salts. 48-h soil selection test showed that isopods were able to choose between NaCl dosed and unamended soil: the isopods preferred to spend time on NaCl spiked soil (up to 5g salt/kg dry soil). In the case of KNO3 and NaNO3 no significant soil selection by isopods was found. In these two exposures isopods spent approximately 50% of their visits on both sides of the soil. The survival of isopods decreased in a dose independent manner when exposed to NaNO3, KNO3, and KCl. We conclude that NaNO3, KCl, and KNO3 affect the isopods, while no effects were caused by NaCl under the exposure conditions of this study. This study implies that also other salts besides commonly investigated NaCl should be tested when the potential hazard of soil salinization to terrestrial organisms is in question.

Draft Genome Sequence of "Candidatus Hepatoplasma crinochetorum" Ps, a Bacterial Symbiont in the Hepatopancreas of the Terrestrial Isopod Porcellio scaber.

“Candidatus Hepatoplasma crinochetorum” Ps is an extracellular symbiont residing in the hepatopancreas of the terrestrial isopod Porcellio scaber. Its genome is highly similar to that of the close relative “Ca. Hepatoplasma crinochetorum” Av from Armadillidium vulgare. However, instead of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system, it encodes a type I restriction modification system.

Does temperature and oxygen affect duration of intramarsupial development and juvenile growth in the terrestrial isopod Porcellioscaber (Crustacea, Malacostraca)?

According to the temperature-size rule (TSR), ectotherms developing under cold conditions experience slower growth as juveniles but reach a larger size at maturity. Whether temperature alone causes this phenomenon is unknown, but oxygen limitation can play a role in the temperature-size relationship. Oxygen may become limited under warm conditions when the resulting higher metabolism creates a greater demand for oxygen, especially in larger individuals. We examined the independent effects of oxygen concentration (10% and 22% O2) and temperature (15 °C and 22 °C) on duration of ontogenic development, which takes place within the maternal brood pouch (marsupium), and juvenile growth in the terrestrial isopod common rough woodlouse (Porcellio scaber). Individuals inside the marsupium undergo the change from the aqueous to the gaseous environment. Under hypoxia, woodlice hatched from the marsupium sooner, but their subsequent growth was not affected by the level of oxygen. Marsupial development and juvenile growth were almost three times slower at low temperature, and marsupial development was longer in larger females but only in the cold treatment. These results show that temperature and oxygen are important ecological factors affecting developmental time and that the strength of the effect likely depends on the availability of oxygen in the environment.

Bioavailability of cobalt and iron from citric-acid-adsorbed CoFe2O4 nanoparticles in the terrestrial isopod Porcellio scaber

The aim of this study was to determine whether citric acid adsorbed onto cobalt ferrite (CoFe2O4) nanoparticles (NPs) influences the bioavailability of their constituents Co and Fe. Dissolution of Co and Fe was assessed by two measures: (i) in aqueous suspension using chemical analysis, prior to application onto the food of test organisms; and (ii) in vivo, measuring the bioavailability in the model terrestrial invertebrate (Porcellio scaber, Isopoda, Crustacea). The isopods were exposed to citric-acid-adsorbed CoFe2O4 NPs for 2weeks, and tissue accumulation of Co and Fe was assessed. This was compared to pristine CoFe2O4 NPs, and CoCl2 and Fe(III) salts as positive controls. The combined data shows that citric acid enhances free metal ion concentration from CoFe2O4 NPs in aqueous suspension, although in vivo, very similar amounts of assimilated Co were found in isopods exposed to both types of NPs. Therefore, evaluation of the dissolution in suspension by chemical means is not a good predictor of metal assimilation of this model organism; body assimilation of Co and Fe is rather governed by the physiological capacity of P. scaber for the uptake of these metals. Moreover, we propose that citric acid, due to its chelating properties, may hinder the uptake of Co that dissolves from citric-acid-adsorbed CoFe2O4 NPs, if citric acid is present in sufficient quantity.

Altered physiological conditions of the terrestrial isopod Porcellio scaber as a measure of subchronic TiO2 effects.

Titanium dioxide nanoparticles (nano-TiO2) show low toxic potential against a variety of environmental organisms when measured by conventional toxicity endpoints. However, the question is whether the conventional measures of toxicity can define the adverse effects of nanoparticles. The aim of this study was to asses the potential toxic and cytotoxic effects of the ingested nano-TiO2 (anatase, <25 nm) on a terrestrial isopod, Porcellio scaber. In addition to conventional toxicity parameters, the physiological condition of the animals was assessed. Following 28-day feeding exposure to nano-TiO2 at concentrations up to 5,000 μg nano-TiO2/g leaf dry weight, no toxic or cytotoxic effects were demonstrated. However, the physiological condition of the animals was affected in a dose-dependent manner. The physiological state of organisms is an important parameter to assess the potential population implications due to the exposure to nanomaterials. Therefore, we suggest that only if both, the physiological state of the animals exposed to nano-TiO2 and the conventional toxicity markers show no effects, the exposure dose can be interpreted as non-hazardous.

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda)

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.

Can terrestrial isopods (Isopoda: Oniscidea) make use of biodegradable plastics?

Biodegradable plastics more and more replace conventional plastics, because they are considered environmentally friendly. Soil macro-invertebrates have been demonstrated to consume some of these biodegradable plastics, but studies usually do not go beyond notice of consumption and possible short-term ecotoxicological effects on organisms. This study uses the terrestrial isopod Porcellio scaber as a soil detritivore model and three biodegradable plastics (starch-, cellulose- and poly(3-hydroxybutyrate) (PHB)-based films) to evaluate both the contribution of isopods to the disintegration of biodegradable plastics and the effects of plastic-feeding on isopod ecology. Consumption rate of starch-based plastic was similar to that of leaf litter (mainly beech) and on average higher than those of the other two plastic types. Digestibility, however, was highest for cellulose-based plastic. HPLC results show that isopods break down starch-based plastic into maltose and glucose, and cellulose-based plastic into cellobiose. No glucose was present in feces of isopods having fed cellulose-based plastic, either for inability of breaking down cellobiose into glucose, or due to a rapid uptake of the glucose by isopods. Growth rates were negative, but not significantly different from zero, for all food sources; cellulose-based plastic caused the highest biomass loss to isopods. Toughness of starch-based plastic diminished over time when litter and/or isopods were present. Cellulose-based plastic increased in toughness over the disintegration experiment, possibly affecting its consumption by isopods. Overall, isopods increased the disintegration rates of starch- and cellulose-based plastics, but no PHB film was consumed, and its disintegration rate was low. We conclude that starch-based plastic is comparable to a natural low-quality food source (e.g., beech litter), and isopods would probably consume starch- and cellulose-based plastics in the field.

Time-related survival effects of two gluconasturtiin hydrolysis products on the terrestrial isopod Porcellio scaber

Glucosinolates are compounds produced by commercial crops which can hydrolyse in a range of natural toxins that may exert detrimental effects on beneficial soil organisms. This study examined the effects of 2-phenylethyl isothiocyanate and 3-phenylpropionitrile on the survival and growth of the woodlouse Porcellio scaber exposed for 28d. 2-Phenylethyl isothiocyanate dissipated from the soil with half-lives ranging from 19 to 96h. Exposure through soil showed toxic effects only on survival. The LC50s after 28d were significantly different at 65.3mgkg−1 for 2-phenylethyl isothiocyanate and 155mgkg−1 for 3-phenylpropionitrile. A toxicokinetic–toxicodynamic (TKTD) approach, however, revealed that both compounds in fact have very similar effect patterns. The TKTD model was better suited to interpret the survival data than descriptive dose–response analysis (LCx), accounting for the fast dissipation of the compounds in the soil. Found effects were within environmentally relevant concentrations. Care should therefore be taken before allowing these natural toxins to enter soil ecosystems in large quantities.

The link between antioxidant enzymes catalase and glutathione S-transferase and physiological condition of a control population of terrestrial isopod (Porcellio scaber)

The aim of this work was to investigate if the activities of catalase and glutathione S-transferase in a control population of terrestrial isopods (Porcellio scaber) are correlated with the physiological condition of the isopods. For this purpose, the activities of these enzymes were analysed in isopods from a stock population and in parallel, the physiological condition of the same specimens was assessed using a histological approach based on epithelial thickness and lipid droplets. We found a correlation between antioxidant enzymes and the physiological condition of the isopods. This implies that these enzymes could be used as predictive indicators of the physiological condition in a stock population before comprehensive toxicological studies are conducted and also in control group after the experiment. When a control group is found to be very heterogeneous in terms of physiological condition, the experiment should be repeated with a larger number of experimental animals. The findings of this study will contribute to more accurate experimental design of toxicity tests when using biomarkers. This should encourage other researchers to increase their effort to know the physiological state of their test organisms.