The effect of lindane on nucleic acids, protein and carbohydrate content in Tetrahymena pyriformis

The effects of four lindane concentrations, i.e. 20, 40, 60 and 80 microg ml(-1), on the nucleic acid, protein and carbohydrate content of Tetrahymena pyriformis have been examined. All the test concentrations inhibited the synthesis of DNA, RNA, protein and carbohydrate contents. The degree of inhibition increased with increasing toxicant concentrations. Protein content was the most sensitive macromolecule affected by lindane.     

DNA content of Tetrahymena pyriformis as a biomarker for different toxic agents

The toxicity of different substances was studied on the protozoan Tetrahymena pyriformis, using as an endpoint the DNA content of the macronucleus. Substances from various chemical classes were administered to the Tetrahymena cultures and then the DNA content of the protozoan macronuclei was measured by means of Image Analysis System. The increase in the DNA content of the nuclei is indicative of the stimulation of the mitotic process. Since mitogenic stimuli can substantially alter susceptibility to chemical carcinogenesis, the results of such experiments, which are cheap and easy to run, may contribute to the investigation of the toxic action of several substances on cellular level.     

Biological treatment of whey by Tetrahymena pyriformis and impact study on laboratory-scale wastewater lagoon process

A procedure based on a biological treatment of whey was tested as part of research on waste treatment at the scale of small cheesemaking units. We studied the potential biodegradation of whey by a protozoan ciliate, Tetrahymena pyriformis, and evaluated the functional, microbiological and physiological disturbances caused by crude whey and the biodegraded whey in laboratory-scale pilots mimicking a natural lagoon treatment. The results show that T. pyriformis can strongly reduce the pollutant load of whey. In the lagoon pilots serving as example of receptor media, crude whey gradually but completely arrested operation, whereas with the biodegraded whey adverse effects were only temporary, and normal operation versus a control was gradually recovered in a few days.     

The effects of dieldrin, dimethoate and permethrin on Tetrahymena pyriformis

The effects of three insecticides, dieldrin, dimethoate and permethrin, on the growth of a holotrichous ciliate, Tetrahymena pyriformis, were studied for 5 days. The ciliate was very sensitive to dieldrin and dimethoate. Both these insecticides produced approximately 81% and 84% inhibition of growth within two days. Dieldrin caused rounding of cells, while dimethoate induced cell lysis. Dimethoate also triggered a general mucocyst discharge. Of the three insecticides, permethrin was the least toxic and induced no morphological alterations.     

Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes

Toxicity of two azo dyes (Reactive Orange 16 (RO16); Congo Red (CR)) and two anthraquinone dyes (Remazol Brilliant Blue R (RBBR); Disperse Blue 3 (DB3)) were compared using bacterium Vibrio fischeri, microalga Selenastrum capricornutum and ciliate Tetrahymena pyriformis. The following respective endpoints were involved: acute toxicity measured as bacterial luminescence inhibition, algal growth inhibition, and the effects on the protozoa including viability, growth inhibition, grazing effect and morphometric effects. In addition, mutagenicity of the dyes was determined using Ames test with bacterium Salmonella typhimurium His(-). DB3 dye was the most toxic of all dyes in the bacterial, algal and protozoan tests. In contrast to other dyes, DB3 exhibited mutagenic effects after metabolic activation in vitro in all S. typhimurium strains used. Of the methods applied, the algal test was the most sensitive to evaluate toxicity of the dyes tested.     

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.     

Comparison of bioassays by testing whole soil and their water extract from contaminated sites

The harmful effects of contaminants on the ecosystems and humans are characterised by their environmental toxicity. The aim of this study was to assess applicability and reliability of several environmental toxicity tests, comparing the result of the whole soils and their water extracts. In the study real contaminated soils were applied from three different inherited contaminated sites of organic and inorganic pollutants. The measured endpoints were the bioluminescence inhibition of Vibrio fischeri (bacterium), the dehydrogenase activity inhibition of Azomonas agilis (bacterium), the reproduction inhibition of Tetrahymena pyriformis (protozoon), and Panagrellus redivivus (nematode), the mortality of Folsomia candida (springtail), the root and shoot elongation inhibition of Sinapis alba (plant: white mustard) and the nitrification activity inhibition of an uncontaminated garden soil used as "test organism". Besides the standardised or widely used methods some new, direct contact ecotoxicity tests have been developed and introduced, which are useful for characterisation of the risk of contaminated soils due to their interactive nature. Soil no. 1 derived from a site polluted with transformer oil (PCB-free); Soil no. 2 originated from a site contaminated with mazout; Soil no. 3 was contaminated with toxic metals (Zn, Cd, Cu, Pb, As). In most cases, the interactive ecotoxicity tests indicated more harmful effect of the contaminated soil than the tests using soil extracts. The direct contact environmental toxicity tests are able to meet the requirements of environmental toxicology: reliability, sensibility, reproducibility, rapidity and low cost.     

Quantitative structure-activity relationships for the inhibition toxicity to root elongation of Cucumis sativus of selected phenols and interspecies correlation with Tetrahymena pyriformis

The comparative toxicities of selected phenols to higher plants Cucumis sativus were measured and the negative logarithm molar concentration of the root elongation median inhibition (IRC50) were derived. Quantitative structure-activity relationships (QSARs) were developed to explore the toxicity influencing factors and for predictive purpose. The toxicity data, fell into two classes: polar narcosis and bio-reactive. For polar narcotic phenols, a highly significant two-parameter QSAR based on 1-octanol/water partition coefficient (logKow) and energy of the lowest unoccupied orbital (E(lumo)) was derived (IRC50 = 0.77 log Kow - 0.39E(lumo) + 2.36 n = 22 r2 = 0.89). The five bio-reactive chemicals proved to show elevated toxicity due to their typical substructure involved diverse reactive mechanisms. In an effort to model all chemicals, a robust multiple-variable QSAR combining logKow, E(lumo) and Qmax, the most negative net atomic charge, was developed (IRC50 = 0.65 logKow - 0.72E(lumo) + 0.23Qmax + 2.81 n = 27 r2 = 0.94), indicating that hydrophobicity, electrophilicity and hydrogen bond interaction contribute mainly to the phytotoxicity. The toxicological data was compared with Tetrahymena pyriformis 2-d population growth inhibition toxicity (IGC50) and excellent interspecies correlations were observed both for the polar narcotics and for five reactive chemicals (for polar narcotics: IRC50 = 0.95IGC50 + 1.07 n = 16 r2 = 0.89; for bio-reactive chemicals: IRC50 = 0.98IGC50 + 2.19 n = 5 r2 = 0.97; and for all: IRC50 = 0.93IGC50 + 1.63 n = 21 r2 = 0.87). This suggested that T pyriformis toxicity could serve as a surrogate of C. sativus toxicity for phenols and interspecies correlation also could be established for reactive chemicals.     

Ecotoxicity prediction using mechanism- and non-mechanism-based QSARs: a preliminary study

In ecotoxicology, mechanism-based quantitative structure-activity relationships (QSARs) are usually developed with higher quality than QSARs without regard to toxicity mechanism. Correctly determining the mechanism of a compound, which is not always easy, is required to use mechanism-based QSARs for toxicity prediction. The mechanism determination step may introduce extra errors in addition to the intrinsic prediction errors of mechanism-based QSARs, thus compromising these QSARs' performance compared with QSARs regardless of mechanism. In this study, the mechanism identification-toxicity prediction (MI-TP) approach was compared with the direct toxicity prediction (DTP) approach using a data set containing phenol toxicity to Tetrahymena pyriformis. A statistical mechanism classification model for mechanism prediction, four mechanism-based QSARs and a single QSAR without discriminating between mechanisms were developed for toxicity prediction. Toxicity of phenols in an external data set was predicted following the MI-TP and DTP approaches. Results indicated that the mechanisms of several phenols in the external test set were incorrectly predicted which led to significant over- or under-estimation of their toxicity. Overall, the MI-TP approach did not yield more accurate toxicity prediction than the DTP approach.     

Classification of chemicals according to mechanism of aquatic toxicity: an evaluation of the implementation of the Verhaar scheme in Toxtree

A number of mechanisms have been identified that can lead to (acute) aquatic toxicity. The assignment of compounds to a particular mechanism of action is important in the development and utilisation of (quantitative) structure-activity relationships ((Q)SARs) for ecotoxicity. Assignment to a mechanism can be difficult; however in 1992 Verhaar et al. published a series of structural rules which aimed to classify compounds according to mechanism of action. Recent interest has seen the Verhaar rules coded into freely available software such as Toxtree available from the European Chemicals Bureau. To date, a complete critical evaluation of these rules has been lacking. Therefore, the aim of this study was to evaluate the Toxtree implementation of the Verhaar rules using two well characterised aquatic toxicity datasets (Pimephales promelas and Tetrahymena pyriformis phenol databases) for which mechanisms of toxic action are well established. The present study highlights rule, and possible coding, errors that may lead to misclassifications. Improvements to both the rules and prediction architecture are suggested. In particular further rules to improve predictions for polar narcosis (class 2) are suggested.     

An evaluation of global QSAR models for the prediction of the toxicity of phenols to Tetrahymena pyriformis

This study presents an analysis of the ability of a two-parameter response surface, a multiple linear regression and a neural network model to produce global quantitative structure-activity relationships (QSARs) to predict the toxic potency of phenols to Tetrahymena pyriformis. The phenolic toxicity data set analysed is characterised by multiple mechanisms of toxic action. The study aimed to evaluate the confidence that can be applied to the modelling of the differing mechanisms of action. Assessment of confidence was decided in terms of whether the statistics for the global models reflect the ability of the QSARs to model the individual mechanisms of toxic action present in the data set. The results showed that the global statistics only reflected the ability of models to predict the two non-covalent mechanisms (polar narcosis and respiratory uncoupling), with the metabolically transformed and electrophilic mechanism (pre-electrophiles and soft electrophiles) being modelled poorly by all three model building methods. The results confirm the difficulty in modelling electrophilic mechanisms of toxic action. The results also highlight the fact that this poor predictivity is often 'hidden' in good statistical fit of some global models. In particular these results emphasise that for practical predictive purposes the mechanistic applicability domain is required to give confidence to estimated toxicity values.     

A novel approach to predict aquatic toxicity from molecular structure

The main aim of the study was to develop quantitative structure-activity relationship (QSAR) models for the prediction of aquatic toxicity using atom-based non-stochastic and stochastic linear indices. The used dataset consist of 392 benzene derivatives, separated into training and test sets, for which toxicity data to the ciliate Tetrahymena pyriformis were available. Using multiple linear regression, two statistically significant QSAR models were obtained with non-stochastic (R2=0.791 and s=0.344) and stochastic (R2=0.799 and s=0.343) linear indices. A leave-one-out (LOO) cross-validation procedure was carried out achieving values of q2=0.781 (scv=0.348) and q2=0.786 (scv=0.350), respectively. In addition, a validation through an external test set was performed, which yields significant values of Rpred2 of 0.762 and 0.797. A brief study of the influence of the statistical outliers in QSAR's model development was also carried out. Finally, our method was compared with other approaches implemented in the Dragon software achieving better results. The non-stochastic and stochastic linear indices appear to provide an interesting alternative to costly and time-consuming experiments for determining toxicity.     

Regression comparisons of Tetrahymena pyriformis and Poecilia reticulata toxicity

The toxicity data of chemicals common to both the Poecilia reticulata mortality assay and the Tetrahymena pyriformis growth impairment assay were evaluated. Two chemicals were not toxic at saturation in the T. pyriformis assay. In addition, due to abiotic transformation, a third chemical was removed from further consideration. Each chemical was a priori assigned a mode of toxic action: neutral non-covalent, polar non-covalent, or electrophilic covalent toxicity. To further investigate comparisons between endpoints, polar and electrophilic chemicals were separated into class-based groups. The polar non-covalent chemicals were separated into phenols and anilines, while the electrophilic chemicals were separated into those reacting via Schiff-base formation (i.e., aldehydes) and those reacting via bimolecular substitution to a nucleophile (i.e., selected nitroaromatics). A comparison of toxic potency as a collective set was statistically described by the relationship; log(LC50(-1)) = 1.05(log(IGC50(-1))) + 0.56, n = 124; r2 = 0.85; s = 0.42; F = 682; Pr > F = 0.0001. The relationship between endpoints was inversely proportional to reactivity associated with the mode of action. While the comparative toxicity for neutral narcotics exhibited an excellent fit (r2 = 0.94), the fits for polar narcotics and electrophiles were poorer, r2 = 0.69 and 0.62, respectively. Investigations into class-based groupings indicated fit of toxic potency data for aldehydes (r2 = 0.85) and phenols (r2 = 0.81) were quite good. However, fits for anilines (r2 = 0.43) and nitroaromatics (r2 = 0.68) revealed that toxicity was not as well related between endpoints for these chemicals.     

酯类化合物对四膜虫毒性的拓扑研究

提出了拓扑方法,即考虑分子图顶点的性质,直接用酯类化合物的分子碎片的特征值为主元来构建邻接矩阵。定义并计算了酯类化合物的修正的氢连接性指数^mHA及其倒指数^mHB,分析了30个酯化合物对四膜虫的毒性与分子结构之间的关系,结果表明由酯化合物对四膜虫毒性与^mH的回归方程得出的估算值与实验测定值之间能较好地吻合,平均误差为0．22。     

Flow cytometry assessment of cytotoxicity and reactive oxygen species generation by single and binary mixtures of cadmium, zinc and copper on populations of the ciliated protozoan Tetrahymena thermophila

In the present study, we have assessed by flow cytometry, the cytotoxicity of the heavy metals Cd, Zn and Cu on populations of the ciliated protozoan Tetrahymena thermophila. The obtained LC(50) for these metals was estimated as 0.195mgCdl(-1), 3.58mgZnl(-1) and 0.47mgCul(-1), respectively. As a result, the toxicity rank for this eukaryotic microorganism is Cd>Cu>Zn. Using the same methodology and the Concentration Addition approach, the toxicity of binary mixtures of these metals was evaluated in order to detect the type of interaction between these metals. Results indicated that antagonism is the predominant interaction but it can change to additivity or even to synergism at high metal concentrations. Besides, the concentration ratio between metals plays also a crucial role in determining the type of metallic interaction, at least in Tetrahymena. Cytotoxicity data from single and bimetallic mixtures have been compared with those from selected microalgae, other species of ciliates, fish and mammalian cell lines and metazoan. By other hand, we have detected the mitochondrial generation of peroxides induced by both single and binary treatments with Cd, Zn and Cu on populations of Tetrahymena, using the specific fluorophore dyhidrorhodamine. The nature and concentrations of metal as well as the metallic ratio were important factors in reactive oxygen species production. All results found in T. thermophila are compared with previous reports in other organisms and, some explanations and hypothesis to support results are given, including the involvement of metallothioneins as antioxidants and their role in the binding of metal cations.     

Effects of the aquatic contaminant human pharmaceuticals and their mixtures on the proliferation and migratory responses of the bioindicator freshwater ciliate Tetrahymena

An increasing attention is paid to the potential harmful effects of aquatic contaminant pharmaceuticals exerted on both biosystems and humans. In the present work the effects of 14 pharmaceuticals including NSAIDs, antibiotics, β-blockers and a frequently used X-ray contrast media on the proliferation and migratory behavior of the freshwater ciliate Tetrahymena pyriformis was investigated. Moreover, the mixture toxicity of four selected pharmaceuticals (diclofenac, ibuprofen, metoprolol and propranolol) was evaluated in binary mixtures using full factorial experimental design. Our results showed that the sensitivity of Tetrahymena to NSAIDs and β-blockers (EC₅₀ ranged from 4.8 mg L⁻¹ to 308.1 mg L⁻¹) was comparable to that of algal or Daphnia bioassays. Based on these elevated EC₅₀ values acute toxic effects of these pharmaceuticals to T. pyriformis are unlikely. Antibiotics and the contrast agent sodium-diatrizoate had no proliferation inhibiting effect. Chemotactic response of Tetrahymena was more sensible than proliferation as significant chemorepellent action was observed in the environmentally realistic concentration range for acetylsalicylic acid, diclofenac, fenoprofen, paracetamol, metoprolol, propranolol, timolol and trimethoprim (Chemotaxis Index ranged from 63% to 88%).Mixture toxicity experiments resulted in a complex, concentration dependent interaction type pattern with antagonism being the predominant interaction type (59%) followed by additivity (37%) and synergism (4%). Hence the concept of concentration addition validated for NSAIDs in other organisms cannot be adopted for this ciliate.In summary authors suggest Tetrahymena as a sensible model of testing aquatic contaminants as well as underline the significance using more specific endpoints to understand the complex mechanisms investigated.     

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.