independent of precipitation as indicated by the fact that the regression slopes are parallel. However, a greater proportion of the total P b was removed during the first 2.5 mm of precipitation in the misting treatment than for either of the rain treatments. During the rain treatments, raindrops landed on the leaf surface with a force sufficient to cause the raindrop to break up and the leaf to rebound from the impact. On the other hand, the much smaller droplets of the mist settled uniformly over the leaf surface and coalesced into larger drops which then ran toward the leaf edge and dropped off without causing very much leaf movement. This indicates that dissolution or suspension and subsequent runoff is a more important mechanism in removal of PbC12 from the surfaces of leaves than bombardment and splashoff by raindrops. Acknouledgment The technical-help of D. Bubenick and D. Lane contributed greatly to the success of this work. Literature Cited (1) Colucci, J. M., Begeman, C. R., Kumler, K., J. Air Pollut. Control Assoc., 19,255 (1969). (2) Chow, T . J., Earl, J. L., Science, 169,577 (1970). (3) Solomon, R. L., personal communication. ( 4 ) Wedding, J. B., Carlson, R. W., Stukel, J. J., Bazzaz, F. A., Enuiron. Sci. Technol., 9, 151 (1975).
(5) Wedding, J. B., MS thesis, University of Illinois, Urbana, Ill., 1972. (6) Berglund, R. N., Liu, B. Y., Enuiron. Sci. Technol., 7, 147 (1973). (7) Wedding, J. B., Stukel, J. J., ibid., 8,456 (1974). (8) Bazzaz, F. A., Boyer, J. S., Ecology, 53,343 (1972). (9) Wedding, J. B., PhD thesis, University of Illinois, Urbana, Ill., 1974. (10) Chow, V. T., Yen, B. C., “A Laboratory Watershed Experimentation System”, Hydraulic Engineering Series No. 27,195 pp, 1974. (11) Chow, T . J., Nature, 225,295 (1970). (12) Lagerwerff, J. V., Specht, A. W., Enuiron. Sci. Technol., 4,583 (1970). (13) Noller, B. N., Smythe, L. E., Search, 5,108 (1974). (14) Page, A. L., Ganje, T. J., Joshi, M. S., Hilgardia, 4 1 , l (1971). (15) Smith, W.H., Forest Sci., 17, 195 (1971). (16) Smith, W. H., Enuiron. Sci. Technol., 7,631 (1973). (17) Bazzaz, F. A,, Rolfe, G. L., Windle, P., J . Enuiron. Qual., 3,156 (1974). (18) Rolfe, G. L., Bazzaz, F. A,, Forest Sci., 21,33 (1976). (19) Steenken, F., 2. Pflanzenkr. Pflanzenschutz, 80,346 (1973). (20) Arvik, J. H., Zimdahl, R. L., J . Enuiron. Qual., 3,369 (1974). (21) Ricks, G. R., Williams, R.J.H., Environ. Pollut., 8,97 (1975). (22) Corn, M., J . Air Pollut. Control Assoc., 11, 523, 566 (1961). (23) Corn, M., in “Aerosol Science”, C. N. Davies, Ed., p 359, Academic Press, New York, N.Y., 1966. (24) Aylor, D. E., Plant Physiol., 55,99 (1975). (25) Aylor, D. E., Parlange, J.-Y., ibid., 56,97 (1975).
Received for review Januar)) 5 , 1976. Accepted May 3,1976. Research supported by N S F R A N N Grant GI-31605 to the Institute for Environmental Studies, C‘niuersity of Illinois, Urbana, Ill.
Partitioning of Mercury and Polychlorinated Biphenyl by Oil, Water, and Suspended Sediment Gary S. Sayler’ and Rita R. Colwell” Department of Microbiology, University of Maryland, College Park, Md. 20742
Freshwater and marine systems comprising mixtures of suspended sediment and/or crude oil were tested for partitioningof mercury and HCB in the suspended sediment and oil. Appropriate media to which radioactively labeled 203HgC12 (HCB) were added and U-14C 2,4,5,2’,4’,5’-hexachlorobiphenyl were analyzed. Following initial mixture and 24-h incubation, the water, oil, and suspended sediment phases were separated, collected, and assayed for accumulation of the radioactively labeled components. Results of the partitioning experiments showed that both oil and suspended sediment could partition mercury and HCB. The relative percent partitioning by oil was ca. three times greater than by suspended sediment. However, suspended sediment was an equally efficient partitioning agent in terms of net concentration of mercury or HCB. About 85% of the radioactive label was removed from the water column within 24 h under optimum conditions. A significant accumulation of heavy metal and chlorinated hydrocarbons can occur in oil and sedimenting material.
Recent investigations in our laboratory have focused on the biodegradation of three rather ubiquitous pollutants: mercury compounds, petroleum hydrocarbons, and polychlorinated biphenyls (PCB) (1-3). These pollutants commonly are lo) Present address, Department of Microbiology, Graduate Program in Ecology, University of Tennessee, Knoxville, Tenn. 37916.
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Environmental Science & Technology
calized in the sediments of the aquatic environment. Resultant concentrations and interactions among these components are relatively unknown. Evidence does exist indicating a propensity of petroleum hydrocarbons to concentrate chlorinated hydrocarbon pesticides ( 4 , 5 )and mercury compounds ( 6 ) ,but the sequestering of both of these pollutants by petroleum in a fresh or marine system has yet to be shown. This investigation was undertaken to determine the interaction of three of the more common pollutants and the potential partitioning effect that may occur in the natural environment. Future investigations are planned, as a result of the findings, on the effects of combinations of these pollutants on microbial degradation. Materials and Methods Chemicals. Isotopically labeled *03HgC12 (Amersham Searle Corp., Arlington Heights, Ill.) and U-14C 2,4,5,2’,4’,5’-hexachlorobiphenyl (HCB) (New England Nuclear Corp., Boston, Mass.), 98% purity as determined by thin-layer chromatography, were employed in all partitioning studies. Artificial seawater was prepared using Tri Sea salts (Sea Lake Systems, Inc., Euclid, Ohio). Suspended sediment was simulated using the 300 mesh diatomaceous earth, Celite (J. T. Baker Chemical Co., Phillipsburg, N.J.). Experimental Design. An experimental outline describing the partitioning of Hg and HCB between three phases of an oil, water, and particulate system is given in Table I. To assess the various partitionings of each phase in the presence of HgC12 and HCB, separately or in combination, experimental
test systems were established which included water, water and oil, water and sediment, and water, oil, and sediment for both fresh water and seawater. The experimental test systems in the laboratory consisted of 130 X 15 mm sterile screw-capped test tubes containing 10 ml of fresh (tap) water or artificial seawater. To appropriate tubes were added 40.0 fig (3.2 fiC1) 203HgC12,or 10.0 fig (0.14 fiCl in 0.1 ml acetone) U-I4C HCB, or both. [The solubility of HCB in water ( < l o 0 fig l.-l) was exceeded in these experiments. The excess I4C-HCBwas necessary to assure statistically significant quantitation of radioactivity in each phase following partitioning.] Control tubes received no amendments. Additional test environments were established by adding 100 mg celite, or 10% (v/v) Kuwait crude oil, or a combination of celite followed by oil to the initial freshwater and seawater systems. The test tubes containing the components were tightly capped and mixed for 5 s in a vortex mixer. Following mixing, the test tubes were placed in a 15 "C chamber and gently shaken a t 100 rpm for 24 h. After incubation, the water, suspended matter, and oil phases were separated, collected, and assayed for radioactivity. Crude oil layers were separated and collected by pipet followed by centrifugation (2100 X g) of the remaining water and sediment, to pellet any suspended sediments. The aqueous phase was removed by pipetting, and the remaining sediment was washed, centrifuged, resuspended, and harvested. One ml of aqueous phase or sediment resuspended in water was placed in 10 ml dioxane-based Omnifluor (New England Nuclear Corp.) cocktails. 14Cradioactivity was measured with an Intertechnique liquid scintillation counter, Model SL-40 (Teledyne Corp., Westwood, N.J.), employing a standard 14C window setting. Counting efficiency was 94%. Beta emission from 20.3Hgwas also measured, using a standard 14Cwindow as a reference for total radioactivity measured in the double label, 203Hg 14C-HCB, test system. One ml of the 1/100 dilutions of oil was placed in toluene-based Omnifluor (NEN) cocktails and counted in the same manner as the water samples. There was no significant quenching effect observed in any of the liquid scintillation counting systems. Gamma emission from the decay of 203HG(279.2 keV) was measured in a Packard Tri-Carb scintillation counter (Packard Instrument Co., Inc., Downers Grove, Ill.) equipped with an autogamma spectrometer. Harvested samples of sediment, oil, and water were placed directly into gamma tubes following appropriate dilution. Quantitative 203Hg determinations were thus based on gamma emission rather than beta emission. By comparing the ratio 203Hg,8:r to I4C ,8 emission in the double label experiments, it was possible to segregate Hg and HCB partitioning in the various phases.
+
ment. There appears to be a significantly greater percent partitioning of HCB by crude oil, compared with HgC12, in both fresh water and seawater. This effect was also observed in the significant variation found for the Phase X Compound interaction (Table 11). In all cases, suspended sediment was much less effective in partitioning mercury and HCB from the water column, compared with the crude oil. The partitioning of HCB and mercury by particulate matter in the presence of oil appeared to be from the oil rather than the water.
Table I. Outline of Experimental Design for Assessment of Partitioning of Mercury and HCB in Water, Oil, and Suspended Sediment I . Water types
a . Fresh b. Marine I I . Isotopic assessment a. HgCI, b. HgCI, in presence of H C B c. H C B d . H C B in presence of HgCI, I I I . Phase a . Water b. Water a n d oil c. Water a n d sediment d . Oil a n d water e. Oil, water, a n d sediment f . Sediment a n d water g. Sediment, water, a n d oil
Table 1 1 . Factorial Analysis of Variance0 ( 2 x 8 x 4)of and U-"T Hexachlorobiphenyl Partitioning of 2@3HgCI, (HCB), Between Water, Oil, and Sedimentb Source of variation df ss MS Fs
Water ( W ) Phase (P) Compound (C) WXP
wxc
PXC WXPXC
Within subgroups Total
1 7 3 7 3 21 21 64 127
879 151769 21 1025 2 16765 4740 2839 178040
879 21681 7 146 0.6 798 226 44
ANO OVA, M o d e l 1 with replication (a= 0 . 0 5 ) . b d f = degrees o f freedom, SS = sum o f squares, M S = mean square, Fs = F statistic. clndicateS variability significantly greater than within subgroup variation.
Table I l l . Relative Percent0 Partitioning of 2@3Hg and I4C-HCB Radioactivity in Three-phase Fresh Water System % radioactivity partitioned
Results and Discussion Results of a factorial analysis of variance, assuming all major sources of variation to be fixed treatment effects, showed significant variation between fresh water and seawater and between the various oil, water, and suspended sediment phases (Table 11).No significant variation was attributable to the individual labeled compounds being partitioned. Variation attributed to all treatment effect interactions, except that between water and labeled compounds was significant. These results refute the null hypothesis that there is no significant change in HCB or Hg concentration in the water column. Furthermore, salinity and partitioning of the various phases were associated with significant difference. A comparison of the mean percent partitioning of 203Hgand 'IC-HCB by the various phases in the freshwater and seawater systems was revealing (Tables I11 and IV). From the data given in Tables I11 and IV, oil is an extremely effective partitioning agent for both HCB and HgC12 even in the presence of sedi-
19.8C 488c __ 0.2 3.3c 0 18.0C 5.8C
2@3HgC12
14C-HCB
I 4 C - H C B (HgCI,)
Water
Oil
Suspended sediment
19.0 23.5 75.7 100.0
72.8 85.5
...
0.9 0.4 74.8 100.0 2.7 2.5 71.2 100.0
8.2
...
24.4
63.1
8.5
...
92.4 99.5
...
6.8
...
...
25.2
93.7 97.5
...
... ... ...
...
2.6
28.8
...
a Mean o f duplicate observations relative t o the total radioactivity in the water column.
Volume 10, Number 12, November 1976
1143
In comparing data given in Tables I11 and IV, a greater partitioning of Hg and HCB occurred in the three-phase, freshwater system, compared with the seawater system. This difference, although significant, is much less apparent than the partitioning effect of the three phases (Table 11). Thus, the ability of seawater to retain both HCB and Hg is somewhat greater than fresh water; however, the effect is secondary to the partitioning of the suspended sediment and oil phases. Data on mean percent loss of radioactive label for both fresh water and seawater were pooled to determine potential loss of both HCB and mercury from the water column to the oil and sediment phases (Table V). Eighty-five percent of the HCB and mercury was removed from the water column by both the oil phase, alone, or the oil in conjunction with sus-
Table IV. Relative Percenta Partitioning of 203HgCI,and I4C-HCBRadioactivity i n Three-phase Seawater System % radioactivity partitioned
O3
HgCl ,
Water
Oil
28.3 17.2 61.5 100.0 30.0 30.9 76.4 00.0 10.7 0.9 69.3 00.0
54.9 82.4
~~
,03HgCI, ( H C B )
I4C-HCB
Suspend ed sediment
16.8
...
...
38.5
...
...
61.9 69.1
8.1
... ...
...
23.6
...
81.1 99.1
I4C-HCB (HgCI,)
uu.u
8.3
...
...
... 30.7 ...
85.4
10.5
...
...
a M e a n o f d u p l i c a t e observ : i o n s r e l a t i v e t o t h e t o t a l r a d i o a c t i v i t y in the water column.
Table V. Mean Percent LOSS^ of 203HgCI,and 14C-HCB Radioactivity from Water Column t o Oil and Suspended Sediment Phasesb Radioactive label
Phases
203HgCl,
I4C-HCB
Oil + sediment
73.6 73.9 23.2 0.0
95.4 98.7 22.2 0.0
Oil
Sediment Water
-
Composite
Y
s
84.5 85.4 28.4
12.4 12.9 18.8
0.0
a R e l a t i v e t o t h e t o t a l r a d i o a c t i v i t y i n t h e water f o r b o t h t h e freshwater a n d seawater systems. b y = mean, s = standard d e v i a t i o n .
Table VI. Concentration of HgCI, and HCB in Oil and on Suspended Sediment Following Partitioning from Water Phasea HCB
HSCIz %b
Phase
mg I.-'
s
Oil Oil (sediment)c Sed i m en t
30.4 25.3 24.8 10.4
0.4 0.4 3.9 1.8
Sediment (oil)d
%b
increase mg
760 630 620 240
1.2
9.5 8.8 28.1 13.6
s
increase
0.01 0.07 1.2 0.3
950 880 2810 1360
a Mean o f e i g h t observations. s, s t a n d a r d d e v i a t i o n . Percent increase r e l a t i v e t o t h e i n i t i a l c o n c e n t r a t i o n o f H g C i ( 4 m g ):'i or h e x a c h l o r o b i p h e n y l (1 m g I.-') i n t h e w a t e r colum'n. c o i l p a r t i t i o n ing in t h e presence o f suspended s e d i m e n t . d S u s p e n d e d s e d i m e n t p a r t i t i o n i n g in t h e presence o f o i l .
1144
Environmental Science & Technology
pended sediment. However, suspended particulates removed only 28.4% of the HCB and mercury. The remainder was found in the water. Greater selective partitioning of HCB by crude oil, compared with mercury, was observed (ca. 97% removal vs. 74%), an expected result, considering the affinity of HCB for nonpolar solvents. The relative percent partitioning or removal of mercury and HCB by suspended sediment was less than for crude oil (Tables 111-V). However, when the data are expressed in terms of net concentration of mercury or HCB in the oil layer, compared with the sediment, suspended sediment was highly efficient in concentrating both mercury and HCB (Table VI). From these data, both Hg and HCB increased approximately ZOO-2800-fold because of the partitioning effect of oil or suspended sediment (Table VI). Efficiency of sediment partitioning was slightly reduced in the presence of oil. Concentrations of HCB in sediment increased 13.6-foldand 28.1-fold, respectively, for suspended sediment in the presence of oil and sediment alone. Approximately a ninefold increase was noted for the oil, as computed from the data given in Table VI. Less dramatic results were obtained for concentration of mercury in oil or suspended sediment (Table VI). Separately, oil and sediment were nearly equal in ability to accumulate mercury. However, in a three-phase system, oil was a better competitor in the partitioning of mercury, as indicated by the significant decrease in the concentration of Hg on suspended sediment in the presence of oil. Primary differences in partitioning efficiency, between suspended sediment and oil, arise from the highly nonpolar nature of crude oil and the resulting partitioning due to solubility differences, compared with partitioning on the basis of surface adsorption occurring on the relatively large surface area of the celite. The possibility may exist that increased adsorption of Hg in oil may occur if organomercurial complexes are formed. Kenega ( 7 ) reported that adsorption of chlorinated pesticides in some environmental systems is 50%complete within a few hours. Thus, the findings of the short-term (24 h) laboratory experiments in this study can be extrapolated to the natural environment. Maximum partitioning of DDT (structurally similar to HCB) has been reported to occur a t ca. 10 days ( 4 ) . Greater partitioning of PCB and mercury would be expected to occur as the time of incubation increased. Hartung and Klingler ( 4 ) reported steady-state oil partition coefficients for DDT that exceed lo6,indicating an even greater potential for chlorinated residues, such as PCB, to accumulate in areas continually receiving oil and PCB. Since there was no significant difference noted between mercury and PCB partitioning, it is doubtful that organic forms of mercury would be accumulated to any less extent than the HgClz and HCB employed in this study. However, HgO volatilization is a microbial metabolic pathway occurring in the aquatic environment and is a mechanism of escape of mercury from a combined Hg-oil environment (8). Also, water-extractable material in oil (9) can be a potential mercury-volatilizing mechanism ( I O ) . From the data presented here, concentration of heavy metals and chlorinated hydrocarbons in sediment and/or oil can result in a highly toxic environment, Le., inhibitory to microorganisms capable of degrading each of the components separately but inhibited by the high concentrations in combination. The relatively exotic substrates thereby occurring in sediment must eventually be mineralized by microorganisms. Thus, spilled, dumped, or seeping oil reaching marine or estuarine sediments may be rendered impervious to microbiological attack (1l ). Furthermore, application of degradation kinetics established for any one of the individual pollutants discussed here to a multiply contaminated envi-
ronment will not be valid. The consequences of impeded or inhibited microbial degradation of the components of a mixed-pollutant system are serious and should be investigated. Literature Cited (1) Nelson, J. D., Jr., Colwell, R. R., Microb. Ecol., 1, 191-218
(1975).
(2) Sayler, G. S., Shon, M., Colwell, R. R., ibid., submitted for pub-
lication.
( 3 ) Walker, J. D., Colwell, R. R., Appl. Microbiol., 27, 1053-60
(1974). Hartung, R., Klingler, G. W., Enuiron. Sci. Technol., 4, 407-10 (1970).
(4)
(5) Seba, D. B., Corcoran, E. F., Pestic. Monit. J., 3, 190-93
(1969). (6) Walker, J. D., Colwell, R. R., Appl. Microbiol., 27, 285-87
(1974).
(7) Kenega, E. E., Proc. Intl. Conf. Transport Persist. Chem. Aquatic Ecosys., Vol 11, pp 19-22, Ottawa, Canada, 1974. (8) Larson, J. O., Tandeski, E. V., Anal. Chem., 47, 1159-61 (1975). (9) Sniegoski, P. J., Water Res., 9,421-23 (1975). (10) Alberts, J. A., Schindler, J. E., Miller, R. W., Nutter, D. E., Jr., Science, 184,895-97 (1974). (11) Walker, J. D., Colwell, R. R., Enuiron. Sci. Technol., 10, 1145
(1976).
Received for review September 2,1975. Accepted May 12,1976. Work supported by Sea Grant Project No 04-5-15811, National Oceanic and Atmospheric Administration, Washington, D.C.
Oil, Mercury, and Bacterial Interactions John D. Walker‘ and Rita R. Colwell” Department of Microbiology, University of Maryland, College Park, Md. 20742 Several strains of bacteria were examined for ability t o utilize oil with and without mercury added t o the culture medium. The majority of the oil-utilizing bacterial strains had been isolated from a n oil-contaminated creek in Baltimore Harbor of the Chesapeake Bay. Relatively low concentrations of mercury inhibited the utilization of oil by the strains of bacteria examined in this study. Concentration of environmental pollutants, viz., mercury and chlorinated pesticides, in oil has been reported by several investigators (1-3). In a study reported elsewhere ( 4 ) , partitioning of mercury and polychlorinated biphenyls in water and oil and on diatomacems earth was examined. If mercury, pesticides, and other pollutants concentrate in oil, effects of such combinations on the microbial degradation of each of the pollutants must be investigated since the potential consequences are of great environmental significance. Hence, as an initial step toward the understanding of the microbial degradation of combinations of selected pollutants, the effect of mercury concentrated in oil on the microbial biodegradation of oil was examined. Materials a n d Methods
Cultures. Procedures employed for the isolation and identification of cultures used in this study have been published previously (5,6). Bacterial strains examined are listed in Table I. A total of 20 strains of bacteria were screened for mercury resistance when grown in heterotrophic and in oil media. The strains were isolated from water, plankton, and sediment samples collected in several areas of Chesapeake Bay: Colgate Creek, a n oil-polluted site located in Baltimore Harbor: Poole’s Island, a dredge spoil disposal site in Chesapeake Bay; and Eastern Bay, a shellfish-harvesting area of the Bay. American Type Culture Collection (ATCC) strains were also examined as reference cultures. C u l t u r e Systems. The strains were cultured in a basal I Present address, Environmental Technology Center, Martin hlarietta Corp., 1430 South Rolling Road, Baltimore, Md. 21227.
Table I. Strains of Bacteria Used in This Study Genus and species name0
Lab n o .
Strain no.
Source
Coryneform
4
...
P s e u d o m o n a s sp.
9s
...
P s e u d o m o n a s sp.
125
P s e u d o m o n a s sp.
215
Coryneform
232
V i b r i o sp.
254
...
A c i n e t o b a c t e r sp.
263
...
Pseudomonas aeruginosa R h i z o b i u m m elilo t i Rhizobium leguminosarum Nocardia otitidis caviaru m Leucothrix mucor Nocardia corallina No cardia asteroides
492
1014s-1
Colgate Creek sed i men t Eastern Bay water Colgate Creek sediment Eastern Bay water Colgate Creek sediment Colgate Creek water Poole’s Island sediment Colgate Creek water Colgate Creek plankton Colgate Creek sediment Eastern Bay sed i men t Colgate Creek water Eastern Bay sediment ATCC f J
495 496
4399 10004
ATCC ATCC
497
14629
ATCC
498 50 1 5 10
25107 4273 14759
ATCC ATCC ATCC
Coryneform
53
P s e u d o m o n a s sp.
1o s
Coryneform
76
...
P s e u d o m o n a s p.
11s
...
P s e u d o m o n a s sp.
101
P s e u d o m o n a s sp.
12s
...
O w h e r e t h e c o m m o n e p i t h e t , i.e., “ C o r y n e f o r m ” i s used o r speCieS given as “sp,!’, i d e n t i f i c a t i o n is p r e s u m p t i v e a n d f u r t h e r Study o f t h e classification o f t h e strain is i n progress. b o b t a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e Collection. 1 2 3 0 1 P a r k l a w n . R o c k v i l l e , Md.
Volume 10, Number 12, November 1976
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