vl.ous 'conctl.. h a s been exp r e s s e d over t h e discharge into the aquatic environment of organic compounds that are potentially persistent or toxic or that accumulate in biota. The production of bleached pulp iuvolves removal of lignin by various chemical treatments. Considerable effort has been given to the possible adverse environmental effects of the effluents hecause these may he discharged into the aquatic environment in substantial quantities. The procedures outlined here could be applied to any organic compound and, with appropriate modification, to the terrestrial environment. Although they are not discussed here, potentially adverse effects on human health , could result both from direct exposure and via consumption of contaminated food harvested from higher levels of the food chain. We present here a strategy evaluating the hazard to I,,, aquatic environment of chlorophenolic components of bleachery effluents. We emphasize lahoratory studies supplemented with selected investigation of field samples. A comprehensive program of research has been carried out and has led to new perspectives on critical aspects of the microbiology, partition, and toxicology of xenohiotics i n t h e aquatic environment. It s h o u l d he underscored that al-
.xmation during conventional bleaching with molecular chlorine has not been clearly resolved ( 3 ) ,and bleaching technology is probably not the main contributor to background levels of the highly chlorinated congeners ( 4 ) .
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though chlorophenolic compounds and a restricted geographical area were chosen to illustrate the principles, their application is restricted neither to these compounds nor to specific ecosystems. We direct readers to a more genera1 review (I) and to an account of chlorinated dibenzo-1,4-dioxins [ Z ) , although the mechanism of their
Environ. Sci. Technol., Val. 28, No. 6, 1994
Constituents of bleachery effluents Pulp produced by the sulfate process is normally "hrightened" by removal of residual lignin. In conventional bleaching processes this is carried out in a number of stages that include reactive chlorine in the form of molecular chlorine or hypochlorite. Bleachery effluents therefore contain a structurally diverse range of low molecular weight organochlorine compounds. Because of concern ahout the discharge of such compounds into the environment, and specific evidence for the toxicity-including the genotoxicity (5-8) of some of them-xtensive effort has been devoted to their identification (9-191. Current bleaching technology is directed to diminishing the load of organochlorine components in the effluents by the replacement of molecular chlorine with chlorine dioxide, or the elimination of chlorine using oxygen prebleaching and peroxide technologies. Current effluents therefore differ from those formerly produced in at least two important ways: the degree of chlorination of individual compounds is lower and the low molecular weight phenolic compounds are dominated by 6-chlorovanillin ( 2 0 , Z I ) .
0013-936W94/0927-278A$04.50/00 1994 American Chemical Society
Environ. Sci. Technol., Vol. 28. No. 6. 1994 279 A
In addition to these low molecu- contains 3-chloro-4-hydroxy- and lected field samples. Analytical and lar weight compounds, a high mo- 3,5-dichloro-4-hydroxyphenyl synthetic studies formed an integral lecular weight component termed structures that may plausibly have a part of all the investigations. In the studies on persistence, at“chlorolignin” is also produced and similar origin (30).It may be provihas been the object of study. Con- sionally assumed that in quantita- tention has been directed excluflicting views have emerged both on tive terms, chlorinated catechols, sively to reactions carried out by its composition, its stability (22, guaiacols, and vanillins originate bacteria. This choice is justified by 23), and its biological effect, but we primarily if not exclusively from the dominance of bacteria as micronote two important developments. the production of bleached pulp us- organisms of metabolic significance First, it has been shown conclu- ing conventional technologies with in most receiving waters and by the quantitatively greater significance sively that the polymeric chlorolig- molecular chlorine. of biotic rather than, for example, nin effectively binds low molecular photochemical reactions in northweight chlorophenolic compounds The basic criteria for hazard assessment ern latitudes. Each of the three types (24) so that the environmental significance of chlorolignin depends It is generally accepted that there of investigation that has been caron the strength of this association are three basic components of a haz- ried out is discussed separately and on the extent to which it is re- ard assessment of organic com- even though in practice, and by deversible. Second, a study of chloro- pounds discharged into the aquatic sign, they overlap considerably. lignin using gel permeation chro- environment: persistence to both abiotic and bi- Exemplification of procedures matography (25) has shown that the Microbiological investigations. molecular weight distribution of the otic degradation, polymer is critically dependent on partition from the aquatic phase Some aspects should be emphathe solvent used, and this has re- into the sediment phase and into sized because they differ significantly from conventional procesulted in revised estimates of the biota, and dures for determining biodegradamolecular weight. Previous views toxicity to biota. on the environmental significance These issues have been incorpo- bility. These have restricted releof chlorolignin may therefore have rated into a composite program (31) vance to the natural environment to be revised. (Figure 1).The first part is devoted for several reasons: They frequently employ inocula We will present an overview of to laboratory experiments, and the from municipal sewage treatment strategies that have been used in second is aimed at verification of this laboratory to assess the impact the conclusions by examining se- systems whose flora is subject to enof bleachery effluents on the aquatic environment. Attention has been directed to the chlorophenolic components because these have attracted considerable attention in view of the toxicity of chlorophenols themselves (26).Emphasis was placed on laboratory-based procedures supplemented by examination of field samples, although monitoring h a s ’ n o t been a major activity. There has been renewed interest in the synthesis of naturally occurring organochlorine compounds and their quantitative significance compared with anthropogenic organochlorine compounds. Whereas there is no doubt that haloperoxidase systems halogenate phenols to Me produce, for example, 2,4,6-trichlorophenol from 2,4-dichlorophenol (271,there seems to be no evidence for the formation of the correspondEwtoxicological studies (toxicity, bioco ing chlorinated guaiacols or catechols. It has also been shown that 2,3,7,8-tetrachlorodibenzo-1,4dioxin can be produced by the reaction of 2,4,5-trichlorophenol with hydrogen peroxide and peroxidase Examinatn.. _.dbstrates and metabolite (28).Although the fungus Bjerkandera sp. produces 3-chloro- and 3,sExistence in biota and Sediments dichloroanisaldehydes (29),the for0 Binding to sediments and release mation of chlorovanillins b y Uptake and metabolism in biota analogous reactions has not so far 0 Microbial transformation in sedim been reported. On the other hand, Nonmicrobial transport processe high molecular weight organic material from uncontaminated ‘areas 280 A
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tirely different selection pressures. The substrate concentrations are determined by the end-points such as the evolution of carbon dioxide or consumption of oxygen, and are environmentally excessive. Only degradation of the substrate is examined, whereas its biotransformation is not taken into account. They cannot evaluate the role of auxiliary substrates (cosubstrates). Biodegradation refers to reactions in which, under aerobic conditions, mineralization of the test compound takes place with the formation of CO,, H,O, SO:-, NH;, PO:-, or C1-; under anaerobic conditions, reduced products such as CH, and S2- are produced. In both situations the substrate serves as the main source of cell carbon. Biotransformation refers to processes in which only a few reactions bring about comparatively minor structural modification to the original substrate; these definitions differ slightly from others (32). There has been an understandable tendency to emphasize the environmental significance of biodegradation at the expense of biotransformation even though this may have profound ecological importance. One of the objectives of the laboratory experiments was to incorporate a maximum degree of environmental realism. The microorganisms that were used have originated exclusively from environmental samples of water or sediment and, to achieve a high degree of reproducibility over extended periods of time, elective enrichment was used. For aerobic bacteria, pure cultures were readily obtained but for anaerobic bacteria this has proved less successful. Therefore metabolically stable enrichment cultures that have undergone at least 10 successive transfers were consistently used. The philosophy behind this methodology has been elegantly summarized by van Niel(33), one of the pioneers of this approach. There are a number of important advantages of this procedure including the following: Relevant environmental parameters such as temperature, salinity, oxygen concentration, or substrate concentration can readily be simulated under controlled laboratory conditions. It is possible to examine in detail the formation of metabolites including those that may be transient or that may inhibit further metabolism of the substrate. In the natural environment organ-
isms are exposed to a single substrate very seldom, and these laboratory experiments are ideally suited to studying the effect of cosubstrates and concurrent metabolism (34). Care should be exercised in extrapolating the conclusions of laboratory experiments to natural ecosystems. Two important issues are the rates at which the various reactions occur and the extent to which the substrate is accessible to the relevant microorganisms. Rates from laboratory experiments can be normalized to the number of cells carrying out the reaction, although complex kinetics may be encountered ( 3 5 ) ,and in natural situations it may be difficult to determine the number of cells carrying out the desired reaction. The application of the relevant DNA probes (36, 37) will certainly facilitate this. All of the investigations that will be described included the kinetics of the various reactions. The biodegradation of chlorophenols has been extensively studied in other laboratories, and the pathways have been delineated. For congeners with only one or two substituents, the reaction proceeds by ring hydroxylation to chlorocatechols followed by ring cleavage and subsequent loss of chloride to form compounds that enter central catabolic pathways of the cell (38). The degradation of pentachlorophenol, however, proceeds by initial hydrolytic dehalogenation followed by reduction before ring cleavage of the resulting 1,2,4-trihydroxybenzene (39),and a similar pathway is used by Rhodococcus chlorophenolicus for the degradation of tetrachloroguaiacol (40). Details of the pathways for the lower chlorinated congeners that have been examined both in this organism and in a strain of Acinetobacter junii (41) have not been delineated but plausibly involve 0-demethylation followed by ring cleavage of the chlorocatechols. Experiments from this laboratory led to a substantially different perspective with important environmental consequences. Aerobic bacteria were obtained by enrichment with a permissive substrate lacking chlorine substituents (42, 43). Initial experiments with dense. cell suspensions of the appropriate bacteria showed that 0-methylation of chloroguaiacols to the corresponding chloroveratroles occurred and that the metabolites were stable to further degradation (42). Experi-
ments at low substrate concentrations (100 p$/L) revealed a quantitative synthesis of metabolites by 0-methylation that exactly mirrored the kinetics of the decrease in the concentration of the substrates. The results of extensive experiments showed the following: Organisms able to carry out 0methylation of chloroguaiacols were widely distributed in the Baltic Sea and Gulf of Bothnia (43). The substrate concentration was of cardinal importance in determining t h e metabolites that were formed (Figure 2). 0-methylation took place during growth with a number of carbon sources and occurred at cell densities comparable with those that could be encountered in natural ecosystems (43). 0-methylation was observed in whole cells of a number of bacteria, and the rates for a series of di- and trichlorophenols illustrated the significance not only of the number of substituents but also their orientation (Table l) (45). 0-methylation was also observed in cell extracts from both Gram-positive and Gramnegative bacteria ( 4 4 ) .Collectively, these results led to the proposal that in natural ecosystems, O-methylation should be considered a significant alternative to biodegradation for halogenated phenolic compounds. Experiments using endogenous microorganisms in sediment samples incubated under conditions resembling those encountered in receiving waters verified these conclusions (46) and led to an examination of the partition of chloroguaiacols and chlorocatechols between the water and the sediment phase. These compounds were effectively partitioned into the sediment phase, and it was therefore concluded that their ultimate fate would be determined by anaerobic reactions in the sediment phase. Extensive investigations were therefore initiated into anaerobic transformations of chloroguaiacols and chlorocatechols. Three reactions were studied: O-demethylation of chloroguaiacols, dechlorinat i o n of chlorocatechols, a n d reductions of chlorovanillins and related compounds. In these investigations stable enrichment cultures have been used and have displayed good repeatability over several years; they may indeed mimic the natural situation more closely than pure cultures.
Environ. Sci. Technol., Vol. 28,No. 6, 1994 281 A
Details have been given (47-49) of the experimental procedures because these differ in important respects from others that have used unenriched sediment slurries. A range of nonchlorinated substrates was used for enrichment: these were chosen on the basis of similarities to structural entities of the organic material plausibly found in natural sediments: tannins and other components of plant detritus. These were the only growth substrates during enrichment because organic material from the sediment would have been removed by dilution. Both the concurrent metabolism methodology outlined above for aerobic biotransformations and dense cell suspensions were used. Experiments with growing cells showed that 0-demethylation of chloroguaiacols occurred rapidly during early stages of cell growth and that subsequently the chlorocatechols formed were dechlorinated ( 4 7 ) . Subsequent experiments using either growing cultures or dense cell suspensions revealed the following: 0-demethylation of chloroguaiacols could be an extremely rapid reaction (47). Only partial dechlorination of the chlorocatechols occurred so that these reactions were strictly biotransformations, and the yield of the dechlorination products was often quantitative ( 4 8 ) . Depending on the substrate used for growth of the cells, 3,4,5-trichlorocatechol could be dechlorinated to any of the possible dichlorocatechols ( 4 8 , 49). These results are consistent with those from other laboratories that have examined a range of chlorinated aromatic compounds including chlorophenols (50), chloroanilines (511,chlorobenzoates (521, chlorobenzenes (531,and polychlorobiphenyls ( 5 4 ) . Three important aspects of the fate of chlorocatechols remained to be addressed: the role of readily degraded substrates, the role of sulfate concentration, and the bioavailability of “aged” chlorocatechols in the s e d i m e n t phase. Experiments showed that the presence of readily degraded components (the carbohydrate portion of glycones) led to cultures with diminished dechlorination capacity ( 4 9 , and that the presence of high concentrations of sulfate in the enrichment media led to populations of organisms that were markedly less effective in dechlorinating chlorocatechols 282 A Environ. Sci. Technol., Vol 28, No. 6. 1994
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of substituent ntation on the rai IO-’’ x pg/h/cells per mL) f Omethylation of hlorophenols by whole cells I
4- Dichlorophenol 4- Dichlorophenol
6-Dichlorophenol 4.5 -Trichlorophenol 3,4 -Trichlorophenol d fi.Tnrhlnmnhmn1
(49). These results are equally relevant to anaerobic biological treatment systems because bleachery effluents contain readily degraded components that could result in selection of a microbial flora deficient in dechlorination capability. Investigations have clearly shown that in contrast to dissolved chlorocatechols, those existing in “aged” sediment phases, or in interstitial water prepared from it, are resistant to anaerobic bacterial dechlorination (55). This important issue appears not to have received sufficient attention. The results of the experiments with chlorovanillins and related hydroxybenzaldehydes were complex ( 5 6 ) . In summary, the aldehyde group underwent a dismutation by oxidation to a carboxylic acid that could then be decarboxylated with formation of chlorocatechols (or chlorophenols) and concomitant reduction to a benzyl alcohol whose hydroxymethyl group could be further reduced to a methyl group. The reaction is formally similar to the oxidation of aromatic aldehydes to the corresponding carboxylic acids that has been confirmed elsewhere (57)using pure cultures of anaerobic sulfate-reducing bacteria. Partition of chlorophenolic compounds and their metabolites. The results of these microbiological experiments raised two further issues: the bioconcentration potential of the neutral bacterial metabolites of chloroguaiacols and chlorophenols, and the water-sediment partition of chloroguaiacols and chlorocatechols. Chloroguaiacols are polar compounds with only low potential for concentration in biota (581. The 0methylated metabolites produced in the experiments discussed above
vere, however, neutral compounds nd less rather than more polar than heir precursors. Their relative moiility on C18 thin-layer chromatography plates showed that all of them had log bioconcentration factor (BCF) values exceeding 3.5 (59). Further details were added using zebra fish (Brochydonio rerio) exposed to low concentrations of 3,4,5-trichloroveratrolefor 28 days or to tetrachloroveratrole for 56 days. From the concentrations in the aqueous phase and in the fish, BCF values could he calculated, and they agreed closely with those obtained using the surrogate procedure. Consistent with their hioconcentration potential, both 3,4,5-trichloroveratrole and tetrachloroveratrole were unambiguously identified by GUMS in samples of wild fish captured from areas putatively polluted with hleachery effluents (59).Chloroveratroles are not, however, persistent in fish: Analysis of fish at an early stage of the hioconcentration experiments before a steady state had been reached showed that extensive metabolism by O-demethylation of the veratroles had occurred a n d that the resulting phenolic compounds were conjugated to form sulfates or glucuronates (60). Metabolism and excretion of the xenobiotic may therefore result in only low levels of accumulation in biota. In assessing the dissemination of a given xenobiotic, attention should he directed both to microbial metabolites and to those excreted by fish as water-soluble conjugates. In the experiments directed to verification of 0-methylation, natural sediment samples were incubated with aqueous solutions of chloroguaiacols. These substrates were rapidly partitioned into the sediment phase (461, and further studies have considerably extended this to a wider range of chloroguaiacols and chlorocatechols (Table 2). Experiments on the recoverability of chloroguaiacols and chlorocatechols from sediments by different chemical procedures showed that the recoverability of these compounds was quite different (61)(Table 3). On the basis of these results, the solvent extractable fraction has been designated “free” and the fraction requiring the use of methanolic alkali “bound,” although it should be emphasized that this division is pragmatic. It was hypothesized that whereas the chloroguaiacols were associated with organic compo-
nents of the sediment phase, possibly involving covalent bonds, the chlorocatechols formed relatively readily dissociated complexes with Fe or AI constituents. The putative role of these metal cations was supported in experiments that showed that there was a linear relation between the release of Fe (and All and tetrachlorocatechol (62); in addition, chlorocatechols i n interstitial water were strongly associated with particulate material that could be removed by ultracentrifugation at 100,000 g. The extent to which they are accessible to higher organisms remains unresolved, although they are apparently inaccessible to cultures of anaerobic dechlorinating bacteria (55). The situation for chloroguaiacols presents a further example of this dilemma because it has been conclusively shown in laboratory experiments with a wide range of mixed cultures that O-demethylation of chloroguaiacols is readily accomplished; this has been confirmed with cell extracts of Acetobacterium woodii and Eubacterium limosum (63). The compounds in the sediment phase are apparently not accessible to the relevant demethylating microorganisms. After deposition and during the “aging” of the sediment, the degree of recoverability changes: this cannot be predicted from the recovery of the analytes from spiked samples in which the substrates are in contact with the sediment phase for only a short time. There is therefore an intrinsic indeterminacy in such analyses that should he considered in interpreting the results of monitoring programs (61). Toxicity of chloroguaiacols, chlorocatechols, and their metabolites. Although the toxicity of chlorophenols is generally established (261, the toxicity of chlnroguaiacols and chlorocatechols to aquatic organisms has been less extensively pursued ( 6 4 ) .It is now appreciated that assays for the acute toxicity of xenobiotics do not possess the rigor required for a hazard assessment at the concentrations that are encountered in the natural environment. Attention has therefore been directed to tests that reveal sublethal effects and, in particular, effects on impairment of reproduction. In this laboratory, the zebra fish emhryolarvae test has been used over many years and provides values of the threshold toxicity: the concentration of the toxicant displaying no observed effect and the lowest con-
Environ. Sci. Technol.. Vol. 28, No. 6, 1994 283 A
artition coefficients K, Jkg organic carbon) between the aqueous and sediment phases for a range of chloroguaiacols and chlorocatechols impound
Chlorocatechol 5- Dichlorocatechol
4,6-TrichlorocatechoI 4,5-TrichlorocatechoI ?trachlorocatechol 5 - Dichloroguaiacol
5,6-TrichloroguaiacoI 4.5 Trichloroguaiacol ?lrachloroguaiacol Chlorovanillin
centration that elicits an observed effect. One striking result was obtained by the application of this test: The neutral chlorinated veratrole metabolites induced an effect that bas not been observed with their phenolic precursors. The larvae were curved and the notochord seriously deformed with evidence of hemolysis (58). and the concentrations required to induce these serious morphological defects were lower than those using the median survival time as an end-point. Although skeletal deformation had already been observed after exposure of fish to structurally different neutral organic compounds (65-67),this was apparently the first time the compounds examined were microbial metabolites produced under plausible environmental conditions. In assessing the effect of toxicant exposure, it is important to evaluate the effect of preexposure because few fish are stationary in their natural habitats, and anadromous fish may be temporarily exposed to plumes of xenobiotics. A laboratory test was designed to quantify the effect of preexposnre and the extent to which any observed effect was annulled after removal of the toxicant. Details of the somewhat complex protocol have been given (68, 691, but the results for two pure chlorophenolic compounds clearly showed an approximately fivefold increase in sensitivity to the toxicant after preexposure. Greater sensitivity has also been observed in experiments with whole effluents (70, 72).In all cases that have been examined hitherto, the effect of these chlorophenolic toxicants (in-
cluding unidentified compounds in effluents) was apparently “reversible’’ and was annulled after postexposure in a dilution medium lacking toxicant. Such experiments could be incorporated at an appropriate level into a hierarchical system for evaluating the toxicity of xenobiotics (69). It is clearly necessary to examine the toxicity of the metabolites of a xenobiotic in addition to that of its mecursor, and it is possible to in:orporate into laboratory bioassays eatnres that considerably increase their environmental relevance. Verification procedures: Mesocosm experiments. Experiments with 4,5,6-trichloroguaiacol were carried out in mesocosms as part of a comprehensive study to evaluate various procedures for advanced hazard assessment in the aquatic environment (72). The mesocosms were designed to simulate the Baltic Sea littoral zone and consisted of 3.6-m-diameter 001s with a volume of 7.5 m containing sediment and bladder wrack (Fucus vesiculosis) with its accompanying microfauna. The pools were maintained in the open at Karlskrona in southeastern Sweden and were continuously treated during several months with 4,5,6-trichloroguaiacol at nominal concentrations of 5 VglL and 50 pglL. Samples of water, algae, microfauna, and sediment were analyzed, and biological effects were assessed from the community structure of the microfauna and from the growth of bladder wrack. The much greater sensitivity of b l a d d e r wrack compared w i t h axenic cultures of algae used in laboratory experiments (69) should be noted. Otherwise, the results fully confirmed the conclusions of the laboratory experiments on biotransformation and partition described above. The overall agreement between the two approaches was grat-
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ifying and illustrated the role of mesocosms as a valuable complement to laboratory investigations and an alternative to in situ experiments. The role of chemical support Identification and quantification of metabolites necessitates access to authentic reference compounds that may not be commercially available. Illustrative examples include the synthesis of chlorocatecbols (48). the metabolites from halogenated vanillins and hydroxybenzaldehydes under anaerobic conditions (56),the range of nitro- and cbloronitroguaiacols and vanillins (73)resulting from interest in bleaching processes using nitrogen dioxide, and the 18(19tnorditerpenes that are discussed later. In experiments such as those discussed above, a number of problems may arise: association of low concentrations of the analyte with bacterial cells that may be circumvented by sonication in acetonitrile (42), interference of high concentrations of the growth substrate with the xenohiotic and the need for mild procedures for removal of t h e former (481, the presence of metabolites in areas of gas chromatograms where serious interference from other comp o u n d s necessitates c l e a n u p procedures using open-column chromatography (48) supplemented by the use of suitable derivatives of monochlorocatechols to increase the GC sensitivity of the metabolites, and the elaborate procedure needed for isolation of the low concentrations of the chloroguaiacol and cblorocatechol conjugates in zebra fish following exposure to cbloroveratroles (60). Two examples will be given to illustrate more extensive investiga-
tions that originated from experiments on partition and toxicity. First, monitoring programs i n Sweden on the environmental distribution of organochlorine compounds from bleachery effluents h a v e s h o w n that s u b s t a n t i a l amounts of organochlorine compounds may be recovered from samples of sediment and biota. This has used analyses for the sum-parameter cyclohexane-extractable organic chlorine (EOCI) (74) or less specific analyses for absorbable organic halogen (75).There are serious limitations in the application of these parameters for environmental hazard assessments because they do not unequivocally reveal the source of the discharge, they may be compromised by interference from longdistance transport via the atmosphere of unrelated compounds, and they cannot be used to determine which compounds (or groups of compounds) are capable of causing environmentally adverse effects. Analysis of the cyclohexane EOCl extracts from sediments (76) included not only chlorophenolic compounds but chlorinated diterpenes and chlorinated long-chain alkanoic acids. Chlorophenolic compounds made only a minor contribution to the total organic chlorine, and the major part of the organic carbon was unchlorinated and comprised long-chain alkanoic acids, diterpenes, triterpenes, and plant sterols that are natural products originating in the plant material used for producing pulp. In view of their quantity, the toxicity of some of them, and the lack of knowledge of their persistence, attention to this group of compounds is possibly justified. Further investigations (77) brought to light another group of hitherto unidentified compounds that resulted from decarboxylation of dehydroabietic acid and 12,14dehydroabietic acid. Authentic samples of the 18I19)nor compounds were synthesized and the configurations determined by NMR. These compounds could then be unambiguously identified a n d quantified in samples of sediment and fish. Second, few pure compounds have been examined in fish using biochemical and physiological parameters, and to evaluate the hierarchical approach to toxicity (78) the same compound should be examined at all levels. Tetrachloro-1,Zbenzoquinone had been suggested
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PxMf.ot-xsnobirrticic not possessthe rigor required for a ham* -a s s e p t at mecalrcentratiomW - a M IiGintesiim~ Ilam mment.
clearly illustrate the value of procedures for specific analytes that were necessary both for the chlorinated benzo-1,2-quinones and for the 2-5dichloro-3,6-dihydroxybenzo-l,4quinone: neither of these would have been revealed by application of conventional procedures.
A wider perspective Two important issues-the evaluation procedure itself and the application of the principles to other Droblem areas-deserve further discussion. The evaluation orocedure: the null hypothesis i n d the role of monitoring. The most difficult part of environmental hazard evaluation is the synthesis of the data on persistence, partition, and toxicity to a consensus about the acceptability of the xenobiotic or effluent. Because truly objective procedures are not available, a balanced subjective assessment is the best that can be attained. Appreciation of the plethora of factors that must be taken into consideration in most cases makes the decision-making process increasingly complex. The uncertainty of cause-effect relationships may be illustrated from examples as a component of bleachery efflu- such as the association between exents (79)and was therefore submit- posure to polychlorinated bipheted to the standard toxicological nyls (PCBs) and the death of seal procedures using zebra fish. For populations (82). the relative imbioassays of pure compounds, toxi- portance of pesticides as carcinocant exposure should be monitored gens in humans (83).or the magniby periodic analysis. A procedure t u d e of t h e threat posed by specific for chlorinated benzo-1,Z- chlorinated dibenzo-l,4-dioxins. quinones was developed by quanti- Environmental monitoring seems tative reaction with diazomethane inevitable and should address a to form the methylenedioxy com- number of issues: analysis should be carried out for pounds (80).It was then found that chlorinated 1,2-benzoquinones specific compounds and should inwere unstable both in organic sol- clude not only the compounds disvents that could be dehydrogenated charged but also possible transfor(tetrahydrofuran and dioxan) and in mation products, the choice of analytes should be aqueous solutions at the pH values used for the zebra fish assay. A based on considerations of persisnumber of transformation products tence, partition-including bioconfrom tetrachlorobenzo-1 ,'&quinone centration potential-and toxicity, were identified (80), but it could be and attention should be given to the shown that the concentration of tetrachlorocatechol alone was suffi- season of sampling, the age, the sex, cient to account for the observed and the fat content of fish (84). Demonstrating the adverse effect toxicity toward zebra fish. An examination of bleachery ef- (if any) of these compounds on natfluents for chlorinated benzo-l,2- ural ecosystems is a retroactive exquinones and their transformation ercise. Can it be persuasively shown that the observed environmental products then showed that one of them (2,5-dichloro-3,6-dihydroxy- perturbations are causally associbenzo-1,4-quinone) was present in a ated with the environmental discharge of a given compound? The range of bleachery effluents and principles of epidemiology (85) ofthat its concentration generally equaled or even exceeded that of fer valuable guidelines, though it should be appreciated that epidemiany other single chlorophenolic c o m p o u n d (81).These s t u d i e s ological procedures are designed
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not to establish unambiguous causal relationships, but to indicate which criteria may validly be used to support a given hypothesis. The five basic criteria are: consistency, strength, specificity, temporal relationship, and coherence of the associations. A probabilistic evaluation may be all that is realistically attainable, but it may be valuable to be able to exclude alternative hypotbeses. Extension of the principles to other problem areas. The procedures for determining persistence and partition may be effectively applied to evaluating biological treatment systems. Assessment of the environmental acceptability of the effluents after treatment is amenable to the principles that have been outlined in this review and, with modification, also to the treatment systems themselves. These may be considered as large-scale continuous enrichment cultures, although they are inherently less stable than laboratory systems because of fluctuating loads and variations in the composition of the effluents. As has been illustrated for procedures simulating natural ecosystems, it is important that two issues be examined because the objective is biodegradation of the organic components a n d not merely biotransformation or phase redistribution: first, the synthesis of stable metabolites that could compromise the effectiveness of the treatment system, and second, the partition of the constituents between the aqueous and sludge phases. It has been clearly demonstrated elsewhere that the latter may be a serious problem at least in aerobic systems (86, 87) and not least because eventually the sludge must be disposed of (88). Although effective incineration procedures under controlled conditions may be implemented, hazards may result from its disposal in landfills or on agricultural land, and this should be carefully examined. There are at least three important issues arising from the foregoing discussion. First, it may be difficult to assess the persistence of the sludge constituents because of association with organic components of the soil: this phenomenon is well established from extensive investigations on the fate of agrochemicals (891 and evidence for the incorporation of xenobiotics into humus structures bv enzvmatic activitv (90,91). Second, the 0-methylation of chlorophenols was originally estab-
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"...J... limitation with hazard issessment of bleacher effluents in the aquatig environment is the liffieultfiitrxtrapolatin -laboratory re& - _to natural eeofy ns, -3. " I . " " .
lished as a result of investigations into the off-flavor in broiler cbickens that was caused by fungal 0-methylation of chlorophenols to chloroanisoles (92). The problem of offflavor caused by the presence of chlorinated anisoles is possibly widespread and includes the tainting of freshwater fisb in Finland (93) and of river water samples in Canada that received bleacbery effluents (94). Methylation has wider metabolic dimensions because fungi are able to methylate chloride ion (95) and mediate transmethylation to a range of aromatic phenols a n d carboxylic acids (96, 971. Clearly, then, there are ramifications of this apparently simple reaction that could have important environmental consequences. Last, by analogy with the situation of cbloroveratroles i n t h e aquatic environment and their concentration in biota, the synthesis of lipophilic metabolites raises the possibility of their transport and mobilization into higher trophic levels. This has been demonstrated with earthworms and chlorophenols (98,99) and, analogous to the biomagnification of PCBs in fisbeating birds [fOO),dissemination by predators such as birds is a possibility. Current evidence ( 1 O f ) suggests, however, that for chlorolignins, monomeric compounds that might have originated in bleachery effluents are not released into the terrestrial environment.
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Although this account has been devoted exclusively to chlorophenolic compounds, the principles are applicable to a wider spectrum of environmental problems with only minor modifications. Two examples are given as illustration. First, attention bas already been drawn to the major contribution of nonchlorinated alicyclic and aliphatic compounds in cyclohexane extracts of sediment samples (76). Adverse effects of pulp mill effluents may be the result of exposure to these or to other hitherto unidentified compounds, and removal of chlorine in any form from bleaching technologies may not therefore necessarily eliminate the hazard from discharge of bleacbery effluents into the aquatic environment. Second, increasing effort has been devoted to the problem of solid waste; this includes landfills that may contain both domestic and industrial waste (102) and the bioremediation of sites historically contaminated with industrial waste (32, 103). The spectrum of compounds that may be implicated is extremely wide, but the principles for effective application of these technologies are encompassed by the procedures outlined in this review. Unresolved issues Some important issues associated with hazard assessment of bleachery effluents in the aquatic environment remain unresolved. First, the greatest single limitation in all such strategies is the difficulty of extrapolating the results of laboratory investigations to natural ecosystems. This applies equally to problems of persistence, partition, and toxicity. All of these require a nice sense of judgment because completely rational procedures do not so far exist. Fuller appreciation of the determinants and departure from exclusively inductive principles (f04) are probably necessary. Second, at least in the Northern Hemisphere the main source of pulp is birch and pine. This account has therefore discussed only problems arising from the use of these. If other sources of pulp are used, the problems may be substantially different. An analysis of the effluents would be worth the investment. Whereas in northern latitudes discharge will often take place to receiving waters such as rivers or lakes with relatively low temperatures and low salinities, the situa-
tion for marine systems with high r a t e s of flushing c a u s e d by tides (106)or i n tropical or subtropical systems w i t h high water temperatures will be significantly different and should be taken into account. Caution s h o u l d therefore be exercised i n uncritical extrapolation of the principles that have been outlined here. Last, it is virtually impossible to avoid errors of judgment; the most i m p o r t a n t t h i n g is to maintain a close watch for environmental perturbations a n d e n s u r e that t h e a p propriate control measures are implemented as soon as possible. All of this merges with t h e m u c h w i d e r i s s u e of e n v i r o n m e n t a l m a n a g e m e n t a n d ecoepidemiology-both of which, however, lie well beyond t h e scope of this account.
R. M.: Luthe. C.E.: Voss. R. H. Enairon. Sci. Technol. 1993.27,1164. ( 5 ) Carlhcrg. G. E.: Urangsholt. H.: Gjiis. N . Sri. 'TofolEnviron. 1986. 4H. 157. (R) Kringst;d. K. P.
