Rita Triebskorn

Wildlife Ecotoxicology of Pesticides: Can We Track Effects to the Population Level and Beyond?

During the past 50 years, the human population has more than doubled and global agricultural production has similarly risen. However, the productive arable area has increased by just 10%; thus the increased use of pesticides has been a consequence of the demands of human population growth, and its impact has reached global significance. Although we often know a pesticide′s mode of action in the target species, we still largely do not understand the full impact of unintended side effects on wildlife, particularly at higher levels of biological organization: populations, communities, and ecosystems. In these times of regional and global species declines, we are challenged with the task of causally linking knowledge about the molecular actions of pesticides to their possible interference with biological processes, in order to develop reliable predictions about the consequences of pesticide use, and misuse, in a rapidly changing world.

The use of histopathological indicators to evaluate contaminant-related stress in fish

As a component of a large research program toevaluate the effects of contaminants on fish healthin the field, histopathological studies have beenconducted to help establish causal relationshipsbetween contaminant exposure and various biologicalresponses. Brown trout (Salmo trutta f. fario)and loach (Barbatula barbatula) were exposedto water diverted from polluted streams undersemi-field conditions at various times during theyear. The histopathological studies revealedseasonal differences in the types and severity oforgan lesions between fish of the two streams. Bothtoxicant-induced alterations and organ lesionsresulting from natural stressors (physicochemicaland limnological water parameters) and secondarystress effects of pollution (diseases) could bedetected. In evaluating the general health ofexperimental and control fish, the use ofhistopathological studies are recommended for makingmore reliable assessments of biochemical responsesin fish exposed to a variety of environmentalstressors. Stereological analysis providesquantitative data on pathological lesions whichhelps to establish correlation with other biomarkersthereby increasing the probability of identifyingcause (stressor) and effect (biomarker) relationships.

Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams

Ultrastructural biomarkers in gill, liver, andkidney of brown trout (Salmo truttaf. fario) and stone loach (Barbatulabarbatula) were investigated over afive year period. The cellular damage of theorgans was assessed semi-quantitatively basedon a three-step classification ofultrastructural responses. Data obtained forfish exposed under semi-field conditions in twodifferently polluted test streams and in thelaboratory demonstrated that the ultrastructureof the organs can be correlated with differentpollutant exposure conditions. Cellular damagewas generally more severe in fish exposed to acomplexly polluted stream than in those exposedto a moderately polluted stream or to tap waterin the laboratory. Histopathological effects inliver and gill of trout were more pronounced inferal fish than in transplanted fish, whereasresponses in the kidney in both species, and inliver and gill of loach, were similar forintroduced and feral fish. In a laboratoryexperiment where trout were exposed todifferent mixtures of pollutants includingpesticides, PAH, and ammonia, only theultrastructure of kidney and liver showedsignificant differences between the threeexperimental set-ups. In a recovery experiment,where trout were transferred from thesemi-field condition back to the laboratory,ultrastructural investigations showed adifferential capacity of the respective organsto recover from stress under field conditions.Kidney and liver fully recovered after threemonths under control condition whereas gillsdemonstrated only partial recovery.

Induction of heat shock proteins, changes in liver ultrastructure, and alterations of fish behavior: are these biomarkers related and are they useful to reflect the state of pollution in the field?

Heat shock protein (hsp70) expression, changes inliver ultrastructure, and alterations of swimmingbehavior were simultaneously investigated in browntrout (Salmo trutta f. fario) exposed towater from test streams in southern Germany undersemi-field conditions during 1995 and 1996. Chemicalanalyses revealed different levels of contamination ofthe two streams by pesticides, PAHs, PCBs, and heavymetals, whereas stream pollution was only partlyreflected by body burdens of contaminants in fish.Biomarker responses were compared after differenttimes of exposure and with respect to differenttemperature conditions. Differences in pollutionstates of the streams were reflected by differentbiomarker responses of the trout. In the highlycontaminated stream, the biomarker responses were morecoincident with one another than in the moderatelypolluted stream in which the biomarkers showed littlerelation to one another. This is primarily due to thedifferential capacities of the biomarkers to integratestress responses over time. Understanding the dynamicsof the stress response curves and the effects oftemperature on biomarker responses is essential forevaluating the effects of environmental stressors suchas contaminants on the health of organisms.

ULTRASTRUCTURAL CHANGES IN FISH GILLS AS BIOMARKER TO ASSESS SMALL STREAM POLLUTION

In order to verify the principal suitability of gill ultrastructure as a biomarker, semi-field studies with two endigoneous fish, trout (Salmo trutta f. fario) and loach (Barbatula barbatula), were performed. The fish were exposed in flow-through systems to one heavily polluted (Körsch) and one lightly polluted small stream (Krähenbach) in South-West Germany. Ultrastructural responses in gills were correlated with limnological and chemical data recorded over a 2 year period in each stream. After 8 weeks of exposure to the heavily polluted stream, fish showed ultrastructural changes in the gills, such as cell proliferation, dilation of the endoplasmic reticulum, hyperplasia, hypersecretion, and epithelial lifting in chloride, epithelial, and mucus cells. The results of the study demonstrate that ultrastructural reactions in the gills of fish kept under semi-field conditions are potentially useful biomarkers indicating small stream pollution.

Acute effects of diclofenac and DMSO to Daphnia magna: Immobilisation and hsp70-induction

To determine the toxicity of the anti-rheumatic drug diclofenac to Daphnia magna, acute toxicity tests according to the OECD guideline 202 were combined with biochemical investigations of the hsp70 level as a biomarker for proteotoxicity. Particular attention was paid to the impact of the solvent DMSO as a confounding factor to diclofenac toxicity by means of testing different variations of producing stock solutions. In the acute immobilisation tests, diclofenac was most toxic as a singular test substance, with indication of a slight antagonistic interaction between the two substances. The highest EC50 values were obtained in those approaches using diclofenac pre-dissolved in DMSO. Thus, the observed antagonism seems to be intensified by pre-dissolution. Hsp70 levels of 12- to 19-days-old D. magna were determined after 48h exposure using a highly reproducible immunological protocol. Hsp70 induction occurred at a LOEC of 30mgl(-1) diclofenac plus 0.6mll(-1) DMSO, and at a LOEC of 40mgl(-1) for diclofenac alone. In summary, DMSO showed only slight confounding effects on diclofenac action in the applied range of concentrations.

B-type esterases in the snail Xeropicta derbentina : An enzymological analysis to evaluate their use as biomarkers of pesticide exposure

The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (K(m)=77.2 mM; V(max)=38.2 mU/mg protein) and 1-naphthyl acetate (K(m)=222 mM, V(max)=1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50=1.35x10(-5)-3.80x10(-8) M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50=1.20x10(-5)-2.98x10(-8) M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions.

The project VALIMAR (Validation of biomarkers for the assessment of small stream pollution): objectives, experimental design, summary of results, and recommendations for the application of biomarkers in risk assessment

Between 1995 and 1999, active and passivebiomonitoring experiments in two small streams,and tests with pollutant mixtures in thelaboratory were performed with brown trout(Salmo trutta f. fario) and stone loach(Barbatula barbatula) in order toevaluate the suitability of biomarkersrepresenting different levels of biologicalorganization for the assessment of pollution insmall streams. The following groups ofbiomarker responses were measured in bothspecies: (1) induction of stress proteins(hsp70) in fish liver, (2) alterations in theactivities of 13 metabolic enzymes and ofacetylcholine esterase in liver or brain in vivo as well as in cultured fish cells, (3)changes in phase I and II biotransformationenzyme activities in liver, (4) responses ofblood parameters, (5) histopathologicalalterations in liver, kidney, gills, andspleen, and (6) ultrastructural effects in theliver, gills, and kidney of individuals, aswell as in fish cell cultures. To supplementthese biomarker studies, (7) behavioral changesof fish and (8) impacts on embryo developmentwere also investigated. In parallel, the teststreams were characterized morphometrically,limnologically and analytically for five years.Furthermore, during the second-half of thisproject, ecological studies characterizingbrown trout and stone loach populationdemography and the fish, macro- andmeiozoobenthos communities in the two teststreams were also included. The present papersummarizes the aims and scopes and the generalresults of this project and provides a detaileddescription of the experimental designs whichare the basis of all related studies reportedin this issue. Recommendations are alsoprovided for the application of biomarkers inrisk assessment of small stream pollution.

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.

Biomarkers as Diagnostic Tools for Evaluating Effects of Unknown Past Water Quality Conditions on Stream Organisms

The following biomarkers were investigated in stream populations of juvenile brown trout (Salmo trutta f. fario) and gammarids (Gammarus pulex) to determine if crayfish mortality could have been confounded by pollutants: (1) alterations of fish liver ultrastructure, (2) fish gill and kidney histopathology, (3) stress protein (hsp70) expression in fish liver and gills and in gammarids, and (4) changes in various blood parameters of brown trout. In addition, the following measurements were conducted in parallel with the biological sampling: (a) chemical analyses including several pesticides, organochlorines, PCBs, and PAHs in sediment and tissue samples of brown trout and crayfish (Astacus astacus), and (b) limnochemical analyses of nutrients, electrolytes, dissolved oxygen content, temperature and pH. Biomarkers together with chemical and limnochemical analyses concomitantly indicated moderate pollution of the stream at all sampling sites. Biological data indicated a transient, episodic event at one sampling site resulting (a) in altered stress protein levels in gills and livers of trout and in whole gammarids as well as (b) in elevated numbers of macrophages in liver tissue. Biomarker responses provided spatial and temporal evidence that a contaminant release was associated with the crayfish mortalities observed in this stream system.