Thamnocephalus Platyurus Research Articles

Topology and enhanced toxicity of bound microcystins in Microcystis PCC 7806

The cyanobacterium Microcystis is a potent producer of microcystins and cyanopeptolins and causes most of the toxicity outbreaks in freshwaters worldwide. Microcystins are mainly stored in the cells and little is found in the water. The intracellular concentration of microcystins in Microcystis PCC 7806 was at least 0.9 mM, although the solubility of microcystins in water was only about 10 μM. This low solubility does not allow the solubilisation of such high amounts of microcystins in the cytosol of Microcystis. Differential fractionation of cell constituents showed that microcystins and cyanopeptolins were bound to a protein fraction primarily composed of phycobilins. The percentage of microcystins and cyanopeptolins found in the thylakoid membranes was very low. Phycobilins may be the major proteins that have binding sites for these oligopeptides. A molar ratio near to 6 was observed for microcystins to the phycobilin ( αβ) monomer. The binding of the microcystins to the protein was rather weak and allowed rapid dissociation of microcystins from the protein-matrix. Toxicity assays with Thamnocephalus platyurus showed that native microcystin when still bound to cyanobacterial protein was more toxic than an equivalent amount that has been desorbed from the protein by treatment with methanol. It is suggested that phycobilins serve in the gut of grazers as carrier molecules for the rapid transport of microcystin from lysed cells of Microcystis to the epithelium where the uptake of microcystins occurs. Because protein-bound microcystin does not bind to C18 cartridges, this behaviour can be the cause of many analytical discrepancies observed. The blue-coloured water observed upon the collapse of Microcystis blooms may be extremely toxic because the released phycobilins may carry the major fraction of microcystins.

Photodegradation and phototoxicity of thioridazine and chlorpromazine evaluated with chemical analysis and aquatic organisms

The photochemical behaviour of chlorpromazine (CPZ) and thioridazine (THR) incubated under VIS light and a UV-A lamp was investigated with a high-performance liquid chromatography photodiode array detector (HPLC-PAD) and two bioassays. VIS light caused the decrease of CPZ and THR to 25% and 34% of the initial level, respectively, while UV-A degraded the drugs almost totally. CPZ and THR were very toxic to the protozoan Spirostomum ambiguum (Spirotox) and anostracan crustacean Thamnocephalus platyurus (Thamnotoxkit F) with 24-h LC50 values of around 0.5 mg l(-1). In spite of the drastic decrease of the concentration of the drugs, the irradiated samples were toxic to the protozoan, especially when a sublethal end-point was taken into consideration. Contrary to the protozoan the crustacean was not sensitive to the products of photodegradation. Mass spectrometry analysis showed the presence of dimers and trimers of the CPZ and mono-, di-, and tri-oxygenated derivatives of THR. The presented data give a strong indication of the importance of the investigation of the environmental fate of drugs, especially those known to be phototoxic.

In Vitro Biotransformation of Amitriptyline and Imipramine with Rat Hepatic S9 Fraction: Evaluation of the Toxicity with Spirotox and Thamnotoxkit F™ Tests

Pharmaceutical products, as well as their related metabolites, end up in the aquatic environment after use. Little is known about the effects and the hazard of exposure to drugs for aquatic organisms. This study was designed to assess the ecotoxicity of amitriptyline (AMI), imipramine (IMI), and their metabolites. The tested drugs were very toxic to the protozoan Spirostomum ambiguum and the crustacean Thamnocephalus platyurus with the LC50 values around 1 mg l(-1). Moreover, simple additivity occurs between the drugs and their N-desmethyl metabolites. Tested compounds were incubated with S9 rat hepatocyte fraction at 37 degrees C for 4 hours. Unchanged drugs and metabolites were determined using high-pressure liquid chromatography-photodiode array detector. AMI and IMI were biotransformed almost completely. Three AMI and IMI metabolites were detected: desmethyl-, didesmethyl-, and N-oxide. The toxicity of the deproteinated reaction mixtures (TU) was compared to the toxicity equivalency units (TEU) calculated based on the concentrations of the drugs and their LC50 values. It has been demonstrated that the toxicity of mixture of metabolites to Spirotox and Thamnotoxkit F is higher than the predicted value calculated from the concentrations of the drugs and their N-desmethylated derivatives in the sample. The results indicate that the harmfulness of the drug metabolites should be taken into consideration in the ecotoxicological studies.

Evaluation of the in vitro biotransformation of fluoxetine with HPLC, mass spectrometry and ecotoxicological tests

Fluoxetine (FLU) is potent and highly selective serotonin-reuptake inhibitor used in the treatment of major depression. The FLU is apparently the most acute toxic pharmaceutical reported so far for aquatic organisms. Very little is known about possible toxicity of its metabolites. Ingested drugs are excreted to the environment in a biologically active form, either as the parent substance or as an active metabolite. This study was designated to assess the ecotoxicity of FLU and its metabolites. FLU and norfluoxetine (NFLU) were very toxic to applied bioassays with LC50 around 0.5 mg l −1. The compounds affected only slightly stronger the protozoan Spirostomum ambiguum than the crustacean Thamnocephalus platyurus in the 24 h lethality tests. NFLU was 50% more toxic than FLU in both bioassays. FLU was metabolised in vitro with S9 rat liver fraction. The composition of the reaction mixtures was assessed with HPLC-PAD and MS, and their toxicity was evaluated with the bioassays. The chemical analysis showed besides FLU and NFLU the presence of 4-trifluoromethylphenol, which was much less toxic to the bioassays. Predicted toxicity values were calculated on the base of the FLU and NFLU concentrations in the samples and EC50, and LC50’s of the pure compounds. The toxicity of the solutions received during the metabolism of FLU can be predicted based on the concept of concentration addition. The results give the strong indication on the importance of investigation not only parent drugs but also their metabolites.

Limb morphogenesis in the branchiopod crustacean, Thamnocephalus platyurus, and the evolution of proximal limb lobes within Anostraca

en

Preparation and ecotoxicity assessment of new biodegradable polyurethane foams

Flexible biodegradable polyurethane foams (BIO-PUR) were prepared by a one-shot process using commercially available coreactants and modified by substituting biodegradable additive based on the acetylated starch (AS), acetylcellulose (AC), 2-hydroxyethylcellulose (HEC) and carboxymethylcellulose sodium salt (CMC) for 5 or 10 wt% of commercial polyether polyol. The BIO-PUR foams were characterized by FTIR, TGA and polarization microscopy. Ecotoxicity of BIO-PUR foams freshwater extracts was evaluated using the alternative crustacean toxicity test Thamnotoxkit F. Values of toxicity were expressed as percentage mortality of the instars II–III larvae of freshwater fairy shrimps (Thamnocephalus platyurus) dependence on the effect criterion of the respective assay. The leaches of BIO-PUR foams modified with 5 or 10 wt% of HEC showed higher toxicity then other BIO-PUR foams, whereas leaches of BIO-PUR with 5wt% of AS and 10wt% of AC were even less toxic than REF. PUR foam.

Unraveling the causes of the toxicity of extremely acid waters of volcanic origin

A river ecosystem in East Java, Indonesia, fed by a volcanic lake with high concentrations of dissolved metals and a low pH, was found to support only few macroinvertebrates. To unravel the causes of toxicity and to determine the level of dilution necessary to obtain non-toxic water, a bioassay was conducted with the fairy shrimp Thamnocephalus platyurus. A partial toxicity identification evaluation (TIE) approach was used with EDTA as a chelating agent to relate toxicity to pH and metal concentrations. Three water samples were tested, with pHs ranging from 0.72 to 4.5, and diluted with water from a neutral river to different degrees. The dilution factor necessary to achieve no more than 50% mortality in the Thamnotox test (Ldf 50) varied from > 300 at the most acidic site, to 7 for water of pH 2.6 and 1.5 for water of pH 4.5. Toxicity was best explained from both low pH and high concentrations of metals, especially Al and Fe. The key role of Al and Fe in the toxicity was confirmed by relating concentrations of the different compounds in the river water to toxicity data from the literature. EDTA addition did not significantly influence Ldf 50 or the lethal pH 50% (LpH 50), suggesting a large effect, besides cationic components and pH, of anions (F, SO 4 and Cl).

The Use of Bioassays for the Risk Assessment of Toxic Leachates: An Experimental Study

Solid wastes from the oil-shale industry produce leachates containing toxic compounds such as heavy metals and persistent polycyclic aromatic hydrocarbons (PAH). The hazard to the environment represented by waste leachates depends not only on their chemical composition, but also on the mobility and bioavailability of toxic contaminants in soils. We evaluated the applicability of bioassays for toxicity assessment of the bioavailable fraction of heavy metals and PAH in soils, in experiments with samples of four different soil types (Rendzina, Brown pseudopodzolic, Typical brown, Sodpodzolic), the pH of which ranged from 6.2 to 7.2. The toxicity of the bioavailable fraction of the soil contaminants was assessed with the dehydrogenase enzyme activity assay, and with a Toxkit microbiotest with the crustacean, Thamnocephalus platyurus, after treatment of the soil samples with an artificial solution containing chromium (III), lead (II), copper (II), cadmium (II) and pyrene. The test results confirm those of earlier experiments, which characterised the sorption potential of investigated soils for the same compounds. Both tests turned out to be sufficiently sensitive, and hence can be recommended as effective and useful tools for the assessment of the bioavailable fraction of soil contaminants.

A rapid microbiotest for the detection of cyanobacterial toxins

Cyanobacteria occur widely in lakes, reservoirs, ponds, and slow flowing rivers. Many species are known to produce toxins (cyanotoxins), a number of which are of concern for health. Cyanotoxins vary in chemical structure and may be found intracellular or released into water. There is not only a wide variation in the toxicity of known cyanotoxins but a substantial number of toxins have to date not been identified chemically. Chemical analysis of cyanotoxins is nowadays not used for routine monitoring because it is time consuming, it requires specialized equipment and expertise, and is hence expensive. There is hence an urgent need for rapid tests in surface waters to detect cyanobacterial toxins because of the need for safe drinking water and safe natural bathing waters, which may be burdened by cyanobacterial blooms or scums. Previous investigations have already shown that larvae of the anostracan crustacean Thamnocephalus platyurus are quite sensitive to neurotoxic and hepatotoxic cyanotoxins. The present paper reports on the sensitivity comparison of the (1 h) Rapidtoxkit (based on a sublethal endpoint) and the (24 h) Thamnotoxkit microbiotest (based on mortality). Both assays make use of larvae of T. platyurus. The Rapidtoxkit is a new microbiotest that determines the decrease of ingestion of colored particles by the crustacean larvae, which are stressed by a short exposure to toxicants. Fifteen cyanobacterial samples composed of laboratory strains and natural bloom samples were tested by both microbiotests. All samples were also analyzed concurrently by HPLC for microcystins and cylindrospermopsin. The correlation coefficient between the two microbiotests (r = 0.82) showed the very good correspondence between the sublethal and the lethal effects. No known toxins could be detected in some samples, although the latter were found highly toxic to the test organisms in both bioassays. These results point to the presence of unknown toxin(s) produced by some cyanobacteria such as e.g., the Cylindrospermopsis raciborskii strain isolated from Lake Balaton in Hungary. This comparative study clearly showed that the 1 h Rapidtoxkit is an attractive rapid alternative to the Thamnotoxkit microbiotest.

Phototransformation products of tamoxifen by sunlight in water. Toxicity of the drug and its derivatives on aquatic organisms

Transformation of tamoxifen has been observed in water by prolonged sunlight irradiation. The main photoproducts, isolated by chromatographic techniques, have been identified by spectroscopic means. Photoisomerization, photocyclization and, to a lesser extent, photooxygenation appear to be involved in the degradation of the drug. The acute and chronic toxicity of the parent drug and its photoproducts were tested on non-target aquatic organisms (Brachionus calyciflorus, Thamnocephalus platyurus, Daphnia magna and Ceriodaphnia dubia). Exposure to all the compounds induced mainly chronic effects without significant differences among the parental and derivative compounds.

Phototransformation of Amlodipine in Aqueous Solution: Toxicity of the Drug and Its Photoproduct on Aquatic Organisms

The phototransformation of amlodipine in water was investigated under various conditions. A quantum yield ΦS2.2×10−4 and a half-life time t1/2 0.419 days were calculated when the drug in water (10−4 M) was exposed to sunlight. The only photoproduct found was its pyridine derivative. Formation of this product was explained on the basis of a radical cation intermediate. The acute and chronic toxicity of the drug and its photoproduct were evaluated on different organisms of the freshwater chain (Brachionus calyciflorus, Thamnocephalus platyurus, Daphnia magna, Ceriodaphnia dubia). The photoproduct exhibited a stronger toxic potential than the parent drug on the long time for C. dubia.

Intra-laboratory evaluation of Microbial Assay for Risk Assessment (MARA) for potential application in the implementation of the Water Framework Directive (WFD)

The Microbial Assay for Risk Assessment (MARA) is an innovative system based on an array of 11 different microbial species freeze-dried in a 96-well micro-titre plate format. Developed for testing the toxicity of chemicals, mixtures and environmental samples, the assay employs species of a taxonomically diverse range. In addition to ten prokaryotic species, a eukaryote (yeast) is included in the range. The MARA's innate scope of a multi-dimensional test allows determination of toxicity based on a unique assay fingerprint or index, numerically expressed as the mean Microbial Toxic Concentration (MTC). The most significant potential of the test is in the additional inference that can be conveyed to the toxicity evaluation because of the presence of each of the constituent species. In view of the fact that conventional aquatic bioassays, like fish or cladoceran tests, are expensive and impractical, the MARA could provide a cost-effective solution for routine ecotoxicological testing. The performance of the MARA was evaluated to ascertain its capability and potential scope. Sensitivity to toxicants and different environmental samples was assessed. Evaluation included comparison with other tests: namely Microtox, invertebrate (Daphnia magna and Thamnocephalus platyurus) microbiotests, and respiration-inhibition and nitrification-inhibition tests. The most sensitive invertebrate test was found to be the T. platyurus microbiotest for three of the four metals tested. The LC(50) values for this test for Cd(ii), Cr(vi) and As(iii) were 0.2, 0.018 and 0.3 mg l(-1), respectively; and the corresponding most sensitive MARA species MTC values were 4.4, 2.8 and 17 mg l(-1), respectively.

Toxicity of Three Polyethoxylated Tallowamine Surfactant Formulations to Laboratory and Field Collected Fairy Shrimp, Thamnocephalus platyurus

Polyethoxylated tallowamine (POEA) is a non-ionic surfactant used in herbicide formulations to increase the efficacy of active ingredients. POEA promotes penetration of herbicide active ingredients into plant cuticles, and in animal species is known to cause alterations in respiratory surfaces. POEA use has increased recently with the advent of "Roundup-Ready" crops; however, its potential effects on aquatic invertebrates are relatively unknown. The aquatic macroinvertebrate Thamnocephalus platyurus (Crustacea, Anostraca) was used to assess the acute toxicity of POEA. Three formulations of POEA consisting of a 5:1, 10:1, and 15:1 average oxide:tallowamine were used in this study. All POEA formulations were found to be extremely toxic to T. platyurus with 48-h LC50 concentrations as low as 2.01 microg/L for 15:1. POEA toxicity increased as the tallowamine chain length was reduced, whereas the oxide chain length appeared to only slightly increase toxicity. Based on these results, POEA has the potential to adversely affect aquatic organisms in areas in which it is used.

Structure and development of setae on the thoracic limbs of the anostracan crustacean, Thamnocephalus platyurus

Setae are a prominent feature of arthropod limbs. In taxa where the limbs develop during the larval phase, developing setae are an integral part of the developing limb bud and their differentiation cannot easily be separated from the early patterning and formation of the overall limb. Here I describe the morphogenesis and adult setae in a branchiopod crustacean, the anostracan, Thamnocephalus platyurus. The majority of the setae on the limbs are non-innervated plumose setae that are formed from six underlying cells. Because branchiopods are often sampled in comparative studies of limb development, the details of the cellular morphogenesis of their limbs provide a necessary basis for studies of limb patterning.

Ecotoxicological properties of wastewater treated using tertiary methods

The objective of this work was the examination of the efficiency of coagulation and ozonation processes for the production of reclaimed wastewater with low toxicity. Municipal secondary effluents were treated by FeCl3, Al2(SO4)3 (alum), and a commercial substance at metal ion concentrations of 0.5 and 1 mmol/L. Alternatively, the effluents were treated by ozonation in a semibatch ozone reactor. The feed gas was introduced at a flowrate of 3 L/min containing ozone at various concentrations, ranging between 2.5 and 8 mg/L; ozone residence times were 2, 5, 15, and 30 min. The toxic effects of the advanced treated effluents were examined by a battery of tests using the marine bacteria Vibrio fischeri, the freshwater crustaceans Daphnia magna, Daphnia pulex, and Thamnocephalus platyurus, and the rotifers Brachionus calyciflorus. The addition of alum decreased the toxic effect of reclaimed wastewater on immobilization of D. pulex, from 90 to 60%. Ozonation was also effective for toxicity removal, which decreased to 25% effect on D. pulex after treatment by 2.5 mg O3/L for 2 min. However, acute toxic effects after ozonation, were observed on V. fischeri and were related to ozone gas concentration and contact time. At the highest ozone dosage, the toxicity reached almost 100% inhibition of bioluminescence after 15 min. The toxicity of the ozonated effluents to bacteria decreased with sample storage time and was almost negligible after 48 h, indicating that the potential adverse effect of reclaimed wastewaters on receiving waters might be reduced by storage for a certain time.

Sensitivity of ecotoxicological tests in analysis of superfund sites

During the analysis of environmental risk posed by hazardous waste disposal sites, ecological impact on whole ecosystems should be assessed. It requires a complex testing scheme where surrogate species represent key elements of the ecosystem. However, different organisms are exposed to a differing degree, also, their sensitivity to the same contaminant may vary. A possible way to determine which test reflects most the actual toxic conditions, correlation can be calculated between the measured ecological parameter (such as growth inhibition, mortality, etc.) and a contaminant gradient. The basic aim of this study was to determine which ecotoxicological test shows the best correlation with the measured analytical parameters. The selected tests were Lemna minor (representing primary producers), Thamnocephalus platyurus (a primary consumer organism) and Vibrio fischeri (decomposer). When testing soil samples, the Thamnocephalus test showed excellent consistency with most contaminants but was oversensitive in the case of groundwater samples. The Vibrio fischeri bioluminescence inhibition test (ToxAlert) behaved in a different way, reflecting well the distribution of most contaminants in groundwater samples. Finally, Lemna test proved to be completely inadequate.

High crustacean toxicity of microcystin congeners does not correlate with high protein phosphatase inhibitory activity

Microcystins are strong toxins and efficient inhibitors of eukaryotic protein phosphatases. To determine structure related properties of six different microcystin congeners, we applied standardized inhibition assays for the protein phosphatases 1 and 2A, and an acute toxicity assay with Thamnocephalus platyurus. Protein phosphatase inhibition and acute toxicity did not correlate with each other. While the inhibition of the protein phosphatases 1 and 2A was much weaker for [ d-Asp 3,( E)-Dhb 7]microcystin-RR than for the other congeners, the toxicity was one of the highest. [ d-Asp 3]microcystin-LR exhibited only small differences to microcystin-LR. The data show that mechanisms other than the inhibition of protein phosphatases, such as uptake, transport, detoxification or other target sites may have a strong modulating effect on the toxicity of a microcystin congener for a particular animal. Structural changes can offset or even reverse the specific toxicity of microcystin congeners.

Consumptive and non-consumptive effects of turbellarian (Mesostoma sp.) predation on anostracans

We assessed the predatory impact of Mesostoma sp. (Rhabditophora) on hatching and survival of Branchipodopsis wolfi (Anostraca) under laboratory conditions. The hatching percentages of resting eggs of B. wolfi were negatively influenced to a significant degree by the presence of Mesostoma sp. or the medium in which the worms were kept before, but only when prey and predator originated from the same pool. This relationship was also not observed when eggs of the American anostracan Thamnocephalus platyurus were exposed to the Turbellaria from Botswana. There was, however, no indication of predation of B. wolfi eggs by Mesostoma sp., either by eating them totally or by sucking them out. When the same number of anostracans of different ages was offered simultaneously to Mesostoma sp., the 1- and 3-day-old specimens were eaten significantly faster than the 5-day-old juveniles and the adult B. wolfi. We conclude that Mesostoma sp. has a potentially strong influence on the population dynamics and life history characteristics of B. wolfi in temporary aquatic systems. Additionally, the feeding behaviour of Mesostoma sp. was described and the ultra structure of its rostral area was investigated.

Comparative study on the environmental risk induced by several pyrethroids in estuarine and freshwater invertebrate organisms

The acute toxicity of permethrin, resmethrin, and cypermethrin to four species of aquatic non-target invertebrate organisms, found in estuarine and freshwater ecosystems, was evaluated. Artemia franciscana and Brachionus plicatilis larvae, as estuarine organisms, and Brachionus calyciflorus and Thamnocephalus platyurus larvae, as freshwater organisms, were exposed for 24 h to concentrations of these pyrethroids, and the LC 50 values were compared. The freshwater organisms were more sensitive to these pyrethroids than estuarine organisms tested. A. franciscana larvae were more tolerant organisms than B. plicatilis larvae. The freshwater organisms tested have demonstrated to be a good alternative to the standard acute toxicity assays using Daphnia, although Brachionus plycatilis larvae were more sensitive to these pyrethroid insecticides than T. platyurus. Analysis of 24 h LC 50 values of these pyrethroids, determined by static bioassays, revealed that the rank order of toxicity was: permethrin < resmethrin = cypermethrin for A. franciscana, B. calyciflorus, and T. platyurus, whereas with B. plicatilis larvae the order of toxicities was: resmethrin < permethrin < cypermethrin.

Phototransformation of carboxin in water. Toxicity of the pesticide and its sulfoxide to aquatic organisms.

Sunlight exposure of aqueous suspensions of carboxin (1) causes its phototransformation to sulfoxide 2 and minor components. Similar effects are observed in the presence of humic acid or nitrate or at different pH values. Photoproducts 2-9 were isolated by chromatographic techniques and/or identified by spectroscopic means. Carboxin 1 and its main photoproduct sulfoxide 2 were tested to evaluate acute toxicity to primary consumers typical of the aquatic environment: the rotifer Brachionus calyciflorus and two crustaceans, Daphnia magna and Thamnocephalus platyurus. Chronic tests comprised a producer, the alga Pseudokirchneriella subcapitata, and a consumer, the crustacean Ceriodaphnia dubia.