Toxicological interest in the CYP1A subfamily was greatly intensified by a 1973 report correlating the level of CYP1A1 inducibility in cigarette smokers with individual susceptibility to lung cancer (Kellermann, Shaw and Luyten-Kellermann 1973). The molecular basis of CYP1A1 and CYP1A2 induction has been a major focus of numerous laboratories. The induction process is mediated by a protein termed the Ah receptor to which dioxins and structurally related chemicals bind. The name Ah is derived from the aryl hydrocarbon nature of many CYP1A inducers. Interestingly, differences in the gene encoding the Ah receptor between strains of mice result in marked differences in chemical response and toxicity. A polymorphism in the Ah receptor gene also appears to occur in humans: approximately one-tenth of the population displays high induction of CYP1A1 and may be at greater risk than the other nine-tenths of the population for development of certain chemically induced cancers. The role of the Ah receptor in the control of enzymes in the CYP1A subfamily, and its role as a determinant of human response to chemical exposure, has been the subject of several recent reviews (Nebert, Petersen and Puga 1991; Nebert, Puga and Vasiliou 1993).
Figure 4B illustrates the systemic microsomal reductive pathway for nitrobenzene, with the formation of a nitroanion free radical (Fouts & Brodie, 1957). NADPH mediates the abstraction of one electron from nitrobenzene, and the nitroanion free radical forms (Mason & Holtzman, 1975a). The nitroanion free radical generated from nitrobenzene has a sufficiently long half-life of 1–10 s that it can be detected by emission of a unique ESR spectrum. The more extended the ring aromaticity in nitroaromatic compounds, the longer the free radical half-life that is expected. This is based on electronic delocalization, which leads to radical longevity. Nitroanion free radicals have sufficient longevity to travel and react; the longer the residence time, the more chances they have to react with cellular macromolecules. The presence of all four free radicals shown in Figure 4B can be detected non-destructively by their specific ESR signal patterns (Mason & Holtzman, 1975a; Maples et al., 1990).
Davis et al. (1981) investigated the degradation of nitrobenzene and metabolite formation during the decomposition of nitrobenzene using seed from industrial wastewater treatment units and from municipal activated sludge. The industrial sludge contained mainly the four bacterial genera , , and and the yeast . The municipal sludge was not classified for microbial genera. In each experiment, the bacterial cell concentration was 18 × 108 cells/ml, and all incubations were carried out at 28 °C. Degradation was monitored by oxygen uptake measurement in a Warburg respirometer and by substance-specific analysis (GC/MS). Respiration was inhibited by nitrobenzene concentrations of 100 and 200 mg/litre (industrial sludge) and 200 mg/litre (municipal sludge). Using the municipal seed, an initial concentration of nitrobenzene of 50 mg/litre was reduced to 0.3 mg/litre within 6 days. After subtracting the volatile fraction, approximately 20% of removal could be attributed to microbial degradation. After 6 days, a further 50 mg nitrobenzene/litre was added to the flask, and 40 mg/litre was found to remain after a further 6 days’ incubation. This was thought to indicate that microbial degradation was occurring mainly via co-metabolism, as the amount of glucose available in the culture was minimal for the second 6-day period. Using the industrial seed (in the endogenous growth phase) and an initial nitrobenzene concentration of 50 mg/litre, approximately 9–10 mg nitrobenzene/litre was biodegraded in 6 days. Aniline and phenol were detected as metabolites. Because of the small decrease in nitrobenzene levels found during the investigations, further experiments were performed to determine whether the applied test substance was adsorbed or absorbed by the bacterial mass. However, after cell digestion, no nitrobenzene could be found in the inocula, indicating that the removal was due to microbial transformation rather than simple adsorption to the culture.
Cultures of several species of were grown at 30 °C with nitrobenzene supplied in the vapour phase above the culture. The bacteria included , , , and several unidentified strains. All the bacteria were known to contain toluene degradative pathways. The cells were harvested and then incubated with nitrobenzene at 30 °C to enable metabolites to be identified. All the strains grew in the presence of nitrobenzene vapour when glucose or arginine were provided as an alternative carbon source, but none grew on nitrobenzene as the sole carbon source. Several metabolites were identified from the various strains, including 3-nitrocatechol, 4-nitrocatechol, -nitrophenol and -nitrophenol, although several strains did not transform nitrobenzene at all. The nitrocatechols were slowly degraded to unidentified metabolites. Results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes (Haigler & Spain, 1991). In contrast to this, Smith & Rosazza (1974) found no phenolic metabolites when nitrobenzene (1000 mg/litre) was incubated at 27 °C for 24–72 h with the following microorganisms with demonstrated aromatic metabolizing ability: , , , , , sp., , , , and .
Patil & Shinde (1988) studied the elimination of nitrobenzene both alone and as a mixture with aniline by activated sludge derived from a domestic sewage treatment plant. The inoculum was acclimated for 15 days to wastewater containing both aniline and nitrobenzene. Decomposition was followed by measurement of the chemical oxygen demand (COD) and substance-specific analysis (GC). Initial nitrobenzene concentrations in the range of 184–250 mg/litre were found to be completely degraded in all experiments within 7–8 h of incubation.
Kincannon & Lin (1985) studied the primary degradation of nitrobenzene in columns containing soil material and waste sludges. Three soil types were used, ranging from clay to sandy soils. Waste sludges (a dissolved air flotation sludge, a slop oil sludge and a wood preserving sludge) were applied to the top of the column and worked into the top 20 cm. The removal of the initial influent concentration of 2400 mg nitrobenzene/kg was monitored by GC analysis in different depths of the soil. Half-lives for nitrobenzene were found to be 56 days in the dissolved air flotation sludge-amended column, 13.4 days in the slop oil sludge-amended column and 196.6 days in the wood preserving sludge-amended column. The contribution of abiotic removal mechanisms remains unclear, as nitrobenzene was found to be removed fairly rapidly from sterilized soil columns, presumably by volatilization, with a half-life of around 9 days.
Elimination of a toxicant by exhaled air in relation to the post-exposure period of time usually is expressed by a three-phase curve. The first phase represents elimination of toxicant from the blood, showing a short half-life. The second, slower phase represents elimination due to exchange of blood with tissues and organs (quick-exchange system). The third, very slow phase is due to exchange of blood with fatty tissue and skeleton. If a toxicant is not accumulated in such compartments, the curve will be two-phase. In some cases a four-phase curve is also possible.
Anderson et al. (1991) studied the removal of nitrobenzene in two soils, a silt loam of 1.49% organic carbon content and a sandy loam of 0.66% organic carbon content. The experiment was carried out using both sterile and non-sterile soils to distinguish biotic losses from abiotic losses. Nitrobenzene was added to the soils at 100 mg/kg dry weight, and the mixture was incubated at 20 °C in the dark. The half-life of nitrobenzene in both soils was around 9 days, and the differences in the rate of disappearance between sterile and non-sterile soils was slight, indicating that the loss was caused by abiotic processes. Being unable to decisively explain the fate of the applied test substance, the authors discussed irreversible partitioning to soil organic matter and losses during preanalysis storage as possible sinks. Very similar results have also been reported by Walton et al. (1989).
The removal of nitrobenzene was determined in a complete-mix, bench-scale, continuous-flow activated sludge reactor fed a synthetic wastewater containing a mixture of readily degradable compounds as well as the compound under study. The activated sludge was sampled from a municipal treatment plant and acclimated to the nitrobenzene-containing wastewater prior to the test. The reactors were operated with a hydraulic retention time of 8 h. Following acclimation, samples were collected over a test period of 60 days and monitored for 5-day biochemical oxygen demand (BOD5), TOC, COD and nitrobenzene (GC analysis). About 76–98% of the concentration of nitrobenzene applied to the influent (100 mg/litre) was removed during the column passage (Stover & Kincannon, 1983).
Incubation of nitrobenzene (50 µg/litre) with activated sludge from a municipal sewage treatment plant (1 g dry weight/litre) was carried out for 5 days at 25 °C. Meat extract and peptone were added as additional substrates, and mineralization of nitrobenzene was monitored by carbon dioxide analysis. No metabolites were identified during the experiment, and only 0.4% of the applied radioactivity was found as carbon dioxide, indicating that nitrobenzene was not metabolized to any significant extent under the conditions of the test (Freitag et al., 1982, 1985).
In summary, several investigations have reported that nitrobenzene was not readily degraded by activated sludge inoculum. However, concentrations of nitrobenzene used in these studies were generally much higher than those detected in effluents and likely to be toxic to microorganisms. A more extensive range of other studies indicated that the use of raw sewage sludge from wastewater treatment plants can lead to complete degradation of nitrobenzene under aerobic conditions.