Laboratory simulation of biodegradation of chemicals in surface waters: closed bottle and respirometric test

Microbial degradation is the most dominant elimination mechanism of organics from the environment. For evaluation of biodegradability of pure chemicals many standardized tests are available, but no standardized procedure for assessment of biodegradability of chemicals in surface water is agreed upon. Rates of in-situ biodegradation are usually estimated in laboratory simulation where environmental factors are reproduced to some extent. The aim of our study was to compare standardised ready biodegradability assessment, test (Closed bottle test) and its modifications employing the basic agreements on test conditions to simulate biodegradation in surface water. Standard test was modified using various natural river waters to simulate the natural environment in a simplified way. The impact of different types and amounts of nutrients and microorganisms on biodegradation was confirmed. The conditions in the recipient should be examined to extrapolate the results from ready biodegradability tests to real surface water.     

Improving ready biodegradability testing of fatty amine derivatives

This study assesses the biodegradation potential of a number of fatty amine derivatives in tests following the OECD guidelines for ready biodegradability. A number of methods are used to reduce toxicity and improve the bioavailability of the fatty amine derivatives in these tests. Alkyl-1,3-diaminopropanes and octadecyltrimethylammonium chloride are toxic to microorganisms at concentrations used in OECD ready biodegradability tests. The concentration of these fatty amine derivatives in the aqueous phase can be reduced by reacting humic, or lignosulphonic acids with the derivatives or through the addition of silica gel to the test bottles. Using these non-biodegradable substances, ready biodegradability test results were obtained with tallow-1,3-diaminopropane and octadecyltrimethylammonium chloride. Demonstration of the ready biodegradability of the water-insoluble dioctadecylamine under the prescribed standard conditions is almost impossible due to the limited bioavailability of this compound. However, ready biodegradability results were achieved by using very low initial test substance concentrations and by introducing an organic phase. The contents of the bottles used to assess the biodegradability of dioctadecylamine were always mixed. False negative biodegradability results obtained with the fatty amine derivatives studied are the result of toxic effects and/or limited bioavailability. The aids investigated therefore improve ready biodegradability testing.     

The use of inert carriers in regulatory biodegradation tests of low density poorly water-soluble substances

Many poorly water-soluble compounds fail regulatory ready biodegradation tests as the method of test material preparation limits the bioavailability of the chemical. The recognised method for delivery of poorly soluble materials into biodegradability tests consists of coating test material inside the test vessel or onto inert substrates (i.e., glass cover slide, boiling beads, filter paper, or Teflon stir bar) that are placed inside the vessels. Volatile solvents are often used to augment this process. Although these substrates work fairly well for delivering many poorly soluble materials into biodegradability tests, they have not been effective in keeping low density, poorly water-soluble substances in the test medium. Soon after medium is added to the test vessels, these chemicals break loose from the substrates and float on the surface where they have limited contact with micro-organisms in the test medium. Hence, there is a reduced potential for measuring substantial biodegradability in the test. This paper describes the work undertaken to establish a standard method of adding low density, poorly water-soluble substances into test vessels of biodegradability studies to ensure these materials remain in contact with micro-organisms in the test medium. The substances are prepared for testing by adsorption onto silica gel followed by dispersion into the culture medium. This method of delivery may provide greater intra- and inter-laboratory consistency in biodegradability test results for low density, poorly water-soluble substances and it may more closely mimic the probable transport and fate of these substances in the environment.     

Evaluation of the toxicity of substances to be assessed for biodegradability

The ability to distinguish between true non-biodegradability, and inhibition of biodegradability due to the toxic effects of the compound tested present problems. It results in possible false identification of substances as non-biodegradable.Six chemicals were evaluated in a variety of tests to assess their toxicity to aerobic sludge organisms. The tests employed were: BOD₅ and Closed Bottle inhibition tests, inhibition of respiration of activated sludge, growth inhibition of activated sludge, light emission from (MICROTOX test) and the repetitive die-away test (RDA). Results from these tests were compared with results obtained from a number of ready biodegradability tests using the compounds listed above at anticipated non-toxic and toxic concentrations.No test method evaluated consistently forecast toxicity due to the chemicals tested. The MICROTOX and nitrification inhibition tests were too sensitive. A combination of sludge respiration rate and/or growth tests seem most appropriate. To avoid toxicity to the sludge, biodegradability testing should be made at 10% of the EC₅₀ value. Compounds with an EC₅₀ value greater than 300 mg/l are unlikely to be toxic in ready biodegradability tests. Compounds with an EC₅₀ value of less than 20 mg/l may pose problems necessitating the use of the stringent closed bottle test, or the use of C¹⁴ labelled test materials. Compounds with an intermediate EC₅₀ that is between 20–300 mg/l, need to be evaluated at a range of concentrations in biodegradability tests, or may need to be evaluated carefully to define the precise no-effect level. Procedure recommendations to distinguish between the inhibition of biodegradability and inertness of the test substance are made.     

High throughput and miniaturised systems for biodegradability assessments

The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.     

Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems

The primary aerobic and anaerobic biodegradability at intermediate concentrations (50-5000 microg/l) of the antibiotics olaquindox (OLA), metronidazole (MET), tylosin (TYL) and oxytetracycline (OTC) was studied in a simple shake flask system simulating the conditions in surface waters. The purpose of the study was to provide rate data for primary biodegradation in the scenario where antibiotics pollute surface waters as a result of run-off from arable land. The source of antibiotics may be application of manure as fertilizer or excreta of grazing animals. Assuming first-order degradation kinetics, ranges of half-lives for aerobic degradation of the four antibiotics studied were 4-8 days (OLA), 9.5-40 days (TYL), 14-104 days (MET) and 42-46 days (OTC). OLA and OTC were degraded with no initial lag phase whereas lag phases from 2 to 34 days (MET) and 31 to 40 days (TYL) were observed for other substances. The biodegradation behaviour was influenced by neither the concentrations of antibiotics nor the time of the year and location for sampling of surface water. Addition of 1 g/l of sediment or 3 mg/l of activated sludge from wastewater treatment increased the biodegradation potential which is believed to be the result of increased bacterial concentration in the test solution. Biodegradation was significantly slower in tests conducted in absence of oxygen. Assessments of the toxic properties of antibiotics by studying the influence on the biodegradation rates of 14C-aniline at different concentrations of antibiotics showed that no tests were conducted at toxic concentrations.     

Standard inocula preparations reduce the bacterial diversity and reliability of regulatory biodegradation tests

OECD ready biodegradability tests have been central to understanding the biodegradation of chemicals from a regulatory perspective for many decades. They are not fit for contemporary prioritisation of chemicals based on persistence, however, due to the low concentration of inocula used, short duration and high variability between tests. Two OECD standard inoculum pretreatment methods (settlement and filtration) were investigated to observe their effect on the probability of biodegradation and associated changes in bacterial community structure and diversity of inocula sourced from the activated sludge process of wastewater treatment plants. Both settlement and filtration were shown to dramatically and significantly reduce the probability and increase the variability of biodegradation of 4-nitrophenol compared to the use of unprocessed inocula. These differences were associated with a significant hundred-fold reduction in cell numbers and solids content and a significant shift in bacterial community structure that was sometimes accompanied by significant reductions in detectable operational taxonomic unit richness and evenness. The natural variation (between different environments) and variation due to differential selection of bacterial communities (by different pretreatment methods) is offered as an explanation for the historical high variability in standard OECD ready biodegradability tests.     

Biodegradation rates in adapted surface water can be assessed following a preadaptation period with semi-continuous operation

The paper presents a semi-continuous preexposure procedure (SCEP) for use with surface water batch simulation biodegradability tests at low test substance concentrations (0.1-100 microg/l). Simple one step batch tests are normally used first of all for determining "initial rates" characteristic of the water as sampled, as by contrast to "adapted" rates obtained as a result of exposure of the microbial community to the test compound. The aim of the SCEP is to facilitate this adaptation and to become able to estimate reproducible "adapted rates" representing a steady state situation. This is accomplished by maintaining the microbial diversity and a supply of test substance and natural substrates. Conducting a SCEP involves regular renewal of part (e.g. one third) of the test suspension (e.g. every two weeks) adding freshly collected natural water with test compound of the initial concentration. An example study was performed with aniline, 4-nitrophenol, 2,4-dichlorophenoxyacetic acid, 4-chloroaniline, and water from the urban river M?lle?. Following preadaptation lag phases were considerably reduced and much more reproducible than obtained with simple batch tests. In tests at 100 microg/l lag phases for aniline decreased from 5.2 to <1 day, 4-nitrophenol from 10 to <1 day, 2,4-dichlorophenoxyacetic acid from 24 to <1 day, and 4-chloroaniline from 88 to 9 days, respectively. Adapted rates obtained with the SCECP were roughly the same as final rates in simple batch tests with successful adaptation. The adapted rate constant is perceived as an inherent characteristic of the test compound at a specific concentration and under environmental influence (temperature, natural substrates, etc.) but with no simple links back to the original microbial population. By contrast, the initial rates in one step batch tests are determined also by the microbial population initially present in the water sampled.     

An examination of the physical properties,fate, ecotoxicity and potential environmental risks for a series of propylene glycol ethers

Propylene glycol ethers (PGEs) are comprised of mono-, di- and tri-PGEs and several of their acetate esters. The nature of the range of applications that use PGEs suggests that there is a potential for both intentional and unintentional entry of the materials into the environment. Selected physical/chemical properties, fate characteristics, aquatic toxicity data and calculated environmental concentrations were used to assess potential risks from the manufacture, handling, use, and disposal of PGEs. In general, the PGEs are low to moderately volatile, have high aqueous solubilities, low octanol-water partition coefficients (Kow), and bioconcentration factor values of <10, which indicate they are unlikely to accumulate in aquatic food chains. Both abiotic and biological degradation processes reduce environmental concentrations of PGEs. In air, vapor-phase PGEs react with photo-chemically produced hydroxyl radicals and have half-lives ranging from 5.5 to 34.4 h. A variety of ready and inherent biodegradation test methods, as well as tests that simulate biodegradation in wastewater treatment plants, surface water and soil have been conducted on PGEs. Significant aerobic biodegradation was generally observed, with a range of biodegradation half-lives on the order of 5-25 d. Acute aquatic toxicity studies with PGEs resulted in LC50 values ranging from approximately >100 to >20,000 mg/l for freshwater fish, the pelagic invertebrate Daphnia magna, green algae Selenastrum capricornutum (now called Pseudokirchneriella capricornutum) and bacteria. Level 3 multi-media modeling (EQC model of Mackay) was used to simulate regional-scale concentrations of PGEs in air, soil, water, and sediment. Toxicity thresholds were then compared with regional-scale water, soil and sediment concentrations to determine hazard quotients. Based upon this analysis, concentrations of PGEs are unlikely to pose adverse risks to the environment.     

Biodegradation of US premanufacture notice chemicals in OECD tests

Biodegradation testing of commercial chemicals other than pesticides is generally performed using test guidelines of the Organization for Economic Cooperation and Development (OECD). We used test data submitted with US Premanufacture Notifications (PMNs) received from 1995 through 2005 to study performance of OECD biodegradation tests, as well as the overall testing strategy and guidance. Among the findings are that (1) ready biodegradation (RB) tests gave fairly consistent results relative to the pass/fail outcome, but not necessarily percent degradation; (2) the Zahn-Wellens test worked well in providing a quick measure of sorption potential, but aside from this, provided little useful information for the investigated chemicals beyond what was already available from RB tests; (3) the SCAS test sometimes gives lower % removal than continuous-feed simulation tests like OECD 303A; and (4) OECD 306 (marine biodegradation test) appeared less conservative than ordinary RB tests. Overall, the PMN data lend support to new OECD guidance that endorses the primary role of RB tests, but emphasizes simulation rather than inherent biodegradation tests as the next step.     

A method for measuring the anoxic biodegradability under denitrifying conditions

A test for assessing the anoxic biodegradability of organic compounds under denitrifying conditions is proposed. The method is based on the recovery and quantification of the CO2 produced, which is evidence of complete biodegradation of the test compound (added as the sole carbon source). The tests were carried out in a mineral medium, with nitrate as electron acceptor. Whole lake sediments, sediment extracts and a commercial inoculum were assayed as a possible inoculum source by means of glucose biodegradability tests. It was found that the sediment extracts constitute a suitable and environmentally-relevant inoculum source, since they add non-significant amounts of carbon to the tests. Two xenobiotic compounds, namely, aniline and phenol, were tested in the aforementioned conditions as well as in a standard aerobic biodegradability test. Both aniline and phenol attained a biodegradation level higher than 60% in a short time period (<28 days) and thus can be considered as readily biodegradable in denitrifying environments. Nevertheless, the kinetics obtained in the anoxic test were slower than in aerobic conditions, and even suggested the accumulation of intermediate metabolites in the case of phenol. The results of this study indicate that the fate of xenobiotic compounds under anoxic conditions differs from that observed in an oxic environment, and therefore it should be considered by standard biodegradability testing procedures.     

Inocula from activated sludge for ready biodegradability testing: homogenization by preconditioning

The use of activated sludge as inoculum source in ready biodegradability tests (RBT) suffers from several drawbacks related to the heterogeneity of these communities. In this work, the ability of a 7-day aeration period in a mineral medium to homogenize the characteristics of various activated sludges, as suggested by some RBT, was studied. The biodegradation potential of three activated sludge supernatants obtained from different wastewater treatment plants was assessed in terms of cultivable cell density, dehydrogenasic activity and a profile of hydrolytic enzymes. After the preconditioning, the homogenization of these characteristics in the supernatants was observed, as well as a decrease. When preconditioned inocula were used in acetate RBT, the biodegradation kinetics were homogenized. However, some preconditioned supernatants lost their ability to degrade an easily-assimilable xenobiotic compound (aniline) during the observation period, showing the effect of inoculum preconditioning on the behavior of complex bacterial communities, specialist populations (e.g. aniline degraders) being more sensitive than generalist populations (e.g. acetate degraders). These results show that preconditioning cannot be an optional inoculum pretreatment in RBT, and emphasize the importance of further studies focusing on inoculum homogenization.     

A new bioseed for determination of wastewater biodegradability: analysis of the experimental procedure

A new bioassay proposed in the patent P201300029 was applied to a pre-treated wastewater containing a mixture of commercial pesticides to simulate a recalcitrant industrial wastewater in order to determine its biodegradability. The test uses a mixture of standardized inoculum of the lyophilized bacteria Pseudomonas putida with the proper proportion of salts and minerals. The results highlight that biodegradation efficiency can be calculated using a gross parameter (chemical oxygen demand (COD)) which facilitates the biodegradability determination for routine water biodegradability analysis. The same trend was observed throughout the assay with the dehydrated and fresh inoculums, and only a difference of 5 % in biodegradation efficiency (E _f) was observed. The obtained results showed that the P. putida biodegradability assay can be used as a commercial test with a lyophilized inoculum in order to monitor the ready biodegradability of an organic pollutant or a WWTP influent. Moreover, a combination of the BOD₅/COD ratio and the P. putida biodegradability test is an attractive alternative in order to evaluate the biodegradability enhancement in water pre-treated with advanced oxidation processes (AOPs).     

Changes in microbiological metabolism under chemical stress

Chemical stress may alter microbiological metabolism and this, in turn, may affect the natural and engineered systems where these organisms function. The impact of chemical stress on microbiological metabolism was investigated using model chemicals 2,4-dinitrophenol (DNP), pentachlorophenol (PCP), and N-ethylmaleimide (NEM). Biological activity of Pseudomonas aeruginosa was measured in batch systems, with and without stressors at sub-lethal concentrations. Stressor DNP, between 49 and 140 mg l(-1), and PCP, at 15 and 38 mg l(-1), caused decreases in biomass growth yields, but did not inhibit substrate utilization rates. These effects increased with stressor concentrations, showing as much as a 10% yield reduction at the highest DNP concentration. This suggests that a portion of carbon and energy resources are diverted from growth and used in stress management and protection. Stressor DNP, between 300 and 700 mg l(-1), and PCP at 85 mg l(-1) caused decreases in growth yields and substrate utilization rates. This suggests an inhibition of both anabolism and catabolism. Stressor NEM was the most potent, inhibiting biological activity at concentrations as low as 2.7 mg l(-1). These findings will ultimately be useful in better monitoring and management of biological treatment operations and contaminated natural systems.     

Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegradation method at an environmentally relevant concentration

The chemical product diaryl-p-phenylene diamine (DAPD), produced by The Goodyear Tire & Rubber Company as POLYSTAY 100® (CAS 68953-84-4), is employed as an antidegradant in polymers used in tires and industrial rubber products. Previous evaluations pertaining to the ecological fate of DAPD indicated a lack of biodegradative activity in aquatic media. In order to further pursue the biodegradation potential of DAPD, it was deemed necessary to enhance the sensitivity of the aquatic biodegradation assay through (a) employment of a radiotracer of the test substance, and (b) optimisation of conditions for achieving maximal solubilisation of test material in the aquatic media of the incubation vessels. Test vessels were prepared according to the OECD ready biodegradability test guidelines, with DAPD added on silica gel at concentrations of 10 or 100 μg L⁻¹, together with a surfactant to aid solubilisation. After 63 d incubation up to 37% mineralisation was measured and up to 29% of the applied radioactivity was incorporated into cell biomass. Also, after 28 d no DAPD could be measured in solution by radio-TLC and HPLC–MS. These three results demonstrate that the antioxidant DAPD undergoes microbiologically mediated biodegradation and is highly unlikely to persist in the environment.     

Fate and effect of monoalkyl quaternary ammonium surfactants in the aquatic environment

The effect of the alkyl chain of quaternary ammonium-based surfactants on their aquatic toxicity and aerobic biodegradability has been studied. Two families of monoalkylquats surfactants were selected: alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium halides. Acute toxicity tests on Daphnia magna and Photobacterium phosphoreum were carried out and EC50 values in the range of 0.1-1 mg/l were obtained for the two series of cationic surfactants. Although the substitution of a benzyl group for a methyl group increases the toxicity, an incremental difference in toxicity between homologs of different chain length were not observed. Biodegradability of the different homologs was determined not only in standard conditions but also in coastal water, both tests yielding similar results. An increase in the alkyl chain length or the substitution of a benzyl group for a methyl group reduces the biodegradation rate. The degradation of these compounds in coastal waters was associated with an increase in bacterioplankton density, suggesting that the degradation takes place because the compound is used as a growth substrate.     

Assessing the aerobic biodegradability of 14 hydrocarbons in two soils using a simple microcosm/respiration method.

The aerobic biodegradability of 14 hydrocarbons in two soils was determined using a simple microcosm/respirometric method based on oxygen consumption. Biodegradability was assessed indirectly by measuring the depletion of oxygen over time in the headspace of microcosms containing soil and test chemicals. The microcosms consisted of small glass vials fitted with valves that allowed headspace gas samples to be collected, essentially resulting in a sealed system. Respiration data from control microcosms were obtained from identically treated microcosms with no test chemical. Control data were necessarily included in all calculations of percent of theoretical oxygen demand (%ThOD) for any given test chemical. Two experiments were performed to verify this simple biodegradation test method. First, an experiment was performed in which disappearance of n-tetradecane from the microcosms was measured directly by standard soil extraction and analytical techniques while simultaneously performing this simple respirometric method based on %ThOD with the same test chemical. Second, the method was compared to a well-established radiochemical technique using 14C-phenanthrene. Results of both comparisons showed that the method is both accurate and reliable. The consistent manner with which the data were produced in two different soils show that the method is also very reproducible. The method described here provides a simple and inexpensive method for determining the aerobic biodegradability of organic compounds in soils.     

Biodegradation of phenolic mixtures in a sequencing batch reactor

GOAL, SCOPE AND BACKGROUND: In this study, attention was focused on substituted phenols because of their widespread presence in industrial effluents originating from many different sources: they are major constituents of wastewater from coal conversion processes, coke ovens, petroleum refineries and petrochemical industries, resin and fibreglass manufacturing and herbicide production. Moreover, for their characteristics of toxicity to humans and aquatic life (1 mgl(-1) is enough to detect the effects), they are included in the USEPA list of priority pollutants. Toxicity is higher in substituted phenols and is dependent on the nature and numbers of substituent groups. Objective of the present paper is to give a contribution to the modelling of phenolic mixture biodegradation by kinetic studies in which the different compounds are followed separately: this can be easily attained with an experimental apparatus such as the Sequencing Batch Reactor (SBR). Two substituted phenols, 4-nitrophenol (4NP) and 3,4-dimethylphenol (3,4DMP), were utilized as substrates and their degradation kinetics were investigated to evaluate the process parameters both in single compound and in mixture tests. METHODS: Single compound and mixture kinetic tests have been carried out during the reaction phase of the working cycle of the SBR reactor. The single substrates and their mixture were utilized as sole carbon and energy sources. Moreover, in order to verify data reproducibility, all kinetic tests have been carried out in at least two replicates under the same operating conditions. RESULTS AND DISCUSSION: Kinetic data showed the presence of substrate inhibition, to model this experimental evidence the Haldane equation, that is usually employed for substrate inhibited kinetics, was rearranged in a different form with parameters which have a precise meaning in relation to the process kinetics and, at the same time, make the integration procedure easier. The derivation of the equation is shown in an Appendix at the end of the paper. Kinetic parameters obtained are suitable for application. It was observed that the 4-nitrophenol removal rate in single compound tests is significantly higher than the 3,4-dimethylphenol removal rate in the whole range of investigated concentrations (up to 80 mg COD l(-1)). A faster 4-nitrophenol biodegradation was also observed in mixture tests. Moreover, it is worth noting that the two compounds were simultaneously degraded and no diauxic growth was observed. The comparison between single compound and mixture degradation kinetics showed that the 4-nitrophenol degradation rate was comparable in the two cases while a significantly beneficial effect (by increase by about 80% of the maximum removal rate) was detected for 3,4-dimethylphenol degradation in the mixture. CONCLUSIONS: Results of this study showed that the biodegradation kinetics of substituted phenols in mixture can be significantly different from that observed in single compound tests: in fact, the presence of a faster degradable compound (the 4NP) seems to exert a positive effect on the removal of a slower degradable compound (the 3,4DMP). The higher removal rate detected for 4NP, both in single compound and mixture tests, confirmed the key role of the biomass acclimatization in determining the biodegradation kinetics of xenobiotic compounds. The experimental approach and the original method applied for data analysis are of general validity and can be extended to the investigation of different classes of compounds. RECOMMENDATIONS AND PERSPECTIVES: A relevant aspect related to the process applicability is the demonstrated possibility of easily adapting an enriched culture grown on a specific xenobiotic (in our case the 4NP) for the removal of similar single compounds or in mixtures. When biological process are considered for xenobiotic removal, this suggests a possible strategy of developing enriched cultures on target compounds that can be efficiently utilized on more complex matrices with reduced start up and acclimatization periods.