Bioconcentration Assessment in Fish Based on In Vitro Intrinsic Clearance: Predictivity of an Empirical Model Compared to In Vitro-In Vivo Extrapolation Models.

To estimate the bioconcentration factor (BCF), the in vitro intrinsic clearance (CLIN VITRO,INT) from rainbow trout liver S9 fractions (RT-S9) can be applied to in vitro-in vivo extrapolation (IVIVE) models, yet uncertainties remain in model parameterization. An alternative model approach is evaluated: a regression model was built in the form log BCF = a × log Kow + b × log CLIN VITRO,INT. The coefficients a and b were fitted based on a training set of 40 chemicals. A high robustness of the coefficients and good accuracy of BCF prediction were found on independent datasets of neutral organic chemicals (measured log Kow 3.3-6.2). BCF predictions were similar to or in better agreement with in vivo BCFs compared to IVIVE models (2.4- to 2.9- vs 2.8- to 3.6-fold misprediction) for training and test sets. Species-matched models (trout, carp) did not result in improvements. This study presents the largest dataset on CLIN VITRO,INT and BCFs to assess predictivity of the RT-S9 assay. The robustness of the regression statistics on different datasets and the high statistical weight of the CLIN VITRO,INT term illustrate the predictive power of the RT-S9 assay as an important step toward regulatory acceptance to replace animal experiments.

Towards the mechanism of spermatotoxicity of p-tert-butyl-alpha-methylhydrocinnamic aldehyde: inhibition of late stage ex-vivo spermatogenesis in rat seminiferous tubule cultures by para-tert-butyl- benzoic acid.

Molecules metabolized to para-tert-butyl-benzoic acid (p-TBBA) affect male reproduction in rats through effects on spermatogenesis. This toxicity is specific to p-TBBA and not observed in meta-substituted analogues. The underlying mode of action was evaluated by comparing effects of p-TBBA and the position isomer m-TBBA (2-50 µM) in an ex vivo 3D primary seminiferous tubule cell culture system from juvenile Sprague Dawley rats (Bio-AlteR®). Treated cultures were evaluated for CoA-conjugate formation, cytotoxicity, blood-testis barrier functionality and different germ cell populations to assess effects on spermatogenesis. In addition, an evaluation of the metabolome of treated cultures was performed by using MxP® Broad Profiling via a LC-MS/MS and GC-MS platform. Para-TBBA decreased germ cell populations of late stages of spermatogenesis and led to the formation of CoA-conjugates in the ex vivo tissue. In addition, p-TBBA had a pronounced effect on the metabolome by affecting lipid balance and other CoA-dependent pathways contributing to energy production and the redox system. Meta-TBBA did not affect germ cell populations and no m-TBBA related CoA-conjugates were detectable. The metabolic profile of m-TBBA treated cells was comparable to vehicle control treated cultures, indicating that formation of CoA-conjugates, inhibition of spermatogenesis, and effects on the metabolome are mechanistically linked events. Thus, for this specific chemical group an adverse outcome pathway can be postulated, including the formation of benzoic acid metabolites, accumulation of CoA-conjugates to a certain threshold and CoA depletion, which affects the metabolic and lipid profile and leads to tissue specific effects with impaired functionalities such as spermatogenesis.

Examining Uncertainty in In Vitro-In Vivo Extrapolation Applied in Fish Bioconcentration Models.

In vitro biotransformation rates were determined for 30 chemicals, mostly fragrance ingredients, using trout liver S9 fractions (RT-S9) and incorporated into in vitro-in vivo extrapolation (IVIVE) models to predict bioconcentration factors (BCFs). Predicted BCFs were compared against empirical BCFs to explore potential major uncertainties involved in the in vitro methods and IVIVE models: (i) in vitro chemical test concentrations; (ii) different gill uptake rate constant calculations (k1); (iii) protein binding (different calculations and measurement of the fraction of unbound chemical, fU); (iv) species differences; and (v) extrahepatic biotransformation. Predicted BCFs were within 0.5 log units for 44% of the chemicals compared to empirical BCFs, whereas 56% were overpredicted by >0.5 log units. This trend of overprediction was reduced by alternative k1 calculations to 32% of chemicals being overpredicted. Moreover, hepatic in vitro rates scaled to whole body biotransformation rates (kB) were compared against in vivo kB estimates. In vivo kB was underestimated for 79% of the chemicals. Neither lowering the test concentration, nor incorporation of new measured fU values, nor species matching avoided the tendency to overpredict BCFs indicating that further improvements to the IVIVE models are needed or extrahepatic biotransformation plays an underestimated role.

Benzoyl-CoA conjugate accumulation as an initiating event for male reprotoxic effects in the rat? Structure-activity analysis, species specificity, and in vivo relevance.

A number of para-substituted benzoic acids (p-BA) and chemicals metabolized to p-BA have been found to confer adverse effects in male rats on sperm viability, motility, and morphology. These effects are putatively associated with the metabolism of p-BA to toxic intermediates. We had shown that p-BA lead to accumulation of high levels of p-alkyl-benzoyl-CoA conjugates in plated primary rat hepatocytes. Here we further investigated the relevance of this metabolic pathway for the reprotoxic effects in rats and rabbits. We extended the structure-activity relationship to a set of 19 chemicals (nine reprotoxic and ten non-reprotoxic) and confirmed a very strong correlation between p-alkyl-benzoyl-CoA accumulation in rat hepatocytes and the toxic outcome. Species specificity was probed by comparing rat, rabbit and human hepatocytes, and p-benzoyl-CoA accumulation was found to be specific to the rat hepatocytes, not occurring in human hepatocytes. There was also very limited accumulation in hepatocytes from rabbits that are a non-responder species in in vivo studies. Tissues of rats treated with 3-(4-isopropylphenyl)-2-methylpropanal were analysed and p-isopropyl-benzoyl-CoA conjugates were detected in the liver and in the testes in animals at toxic doses indicating that the metabolism observed in vitro is relevant to the in vivo situation and the critical metabolite does also occur in the reproductive tissue. These multiple lines of evidence further support benzoyl-CoA accumulation as a key initiating event for a specific group of male reproductive toxicants, and indicate a species-specific effect in the rat.

PeBiToSens™: A Platform for PBT Screening of Fragrance Ingredients Without Animal Testing.

The determination of persistence (P), bioaccumulation (B) and toxicity (T) plays a central role in the environmental assessment of chemicals. Persistence is typically evaluated via standard microbial biodegradation tests. Bioaccumulation refers to the accumulation of chemicals in organisms and is usually assessed in fish exposed to the test chemical. Toxicity is determined at three trophic levels, with fish toxicity as the highest trophic level assessed. Thus, animal tests are classically needed for both B and T assessment. In vitro systems based on fish liver cells or liver S9 fractions ('RT-S9 assay') have been recently adopted by OECD to measure the biotransformation rates for the chemicals for B assessment. Biotransformation drives clearance from the body and reduces bioaccumulation. For T assessment, an assay based on in vitro toxicity on fish gill cells has been established ('RTgill-W1 assay'). Here we summarize our findings indicating that these tests are highly predictive for fragrance ingredients, and show with two case studies of our latest new registered substances how we apply these tests in particular during development and also for chemical registration. This platform of tests (PeBiToSens™) could fully replace animal tests in ecotoxicological assessment and is key in the Givaudan Safe by Design™ approach to develop safer and environmentally compatible novel fragrance ingredients.

Predicting the bioconcentration of fragrance ingredients by rainbow trout using measured rates of in vitro intrinsic clearance.

Bioaccumulation in aquatic species is a critical end point in the regulatory assessment of chemicals. Few measured fish bioconcentration factors (BCFs) are available for fragrance ingredients. Thus, predictive models are often used to estimate their BCFs. Because biotransformation can reduce chemical accumulation in fish, models using QSAR-estimated biotransformation rates have been developed. Alternatively, biotransformation can be measured by in vitro methods. In this study, biotransformation rates for nine fragrance ingredients were measured using trout liver S9 fractions and used as inputs to a recently refined in vitro-in vivo extrapolation (IVIVE) model. BCFs predicted by the model were then compared to (i) in vivo BCFs, (ii) BCFs predicted using QSAR-derived biotransformation rates, (iii) BCFs predicted without biotransformation, and (iv) BCFs predicted by a well-known regression model. For fragrance ingredients with relatively low (<4.7) log K(OW) values, all models predicted BCFs below a bioaccumulation threshold of 1000. For chemicals with higher (4.7-5.8) log K(OW) values, the model incorporating measured in vitro biotransformation rates and assuming no correction for potential binding effects on hepatic clearance provided the most accurate predictions of measured BCFs. This study demonstrates the value of integrating measured biotransformation rates for prediction of chemical bioaccumulation in fish.

Relating skin sensitizing potency to chemical reactivity: reactive Michael acceptors inhibit NF-κB signaling and are less sensitizing than S(N)Ar- and S(N)2- reactive chemicals.

The skin sensitization potency of chemicals is partly related to their reactivity to proteins. This can be quantified as the rate constant of the reaction with a model peptide, and a kinetic profiling approach to determine rate constants was previously proposed. A linear relationship between the skin sensitization potency in the local lymph node assay (LLNA) and the rate constant for Michael acceptors was reported, characterized by a relatively flat regression line. Thus, a 10-fold increase of reactivity correlates to an increase of the sensitization potential of only 1.7-fold. Here, we first validate this model by repeating previous data and testing additional Michael acceptors and prove that the model is both reproducible and robust to the addition of new data. Chemicals of different mechanistic applicability domains, namely, S(N)Ar- and S(N)2-reactive sensitizers, were then tested with the same kinetic profiling approach. A linear relationship between sensitization potency in the LLNA and rate constants was also found, yet with a much steeper slope, i.e., for S(N)Ar- and S(N)2-reactive sensitizers, increasing reactivity correlates to a much stronger increase in sensitization potency. On the basis of the well-known inhibitory activity of some Michael acceptors on IKK kinase, it was hypothesized that the difference in the slopes is due to the specific anti-inflammatory potential of Michael acceptor chemicals. Therefore, all chemicals were tested for anti-inflammatory activity in a reporter gene assay for the inhibition of NF-κB activation. Increasingly reactive Michael acceptors have increasing anti-inflammatory potential in this assay, whereas no such biological activity was detected for the S(N)Ar and S(N)2 reactive sensitizers. Thus, the increasing reactivity of Michael acceptors confers both anti-inflammatory and skin sensitizing/pro-inflammatory potential, which may partially neutralize each other. This may be the reason for the relatively weak relationship between the potency in the LLNA and the rate constant of this particular group of chemicals.

A critical assessment of the estrogenic potency of benzyl salicylate.

Benzyl salicylate (BS) is a natural ingredient of essential oils and a widely used fragrance chemical. A number of in vitro screening studies have evaluated the estrogenic potential of BS with ambiguous results. Lack of dose-response information for the positive control 17ß-estradiol (E2) in most studies makes an assessment of the relative potency and efficacy challenging. Notwithstanding this difficulty, BS has been added as the only fragrance ingredient to the list of the first 14 substances to be screened as potential endocrine disruptors by the European Scientific Committee for Consumer Safety (SCCS) and it is included in the Community rolling action plan (CoRAP) of the European REACH regulation to be assessed for the same property. Here we review all literature evidence and present new data to quantify the in vitro potency and efficacy of BS vs. E2 with full dose response analysis in both an estrogen response element (ERE) depending reporter gene assay and in the MCF7 cell proliferation (E-screen) assay. In both assays, very similar results for BS were found. BS is a partial agonist exhibiting 35-47 % maximal efficacy and it is active only close to the cytotoxic concentration. The extrapolated concentration to achieve 50 % efficacy is 21'000'000 higher as compared to E2 in the reporter gene assay. A ca. 36'000'000 higher concentration of BS as compared to E2 is required to reach equivalent partial cell proliferation stimulation in the MCF7 proliferation assay. This potency is significantly below the agonistic activity of known chemicals which cause estrogenic effects in in vivo assays. Importantly, in this study the weak agonistic activity is for the first time directly related to the activity of E2 in a full quantitative comparison in human cell lines which may help ongoing evaluations of BS by regulatory bodies.

A species specific metabolism leading to male rat reprotoxicity of Cyclamen aldehyde: in vivo and in vitro evaluation.

Cyclamen aldehyde (CA; 3-(4-isopropylphenyl)-2-methylpropanal) is a widely used fragrance material. Repeated dose studies in rats revealed adverse effects on sperm maturation. Here we review all the mechanistic and in vivo evidence, to determine relevancy to human health. The effect on spermatogenesis appears to be linked to the metabolite p-isopropyl-benzoic acid (p-iPBA). Studies in rat, rabbit and human suspended hepatocytes indicated species differences with p-iPBA detected in rat hepatocytes only. In plated rat hepatocytes, p-iPBA is conjugated to Coenzyme A (CoA) and p-iPBA-CoA accumulates to stable levels over 22 h. In vitro accumulation of CoA conjugates is a metabolic hallmark correlated to male rat reproductive toxicity for related compounds. p-iPBA-CoA is formed in vivo in liver and testes of rats dosed with CA. In plated rabbit and human hepatocytes p-iPBA-CoA doesn't accumulate. Correlating to this lack of metabolite accumulation, no effects of CA on spermatogenesis were observed in a rabbit in vivo study. A species specific metabolic fate linked to CA toxicity in male rats is postulated which appears not relevant to the rabbit as non-responder species. Lack of accumulation of p-iPBA-CoA in human hepatocytes indicates that like rabbits, humans are unlikely to be vulnerable to p-iPBA hepatic and testicular toxicity.

In vitro bactericidal activity of iclaprim in human plasma.

This study evaluated the effect of human plasma on the in vitro bactericidal activity of the novel diaminopyrimidine iclaprim against methicillin (meticillin)-susceptible and -resistant Staphylococcus aureus strains. MICs and minimal bactericidal concentrations (MBCs) of iclaprim, with approximately 93% protein binding, were similar in the absence and in the presence of 50% human plasma; MICs and MBCs ranged from 0.06 to 0.125 microg/ml. Furthermore, the activity of iclaprim was not affected by plasma, with > or = 99.9% reduction in CFU after 5.0 to 7.6 h.

Increased hydrophobic interactions of iclaprim with Staphylococcus aureus dihydrofolate reductase are responsible for the increase in affinity and antibacterial activity.

OBJECTIVES: Iclaprim is a novel 2,4-diaminopyrimidine that exhibits potent, rapid bactericidal activity against major Gram-positive pathogens, including methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus, and is currently in clinical development for the treatment of complicated skin and skin structure infections. An understanding of the known mechanism of resistance to trimethoprim led to the design of this new inhibitor, with improved affinity towards dihydrofolate reductase (DHFR) from S. aureus and clinically useful activity against S. aureus including isolates resistant to trimethoprim. The objective of this study was to characterize the mode of action of iclaprim and its inhibitory properties against DHFR. METHODS: The mode of action of iclaprim was assessed by enzymatic analysis, direct binding studies, macromolecular synthesis profiles, synergy and antagonism studies to define its role as an inhibitor of DHFR. The binding properties of iclaprim to DHFR were compared with those of trimethoprim by X-ray crystallography. RESULTS: The enzymatic properties, direct binding and X-ray crystallographic studies delineated the mode of interaction with DHFR and the reason for the increased affinity of iclaprim towards the enzyme. The effect of iclaprim on bacterial physiology suggests that iclaprim behaves as a classical antibacterial DHFR inhibitor, as previously documented for trimethoprim. CONCLUSIONS: Iclaprim binds and inhibits bacterial DHFR in a similar manner to trimethoprim. However, the increased hydrophobic interactions between iclaprim and DHFR account for increased affinity and, unlike trimethoprim, enable iclaprim to inhibit even the resistant enzyme with nanomolar affinity, thus overcoming the mechanism of trimethoprim resistance. The increased antibacterial activity and lower propensity for resistance make iclaprim a clinically promising and useful inhibitor.

Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity.

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

Accurate prediction of acute fish toxicity of fragrance chemicals with the RTgill-W1 cell assay.

Testing for acute fish toxicity is an integral part of the environmental safety assessment of chemicals. A true replacement of primary fish tissue was recently proposed using cell viability in a fish gill cell line (RTgill-W1) as a means of predicting acute toxicity, showing good predictivity on 35 chemicals. To promote regulatory acceptance, the predictivity and applicability domain of novel tests need to be carefully evaluated on chemicals with existing high-quality in vivo data. We applied the RTgill-W1 cell assay to 38 fragrance chemicals with a wide range of both physicochemical properties and median lethal concentration (LC50) values and representing a diverse range of chemistries. A strong correlation (R2 = 0.90-0.94) between the logarithmic in vivo LC50 values, based on fish mortality, and the logarithmic in vitro median effect concentration (EC50) values based on cell viability was observed. A leave-one-out analysis illustrates a median under-/overprediction from in vitro EC50 values to in vivo LC50 values by a factor of 1.5. This assay offers a simple, accurate, and reliable alternative to in vivo acute fish toxicity testing for chemicals, presumably acting mainly by a narcotic mode of action. Furthermore, the present study provides validation of the predictivity of the RTgill-W1 assay on a completely independent set of chemicals that had not been previously tested and indicates that fragrance chemicals are clearly within the applicability domain. Environ Toxicol Chem 2018;37:931-941. © 2017 SETAC.

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial.

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (<4-fold difference for all chemicals). Hepatic clearance rates (CLH; l/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.

p-Alkyl-Benzoyl-CoA Conjugates as Relevant Metabolites of Aromatic Aldehydes With Rat Testicular Toxicity-Studies Leading to the Design of a Safer New Fragrance Chemical.

Several aromatic aldehydes such as 3-(4-tert-butylphenyl)-2-methylpropanal were shown to adversely affect the reproductive system in male rats following oral gavage dose of ≥ 25 mg/kg bw/d. It was hypothesized that these aldehydes are metabolized to benzoic acids such as p-tert-butylbenzoic acid as key toxic principle and that Coenzyme A (CoA) conjugates may be formed from such acids. Here we performed a detailed structure activity relationship study on the formation of benzoic acids from p-alkyl-phenylpropanals and related chemicals in rat hepatocytes in suspension. Formation of CoA conjugates from either p-alkyl-phenylpropanals directly or from their benzoic acid metabolites was further assessed in plated rat hepatocytes using high resolution LC-MS. All of the test chemicals causing reproductive adverse effects in male rats formed p-alkyl-benzoic acids in rat hepatocytes in suspension. Compounds metabolized to p-alkyl-benzoic acids led to accumulation of p-alkyl-benzoyl-CoA conjugates at high and steady levels in plated rat hepatocytes, whereas CoA conjugates of most other xenobiotic acids were only transiently detected in this in vitro system. The correlation between this metabolic fate and the toxic outcome may indicate that accumulation of the alkyl-benzoyl-CoA conjugates in testicular cells could impair male reproduction by adversely affecting CoA-dependent processes required for spermatogenesis. This hypothesis prompted a search for new p-alkyl-phenylpropanal derivatives which do not form benzoic acid metabolites and the corresponding CoA conjugates. It was found that such metabolism did not occur with a derivative containing an o-methyl substituent, ie, 3-(4-isobutyl-2-methylphenyl)propanal. This congener preserved the fragrance quality but lacked the male reproductive toxicity in a 28-day rat study, as predicted from its in vitro metabolism.

Identification of Bilophila wadsworthia by specific PCR which targets the taurine:pyruvate aminotransferase gene.

The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.

Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp.

Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), DL-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate.

A new sulfate-reducing bacterium was isolated from marine sediment with phosphite as sole electron donor and CO(2) as the only carbon source. Strain FiPS-3 grew slowly, with doubling times of 3-4 days, and oxidized phosphite, hydrogen, formate, acetate, fumarate, pyruvate, glycine, glutamate, and other substrates nearly completely, with concomitant reduction of sulfate to sulfide. Acetate was formed as a side product to a small extent. Glucose, arabinose, and proline were partly oxidized and partly fermented to acetate plus propionate. Growth with phosphite, hydrogen, or formate was autotrophic. Also, in the presence of sulfate, CO dehydrogenase was present, and added acetate did not increase growth rates or growth yields. In the absence of sulfate, phosphite oxidation was coupled to homoacetogenic acetate formation, with growth yields similar to those in the presence of sulfate. Cells were small rods, 0.6 - 0.8 x 2-4 microm in size, and gram-negative, with a G+C content of 53.9 mol%. They contained desulforubidin, but no desulfoviridin. Based on sequence analysis of the 16S rRNA gene and the sulfite reductase genes dsrAB, strain FiPS-3 was found to be closely related to Desulfotignum balticum. However, physiological properties differed in many points from those of D. balticum. These findings justify the establishment of a new species, Desulfotignum phosphitoxidans.