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Biodegradation in water: screening tests

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No reliable guideline study has been identified, therefore a weight of evidence assessment has been used for this endpoint. Based on a weight of evidence assessment 1, 2, 4-trimethylbenzene does not meet the criteria for ready biodegradability but is considered to be biodegradable and would not be persistent in the environment.

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Additional information

Hutchins (1992) is a non-GLP, non-guideline, experimental study, published in peer reviewed literature. The procedure and means of analysis suggest that the methodology was appropriate to evaluate the biodegradability of 1,2,4-trimethylbenzene under the specific conditions described. The test conditions are very specific and non-standard and therefore should not be considered as showing ready biodegradability of the substance. However, the study is used here in a weight of evidence assessment to conclude on the persistence of the substance in the environment. 1,2,4-trimethylbenzene was biodegraded under denitrifying conditions in an aquifer microcosm to an extent of 96% in 13 days at 12C in the dark.

Marion and Maleny (1963) is a non-GLP, non-guideline, experimental study which predates implementation of GLP and the development of harmonised study guidelines. However, the procedure and means of analysis suggest that the methodology was appropriate to evaluate the required endpoint. As the study did not follow recommended guidelines, the interpretation of the results cannot be fully assessed against the guideline criteria and therefore the result is only an indication of biodegradation properties. However, the study has been used in a weight of evidence assessment to conclude on the persistence of the substance. After 120 hours exposure, 1, 2, 4-trimethylbenzene was significantly biodegraded, with the extent of the biodegradation depending on the source of activated sludge used. The actual results showed biodegradation levels for each source of: Brookside: 12.5%, Hilliard: 34.7% and Columbus 75%. The authors concluded that of all the polymethylated benzenes, 1,2,4-trimethylbenzene was the only one to be significantly biologically oxidized and is more susceptible to oxidation than o-xylene. The study also included aniline, which is used as a reference substance in current guideline studies. Degradation of aniline indicates that the test system was performing acceptably.

Leahy (2003) is a non-GLP, non-guideline, experimental study, published in peer reviewed literature and is considered suitable as part of a weight of evidence assessment. The status of the study’s GLP-compliance is not considered to be applicable as it was not a standard Contract Research Organisation experimental study report but consisted of research undertaken for a published journal article. 1, 2, 4-trimetylbenzene, as part of a condensate resulting from steam classification of solid waste, was 100% degraded in one day at 30ºC by a mixture of bacteria including Pseudomonas putida 1,2,4-TMB-2, a bacteria strain cultured to specifically degrade 1, 2, 4-trimethylbenzene.

CITI (1977) was given a reliability of 4 because the primary source was unavailable for review. The information on the substance was published in the Official Bulletin of Economy, Trade and Industry (formerly: The Official Bulletin of the Ministry of International Trade and Industry) but pre-dates the implementation of GLP. Accordingly the study was classified as non-GLP, information from a reference text, reliability not known but contributing to a weight of evidence assessment. The statement published concluded a judgement of “non-biodegradability” on the basis of a MITI-I (OECD TG 301C) study with improved type test equipment for a volatile substance. The study was conducted over 28 days using concentrations of 100 ppm 1,2,4-trimethylbenzene and 30 ppm activated sludge. The results showed biodegradation levels of 4 - 18% by indirect analysis of BOD and 0% by direct analysis of the test substance using gas chromatography. There is no information on the conditions under which the study was conducted or the source or type of micro-organisms used. No information was provided on the results page describing the adaptation of the test method and equipment for handling volatile substances. Due to the significant limitations in study reporting, the study is considered not to be a reliable source of information.

The use of QSAR estimates to predict the biodegradability of 1,2,4-trimethylbenzene is considered valid as the substance was included in the training set of the BIOWIN model and the validation set of the BioHC model. A structurally similar compound, o-xylene, was also used in the training set for the development of the BIOWIN model and other structurally similar compounds including o-xylene, 1,2,3-trimethylbenzene, 1,3,5-trimethylbenzene, 1,2,3,4-tetramethylbenzene and 1,2,4,5-tetramethylbenzene were used in the training set for the BioHC model, with 1,2,3,5-tetramethylbenzene used in the validation set. The databases were created using experimental biodegradation data for compounds found in crude oil and its products, with biodegradation references obtained for each of these compounds, mainly from the BIOLOG and DATALOG files of EFDB, TOXLINE and American Chemical Society Chemical Abstracts as well as literature searches. For the BioHC model, a single recommended biodegradation half-life was chosen for use in the regression analysis from the primary biodegradation data that were compiled for each hydrocarbon structure. Biodegradation data for o-xylenes and several PAHs were taken from the Syracuse Research Corporation database.

The BIOWIN results page indicates that 1,2,4-trimethylbenzene biodegrades fast (linear and non-linear model prediction) with a primary biodegradation timeframe of days-weeks and an ultimate biodegradation timeframe of weeks-months. The MITI linear and non-linear model predicts the substance to be readily degradable but 1,2,4-trimethylbenzene is estimated not to biodegrade fast under anaerobic conditions and to have an overall ready biodegradability prediction of “no”. As with the other weight of evidence arguments, the results indicate that 1,2,4-trimethylbenzene is not expected to be readily biodegradable, but is expected to degrade in the environment and would not be persistent. The BioHC results estimate the half-life of 1,2,4-trimethylbenzene to be 4.39 days, indicating that the substance would degrade rapidly in the environment.

A screening assessment for persistence can be made using the combined results of some of the Biowin models (ECHA (2012) Guidance on information requirements and chemical safety assessment Chapter R11: PBT assessment). The non-linear biodegradation probability (Biowin 2) for 1,2,4-trimethylbenzene is 0.95 indicating a high probability of the substance being biodegradable and the survey model for ultimate biodegradation (Biowin 3) indicates a degradation time in the order of weeks (2.71). As the prediction of Biowin 2 is >0.5 and that of Biowin 3 is >2.2, the substance is likely to be biodegradable. Similarly, the non-linear biodegradation probability using the MITI model (Biowin 6) is 0.58, also indicating a high probability that the substance is likely to be biodegradable. According to this assessment, 1,2,4-trimethylbenzene is not considered to be persistent in the environment but may not be classified as readily biodegradable.

 

Additional weight of evidence for the biodegradation properties of 1,2,4-trimethylbenzene have been read across from m-, o-, and p-xylene. The data show that m- and p-xylene are readily biodegradable and o-xylene is readily biodegradable but failing the 10 day window. The read across supports the conclusion that although 1,2,4-trimethylbenzene cannot be considered to be readily biodegradable, it is not expected to persist in the environment.

Exxon Biomedical Sciences (1995 and 1996) are GLP compliant, guideline studies following OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test). The non-adapted sludge was exposed to 41 mg/L p-xylene and 36 mg/L o-xylene, respectively, for 28 days and the oxygen consumption was measured. Both o- and p-xylene were degraded by >60% in 28 days under the test conditions, though the validity criteria were not met in either test as there was greater than 20% variability between replicates. Despite this limitation, in the study with p-xylene, all three replicates reached >60% biodegradation by day 11 so this is not considered to have invalidated the results of this test. In the study with o-xylene, only one of the three replicates met the 10 day window criteria and due to the variability between replicates at the start and end of the 10 day window and at the end of the test, o-xylene is considered to degrade rapidly but cannot be considered readily biodegradable.

Bridie et al. (1979) predates the implementation of GLP and the OECD guidelines for biodegradation screening tests. The study authors report that they followed guidelines in operation at the time, although the level of detail in the publication is very limited. In particular the concentration of the test substance is not reported. Despite these limitations, this study can be used as a supporting study as it does demonstrate that p-xylene, o-xylene and m-xylene were biodegraded 44%, 52%, 80% respectively by non-adapted sewage sludge within 5 days. This study is used in the draft SIDS for xylenes.

MITI (2001a, b) have been given a reliability of 4 because the primary source was unavailable for review. The information on the substances was published in the Official Bulletin of Economy, Trade and Industry (formerly: The Official Bulletin of the Ministry of International Trade and Industry). Accordingly the studies were classified as non-GLP, information from a reference text, reliability not known but contributing to a weight of evidence assessment. The statement published concluded a judgement of “biodegradable” for p-xylene and “readily biodegradable” for m-xylene on the basis of the MITI-I (OECD TG 301C) studies. The studies were conducted over 28 days using concentrations of 100 ppm and the results showed biodegradation levels of 38% by indirect analysis of BOD and 92% by direct analysis using gas chromatography of p-xylene and 100% for both direct and indirect analysis for m-xylene. There is no information on the conditions under which the studies were conducted or the source or type of micro-organisms used. Due to the significant limitations in study reporting, the studies cannot be assessed as reliable sources of information, but are used here as part of a weight of evidence assessment.

m-, o- and p-xylene are structurally similar to 1,2,4-trimethylbenzene, consisting of a benzene ring with two and three methyl functional groups attached respectively. The inclusion of an additional methyl functional group on the benzene ring is not expected to change the biodegradation properties of the substance significantly, and this is supported by experimental observations. The similarity in structures, water solubilities and behaviours in the aquatic environment indicate that the biodegradation of the substances is likely to be similar; all substances are expected to be relatively soluble in water, with limited potential to partition to soils or sediment. The water solubility and the log partition coefficient of 1,2,4-trimethylbenzene are 56 mg/L and 3.63 while those of m-xylene are 146 mg/L and 3.2, of o-xylene are 170.5 mg/L and 3.12 and p-xylene are 156 mg/L and 3.15, respectively. The estimated log adsorption coefficient for 1,2,4-trimethylbenzene is 3.04 while the measured value for o-xylene, also read across to m-xylene and p-xylene, is 2.73. m-xylene and p-xylene are considered to be readily biodegradable, while o-xylene is considered to be readily biodegradable but failing the 10 day window. Marion and Melaney (1963) investigated the biodegradation of a range of polymethylated benzenes and concluded that 1,2,4-trimethylbenzene is more susceptible to oxidation than o-xylene. Given the similarity in structure and environmental fate properties, it is concluded that 1,2,4 -trimethylbenzene would not persist in the environment.