Effect of glyphosate, its metabolite AMPA, and the glyphosate formulation Roundup® on brown trout (Salmo trutta f. fario) gut microbiome diversity.

Glyphosate is used worldwide as a compound of pesticides and is detectable in many environmental compartments. It enters water bodies primarily through drift from agricultural areas so that aquatic organisms are exposed to this chemical, especially after rain events. Glyphosate is advertised and sold as a highly specific herbicide, which interacts with the EPSP synthase, an enzyme of the shikimate metabolism, resulting in inhibition of the synthesis of vital aromatic amino acids. However, not only plants but also bacteria can possess this enzyme so that influences of glyphosate on the microbiomes of exposed organisms cannot be excluded. Those influences may result in subtle and long-term effects, e.g., disturbance of the symbiotic interactions of bionts with microorganisms of their microbiomes. Mechanisms how the transformation product aminomethylphosphonic acid (AMPA) of glyphosate might interfere in this context have not understood so far. In the present study, molecular biological fingerprinting methods showed concentration-dependent effects of glyphosate and AMPA on fish microbiomes. In addition, age-dependent differences in the composition of the microbiomes regarding abundance and diversity were detected. Furthermore, the effect of exposure to glyphosate and AMPA was investigated for several fish pathogens of gut microbiomes in terms of their gene expression of virulence factors associated with pathogenicity. In vitro transcriptome analysis with the fish pathogen Yersinia ruckeri revealed that it is questionable whether the observed effect on the microbiome is caused by the intended mode of action of glyphosate, such as the inhibition of EPSP synthase activity.

Endocrine effects of contaminated sediments on the freshwater snail Potamopyrgus antipodarum in vivo and in the cell bioassays in vitro.

Lake Pilnok located in the black coal-mining region Ostrava-Karvina, Czech Republic, contains sediments highly contaminated with powdered waste coal. Moreover, population of the endangered species of narrow-clawed crayfish Pontastacus leptodactylus with high proportion of intersex individuals (18%) was observed at this site. These findings motivated our work that aimed to evaluate contamination, endocrine disruptive potency using in vitro assays and in vivo effects of contaminated sediments on reproduction of sediment-dwelling invertebrates. Chemical analyses revealed low concentrations of persistent chlorinated compounds and heavy metals but concentrations of polycyclic aromatic hydrocarbons (PAH) were high (sum of 16 PAHs 10 microg/g dw). Organic extracts from sediments caused significant in vitro AhR-mediated activity in the bioassay with H4IIE-luc cells, estrogenicity in MVLN cells and anti-androgenicity in recombinant yeast assay, and these effects could be attributed to non-persistent compounds derived from the waste coal. We have also observed significant in vivo effects of the sediments in laboratory experiments with the Prosobranchian euryhaline mud snail Potamopyrgus antipodarum. Sediments from Lake Pilnok as well as organic extracts of the sediments (externally added to the control sediment) significantly affected fecundity during 8 weeks of exposure. The effects were stimulations of fecundity at lower concentrations at the beginning of the experiment followed by inhibitions of fecundity and general toxicity. Our study indicates presence of chemicals that affected endocrine balance in invertebrates, and emphasizes the need for integrated approaches combining in vitro and in vivo bioassays with identification of chemicals to elucidate ecotoxicogical impacts of contaminated sediment samples.

Effects of nickel chloride and oxygen depletion on behaviour and vitality of zebrafish (Danio rerio, Hamilton, 1822) (Pisces, Cypriniformes) embryos and larvae.

We examined acute (2 h exposure of 5-day-old larvae) and subchronic (exposure from fertilization up to an age of 11 days) effects of NiCl(2).6H2O on embryos and larvae of zebrafish (Danio rerio), both alone and in combination with oxygen depletion. The following endpoints were recorded: acute exposure: locomotory activity and survival; subchronic exposure: hatching rate, deformations, locomotory activity (at 5, 8 and 11 days) and mortality. In acute exposures nickel chloride (7.5-15 mg Ni/L) caused decreasing locomotory activity. Oxygen depletion (or=10 mg Ni/L resulted in delayed hatching at an age of 96 h, in decreased locomotory activity at an age of 5 days, and increased mortality at an age of 11 days (LC20=9.5 mg Ni/L). The observed LOEC for locomotory activity (7.5 mg Ni/L) is in the range of environmentally relevant concentrations. Since locomotory activity was already affected by acute exposure, this parameter is recommended to supplement commonly recorded endpoints of toxicity.

An invertebrate embryo test with the apple snail Marisa cornuarietis to assess effects of potential developmental and endocrine disruptors.

A novel invertebrate embryo test with the apple snail, Marisa cornuarietis, comprising a test protocol for the following developmental endpoints is described: formation of eyes and tentacles, heart rate, hatching, weight after hatching. To evaluate effects on embryonic development, the snails were treated in a first step with 250 or 500 microg/l cadmium. Sublethal effects in terms of a significant delay in hatching could be found in the 250 microg/l treated animals, whereas 500 microg/l Cd were lethal for the snail embryos. To test endocrine disrupting chemicals with this protocol, experiments with bisphenol A (50 microg/l, 100 microg/l) and 17alpha-ethinylestradiol (10 microg/l) were performed. In both treatments an increase of weight after hatching was observed as well as a significant decline in the heart rate of the embryos. As shown here, the sensitivity of M. cornuarietis embryos test is equal or even higher than other test species like zebrafish embryos and, therefore, this test can be regarded as an alternative or supplement for ecotoxicological studies.

Assessing contamination levels of Laguna Lake sediments (Philippines) using a contact assay with zebrafish (Danio rerio) embryos.

The present study investigated the suitability of a sediment contact assay using zebrafish (Danio rerio) embryos to evaluate the degree of lake sediment contamination. As endpoints, developmental parameters (mortality, abnormality, heart rate, and hatching rate) as well as stress protein responses (hsp 70 levels) in the developing embryos were recorded during a 96-h exposure. Fertilized zebrafish eggs were exposed to both the whole as well as organic extract concentrations prepared from collected sediments from 5 sites along Laguna Lake, Philippines. Compared to whole sediment exposure, more severe embryotoxic and teratogenic responses were elicited in embryos exposed to organic extracts. However, since whole sediment-exposed embryos also revealed significant developmental defects, this exposure phase served as the more realistic exposure scenario in our study. Weak to strong upregulation of hsp 70 levels was also registered among embryos exposed to both whole sediments and organic extracts. The observed embryotoxic and proteotoxic responses by zebrafish embryos to Laguna Lake sediment exposures were discussed in relation to the analyzed contaminants in the sediments (heavy metals, PAHs (perylene), solvent). Overall, the present study points out that the sediment contact assay with zebrafish embryos offers a practicable and highly sensitive bioassay for the general assessment of sediment toxicity.

Hsp70 and lipid peroxide levels following heat stress in Xeropicta derbentina (Krynicki 1836) (Gastropoda, Pulmonata) with regard to different colour morphs.

Terrestrial snails which live under dry and hot conditions need efficient mechanisms of adaptation to counteract the problems of desiccation and over-heating. A profoundly heat tolerant snail species is the Mediterranean Xeropicta derbentina, exhibiting different shell colour morphs ranging from pale white to darkly banded. Considering that dark-pigmented snails are believed to have a disadvantage due to faster heating, we investigated possible differences in the stress markers Hsp70 and lipid peroxideation between four pre-defined colour morphs which were exposed to different temperatures for eight hours. The highest Hsp70 levels were observed in response to 38-40 °C. Levels decreased when this temperature was exceeded. Snails of a pre-defined colour category 3 (with a large black band at the umbilicus side of the shell) showed the most prominent Hsp70 response. Lipid peroxideation levels also showed a maximum at 38 °C but displayed a second peak at rather high temperatures at which the Hsp70 level already had decreased (45-48 °C). Particularly pure white snails (category 1) and the most pigmented ones (category 4) were found to have different levels of lipid peroxidation at 38 °C and 45 °C compared to the other morphs. A hypothesis involving a combined two-phase defence mechanism, to which both, the Hsp70 protection system and the antioxidant defence system, may contribute, is discussed.

Biomarkers in terrestrial invertebrates for ecotoxicological soil risk assessment.

This review has served to present the most recent information on a selected series of biomarker studies undertaken on soil invertebrates during two extensive European-funded scientific consortia, BIOPRINT and BIOPRINT-II. The goals were to develop and validate methods for the analysis of markers of stress in a range of soil-dwelling organisms. We have discussed the potential and limitations of the following invertebrate biomarkers for soil risk assessment purposes: heat shock proteins, histological and ultrastructural markers, metallothioneins and metal-binding proteins, esterases, lysosomal integrity, and the novel biomarker histidine. The hsp response in soil invertebrates is especially suitable to indicate the effects of exposure to comparatively low concentrations for a range of toxicants and can be regarded as a biomarker of general stress. The application of MTs and other metal-binding proteins as biomarkers for exposure in soil invertebrates has been well described, and new methods are being developed for analyzing MT induction both at the protein and molecular level, and reliable and reproducible methods are now available. (Cd)-MT is well characterized for the springtails and its MT concentration is a useful biomarker for exposure as well as for effect. For snails, (Cd)-MT can accumulate in the midgut gland over extended periods of time and therefore its concentration is a biomarker not only for recent intoxication but also for events of cadmium exposure that snails may have experienced a long time before the measurement took place. Cellular and histological alterations can be regarded as reflecting the "health" state of a cell, which may be a measure for the presence of toxicants. Histopathological work on terrestrial invertebrates, however, is still scarce. Isozymes have been poorly studied in soil invertebrates despite their promising role as potential biomarkers in aquatic organisms. Among the large diversity of isozymes, the most well studied are esterases that are frequently used a biomarkers of exposure to various classes of pesticides. Many other isozymes offer potentials for biomarker research, such as glucosephosphate isomerase and phosphoglucomutase, both enzymes necessary for the glycolytic pathway. The lysosomal system has been identified as a particular target for the toxic effects of xenobiotics, although it has yet a limited application in soil invertebrates. This marker is nonspecific, responding equally sensitively to organic or inorganic contamination; however, if used in combination with an earthworm immnunocompetence assay such as total immunoactivity of the coelomocytes, then it is possible to be more specific as to the likely nature of contamination. Free histidine was positively correlated with increasing copper exposure and total copper burden in earthworms from a semifield study. Histidine may thus act as a biomarker of exposure. The transient responses and confounding factors of biomarkers obscure a proper interpretation of biomarker responses under field conditions. These factors are still very poorly understood and require more study. For risk assessment purposes it is recommended that the aforementioned biomarkers may show promise when included in a suite of biomarkers among different soil invertebrate species. It is recommended that a risk assessment protocol draw upon ranking of biomarker responses on a defined scale. It is also hoped that the problems outlined in this review will aid the direction of future research on soil invertebrate biomarkers.

Toxic effects of the non-steroidal anti-inflammatory drug diclofenac. Part II: cytological effects in liver, kidney, gills and intestine of rainbow trout (Oncorhynchus mykiss).

In the present study, cytopathology was investigated in the liver, kidney, gills and gut of rainbow trout (Oncorhynchus mykiss) exposed to five different concentrations (1, 5, 20, 100 and 500 microg/L) of the anti-inflammatory drug diclofenac under laboratory conditions. The lowest observed effect concentration (LOEC) for cytological alterations in liver, kidney and gills was 1 microg/L. In the gut, however, no diclofenac-induced cytopathology occurred. As the most prominent reactions induced by diclofenac (1) in the kidney, a severe accumulation of protein in the tubular cells (so called hyaline droplet degeneration), macrophage infiltration and structural alterations (dilation, vesiculation) of the endoplasmic reticulum (ER) in the proximal and distal renal tubules were observed. Furthermore, shortening of podocytes and their retraction from the basal lamina, a thickening of the basal lamina, the formation of desmosomes, and necrosis of endothelial cells in the renal corpuscles occurred; (2) in the liver, the most striking reactions were the collapse of the cellular compartmentation as well as the glycogen depletion of hepatocytes; (3) in the gills, pillar cell necrosis, hypertrophy of chloride cells, and epithelium lifting became evident in the secondary lamellae.

Lead impact on nutrition, energy reserves, respiration and stress protein (hsp 70) level in Porcellio scaber (Isopoda) populations differently preconditioned in their habitats.

The impact of lead on food consumption, energy metabolism and the stress protein (hsp 70) level was investigated in the woodlouse Porcellio scaber (Isopoda), a common representative of the saprophagous soil macrofauna. To examine possible acclimation or tolerance to lead in woodlice from a contaminated habitat, animals of two populations, one deriving from a lead-contaminated artillery range and one from an uncontaminated control stand, were exposed to a series of lead concentrations under otherwise constant laboratory conditions for a maximum of 80 days. The applied lead concentrations (at a maximum 7945 mg/kg food dry wt) did not have any significant quantitative effect on the food consumption of the isopods, although the population pre-exposed in the artillery range showed a tendency toward a higher food uptake than the control population. After 80 days of exposure, both populations showed an equal trend toward increasing their respiration as lead concentrations, that they had been fed on, were increased. Accordingly, the glycogen content of the body, in both populations, was elevated with increasing lead concentrations in the food. This effect was more pronounced in the pre-exposed isopod population than in the one from the control stand. The non-pre-exposed isopods showed a general tendency toward a lower protein content of their bodies than the pre-exposed ones, although no effect of the lead on this parameter could be statistically proven. The ability of the artillery range isopods to synthesise stress proteins (hsp 70) in response to lead contamination decreased at much lower lead concentrations in their food than in the non-pre-exposed control population, even though the artillery range isopods seemed to be equally or even slightly better equipped with energy storage products. Even though the better nutrient status of these animals might refer to some lead tolerance of the pre-exposed population, the stress protein data suggest that a metal-resistant Porcellio population did not evolve in this lead-contaminated site.

The impact of heavy metals on the grey garden slug, Deroceras reticulatum (Müller): Metal storage, cellular effects and semi-quantitative evaluation of metal toxicity.

Laboratory-reared grey garden slugs, Deroceras reticulatum, were exposed to soil and food treated with solutions of three metal salts (CdCl2, ZnCl2, PbCl2) in three environmentally relevant concentrations, each for 21 days. Metal concentrations were determined in the soil, food and slugs by atomic absorption spectrophotometry (AAS). Zinc was localized ultrastructurally in the hepatopancreatic cells by means of energy-filtering transmission electron microscopy (EFTEM). Zinc, and also high amounts of copper, could be detected by electron spectroscopical imaging (ESI) and electron energy loss spectroscopy (EELS) in spherites of the basophilic cells. Ultrastructural responses to metal impact were investigated in two cell types of the hepatopancreas (digestive and basophilic cells) and the cellular responses were found to be dose- and metal-dependent. In order to evaluate the toxicity of the respective metal concentrations to the slugs, the ultrastructural reactions were semi-quantified and summarized as complex reaction patterns of numerous organelles. This novel approach provides a basis for the use of data from standardized tests as a background for risk assessment studies in the field.

Cellular alterations in collembolan midgut cells as a marker of heavy metal exposure: ultrastructure and intracellular metal distribution.

The ultrastructure of the midgut epithelial cells of Tetrodontophora bielanensis (Collembola) fed either with lead-, cadmium- or zinc-enriched food or kept under control conditions was compared by transmission electron microscopy (TEM). Atomic absorption spectrophotometry (AAS) showed accumulation of these three metals in the body tissues of the collembolans. Intracellular localization of zinc in the midgut epithelial cells was investigated by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The presence of lead in the midgut tissue was shown by laser microprobe mass spectrometry (LAMMS). Under heavy metal conditions, the midgut cells showed different ultrastructural alterations, the degree of which was found to be dose-dependent. Independent of the type of the metal in the food, the endoplasmic reticulum appeared fenestrated or vesiculated, and often large vacuoles, which were shown to be ER-derived, occurred throughout the cells. Also the mitochondrial membranes were affected by heavy metal stress. In epithelial cells of individuals exposed to either lead or cadmium, an increase of myelin-like structures could be observed. In the case of exposure to zinc in the highest applied concentration, the cytoplasm showed condensation and portions of the microvillous border appeared destroyed. Additionally, an increase of mineral congregations (type-A spherites) could be observed under heavy metal influence. Intracellularly stored zinc could be localised at highly affected mitochondrial membranes, in the microvillous border, and in the heterochromatin.

Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO.

One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 microg l(-1)) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 microg l(-1). Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSO-triggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish.

Daily and seasonal changes in heat exposure and the Hsp70 level of individuals from a field population of Xeropicta derbentina (Krynicki 1836) (Pulmonata, Hygromiidae) in Southern France.

The Mediterranean land snail Xeropicta derbentina forms huge populations in Southern France. In order to characterize heat exposure and the induction of the 70-kD heat shock protein (Hsp70) response system during the life cycle of this snail, a selected population from the Vaucluse area, Provence, was investigated encompassing the issues of morphological life cycle parameters (shell size and colouration), the daily courses of heat exposure at different heights above the ground, of shell temperature, and that of the individual Hsp70 levels. The study covered all four seasons of the year 2011. Snails were found to be annual, reaching their final size in August. The shell colouration pattern showed high variation in juveniles (spring) with a strong tendency towards becoming uniformly white at old age in autumn. In all seasons, ambient air temperature decreased with increasing distance from the ground surface during daytime while remaining constantly low in the night. Overall, the Hsp70 level of individuals followed the ambient temperature during diurnal and seasonal variations. Correlation analysis revealed a positive association of individual shell temperature and Hsp70 level for the most part of the life cycle of the snails until late summer, whereas a negative correlation was found for aged animals indicating senescence effects on the capacity of the stress response system.

Population structure and dynamics of Gammarus fossarum (Amphipoda) upstream and downstream from effluents of sewage treatment plants.

Two streams in Germany (Körsch and Lockwitzbach), each with two sampling sites above and below a sewage treatment plant (STP) discharging effluent, were investigated. Sampling sites were characterized, and exposure monitoring for chemicals with known or assumed endocrine disrupting potential was carried out. Both the population structure and the population dynamics of Gammarus fossarum were examined. The physicochemical parameters measured at the sampling sites of the Lockwitzbach and Körsch streams were found not to reach levels having an acute toxic impact on the development of gammarids. The calculated estrogenic potential in the stream water was 22- to 35-fold higher at the downstream site of the Körsch compared with the other sampling sites, mainly because of the concentrations of 17alpha-ethinylestradiol on two sampling dates. At both streams, an influence of the respective STP effluent on the sex ratio of G. fossarum was not observed. Moreover, intersexuality was not induced by these effluents. Differences in the structure and dynamics of G. fossarum populations were more pronounced at the Körsch than at the Lockwitzbach. At the downstream sampling site at the Körsch, gammarids reached their highest abundances. Particularly at the downstream sampling site of the Körsch, the proportion of breeding female gammarids and the proportion of juvenile gammarids in the smallest body length class were decreased compared with upstream. Adult gammarids were larger from the Lockwitzbach downstream site, but they were smaller from the Körsch downstream site compared with the respective upstream site. At the Körsch, the earlier onset of the autumnal reproductive resting period could be caused by the influence of the STP effluent.

Assessing water quality in a tropical lake using biomarkers in zebrafish embryos: developmental toxicity and stress protein responses.

In order to achieve a more substantial appraisal of lake water quality, the assessment must not be based only on chemical measurements and analyses of the water itself, but even more so on the impact of existing conditions on aquatic biota. This is possible with the use of biotests or biomarkers, e.g. investigations of the developmental parameters (96-h embryotoxicity evaluate) or of the induction of heat shock proteins (proteotoxicity evaluate). To evaluate the suitability of these tests for environmental screening, fertilized zebrafish eggs were exposed to water samples collected from five sites of varying levels of stress from Laguna Lake, Philippines. Reconstituted water was used as laboratory control while water samples from a highly polluted freshwater source was used as positive control. Developmental parameters were noted and described within 48 and 96 h of exposure. Dilution experiments of the positive control were also done to further assess and compare toxicity potentials of Laguna Lake waters with those originating from a polluted freshwater. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in embryos from all exposure groups. Results showed 100% mortality in embryos exposed to undiluted positive control (PC) within only 12 h. Increasing dilution levels, however, resulted in lower mortality and lower abnormality rates. No detectable developmental differences were noted among embryos exposed to either the laboratory control or Laguna Lake waters at the end of 96 h, regardless of the source. Very high survival rates and high hatching success rates were observed in embryos exposed to lake waters as well as laboratory control, and the data did not differ significantly among the groups. Likewise, no significant malformations were noted among all developing embryos throughout the exposure period. However, the levels of heat shock proteins in the two sites located closest to Manila, the Philippine capital (Northern West Bay and Central West Bay), showed a pronounced elevation relative to the control, indicating that these stress proteins protect the embryos from the detrimental effects of pollutants in the water. Based on the 96-h early life stage (ELS) test, the water quality of the lake is good for fishery propagation despite the current levels of pollutants in the water. This finding is in accordance with the Class C status (i.e. suitable for fish growth and propagation) as given to the lake by the local environmental agency. On the other hand, data on proteotoxicity showed that the fish are under stress, presumably deriving from pollutants. This calls for a continuous monitoring and improvement of the lake water. The present study indicates that the two biomarker methods are very easy to use, practical, rapid, and sensitive for assessing water quality in a tropical lake and recommends for their incorporation into the future monitoring program of Laguna Lake.

Endocrine effects in Gammarus fossarum (Amphipoda): influence of wastewater effluents, temporal variability, and spatial aspects on natural populations.

In a field study, individuals of autochthounous populations of the amphipod Gammarus fossarum were examined for their maturity status, oocyte development, and biochemical parameters associated with their reproductive cycle. Variability in these parameters was related to abiotic exposure parameters varying in accordance to wastewater discharges, stream identity, and time. Patterns of all investigated parameters showed predominantly seasonal rather than spatial influence for both exposure and biologic effects. Single selected-effect parameters, however--such as the maturity index, late vitellogenic oocyte size and atresia, and the hsp90 level--responded to a sewage treatment plant discharge showing an estrogenic potential and also correlated significantly with the concentration of potential xenoestrogens at the different locations.

Colour change: A novel biomarker indicating sublethal stress in the millipede Julus scandinavius (Diplopoda).

Abstract During investigations on the induction of the 70 kD stress protein family (hsp70, stress-70) in Julus scandinavius following exposure to different biohazards, the colour of the supernatant of the homogenate was closely correlated to the hsp70 level. Hsp70 has recently been shown to be a suitable biomarker for sublethal toxicity in soil animals. Control millipedes typically exhibited red or red-orange supernatants whilst the supernatant of starved or toxin-exposed diplopods was orange, orange-yellow, or even bright yellow. Based on these observations, a quantitative colour test was established which was found to be able to indicate the degree of stress situations caused by exposure to heavy metals (cadmium, zinc), organic pollutants (lindane, PCB 52), or by food deprivation in laboratory tests. It is suggested that this is caused by a breakdown of the red-orange bilirubins into orange-yellow urobilins.

The 70 kD heat shock protein (hsp 70) in soil invertebrates: a possible tool for monitoring environmental toxicants.

The expression of hsp 70 after heat shock or exposure to heavy metals/molluscicides was investigated by fluorography or immunoblot in three diplopods (Glomeris marginata, Cylindroiulus punctatus, Tachypodoiulus niger), two slugs (Deroceras reticulatum, Arion ater), and one isopod (Oniscus asellus). In O. asellus, hsp 70 expression occurred after heat shock and also after lead treatment, whereby a solution of 100 mg/kg Pb2+ was sufficient. Animals of the same species taken from a heavy metal polluted site in the vicinity of a lead/zinc smelter also showed the presence of hsp 70. The comparison of laboratory and field experiments demonstrated the suitability of O. asellus for monitoring tests. In contrast, the blot pattern after contamination with 1,000 mg/kg Pb2+ (in the mentioned diplopods) or different concentrations of the molluscicide Cloethocarb (BASF, FRG) (slugs) showed no differences compared to the respective control group.