Neilson, Alasdair H. "Persistence: General Orientation" Organic Chemicals : An Environmental Perspective Boca Raton: CRC Press LLC,2000 ©2000 CRC Press LLC 4 Persistence: General Orientation SYNOPSIS An overview is presented of the factors that determine the per- sistence of xenobiotics including the role of both abiotic and biotic reactions. Examples are given of photochemical reactions including those that take place in the troposphere and transformation products that may subsequently enter aquatic or terrestrial systems, and of chemical transformations includ- ing hydrolysis, dehalogenation, oxidation, and reduction. It is pointed out that combinations of abiotic and biotic processes may be of determinative sig- nificance, and the significance of these reactions in determining the analytes that may be included in monitoring programs is emphasized. Biotic reactions are discussed in detail and the important distinction between biodegradation and biotransformation is emphasized. Attention is directed to the metabolic potential of groups of microorganisms that have been less extensively exam- ined; these include enteric bacteria, ammonia oxidizers, marine and lithotrophic bacteria, algae, and anaerobic phototrophic bacteria. The signifi- cance of electron acceptors other than oxygen is noted, and examples are illustrated with organisms using nitrate and related compounds, and those growing anaerobically by reduction of Fe(III), Mn(IV), or U(VI). Some impor- tant reactions mediated by yeasts, fungi, and algae are outlined. The mecha- nisms whereby oxygen is introduced into xenobiotics are discussed, and brief accounts of the enzymology are included. Attention is directed to metabolic interactions where several organisms and a single substrate are present, or where several substrates and a single organism occur. Examples are given of metabolic limitations imposed by enzyme regulatory mechanisms and of metabolic situations where a single readily degraded substrate is present in addition to a more recalcitrant xenobiotic. Factors that may critically deter- mine the biodegradability of xenobiotics in natural systems are summarized; these include temperature, the oxygen concentration, the substrate concen- tration, the synthesis of natural emulsifying agents, the nature of transport mechanisms, and the cardinal issue of the bioavailability of the xenobiotic. A number of incompletely resolved issues are discussed including biodegrada- tion in pristine environments, natural enrichment in contaminated environ- ments, estimation of the rates of metabolic reactions both in laboratory and natural systems, and the significance of toxic metabolites. Brief comments are given on the role of catabolic plasmids. ©2000 CRC Press LLC Introduction Procedures for the analysis of environmental samples have been outlined in Chapter 2, and the processes that determine the dissemination of xenobiotics after discharge from point sources have been discussed in Chapter 3. In the next three chapters, the factors that determine the ultimate fate of xenobiotics will be discussed. This chapter attempts to present an overview of the factors that determine the persistence of xenobiotics, while Chapter 5 will be devoted to experimental procedures for carrying out the relevant investigations, and Chapter 6 to a detailed examination of the pathways taken for the degradation and transformation of a wide range of structurally diverse xenobiotics. Atten- tion will be focused on microorganisms, and in particular on bacteria that are the most important degradative organisms in virtually all aquatic ecosystems. A certain degree of overlap between this chapter and Chapter 6 is inevitable, but an attempt has been made to minimize this by inclusion of cross-references. It was the persistence of DDT which raised the greatest alarm over its extensive use during the years 1940 to 1968. Although levels since its banning have decreased dramatically, those of its metabolite DDE may still be appre- ciable and serve to sustain the initial concern. Many organic compounds have become environmentally suspect, but it is especially the highly chlorinated ones such as the polychlorinated biphenyls, polychlorinated camphenes, and mirex which have acquired the reputation of being unacceptable due to their apparent persistence. As a result of these fears, there has emerged a general concern with all synthetic chlorinated organic compounds (Hileman 1993) which may possibly have deflected interest from other groups which merit comparable attention. It should, of course, be appreciated that on the other hand a number of compounds and products such as modern plastics have been developed for their stability under a variety of conditions — and are produced with this end in view. For these reasons, studies on biodegradation began to occupy a central position in discussions on the environmental impact of organic chemicals, and the complexities have been clearly presented (Landner 1989). It should be appreciated at the outset that the terms persistent and recalcitrant are relative rather than absolute since probably most chemical structures can be degraded or transformed by microorganisms. The crucial issue is the rate at which the reactions occur, and the area between slowly degradable compounds and truly persistent ones is often unresolved. For example, in spite of the fact that degradation of some PCB congeners has been demonstrated under aerobic conditions, and biotransformation (dechlorination) under anaerobic condi- tions, these compounds are still recoverable from many environmental sam- ples; they should therefore be regarded as persistent. The critical questions are both what reactions take place and the rate at which they occur in the environment into which the compound is discharged. Both of these should be addressed in investigations aimed at incorporating environmental relevance. ©2000 CRC Press LLC Two essentially different processes determine the persistence of an organic compound in the aquatic environment. The first are abiotic reactions, and for some groups of compounds these reactions may be dominant in determining their fate. The second are biotic reactions mediated by a wide range of organ- isms. Only microorganisms will be discussed in the next three chapters, although a brief discussion of the metabolism of xenobiotics by higher organ- isms is given in Chapter 7 (Section 7.5). 4.1 Abiotic Reactions Virtually any of the plethora of reactions known in organic chemistry may be exploited for the abiotic degradation of xenobiotics. Hydrolytic reactions may convert compounds such as esters, amides, or nitriles into the corresponding carboxylic acids, or ureas and carbamides into the amines. These abiotic reac- tions may therefore be the first step in the degradation of such compounds; the transformation products may, however, be resistant to further chemical transformation so that their ultimate fate is dependent upon subsequent microbial reactions. For example, for urea herbicides the limiting factor is the rate of microbial degradation of the chlorinated anilines which are the initial hydrolysis products. The role of abiotic reactions should, therefore, always be taken into consideration, and should be carefully evaluated in all laboratory experiments on biodegradation and biotransformation (Section 5.3). It should be appreciated that the results of experiments directed to microbial degrada- tion are probably discarded if they show substantial interference from abiotic reactions. A good illustration of the complementary roles of abiotic and biotic processes is offered by the degradation of tributyl tin compounds. Earlier experiments (Seligman et al. 1986) had demonstrated the transformation of tributyltin to dibutyltin primarily by microbial processes. It was subsequently shown, however, that an important abiotic reaction mediated by fine-grained sediments resulted in the formation also of monobutyltin and inorganic tin (Stang et al. 1992). It was therefore concluded that both processes were impor- tant in determining the fate of tributyl tin in the marine environment. A study of the carbamate biocides, carbaryl and propham, illustrates the care that should be exercised in determining the relative importance of chem- ical hydrolysis, photolysis, and bacterial degradation (Figure 4.1) (Wolfe et al. 1978a). For carbaryl, the half-life for hydrolysis increased from 0.15 day at pH 9 to 1500 day at pH 5, while that for photolysis was 6.6 day: biodegradation was too slow to be significant. On the other hand, the half-lives of propham for hydrolysis and photolysis were >10 4 and 121 day - so greatly exceeding the half-life of 2.9 day for biodegradation that abiotic processes would be considered to be of subordinate significance. Close attention to structural fea- tures of xenobiotics is therefore clearly imperative before making generaliza- tions on the relative significance of alternative degradative pathways. ©2000 CRC Press LLC 4.1.1 Photochemical Reactions in Aqueous and Terrestrial Environments Photochemical reactions may be important especially in areas of high solar irradiation, or on the surface of soils, or in aquatic systems containing ultravi- olet (UV) absorbing humic and fulvic acids (Zepp et al. 1981), and they may be especially relevant for otherwise recalcitrant compounds. It has also been shown (Zepp and Schlotqhauer 1983) that, although the presence of algae may enhance photometabolism, this is subservient to direct photolysis at the cell densities likely to be encountered in rivers and lakes. It should be noted that different products may be produced in natural river water and in buffered medium; for example, photolysis of triclopyr (3,5,6-trichloro-2-pyridyloxy- acetic acid) in sterile medium at pH 7 resulted in hydrolytic replacement of one chlorine atom, whereas in river water the ring was degraded to form oxamic acid as the principal product (Woodburn et al. 1993). Particular atten- tion has understandably therefore been directed to the photolytic degradation of biocides — including agrochemicals — that are applied to terrestrial sys- tems. There has been increased interest in the phototoxicity toward a range of biota (references in Monson et al. 1999), and this may be attributed to some of the reactions and transformations that are discussed later in this chapter. It should be emphasized that photochemical reactions may produce molecules structurally more complex and less susceptible to degradation than their pre- cursors, even though the deep-seated rearrangements induced in complex compounds such as the terpene santonin during UV irradiation (Figure 4.2) are not likely to be encountered in environmental situations. The Diversity of Photochemical Transformations In broad terms, the following types of reactions are mediated by the homolytic fission products of water (formally, hydrogen and hydroxyl radi- cals) and molecular oxygen or its excited states: hydrolysis, elimination, oxi- dation, reduction, and cyclization. The Role of Hydroxyl Radicals The hydroxyl radical plays two essentially different roles: (1) as a reactant mediating the transformations of xenobiotics and (2) as a toxicant operating by damaging DNA. Hydroxyl radicals are important in a number of FIGURE 4.1 Carbaryl (A) and propham (B). ©2000 CRC Press LLC environments: (1) in aquatic systems under irradiation, (2) in the troposphere that is discussed later in this section, and (3) in biological systems that are noted in the context of superoxide dismutase and the role of Fe in Section 4.6.1.2 and in Sections 5.2.4 and 5.5.5. Hydroxyl radicals in aqueous media may be generated by (1) photolysis of nitrite and nitrate (Brezonik and Fulk- erson-Brekken 1998), (2) the Fenton reaction with H 2 O 2 and Fe 2+ in the pres- ence of light that is noted later, and (3) photolysis of fulvic acids under anaerobic conditions (Vaughan and Blough 1998), and (d) reaction of Fe(III) or Cu(II) complexes of humic acids with hydrogen peroxide (Paciolla et al. 1999). For the sake of completeness, attention is drawn to the following: (1) the interactive role of hydroxyl radicals, superoxide, and Fe levels in wild and mutant strains of Escherichia coli lacking Fe and Mn superoxide dismutase is discussed in Sections 5.2.4 and 5.5.5 and (2) the possible role of hydroxyl rad- icals in mediating the transformations accomplished by the brown-rot fun- gus Gleophyllum striatum which is supported by the overall similarity in the structures of the fungal metabolites with those produced with Fenton’s reagent (Wetzstein et al. 1997). Analytical procedures for hydroxyl radicals noted in Section 2.3 and have been used to demonstrate the role of the anticancer drug 2,5-bis(1-azacyclo- propyl)-3,6-bis(carboethoxyamino)benzo-1,4-quinone in mediating the pro- duction of hydroxyl radicals in JB6 mouse epidermal cells (Li et al. 1997). Illustrative Examples of Photochemical Transformations in Aqueous Solutions 1. Atrazine is successively transformed to 2,4,6-trihydroxy-1,3,5- triazine (Pelizzetti et al. 1990) by dealkylation of the alkylamine FIGURE 4.2 Photochemical transformation of santonin. ©2000 CRC Press LLC side chains and hydrolytic displacement of the ring chlorine and amino groups (Figure 4.3). A comparison has been made between direct photolysis and nitrate-mediated hydroxyl radical reactions (Torrents et al. 1997). The rates of the latter were much greater under the conditions of this experiment, and the major difference in the products was the absence of ring hydroxylation with loss of chloride. 2. Pentachlorophenol produces a wide variety of transformation products including chloranilic acid (2,5-dichloro-3,6-dihydroxy- benzo-1,4-quinone) by hydrolysis and oxidation, a dichlorocyclo- pentanedione by ring contraction, and dichloromaleic acid by cleavage of the aromatic ring (Figure 4.4) (Wong and Crosby 1981). 3. The main products of photolysis of 3-trifluoromethyl-4-nitrophe- nol are 2,5-dihydroxybenzoate produced by hydrolytic loss of the nitro group and oxidation of the trifluoromethyl group, together with a compound identified as a condensation product of the orig- inal compound and the dihydroxybenzoate (Figure 4.5) (Carey and Cox 1981). FIGURE 4.3 Photochemical transformation of atrazine. FIGURE 4.4 Photochemical transformation of pentachlorophenol. ©2000 CRC Press LLC 4. The potential insecticide that is a derivative of tetrahydro-1,3-thi- azine undergoes a number of reactions resulting in some 43 prod- ucts of which the dimeric azo compound is the principal one in aqueous solutions (Figure 4.6) (Kleier et al. 1985). 5. The herbicide trifluralin undergoes a photochemical reaction in which the n -propyl side chain of the amine reacts with the vicinal nitro group to form the benzopyrazine (Figure 4.7) (Soderquist et al. 1975). 6. Heptachlor and cis- chlordane both of which are chiral form caged or half-caged structures (Figure 4.8) on irradiation and these prod- ucts have been identified in biota from the Baltic, from the Arctic, and from the Antarctic (Buser and Müller 1993). 7. Methylcyclopentadienyl manganese tricarbonyl that has been sug- gested as a fuel additive is decomposed in aqueous medium pri- marily by photolysis. This resulted in the formation of methylcyclopentadiene that may plausibly be presumed to poly- merize, and a manganese carbonyl that decomposed to Mn 3 O 4 (Garrison et al. 1995). FIGURE 4.5 Photochemical transformation of 3-trifluoromethyl-4-nitrophenol. FIGURE 4.6 Photochemical transformation of a tetrahydro-1,3-thiazine. FIGURE 4.7 Photochemical transformation of trifluralin. ©2000 CRC Press LLC 8. Stilbenes that are used as fluorescent whitening agents are pho- tolytically degraded by reactions involving cis-trans isomerization followed by hydration of the double bond or oxidative fission of the double bond to yield aldehydes (Kramer et al. 1996). 9. The photolysis of chloroalkanes and chloroalkenes has received attention and results in the formation of phosgene as one of the final products. The photodegradation of 1,1,1-trichloroethane pro- ceeds by hydrogen abstraction and oxidation to trichloroacetalde- hyde that is degraded by a complex series of reactions to phosgene (Nelson et al. 1990; Platz et al. 1995).Tetrachloroethene is degraded by reaction with chlorine radicals and oxidation to pentachloropro- panol radical which also forms phosgene (Franklin 1994). Attention has already been drawn to the significance of these reactions in the context of environmental analytes (Section 2.5), and the atmo- spheric dissemination of xenobiotics (Section 3.5.3). 10. Although EDTA is biodegradable under specific laboratory condi- tions (Belly et al. 1975; Lauff et al. 1990; Nörtemann 1992; Witschel et al. 1997), the primary mode of degradation in the natural aquatic environment involves photolysis of the Fe complex (Lockhart and Blakeley 1975; Kari and Giger 1995). Other metal complexes are relatively resistant, so that its persistence is critically determined not only by the degree of insolation but by the concentration of Fe in the environment. The available evidence suggests that, in con- trast to NTA that is more readily biodegradable, EDTA is likely to be persistent except in environments in which concentrations of Fe greatly exceed those of other cations. 11. The photolytic degradation of the fluoroquinolone antibiotic enro- floxacin involves a number of reactions that produce 6-fluoro-7- amino-1-cyclopropylquinolone 2-carboxylic acid that is then degraded to CO 2 via reactions involving fission of the benzenoid ring with loss of fluoride, dealkylation, and decarboxylation (Burhenne et al. 1997a,b) (Figure 4.9). 12. Photolysis of the oxime group in the pyrazole miticide fenpyroxi- mate resulted in the formation of two principal transformation products: the nitrile via an elimination reaction and the aldehyde by hydrolysis (Swanson et al. 1995). FIGURE 4.8 Photochemical transformation of chlordane. ©2000 CRC Press LLC 13. Photochemical transformation of pyrene in aqueous media pro- duced the 1,6- and 1,8-quinones as stable end products after initial formation of 1-hydroxypyrene (Sigman et al. 1998). Irrespective of mechanism, these reactions are formally comparable to those oper- ating during the transformation of benzo[ a ]pyrene by Phanerochaete chrysosporium (Chapter 6, Section 6.2.2). 14. The transformation of isoquinoline has been studied both under photochemical conditions with hydrogen peroxide, and in the dark with hydroxyl radicals (Beitz et al. 1998). The former resulted in fission of the pyridine ring with formation of phthalic dialdehyde and phthalimide whereas the major product from the latter involved oxidation of the benzene ring with formation of the 5,8- quinone and a hydroxylated quinone. 15. In the presence of both light and hydrogen peroxide, 2,4-dinitro- toluene is oxidized to the corresponding carboxylic acid; this is then decarboxylated to 1,3-dinitrobenzene which is degraded further by hydroxylation and ring fission (Figure 4.10) (Ho 1986). Comparable reaction products were formed from 2,4,6-trinitrotoluene and hydroxylated to various nitrophenols and nitrocatechols before cleavage of the aromatic rings, and included the dimeric 2,2 ′ car- boxy-3,3 ′ ,5,5 ′ -tetranitroazoxybenzene (Godejohann et al. 1998). Hydroxyl Radicals in the Destruction of Contaminants The use of hydroxyl-radical mediated reactions has attracted interest in the context of destruction of contaminants, and two are provided as illustration. These reactions should be viewed against those with hydroxyl radicals that occur in the troposphere that are considered in Section 4.1.2. FIGURE 4.9 Photochemical degradation of enrofloxin. [...]... degradation of many other terpenes has been examined including the β-pinene, D-limonene, and trans-caryophyllene (Grosjean et al 1993b) 6 The products formed by reaction of NO3 radicals with α-pinene have been identified and include pinane epoxide, 2-hydroxypinane-3-nitrate, 3-ketopinan-2-nitrate formed by reactions at the double bond, and pinonaldehyde that is produced by ring fission between C2 and C3 (Wängberg... that produced a range of cyclobutane carboxylic acids (Kamens et al 1999); 3 The rapid reactions of linalool with OH radicals, NO3 radicals and ozone in which the major products were acetone and 5-ethenyldihydro-5-methyl-2(3H)-furanone (Shu et al 1997); 4 The plant metabolite cis-hex-3-ene-1-ol that is the precursor of peroxypropionyl nitrate (Grosjean et al 1993b) analogous to peroxyacetyl nitrate;... rinoids and porphyrins in the presence of a chemical reductant (references in Workman et al 1997), and an illustration is provided by the dechlorination and elimination reactions carried out by titanium(III) citrate and hydroxocobalamin (Bosma et al 19 94) ; hexachlorobuta-1,3-diene was dechlorinated to the pentachloro compound, and by dechlorination and elimination successively to trichloro-but-1-ene-3-yne... bacteria and it therefore seems plausible to assume that such processes might be especially important in warm-water marine environments 1 The degradation of pyridine dicarboxylates (Amador and Taylor 1990) 2 The degradation of 3- and 4- trifluoromethylbenzoate: the microbial transformation resulted in the formation of catechol intermediates that were converted into 7,7,7-trifluoro-hepta-2 , 4- diene-6-one carboxylate... toluene, the major product was benzaldehyde with lesser amounts of 2-nitrotoluene > benzyl alcohol nitrate > 4- nitrotoluene > 3-nitrotoluene (Chlodini et al 1993) An interesting example is the formation of the mutagenic 2-nitro- and 6-nitro-6H-dibenzo[b,d]pyran-6-ones (Figure 4. 16) from the oxidation of phenanthrene in the presence of NOx and methyl nitrite as a source of hydroxyl radicals (Helmig et al... reactions are important in the photochemical transformation of PAHs: those with molecular oxygen, and those involving cyclization Illustrative examples are provided by the photooxidation of 7,12-dimethylbenz[ a ]anthracene-3 , 4- dihydrodiol (Lee and Harvey 1986) (Figure 4. 12a) and benzo[a]pyrene (Lee-Ruff et al 1986) (Figure 4. 12b), and the cyclization of cisstilbene (Figure 4. 12 c) FIGURE 4. 12 Photooxygenation... again in Chapter 6, Section 6.1.3; FIGURE 4. 27 Biotransformation of dehydroabietic acid by Mortierella isabellina ©2000 CRC Press LLC 3 The formation of 16-chlorohexadecyl-16-chlorohexadecanoate from hexadecyl chloride by Micrococcus cerificans (Kolattukudy and Hankin 1968) CH3[CH2] 14 CH2Cl → ClCH2[CH2] 14 CH2·O·CO·[CH2] 14 CH2Cl 4 The O-methylation of chlorophenols to anisoles by fungi (Cserjesi and Johnson... the 1-position followed by transannular dioxygenation at the 2- and 5-positions followed by ring fission (Brubaker and Hites 1998) Reactions of hydroxyl radicals with polychlorinated dibenzo[1 ,4] dioxins and dibenzofurans also play an important role for their removal from the atmosphere (Brubaker and Hites 1997) The gas phase and the particulate phase are in equilibrium, and the results show that gas-phase... oxidation and cleavage of ring A, ring C, ring D, and rings C and D, and rings B, C, and D (Jang and McDow 1997) b 1-Nitropyrene is a widely distributed contaminant produced in the troposphere by reaction of nitrate radicals with pyrene that is discussed in Section 4. 1.2 A solution in benzene was ©2000 CRC Press LLC photochemically transformed into 9-hydroxy-1-nitropyrene that is less mutagenic than its... mutagenic than its precursor (Koizumi et al 19 94) 3 The photochemical transformation of phenanthrene sorbed on silica gel (Barbas et al 1996) resulted in a variety of products including cis-9,10-dihydrodihydroxyphenanthrene and phenanthrene9,10-quinone, and a number of ring fission products including biphenyl-2,2′-dicarboxaldehyde, naphthalene-1,2-dicarboxylic acid, and benzo[c]coumarin This may be compared . 5-ethe- nyldihydro-5-methyl-2(3H)-furanone (Shu et al. 1997); 4. The plant metabolite cis-hex-3-ene-1-ol that is the precursor of per- oxypropionyl nitrate (Grosjean et al. 1993b) analogous to peroxy- acetyl. cis -9 ,10-dihydrodihydroxyphenanthrene and phenanthrene- 9,10-quinone, and a number of ring fission products including biphenyl-2,2 ′ -dicarboxaldehyde, naphthalene-1,2-dicarboxylic acid, and. presumed to poly- merize, and a manganese carbonyl that decomposed to Mn 3 O 4 (Garrison et al. 1995). FIGURE 4. 5 Photochemical transformation of 3-trifluoromethyl -4 - nitrophenol. FIGURE 4. 6 Photochemical