viously not random. I t can be related to the purification method, where radium is isomorphously coprecipitated with barium sulfate. If certain limiting conditions (see below) are fulfilled, the distribution of radium and barium between solution and barium sulfate crystals can be described by an equation analogous to that derived by Henderson and Kracek (11): (CRa/CBa)diss
= KRa,BaS04 (CRa/CBa)particulate
Here K R ~ , Bis~the s ~so-called ~ homogeneous distribution coefficient or, more correctly, separation factor (12),and C R ~ and C B are ~ concentrations of radium and barium. The values of K R a , B a s o 4 calculated from the average ratios found a t individual sampling places are given in Table 111. Except for sampling places 3 , 7 , and 9, the calculated values (for 2-13 OC) are close to the reported value for pure solutions ~ 1.8 s ~ (13). ~ The different value found at and 20 “C, K R ~ , B = sampling place 7 is without doubt due to an unequal dilution of soluble forms of radium and barium at the confluence, which is also evident from the lower Value of (CRa/CBa)diss found at this place. As already discussed above, the equilibrium cannot reestablish on the short distance between the confluence and sampling place 7 ; 12 km downriver, at sampling place 8, the proper ratio of dissolved and suspended forms of radium and barium is reached again. Two important conclusions can be drawn from these results. First, the predominant particulate form of both of the elements a t sampling places 4-8 appears to be Ba(RaIS04, whereas the nature of the insoluble forms of radium in mining waters and in the river water upstream at the confluence is at least partially different. Verification of this conclusion using the method of selective dissolution (14) is in progress. Second, the quantitative theory of homogeneous distribution (12,131 can be used for the description of isomorphous coprecipitation even in such complicated systems as surface waters. A closer examination of the individual results proved that Doerner and Hoskins’ (15)logarithmic distribution law is not applicable here. The concentration of dissolved barium in the studied waters should be controlled by the concentration of dissolved sulfates (present in excess) according to the solubility product of barium sulfate, log K , = -10 (16). Table I contains the log K,, values calculated from the average concentrations of dissolved barium and sulfates found at the corresponding sampling places. The values range from -8.95 to -9.23 (sampling place 4 cannot be considered in this connection as the precipitation
of barium sulfate was not finished there). The difference between the tabulated value and the values found here points to a very small particle size of the suspended barium sulfate or to its imperfect crystalline structure, since it is known that solubility of precipitates increases with decreasing particle size and with decreasing perfectness of their structure. It is the small particle size or the imperfect structure of crystals which is known to be a necessary condition for a quick establishment of coprecipitation equilibrium that can be described by the equation given above (12). Acknowledgment
We thank Dr. I. Rubeska for the atomic absorption spectrophotometry analyses. Literature Cited (1) Tsivoglou, E. C.; Stein, M.; Towne, W. W. J. Water Pollut. Control Fed. 1960,32,262. (2) Justjrn, J.; Stangk, Z. Prague, Czechoslovakia, 1968, Water Re-
search Institute Final Report No. S-R-30-238/4. (3) Iyengar, M. A. R.; Markose, P. M.Proc. Natl. Symp. Rarliat. Phys. 1970 1971,473. (4) Hanslik, E.; Mansfeld, A. Prague, Czechoslovakia, 1973, Water Research Institute Final Report No. R-502004. (5) “Standard Methods for the Examination of Water and Wastewater.” 13th ed.: APHA. AWWA. and WPCF, Washington, - D.C., 1971; pp 331 and 611. (6) Veimiiovskjr, J.; BuEina, I. Jaderlui Energie 1966,12, 413. (7) Sixta, V.; Mikiovskjr, M.; Sulcek, Z. Collect. Czech. Chem. Commun. 1973,38, 3418. ( 8 ) Brinck. W. L.: Schliekelman. R. J.: Bennett. D. L.: Bell. Ch. R.: Markwood, I. M. Washington, D.C., Dec. 1976,U.S. Environmental Protection Agency Technical Note ORP/TAD-76-5. (9) Canet, A.; Zettwoog, P. ‘‘Removalof Radium from Uranium Mill and Mine Effluents using Barium Chloride Method” (in French), Report to the French Atomic Energy Commission, Fontenayaux-Roses,1977. (10) Bene;, P.; Steinnes, E. Water Res 1974,8, 947. (11) Henderson, L.; Kracek, F. J A m Chem SOC 1927,49, 738. (12) Benei, P.; Majer, V. “Trace Chemistry of Aqueous Solutions”; Elsevier: Amsterdam, 1980. (13) Starik, I. E. “Principles of Radiochemistry” (in Russian), 2nd ed.; Nauka: Leningrad, 1969; p 310. (14) Bene;, P.; SedlPEek, J.; Sebesta, F.; Sandrik, R.; John, J. Water Res., in press. (15) Doerner. H. A.: Hoskins. W. M. J . Am. Chem. SOC.1925.47. 662. (16) SillBn, L. G.; Martell, A. E. “Stability Constants;” Chemical Society, London, p 236. Received for review March 26,1979. Accepted August 15,1980.Financial support from the International Atomic Energy Agency under research contract No. 1729/RB is gratefully acknowledged.
Kinetic Study of the Biodegradation of Biphenyl and Its Monochlorinated Analogues by a Mixed Marine Microbial Community Paul B. Reichardt,” Barbara L. Chadwick, Maureen A. Cole, Betsy R. Robertson, and Don K. Button Institute of Marine Science and Department of Chemistry, University of Alaska, Fairbanks, Alaska 9970 1
The first comparative biodegradation rates of biphenyl (BP) and chlorobiphenyls (ClBP) by microbial communities in seawater is presented. Rates were in the order BP > 2-ClBP > 4-C1BP > 3-ClBP. The total observed range in these relative rates was only a factor of 3.7. The turnover time of monochlorobiphenyls in an Alaskan estuary is estimated to be on the order of 1 yr a t a concentration of 0.1 pg/L or less with longer turnover times probable a t higher concentrations. No significant buildup of partially degraded products was observed.
0013-936X/81/0915-0075$01.00/0
@ 1981 American Chemical Society
The polychlorinated biphenyls (PCBs) are now generally recognized as one of the most ubiquitous and persistent types of environmental contaminapts. These synthetic mixtures of chlorinated derivatives of biphenyl have found widespread industrial use due to their physical and chemical stability and their dielectric properties. Inadequate waste disposal procedures have led to their release into the environment where they have been routinely detected in soil, water, and biota. Environmental concern about PCBs centers on their toxic effects toward a wide range of organisms ( I ) and their modulating effects on microbial species composition (2,3). Volume 15, Number 1, January 1981 75
The initial view that these persistent contaminants are not significantly degraded in the environment has been replaced by a model involving both photochemical ( 4 ) and biological ( 5 )degradation. Whereas the major features of the pathways for chlorinated biphenyl metabolism have been elucidated ( 6 ) , less is known about the factors which control the rates at which individual chlorobiphenyls are biodegraded. In general, lightly chlorinated biphenyls disappear from the environment much more rapidly than their more highly chlorinated counterparts (7). Several studies (8-10) on microbial degradation of individual chlorinated biphenyls have corroborated the relationship between degree of chlorination and biodegradability, but they have also demonstrated important biodegradative effects due to the position of chlorination. The oceanic regime, acknowledged to be a major environmental sink for PCBs ( I l ) , has not yet been integrated into this biodegradative framework. Harvey has noted a decline in oceanic PCB levels since their industrial use has been curtailed (12), and Colwell has demonstrated the widespread occurrence of potential PCB metabolizers in estuarine bacteria ( 5 , 1 3 ) . However, the processes of PCB biodegradation in the marine environment remain largely unknown. This study was designed to provide the first information on the relative rates of catabolism of individual monochlorobiphenyls and the nature of the products in mixed cultures representative of marine microbial systems. The monochlorobiphenyls were chosen as substrates since they are expected to be the most labile of the chlorinated biphenyls and structure-biodegradability relationships are more easily assessed than with multiply chlorinated analogues.
400
I
-0
a, + I a,
> C
E-J a,
c
3001 200
‘2 c cn
n Y
cn
I
1
20
40
I
i 60
DAY Figure 1. [ ’‘C]C02 production from labeled chlorobiphenyls in Valdez seawater. Initial concentrations were as follows: biphenyl I, 0.72 mg/L; biphenyl 11, 0.68 mglL; 2-chlorobiphenyl, 0.28 mg/L; 4-chlorobiphenyl, 0.54 mg/L; and 3-chlorobiphenyl, 0.68 mg/L.
H2S04) a stream of nitrogen was bubbled from a glass frit through the solution (ca. 400 mL/min). The gas stream was passed through a tube containing glass wool and concentrated HzS04 to remove most of the water. The CO2-containing E x p e r i m e n t a l Section stream was then passed through a chilled (-78 OC) tube containing glass beads followed by a chilled (-78 “C) 10 X 0.5-cm Reference Compounds, The following compounds were column of Tenax-GC (Applied Science, 60-80 mesh) to repurchased commercially and recrystallized from aqueous move traces of volatile organics. The purified stream was then EtOH before use: biphenyl (BP), benzoic acid (BA), monopassed through a series of three scintillation vials fitted with phenylphenols (2-, 3-, 4-PP), and monochlorobenzoic acids bubbling tubes and filled to half-volume with phenethylamine (2-, 3-, 4-ClBA). The three monochlorobiphenyls were synscintillation cocktail. After 30 min bubbling was discontinued, thesized and purified according to published procedures and the vials were analyzed for radioactivity. Controls showed (14). absorption of >99% of the COz and removal of >99.99% of Radioactive Compounds. BP-U-14C (0.15-2.4 mCi/mmol) residual hydrocarbon. was prepared by diluting commercial BP-U-I4C with unlaAnalysis for Nonvolatile Organics. The solution which beled material and recrystallizing from aqueous EtOH or by had been stripped of COz was further acidified to pH 2 vacuum transfer from powdered NaOH onto a cold finger. Labeled monochlorobiphenyls (2-C1BP-I’,2’,3’,4’,5’,6’-14C~, (HzSO4) and sonicated for 2 min. After continuous extraction (15 h) with ether, the ethereal extract was dried (MgS04), 0.20 mCi/mmol; 3-ClBP-I’,2’,3’,4’,5’,6’-14C6, 0.20 mCi/mmol; filtered, and concentrated ( 2-C1BP > 4-C1BP > 3-ClBP. Potential influences on the shapes of Figure 1curves include administered substrate depletion, oxygen depletion, 14C02 loss (BP data at 40 days), induction, and selective cell growth during incubation. In view of a K m of 1.5 nM for biphenyl in this system (Table 111) and with the assumption that K ,
Bioconcentration of nonpolar substrates by aquatic microorganisms is governed by an equilibrium process (1719),established in minutes or less (17,19),with large partition coefficients favoring lipophilic vs. aqueous medium. Since environmental chlorobiphenyl metabolism half-lives are many months (see below), slow steps occur which could lead to the accumulation of partially degraded (oxidized) substrates. Since any such product accumulation is of environmental importance, a search for significant quantities of likely metabolic intermediates in these incubations was conducted. The search was concentrated on phenylphenols and benzoic acids. Phenylphenols have been detected in microbial degradations of biphenyl, probably as artifacts coming from dihydrodiols during the isolation procedure (20). Chlorinated benzoic acids have been identified as microbial degradation products from a variety of chlorinated biphenyls ( 2 1 , 2 2 ) . Incubations of biphenyl-14C with water from Port Valdez were analyzed for the presence of 14Cin nonvolatile organics. Autoradiographic analyses of chromatographs of nonvolatile organics from incubation of biphenylJ4C revealed significant radioactivity only at spots corresponding to biphenyl and the origin (total recovery of radioactivity I80%).Radiochemical analysis (liquid scintillation counting) of benzoic acid and phenylphenols obtained from these chromatographs confirmed the low radioactivity in these substances (Table IV). Similar autoradiographic analyses of incubation mixtures from monochlorobiphenyls revealed radioactivity only in unaltered substrate and at the origin. Isolation of benzoic and chlorobenzoic acids and recrystallization to constant specific activity demonstrated insignificant accumulation of radioactivity in these substances (Table V). These data allow the conclusion that the only significant carbonaceous product from biphenyl and its monochlorinated derivatives is COz. This finding simplifies the analysis of kinetic data in that the determination of the rate of COZ production approximates the rate of disappearance of chlorobiphenyl. Furthermore no corrections are needed in comparing rate data from substrates having nonidentical radiochemical labeling patterns. Discussion
This study has demonstrated the ability of the microbial community from Alaskan coastal waters to completely metabolize biphenyl and monochlorobiphenyls at measurable Volume 15, Number 1, January 1981
77
rates. While biphenyl is metabolized more rapidly than any of the monochlorobiphenyls, there are significant differences in the maximum rates of biodegradation of the individual monochlorobiphenyls. This situation is consistent with the generalization (7) that the more highly chlorinated biphenyls are more recalcitrant to biodegradation, but it also demonstrates the position of chlorine substitution to be important. Thus the former generalization is reliable as a predictive tool only when carefully chosen substrates are compared. Although several studies on the relative rates of metabolism of chlorinated biphenyls have been reported, there are only two previous reports of the rates of microbial metabolism of the monochlorobiphenyls. Ahmed and Focht (21) measured the rates of oxygen uptake by Achromobacter-pCB (isolated from sewage effluent by enrichment techniques with 4-chlorobiphenyl as the sole carbon source) when biphenyl and monochlorobiphenyls were added. Wong and Kaiser (23) measured the rates of disappearance of biphenyl and 2- and 4-chlorobiphenyl when incubated with water from Lake Ontario. The relative rates calculated from their data along with our findings are presented in Table VI. Comparison of the data in Table VI indicates that Achromobacter-pCB is able to metabolize two of the three monochlorobiphenyls tested at about the same rate as biphenyl. However the whole microbial communities from both freshwater and marine environments metabolize monochlorobiphenyls at rates significantly below that for biphenyl. Thus Achromobacter may have particularly high levels of dehalogenase so that chlorination does not effect the rate-limiting step, or else the oxidase and associated enzymes may be differently situated in order to take advantage of the higher lipid solubility of chlorinated compounds. Alternatively one can explain the kinetic differences by postulating that natural waters contain low populations of efficient chlorobiphenyl metabolizers and higher populations of microorganisms which metabolize aromatic hydrocarbons much more rapidly than their chlorinated analogues. Probably more important than this observation, however, is the realization that all three systems show qualitatively the same relative rates for biodegradation of the isomeric monochlorobiphenyls (possibly equivocated by the fact that Wong and Kaiser did not include 3-chlorobiphenyl in their study). Thus the chlorobiphenyl metabolizing enzymes of these three microbial systems apparently face the same kinetic restraints in oxidizing chlorinated biphenyls. We have then, for the first time, enough biodegradative rate data from a variety of microorganisms and the same set of chlorobiphenyls to justify attempts to define structure-biodegradability relationships. While the monochlorobiphenyls are not completely representative of PCBs causing environmental concern in that they contain a nonchlorinated ring which provides the site for initial enzymatic attack (21, 22), any proposed structurebiodegradability relationship must take into account the differences in rates observed for the isomeric monochlorobiphenyls. Of the various proposed models for structure-biodegradability relationships proposed for PCBs (7, 9, 24), two are applicable to the monochlorobiphenyls. The earliest proposal (7)-rate of biodegradation inversely proportional to degree of chlorination-remains useful as a generality. However the rate data of Table I as well as data obtained in other studies (e.g., ref 8, 9,21, and 23) have demonstrated the limitations of this generalization. Schulte and Acker (24) proposed that nonchlorinated para and meta positions on the biphenyl template are required for biodegradation. The substrate (2ClBP) with the greatest number of these is indeed metabolized most quickly. However it also appears that substitution of a single meta position with chlorine has a greater rate-retarding effect than does a similar substitution of a para position. 78
Environmental Science & Technology
Table IV. Analysis of Residual Organics from [14C]Biphenyl Incubation at 14% Conversion to COP % of radioactlvlty in ether-extractable organlcs
compd
biphenyl benzoic acid 2-phenylphenol 3- and 4-phenylphenol
83 1.5
1.4 1.5
Table V. Analysis of Residual Organics from [14C]Monochlorobiphenyl Incubations at 5-10% Conversion to COz substrate
2-chlorobiphenyl 3-chlorobiphenyl 4-chlorobiphenyl a
% of radloactlvlty in etherextractable organics
compd
benzoic acid 2-chlorobenzoic Acid benzoic acid 3-chlorobenzoic Acid benzoic acid 4-chlorobenzoic Acid
0 0
