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Environmental fate & pathways

Biodegradation in water: screening tests

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Peer reviewed publications are used to show the wide distribution (aerobic and anaerobic environments) of dichloromethane degrading micro-organisms, the very high growth rate of competent micro-organisms, and the evidence of ultimate degradation of dichloromethane which leads to the conclusion that dichloromethane should be classified as readily biodegradable. A biodegradation percentage of 68%  at day 28 in a valid GLP ready biodegradability test (closed bottle test; OECD 301D) confirms the ready classification.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Aerobic and anaerobic biodegradation of dichloromethane has been studied very comprehensively. Degradation can be easily demonstrated with appropriate methodology. A ready biodegradability test result informs about the following aspects of biodegradation a) ultimate (complete) biodegradation by micro-organism capable of utilizing the test substance as sole carbon and energy source, b) rate of biodegradation by micro-organisms growing on dichloromethane and c) the number and occurrence of competent micro-organisms present in “unacclimated” ecosystems and biological treatment plants. Below all three aspects are dealt with to enable classification using evidence presented in peer reviewed scientific journals. Subsequently, the results from two ready biodegradability test guideline studies with dichloromethane are presented which confirm the classification based on scientific evidence. Finally, scientific evidence (peer reviewed) of anaerobic dichloromethane biodegradation is given.

A) ultimate (complete) biodegradation; ultimate biodegradation of dichloromethane has been proven with pure culture studies The micro-organisms utilise dichloromethane as sole carbon and energy source. These bacteria utilize dichloromethane with stoichiometric release of chloride (Brunner et al. 1980; Emanuelsson et al. 2009, Nikolausz et al. 2005, Janssen et al. 1995, Leisinger et al. 1994). Mineralisation of dichloromethane is initiated in the presence of reduced glutathione by a dichloromethane dehalogenase (Figure, see attachment). This enzyme produces the intermediate S-chloromethylglutathione and HCl from dichloromethane. The intermediate is spontaneously converted to S-hydroxymethylglutathione which breaks down to formaldehyde, HCl and reduced glutathione (Stucki et al. 1981). The formaldehyde is further mineralized by formaldehyde dehydrogenase via formate to carbon dioxide. Two different groups of dichloromethane dehalogenases have been identified. Dichloromethane dehalogenases of group A have a low catalytic activity but upon induction will represent 20 to 50 % of the suspended protein(Gisi et al. 1998). Dichloromethane dehalogenases of group B are almost six times more active than dichloromethane dehalogenases of group A (Vuilleumier and Leisinger 1996).

The proposed biodegradation pathway and the chlorine mass balances demonstrate that dichloromethane is ultimately biodegraded by micro-organisms. Complete (ultimate) biodegradation in OECD 301 tests results in biodegradation percentages of ≥60.


B) rate of biodegradation;The maximum growth rates of the bacteria isolated ranged from 0.028 to 0.22 h-1(Stucki et al. 1981; Galli and Leisinger 1985; Scholtz et al. 1988; Kästner 1989; Emanuelsson et al. 2009; Brunner et al. 1980). Painter and King (1983) used a model based on the Monod equation to interpret the biodegradation curves in ready biodegradability tests. According to this model growth rates of competent micro-organisms of 0.025 h-1or higher will result in a ready biodegradation of the test substance (degradation within 28 days).

Dichloromethane biodegrades completely under aerobic conditions using micro-organisms originating from activated sludge plants within 6 hours to 7 days (Klecka 1982; Stover and Kincannon 1983; Davis et al. 1981). Activated sludge treatment was rapidly acclimated to dichloromethane removing dichloromethane influent concentrations of up to 100 mg/L. The time required to obtain acclimatization was less than 2 weeks (Klecka, 1982). Davis et al (1981) did find comparable biodegradation potentials for benzene and dichloromethane in their experiments. Benzene is regarded as a readily biodegradable substance. Stover and McKinnon (1983) operated their activated sludge units at sludge retention times of ≤6 days. Biodegradation of dichloromethane in systems with these low sludge retention times shows the high biodegradation potential of dichloromethane. The median value for biodegradation rate constant in soil of dichloromethane is 0.05 day-1(Davis and Madsen 1991). This corresponds to a half-life of 14 days, which is well below the default half-live of 30 days for ready biodegradable substances.

C) Number of competent micro-organisms (ubiquitousness); Dichloromethane utilizing bacteria have been found in soils, sub-soil, an estuary, air and wastewater treatment plants through the use of enrichment cultures. The following organisms were isolated from waste water treatment plants processing domestic or industrial waste water; a Hyphomicrobiumsp. KDM2(Kästner 1989), a Xanthobactersp. strain TM1 (Emanuelsson et al. 2009) and a Lysinibacillus sphaericus (Wu et al. 2009). Hyphomicrobium sp. KDM4 was enriched from contaminated groundwater (Kästner 1989). Pseudomonas DM1 enriched in a chemostat originated from air (Brunner et al. 1980). A Pseudomonas sp., a Brevundimonas sp. and an Acinetobacter sp were isolated from estuarine waters (Krausova et al. 2006). This range of microbial genera indicates a high potential for dichloromethane degradation. These findings demonstrate the ubiquitousness of micro-organisms capable of degrading dichloromethane. The aerobic isolates are also able to grow on methanal and formate, both readily biodegradable substances also indicating that dichloromethane degrading micro-organisms occur in the environment.

Biodegradation guideline studies. Two ready biodegradation guideline studies are available for dichloromethane. Biodegradation percentages of 9 -26% were achieved in an OECD 301C MITI (I) test (CITI, 1985). The MITI test is less reliable for volatile substances (table I, OECD Guideline for testing chemicals, ready biodegradability (1992)). No precaution measures to prevent volatilization are reported therefore the low biodegradation percentages can probably be explained due to losses of dichloromethane. A more suitable ready biodegradability test for volatile substances is the Closed Bottle test (OECD 301D). Dichloromethane was biodegraded 68% at day 28 in the Closed Bottle test (Ginkel van, 2012). Over 60% biodegradation was achieved in a period of approximately 10 days immediately following the attainment of 10% biodegradation. The 14 -day window criteria was therefore met. Hence, dichloromethane should be classified as readily biodegradable.

Anaerobic biodegradation. Dichloromethane utilizing bacteria have been also found in the anaerobic environment. An anaerobic enrichment culture biodegrading dichloromethane was obtained from a fixed bed packed with activated charcoal treating continuously an industrial polluted anaerobic groundwater (Stromeyer et al. 1991; Braus-Stromeyer et al. 1993). Dichloromethane degradation, with the inoculum derived from an anaerobic digester, by an enrichment culture under methanogenic conditions was shown by Freedman and Gosset (1991). Nonmethanogenic organisms mediated the dichloromethane degradation oxidizing a part to CO2 and fermenting the remainder to acetate (acetogens). Methanogens in the enrichment culture then converted the products of dichloromethane degradation to CH4. Magli et al 1998 investigated the metabolism of dichloromethane by a pure culture of Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts. Dehalobacterium formicoaceticum is a strictly anaerobic gram-positive bacterium that exclusively utilizes dichloromethane as a growth substrate and ferments this compound to formate and acetate.