Christian E. W. Steinberg

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.

Effects of dissolved organic matter (DOM) on the bioconcentration of organic chemicals in aquatic organisms — a review —

Current knowledge on the effects of dissolved organic matter (DOM) on the bioconcentration of organic chemicals in aquatic animals (water fleas, mussels, amphipods and fish) is summarized. A graphical representation of the available data gives an overview of the magnitude of the observed effects. Most of the studies have shown decreases in bioconcentration in the presence of DOM (2 to 98% relative to DOM-free controls). However, at low DOM levels, up to 10 mg/L, also enhancements of bioconcentration due to DOM, ranging from 2 to 303% have been reported. Generally, the change in BCFW (Bioconcentration factor on a wet weight basis) per mg/L DOC was most pronounced at low levels of DOC. The data also show that DOM from different sources with different characteristics and quality can lead to substantial variations in the bioconcentration of organic compounds at comparable levels of DOC. While decreases in bioconcentration have generally been attributed to a lack of bioavailability of DOM-bound chemical, no mechanisms have been proposed to explain increased uptake of xenobiotics caused by DOM.

Uptake and effects of microcystin-LR on detoxication enzymes of early life stages of the zebra fish (Danio rerio)

The effects of cyanotoxins on fish have been studied mainly in adults, rather than in early life stages which could be more sensitive or, in view of their immobility, more readily affected. The uptake of microcystin‐LR by different early life stages of the zebra fish (Danio rerio) was investigated using 14C‐labelled microcystin‐LR. The effects on the activity of the detoxication enzymes, microsomal and soluble glutathione S‐transferases (GST), and glutathione peroxidase (GP‐X) were examined. There was a detectable uptake of microcystin from the first day of embryonic development up to 5 day old larvae. On average, an absorption of 0.5 ng microcystin for eggs and eleuthero‐embryos was calculated over the entire exposure time. Because of the differences in volume of the eggs and eleuthero‐embryos, there was an increase in the microcystin‐LR concentration between these stages. In the eggs, approximately 25% of the medium concentration was found, and in eleuthero‐embryos an equilibrium between fish and medium was reached.

Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians

The effects of cyanobacterial hepatotoxins microcystin‐LR, ‐RR, and ‐YR, of the neurotoxins saxitoxin and anatoxin‐a, and of crude aqueous extracts of cyanobacteria were determined on the embryos of fish and amphibians from cleavage up to advanced stages of embryonic development. No acute toxic effects were observed after exposure to microcystins at 0.5, 5, and 50 μg/L, but the timing of hatching was altered in rainbow trout: earlier hatching occurred with 0.5, 5, and 50 μg/L microcystin‐RR, 5 and 50 μg/L microcystin‐YR, and 50 μg/L microcystin‐LR; a minor delay in hatching was observed at 0.5 and 5 μg/L microcystin‐LR. In axolotl, a delay in feeding was observed (microcystin‐LR at 5 and 50 μg/L; microcystin‐YR at 50 μg/L). After termination of exposure and rearing in toxin‐free tanks, survival rate and growth were adversely affected in zebrafish larvae preexposed to microcystin‐LR at 5 and 50 μg/L. At the highest applied concentration of microcystin‐LR (10 mg/L), morphological effects were detected. Saxitoxin at 10 μg/L and above delayed hatching in zebrafish and led to malformations and mortalities at 500 μg/L. Hatching was also delayed in axolotl at 500 μg/L saxitoxin. Anatoxin‐a (400 μg/L) altered the heart rate in zebrafish, but no chronic effects were observed. Far more pronounced effects, compared to those obtained with pure toxins, were observed following exposure to various aqueous crude extracts of cyanobacteria from field samples and batch cultures: similar malformations combined with high mortalities and adverse effects on outer egg structures were observed concomitantly in all species. HPLC data showed that these effects cannot be attributed to microcystin content alone. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 77–88, 1999

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.

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.

Effects of microcystin-LR and cyanobacterial crude extracts on embryo-larval development of Zebrafish (Danio rerio)

Abstract Eggs of zebrafish ( Danio rerio ) were exposed to microcystin-LR at concentrations of 0.5, 5 and 50 μg litre −1 from blastula stage up to the end of embryonic development. No effects were observed during this period. After termination of exposure and transfer into microcystin-LR-free water, the evaluation at the end of the larval period showed decreased survival at 5 and 50 μg litre −1 microcystin-LR. Furthermore, concentrations of 0.5, 5 and 50 μg litre −1 microcystin-LR retarded larval growth. Far more pronounced effects occurred following exposure with various aqueous cyanobacterial crude extracts from field samples and batch cultures: gross malformations and high mortalities during embryonic development were observed.

Effects of atrazine on swimming behavior of zebrafish, Brachydanio rerio

Effects of atrazine (5–3125 μg l−1) on the swimming behavior of zebrafish, Brachydanio rerio, were studied by recording and quantifying the spontaneous locomotor activity. Exposure to atrazine significantly (P < 0.0001) altered the behavior: the zebrafish preferred habitats with dark substratum of the aquaria. Since the different exposure concentrations did not reveal clearly different responses, we assume a threshold effect rather than a dose-effect relationship. Possible ecological consequences of these findings are outlined.

Impact of the cyanobacteria toxin, microcystin-LR on behaviour of zebrafish, Danio rerio

Abstract Quantitative behaviour of zebrafish, Danio rerio, was recorded to assess and predict long-term sublethal effects of the cyanobacteria toxin microcystin-LR (MCYST-LR; nominal concentrations of 0.5, 5, 15 and 50 μg litre−1) by using the automated BehavioQuant®. The exposure to MCYST-LR caused dose–effect related changes in spontaneous locomotor activity. Whereas the two lower exposure concentrations (0.5 and 5 μg litre−1) caused an increase in daytime motility, elevated exposures led to significantly decreased motilities. The highest exposure (50 μg litre−1) also reduced the spawning activity and success. In contrast to daytime activities, night-time swimming activity was significantly greater at the higher MCYST-LR exposures. The chronobiological analysis indicated a phase shift of maximum swimming activities and a lowered reaction on trigger points like feeding, at dusk and dawn. Furthermore, the results indicate some adverse consequences in reproduction success and in the spatial and temporal fit of the fish into its habitat.

Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp Artemia salina.

The toxicity and metabolism of the cyanobacterial toxins microcystin-LR (MCLR), Dhb-microcystin-HtyR and nodularin were investigated in the cysts, nauplii and adults of the brine shrimp Artemia salina. The presence of the phase II detoxication system glutathione S-transferase (sGST) in these stages was shown using different substrates. Exposure of adult A. salina to the toxins led to an elevation of GST activity in vivo. All three toxins were conjugated to glutathione via GST, which has been shown as an initial step of microcystin and nodularin detoxication.