NOTES
An Investigation of the Heavy Metal Content of the Water and Sediments in a Reservoir Supplying Drinking Water to a Major Mining Center Alan J. Coggins, Kevin D. Tuckwell, and Robert E. Byrne" W.S. & L.B. Robinson University College, University of New South Wales, Broken Hill, New South Wales, Australia
*_----.__
Water and sediment samples were collected from a reservoir that supplies 30S'o of the domestic water use of a major leadsilver-zinc mining region. The samples were analyzed for cadmium, lead, zinc, and copper. Samples showed varying degrees of contamination by these elements, although measured levels in the water were below the World Health Organization limits for drinking water. Solid%ion-sediment interactions in the catchment and reservoir were examined and showed a marked tendency for metals to be removed from solution into the underlying sediment. The relative effects of iron and manganese hydrous oxides, organic material, and clays on the distributions of each metal were examined.
,I'
B
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Broken Hill is a relatively isolated city set in a semiarid environment in the center of Australia. It owes its existence to the discovery, in 1883, of massive lead-zinc deposits that have been mined continuously since that time, producing in excess of 120 M t of lead, silver, and zinc concentrates. The city shows tangible evidence of more than 85 years of mining activity. The main line of lode is marked by massive slag heaps from early smelting of ore concentrates and residue dumps from more recent mineral extraction. The average annual rainfall is 220 mm, and, until 1952, the city was dependent on two reservoirs a t Stephens Creek and Umberumberka for its total supply of domestic and industrial water. Because of severe shortages in the early 1940s, a pipeline was constructed to the Darling River some 112 km from the city. Because of the costs involved in pumping water over this distance, Stephens Creek reservoir remains a major water supply providing 30% of the average annual use of 7000 ML. This reservoir lies approximately 17 km northeast of Broken Hill and has a water storage area of 8.5 km2, giving a capacity, a t overflow level, of 20 000 ML. The large surface area of the reservoir results in high evaporation losses. It has been estimated that for ekery liter of water pumped almost 3 L is lost by evaporation and seepage ( 1 ) . The reservoir has a total catchment area of 513 km2,which can be divided into three smaller catchments as shown in Figure 1. Catchment A has an area of about 107 km2 and includes a large portion of the city. Catchments B and C are relatively nonmirceralized areas that supply about 80%of the intake to the reservoir. Because this reservoir is used as a domestic water supply, it is essential that World Health Organization limits on specific metal contents should not be exceeded. The maximum allowable limits set by this body for cadmium, lead, copper, and zinc are 10,100,1500, and 15 000 pg L-l, respectively (2). No detailed investigation has ever been carried out on the levels of these four metals in the water and sediments of the Stephens Creek reservoir. An extensive project was undertaken to determine levels and distribution patterns, sources, mode of transport, and speciation of these metals within the catchment-reservoir system. This report deals specifically with the water and sediments of the reservoir itself. An outline of other work related to the project is given elsewhere ( 3 ) . 0013-936X/79/0913-1281$01.00/0
Broken Hill
Figure 1. Broken Hill and Stephens Creek Reservoir showing the main creeks in catchments A, B, and C
Experimental Sampling Procedure. Sampling sites were chosen over the reservoir in an approximate grid pattern. Plastic buoys, attached by polypropylene rope to concrete weights, were used to mark the sites shown in Figure 2. The water samples were taken on Dec 22, 1976. They were classified as surface, bottom, or total. A total sample was taken when the water was too shallow to obtain discrete surface and bottom samples. A 2-L sample was taken a t each site. Polyethylene containers were soaked in 2% v/v nitric acid for 1 week and rinsed once with mains water and three times with deionized water. After drying, the containers were stored in sealed polyethylene bags until needed. Depth samples were taken with sealed and weighted borosilicate glass containers that were slowly lowered until the weight was resting on the bottom of the reservoir. The mouth of the bottle was then about 50 cm from the bottom. The container was allowed to fill and then slowly retrieved. The water sample was immediately acidified to a pH of about 1.5 to 2.0 with analytical grade nitric acid as recommended in the literature ( 4 ) . Fortuitously, the water contained in the reservoir became so saline (due to a poor rainfall and high evaporation rate) that the controlling authority decided to release the water held in storage. This presented a unique opportunity to study the sediments. The sediment samples were taken on April 14 and 15, 1977, with a corer designed specifically for the task. It
@ 1979 American Chemical Society
Volume 13, Number IO, October 1979
1281
\
i, /‘L-)’
water Iwrl
(22-12-76)
0
wmplr #it.
+
datum p-8
Figure 2. Stephens Creek Reservoir showing sample site localities
consisted of a barrel, 60 cm long and 3 cm in diameter, which was lined with a polypropylene tube. The lining was designed to minimize metal contamination and to facilitate core removal and subsequent handling. Most cores were between 45 and 55 cm in length with the exception of sites F and N, which had dried out substantially, and site G, which was underlain with rock. Cores obtained a t these locations were 10,15, and 20 cm, respectively. Sediment cores were transported and stored in the polypropylene linings. On arrival a t the laboratory the tubes and cores were sliced in half. Both halves were divided into 5-cm segments while still moist. These were stored in clean acid-washed polystyrene jars. Sample Preparation. Water samples, when received at the laboratory, were filtered through a Millipore 47-mm membrane filter (0.45-wm pore size) using a vacuum pump to speed filtration. Obviously, the acid preserving agent would have extracted some metal from the suspended particles in the water, This seemed preferable to filtration in the field with a concomitant high risk of contamination. The membrane filters were tested with distilled, deionized water acidified to pH 1.5 with nitric acid to determine if any of the four metals being analyzed were released from the filter. None of the four metals was detected in the filtrate, The filter was similarly tested for adsorption of the ions using an acidified standard solution containing each of the ions a t 1.00 mg L-’ concentration. No significant adsorption was detected. Hawkes and Webb ( 5 ) recommended the use of the -80 mesh fraction in the analysis of sediments, and this fraction is widely used in geochemical surveys. The reservoir sediments were all silts, loams, or clays with grain size less than 80 mesh, and sizing was unnecessary. One set of 5-cm segments for total metal analysis was air-dried, ground, and mixed in a ceramic mortar to ensure homogeneity. The other half of the section was gently disaggregated prior to separate partial extractions with aqueous solutions of calcium chloride and the disodium salt of ethylenediaminetetraacetic acid. Preconcentration Procedure. Preliminary testing indicated that levels of metals present in the water samples were below the limits of detection for conventional atomic ab1282
Environmental Science & Technology
sorption flame spectroscopy. Solvent extraction was selected as a preconcentration procedure. Ammonium l-pyrollidinecarbodithioate (APCD) and methylisobutyl ketone (MIBK) have been used extensively in studies of this nature (6-9). Because of conflicting data in the literature, the optimum pH of extraction was determined experimentally and found to be p H 4.0 for the four metals being determined. The effects of equilibration and settling time were examined, and an equilibration time of 3 min on a mechanical shaker was found t o be optimum. Absorbance readings were found to be independent of settling time, and a period of 15 min was used. Extractions were carried out in acid-washed, 250-mL separating funnels, with a “run” consisting of one blank, three standards, and six samples. A tenfold concentration was achieved by extracting 200 mL of solution with 20 mL of MIBK using 10 mL of 2% v/v APCD solution as the chelating agent; 20 mL of citrate buffer, p H 4.0, was used to maintain p H control. Sediment Digestion. Total Metal Content. The nitricperchloric-hydrofluoric acid digestion of Agemian and Chau (IO) was applied to the total extraction of metals from the sediments. Approximately 2 g of the sediment sample was weighed accurately into a Teflon beaker, and 20 mL of hydrofluoric acid, 10 mL of nitric acid, and 2 mL of perchloric acid were added. The major portion of the digesting acids was removed on a water bath at 100 “C. The samples were then taken to dryness on a hot plate a t 120 “C. The digestion procedure was repeated, and the resulting dry residue was dissolved and made up to a volume of 200 mL with 2% v/v hydrochloric acid. The solutions contained a small amount of acid-resistant minerals such as zircon, corundum, sillimanite, tourmaline, and rutile ( 1 2 1. Calcium Chloride Extraction. The determination of exchangeable metals in the sediments was based on a procedure used by Suarez and Langmuir (12). Accurately weighed sediment samples (from 1 to 16 g, depending on the amount available) were placed in nitric acid washed flasks. Onehundred milliliters of 0.5 M aqueous calcium chloride was added, and the solutions were stirred for a total of 8 h over 5
Table 1. Comparison of Metal Concentrations in the Reservoir Water Samples with Mean Blank Values and WHO Limits metal
Cd Pb Zn
cu
Table II. Total Metal Content of Sediment Core Sections metal concn, pg g-1
metal concns, pg L-1
WHO llmlts
blanka value
10 100 15 000 1500
0.1 6.7 1.6 0.4
minb cOncn
0.4 5.0 31 1.2
max concn
3.7 69 240 16
Mean value of ten replicate determinations. Minimum of ail samples analyzed. = All maximum values found for total sample at site N.
days, the p H being maintained at 5.0. The samples were allowed to stand for 4 days, and the clear supernatant liquid was removed for analysis. E D T A Extraction. The cold extraction of the metals from the sediments by an aqueous solution of the disodium salt of ethylenediaminetetraacetic acid (EDTA) was based on the method of Agemian and Chau (10). Samples of up to 5 g were weighed accurately into 250-mL conical flasks, and 100 mL of 0.05 M EDTA was added a t pH 4.8. The mixture was stirred overnight a t roorn temperature and then allowed to settle for 4 days. The clear supernatant liquid was removed for analysis. Loss o n Ignition. Davenport used loss on ignition measurements to estimate the organic matter content of sediments (13).This method may be questionable as the mass loss to 110 OC may be a measure of the low-temperature dehydration of clays. Loss on ignition studies were undertaken in the temperature ranges ambient to 110 O C and 110 to 500 OC. Metal Analysr’s. A Perkin-Elmer, Model 460, atomic absorption spectroineter was used to determine metal concentrations in the water and sediment samples. Air and acetylene were used as the oxidant and fuel mixture with flow rates of 22 and 1.8 L min--l, respectively. All readings were taken over an integration time of 10 s with a t least three readings being taken on each sample. Reservoir water samples were analyzed in triplicate to check precision.
Analytical Results and Discussion Water Samples. Blank values were determined for each of the four metals. Mean values for each element are listed in Table I. Precision of the blanks was in the range 30-40% relative standard deviation. The highest metal concentrations were recorded in the surface sample taken a t site N, the Nurses Creek inlet. However, all metal concentrations a t this location were below the World Health Organization limits, as shown in Table I. Concentrations of metals in surface and bottom waters were contoured, but values for top and bottom samples a t individual sites were generally similar. A decrease in aqueous metal concentrations away from the Nurses Creek inflow indicates that the metals were introduced to the reservoir from Catchment A. The progressive decrease in concentration is probably due to dilution by the inflow from Stephens Creek and to a lesser extent Lindsays Creek or by removal from solution into the underlying sediments as proposed by Shapiro and Connell (14). Sediment Samples. A total of 118 sediment core sections, sampled a t 14 different sites. were analyzed for total cadmium, lead, zinc, copper, iron, and manganese content. Mean values, standard deviations, and sample ranges are given for each element in Table 11. The values were computed using data from all of the 118 core sediments. Twenty-six segments from cores A, G, H, and N were extracted with aqueous solutions of EDTA and calcium chloride
metal
Cd Pb Zn
cu Mn Fe
mean value
4.3 150 250 49 590 38 500
SD
range 01 values
0.9 90 140 9 190 35 500
2.2-6.9 30-650 61-990 25-60 340-1 480 22000-72000
Table 111. Mean and Standard Deviation Values for the Percentage Extraction of Metals in the Reservoir Sediments by EDTA and CaCI2 Solutions metal
Cd Pb Zn
cu
% extractton of total metal content EDTA CaC12 mean value SD mean value
26 67 34 36
18 14 22
a
60 23 2 7
SD
22 13 3 3
as previously described. The supernatant liquors were analyzed for cadmium, lead, zinc. and copper. Mean values and standard deviations are expressed in Table 111. Geologic trend analysis is commonly used to define less obvious distribution patterns of trace elements in surveyed areas (15).In this study the trend surfaces were generated for first, second, third, and fourth order polynomials using a Fortran IV multiple regression program ( 1 6 ) .The trend surface maps were printed, by a computer subroutine, in the form of a box. A pictorial representation of the third-order trends is given in Figure 3. The orientation of the boxes with respect to the reservoir is shown a t the top of the figure. Contours of cadmium, lead, and zinc showed decreasing concentration to the north with little or no variation with depth. Copper decreased downwards and to the west from a point near the main wall, probably due to a highly anomaloiis value at the top of core B. The second-order copper trends provided a more general fit and indicated a decrease in metal concentration to the north and with depth. F ratios were significant a t the 99% confidence level for all trends except first-order copper, and the degree of correlation increased with order. The vertical distribution of the metals within each core showed no obvious trends. Horizontal variations between cores were evident, although not well defined. Metal levels were generally higher in those cores nearer to the Nurses Creek inlet, Le., cores L, M, and N, and lower in the more distant cores such as A and B. “Anomalous” metal values, defined geochemically, are those that vary from the mean of the values by more than two standard deviations (5,171. Results showed that lead levels were anomalously high in cores L and N and zinc in cores G, L, and N. For both of these metals, core N showed concentrations almost double the value of X f 2s. The levels of’ cadmium and copper were less variable, cadmium showing slightly higher values in cores 1, M, and L and copper in the top of core B. Low levels of both of these elements occur in core A and in the bottom of core B. Mechanisms for the removal of heavy metals from solution may be found in the literature. Soldatini and co-workers ( 1 8 ) found that organic matter and clay were the main factors controlling lead adsorption in soils. Leland discussed the relationship between heavy metals and organic matter, hydrous oxides, clay minerals, and calcite in sediments (19). Jenne proposed that iron and manganese hydrous oxides were the Volume 13, Number 10, October 1979
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Table IV. lnterelement Correlation Coefficients in Reservoir Sediments Cd Pb Zn Cu Fe Mn I
Cd
Pb
Zn
cu
Fe
Mn
1.oo 0.48 0.69 0.50 0.26 0.52
1.00 0.81 0.33 0.09 0.10
1.00 0.37 0.17 0.44
1.00 0.59 -0.02
1.00 0.12
1.00
.
Table V. Correlation Coefficients between Metals and Loss on Ignition Values Cd
Pb
Zn
cu
Fe
Mn
0.55 0.61
0.45 0.20
0.49 0.46
0.47 0.48
0.48 0.45
0.03 0.22
range, OC
ambient to 110 110 to 500 Cadmium
Laod
Zinc
Copper
d ecrao 8 ing concant ro t ion
Figure 3. Third-order trend surfaces for cadmium, lead, zinc, and copper in reservoir sediments
principal controls on copper and zinc fixation in fresh water sediments (20).Gardiner showed that, for cadmium, rates of adsorption and desorption were rapid and that concentration factors in river muds varied between 5000 and 50 000 ( 2 1 ) . The three main controls on heavy metal interaction with sediments appear to be: (a) adsorption onto, or coprecipitation with, iron and manganese hydrous oxides; (b) formation of metal-organic complexes or adsorption onto organic material; (c) association with clays by processes such as adsorption or ion exchange. In order to estimate the relative importance of each of these mechanisms, covariance correlations between metal concentrations and relevant sediment parameters are often used (13, 22-24). Table IV shows the interelement correlation coefficients between cadmium, lead, zinc, copper, iron, and manganese in the 118 reservoir sediment sections. The coefficients for these six metals and the loss on ignition values are given in Table V. Values greater than 0.30 are significant a t the 99.9% confidence level. All correlation coefficients for the matrix containing cadmium, lead, zinc, and copper are significant. This may indicate a similar source for all metals as suggested by Holmes ( 2 4 ) . The coefficients for iron and manganese and the other four metals show some variation. It appears that manganese hydrous oxides play a significant role in the fixation of cadmium and zinc and the iron hydrous oxides in the fixation of copper, but neither affects lead markedly. Interpretation of the correlation coefficients with loss on ignition should be treated with caution because of the possibility of low-temperature dehydration of the clay substrate. Results presented in Table V suggest that the organic matter has a significant control over cadmium, zinc, copper, and iron in the sediments. Manganese has poor correlation in all three ranges, and lead is significant only in the first range, suggesting 1284
Environmental Science & Technology
that it is associated with clays but not with organic materials. The behavior of the metals in the reservoir involves a complex relationship between metals in the water and the sediments. Previous work ( 3 ) had indicated that the movement of metals from their source to the reservoir was due to syngenetic dispersion, while the distribution of metals in the reservoir was attributable to epigenetic dispersion. Extraction of selected sediment portions with EDTA and calcium chloride solutions was undertaken in an attempt to define the possible origins of the metals in the sediments. Calcium chloride extractions were used to indicate the importance of metal ion-clay interactions. In view of the results obtained, as shown in Table 111, it is only possible to make certain generalizations. When we assume a relationship between calcium chloride exchangeability and metal adsorption on clays, the degree of adsorption follows the order Cd < P b < Cu < Zn. The marked difference between cadmium and zinc adsorption may be a measure of the mobility of zinc (25). The high value for zinc is in keeping with the findings of Gardiner (21). It would seem that copper-clay and zinc-clay interactions are negligible. I t is also surprising that the calcium chloride extractable fraction is consistently lower than the EDTA-extractable fraction for lead, zinc, and copper, while the reverse occurs for cadmium. This result may be linked with the cadmium speciation, since the extraction pH and stability constants of both zinc and cadmium-EDTA complexes are similar in magnitude.
Conclusion Despite the uncertainty associated with some of the results of this work, some facts have emerged that are highly significant from an environmental viewpoint. There is obviously an input of heavy metals into the reservoir from catchment A. However, there is a decrease in concentration of the metals away from the source indicating either removal or dilution. Thus, although the water supply is being polluted, there exists a natural sink for the metals, Le., a natural source is establishing equilibrium concentrations of the four metals in the water that are well below World Health Organization limits. The range of pH values recorded in routine analysis of the reservoir water is 6.6 to 8.1. Provided that the pH does not fall markedly in the future, there appears to be no immediate danger of metal concentrations in the water changing significantly. A c k nou11e dg m e n t
The assistance of Ivan Pauncz from the School of Applied Geology, The University of New South Wales, is gratefully appreciated. Mr. Pauncz initiated and interpreted the com-
puter results for the 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, and 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. The majority of isolates were classified as belonging to the genus Pseudomonas and t o the Flauobacterium-Cytophaga group. A high percentage of the bacteria exhibited denitrifying ability. Nematoda were found in t h e lead column of the 25 “C set of columns, while Mastigophora and Amoeba were present in the lead column of t h e 5 “ C set of columns. The nature of microbial life in activated carbon adsorbers appears to be similar in nature and number to that of the 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, and 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 the 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 that 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 the 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 the 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, the 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. The 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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