Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus.

Based on the chemical features of natural organic matter (NOM) with its variety of functional groups, we hypothesized that NOM will modify the multixenobiotic-resistance (MXR) of an organism as xenobiotic chemicals do. The MXR system is a general first rather non-specific line of defense against environmental contaminants. The aim of this study was to compare the impacts on MXR activity in amphipod species (Eulimnogammarus cyaneus and E. verrucosus, from Lake Baikal) stressed by cadmium chloride or dissolved NOM for 24 h. NOM exposure concentrations were environmentally realistic. MXR activity was assessed based on rhodamine B efflux; its specificity was proven by a verapamil inhibition assay. It was shown that both NOM and CdCl(2) lead to substantial reduction of the rhodamine B efflux. This suggests that NOM may be regarded as a chemosensor which is able to reduce the efficiency of the MXR system. Possible mechanisms of direct NOM impact on MXR processes are discussed, such as peroxidation of the membranes (including P-glycoproteins) or internal blockage of the MXR pump by bioconcentrated NOM. In general, our results show that well-developed depuration pathways of freshwater organisms in contaminated environments may be impaired by strong chemical stressors and, more important, by natural biogeochemical matrices such as humic substances--humic substances are present in all freshwater systems.

Antioxidant response to natural organic matter (NOM) exposure in three Baikalean amphipod species from contrasting habitats.

The aim of the present work is to comparatively evaluate the oxidative stress response on exposure to natural organic matter (NOM) in three amphipod (Crustacea, Amphipoda) species from different taxonomic groups and different habitats of Lake Baikal. Endemic species from Lake Baikal were used: the shallow-water dwelling Gmelinoides fasciatus (Dyb.), Pallasea cancelloides (Gerstf.), and the deep-layer inhabitant Ommatogammarus flavus (Dyb.). Three key enzymes, catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST), were studied. The applied NOM from Lake Schwarzer (Germany) directly impacts the two littoral species which quickly respond. The response is characterized by a significant decrease of POD and an increase of CAT activities. GST activity remains stable or decreased slightly. In contrast to the littoral amphipods, the deep-layer inhabitant O. flavus showed no significant reaction to NOM exposure, probably due to decreased adaptive ability of this species. The stable environment of the Baikalean deep zones obviously does not provide triggers for the development of flexible antioxidant or general defense systems.

Dissolved natural organic matter (NOM) impacts photosynthetic oxygen production and electron transport in coontail Ceratophyllum demersum.

Dissolved natural organic matter (NOM) is dead organic matter exceeding, in freshwater systems, the concentration of organic carbon in all living organisms by far. 80-90% (w/w) of the NOM is made up of humic substances (HS). Although NOM possesses several functional groups, a potential effect on aquatic organisms has not been studied. In this study, direct effects of NOM from various origins on physiological and biochemical functions in the aquatic plant Ceratophyllum demersum are presented. Environmentally relevant concentrations of NOM cause inhibitory effects on the photosynthetic oxygen production of C. demersum. Various NOM sources and the synthetic humic substance HS1500 inhibit the photosynthetic oxygen production of the plant as observed with 1-amino-anthraquinone, a known inhibitor of plant photosynthesis. 1-Aminoanthraquinone may serve as an analogue for the quinoid structures in NOM and HS. Most likely, the effects of NOM may be related to quinoid structures and work downstream of photosynthesis at photosystem (PS) II.

Direct and interacting toxicological effects on the waterflea (Daphnia magna) by natural organic matter, synthetic humic substances and cypermethrin.

Humic substances are the main component of dissolved organic matter in all aquatic ecosystems, comprising a variety of molecular structures and functional groups. They bind organic pollutants and metals, thereby decreasing the bioavailability and consequently the toxicity of these substances in most instances. Recent studies also describe direct interactions and effects on organisms. Current studies also show that in some cases mitigation effects are stronger at lower concentrations of natural organic matter (NOM) and that toxicity increases with increased NOM concentrations. We hypothetise that at higher concentrations the mitigating effects are overlayed by direct effects of NOM themselves, thus these aspects were investigated in combination. So, on the one hand, this study demonstrates direct effects on toxicological parameters and activities of transformation enzyme systems of Daphnia magna, provoked by two NOM and one synthetic humic-like substance, HS1500. On the other hand, capacities of NOM and synthetic HS1500 to mitigate effects of the insecticide cypermethrin were investigated. Taken together, mitigation effects were overlayed by direct own effects of the NOM and HS1500. The NOM used were isolates from Suwannee River (XAD, Spring 2000) and from streams of the Svartberget forest (reverse osmosis, Spring 2000). The HS1500 was synthetically produced by radicalic autoxidation of hydroquinones. Suwannee River NOM at concentrations between 50 and 100 mg/l, and HS1500 (10-50 mg/l), but not Svartberget NOM increased immobility and lethality of the daphnids. All elevated the activity of the soluble glutathione S-transferase from 0.5 mg/l (HS1500), 1.0 mg/l (Suwannee River NOM) and 10 mg/l (Svartberget NOM) onwards, the microsomal glutathione S-transferase did not react. The glutathione peroxidase tended to increase. In the single exposure, the insecticide cypermethrin increased all toxicological parameters, elevated soluble and tendentially microsomal glutathione S-transferase activity between 0.001 and 1.0 microg/l and tendentially increased glutathione peroxidase. In contrast to that, 50 mg/l of the NOM and HS1500 did not mitigate toxicological effects. HS1500 in combination with cypermethrin even increased immobility, compared to cypermethrin alone. Increase of the NOM or HS1500 concentrations in combination with cypermethrin did not increase mitigation as seen in the activity of soluble glutathione S-transferases, activities of microsomal glutathione S-transferase and glutathione peroxidase even decreased, relative to control, which can be a hint of enzyme disfunction or further damages in the cell. An increase of concentration did not increase mitigation. Mitigation was higher at lower NOM or HS1500 concentration, probably as a consequence of the direct effects caused by themselves. Consequently, direct effects are relevant for analysing the mitigation qualities.

Sustainable development--assessment of water resource management measures.

An approach is presented which supports decision makers in the field of water resource management to create and evaluate alternative strategies in the context of goals based on in sustainable development. The broad range of water resource management activities demand a highly structured methodology. The building blocks of the assessment procedure range from the characterization of the status quo to the development and application of assessment aids to perform exemplary evaluations.

Applying the concept of partially ordered sets on the ranking of near-shore sediments by a battery of tests.

When a ranking of some objects (chemicals, geographical sites, river sections, etc.) by a multicriteria analysis is of concern, then it is often difficult to find a common scale among the criteria, and therefore even the simple sorting process is performed by applying additional constraints, just to get a ranking index. However such additional constraints, often arising from normative considerations, are controversially discussed. The theory of partially ordered sets and its graphical representation (Hasse diagrams) does not need such additional information just to sort the objects. Here, the approach of using partially ordered sets is described by applying it to a battery of tests, developed by Dutka et al. In our analysis we found the following: (1) The dimension analysis of partially ordered sets suggests that, at least in the case of the 55 analyzed samples and the evaluation by the scores, developed by Dutka et al., there is a considerable redundancy with respect to ranking. The visualization of the sediment sites can be performed within a two-dimensional grid. (2) Information, obtained from the structure of the Hasse diagram: For example six classes of sediment sites have high priority, and each class exhibits a different pattern of results. (3) Loss of information, when an aggregation of test results is used in order to guarantee complete comparability among all objects. A relation between information drawn from the graphic and the uncertainty of ranking after using an aggregation is given. (4) The sensitivity analysis identifies one test as most important, namely the test for Fecal Coliforms/Escherichia coli. This means that the ranking of samples is heavily influenced by the results of this specific test.

Interaction of cadmium toxicity in embryos and larvae of zebrafish (Danio rerio) with calcium and humic substances.

The influence of humic substances (HS) and calcium (Ca) on cadmium (Cd) toxicity was determined using zebrafish (Danio rerio). Embryo and larvae of the zebrafish were exposed to various Cd concentrations (1.8; 2.8; 4.2; 6.2; 9.3 mg/l Cd) for 144 h. Combinations of low (0.2 mmol/l) and high (2 mmol/l) Ca, +HS (5 mg/l C) or -HS were used during Cd exposure. The toxicity of Cd was affected by (1) exposure concentration; (2) exposure time; (3) presence of HS; and (4) the Ca concentration. The results show that Ca and HS protect against Cd toxicity in zebrafish embryos. The best protection was in the high Ca-HS group, followed by high Ca+HS group and low Ca+HS group. The survival in the low Ca-HS group was the worse. Survival in the high Ca-HS group and the high Ca+HS group was similar with the exception of the highest Cd concentration (9.3 mg/l) where the survival of the high Ca+HS group was less than in the high Ca-HS group. The exposure system was modelled using a chemical equilibrium program (MINEQL+) to determine if the likely mechanism causing the anomalous result in the highest Cd concentration. The equilibrium model cannot explain these results, which suggests that this effect has a kinetic basis, such as time needed for Cd to displace Ca already bound by HS.

Influence of aqueous sediment extracts from the Oder River (Germany/Poland) on survival of Diplostomum sp. (Trematoda: Diplostomidae) cercariae.

Laboratory experiments were carried out to test whether the life span of Diplostomum sp. cercariae is affected by contamination of its aquatic environment. Freshly emerged cercariae therefore were exposed to either standardized water, Oder River (Germany/Poland) or Lake Odersee (Germany) extracts of aqueous sediments; mortality patterns and mean survival times (MSTs) were compared. Cercariae added to extracts of aqueous sediments from the Oder River and Lake Odersee exhibited impaired survival with significantly different mortality pattern and reduced MST compared to that of controls. A significant negative correlation (r = -0.843, p < 0.05) was found between MST and mercury content of sediments, indicating a potential impact of mercury on survival of Diplostomum sp. cercariae. The results indicate that contamination of freshwater habitats may influence the viability of free-living transmission stages of helminth parasites of fish and may thus be of epizootiological significance.

Uptake, effects, and metabolism of cyanobacterial toxins in the emergent reed plant Phragmites australis (cav.) trin. ex steud.

The commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) was rapidly taken up by the emergent reed plant Phragmites australis with clear distribution in the different cormus parts of the plant. Highest uptake was detected in the stem, followed by the rhizome. Enzyme extracts of the rhizome system, the stem, and the leaf revealed the presence of soluble glutathione S-transferases (sGST) measured with the model substrate 1-chloro-2,4-dinitrobenzene. A significant elevation of sGST activity in the rhizome and stem parts of P. australis was detected after a 24-h exposure to 0.5 microg/L MC-LR. Rhizome, stem, and leaf tissues were also able to conjugate several microcystin toxins. However, no conjugation, either chemical nor enzymatic, was detected using the related cyanobacterial toxin nodularin as substrate. Highest glutathione S-transferase activity for the toxin substrates was detected in the pkat/mg range in the stem of P. australis. For MC-LR, a complete metabolism from the formation of a glutathione conjugate to the degradation of a cysteine conjugate in all cormus parts of the plant is reported. The stepwise degradation of the MC-LR-glutathione conjugate to a gamma-glutamylcysteine and a cysteine conjugate was demonstrated by comparison with chemically formed reference compounds and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This is the first evidence for the uptake and metabolism of cyanobacterial toxins by an emergent aquatic macrophyte.

Toxicity of cadmium to Caenorhabditis elegans (Nematoda) in whole sediment and pore water--the ambiguous role of organic matter.

A bioassay using the nematode Caenorhabditis elegans was performed with natural sediment that had been spiked with organic matter (36-117 g total organic carbon/kg dry wt) and cadmium (Cd; 10-1,200 mg/kg wet wt). Whole sediment and pore water were tested to study the influence of particulate organic matter (POM) and dissolved organic matter (DOM) on Cd toxicity and to compare the toxicity of the two sediment phases. Toxicity was measured with nematode growth as test parameter. No toxicity was observed if sediment concentrations of Cd were below concentrations of acid-volatile sulfides (AVS). At higher Cd concentrations, toxicity in whole sediment and pore water increased with increasing organic content. This phenomenon was explained by an increase of DOM concentrations in organically enriched treatments and a resulting solubilization of Cd due to Cd complexation by DOM. Because DOM did not alter the bioavailability of Cd for the nematodes, bacteria, serving as food, might have functioned as vectors for Cd-DOM complexes, so that Cd could have become available in the gut of the nematodes. A higher toxicity in whole sediment compared to in pore water in the organically enriched treatments indicated that POM-bound Cd may have contributed to the toxicity of Cd to C. elegans.

In vivo laser-induced fluorescence detection of pyrene in nematodes and determination of pyrene binding constants for humic substances by fluorescence quenching and bioconcentration experiments.

The bioconcentration of pyrene by bacterivorous thread worms (nematodes) of the species Caenorhabditis elegans was studied with laser-induced fluorescence (LIF) spectroscopy, fluorescence imaging and a radiotracer method. The vibronic band intensities of the LIF spectra indicated that the microenvironment of pyrene in the nematodes was similar to a low-polarity solvent, and thus provided direct evidence that pyrene was accumulated in lipid-rich areas inside the nematodes. The concentration of pyrene in the nematodes was estimated from the monomer/excimer fluorescence intensity ratio. Results from this method were in fair agreement with results using 14C labeled pyrene for measuring pyrene bioconcentration. Preliminary results indicated that LIF measurements of pyrene may be possible even in single nematodes. Fluorescence microscopic observations revealed that pyrene was not adsorbed on the outside of the organisms, but was strongly concentrated in restricted areas inside the worms. In the second part of the study, the effects of six different humic substances (HS) on the bioconcentration of pyrene were investigated and sorption coefficients (KDOC) calculated from reductions in bioconcentration (KDOC(biol)) were compared with sorption coefficients measured with a fluorescence quenching technique (KDOC(flu)). The results of these two different experimental methods agreed well (with KDOC(biol) being slightly lower than KDOC(flu), indicating that the fraction of pyrene that was determined as freely dissolved by the fluorescence quenching method was comparable to the bioavailable fraction.

Predicting bioconcentration factors (BCFs) of polychlorinated bornane (Toxaphene) congeners in fish and comparison with bioaccumulation factors (BAFs) in biota from the aquatic environment.

Polychlorinated bornanes, the main components of Toxaphene, are bioconcentrated in aquatic organisms to a high extent. However, up to this time no bioconcentration tests with individual chlorinated bornanes in aquatic organisms have been performed. Therefore, the bioconcentration factors (BCFs) of seven selected persistent chlorinated bornane congeners which are regularly found in aquatic organisms, were predicted from their n-octanol/water partition coefficients (log Kow). Furthermore, these BCF values were compared with the measured bioaccumulation factors (BAFs) in zooplankton and different fish species from the aquatic environment.

Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication.

Cyanobacterial toxins have adverse effects on mammals, birds and fish and are being increasingly recognised as a potent stress factor and health hazard factor in aquatic ecosystems. Microcystins, cyclic heptapeptides and a main group of the cyanotoxins are mainly retained within the producer cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. Any toxin present could then come into contact with a wide range of aquatic organisms including phytoplankton grazers, invertebrates, fish and aquatic plants. Recent studies showed the conversion of microcystin in animal liver to a more polar compound in correlation with a depletion of the glutathione pool of the cell. The present study shows the existence of a microcystin-LR glutathione conjugate formed enzymatically via soluble glutathione S-transferase in various aquatic organisms ranging from plants (Ceratophyllum demersum), invertebrates (Dreissena polymorpha, Daphnia magna) up to fish eggs and fish (Danio rerio). The main derived conjugate was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry yielding a mass of m/z 1302, which is equivalent to the mass assumed for a glutathione microcystin-LR conjugate. This conjugate appears to be the first step in the detoxication of a cyanobacterial toxin in aquatic organisms.

Pyrene degradation by Mycobacterium sp. strain KR2.

A Mycobacterium sp., strain KR2 which was able to utilise pyrene as sole source of carbon and energy was isolated from a polycyclic aromatic hydrocarbon (PAH) contaminated soil originating from the area of a former gaswork plant. The isolate metabolised up to 60% of the pyrene added (0.5 mg/mL) within 8 days at 20 degrees C. Cis-4,5-pyrene dihydrodiol, 4,5-phenanthrene dicarboxylic acid, 1-hydroxy-2-naphthoic acid, 2-carboxybenzaldehyde, phthalic acid, and protocatechuic acid were identified as degradation products. Based on these findings a degradation pathway for pyrene is suggested which is in good accordance with the data published so far on bacterial pyrene metabolism.

Glutathione S-transferase activity in aquatic macrophytes with emphasis on habitat dependence.

Glutathione S-transferase activity of both the microsomal and soluble fractions was determined in a variety of aquatic macrophytes. The examined enzyme extract was prepared from a combination of leaves and shoots. Four different model substrates were used. The highest conjugation rate was obtained for 1-iodo-2,4-dinitrobenzene, followed by 1-chloro-2,4-dinitrobenzene, p-nitrobenzoyl chloride, and 1,2-dichloro-4-nitrobenzene. Comparison of several samples of Nuphar lutea L. from two different lake areas revealed increased glutathione S-transferase activity in plants from the site contaminated with polyaromatic hydrocarbons.

Metabolism of phenanthrene in cell suspension cultures of wheat and soybean as well as in intact plants of the water mossFontinalis antipyretica: A comparative study.

The metabolism of phenanthrene was studied both in cell suspension cultures of wheat (Triticum aestivum) and soybean (Glycine max), and in intact plants of the water mossFontinalis antipyretica. Metabolism in cell suspension cultures strongly differed between the monocotyle and the dicotyle plant. Only small amounts oftrans-phenanthrene-9,10-dihydrodiole and phenanthrene-9,10-dione were detectable in the wheat culture. Soybean cultures, in contrast demonstrated a strong turnover resulting in a 75% reduction of the initial phenanthrene concentration. Metabolites were phenanthrene-9,10-dione, not further characterized polar metabolites and bound residues. Intact plants ofFontinalis antipyretica metabolized only small amounts of phenanthrene. Data obtained from cell cultures did not provide information for the metabolic potential in intact plants. Therefore standardized tests with model systems like suspension cultures lead to inadequate assessment of the ecological risk of certain xenobiotics.

Considerations on genetic and environmental factors that contribute to resistance or sensitivity of mammals including humans to toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Part 1: Genetic factors affecting the toxicity of TCDD.

The marked species differences in short-term toxicity (30-day LD50) of ca. 10,000 (LD50: guinea pigs ca. 1 microgram/kg body wt and Han/Wistar Kuopio rats more than 9600 micrograms/kg body wt) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the central issues of the controversies that have developed on the validity of risk assessment strategies for TCDD and related compounds. One of the most challenging issues that toxicologists face today is the identification of genes that contribute to or are responsible for increased resistance or sensitivity to TCDD and related compounds. It is assumed that most, if not all, toxic effects of TCDD are mediated more or less through the binding affinity to the Ah receptor. This hypothesis was extended and tries to explain the differences in sensitivity/resistance of animals including humans to TCDD by their total fat (lipid) content. In this respect the gene or genes which is or are responsible for obesity of mammals including humans are of great interest. An obvious linear positive logarithmic relationship between the oral 30-day LD50 (microgram/kg) of TCDD in different species and strains of mammals and their total body fat content (TBF%) was found: log LD50 = 5.30 x log (TBF)-3.22, or LD50 = 0.000603 x (TBF)5.30. By means of this regression the toxicity of TCDD in mammals including humans of different age and/or body weight can be predicted if their total body fat content is known. Examples of single-gene and polygenic disease models in different mammals, such as nonobese diabetic, diabetic, viable yellow, obese, and fat mice, as well as transgenic mice, and other suitable animal models, such as fatty Zucker rats, Han/Wistar (Kuopio) rats, and minipigs, are discussed, and predicted LD50 values of TCDD in these animals and humans are presented.

PCBs and PCDD/Fs in lake sediments of Grosser Arbersee, Bavarian Forest, South Germany.

Despite their environmental importance, there are still relatively few historical studies of the environmental occurrence of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), especially in middle Europe. Trends in PCBs and PCDD/Fs were, therefore, assessed in lake sediments of the Grosser Arbersee over the past 130 years (1860-1990). Ballschmiter-PCB concentrations (congeners # 28, 52, 101, 138, 153, 180) increased between 1946 and 1972 from 4.2 to 32.0 microg kg(-1) dry wt, but have since decreased to 25.6 microg kg(-1) dry wt. High chlorinated PCB congeners reached their maxima earlier (1968-1972) than low chlorinated congeners (1985-1991). These trends were consistent with patterns expected from the production and use of PCBs and their precursors. PCDD/F concentrations increased between 1950 from 0.6 microg kg(-1) dry wt to 2.3 microg kg(-1) dry wt in 1977, falling to 1.7 microg kg(-1) dry wt by 1993. PCDF concentrations exceeded those of PCDD until 1968, but afterwards PCDDs (especially OCDD) were predominant. These patterns suggest that domestic heating and waste combustion were the most likely sources, but pollution from local industries, such as metal smelting and glass production, might also have been involved. The data provide a valuable case study from central Europe which confirms the overall declining trends of PCB and PCDD/F contamination shown elsewhere.

Ataxonomic assessment of phytoplankton integrity by means of flow cytometry.

Flow cytometry, a method well established in medicine and biotechnology, can also make an important contribution to (applied) limnological as well as ecotoxicological studies on phytoplankton. Flow cytometry can, for instance, contribute to the ataxonomic structural and functional assessment of phytoplankton. This approach may serve as a supplement to the well-established taxonomic evaluation by means of various microscope techniques. We present some examples for such ataxonomic phytoplankton evaluation. These examples include phytoplankton of eutrophicated and acidified water bodies as well as slowly flowing rivers. Phytoplankters may be differentiated by their pigment contents into carotinoid-rich ones (such as Chrysophyceae, Bacillariophyceae, and Dinophyceae) and carotinoid-poor ones (such as Euglenophyceae and Chlorophyceae). As a useful biomass parameter of phytoplankton algae we tested successfully protein staining by fluorescein isothiocyanate. We discuss the advantage of this approach as compared with results obtained by Coulter counter or by biomass calculations from microscope analyses. Up to now, evaluation of the biological quality of pelagic water bodies is still laborious and time consuming because of the microscopical examination of planktic communities usually practiced. As a possible improvement we present a structural ataxonomic approach for assessing the integrity of individual phytoplankters (on the basis of physiological parameters) as well as of the phytoplankton communities that is based on annual means of biomass spectra. Flow cytometry can provide considerable relief.

The relevance of aquatic organisms' lipid content to the toxicity of lipophilic chemicals: toxicity of lindane to different fish species.

The acute toxicity (48-hr LC50) of lindane (gamma-HCH) to 16 fish species, belonging to eight families, ranges from 22 to 900 micrograms/liter (mean: 150 micrograms/liter). A significant positive linear relationship between the lipid content (% on a wet weight basis) of the fishes and their toxicity to gamma-HCH was found. If the toxicity is referred to 1% lipid, 48-hr LC50 values range between 13.2 and 32 micrograms/liter, and thus the coefficient of variation of the mean is reduced from 139 to 22%. It is concluded that the lipids of aquatic organisms serve as a protective reservoir against the toxic effects of lindane and other lipophilic, relatively persistent organic chemicals, because they are bioconcentrated mainly in the body lipids. Therefore, in organisms with high lipid content, only a relatively small fraction of the hydrophobic chemical can reach target organs (nerves, liver, etc.) and/or receptors. For comparing toxicity data of organic chemicals to aquatic organisms, the total lipid content of the organisms must be considered. The results of this investigation are important in comparative environmental toxicology for risk assessment of freshwater and marine organisms.