Spirostomum Ambiguum Research Articles

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.

Chemical toxicity correlations for several protozoas, bacteria, and water fleas based on the Abraham solvation parameter model

Chemical toxicity data for three species of water fleas (Daphnia magna, Ceriodaphnia dubia, and Daphnia pulex), two protozoas (Tetrahymena pyriformis and Spirostomum ambiguum), and one bacterium (Pseudomonas putida) were retrieved from the published chemical literature. The compiled data were correlated with the Abraham solvation parameter model. Derived mathematical correlations described the observed published toxicity data to within an overall average standard deviation of approximately 0.27 log units. Principal component analysis of the correlation equations showed that the water–octanol system is a poor model for nonspecific aquatic toxicity, but that the water–isobutanol and water–pentanol systems are much better models.Key words: aquatic toxicities, median lethal molar concentration, polar narcosis, nonpolar narcosis, mathematical correlation, solvation parameter model, principal component analysis.

Spirotox-Spirostomum ambiguum acute toxicity test-10 years of experience.

Spirotox is a short-term acute toxicity test with a large ciliated protozoan Spirostomum ambiguum. The test was created in the Department of Environmental Health Sciences, Medical University of Warsaw, Poland, during the early 1990s. It was presented for the first time during the 6th International Symposium on Toxicity Assessment, in Berlin in 1993. S. ambiguum was very resistant to a wide range of environmental conditions, especially to pH and dissolved oxygen. Over the last 10 years the sensitivity of S. ambiguum to many classes of toxicants was evaluated. Spirotox was found to be very sensitive to heavy metals, fungicides, and pharmaceuticals used to cure diseases of the human nervous system. On the other hand, it was generally less sensitive than standard bioassays to simple organics. Spirotox was also used for analysis of cyanobacterial blooms. Though it was moderately sensitive to hepatotoxins, the test seems to be a good tool for evaluation of the entire toxicity of blooms. The last but not the least of the applications of the Spirotox test was evaluation of the toxicity of extracts from medical devices. Protozoa, which are the simplest eukaryotes, seem to be good screening bioassays for monitoring the technology of medical device production.

645 Effect of bromadiolone on biochemical parameters of blood plasma of the common pheasant ( Phasianus colchicus)

Six muniaturized alternative assays (called microbiotests) and three standard toxicity tests were used for a comparative study based on the evaluation of acute toxicity of fifty environmental samples. The test species used in the alternative assays were microalga Raphidocelis subcapitata, crustaceans Thamnocephalus platyurus and Ceriodaphnia dubia, rotifer Brachionus calyciflorus,protozoan Spirostomum ambiguum and bacterium Vibrio fischeri. The standard toxicity tests utilized microalga Raphidocelis subcapitata, crustacean Daphnia magna, and fish Poecilia reticulata as the test organisms. The study compared the ability of bioassays to detect acute toxicity, relative sensitivity of the six microbiotests with regard to three standard toxicity tests, and similarity in their sensitivity to fifty samples. Algal bioassays were the most sensitive tests. Ceriodaphnia dubia and Spirostomum ambiguum detected acute toxicity in the majority of samples (in 62–72%). Vibrio fischeri showed a specific sensitivity pattern that was associated neither with algal nor with animal tests. The other species formed the least sensitive organisms, having similar sensitivities. A battery of three to four alternative assays was selected on the basis of the statistical analyses, sensitivity comparisons and general conditions for the selection of a test battery member like incorporation of different trophic levels or complementation of assays in a battery. Therefore, miniaturized algal assay, rotifer or crustacean microbiotest, bacterial test and possibly protozoan microbiotest could represent an optimal battery of alternative asaays for the toxicity evaluation of fifty environmental samples presented in this study.

Influence of pH on the toxicity of nitrophenols to Microtox® and Spirotox tests

The toxicity of mononitrophenols and dinitrophenols (DNP) to luminescent bacteria Vibrio fischeri (Microtox® test) and ciliated protozoan Spirostomum ambiguum (Spirotox test) was evaluated. Spirotox was more sensitive to the tested nitrophenols (NPs) than the Microtox® test. 2,5-DNP was the most toxic and 2-NP was the least toxic to the both bioindicators. The toxicity depended greatly on the pH of the medium. The highest changes were observed for DNPs, where the toxicity decreased more than 20-times when the pH increased from 6 to 8. No significant decrease of the toxicity was found for NPs, when the pH increased from 6 to 7. Greater increase of the pH to 8 caused from 1.5 to 4-fold decrease of the toxicity.

Long‐term changes in the structure of ciliate communities in a small isolated pond

The ciliate community was studied for a one‐year cycle in a shallow freshwater pond covered by a dense layer of floating macrophytes (Lemna minor) in northern Italy. In all, 38 ciliate species were found with segregation between aerobic (surface layer) and anaerobic (bottom) habitats. Some species, such as Spirostomum ambiguum, and 5. teres, were frequent in both sites and proved the most important component of the ciliate assemblage of the explored pond. The results obtained in this study were compared with those from a previous one carried out in 1987. Even if the number of species found during the first series of samples (47) was higher with respect to the second (38), a large proportion of the ciliate assemblage was maintained, since 31 species were common to both periods. The benthic ciliate communities in the pond were more stable in time than those observed at the surface layer of floating macrophytes. Nevertheless, the latter were substantially differentiated from the former, in spite of the low level of the water column.

The toxicity of cationic surfactants in four bioassays

The purpose of this study was to investigate the toxicity of 15 quaternary ammonium compounds (QACs) in a battery of four bioassays comprising the bacterium Vibrio fischeri, two ciliated protozoa Spirostomum ambiguum and Tetrahymena thermophia, and the anostracean crustacean Artemia franciscana. The compounds were prepared by Professor Pernak's group at Poznań University of Technology (Poland). The toxicity of the test compounds was very high, with EC(LC) 50 values varying from 0.11 to 70 μmol/L. Microtox was the most sensitive bioassay, while the crustacean test was the least sensitive. Among the protozoa T. thermophila was 5–30 times less sensitive than S. ambiguum. The toxicity of the QACs depended on their structure, but no simple correlation was found for all the bioassays applied.

The toxicity of tri-substituted benzenes to the protozoan ciliate Spirostomum ambiguum

The Spirotox test utilises a large ciliate protozoan Spirostomum ambiguum as a test organism. The goal of the present study was to evaluate the toxicity of tri-substituted benzenes in the Spirotox test. Twenty-six organic compounds were tested in this study and included: dimethylphenols (DMPs), dichlorophenols (DCMs), trichlorobenzenes (TCBs), dichloroanilines (DCAs), dinitrophenols (DNPs), dinitroaniline (DNA), dinitrochlorobenzene (DNCB) and dinitrofluorobenzene (DNFB). The toxicity of the compounds tested varied almost four orders of a magnitude. DMPs and DCAs were the least toxic, whereas dinitro derivatives were the most toxic to S. ambiguum. When chlorine or fluorine atoms were replaced by amino or hydroxy substituents, the toxicity increased dramatically. The results of the Spirotox test were compared with three other bioassays that are widely used around the world: Microtox ®, Tetrahymena pyriformis and Daphnia magna. The Spirotox was less sensitive than these other bioassays for the majority of these compounds, with an exception found for the dinitro derivatives.

Tests for the toxicity assessment of cyanobacterial bloom samples.

Cyanobacterial (blue-green algal) blooms are one of the common consequences of the increasing eutrophication of surface waters. The production of cyanobacterial toxins and their presence in drinking and recreational waters represents a growing danger to human and animal health. Due to a lack of toxin standards and to resource limitations on the wide-scale use of analytical methods (e.g., high-performance liquid chromatography, enzyme-linked immunosorbent assay (ELISA)) in cyanobacterial toxin monitoring, it is necessary to assess and to develop additional methods for their detection and estimation. Microbiotests using invertebrates offer a possible approach for the inexpensive and straightforward detection and assessment of cyanobacterial bloom toxicity. Three microbiotests with: Thamnocephalus platyurus, Daphnia magna, and Spirostomum ambiguum were examined with bloom samples containing hepatotoxic microcystin-LR and up to five additional microcystin variants. Two kinds of cyanobacterial bloom sample preparations were tested: crude extracts (CE) and purified extracts (PE). The highest toxicity was found when CE was used for microbiotests. The sensitivity of microorganisms decreased from S. ambiguum to T. platyurus and to D. magna. A statistically significant correlation was found between microcystin concentration and T. platyurus biotest, and between mouse bioassay and S. ambiguum results. Addition of Me2SO (1%, v/v) is a possible method to increase the sensitivity of the microorganisms for microcystin-LR.

Studies of the Ossicles from the Ciliate Protozoan Spirostomum ambiguum

Spirostomum ambiguum is able to concentrate intracellular deposits of calcium phosphate. This mineralization process is dependent on the age of the culture and can be stimulated by an applied piezoelectric potential. Crystalline deposits induced to form in laboratory cultures were analysed both in situ and after isolation from the cells, using transmission and scanning electron microscopies, energy dispersive microanalysis, and optical spectrophotometry. The ratio of Ca:P (1.57±0.01) fits with the expected value for hydroxyapatite.

Spirotox — A new tool for testing the toxicity of volatile compounds

A new method for estimating the toxicity of volatile compounds was developed. The test was carried out in the disposable polystyrene multiwells. After the organisms, protozoa Spirostomum ambiguum, were added to the wells, microplate was tightly closed using silicone grease and polyethylene film. The toxicities of 21 organic compounds were estimated. No control mortality was observed in all cases. Transparent PE film enabled good observation of test response. The toxicity of tested compounds varied over 4 orders of magnitude. Deformations were 2–4 more sensitive toxic response then lethality. The toxicity of tested compounds in Spirotox test correlates well with the log Kow and toxicity results from other bioassays: Microtox™, D. magna and T. pyriformis.

Evaluation of alternative and standard toxicity assays for screening of environmental samples: Selection of an optimal test battery

Six muniaturized alternative assays (called microbiotests) and three standard toxicity tests were used for a comparative study based on the evaluation of acute toxicity of fifty environmental samples. The test species used in the alternative assays were microalga Raphidocelis subcapitata, crustaceans Thamnocephalus platyurus and Ceriodaphnia dubia, rotifer Brachionus calyciflorus,protozoan Spirostomum ambiguum and bacterium Vibrio fischeri. The standard toxicity tests utilized microalga Raphidocelis subcapitata, crustacean Daphnia magna, and fish Poecilia reticulata as the test organisms. The study compared the ability of bioassays to detect acute toxicity, relative sensitivity of the six microbiotests with regard to three standard toxicity tests, and similarity in their sensitivity to fifty samples. Algal bioassays were the most sensitive tests. Ceriodaphnia dubia and Spirostomum ambiguum detected acute toxicity in the majority of samples (in 62–72%). Vibrio fischeri showed a specific sensitivity pattern that was associated neither with algal nor with animal tests. The other species formed the least sensitive organisms, having similar sensitivities. A battery of three to four alternative assays was selected on the basis of the statistical analyses, sensitivity comparisons and general conditions for the selection of a test battery member like incorporation of different trophic levels or complementation of assays in a battery. Therefore, miniaturized algal assay, rotifer or crustacean microbiotest, bacterial test and possibly protozoan microbiotest could represent an optimal battery of alternative asaays for the toxicity evaluation of fifty environmental samples presented in this study.

Evaluation of toxicity of medical devices using Spirotox and Microtox tests: I. Toxicity of selected toxicants in various diluents

Significant effort has been directed toward developing in vitro alternatives, which can be the first step of toxicity analysis. Tissue culture assays are currently the most popular in vitro tests for evaluating acute toxicity. The possibility of applying two bioassays using microorganisms in assessing the toxicity of extracts of medical devices was investigated. The Microtox test system—a luminescent bacteria toxicity test—assesses changes in light output from a luminescent bacteria, Vibrio fischeri. Spirotox used a large ciliate protozoan: Spirostomum ambiguum. The most widely used extraction solvent, 0.9% NaCl, must be concentrated up to 2% for Microtox and diluted nine times for the Spirotox test. The organic solvents ethanol, DMSO, and polyethylene glycol 400 were not toxic in either test in concentrations of 1–2%. The toxicity of reference compounds Hg, Cd, Zn, Pb, and SDS was examined in various diluents. The sequence of toxicity of the tested compounds in Spirotox and Microtox was: Hg > Cd > Zn > Pb > SDS, and Hg > Pb = Zn > SDS > Cd, respectively. Addition of organic solvents changed the toxicity of compounds tested in 60% of Spirotox tests and only in 25% of Microtox tests. Changes were low, not exceeding 100% in almost all cases. No correlation was observed between diluent and toxicant in either bioassay. © 1997 John Wiley & Sons, Inc.

Evaluation of toxicity of medical devices using Spirotox and Microtox tests: I. Toxicity of selected toxicants in various diluents.

Significant effort has been directed toward developing in vitro alternatives, which can be the first step of toxicity analysis. Tissue culture assays are currently the most popular in vitro tests for evaluating acute toxicity. The possibility of applying two bioassays using microorganisms in assessing the toxicity of extracts of medical devices was investigated. The Microtox test system--a luminescent bacteria toxicity test--assesses changes in light output from a luminescent bacteria, Vibrio fischeri. Spirotox used a large ciliate protozoan: Spirostomum ambiguum. The most widely used extraction solvent, 0.9% NaCl, must be concentrated up to 2% for Microtox and diluted nine times for the Spirotox test. The organic solvents ethanol, DMSO, and polyethylene glycol 400 were not toxic in either test in concentrations of 1-2%. The toxicity of reference compounds Hg, Cd, Zn, Pb, and SDS was examined in various diluents. The sequence of toxicity of the tested compounds in Spirotox and Microtox was: Hg > Cd > Zn > Pb > SDS, and Hg > Pb = Zn > SDS > Cd, respectively. Addition of organic solvents changed the toxicity of compounds tested in 60% of Spirotox tests and only in 25% of Microtox tests. Changes were low, not exceeding 100% in almost all cases. No correlation was observed between diluent and toxicant in either bioassay.

Effect of Mg 2+ on Ca 2+-dependent contraction of a Spirostomum cell model

Summary A detergent-extracted cell model of the heterotrichous ciliate Spirostomum ambiguum showed ATP-independent contraction on addition of > 10−7 M Ca2+, which is caused by contraction of the myoneme. To further understand the mechanism of cell contraction, the effects of Mg2+ and other cations were examined. Among various kinds of cations tested, Ba2+, Mn2+, Sr2+ and Cd2+ induced slight contraction, but they required higher concentrations (> 10−3 M). Contraction of the cell model was not induced by Mg2+. However, it shifted the threshold of Ca2+ concentration for inducing contraction to higher levels. The Hill constant, denoted as the number of calcium ions cooperatively bound to a contractile element in the myoneme, was 2–3 at 0–1 mM Mg2+, while it decreased with increasing Mg2+ concentration. The Ca2+-induced contraction was not influenced by changing ionic strength of the reactivation medium. These results suggest that Mg2+ ions interact with the Ca2+-binding sites which induce contraction of the myoneme.

A new low cost microbiotest with the freshwater ciliate protozoan Spirostomum ambiguum

This paper reports on preliminary experiments on the controlled culturing of the freshwater ciliated protozoan Spirostomum ambiguum, as a first step in the development of a new low cost microbiotest, to complement the limited battery of standardized cost-effective toxicity tests presently available, with a representative of unicellular biota.

Relative contribution of bacteria, algae and ciliates to the microplankton biomass in an eutrophic lake

AbstractBiomass in size‐fractionated plankton (0.2–1.2 μm; 1.2–12/μm; 12–45 μm and 45–160 μm), together with that of ciliated protozoans (size fraction 1–160 μm), were assessed during stratification, in an eutrophic lake (Lake Aydat, Massif Central, France). Samples (51) were gathered from depths of 2, 7 and 14 m during 17 weekly samplings in the centre of the lake. Three biomass estimations were performed: cell‐counts, chlorophyll a and ATP assays. Ciliates were counted live.Five species groups dominated the ciliate community: Loxodes striatus, Frontonia spp., Spirostomum ambiguum, Strombidium spp. and Dexiotrica plagia. Frontonia always had the highest cell concentration (54–64% of total cell counts), while the bulk of biomass was divided between Loxodes (42–70% of total) and Frontonia (29–53% of total).In terms of carbon, both the bacteria and ciliate contributions to the total microplanktonic biomass (size fraction 1–160 μm, i.e. bacteria + phytoplankton + ciliates) depends on the choice of the phytoplankton biomass estimation technique used. Thus, the contributions increase respectively from 16% and 19%, when phytoplankton biomass is estimated from chlorophyll α concentrations, to 24% and 27%, when phytoplankton biomass is based on biovolume.Relationships between bacteria, phytoplankton and ciliated protozoans reveal that ATP data are greatly overestimated by bacteria and ciliate interferences, especially in the metalimnion and hypolimnion. In the epilimnion, this interference is less. Interpretations of phytoplankton dynamics and metabolic activity from ATP assays should be made with extreme care, especially in eutrophic environments.

Adaptation of inducible defense in Euplotes daidaleos (Ciliophora) to predation risks by various predators

The extent of induced morphological defense in Euplotes daidaleos correlates to this ciliate's predation risk from the defense-inducing predator species. Euplotes daidaleos responded by morphological transformation only to organisms that are able to feed on typically formed Euplotes cells (63 ± 5 μm cell width in E. daidaleos). Three of those potential predator species caused defensive changes to various degrees (Student's t-test, P < 0.1 to P < 0.0001): Lembadion bullinum (Ciliata) induced 82 ± 6 μm cell width in E. daidaleos; Chaetogaster diastrophus (Oligochaeta) induced 85 = 6 μm width; and Stenostomum sphagnetorum (Turbellaria) induced 89 ± 8 μm width (at a density of 10 predators per milliliter, respectively). At higher predator densities (50 or 100 organisms per milliliter), Euplotes developed a correspondingly larger width (to a maximum of 103 ± 10 μm in the presence of S. sphagnetorum). Euplotes did not respond to organisms (e.g., Blepharisma japonicum, Colpidium campylum, Didinium nasutum, Paramecium caudatum, Spirostomum ambiguum, Stentor coeruleus) that cannot feed on this ciliate species. Daphnia longispina and Bursaria truncatella predators, which can feed on large prey of ≥125, or ≥200 μm in diameter, respectively, also had no effect on the morphology of Euplotes. The extent of defense in Euplotes that was induced by 10 predators per milliliter during 24 h decreased the predation risk from those predators to 67% in the presence of S. sphagnetorum, to 50% with L. bullinum, and to 15% with C. diastrophus, compared to the typical form of Euplotes. In a natural population, the defensive form of E. daidaleos was found with average cell widths of 88 ± 8 μm. The results indicate that predator-induced defense in natural Euplotes populations is beneficial to this prey and that it is adapted to the predation abilities of Euplotes predators, whereby energetical costs related to defensive changes may be saved.

Phylogenetic Relationships among Karyorelictids and Heterotrichs Inferred from Small Subunit rRNA Sequences: Resolution at the Base of the Ciliate Tree

Ciliate protozoa are among the most diverse and complex cells that have been described. Ciliates are characterized by nuclear dimorphism, possessing a macronucleus and a micronucleus which share the same cytoplasm. An understanding of the evolution of ciliate diversity depends upon knowledge of their phylogeny. In this study we attempted to resolve some of the relationships at the base of the ciliate tree by determining the phylogenetic position of a sample of heterotrich and hypothesized primitive karyorelictid ciliates. Karyorelictids are considered primitive because they possess a "simple" form of nuclear dualism whereby the macronucleus does not divide once it has differentiated from a micronucleus. We micromanipulated cells of two heterotrichs, Spirostomum ambiguum and Gruberia sp., and two karyorelictids, Loxodes magnus and Tracheloraphis sp., and amplified their small subunit (SSU) rDNA using PCR. The primary structure of the SSU rDNA was determined for each species and used to infer their positions in the ciliate phylogenetic tree. The results indicate, with strong support, that the aerobic heterotrichs and the karyorelictids sampled constitute a monophyletic group. The most parsimonious interpretation of the form of nuclear dimorphism in karyorelictids is that it is derived from the general condition as found in its sister group the aerobic heterotrichs. The two anaerobic heterotrichs, Metopus contortus and Metopus palaeformis, comprise a distinct clade, so that the subclass Heterotrichia, as currently conceived, is not a monophyletic group. The complex mouth architecture which characterizes all heterotrichs must be reassessed in light of this finding.

Protozoan Spirostomum ambiguum as a highly sensitive bioindicator for rapid and easy determination of water quality

A bioassay technique utilizing a new species of ciliated protozoa Spirostomum ambiguum is discussed. S. ambiguum is a very large, unicellular organism (2-3 mm long), so observation of the test responses is very easy. It can be cultured and tested under non-sterile conditions. Toxicity tests can be performed in a wide range of water pH (6.0–8.5) and hardness (2.8–250 mg CaC0 3/1). Five toxicants were tested: metal ions Hg 2+, Ag +, Cu 2+, Cd 2+ and a surfactant SDS (sodium dodecyl sulphate). Two kinds of dilution water were used: a soft water (total hardness 2.8 mg CaCO 3/1) and a hard one (total hardness 250 mg CaC03/1). Two test responses were observed: various cell deformations and lethality. The toxicity was evaluated by the static, acute plate assay. The most toxic substances were Cu and Ag (24-h LC50 values were 5.49 and 15.3 ppb, respectively). Their toxicity depended on time. However, short 1 h tests are sufficiently sensitive to estimate the toxicity of water polluted by those heavy metals. Slightly less toxic was Hg. Sensitivity of S. ambiguum to the three metals Ag, Cu and Hg did not depend on hardness of water. On the other hand Cd was 2.5-times less toxic than Hg in the soft water and more than 220-fold less toxic in the hard (24-h LC 50 were: 78.1, 30.4, 5270 and 23.8 ppb, respectively). In contrast, the surfactant SDS was about 5–7-times more toxic to S. ambiguum in the hard water than in the soft. 24-h LC50 was 1.6 and 12.1 ppm, respectively. Deformations of the cells are a faster and more sensitive test response as compared to lethality. The presented data clearly indicate that S. ambiguum is an excellent, sensitive and convenient bioindicator of evaluation of toxicity of water polluted by heavy metals and surfactants. The short time (1 h) and simplicity of the test procedure enable this test to be used in semifield studies.