Tetrachloromethane in the living environment: recalcitrance, toxicity and transformation
Tetrachloromethane (carbon tetrachloride, CCl4) is a volatile chlorinated solvent with biocidal properties, which has been used widely over decades as an industrial degreasing agent, as a pesticide, for dry cleaning and in fire extinguishers (Doherty, 2000). It is toxic and predicted to be carcinogenic, with deleterious effects on stratospheric ozone (Table 1). As a consequence, commercial production and use of CCl4 has been progressively restricted. Its use as a pesticide and grain fumigant was banned in 1986 (ITRC-In Situ Bioremediation Team, 2002). The Montreal protocol on substances that deplete the ozone layer (1987) and its four amendments (London, 1990; Copenhagen, 1992; Montreal, 1997; Beijing, 1999) have implemented a complete phase-out of the use of CCl4, by 1996 for developed countries and by 2010 for developing countries [United Nations Environment Programme (UNEP), 2006]. Currently, CCl4 is still produced, but only as an intermediate in the production of other chemical compounds. Prolonged large-scale use of CCl4 has led to substantial soil and subsurface aquifer contamination and CCl4 is at the top of the priority list of hazardous groundwater contaminants (Knox & Canter, 1996). With an estimated half-life for abiotic hydrolysis of 7000 years in water at 20 °C (Vogel et al., 1987), CCl4 is highly persistent in the environment compared with other halogenated aliphatic compounds. In the case of dichloromethane, for example, published estimates range from 1.5 to 704 years (Vogel et al., 1987). Moreover, the low water solubility of CCl4 (Table 1) leads to its accumulation in subsurface aquifers as a poorly bioavailable, dense non-aqueous-phase liquid (DNAPL), which dissolves only slowly into groundwater (ITRC-In Situ Bioremediation Team, 2002).
|Natural||Marine algae, oceans, volcanoes, drill wells. Mean concentrations in volcanic gases: 2.0 ± 1.0 p.p.b.||Isidorov et al. (1990), Butler et al. (1999), Gribble (2003)|
|Anthropogenic*||Industrial production. Net production: +73 000 ton (1990); +48 000 ton (2000); −9500 ton (2007)||UNEP website, http://ozone.unep.org/Data_Reporting/Data_Access/|
|Toxicity for human health||Classified in group 2B (possibly carcinogenic; nongenotoxic; causes hepatic, renal and neurological damage)||IARC (1999), WHO (2004)|
|Drinking water guideline value||4 μg L−1||WHO (2004)|
|Subsurface half-life||7000 years (hydrolysis)||Vogel et al. (1987)|
|Stratospheric lifetime||34 ± 5 years (photolysis)||Allen et al. (2009)|
|Atmospheric concentration||100–130 p.p.t.||Allen et al. (2009)|
|Global warming potential (GWP)†||1400||Allen et al. (2009)|
|Ozone-depleting potential (ODP)‡||1.1||UNEP (2006)|
|Chlorine equivalents contribution to ozone depletion||9%||Butler (2000)|
|Molecular weight||153.8 g mol−1||WHO (2004)|
|Density||1.594 at 20°C||WHO (2004)|
|Octanol/water partition coefficient (logPow)§||2.64||WHO (2004)|
|Water solubility||800 mg L−1 at 20°C||WHO (2004)|
|Boiling point||76.5°C||WHO (2004)|
|Henry's law constant||29.5 atm L mol−1 at 25°C||Dolfing & Janssen (1994)|
|Gibbs free energy values (ΔG°′) and redox potential||Dolfing & Janssen (1994)|
|Reductive hydrogenolytic dechlorination¶|
|Tetrachloromethane→Trichloromethane||−192.6 kJ/584 mV|
|Trichloromethane→Dichloromethane||−170.8 kJ/471 mV|
|Dichloromethane→Chloromethane||−157.4 kJ/402 mV|
|Chloromethane→Methane||−153.2 kJ/380 mV|
|Tetrachloromethane→CO2 (with H2O as an electron donor and O2 as an electron acceptor)||−551 kJ|
The toxicity of CCl4 to living organisms is well documented (IARC, 1999; WHO, 2004; Eastmond, 2008), and this also applies to microorganisms. Exposure of bacteria to CCl4 was shown to cause inhibition of a variety of environmentally significant metabolic processes, such as methanogenesis and autotrophy, even at very low concentrations (3 and 80 μM, respectively; Bauchop, 1967; Egli et al., 1988). As with other chlorinated methanes, CCl4 may exert a biostatic effect on methanogenic Archaea due to its structural similarity to other C1 compounds, which is likely to affect methane formation through competitive inhibition of enzymatic reactions or interaction with key cofactors of the pathway (Zhao et al., 2009). As a lipophilic compound with a high octanol/water partition coefficient (Table 1), CCl4 may also cause damage to cellular membranes. As reviewed by Sikkema et al. (1995), cytotoxic organic solvents disturb membrane permeability, thereby disrupting critical functions, for example by dissipation of the membrane potential and through loss of valuable cellular components. For example, cytoplasmic enzymes involved in Escherichia coli central metabolism were released from Mg2+-depleted cells treated with toluene due to structural alterations of the cytoplasmic membrane (de Smet et al., 1978).
Strategies described for microorganisms that tolerate organic solvents involve mechanisms that prevent intracellular exposure to the toxicants, such as membrane adaptation, for example through alterations of phospholipid fatty acid and headgroup composition, to ensure homeostasis of membrane fluidity. Sequestration mediated by membrane vesicles (Kobayashi et al., 2000), active extrusion with energy-driven efflux pumps (reviewed by Nicolaou et al., 2010) and membrane proton-motive force maintenance upon solvent-damaged inner membrane involving phage shock protein synthesis (Engl et al., 2009) may also afford cell protection against the toxic effects of halogenated solvents. In the specific case of CCl4, modifications in the saturated phospholipid content were observed in the aerobic methylotroph Methylobacterium extorquens DM4 (Vuilleumier et al., 2009) exposed to very low (0.13 mM, 20 mg L−1) concentrations of CCl4 (C. Penny, F. Bringel, C. Gruffaz, T. Nadalig, H. Heipieper & S. Vuilleumier, unpublished data). However, it is striking that both CCl4-degrading and -non-degrading bacteria were equally insensitive to the deleterious effects of CCl4 at concentrations near or exceeding its water solubility (Table 2). Clearly, many aspects of the bacterial tolerance to CCl4, as for other halogenated compounds, have yet to be investigated.
|Characteristic metabolic trait||Phylogenetic affiliation||MIC (mg L−1)*|
|Methylobacterium extorquens DM4 (DSM 6343)||Dichloromethane degradation||Alphaproteobacteria||400|
|Herminiimonas arsenicoxydans ULPAs1 (DSM 17148)||Arsenic resistance||Betaproteobacteria||400|
|Pseudomonas putida (DSM 291)||Degrades many organic pollutants||Gammaproteobacteria||>800†|
|Pseudomonas putida (DSM 3602)||Degrades many organic pollutants||Gammaproteobacteria||600|
|Pseudomonas stutzeri KC (DSM 7136)||Tetrachloromethane degradation||Gammaproteobacteria||>800†|
Degradation or transformation of CCl4 is the other major source of toxicity of the compound, as some dechlorination pathways generate toxic intermediates and products (Fig. 1; more details in Tetrachloromethane-degrading bacteria: why not better? and Cometabolism galore: a large panel of low-molecular-weight molecules enhances CCl4 degradation). This mainly seems to be due to intracellular CCl4 transformation by nonspecific reactions, leading to the formation of reactive radicals that, by promoting nonspecific oxidation, can detrimentally affect and inactivate key cellular components, including proteins, DNA and lipids (McGregor & Lang, 1996). This was most clearly shown in investigations involving the Ames test, in which exposure to gaseous CCl4 was shown to have mutagenic effects on Salmonella typhimurium and E. coli tester strains (Araki et al., 2004).
This paper presents an overview of the prokaryotic organisms mediating CCl4 dechlorination, describes a large panel of reactions and catalysts as well as the thermodynamic and kinetic aspects of this dechlorination, and discusses the physicochemical conditions necessary for microorganism-mediated CCl4 degradation. Perspectives for research to discover new, more efficient bacterial strains and to apply bacterial metabolism for the treatment of sites contaminated with tetrachloromethane are then proposed.