Biodegradation Screening Tests Research Articles

Enhanced ready biodegradability screening tests for the evaluation of potential PBT substances

In persistence assessment enhanced ready biodegradation tests (eRBT) are aimed to close the gap between screening tests and complex simulation tests. However, only few data from these tests are available and neither guidance on the design and interpretation of eRBTs, nor suitable validity criteria have been established so far. In a practical testing programme 5 compounds with controversial degradation data have been tested in 4 test series including prolongation to 60 days and use of different inocula (activated sludge, final effluent from a STP), flask sizes, and endpoints (CO2, O2, DOC). The drug ibuprofen and the intermediate 4-fluorophenol were biodegraded by >60% within 28 days within a 10-day-window and therefore are considered as readily biodegradable and in conclusion fulfilling the criteria for “not persistent”. The mean mineralization of the pesticide synergist piperonylbutoxide and the antioxidant octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate achieved 20%–50% (=“potentially P”). The mineralization of the cosmetic ingredient cis-13-docosenonamide (Erucamide) was between 36%–64% after 60 days with activated sludge and 21% with the effluent from the STP. Diethylene glycol reached the pass level of 60% mineralization within 28 days in all test series without always meeting the 10-day window, and thus proved to be a suitable reference substance for eRBTs. Based on the results of the study several recommendations for the test design, the evaluation and the interpretation of eRBTs are made. However, a broader data set is required and further enhancements such as the quality and amount of the inoculum should also be considered in future research.

Biodegradability of Polyvinyl Alcohol Based Film Used for Liquid Detergent Capsules

Abstract Questions and potential misperceptions have arisen about the potential contribution of liquid detergent capsules to the environmental microplastics issue. The film of these detergents is highly water soluble, also in cold water, as it must fully dissolve during every type of washing process. Water-soluble grades of polyvinyl alcohol, the most commonly used detergent capsule film material, are recognised to be biodegradable. In the current paper, adequate biodegradability is confirmed by means of ready biodegradation screening tests, across a range of polyvinyl alcohol detergent grade films. The high water solubility in itself implies that detergent capsule films are not within the microplastic scope. Furthermore, their biodegradability ensures there is no concern for persistence or accumulation in the environment.

Multi-laboratory Validation of a New Marine Biodegradation Screening Test for Chemical Persistence Assessment.

Current biodegradation screening tests are not specifically designed for persistence assessment of chemicals, often show high inter- and intra-test variability, and often give false negative biodegradation results. Based on previous studies and recommendations, an international ring test involving 13 laboratories validated a new test method for marine biodegradation with a focus on improving the reliability of screening to determine the environmental degradation potential of chemicals. The new method incorporated increased bacterial cell concentrations to better represent the microbial diversity; a chemical is likely to be exposed in the sampled environments and ran beyond 60 days, which is the half-life threshold for chemical persistence in the marine environment. The new test provided a more reliable and less variable characterization of the biodegradation behavior of five reference chemicals (sodium benzoate, triethanolamine, 4-nitrophenol, anionic polyacrylamide, and pentachlorophenol), with respect to REACH and OSPAR persistence thresholds, than the current OECD 306 test. The proposed new method provides a cost-effective screening test for non-persistence that could streamline chemical regulation and reduce the cost and animal welfare implications of further higher tier testing.

Increased cell numbers improve marine biodegradation tests for persistence assessment

Currently available OECD biodegradation screening tests (BSTs) are not particularly suited for persistence screening. Their duration can be much less than international half-life thresholds for persistence and they are variable and stringent, therefore prone to false negatives. The present study extended test durations beyond 28 days and increased biomass concentrations for marine BSTs to better represent the microbial diversity inherent in the sampled environment. For this so-called environmentally relevant BST (erBST) marine cell concentrations were nominally increased 100-fold by tangential flow filtration. The marine erBST was validated against a standard BST using five 14C labeled reference compounds with a range of biodegradation potentials (aniline, 4-fluorophenol, 4-nitrophenol, 4-chloroaniline and pentachlorophenol) in a modified OECD 301B test. A full mass balance was collated to follow chemical fate in the tests. The erBST was more accurate and less variable than the comparator BST in assigning the reference compounds to their expected biodegradation classifications (non-persistent or potentially persistent). According to the REACH non-persistence criterion of ≥60% biodegradation over 60 days, the erBST correctly classified 60% of chemical replicates according to their expected biodegradation classification and had a coefficient of variation of 21% between replicates. In contrast, the BST correctly assessed 40% of reference chemicals in regards to their expected biodegradation classification with a coefficient of variation of 36%. All non-persistent chemicals showed increased degradation in the erBST, except for 4-chloroaniline, which did not degrade in either BST or erBST. Both tests showed no false positive results, correctly classifying the negative control pentachlorophenol as potentially persistent. Next, it is recommended to further validate the marine erBST in an inter-laboratory study incorporating different seawater sources to fully assess its variability and reliability.

Monod-based ‘single-data’ strategy for biodegradation screening tests

Environmental contextObtaining biodegradation data over time can be difficult, especially when dealing with environmental compartments of increasing complexity. We evaluated the possibility of obtaining a full biodegradation depletion curve from a single biodegradation-time experimental measurement, and found that environmental information related to potential chemical persistence can be derived. The applicability of this ‘single-data’ strategy is illustrated using simulated and experimental data for several compounds. AbstractInformation obtained from biodegradability tests, e.g. half-life (t50) or kinetics parameters, is relevant in environmental risk assessment of new chemicals. In these tests, the removal of the tested compound is measured over a prefixed period of time (e.g. 28 days in ready biodegradability tests) to derive a substrate depletion curve. The implementation can be time-consuming, costly and difficult, especially when the complexity of the environmental compartment increases. In this work, the possibility of obtaining a full biodegradation depletion curve from a single biodegradation-time experimental data point (‘single-data’ strategy) was evaluated. Monod kinetics are assumed to avoid the limitations related to first-order kinetics (only valid for very low substrate concentrations). Experimental and simulated data were used to illustrate the potential of the proposed strategy. The effects on the estimates of several variables (e.g. Monod kinetics parameters, compound concentration or variability in biodegradation data) and the errors introduced to some of the variables were also evaluated. The results suggest that the proposed strategy can be used as a rapid (based on data measured at day 7) and low-cost screening approach to anticipate the result of a biodegradability test for new chemicals. The applicability and practical limitations of the ‘single-data’ strategy have been illustrated using experimental data for several compounds ranging from readily biodegradable (e.g. benzoic acid, acetylsalicylic acid, p-toluic acid) to potentially persistent compounds (e.g. bupivacaine, p-phenitidine, phtadinitrile).

Implications of microbial adaptation for the assessment of environmental persistence of chemicals

Persistency of organic chemicals is a key property in their environmental risk assessment. Information on persistency is often derived from the results of biodegradability screening tests such as the ready biodegradability tests (RBTs). RBTs are, however, not designed for this purpose and suffer from several problems that lead to a high variability of the results and, hence, to difficulties in their interpretation. The origin and exposure history of the inocula used for biodegradability testing can lead to highly variable outcomes. Microbial adaptation to chemicals and its impact on biodegradation needs further investigation in order to have a better understanding of their effects on persistency assessments of chemicals. It is well described that microbial adaptation stimulates biodegradation of organic chemicals. Several mechanisms responsible for these phenomena have been described, amongst which are i) shifts in community composition or abundances, ii) mutations within populations, iii) horizontal gene transfer or iv) recombination events. These adaptation processes may well be mimicked under laboratory conditions, but the outcome remains difficult to predict as we lack a fundamental understanding of the adaptive responses. This review aims to bring together our current knowledge regarding microbial adaptation and its implication for the testing of biodegradation of chemicals.

Improving the biodegradability in seawater test (OECD 306)

Growth and extensive urbanisation of the human population has been accompanied by increased manufacture and use of chemical compounds. To classify the fate and behaviour of these compounds in the environment, a series of international standardised biodegradation screening tests (BSTs) were developed over 30 years ago. In recent years, regulatory emphasis (e.g. REACH) has shifted from measuring biodegradation towards prioritisations based on chemical persistence. In their current guise, BSTs are ineffective as screens for persistence. The marine BST OECD 306 in particular is prone to high levels of variation and produces a large number of fails, many of which can be considered false negatives. An ECETOC funded two-day workshop of academia, industry and regulatory bodies was held in 2015 to discuss improvements to the marine BSTs based on previous research findings from the Cefic LRI ECO11 project and other foregoing studies. During this workshop, methodological improvements to the OECD 306 test were discussed, in addition to clarifying guidance on testing and interpretation of results obtained from marine BSTs (such as pass criteria, lag phases, freshwater read across and complex substances). Methodologically: (i) increasing bacterial cell concentrations to better represent the bacterial diversity inherent in the sampled environments; and (ii) increasing test durations to investigate extended lag phases observed in marine assessments, were recommended to be validated in a multi-institutional ring test.

High Throughput Biodegradation-Screening Test To Prioritize and Evaluate Chemical Biodegradability.

Comprehensive assessment of environmental biodegradability of pollutants is limited by the use of low throughput systems. These are epitomized by the Organisation for Economic Cooperation and Development (OECD) Ready Biodegradability Tests (RBTs), where one sample from an environment may be used to assess a chemical's ability to readily biodegrade or persist universally in that environment. This neglects the considerable spatial and temporal microbial variation inherent in any environment. Inaccurate designations of biodegradability or persistence can occur as a result. RBTs are central in assessing the biodegradation fate of chemicals and inferring exposure concentrations in environmental risk assessments. We developed a colorimetric assay for the reliable quantification of suitable aromatic compounds in a high throughput biodegradation screening test (HT-BST). The HT-BST accurately differentiated and prioritized a range of structurally diverse aromatic compounds on the basis of their assigned relative biodegradabilities and quantitative structure-activity relationship (QSAR) model outputs. Approximately 20 000 individual biodegradation tests were performed, returning analogous results to conventional RBTs. The effect of substituent group structure and position on biodegradation potential demonstrated a significant correlation (P < 0.05) with Hammett's constant for substituents on position 3 of the phenol ring. The HT-BST may facilitate the rapid screening of 100 000 chemicals reportedly manufactured in Europe and reduce the need for higher-tier fate and effects tests.

Environmentally Relevant Inoculum Concentrations Improve the Reliability of Persistent Assessments in Biodegradation Screening Tests.

Standard OECD biodegradation screening tests (BSTs) have not evolved at the same rate as regulatory concerns, which now place an increased emphasis on environmental persistence. Consequently, many chemicals are falsely assigned as being potentially persistent based on results from BSTs. The present study increased test duration and increased inoculum concentrations to more environmentally relevant levels to assess their impact on biodegradation outcome and intratest replicate variability for chemicals with known environmental persistence. Chemicals were assigned to potential persistence categories based on existing degradation data. These more environmentally relevant BSTs (erBSTs) improved the reliability of persistence assignment by reducing the high variability associated with these tests and the occurrence of failures at low inoculum concentrations due to the exclusion of specific degraders. Environmental fate was determined using a reference set of 14C-labeled compounds with a range of potential environmental persistences, and full mass balance data were collated. The erBST correctly assigned five reference chemicals of known biodegradabilities to their appropriate persistence category in contrast to a standard OECD Ready Biodegradation Test (RBTs, P < 0.05). The erBST was significantly more reproducible than an OECD RBT (ANOVA, P < 0.05), with more consistent rates and extent of biodegradation observed in the erBST.

Current limitations of biodegradation screening tests and prediction of biodegradability: A focus on fragrance substances

Abstract Key parameters of OECD biodegradation screening tests were analyzed for impact on the assessment of biodegradation potential of selected fragrance compounds. Comparison of official Japanese OECD 301C test results for 48 predominantly readily biodegradable chemicals with recent OECD 301F screening data demonstrated a significantly higher occurrence of false negatives potentially attributable to inoculum limitations for the former, while for the latter, restriction to 28 days was frequently limiting. Influence of test concentration was studied for two groups of quaternary carbon-containing compounds under high (OECD 301F) and low (OECD 301D) concentrations and extended incubations. Ionones were generally ultimately biodegradable within 28 days or readily biodegradable, whereas damascones reached ultimate biodegradation in OECD 301D tests only and required extended incubations. Predictions from Biowin and Catalogic models were analyzed for ionones, damascones and other selected structural groups of fragrance compounds. Comparison with previously published experimental data revealed significant differences in results between OECD screening tests, thus illustrating the shortcomings of currently available data collections and the potential risk of training prediction models with false negative results, particularly if used for models that are primarily based on data from one specific test. In addition, the analysis revealed specific limitations for prediction models that cannot take into account positioning of fragments within a structure. As a result, it appears that the presence of more than one unfavorable fragment usually does not allow reaching currently accepted threshold scores that would identify a given compound as biodegradable. The outcome of the analyses of this study underlines the need to take into account results from OECD screening tests that are best adapted to physico-chemical properties of fragrances (hydrophobicity, volatility and inhibition potential at high test concentration) and conducted at reduced stringency compared to OECD guidelines (extended incubation owing to reduced bioavailability). Negative MITI database results should be critically reviewed, particularly for quaternary carbon-containing compounds. For future efforts aimed at designing more environmentally benign chemicals, the apparent underestimations will have to be overcome by providing more realistic experimental data that should subsequently be used to improve current biodegradation prediction models for better guidance.

English

Palm-based DHSA is a new chemical developed in MPOB that has three reactive sites that would enable the preparation of many useful derivatives. New chemicals introduced into the market have to be registered and information on their environmental performance such as biodegradability has to be supplied as required under various national and international laws. Biodegradability screening tests, such as the OECD 301 series, are basic tools for assessment of the environmental fate of chemicals in aquatic environments. A substance is considered readily biodegradable when its biodegradability reaches the pass levels of 60% carbon dioxide (CO2)/theoretical carbon dioxide (ThCO2) evolution, 60% biochemical oxygen demand (BOD)/theoretical oxygen demand (ThOD) or 70% dissolved organic carbon (DOC) removal within 28 days. Palm-based DHSA was found to be readily biodegradable where its biodegradability reached 65% within 28 days test period determined via the OECD 301C Modified MITI (I) test method, which passed the 60% level of BOD/ThOD requirement as stipulated in the OECD Guidelines for testing of chemicals. This result is indicative of DHSA’s rapid degradation in aquatic environments. Hence, DHSA is not expected to accumulate in the food chains and will not pose problems in the aquatic environments although it is poorly soluble in water.

Chemical structure‐based predictive model for methanogenic anaerobic biodegradation potential

Many screening-level models exist for predicting aerobic biodegradation potential from chemical structure, but anaerobic biodegradation generally has been ignored by modelers. We used a fragment contribution approach to develop a model for predicting biodegradation potential under methanogenic anaerobic conditions. The new model has 37 fragments (substructures) and classifies a substance as either fast or slow, relative to the potential to be biodegraded in the "serum bottle" anaerobic biodegradation screening test (Organization for Economic Cooperation and Development Guideline 311). The model correctly classified 90, 77, and 91% of the chemicals in the training set (n = 169) and two independent validation sets (n = 35 and 23), respectively. Accuracy of predictions of fast and slow degradation was equal for training-set chemicals, but fast-degradation predictions were less accurate than slow-degradation predictions for the validation sets. Analysis of the signs of the fragment coefficients for this and the other (aerobic) Biowin models suggests that in the context of simple group contribution models, the majority of positive and negative structural influences on ultimate degradation are the same for aerobic and methanogenic anaerobic biodegradation.

The value of biodegradation screening test results for predicting the elimination of chemicals’ organic carbon in waste water treatment plants

Based on the test results of a large number of chemical substances, the comparability of different biodegradation test methods was assessed in terms of passing or failing threshold values commonly used for the biodegradability characterisation of chemicals. The comparison revealed a high consistency of the ready biodegradability evaluations in OECD 301 tests regardless of the analytical parameter used, i.e., by mineralisation (CO 2 evolution or biological oxygen demand/BOD) or by organic carbon removal measurements. Substances meeting the ready biodegradability threshold values in OECD 301 tests exhibited a high (>80%) carbon removal in model waste water treatment plants (OECD 303A). Results from the Modified Zahn–Wellens test (OECD 302B) representing a screening test with high sludge concentration, were also shown to be useful for the prediction of the organic carbon removal under WWTP conditions provided some prerequisites are met. The analysis of the compared data pairs formed a solid basis for the conclusion that positive results of a chemical in biodegradability screening tests can be used for a reliable prediction of their carbon-based removal in waste water treatment plants.

Behavior of aliphatic alcohol polyethoxylates and their metabolites under standardized aerobic biodegradation conditions

AbstractAerobic biodegradation of aliphatic alcohol polyethoxylates (AEs) was tested under screening test conditions (Organization for Economic Cooperation and Development [OECD] 301E protocol) using recently developed analytic methodologies for the specific determination of AEs and their neutral (polyethylene glycols [PEGs]) and carboxylic (carboxylated PEGs and AEs) aerobic metabolites. Biodegradation screening tests were performed under the same conditions on three typical, commercial AE blends and an individual, linear AE ethoxymer. The linear and monobranched AEs underwent a fast primary biodegradation, whereas the multibranched AEs underwent a slower biodegradation. Based on the formation and oligomeric distribution of PEGs and the lack of detection of other biointermediates before the formation of PEGs, the central cleavage of the AE molecule appeared to be the mechanism for primary biodegradation of the linear and monobranched AEs in the tested commercial blends. As a result, the shorter AE ethoxymers biodegraded faster than the longer ones. No PEGs were detected during biodegradation of the multibranched AEs. In addition, PEGs biodegraded more slowly than the parent AEs and were removed by hydrolysis, thus leading to shorter PEG oligomers, and by oxidative hydrolysis, thus forming carboxylated PEGs.

High-performance liquid chromatographic-fluorescence determination of aliphatic alcohol polyethoxylates and polye(thylene glycol)s in aqueous samples

New derivatization and separation procedures for the routine determination of detergent alcohol polyethoxylates (AEs) and poly(ethylene glycol)s (PEGs) in aqueous environmental and biodegradation screening test samples are proposed. The AE derivatives were separated both homolog-by-homolog or ethoxymer-by-ethoxymer by reversed-phase HPLC-fluorescence (RP-HPLC-FL) after derivatization with naphthyl isocyanate (NIC) and naphthoyl chloride (NC), respectively. The ethoxymer-by-ethoxymer separation of PEGs was obtained by RP-HPLC-FL after derivatization with NC. The fluorescence response of the various AE homologs and AE, PEG ethoxymers were evaluated. AE, PEG as well as nonylphenol polyethoxylates were cumulatively extracted from biodegradation test liquors and sewage-treatment plant influent and final effluent samples by solid-phase extraction on graphitized carbon black (GCB), and then derivatized and analyzed by RP-HPLC-FL. The recovery was systematically >90%, with relative standard deviations of 3–6%, for all compounds considered. The proposed derivatization, separation and detection procedures showed a sensitivity of 5–10 ng of injected AE or PEG derivatives leading to a S/ N ratio of 10.

Metabolic efficiency and turnover of soil microbial communities in biodegradation tests.

Biodegradability screening tests of soil commonly measure 14CO2 evolution from radiolabeled test compounds, and glucose has often served as a positive control. When constant amounts of radiolabel were added to soil in combination with increasing amounts of unlabeled substrates, glucose and some related hexoses behaved in an anomalous manner. In contrast to that of formate, benzoate, n-hexadecane, or bis(2-ethylhexyl) phthalate, dilution of glucose radiocarbon with unlabeled glucose increased rather than decreased the rate and extent of 14CO2 evolution. [14C]glucose incorporation into biomass and Vmax values were consistent with the interpretation that application of relatively high concentrations of glucose to soil shifts the balance of the soil microbial community from the autochthonous (humus-degrading) to the zymogeneous (opportunistic) segment. The higher growth and turnover rates that define zymogeneous microorganisms, combined with a lower level of carbon incorporation into their biomass, result in the evolution of disproportionate percentages of 14CO2. When used as positive controls, glucose and related hexoses may raise the expectations for percent 14CO2 evolution to levels that are not realistic for other biodegradable compounds.

Comparison of 20‐d bod and oecd closed‐bottle biodegradation tests

AbstractThis study correlated the 20‐d Biochemical Oxygen Demand (BOD) test widely used in the United States with the closed‐bottle test selected and refined by the Organization for Economic Cooperation and Development (OECD) for determining biodegradability of a product. Many chemical producers in the United States have 20‐d BOD data on their products; it is important to use this information when valid both in the interim until OECD tests can be completed on these myriad products and in support of the OECD data when available. This paper presents comparative biooxidation data on 16 chemicals using both sealed‐bottle tests. Similar data were obtained at the end of each test, after 20 d in the BOD test and 28 d in the closed‐bottle test. The reduced microbial seed concentration in the closed‐bottle test required longer incubation time to achieve these similar results. The best correlations were obtained at days 10 and 15, and the poorest was indicated at day 5. The environmental classification specified by the European Community is the same for these chemicals, &gt;60% biooxidation for readily biodegradable classification or not, regardless of the biooxidation test. OECD biodegradation screening test data are included in this report; results from this test, which monitors dissolved organic carbon (DOC), support the biooxidation values obtained in both the OECD closed‐bottle test and the 20‐d BOD test.

COMPARISON OF 20-d BOD AND OECD CLOSED-BOTTLE BIODEGRADATION TESTS

This study correlated the 20-d Biochemical Oxygen Demand (BOD) test widely used in the United States with the closed-bottle test selected and refined by the Organization for Economic Cooperation and Development (OECD) for determining biodegradability of a product. Many chemical producers in the United States have 20-d BOD data on their products; it is important to use this information when valid both in the interim until OECD tests can be completed on these myriad products and in support of the OECD data when available. This paper presents comparative biooxidation data on 16 chemicals using both sealed-bottle tests. Similar data were obtained at the end of each test, after 20 d in the BOD test and 28 d in the closed-bottle test. The reduced microbial seed concentration in the closed-bottle test required longer incubation time to achieve these similar results. The best correlations were obtained at days 10 and 15, and the poorest was indicated at day 5. The environmental classification specified by the European Community is the same for these chemicals, >60% biooxidation for readily biodegradable classification or not, regardless of the biooxidation test. OECD biodegradation screening test data are included in this report; results from this test, which monitors dissolved organic carbon (DOC), support the biooxidation values obtained in both the OECD closed-bottle test and the 20-d BOD test.

Degradation of sodium polyglyoxylate, a non-persistent metal sequestrant, in laboratory ecosystems

Detergent builders such as zeolites, silicates and most organic polymers present concerns to some because of their environmental persistence. Sodium polyglyoxylate (SPG), on the other hand, is classified as readily biodegradable in a variety of aerobic and anaerobic biodegradation screening tests because it is extensively mineralized. Its persistence, however, is dependent on its rate of chemical hydrolysis to sodium glyoxylate, which is in turn controlled by parameters such as pH, temperature, metal ions and end capping group. The time for SPG's degradation ranges from a few hours at pH 5 to a few weeks at pH 9. Even though SPG is more persistent at alkaline pH values, it is rendered less bioavailable via precipitation/adsorption mechanisms. SPG's removal from and degradation in practically all ecosystems indicates that it will not have a significant impact on the environment with widespread use.

A method for measuring the biodegradation of organic chemicals

AbstractAn automated gas‐chromatographic measurement of carbon dioxide is described and its application to a biodegradation screening test procedure is demonstrated. The biodegradation test system consists of 20‐ml glass vials containing aqueous solutions of a test chemical and an acclimated microbial inoculum. The vials and their contents are sealed with rubber septa. At specified time intervals, the contents of selected vials are acidified. Subsequent automated headspace determination of carbon dioxide provides a measure of cumulative biological degradation. To demonstrate the validity of the overall test procedure, the biodegradabilities of eight organic compounds were measured: aniline, benzaldehyde, ethylenediaminetetraacetic acid, ethylene glycol, linear alkylbenzene sulfonate, nitrilotriacetic acid, p‐nitrophenol and N‐methylaniline. Biological degradation was also monitored by measuring residual test chemical concentrations. There was excellent internal consistency of results by comparison of carbon dioxide evolution values with specific chemical analyses. Both the results of carbon dioxide measurements and specific chemical analyses are discussed in relation to literature values derived from similar screening test procedures. It is concluded from this study that automated headspace determination of carbon dioxide has several advantages over existing test methods. It is further concluded that application of this headspace procedure in the screening test described provides an accurate, albeit conservative, estimate of biodegradability, with positive results indicating a low potential for environmental persistence.