Toxicity assessment of the herbicides sulcotrione and mesotrione toward two reference environmental microorganisms: Tetrahymena pyriformis and Vibrio fischeri.

The potential toxicity of sulcotrione (2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione) and mesotrione (2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione), two selective triketonic herbicides, was assessed using representative environmental microorganisms frequently used in ecotoxicology: the eukaryote Tetrahymena pyriformis and the prokaryote Vibrio fischeri. The aims were also to evaluate the toxicity of different known degradation products, to compare the toxicity of these herbicides with that of atrazine, and to assess the toxicity of the commercial herbicidal products Mikado and Callisto. Toxicity assays involved the Microtox test, the T. pyriformis population growth impairment test, and the T. pyriformis nonspecific esterase activity test. For each compound, we report original data (IC(50) values) on nontarget cells frequently used in ecotoxicology. Analytical standards sulcotrione and mesotrione showed no toxic effect on T. pyriformis population growth but a toxic influence was observed on nonspecific esterase activities of this microorganism and on metabolism of V. fischeri. Most of the degradation products studied and the two commercial formulations showed a greater toxicity than the parent molecules. Compared with the effect of atrazine, the toxicity of these triketonic herbicides was less than in T. pyriformis and greater than or the same as in V. fischeri. Additional work is needed to obtain a more accurate picture of the environmental impact of these herbicides. It will be necessary in future experiments to study the ecosystemic levels (aquatic and soil compartments) and to assess the potential toxicity of the newly discovered degradation products and of the additives accompanying the active ingredient in the commercial herbicidal formulations.

Assessment of the potential toxicity of herbicides and their degradation products to nontarget cells using two microorganisms, the bacteria Vibrio fischeri and the ciliate Tetrahymena pyriformis.

The potential toxicity of several herbicides-alachlor, diuron and its photo and biotransformation products, glyphosate and its metabolite aminomethyl phosphonic acid (AMPA)-to nontarget cells was assessed using two microorganisms frequently used in ecotoxicology, Vibrio fischeri and Tetrahymena pyriformis. Toxicity assays involved the Microtox test, the T. pyriformis population growth impairment test employing three different processes (flasks, tubes, microplates), and the T. pyriformis nonspecific esterase activities test. Several IC(50) or EC(50) values are reported for each molecule. Alachlor exerted a toxic effect on the two nontarget cells used. The results for diuron and its photo and biotransformation products indicated that most of the metabolites presented nontarget toxicity higher than that of diuron. Glyphosate and AMPA had a less negative effect on T. pyriformis than on V. fischeri. Nevertheless, in all cases, glyphosate was found to be more toxic than AMPA. Comparison analysis of the sensitivity of the different tests showed that, in general, tests using the eukaryotic cell (T. pyriformis) were more sensitive than test using the prokaryotic cell (V. fischeri), and that a population growth criterion is more sensitive than an enzymatic criterion. The three different processes that could be used to evaluate effects on population growth rate were equally sensitive for the herbicides tested. A significant correlation between toxicity data and the hydrophobicity of the chemicals could only be established with the growth population test. This study demonstrates that it is essential to assess the toxicity of the metabolites formed to complete a more comprehensive study of the environmental impact of a polluting agent.

Influence of UV irradiation on the toxicity of phenylurea herbicides using Microtox test.

Halogenated phenylurea herbicides are not very toxic by themselves to animals, but their exposure to UV light may significantly increase the toxicity of their solutions. Absorption of light may indeed induce a phototransformation of the herbicide with a possible formation of more toxic intermediate photoproducts. Fortunately in environmental conditions photolysis is usually slow and photoproducts do not accumulate appreciably. Microtox was used for the evaluation of the toxicity of the crude irradiated solutions of some phenylurea herbicides. The sharp initial increase of toxicity shown by metobromuron solutions is mainly due to intermediate photoproducts which rapidly disappear. In the case of diuron and metoxuron toxicity is due to minor photoproducts and it does not disappear so rapidly. Hence the decrease of herbicide concentration is not necessarily associated to a lower toxicity of the solution.

Degradation products of a phenylurea herbicide, diuron: synthesis, ecotoxicity, and biotransformation.

The degradation products of diuron (photoproducts and metabolites), already described in the literature, were synthesized in order to carry out further investigations. Their ecotoxicity was determined using the standardized Microtox test, and most of the derivatives presented a nontarget toxicity higher than that of diuron. Therefore, the biotransformation of these compounds was tested with four fungal strains and a bacterial strain, which were known to be efficient for diuron transformation. With the exception of the 3,4-dichlorophenylurea, all the degradation products underwent other transformations with most of the strains tested, but no mineralization was observed. For many of them, the biodegradation compound for which the toxicity was important was 3,4-dichlorophenylurea. This study underlines the importance of knowing the nature of the degradation products, which has to be kept in mind while analyzing natural water samples or soil samples.

Use of the ciliated protozoan Tetrahymena pyriformis for the assessment of toxicity and quantitative structure--activity relationships of xenobiotics: comparison with the Microtox test.

Cytotoxicity and quantitative structure-activity relationships of 13 inorganic and 21 organic substances were determined using three bioassays performed on the ciliated protozoan Tetrahymena pyriformis and the luminescent bacterium Vibrio fischeri. The best concordance of toxicity results was observed between the T. pyriformis FDA--esterase activity and population growth inhibition tests for the organic compounds. The sensitivity of these two assays is compared with that of the Microtox test. The T. pyriformis FDA test showed a high sensitivity is most cases. The aim of the current research was to determine whether the relative toxicity of metal ions and organic molecules, with these three bioassays, was predictable using three ion characteristics and hydrophobicity, respectively. For metal ions, the variable that best modeled the toxicity data obtained with the two T. pyriformis tests was the softness index [sigma(p), i.e., (coordinate bond energy of the metal fluoride--coordinate bond energy of the metal iodide)/(coordinate bond energy of the metal fluoride)]. No correlation was found with the Microtox test. For organic compounds, a significant correlation was observed between the hydrophobicity coefficient and the toxicity data. This correlation is closer with the two tests using Tetrahymena.

A new low-cost microbiotest with the Protozoan spirostomum teres: culture conditions and assessment of sensitivity of the ciliate to 14 pure chemicals.

This paper defines the culture conditions of the ciliate Spirostomum teres and assesses its sensitivity to some xenobiotics for the development of a new low-cost microbiotest. The model was selected for its ubiquitous distribution, large size for a unicellular species, easy culture in holoxenic medium, moderate generation time, and high sensitivity to pure toxicants. The influence of different culture waters, inocula of ciliates, food, temperature, light, and darkness on the growth of the ciliate population was tested. The shortest generation time (average 39 h) was obtained for cultures incubated at 25 degreesC in the dark with an inoculum of 4 ciliates per ml in 25 ml of Volvic mineral water containing 8 boiled wheat grains, when preincubated without ciliates for the previous week. Under these conditions, it was possible to obtain about 3000 ciliates/ml 3 weeks later. Acute toxicity tests (24-h LC50) were carried out for CuSO4, HgCl2, CdCl2, K2Cr2O7, ZnSO4, Pb(NO3)2, thiram, carbaryl, lindane, parathion, parathion methyl, paraoxon, 2, 4,6-trichlorophenol, and sodium pentachlorophenolate (Na-PCP). Very high sensitivity of the model to Hg2+, Cu2+, Cd2+, thiram, and Na-PCP was established. Comparison of its sensitivity with that of Microtox (current results), Daphnia Magna, Tetrahymena pyriformis, Colpidium campylum, and murine fibroblasts (data from literature) confirms the high sensitivity of the model, especially to heavy metals. Easy-to-perform, cost-effective, and sensitive bioassays using S. teres are suitable for risk assessment and early detection of toxicity in fresh water.

Effect of Methanobrevibacter sp MF1 inoculation on glycoside hydrolase and polysaccharide depolymerase activities, wheat straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically-reared lambs.

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 of Ruminococcus flavefaciens and at the age of 4 months with a pure culture of Methanobrevibacter sp. MF1. Following the establishment of MF1, the population of R. flavefaciens slightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.

Quantitative determination of H2-utilizing acetogenic and sulfate-reducing bacteria and methanogenic archaea from digestive tract of different mammals.

Total number of bacteria, cellulolytic bacteria, and H2-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogenes were not detected in some samples of each animal. Archaea methanogens were the largest H2-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 x 10(4) and 4 x 10(4) cells ml-1 respectively).

Effect of anaerobic fungi on glycoside hydrolase and polysaccharide depolymerase activities, in sacco straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically reared lambs.

Four naturally born lambs were placed in sterile isolators 24 h after birth, before the natural establishment of the cellulolytic microorganisms. At the age of 4 weeks, a cellulolytic bacterial population of approximately 10(8) cells g-1 of rumen contents was established by inoculation with a 10(-6) dilution of ruminal contents taken from an adult sheep. A pure culture of Neocallimastix frontalis MCH3 and Piromyces communis FL was inoculated into the rumen 5 months after birth and a stable population of 10(3)-10(4) zoospores g-1 developed; the cellulolytic bacteria and fungi established populations in the 4 lambs that were similar to those observed in conventional animals. The presence of fungi led to an increase in the activity of most of the glycoside and polysaccharide hydrolases of the particle-associated microbial populations. However, this effect was not accompanied by an increase in the in sacco degradation of wheat straw or an increase in the volatile fatty acid concentration in the rumen contents.

In vitro study of the proteolytic activity of rumen anaerobic fungi.

Proteolytic, aminopeptidase, endopeptidase and carboxypeptidase activities of seven strains of rumen anaerobic fungi, selected to represent the fungal population commonly found in the rumen, were investigated in vitro. Whatever the nitrogen source included in the culture medium, a proteolytic activity against the 14C-labelled casein was detected in only one fungal strain. This strain belonged to the genus Piromyces. The activity was extracellular and was found both in the culture supernatant and bound to the mycelium. No carboxypeptidase activity was detected in the seven strains. In contrast, all the strains exhibited aminopeptidase activity. Two strains had an endopeptidase activity.

Inhibition of the cellulolytic activity of Neocallimastix frontalis by Ruminococcus flavefaciens.

A study was made of the antagonistic effect of Ruminococcus flavefaciens on the cellulolytic activity of Neocallimastix frontalis. An extracellular factor inhibiting the cellulolytic activity of the fungus was detected in the bacterial supernatant. The antagonistic factor, which precipitated with ammonium sulphate at 40% saturation, was temperature-sensitive and was destroyed at temperatures above 60 degrees C. After separation by anion-exchange chromatography, sequential precipitation, dialysis and SDS-PAGE, two protein species of 100 and 24 kDa were identified as being involved in this antagonistic effect. It is known whether the proteins are two subunits of a single protein or represent two different proteins. The inhibitory factor, which is not a bacterial cellulase, did not affect fungal growth, but it inhibited the activity of the fungal cellulases.

Effect of anaerobic fungi on the ruminal proteolysis in gnotobiotic lambs.

The establishment of a fungal population composed of the main species usually found in ruminants in the rumen of gnotobiotic lambs did not significantly alter the in sacco digestibility of meat meal and soybean cake. The proteolytic activity of the rumen fluid against 14C-casein was not affected by the fungi. Therefore, these microorganisms probably do not play an important role in the degradation of proteins in the rumen.

Effect of Eubacterium limosum, a ruminal hydrogenotrophic bacterium, on the degradation and fermentation of cellulose by 3 species of rumen anaerobic fungi.

The degradation and fermentation of cellulose filter paper were studied in axenic cultures of 3 species of rumen anaerobic fungi, Neocallimastix frontalis, Piromyces communis and Caecomyces communis, and in cocultures containing 1 of these fungal strains and Eubacterium limosum, a hydrogenotrophic rumen bacterial species. When E limosum was introduced into fungal cultures a slight decrease in fungal cellulolytic activity was observed. The end products of the fermentation of cellulose found in the cocultures were different from those found in the fungal monocultures. E limosum used formate and part of the hydrogen produced by the fungi and probably created a shift in the metabolism of the fungi resulting in a reduction of lactate and ethanol production.

Development of the rumen digestive functions in lambs placed in a sterile isolator a few days after birth.

The development of the rumen digestive functions was studied in lambs placed in sterile isolators at 1, 4, 8 or 9 days of age to define the role of the bacterial species that colonize the rumen just after birth. The values of the main rumen digestive parameters (pH, concentrations of volatile fatty acid, ammonia, lactic acid) in these lambs were close to those observed in conventional controls. Likewise, the digestive utilisation of the dry matter and starch was comparable in isolated and control animals but the digestibility of crude cellulose was higher in isolated lambs, which harboured only Fibrobacter succinogenes as the sole cellulolytic bacterial species. These results suggest that the rumen flora of the very young lamb play an essential role in the establishment of the rumen ecosystem and in the setting up of the digestive functions.

Influence of diet and monensin on development of anaerobic fungi in the rumen, duodenum, cecum, and feces of cows.

Three cows with fistulated rumens, duodenums, and ceca were fed five different diets: lucerne hay, lucerne hay plus whey (40:60), lucerne hay plus beets (50:50), corn silage plus monensin (40 ppm [40 g/kg] of dry matter intake), and lucerne hay plus monensin (80 ppm of dry matter intake). The fungal population was observed in the rumen, duodenum, cecum, and rectum and varied with diet; it was most abundant with lucerne hay alone and with corn silage plus monensin. The proportion of particles colonized by fungi in the duodenum, the cecum, and feces was measured by microscopic observation and varied from 5 to 50%, depending on the diet. The further sporangia attached to the plant particles were from the rumen, the more likely they were to be devoid of spores. Results confirmed the influence of diet on the development of the ruminal fungal population and showed that monensin does not eliminate these microorganisms. They also confirmed the presence of anaerobic fungi in the ruminant intestine. It is likely that anaerobic fungi leave the rumen attached to plant particles. However, large colonies of nonrhizoidal-type fungi were observed in cecum samples and in feces; at these sites, environmental conditions are perhaps more favorable for this type of fungus than they are in the rumen.

Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii.

A new strain of strictly anaerobic fungi was isolated from the rumen of sheep. This strain is characterized by a polycentric thallus, an extensive and polynuclear rhizomycelium, polyflagellated zoospores with gamma particle-like bodies. We propose to assign this strain in a new species: Neocallimastix joyonii.