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A dynamic river model for biodegradability studies: investigations with selected aromatic compounds at low concentrations and comparison with aquatic batch tests.

The objective of this publication is to present a new dynamic aerobic biodegradation test method simulating a river. A laboratory cascade test system and standardized batch shake flask tests were used for biodegradation studies with the non-volatile and non-sorbing model compounds 2,4-dinitrophenol, naphthalene-1-sulphonic acid and sulphanilic acid. To be closer to the often very low concentrations of substances in the environment the concentrations of the compounds used were standard test concentrations and lower. 14C labelled compounds were measured at 50 micrograms/l, capillary electrophoresis at 5000 micrograms/l and the removal of dissolved organic carbon at 50000 micrograms/l. The test results obtained confirmed the known ultimate biodegradability of the test compounds and showed that biodegradation degrees, rates and degradation durations depended on the test systems, the concentrations of test compounds and the inocula. The river model is a suitable simulation test for natural dynamic surface waters which can be used to perform biodegradability studies at low test concentrations if adequate analytical tools, preferably radioactive-labelled substances, are available.

2,4-Dinitrophenol↗

Adsorption of polycyclic aromatic hydrocarbons (PAHs) by soil particles: influence on biodegradability and biotoxicity.

Polycyclic aromatic hydrocarbon (PAH) biodegradation was investigated in contaminated soils from two different industrial sites under simulated land treatment conditions. Soil samples from a former impregnation plant (soil A) showed high degradation rates of PAHs by the autochthonous microorganisms, whereas PAHs in material of a closed-down coking plant (soil B) were not degraded even after inoculation with bacteria known to effectively degrade PAHs. As rapid PAH biodegradation in soil B was observed after PAHs were extracted and restored into the extracted soil material, the kind of PAH binding in soil B appears to completely prevent biodegradation. Sorption of PAHs onto extracted material of soil B follows a two-phase process (fast and slow); the latter is discussed in terms of migration of PAHs into soil organic matter, representing less accessible sites within the soil matrix. Such sorbed PAHs are suggested to be non-bioavailable and thus non-biodegradable. By eluting soil B with water, no biotoxicity, assayed as inhibition of bioluminescence, was detected in the aqueous phase. When treating soil A analogously, a distinct toxicity was observed, which was reduced relative to the amount of activated carbon added to the soil material. The data suggest that sorption of organic pollutants onto soil organic matter significantly affects biodegradability as well as biotoxicity.

Adsorption↗

Toxicological assessment of biodegraded pentachlorophenol: Microtox and fish embryos.

A Gram-negative bacterium, Pseudomonas sp. strain SR3, was isolated from soil at a former wood treatment plant in north central Florida. The ability of this bacterium to degrade pentachlorophenol (PCP) was confirmed by growing cells in a basal salts medium in which PCP was the only source of carbon and energy. Degradation from a measured concentration of 39-40 micrograms PCP/ml to 0.0006 micrograms PCP/ml was observed within 120 h of incubation in the presence of PCP-induced cells of Pseudomonas sp. strain SR3. The initial cell density in these cultures was 6 x 10(6) cfu/ml. Microtox 5 min EC50 toxicity tests revealed that aqueous solutions of PCP, measured concentrations 39-40 micrograms/ml were toxic but that final biodegraded samples, 0.0006 micrograms PCP/ml were nontoxic. However, bioassays with embryonic inland silversides, Menidia beryllina, showed that the biodegraded samples were embryotoxic or teratogenic. Water containing added PCP at concentrations up to 30 times higher than measured in the final biodegraded samples was less toxic/teratogenic. These results indicate that while biodegradation of PCP was nearly complete, intermediate metabolites of the degradation process or undegraded impurities in PCP were toxic or teratogenic. Thus, the M. beryllina bioassay allows extremely sensitive assessment of toxicity associated with biodegraded environmental pollutants and may be a useful criterion for determining whether bioremediated water or soil is safe for discharge back into the environment.

Abnormalities, Drug-Induced↗

Substrate availability in phenanthrene biodegradation: transfer mechanism and influence on metabolism.

The mechanism of phenanthrene transfer to the bacteria during biodegradation by a Pseudomonas strain was investigated using a sensitive respirometric technique (Sapromat equipment) allowing the quasi-continuous acquisition of data on oxygen consumption. Several systems of phenanthrene supply, crystalline solid and solutions in non-water-miscible solvents (silicone oil and 2,2,4,4,6,8,8-heptamethylnonane) were studied. In all cases, analysis of the kinetics of oxygen consumption demonstrated an initial phase of exponential growth with the same specific growth rate. In order to analyze the second phase of growth and phenanthrene degradation, a study of the kinetics of phenanthrene transfer to the aqueous phase was conducted by direct experimentation, with the crystal and silicone oil systems, in abiotic conditions. The data allowed the validation of a model based on phase-transfer laws, describing the variations, with substrate concentrations, of rates of phenanthrene transfer to the aqueous phase. Analysis of the biodegradation curves then showed that exponential growth ended in all cases when the rates of phenanthrene consumption reached the maximal transfer rates. Thereafter, the biodegradation rates closely obeyed, for all systems, the transfer rate values given by the model. These results unambiguously demonstrated that, in the present case, phenanthrene biodegradation required prior transfer to the aqueous phase. With the silicone oil system, which allowed high transfer and biodegradation rates, phenanthrene was directed towards higher metabolite production and lower mineralization, as shown by oxygen consumption and carbon balance determinations.

Biodegradation, Environmental↗

Primary biodegradation of amine oxide and quaternary ammonium amphiphiles.

Biodegradation of two amphiphilic "soft" antimicrobially active derivatives of lauric (dodecanoic) acid, a quaternary ammonium salt and an amine oxide bearing an amide or ester group, was followed using microorganisms from activated sludge. Primary biodegradation was determined by ion-selective electrodes, total biodegradation as the chemical oxygen demand. Though organic ammonium salts quickly undergo primary biodegradation, the rest of the molecule is difficult to destroy. In contrast, amine oxides are easily biodegradable.

Amines↗

Elucidation of n-butyl benzyl phthalate biodegradation using high-performance liquid chromatography and gas chromatography-mass spectrometry.

n-Butyl benzyl phthalate (BBP) is an endocrine-disrupting chemical. A bacterium species capable of using BBP as the sole source of carbon and energy was isolated from mangrove sediment. Effects of BBP concentration, pH, temperature, and salinity on BBP biodegradation were studied. The optimum pH, temperature, and salinity for the BBP biodegradation were 7.0, 37 degrees C, and 15 g L(-1), respectively. BBP was completely degraded within 6 days under optimum conditions, and the biodegradation of BBP could be fitted to a first-order kinetic model. The major metabolites of BBP biodegradation were identified as mono-butyl phthalate, mono-benzyl phthalate, phthalic acid, and benzoic acid by using high-performance liquid chromatography and gas chromatography-mass spectrometry. A preliminary metabolic pathway was proposed for the biodegradation of BBP.

Benzoic Acid↗

The effects of cyanobacterial exudates on bacterial growth and biodegradation of organic contaminants.

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.

2,4-Dichlorophenoxyacetic Acid↗

Anaerobic biodegradability of alkylphenols and fuel oxygenates in the presence of alternative electron acceptors.

Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l(-1)) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l(-1)) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge.

Anaerobiosis↗

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture.

The biodegradation of tributyl phosphate (Bu3-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu3-P biodegradation by growing cells is described. Growth at the expense of Bu3-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu3-P biodegradation was optimal at 30 degrees C, reduced at 20 degrees C and negligible at 4 degrees C and 37 degrees C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu3-P biodegradation significantly, but nitrate had no effect. Sulphate (10-100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 mumol h-1 mg protein-1 and 37 mumol h-1 mg protein-1 respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu3-P hydrolysis in the presence of 35 mM SO(4)2-. At pH 4.5, 79% of the UO2(2)+ was removed at a flow rate of 1.4 ml/h on a 7-ml.test column.

Anions↗

Kinetics of biodegradation of gasoline and its hydrocarbon constituents.

Aerobic biodegradation of gasoline and its constituents, benzene, toluene and ethylbenzene were studied by an enrichment from soil indigenous microbial population. The enrichment culture completely degraded 16.1-660 mg/l gasoline in 2.5-16 days respectively, without accumulation of any by-products. The kinetics of gasoline as well as benzene, toluene and ethylbenzene biodegradation was investigated with initial gasoline concentrations of 16.1-62.6 mg/l. The maximum specific rates of biodegradation of benzene, toluene and ethylbenzene were 0.12, 0.38 and 0.19 mg mg biomass-1 day-1 respectively. When benzene and toluene were used as sole substrate, the maximum specific rates of their biodegradation were 62.9 and 16.4 times greater than the corresponding values for a mixture (gasoline). The microbial culture was able to mineralize up to 200 mg/l pure toluene and benzene. Maximum mineralization efficiencies of benzene and toluene were 76.7 +/- 5.1% and 76.8 +/- 1.3% respectively. Self-inhibition and competitive inhibition patterns were observed during the biodegradation of benzene and toluene alone and in the mixture respectively. The observed kinetics was modeled according to Andrews' inhibition model.

Benzene↗

Biodegradation and bioremediation of hydrocarbons in extreme environments.

Many hydrocarbon-contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, high salt concentrations, or high pressure, Hydrocarbon-degrading microorganisms, adapted to grow and thrive in these environments, play an important role in the biological treatment of polluted extreme habitats. The biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated and nitrated compounds, has been shown to occur in various extreme habitats. The biodegradation of many components of petroleum hydrocarbons has been reported in a variety of terrestrial and marine cold ecosystems. Cold-adapted hydrocarbon degraders are also useful for wastewater treatment. The use of thermophiles for biodegradation of hydrocarbons with low water solubility is of interest, as solubility and thus bioavailability, are enhanced at elevated temperatures. Thermophiles, predominantly bacilli, possess a substantial potential for the degradation of environmental pollutants, including all major classes. Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-polluted desert soil. Some studies have investigated composting as a bioremediation process. Hydrocarbon biodegradation in the presence of high salt concentrations is of interest for the bioremediation of oil-polluted salt marshes and industrial wastewaters, contaminated with aromatic hydrocarbons or with chlorinated hydrocarbons. Our knowledge of the biodegradation potential of acidophilic, alkaliphilic, or barophilic microorganisms is limited.

Bacteria↗

On-line estimation of biodegradation in an unsaturated soil.

The objective of this study was to develop a model-based estimator of biodegradation in unsaturated soil. This would allow real-time assessment of the efficiency of treatment bioprocesses, such as bioventilation and biopile, and eventually permit optimization through the implementation of control strategies. Based on a reduced-order model, an asymptotic observer was designed to estimate on-line the contaminant concentration, using carbon dioxide measurement. Two observer-based estimators were built to approximate: (1) the specific microbial growth rate; and (2) the biocontact kinetics representing the soil resistance to contaminant biodegradation. State observers and parameter estimators were confronted with the experimental results of biodegradation in microcosms. Hexadecane was used as the model compound, representing petroleum hydrocarbons. Three water contents, corresponding to 20%, 50% and 80% of the water-holding capacity, were tested. The asymptotic observer is able to predict hexadecane depletion with an error on the overall time trajectories of 13%, 8% and 4% for the dry, intermediate and wet soils, respectively, which is acceptable given that all the biokinetic parameters were identified from a biodegradation experiment in liquid phase. The observer-based estimator of the specific microbial growth rate, based on the CO(2) measurement, was successfully calibrated using the off-line measurements of hexadecane as validation data, and allowed estimation of the time when biodegradation switched from a microbial to a biocontact limitation. The biocontact kinetics was also identified on-line, using an estimator based on the hexadecane not in biocontact. These results are very encouraging with respect to the potential for on-line assessment of the performance of treatment bioprocesses in unsaturated soils.

Alkanes↗

The influence of structural components of alkyl esters on their anaerobic biodegradation in marine sediment.

Ester-based organic compounds are one type of synthetic base fluid added to drilling mud used during off-shore oil-drilling operations in the Gulf of Mexico. Concern over the environmental impact of synthetic base fluid (SBF) contaminated rock cuttings discharged into the Gulf of Mexico has prompted the promulgation of EPA regulations requiring that all SBF be tested for biodegradability in marine sediment prior to their use in the Gulf. In order to allow the design or selection of suitably biodegradable esters, the anaerobic biodegradability of a variety of ester compounds was tested using a marine sediment inoculum to reveal the effect of: (a) increasing the chain length of the acid moiety, (b) increasing the chain length of the alcohol moiety; (c) alternating the relative size of the alcohol and acid moieties, (d) branching in the alcohol moiety, and (e) the presence of an unsaturated bond in the acidic moiety. The chemical structure of esters was found to affect the completeness and rate of anaerobic biodegradation, and would affect their ability to be certified for use as an SBF in the Gulf of Mexico. Recommendations for ester usage include using esters that have a total carbon number of between 12 and 18 and avoiding the use of branched alcohols (or acids by inference). The presence of an unsaturated bond in the acid (or alcohol by inference) increased biodegradability of the ester.

Biodegradation, Environmental↗

Estimation of kinetic rate constants for biodegradation of chemicals in activated sludge wastewater treatment plants using short term batch experiments and microgram/L range spiked concentrations.

Biodegradation rate constants that are believed to be predictive for activated sludge sewage treatment plants have been determined at microgram/L concentration levels using short term (hours) laboratory scale batch experiments with activated sludge. Rate constants were estimated for four model chemicals with widely different biodegradability characteristics, and experiments were conducted with sludges of various origin and treatment. Test substances were applied at concentrations ranging from a few microgram/L for deriving first order rate constants and up to several mg/L for full investigation of the kinetics. Model substances were acetate, aniline, 4-chloroaniline and pentachlorophenol and their biodegradation was assessed by means of 14C tracer technique. Some experiments included test concentrations equal to those prescribed in standard biodegradability tests (20 mg DOC/L). Sludge types investigated included adapted and non-adapted sludge from laboratory scale semicontinuous reactors as well as sludges collected from a pilot scale sewage treatment plant loaded with predominantly domestic sewage. At low chemical concentrations ( < approx. 100 micrograms/L) first order degradation rate constants were reasonably constant and varied only little with the applied concentration. With aniline, however, elimination rates increased at concentrations below about 20 micrograms/L, probably because transient sorption became significant. At higher concentrations absolute (linear) degradation rates could be described by saturation kinetics, and for aniline a half saturation constant, K(S), was estimated at 3 mg/L. "Best estimates" of average first order rate constants in the low concentration regime measured with 3 g SS/L and at 22 degrees C were: acetate, 8 h-1; aniline, 0.8 h-1, 4-chloroaniline, 0.15 h-1, and pentachlorophenol, 0.01 h-1 (non adapted sludge) or 0.02 h-1 (adapted sludge). These figures seem to agree well with standard or default biodegradation rate constants for sewage treatment plants suggested in a European Union technical guidance document for chemical risk assessment, which is currently under preparation.

Acetates↗

Carbon dioxide recovery in ready biodegradation tests: mass transfer and kinetic considerations.

The kinetics and efficiency of carbon dioxide recovery in modern versions of the Sturm Ready Biodegradation Test were examined to determine the ability of CO2 evolution measurements to accurately estimate the rate and extent of ultimate biodegradation (mineralization). Kinetic data were analyzed by nonlinear regression techniques using an automated curve-fitting package available from commercial sources. The kinetics of CO2 recovery in standard 3.8 L glass carboys containing 2 L of medium were rapid when headspace aeration (approximately 6 ml/min) and moderate agitation (140 rev/min) on a rotary platform shaker were used to ensure adequate aeration and mixing. The time (half-life) for 50% CO2 recovery in external base traps was 4-5 hours, and stoichiometric recoveries of CO2 equivalents added as bicarbonate were obtained within 24 hours. The kinetics of CO2 evolution during biodegradation of several test compounds were significantly slower than the kinetics of CO2 recovery, with half-lives between 65 and 191 hours. Our results indicate that mass transfer limitations do not impact CO2 recoveries or biodegradation kinetic measurements in modern versions of the Sturm Test, even in test vessels with relatively low surface area to volume ratios (1:1). The use of headspace aeration and mixing generates reliable kinetic data, which can be analyzed by commercially-available nonlinear regression packages to provide rate information for the classification of chemicals with different biodegradation profiles.

Biodegradation, Environmental↗

Expert systems survey on biodegradation of xenobiotic chemicals.

To determine the feasibility of developing an expert system for biodegradability assessment, a survey was conducted in which biodegradation experts were asked to estimate rates and products of degradation for 50 chemicals. These chemicals, which varied widely in structure, were considered representative of the spectrum of premanufacture notice chemicals subject to EPA review under the Toxic Substances Control Act. There was substantial agreement among the 22 experts on both sites of initial attack and rates of degradation. The approximate order in which various groups were viewed as contributing to aerobic biodegradability is as follows: ester, amide, anhydride greater than hydroxyl greater than carboxyl, epoxide, site of unsaturation greater than benzene ring, methyl, methylene. Hydrolyzable groups, azo bonds, halogens, and nitro groups were preferred sites of anaerobic attack. Among the negative influences on aerobic biodegradability were molecular mass, branching, halogenation, and nitrogen heterocycles. Results also indicate that estimates of removal by biodegradation in aerobic wastewater treatment and time for aerobic ultimate and primary degradation were well correlated, and that the predictive value of such correlations could be improved using correction factors for certain classes of chemicals. The results lend support to existing rules of thumb, but also offer additional insight that will prove useful in designing a prototype system.

Biodegradation, Environmental↗

Quantitative structure-activity relationships for biodegradation.

Quantitative structure-activity relationships (QSARs) between biodegradation rates of organic compounds and chemical structure parameters are reviewed. Although a number of such relationships have been developed, they in general only apply to restricted ranges of compounds, limiting their value as predictors of biodegradation rates. For many of these classes of chemicals relationships have been reported with different structural descriptors, varying from macroscopic physical properties to molecular structure parameters. More information on the mechanism and rate-determining steps of biodegradation, which can lead to a better-founded choice of descriptors, and more biodegradation rate data are required to further develop QSARs for biodegradation.

Biodegradation, Environmental↗

Screening methods for assessment of biodegradability of chemicals in seawater--results from a ring test.

An international ring test involving 14 laboratories was organized on behalf of the Commission of the European Economic Communities (EEC) with the purpose of evaluating two proposed screening methods for assessment of biodegradability in seawater: (a) a shake flask die-away test based primarily on analysis of dissolved organic carbon and (b) a closed bottle test based on determination of dissolved oxygen. Both tests are performed with nutrient-enriched natural seawater as the test medium and with no inoculum added other than the natural seawater microflora. The test methods are seawater versions of the modified OECD screening test and the closed bottle test, respectively, adopted by the Organization for Economic Cooperation and Development (OECD) and by the EEC as tests for "ready biodegradability." The following five chemicals were examined: sodium benzoate, aniline, diethylene glycol, pentaerythritol, and 4-nitrophenol. Sodium benzoate and aniline, which are known to be generally readily biodegradable consistently degraded in practically all tests, thus demonstrating the technical feasibility of the methods. Like in previous ring tests with freshwater screening methods variable results were obtained with the other three compounds, which is believed primarily to be due to site-specific differences between the microflora of the different seawater samples used and to some extent also to differences in the applied concentrations of test material. A positive result with the screening methods indicates that the test substance will most likely degrade relatively rapidly in seawater from the site of collection, while a negative test result does not preclude biodegradability under environmental conditions where the concentrations of chemicals are much lower than the concentrations applied for analytical reasons in screening tests. Nevertheless, the screening tests are considered useful and cost-effective tools for an initial assessment of biodegradability in marine environments.

Aniline Compounds↗