puter results for t h e polynomial correlations of t h e sediment analyses.
Literature Cited (1) Woodward, 0. H., “A Review of the Broken Hill Lead-Silver-Zinc
Industry”, 2nd ed., Parsons, K. P. W., Ed., West Publishing Corporation Pty. Ltd., Sydney, 1965, p 38. (2) World Health Organization, “International Standards for Drinking Water”, 3rd ed., WHO, Geneva, 1971, pp 32,40. 131 Cowins. A. J.. BSc. Honours Thesis, The University of New SoutuhYWales, Sydney, 1977. (4) “Methods for the Analvsis of Water and Wastes”, Environmental Protection Agency, Marbourne, 1973, p 7. ( 5 ) Hawkes, H. E., Webb, J. S., “Geochemistry in Mineral Exploration”, Harper and Row, New York, 1962,p 205. (6) Malissa, H., Schoffman, E., Mikrochim. Acta, 1, 187 (1955). (7) Mulford, C. E., At. Absorpt. Nerusl., 5,88 (1966). (8) Childs, E. A,, Gaffke, J. N., J . Assoc. Off. Anal. Chem., 57, 360 (1974). (9) Kinrade, J. D., Van Loon, J. C., Anal. Chem., 46,1894 (1974). (10) Agemian, H., Chau, A. S., Anal. Chim. Acta, 80,61(1975);Analyst, 101,761 (1976). (11) Jeffery, P. G., “Chemical Methods of Rock Analysis”, Pergamon Press, Oxford, 1975, p 22. (12) Suarez, D. L., Langmuir, D., Geochim. Cosmochim. Acta, 40, 589 (1976). (13) Davenport, F’. H., Hornbrook, E. H., Butler, A. J., in “Geo-
chemical Exploration 1974”, Elliott, I. L., Fletcher, W. K., Eds., Elsevier, Amsterdam, 1975, p 555. (14) Shapiro, M. A., Connell, D. W., Proc. R Aust. Chem. Inst., 42, 113 (1975). (15) Davis, J. C., “Statistics and Data Analysis in Geology”, Wiley, New York, 1973, p 322. (16) Pauncz, I., “A Fortran I\’ Program for Multiple Regression and Geologic Trend Analysis”, after Esler, cJ. E., Smith, P. F., Davis, J. C., University of New South Wales, 1977. (17) Oliver, B. G., Enuiron. Sci. Technol., 7, 135 (1973). (18) Soldatini. G. F.. Riffaldi. R.. Levi-Minzi. R.. Water. Air Soil Pollut., 6, 111 (1976). (19) Leland, H. V.. Shukla. S.S., Shimp, N. F., in “Trace Metals and Metal-Organic Interactions in Natural Waters”, Singer, P. C., Ed., Ann Arbor Science, Ann Arbor, 1973, p 89. (20) Jenne. E. A,. Adu. Chem. Ser.. No. 73.337 11968). (21) Gardiner, J.; Water Res., 8,157 (1974). (22) Shimp, N. F.. Leland, H. V.. White. W. A,, Illinois Geological Survey Environmental Geology Note 32, 1970. (23) Shimp, N. F., Schleicher, J. A,, Ruch, R. R., Heck, D. B., Leland, H. V., Illinois Geological Survey Environmental Geology Note 41: 1971. (24) Holmes, C. W.,Slade, E. A,, McLerran, C. J., Enciron. Sci. Technol., 8, 225 (1974). (25) Giblin, A. M., Chem. Geol., 23,215 (1978). Received for review July 24, 1978. Accepted June 4 , 1979.
Some Aspects of the Microbiology of Activated Carbon Columns Treating Domestic Wastewater Alina Latoszek and Andrew Benedek‘ Department of Chemical Engineering, McMaster University, Hamilton, Ontario, L8S 4L7 Canada
Activated carbon samples from two sets of pilot plant columns treating coagulated, settled, a n d sand-filtered domestic wastewater at 25 and 5 “C were analyzed by microscopic observations and cultural investigations. Extensive growth of bacteria u p t o lo9 viable cells per g of wet drained carbon was detected. T h e majority of isolates were classified as belonging to t h e genus Pseudomonas and t o the Flauobacterium-Cytophaga group. A high percentage of t h e bacteria exhibited denitrifying ability. Nematoda were found in t h e lead column of t h e 25 “ C set of columns, while Mastigophora a n d Amoeba were present in t h e lead column of t h e 5 “ C set of columns. T h e nature of microbial life in activated carbon adsorbers appears to be similar in nature and number to t h a t of t h e mixed liquor in activated sludge plants. In recent years, the “physical-chemical treatment” (PCT) of sewage has been gaining increasing prominence as a n alternative t o conventional biological treatment. In PCT, activated carbon columns are used to remove soluble organics. Initially, removal was attributed to physical adsorption alone, a n d efforts were made t o minimize microbial growth. These efforts, however, usually failed, and reports on pilot plant operations noted considerable biological activity in such columns. As pilot plant investigations continued, carbon column loadings were observed to be many times higher than t h e maximum obtainable by nonbiological adsorption alone (1-3), and, as a result, biological processes are now considered to play a n extremely important part in making carbon adsorption cost effective. Furthermore, there have been claims t h a t AC presents an excellent support medium for bacterial growth, as the carbon surface can concentrate substrates for subsequent microbial decomposition. Kalinske ( 4 ) and later Pirotti and Rodman ( 5 ) claimed that organisms show increased substrate 0013-936X/79/0913-1285$01.00/0
@ 1979 American Chemical Society
decomposition rates when grown on carbon. Hals (6) disputed increased kinetics rates; however, he observed that bacteria tend t o adsorb onto activated carbon rather than remain in a dispersed state. At t h e present time, our understanding of the nature of microbial life in carbon columns is still extremely limited. Two recent publications (7, 8) indicate that bacteria in numbers ranging from lo5to 1O’O viable cells per g of carbon are present in carbon contactors used to treat potable water. Furthermore, a recent publication in this journal (9) presented scanning electron micrographs of the microbial life around carbon particles. This paper extends our understanding of the viable cell levels t o columns treating domestic wastewater. Furthermore, the microbes from t h e carbon contactors are also studied in a taxonomic sense.
Experimental Activated Carbon Source. Activated carbon was removed from pilot carbon columns after 2 months of operation on chemically clarified Dundas, Ontario, domestic wastewater. T h e details of the pilot plant are presented by Maqsood (10). There were two sets of three columns used in the study; one set was operated a t 25 “ C and the other at 5 “C. The first column in each series was 0.31 m and the subsequent two were 1.5 m each. Based on dissolved oxygen (DO) measurements, t h e lead columns in each set were aerobic (effluent DO? were typically around 2 mg/L). Backwashed carbon (type Filtrasorb 400, made by Calgon Co., Pittsburgh, Pa.) samples were removed from the lead and final columns of each set. T h e samples were identified as HI (lead) and H3 (final) of the 25 “C columns and as L1 (lead) and L.3 (final) for the 5 “C columns. Microbial Investigations. Microscopic observations of the Volume 13, Number 10, October 1979
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Table 1. Results of Viable Cell Counts per Gram of Wet Carbon no. of bacteria/
no. of bacteria/
g of wet C sample
x
10-8
sample
HI
1060
L1
H3
56
L3
g of wet C x 10-6
226 10.7
carbon were performed under the phase-contrast microscope at magnifications of 100 and 400. Viable cell counts were determined after homogenizing 2.5 g of each sample in 250 m L of phosphate buffer for 10 min a t t h e highest speed of a Waring Futura 750 blender. The homogenized sample was then diluted 10 times and plated onto tryptone-glucose-yeast agar by the pour plate technique. Incubation for Hi and H3 samples was a t 27 f 1 “C for 9 days and for L1 and L3 samples at 5 f 1 O C for 32 days. Incubation was ended when t h e growth of new colonies was no longer observable. After incubation, colonies were counted and the number of bacteria calculated per g of wet, drained carbon. Prior to weighing, the samples were allowed to drain on a sieve until free water was no longer visible. Typically, such samples still contain approximately 50% water. Isolates for identification were picked from the above plates of samples H1 and L1 only at the end of the incubation period, tr.ansferred onto nutrient agar slants, and incubated a t 27 f 1 “C for H I and 7 f 2 “C for L1. Twenty-one strains were selected randomly from H1 and 18 strains from L1 plates. After checking for purity, t h e following tests were performed on the isolates: (i) macroscopic observation of growth (color, transparency, etc.); (ii) microscopic examination of morphology and motility under phase contrast; (iii) Gram staining; (iv) oxidase test (11); and (v) dissimilation of carbohydrates in Hugh and Leifson based medium (Difco O/F medium), incubation for 1 month at 27 f 1 “ C for HI and 20 f 1 “C for L1. Using the above tests, isolates were differentiated on the generic level following the determinative scheme proposed by Shewan et al. (12),as supported by Bergey’s Manual (13)and other relevant publications (14-16).
Results a n d Discussion Direct Microscopic Observations a n d Viable Cell Counts. I n the HI sample, activated carbon particles were overgrown by an extensive mass of bacterial growth. Zoogleal or slimy aggregates and bacterial filaments were observed and some Nematoda were present. In the H 3 sample, bacterial growth was considerably less than in the HI sample, Macrofauna were not detected. Bacterial growth on the activated carbon in the L1 sample was less abundant than in the HI column. Sphaerotillus-like threads and a significant quantity of free suspended bacterial rods were observed along with minute Mastigophora and Amoeba. Sample LB showed considerably less bacterial growth than the Ll sample. Once again, macrofauna were not detected. Some of the above observations were confirmed by viable cell counts reported in Table I. According to this table there were approximately 20 times more bacteria in the leading columns (HI and L1) than the respective final columns. The ratio for bacterial populations for different temperatures was about 5:l a t both positions. During the operation of the columns, about 50% of the organic matter was removed in the lead columns with less than 10%removed in the final columns ( 3 ) . T h e highest viable cell count (obtained for t h e H1 sample) 1286
Environmental Science & Technology
is on the order of magnitude of those obtained in the mixed liquor of activated sludge systems. Hawkes (171,for example, reports maximum cell counts for the latter system of 2.2 X lo9 per mL. Assuming 50% wetness and a packed dry carbon density of 0.44 g/cm3, t h e 25 O C lead column contained approximately 0.9 X lo9 viable bacteria per m L of column volume. Thus, activated carbon beds have potential biodegradation capacities on the order of activated sludge systems. Taxonomy. The 21 strains isolated from the H1 column sample were classified as follows: 13 strains belonging to the genus Pseudomonas, 6 strains belonging to Flauobacterium-Cytophaga groups, 1 strain belonging to the genus Achromabacter, and 1 strain defined as an Arthrobacter-like organism. All but one of the Pseudomonas strains produced alkaline reactions in O/F medium. Five of the strains were denitrifiers. The 18 strains isolated from the L1 column were classified as follows: 8 strains belonging to the genus Pseudomonas, 8 strains belonging to Flauobacterium-Cytophaga group, 1 strain defined as an Arthrobacter-like organism, and 1 strain unidentified. Four of the eight Pseudomonas strains produced alkaline reactions in O/F medium and showed denitrification ability. Three (each of different colonial appearance) produced oxidative reaction in O/F medium. One strain did not ‘produce a change in the reaction and possessed denitrifying ability. These data merely indicate the potential bacterial denitrification capability. T h e paper by Maqsood and Benedek ( 3 )reports that nitrate removal did indeed take place during the operation of the adsorption beds. T h e taxonomy of the isolated bacterial strains from the columns is similar to the main bacterial group characteristic of activated sludge and bacteria beds (17, 18). Conclusions Activated carbon columns treating domestic wastewater possess very high bacterial densities that approach those of the mixed liquor of the activated sludge process. T h e determined bacterial genera and group (Pseudomonas, Achromobacter, Flavobacterium-Cytophaga group, and Arthrobacter-like organisms) resemble the bacterial populations found in the activated sludge and trickling filter processes. Literature Cited (1) Bishop, D. F.; Marshall. L. S.; O’Farrell, T . P.; Dean. R. B.; O’Connor, B.; Dohbs, R. A,; Griggs, S. H.; Villiers, R. L‘. J , Water Poiiut. Controi F e d . 1967, 39, 188-203. (2) \Veher, W. ,J., dr. “Biologically Extended Physico-chemical
Treatment”, Advances in LVater Pollution Control, Jerusalem Meeting of the International Association of Vv-ater Pollution Control, 1972 ( 3 ) Maqsood, R.; Benedek, A., “The Effect of Low Temperature on Organic Removal and Denitrification in Activated Carbon Columns”, paper presented a t the 47th Annual Conference, Denver, Colo., 1977. (4)Kalinske, A. A. Water Seu age Works 1972, l l 9 ( 6 ) ,62-4. (5) Pirotti, A. E.; Rodman, C. A. “Factors Involved with Biological Regeneration of iZctivated Carbon”, paper presented a t the 75th National Meeting of the American Institute of Chemical Engineers, Detroit, 1973. (6) Hals, 0. M.Eng. Thesis, McMaster University. Hamilton, Ontario, Canada, 1974. ( 7 ) Klotz. M.; Werner, I’.. Sweisfurth, R. I n “Translation of Reports on Special Problems of Water Technology”; Sontheimer, H., Ed., L.S. Government Report EPA-600/0-76-030. ( 8 ) Benedek. A. Proceedinns of’ the Toronto Meetinr of the International Ozone Institute, 1977. (9) LVeber, W.d.. Jr.; Pirbazari, M.; Nelson, G. L. Enoiron. Sci. Trchnoi. 1978, 12. 817. (10; Maqsood, R., M.Eng. Thesis. Mchlaster University, Hamilton, Ontario, Canada. 1976. (11) Kovacs, N.JVature (London) 1956, 178, 703. (12) Shewan, I. M.; Hohhs, G.: Hodgkiss, W. J . Appi. Rncterioi. 1960,
23, 379-90. (13) Breed, R. S.; Murray, E. G. D.; Smith, N. R. “Bergey’s Manual of Determinative Bacteriology”, 7th ed.; Williams & Wilkins Co.: Baltimore, 1957. (14) Hendrie, M. S.; Mitchell, F. G.; Shewan, J. M. In “Identification Methods for Microbiologists-Part B”; Gibbs, B. M., Shapton, D. A,, Eds.; Academic Press: New York, 1968; pp 67-8. (15) Mulder, E. G. In “Principles and Applications in Aquatic Microbiology”; Henkelekian, H. E., Dondero, N. C., Eds.; Wiley: New
York, London, Sydney, 1964; pp 257-79. (16) Skerman, V. B. D. “ A Guide to t h e Identification o f the Genera of Bacteria”, 2nd ed.: Williams and Wilkins Co.: Baltimore, 1967. (17) Hawkes, H. A. “The Ecology of Waste Lt’ater Treatment”; Macmillan: New York, 1963. (18) Benedict, R. G.; Carlson, D. A. Water Res. 1971,5, 1023-30.
Received for reuieu: February 16, 1979. Accepted July 6, 1979.
Effect of Chemical Speciation on the Uptake of Copper by Chironomus fenfans Elizabeth E. Dodge’ and Thomas L. Theis” Department of Civil Engineering, University of Notre Dame, Notre Dame, Ind. 46556
Laboratory studies of copper uptake by the midge larvae Chironomus tentans indicated that chemical speciation is an important consideration. Copper was taken up from solutions in which the dominant aqueous forms were free cupric ion and a copper hydrox:y complex. No uptake was observed when copper-glycine a.nd copper-NTA were dominant. Studies conducted with freshly killed larvae suggest t h a t a relatively large portion of the copper taken up is due to nonbiological processes. Heavy metal toxicity to aquatic organisms has been shown to be a function of the activity of certain chemical species rather than total inetal concentration. Metal ions in particular have been shown to exert detrimental effects on a wide range of organisms. St’eemann-Nielsen and Wium-Anderson (1) found that ionic copper reduced growth and photosynthesis in the green alga Chlorella pyrenoidosa and diatom Nitzschia palea a t concentrations of total copper found in natural waters and concluded that copper toxicity in aquatic systems is reduced by complexation. By altering p H and chelator concentration, Sunda and Guillard ( 2 ) also linked growth inhibition and cell copper content in the marine diatom T h a lassiosira pseudcnana to cupric ion activity. Anderson and Morel ( 3 )noted similar growth inhibition in the dinoflagellate Gonyaulax tamarensis by varying the cupric ion activity in artificial seawater medium with the chelators tris(hydroxymethy1)aminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA). Andrew et al. ( 4 ) found a direct correlation between cupric ion activities and mortality rates in Daphnia magna, as did Pagenkopf et al. ( 5 ) in the fathead minnow Pimephales promelas and rainbow trout S a l m o gairdnerii. Most of the other metal species studied have been found not to exert harmful effects on aquatic organisms. All studies on metal carbonate complexes indicate that they are not harmful (4-6). Biesinger et a1 ( 7 ) reported a decrease in the toxicity of copper and zinc to D . magna when chelated by nitrilotriacetate (NTA). Barica et al. (8) found no significant uptake of iron, manganese, zinc, copper, lead, or chromium by Chiron o m u s t e n t a n s in the presence of NTA, EDTA, or TPP. Conflicting conclusions are reached in two studies on the effects of copper hydroxy complexes. Pagenkopf et al. conclude that CuOH+ and Cu(OH)zz+probably are not toxic to fathead minnows or rainbow trout, while Andrew et al. found a positive correlation between Cu(OH), activities and Daphnia magna mortalities. The objective of this study was to further investigate this subject area on the consequences of chemical speciation to aquatic organism:; by investigating the uptake of different
’ Present
-
a d d r e s , CH2M Hill, Milwaukee Water Pollution Ahatement Program. 743 Water St., Milwaukee, Wis. ,53202.
0013-936X/79/091?~-1287$01.00/0 @ 1979 American Chemical Society
copper species by larvae of the midge Chironomus tentans.
Experimental Uptake studies were conducted in test solutions in which the dominant forms of copper were cupric ion (Cu2+),the copper hydrolysis product C U ( O H ) ~ ( ~ ~copper ) O , glycine (Cu(Gly2O),and copper nitrilotriacetic acid (CuNTA-). Solutions containing these specific complexes of copper were prepared with the aid of the program MINEQL (9)to compute equilibrium chemical speciation. Stability constants used were obtained from Sillen and Martell ( I O ) , Smith and Martell ( I I ) , and Baes and Mesmer (12).The desired solution characteristics were obtained by adding copper sulfate and the appropriate ligand to double distilled water and adjusting the p H with strong base. The total soluble copper concentration of all test solutions was 325 pg/L as Cu (5.1 X lop6 M) except for the copper hydroxy solution, which had a total copper concentration of 85 pg/L. Partial pressure of carbon dioxide was assumed to be atm. Total ligand concentrations for the glycine and NTA solutions were and M , respectively. The computed speciation of major complexes in all test solutions a t initial conditions is given in Table 1. Two types of experiments were conducted to investigate copper uptake by chironomid larvae. T h e first was designed to determine which of the copper species, if any, were taken up. This was accomplished by measuring copper accumulation in midge larvae over time in short term static tests for each copper species. Residual copper levels in the treatment solutions were measured and acceptable mass balance closures made. Five replicates were run for each treatment combination with each replicate consisting of 10 laboratory reared fourth instar Chironomus t e n t a n s larvae placed in plastic vessels containing 50 mL of test solution. I t was judged that the addition of sediment material, which midge larvae normally inhabit, would change the desired chemical composition of the solutions; thus 2.5-cm sections of small diameter tygon tubing were added to each cup t o promote more natural behavior. All experiments were run a t 25 “C in a darkened, controlled incubator. T h e second experiment was carried out to obtain more insight into possible mechanisms controlling metal uptake. This was done by exposing living and freshly killed C. t e n t a n s larvae to the different test solutions. In this experiment, independent variables were copper species and physiological state (alive or dead), and the dependent variable was total copper taken up. Exposure time and temperature were kept constant a t 11 h and 25 “C, respectively. Four replicates, set up as those in the first experiment, were run for each treatment combination. Larvae were killed by dipping them briefly in hot (80 “C)water. After treatment, midge larvae were rinsed in distilled water and dried a t 80 “C for 24 h. The dried larvae were then Volume 13, Number 10, October 1979
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