Fate and ecotoxicological impact of new generation herbicides from the triketone family: An overview to assess the environmental risks.

Triketones, derived chemically from a natural phytotoxin (leptospermone), are a good example of allelochemicals as lead molecules for the development of new herbicides. Targeting a new and key enzyme involved in carotenoid biosynthesis, these latest-generation herbicides (sulcotrione, mesotrione and tembotrione) were designed to be eco-friendly and commercialized fifteen-twenty years ago. The mechanisms controlling their fate in different ecological niches as well as their toxicity and impact on different organisms or ecosystems are still under investigation. This review combines an overview of the results published in the literature on ß-triketones and more specifically, on the commercially-available herbicides and includes new results obtained in our interdisciplinary study aiming to understand all the processes involved (i) in their transfer from the soil to the connected aquatic compartments, (ii) in their transformation by photochemical and biological mechanisms but also to evaluate (iii) the impacts of the parent molecules and their transformation products on various target and non-target organisms (aquatic microorganisms, plants, soil microbial communities). Analysis of all the data on the fate and impact of these molecules, used pure, as formulation or in cocktails, give an overall guide for the assessment of their environmental risks.

An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.

The aim of this study was to assess the presence and seasonal frequency of various enteric viruses in wastewater treatment. The detection of astrovirus, norovirus, enterovirus, hepatitis A virus (HAV) and rotavirus was carried out by molecular analyses in concentrated water samples collected over 18 months at the entrance and exit of an activated sludge sewage treatment plant. The reverse transcriptase-polymerase chain reaction (RT-PCR) results were confirmed by sequencing, and comparative phylogenetic analysis was performed on the isolated strains. Genomes of human astrovirus and human rotavirus were identified in 26/29 and 11/29 samples of raw sewage, respectively, and in 12/29 and 13/29 treated effluent samples, respectively. Some rotavirus sequences detected in environmental samples were very close to those of clinical strains. Noroviruses, enteroviruses and HAV were not detected during the study period. This could be related to the small sample volume, to the sensitivity of the detection methods or to local epidemiological situations. Frequent detection of viral RNA, whether infectious or not, in the exit effluent of sewage treatment indicates wide dispersion of enteric viruses in the environment. Consequently, viral contamination resulting from the use of these treated waters is a risk that needs to be addressed.

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.

Involvement of Tetrahymena pyriformis and selected fungi in the elimination of anthracene, and toxicity assessment of the biotransformation products.

Anthracene (AC) is a non-mutagenic and non-carcinogenic, low-molecular-weight polycyclic aromatic hydrocarbon present in the environment. Its toxicity can be dramatically increased after solar-light exposure. Biotransformation capacities of AC by Tetrahymena pyriformis and a selection of eight micromycetes were studied, and the ability of these microorganisms to detoxify the polluted ecosystems was assessed. We showed that T. pyriformis was able to accumulate high amounts of AC without any transformation. In contrast, the fungi Cunninghamella elegans, Absidia fusca, Absidia cylindrospora, Rhodotorula glutinis, and Aspergillus terreus were able to transform AC with a high efficiency. Cytotoxicity assays conducted on HeLa cells and T. pyriformis showed that crude extract from A. fusca culture medium obtained after AC biotransformation was not toxic. For A. fusca and A. cylindrospora, 1-4 dihydroxyanthraquinone was shown to be the major product during the biotransformation process. This compound seemed to be a dead-end metabolite at least for the Absidia strains. The cytotoxicity of 1-4 dihydroxyanthraquinone was higher than that of AC to T. pyriformis but lower to HeLa cells. On the whole our results showed that the microorganisms studied were all able to decontaminate an AC-polluted ecosystem, either by accumulating or transforming the compound. A possible detoxification process resulting from AC biotransformation can be considered only using the human cell model.

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.

Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants.

AIMS: The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. METHODS AND RESULTS: The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for PhiX174. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system.

Assessment of anthracene toxicity toward environmental eukaryotic microorganisms: Tetrahymena pyriformis and selected micromycetes.

The toxicity of anthracene, a nonmutagenic, noncarcinogenic, low-molecular-weight polycyclic aromatic hydrocarbon present in the environment, was assessed using a ciliated protozoan, Tetrahymena pyriformis, and a selection of 10 micromycetes from different taxonomic groups living in two environmental compartments, namely aquatic and soil ecosystems. With T. pyriformis, a concentration-dependent inhibitory effect was shown on the cell proliferation rate, IC(50) = 33.40+/-4.84 mg/L (kinetic method). Phagocytosis of nonsoluble anthracene was confirmed by the presence of digestive vacuoles with photon microscopy. In fungi, anthracene did not exhibit a fungicide effect but showed a fungistatic action. Except for Absidia fusca and Cladosporium herbarum, the micromycetes showed a concentration-dependent decrease in growth. From IC(50) values determined by endpoint or kinetic methods, Rhodotorula glutinis and all of the Ascomycotina (except for Penicillium chrysogenum) were the most sensitive species, while Phanerochaete chrysosporium, P. chrysogenum, and the two Deuteromycotina were more resistant to anthracene. Our discussion focuses on the evaluation of toxicity by the two methods used for the calculation of the IC(50) values (endpoint and kinetic), the advantages of studying growth by a kinetic method (the dynamic aspect), and a comparison of toxicity to T. pyriformis and the different micromycetes.

Effect of Diuron on aquatic bacteria in laboratory-scale wastewater treatment ponds with special reference to Aeromonas species studied by colony hybridization.

Six laboratory-scale wastewater treatment ponds were filled with sediment and water obtained from a reference pond (a wastewater treatment plant located in a rural environment at Montel-de-Gelat, Puy-de-Dôme, France). They were kept at 20 degrees C, with alternative light and dark periods (12 h-12 h), and fed with raw effluent supplied weekly. Three of them were treated with Diuron (dissolved in DMSO) at a final concentration 10 mg/l, while the other three received only DMSO. Physico-chemical parameters, total bacteria, cultivable bacteria, and Aeromonas spp. were measured periodically until 41 days after the Diuron contamination. Total bacteria were treated with 4,6-diamidino 2-phenylindole (DAPI) and counted by epifluoroscence microscopy. The cultivable bacteria were quantified on plate count agar medium and Aeromonas spp. using colony hybridization. In the contaminated pilots, biochemical oxygen demand (BOD5), chemical oxygen demand (COD), suspended solids (SS), volatile suspended solids (VSS), ammonium, phosphorus, and bacteria increased, but dissolved oxygen decreased. The abundance of total bacteria, cultivable bacteria (multiplied by 30), and Aeromonas spp. increased for two weeks after Diuron introduction, reverting to initial values three weeks later. The percentage of cultivable bacteria relative to total bacteria was 0.2% in controls and 1.2% in treated pilots, while the percentage of Aeromonas spp. relative to cultivable bacteria decreased from 6-10% to 2%. Our results suggest that Diuron, which acts on the photosystem II of phototrophs, supports the development of cultivable bacteria through new carbon sources derived from the decomposition of photosynthetic micro-organisms, but does not specifically support Aeromonas spp.

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.

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.

Effects of cadmium on the performance and microbiology of laboratory-scale lagoons treating domestic sewage.

Two experiments were performed to assess the impact of cadmium on the sewage lagoon wastewater treatment process. For each one, three laboratory-scale pilot plants with one tank receiving the same raw effluent were used; one plant served as control and the other two were contaminated once only with cadmium. In the first study, the effects of a shock load of two concentrations of cadmium chloride (60 and 300 micrograms/l) on the plant performance, microbial populations (protists and bacteria) and enzyme activities were determined. Initially, most of the performance parameters were affected concentration-dependently. A reduction in the protist population density and some influence on the total bacterial population were observed, and the potential enzymatic activities were also modified. A second experiment with a lower cadmium concentration (30 micrograms/l), supplied as chloride or sulphate, still perturbed most of the parameters studied, and the effects of the two cadmium salts were identical.

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.

The effect of Saccharomyces cerevisiae and Aspergillus oryzae on the digestion of the cell wall fraction of a mixed diet in defaunated and refaunated sheep rumen.

The objective of this study was to determine the effect of two probiotics, Saccharomyces cerevisiae (SC) and Aspergillus oryzae (AO), without their culture medium, on the digestion of plant cell wall components in sheep that had been successively defaunated and refaunated. Six sheep fitted with large rumen cannulae were used to study 1) defaunated sheep with no probiotic, 2) defaunated sheep with SC or AO, 3) refaunated sheep with no probiotic, 4) refaunated sheep with SC or AO. The apparent digestibility of the plant cell walls was not altered (P > 0.05) by the probiotics in defaunated sheep but was increased (P < 0.05) with SC (+16%) in refaunated sheep. Simultaneously, SC stimulated the growth of the protozoal population in the rumen. As noted in most previous experiments, the positive effect of the presence of protozoa on plant cell wall digestion (P < 0.001) was confirmed here. The effect (P > 0.05) of SC or AO on in situ ADF digestion was either not significant or negative in defaunated rumens, whereas it became positive in refaunated rumen after a residence time of 12 h. The improvement of in situ ADF digestion due to the presence of protozoa was significant (P < 0.05 for NDF; P < 0.001 for ADF). However, we could not determine whether this was a direct effect of protozoa or an indirect effect operating via bacteria. The limits of the nylon bag technique for evaluating the microbial activity are discussed in relation to the ability of protozoa to enter and grow in the bags, and the pH regulation inside the bags. The pH values below 5.5 noted inside the bags can significantly alter the bacterial and protozoal populations and limit the validity of the technique. SC increased (P < 0.05) the specific activity of CMCase and xylanase of the solid-adherent bacteria (SAB) isolated from the rumen digesta of refaunated sheep. Stimulation of both the bacterial activity and protozoa numbers could explain the positive effect of SC on cell wall digestion in the rumen since the modelling of marker excretion in faeces showed that the ruminal mean retention time of hay measured from the model of Danhoa et al. was not modified (P > 0.05) by either refaunation or the presence of probiotics. AO increased (P < 0.05) the total retention time of the solid particles in the whole digestive tract and increased the ruminal liquid volume in the refaunated animals but it had no effect on the protozoa population or on the polysaccharidase activity of the SAB, which could explain the absence of effect of AO on the total digestibility of plant cell walls. Both probiotics decreased the liquid turnover in defaunated rumens (P < 0.05) but neither had any effect (P > 0.05) on this parameter after refaunation. The improvement of plant cell wall degradation in the whole digestive tract is probably due to a stimulation of digestion at the rumen level as indicated by the higher activity of the SAB in rumen digesta and the growth of protozoa.

Toxicity assessment of 16 inorganic environmental pollutants by six bioassays.

The relative toxicity of 16 environmental pollutants, such as inorganic elements (Ba, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Nb, Pb, Sb, Sn, Ti, V, and Zn), is evaluated on the L-929 established cell line of murine fibroblasts, with five bioassays [RNA synthesis rate assay (RNA), MTT reduction assay (MTT), neutral red incorporation assay (NRI), Coomassie blue assay, and cellular growth rate assay], and on the ciliated protozoa Tetrahymena pyriformis GL [doubling time of T. pyriformis GL population assay (DTP)]. For each inorganic substance, the six bioassays allowed the toxicological index IC50 ("inhibitory concentration 50%") to be calculated. The IC50 values are useful to rank the tested elements and to compare the features of the six bioassays. The most sensitive assays were the RNA, MTT, NRI, and DTP assays. Moreover, the in vitro IC50 values correlated with the in vivo LD50 values; these results were close to those obtained with established lines of human, murine, or fish cells. The sensitivity and the complementarity of these bioassays would be in favor of their incorporation in a "battery" of tests used for toxicological screening studies of xenobiotics.

Uptake and efflux of polycyclic aromatic hydrocarbons by Tetrahymena pyriformis: evidence for a resistance mechanism.

The action of benzo(a)pyrene (BP), 3-methylcholanthrene (3MC), benzanthracene (BA), and 7,12-dimethylbenzanthracene (DMBA), four polycyclic aromatic hydrocarbons (PAHs), was studied on the unicellular protozoan Tetrahymena pyriformis. This ciliate was exposed to the PAHs at 1, 15, and 37 microM for up to 6 h. BP and BA caused a slight inhibition of cell growth, whereas 3MC and DMBA showed no detectable effect. Cell viability remained unaffected by the PAHs at all concentrations and exposure times tested. Cellular accumulation of PAHs was studied using flow cytometry. The results show immediate accumulation followed by rapid elimination of the compounds. BP uptake was also studied in the presence of verapamil and cyclosporin, compounds known as inhibitors of the multidrug resistance (MDR) pump. In the presence of verapamil, BP was accumulated in larger amounts in cells. With cyclosporin, the accumulation of the PAH was several times higher than under control conditions. The results of GC/MS analysis show that PAH elimination was not linked to biotransformation. These results suggest that the resistance of Tetrahymena against PAH cytotoxicity may be attributed to the rapid efflux of these agents from the cells via an efflux pump probably of the MDR type.

The effect of Saccharomyces cerevisiae and Aspergillus oryzae on fermentations in the rumen of faunated and defaunated sheep; protozoal and probiotic interactions.

We measured the effect of the direct addition to the rumen of Saccharomyces cerevisiae (SC 50 mg/day) and Aspergillus oryzae (AO 3 g/day) on the fermentation processes in fistulated sheep. The measurements were carried out on animals whose rumens were first defaunated and then refaunated. The animals received a ration composed of hay (600 g/day), barley (600 g/day) and soybean meal (150 g/day), fed twice daily in two equal meals. The number of fungi and total, viable or cellulolytic bacteria were lower after the inoculation of protozoa in defaunated rumens. The probiotics stimulated the development of total bacteria but reduced the population of cellulolytic bacteria. The addition of the probiotics and the presence of protozoa each incurred a decrease in the redox potential values. The association of both treatments had an additive effect on this parameter. The two probiotics and the protozoa stabilized the rumen pH after the meal, maintaining it above the value of 6 for a longer period of time. The positive effects on pH were accumulated in the refaunated animals receiving probiotics. The ammonia nitrogen concentration was considerably increased by the presence of the protozoa; the probiotics increased the ammonia concentration only in the refaunated sheep. The methane and hydrogen proportions in the fermentation gases were invariably higher in the refaunated animals. The probiotics had no clear effect either on the gas composition or the concentration and the composition of the mixture of volatile fatty acids; only the concentration of isovalerate was significantly increased by probiotics and only in refaunated animals. The protozoa did, however, considerably increase the concentrations of acetate, butyrate and isoacids and decreased the concentration of caproate.

Microplate technique for screening and assessing cytotoxicity of xenobiotics with Tetrahymena pyriformis.

The microplate technique (MT) is developed and compared to the usual flask technique (FT) of Tetrahymena pyriformis GL cultures for assessing toxic effects of organic and inorganic substances on this ciliated protozoa model. Applied to the IC50 determination of 29 xenobiotics, the MT demonstrated good correlation with the FT (Y = 0.991X + 0.012, r = 0.94, P < 0.0001). The MT is rapid, easy-to-handle, inexpensive, and statistically reliable and, so, may be helpful for screening investigations of water-soluble xenobiotics.

Chemical and in vitro toxicological evaluations of water packaged in polyvinyl chloride and polyethylene terephthalate bottles.

This study proposed a new strategy assessing the health risks of mineral water packaging and compared the chemical analytical techniques and some in vitro cytotoxicological assays for the study of PVC and PET materials at the main stages of the manufacturing process of bottles. These evaluations were carried out with food simulant (deionized-endotoxin-tested water) and with natural mineral water in real conditions of packaging and storage (from 0 to 24 months). The complementarity of these two approaches is discussed. Some analytical and cytotoxic abnormalities were detected in the food simulant after contact with the batches of powdered PVC compound, PET resin and their intermediate steps of transformation (PVC-'paraison', PET-'perform'). But these results did not reflect the actual behaviour of the finished PVC and PET bottles, for which no major abnormality was detected in the natural mineral water.

Effect of ruminal inoculation of Isotricha alone or a mixed B-type fauna in a defaunated rumen on the digestion of a hay-maize diet (70:30) in sheep.

Two adult sheep (75 kg live weight) fitted with rumen cannulas were defaunated by the emptying method during the first period of the experiment. They were inoculated with the genus Isotricha alone during the second period, and with a mixed ciliate population (Entodinium, Eudiplodinium, Epidinium) during the third. They were fed a diet of grass hay (840 g) and pelleted maize grains (360 g) in 8 meals per day, every 3 h. Defaunation was successful and no accidental contamination occurred during the experiment. The protozoa had no significant effect on the volume of rumen digesta, nor on the turnover of the particulate phase. The addition of Isotricha and of the mixed fauna increased the ADF digestibility of the diet but, in the same animals, lowered the in sacco degradation of wheat straw. The ruminal pool sizes of dry matter (DM), organic matter (OM), nitrogen (N), neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid lignin detergent (ADL) remained unchanged after protozoa inoculations. The concentration of total volatile fatty acids (VFA) was not altered by faunation with Isotricha or a mixed fauna. The molar proportion of acetate increased at the expense of all the other VFAs (mainly propionate with the mixed fauna). Correspondingly, the proportion of methane in the rumen gases increased and that of CO2 decreased in inoculated animals. The ammonia concentration was highest in animals with a mixed fauna and lowest in those inoculated with Isotricha alone. This trend is explained in terms of the specific effect of the different genera of protozoa on nitrogen metabolism.