Document MJeoOrJYz3vZ328XQEX76GK17

Activ;it:`(l SJinI"<` I', nnarv by C. S<.<n l Tucklii, Vu.iui: W Svi 'i u and (luvli l.i l(u i.a Mnntuulti ( tnnfmry St. /.</< , */ A il (i(j March Jo, lf/i J Since the identification of polychlorinated biphenyl (PCD) residues in the environment in Sweden (Jensen. 1966) an effort has been made to determine the level and distribution of these materials as well as their effects upon living organisms. The widespread distri bution of low but detectable levels of some PCOs in the environment is now well documented and a substantial amount of data have been accumulated regarding their acute, subacute, and chronic toxicity (Peakali and Lincer, 1970; Edwards, 1970). ` While much is known about the effects of PCDs on living organisms, there is relatively little informa tion available regarding the effects that living organisms have upon PCSs, i.e. their biodegradability. The biodegradab i 1ity or susceptibility of an organic compound to biological degradation, especially by bacteria, is a prime determinant of its environmental residence tine. The finding of PCD residues in the environment suggests to many that PCBs as a class of compounds are resistant to microbial degradation. However, in order to fairly evaluate the persistence of rC0s it must be understood that PCSs are not a single entity, but complex mixtures made up of many entities which may undergo biological degradation at different rates. Chlorinated biphenyls are commercially produced by the direct chlorination of biphenyl. The resultant mix tures can theoretically have as many as 210 different components containing 0-10 chlorine atoms per biphenyl molecule. Of the possible isomers, 103 are considered nest probuole (WidmarU, 1968). The complexity of the PC us, therefore, complicates the determination of both environmental levels and their impact on the biota. Environmental monitoring programs have demonstrated that, with the exception of direct high level controll able contamination near points of manufacture or use, vi. 11, s, 4 nrj if VerUg 70S i***. ACM C05AS WATER PCB-SD0000055145 I lie PCI'i1. gene;a I ly found in the cm v i rein -en L (ire the nnre highly eh ! or i nt! Led h i |> !i c ny 1r. , i.c., those c or. t a i n i ng S or no re chlorine a terns per biphenyl mo 1 n >. n ] < (Jenson and 1! i (li.ij rl- , 190/; Koines, et a 1 , 19G7: Kocinan, ct <i 1 , I960). This is true oven t h cm cj h the more' highly c li 1 o r i na 1 ed biphenyls cons I i Into only a hunt 30-'of all the I'Clis iiianufoc. lured over t!ie years (Monsant o Company, 19/1). This is strong evidence that the less chlorinated materials degrade more rapidly Ilian the more highly chlorinated ones. It also suggests that even under conditions of unrestricted use and withon l special precautions to prevent entry into the environment that the less chlor inated r.'ai.prials degraded rapidly enough to prevent accvc'j l.a t i on . It is obvious, t. he re fere , that to oh t a-in a more com plet'1 u nde i s t a .!'! . n g of the en v i ro n.ap n L a 1 behavior of I'CDs t.hit i n forr'a t i on concerning their susceptibility to i.i i c roll i a ) degradation is needed. It has been shown that biphenyl can be degraded by grc---r aria t i vo bacteria through 2, 3-di hydro -2,3-dihydr c ,\y b i pl.eny 1. ci - by d ro >;y - f.- pheny 1 r.iuc on i r. s ein i - a 1 de hyde and phenol pyruvate (Liint and Fvans, 1 9 70 ), and it h =. s beei' repr. rted that Pseudomonas pu_l_i cj_n oxidizes hiih-.T.yl througn 2,3-dihydro - 2,3-d i hy(iroxy hi phony 1 and benzoic acid ( Ca tol an i , et a 1 , 1971). More rec-i'lly Gibson cr.d co-v.'orhm s isolated a bacterium, tent/t i vc-ly idsrtificd es a 0 e i j c- r i n c I i a species from a pelleted sIit: capable of u't i 1 i zTe.if !> i pheny 1 as a sole sc.iccc- of cwcor and energy for growth (Gibson, e t cl. 1 973 ; a d c rea r:i ss capable of oxidizing b i .* i,y 1 end p-culcro biphenyl have been shown to be widely distributed' in the natural environment (Olmori, e t a 1 . 1 9 7 3 ). De('da Li on studies have also been carriid out on some selected PCU isomers with two specie1- of Achreriohacler isolated from sc.w.ge (Alined and Tocht. lf/3a,b) and with Aroclor I 2s? (Kaiser and l.' o n t; , 19/'l. To date, no . ii r I. hr.s ! ! c- n reported or. the b i oovgra^c t i on of co;v rc i a 1 f'CP ciix lures by activated 11; d g e . In Ihv steely reported here, the s u s c < p M i. i 1 i t y of cor-wc rc i1 po 1 ych 1 n i i n j t.od biphenyl m i > tu e s to pri mary ileijr.il; t i on by activated sludge i.i i c i oorg.j n i sins was investigated. \ 706 *_ u ADM OC 51 WATER PCB-SD0000055146 Pd 1 ycl: 1 cm- i r. a Lc d N i licnvl Mixtures llio f o 11 o\/i ny I'Olych 1 ori na Loci biphenyl mixtures i'inufactuied by Monsanto Coi.spny wore studied: Aroclor1 1 2;iA , Aroclor 12A2, Aroclor 1016, MCS 10-13 (not a commercial i.iixtuic), and Aroclor 1221. In T t- ti 1 o I, tn-? typical per cent composition of the mixtures studied is given in terms of the number of chlorine a t oms per biphenyl. T/Vi! !- I TYPICAL CwibMir. t t t o;; ok PCI YCi: lc; ii tL nw j:_i Plj.LjijJ;. .i'j.uQi;|bC r_3_'_` f Cl Amc 1 or MCS Aroclor Aroic 1 or A r o e 1 in - 1 221 1 0 T 3 1016 1 2A2 1 2 54 phony-1 (? i:: Cl) ( ''O'- Cl ) (Alf. Cl) (fl2::L_c.U 1 6 A ` ' C 0 11 0.1 <0.1 <0 . 1 <0.1 1 51 22 1 1 <0.1 2 32 72 20 16 <0.5 3 4 6 57 A 9 1 A 2 i;o 21 25 21 5 <0.5 f.'D 1 8 A8 6 r:i) HD <0.1 1 23 7 ;;u NO r;i) <0 . 1 6 8 'i 0 t;o NO NO NO *l'cr ce at fw/w) \ij uC/i * T* s using ai r e a correc L 1 t. M f actor s . Nil - l.'o . i JiiCC r Ao 91 Uiodeurd.. *. i on Mothod The primary do g r a cl .1 l i n n rale of the PCC r,i >: t ere e / <i s tie ten., i nod using the Soap and Detergent Association s cm i - con f. i n-.iou s activated sludge (SC/.S) pre-codm o end modified feed (S.D.A., 1965, 1959). The sludge unit used i as ,i cy 1 i nd r i c a 1 glass chamber (06 r.r 0.:,.) of lf-On i: 1 working volume v;itl. provisions for aeration, megm. tic stirring, sampling and draining. The a: l iv.iteb sludge cultme was ol> if. i nod f ror.i a local uni ci lia 1 sewage treatment plant and a c c 1 i; I 2 d on synthetic sewage (300 mg of glucose .* 200 r.iij nutrient hrnth * 130 my KII?P0., por iilor) for several weeks prior l.o the start of iicLu.il feeding of the po 1 yc h 1 o r i n .1 Lc cl l.ip'ienyl mixtures. Tlie mixed liquor (sludge -f- aqueous phase) 11'.eg i s lered trademark of Monsanto Company 707 ACM CC5<* WATER PCB-SD0000055147 was initially adjusted in each sludge unit to a sus pended solids concentration of about 2500 mg/liter and during the course of the tests readjusted to this value on a weekly basis. Each cycle was initiated by the addition of synthetic sewage and the PC8 being studied. Because of their low water solubility, the PCB mixtures being tested were fed via syringe injection of 200 ul of an ethanol solution. In this manner, homogeneous dispersions of the PCBs on the bacterial sludge were ob ta Tned. After about 30 minutes of aeration, a 20 ml aliquot of the nixed liquor was withdrawn and analyzed for the PCB mixture in question. Aeration was continued until the end of the cycle when a second 20 ml sample of mixed liquor was withdrawn for analysis. At this point, the aeration was stopped and the sludge allowed to settle. After noting the sludge volume, two-thirds (1000 ml) of the superna.tant was withdrawn and replaced with tap water. Monitoring of the sludge volume and supernatant pH provided some indication of satisfactory operation . of the unit. The units were generally operated on two 4S-hour and one 72-hour cycle per week. Analytical Methods The PC6s in the mixed liquor samples were isolated from the sample matrix by solvent extraction using either nanograde or spectrograde hexane. After concentrating in a Kuderna-Cani sli evaporative concentrator, the ex tracts were analyzed for PCBs by either e1ectron-capture gas chromatography (EC/GC) or ultraviolet (UV) spectro photometry. The UV analyses were made with a Cary Model ]4 recording spectrophotometer and matched 2.0 cm quartz cells. The gas chromatographic analyses were carried out with a Hewlett-Packard 5750 chromatograph equipped with a hi63 e 1 ec t ron - cap : u r e detector. A 2m X 4 mm glass col umn packed with XE-60 on C0/100 mesh Cliromosorb 1/, II. P . , was employed. The tempera tures of the injection port, column and detector were 220"C, 170-220C, and 300"C, respectively. Calibration curves were prepared using standard solutions of the appropriate PCB mixture. In Table II, the wavelength of the absorption maxima and absorpti v i ti es employed in the l'V analyses are given. Aroclor 1254 did not have sufficient UV absorp tion for analytical purposes. 7na ACM CC 5 5 WATER PCB-SD0000055148 TAULE II ULTRAVIOLET ABSCRPT I Of,I Ar;u RECOVERY DATA FOR BIPHENYL AND POLYCHLOli; HATED BIPHEiiYL MlkTURES Material Biphenyl Aroclor 1221 MCS 1043 A roc 1o r 1016 Aroclor 1242 Aroclor 1254* A Ma x . , nm 246 245 244 24 6 245 - Abs o rptivity Liters/g-cm * 110.9 66.4 48.9 36.1 37.0 - . Per Cent Recnvc- ry 93 * 3 96 + 1 95 + 1 92 + 1 04 + 1 76 + 2 + Analyzed by EC/GC In order to demonstrate the efficiency of the extrac tion procedure, samples of activated sludge were spiked in duplicate at two levels, 2.5 and 5 ppm, with each of the materials and then carried through the entire analytical procedure. The recovery data for each PCG fluid studied are given in Table II. No isomer dis tribution changes were observed upon comparison of the e1ectron-c2pture chromatograms of the PCB reference materials to those of the spiked sludge extracts. In order to show that the PCBswere not irreversibly ad sorbed cn and/cr stored within the bacterial cells of the activated sludge and therefore not recovered via extraction, a sample of acclimated Aroclor 1016 nixed liquor was homo gen i zed with a Polytron Sonic Homogen ize r to lyse the bacterial cells and then extracted. A 100 ml sample of homogenized mixed liquor gave a PCB level of 0.51 mq compared to 0.53 mg for an identical mixed liquor sample treated in the normal manner. RESULTS AND DISCUSSION .. In Figure 1, the per cent degradation rates and 95% confidence limits obtained in this study are given for biphenyl and the PCS fluids. It is apparent from the plot of degradation rate vs weight per cent chlorine that the level of chlorination of the mix ture is the most significant factor in the relative degradability of the PCBs. The .degradation rates reported here were obtained after the sludge units had been acclimated for abnut 5 months to the appropriate PCB. * Initially the PCB mixtures were fed at a rate of 1 mg per 24-hour cycle, but be cause of the relatively slow rate of degradation observed in spot checks, the cycle time was increased to 48 hours. v 709 ACM OC 5 5 WATER PCB-SD0000055149 . % Chlorine (w/w) Figure 1. SCAS primary biodegradation rates of commercial PCBs as a function of the weight per cent chlorine. Since Aroclor 1221 was found to be quite degradable, the effects of both time cycle and feed level were studied brief1.y. In Table III disappearance rate data obtained for 24 and 48-hour tine cycles at 1 and 5 mg feed levels ere given. It is apparent that for Aroclor 1221, most of the degradation occurs during the first 24 hours. This can he explained by the fact that the lower chlor inated biphenyls degrade more rapidly than the higher chlorinated biphenyls. '> To verify that the disappearance of the PCBs -was due predominantly to degradation and not to volatilization, off-gases from the Aroclor 1221, MCS 1043, end Aroclor 1015 units were passed through .i train of three hexane scrubbers during several complete cycles. At the 0.1 cubic foot per hour aeration rare, the d i s a;.'pea rar.ee rates due to volatility were 4.2, G.l, and 2-.G- for Aroclor 1221, MCS 1 043, and Arcelor 10 16, res pectivelv . These losses are well within the 95;; confidence limits of the overall disappearance rates. V 710 ADI* 00 5 WATER PCB-SD0000055150 TABLE III EFFECT OF T I ME CYCLE _A'\' li _T_EEO_ L_C7 L_L (J N THE AROCLOK 122 1 D ['SAP PL I Cl ::.M~E Feed Level Disappearance Rats, 2A-Hr cycle a 3 - H r Cycle 1 P19 5 mg 73 + 2) 89+17 81+6 87+5 - In order to observe changes in the distribution of the chlorinated biphenyls in the PCDs mixtures after exposure to the activated sludge, selected samples of Aroclor 1221, MCS 1043, Aroclor 1016 and Aroclor 1242 were analyzed by EC/GC. The chromatogram for Aroclor 1221 is shown in Figure 2. The top trace is a chro matogram of the Aroclor 1221 standard, representative of the feed material. The center trace is. tnat of a concentrated sludge extract taLen at the end of a degradation cycle. At the bottom is a trace of an Aroclor 12A2 standard run under equivalent conditions. The numbers above each peak indicate the dominant PCD represented by the peak as determined by GC/Mass spectrometry. Comparison of the extract chruuatograms to that of the Aroclor 1 242 standard shows that the minor components in Aroclor 1221, which do not degrade as rapidly, are the major components in Aroclor 1242, MINUTES Figure 2. Typical electron capture chromatograms for Aroclor 1221, after exposure to activated sludge for 24 hours, and Aroclor 1 242v. 711 A lM CCO WATER PCB-SD0000055151 It is important to note that the electron-capture detec tor does not have the same response for all components. With PClls, the sensitivity of the detector generally increases as the degree of chlorination increases. From the Aroclor 1221 chromatograms, it is apparent that the dominant mono ch 1 orob i pheny 1 and d i ch 1 o rob i plieny 1 compo nents are readily degraded. Once the major components of Aroclor 1221 are degraded, the minor more slowly degrading components are easily observed after concen tration of the sludge extracts. . Similar, less dramatic, alterations were noted for KCS 1043 and Aroclor 1242. Uo differences were observed in the extract and Aroclor 1254 standard chromatograms. COflCLUS IONS The results of this study demonstrate that commercial PC3 mixtures which contain predominantly mono- and dich1orobipheny1s readily undergo primary biodegradation under the experimental conditions employed. The data also illustrates that as the levels of tri-, tetra-, and pentach1orobipheny1s increase, the degra dation rates decrease accordingly. This resistance of the more highly chlorinated biphenyls, particularly those containing 5 or more chlorine atoms per molecule, explains in part their detection as residues in weathered biological and environmental samples. \ 712 ACM CC55C' WATER PCB-SD0000055152 ACKNOWLEDGE.'!!:.'/! We are grateful to J. P. Mieure for determining the typical composition of the PCD products studied. REFERENCES AH MEL), M. , and FOCHT, D.D., Bull. Environ. Contain, and Toxicol., lj), 70 (1973a). AHMED, M., and FOCHT, D.D., Can. J. Microbiol. 1_9 , 4 7 ( 1 9 7 3b). CATSLINI, D., SORLINI, C., and TRECEAli I , V., E x p er i e n t i a , 2_7, 1 1 74 ( 1 9 7 1 ). EDWARDS, R., Chemistry and Industry, .20, 1 340 (1970). EVANS , W.C. and LU.`/T, D., Diochem. J. 118, 54P (1970). GIBSON, D.T., ROBERTS , R.L., WELLS, M.C. and KOBAL, V. V. , Biochem. Biophys. Res. Comm., 50 , 2 1 1 (1973 ). HOLMES, D.C., SIMMONS, J.H., and TATTOIJ, J.O'G., ' Nature , 2J_6, 1 274 ( 1967 ) . JENSEN, S., and WIDMARK, G., "Unintended Residues in the Environment", O.E.C.D. Report (1967). JENSEN, S., New Sci., 3 2, 6 1 2 ( 1 966 ). KOEMAN, J.H., TEN NOCVER UE BRAU, M.D., and DE VOS, R.H., Nature 22.1, n?6 (1969). Monsanto Company, Chen. Eng. News ,4_9, 15 (1971). OHMO.Ri, T., I :<A! , T., MINOOA, Y. and YAMADA, K. , Aqr . Biol . Cr.em. 7 , 1 599 (1973 ). PEAr'.ALL, D.B., and LI/TCER, J.L., Bio Science, 20, 958 (1970).- Soap and Detergent Association Sub-Committee on Biodegradation Test Methods, J. Amer. Oil Cham. Soc. 46, 432 (1969). So jo and Detergent Association Su b - Con:ni 11 ee on Biodegradation Test Methods, J. Amor. Oil Chem. Soc. 4j>. 986 (196 5). WIOMARi'., G., "Determination of the Number or Com pounds Which Can Result From the Chlorination of Biphenyl and De ve 1 opi,,f,n t of a Simple System by Which These May be Codified", O.E.C.D. ' Report, Sweden (1958). \ 713 .i \ ADM C-C55C i WATER PCB-SD0000055153