COM M UN ICAT I ON
On the Significance of Metal Complexing Agents in Secondary Sewage Effluents Michael E. Bender, Wayne R. Matson, and Robert A. Jordan Department of Environmental Health, University of Michigan, Ann Arbor, Michigan 48104
Preliminary experiments in this laboratory using recent advancements in anodic stripping techniques have demonstrated that two distinct molecular weight fractions of secondary sewage effluents complex metals. One fraction has a molecular weight similar to that of synthetic chelators and has proved effective in stimulating algal growth.
A
t present, control of eutrophication in natural waters is being attempted by reducing nutrient inputs (i.e., phosphorus and nitrogen) to these waters from sewage effluents. However, there is some question whether the growth stimulation caused by secondary sewage effluents can always be totally accounted for by the nitrogen and/or phosphorus levels in these effluents (McGauhey, Rohlich, et ul., 1968; Stewart and Rohlich, 1967). Many compounds, both inorganic and organic, could be responsible for the effect. Vitamin B,? has been identified by Hoover, Jasewicz, et a/. (i951) as one of the compounds present in sewage which could be effective in stimulating algal growths. Other compounds which could be responsible include : trace metals, organic growth factors, and metal complexing agents. The chelation or complexing of metals by organic compounds has been demonstrated by several workers to cause significant increases in algal production (Johnston, 1961; Shelske, 1962; Shapiro, 1964). While some of these experiments have been conducted utilizing synthetic chelating agents such as EDTA, the chelation of iron by naturally occurring humic substances has been demonstrated by Shapiro (1964) to be significant in increasing algal growth. Prakash and Rashid (1968) have demonstrated that humic s~ibstancesof natural origin increase growth rates of marine algae. It is reasonable to assume that organic chelating substances, if present in sewage effluents, could stimulate algae growth by making trace metals already present in the receiving water more available or reduce growth by forming stable unavailable complexes. We have attempted, as described below, to find such substances. Methods
Secondary sewage effluent was collected from the Ann Arbor activated sludge plant prior to chlorination. The samples were filtered through 0.45-p membrane filters to remove bacteria and other solids. Spikes of copper containing 700 ng. were added to 10-ml. aliquots of the filtrate. Molecular weight fractionations were then made on the 10-ml. samples using 2.5 X 45 cm. columns of Sephadex G-50 medium. To prevent problems of coagulation and precipitation within the column, the 520 Environmental Science & Technology
columns were eluted at a flow rate of 1.3 to 1.7 ml./min. with approximately 0.02 N NaCl of the same conductivity and pH as the sewage. Eluant fractions of 8 ml. were collected for copper and total carbon analysis by infrared absorption. Copper was determined as free or bound using the composite mercury graphite electrode and anodic stripping technique described by Matson, Roe, et uI. (1965, 1967). Each 8ml. fraction was plated at a potential of -0.7 V ts.SCE for 3 to 15 min. and then stripped at a sweep rate of 40 mV/sec., giving a value for free or labile copper ion. After this plating step, 50 pl. of perchloric acid was added to the sample to release the complexed copper, and the plating and stripping steps were repeated giving a value for acid-exchangeable copper. Digestion experiments with ammonium persulfate indicate that acid-exchangeable copper is equivalent to total copper in this type of sample. By use of a modification of techniques reported previously by Allen, Matson, el a/. (in press), various sewage fractions and samples of raw sewage were titrated by adding successive 100-ng. aliquots of copper to the sample at a plating potential of -0.7 V L'S. SCE until a copper peak was observed. Exchange experiments were carried out by following the technique of Matson (1068) with Fe4 on sewage fractions saturated with copper to determine the extent and rate of exchange. Preliminary algul growth studies were conducted with each of the molecular weight fractions, isolated in quantity using larger 4.5-in.-diameter columns, to test their biological significance. The studies were conducted with natural algal populations enclosed in borosilicate glass containers to which the nutrient materials in question were added. In the Third Sister Lake experiment, the containers were suspended in the lake for incubation, while in the Lake Michigan experiment, the containers were incubated on shipboard in a water bath at lake temperature. C-14 uptake was used as a measure of response to the additions.
Results and Discussion Figure 1 shows a typical example of the distribution of copper and the associated quantities of total carbon obtained from the Sephadex separations. Two rather distinct molecular weight fractions are apparent. The large molecular weight fractions at the breakthrough volume of the column (-10,000 mol. wt.), contained about 100 ng. of copper while the smaller molecular weight fractions (500 to 1000 mol. wt.) contained about 700 ng. No free copper was found in any fractions using the 700 ng. spike. The concentration of ligands determined by titration in this and eight other sewage samples ranged from 8 X to 1 X IO-EM equivalent binding sites for copper. Copper made unavailable for electrochemical plating through complexa-
IO
30
so
70
90
I IO
130
150
L 170
190
ELUTION VOLUME in rnl.
Figure 1. Distribution of carbon and of metal binding capacity in secondary sewage fractions obtained from Sephadex separations Table I. Algal C-14 Uptake in CPM (Light-Dark) Third Sister Lake Sewage ligands (low mol. wt.) 1 X lo-’ M (equivalents of copper) No. sewage ligands No. Fe F e ( 5 x 1o-j No. Fe Fe(5 X M)
Date
21 5 586 209 598 49 5 319 5 32 312 Lake Michigan Fe EDTA F~ (3 x 1 0 - 7 ~ ) ( 1x --Low ligands ___-mol. wt. -- _- ( 3 X 10-7M) No. Fe Fe (3 X lO-’M)
780 717 620 406
250 256 343 293
4-25-69 4-28-69 4-30-69 5-2-69
Date
Control no. Fe
4-20-69 4-21 -69
596 485
+
518 658
729 592
tions with ligands in the sewage was released by Fei3 as well as by acid, indicating that the ligands arealsoavailable for iron complexation. Lead in the sewage also approximately followed the copper but was not quantitatively determined. In one unreplicated trial (Table I) on Third Sister Lake in the Saginaw Forest, Ann Arbor, Michigan, the lower molecular weight fraction, both alone and when combined with iron, had a n effect in stimulating algal growth. A second trial (Table I) in Southern Lake Michigan water approximately 15 miles from Chicago showed a smaller but still measurable effect for low molecular weight sewage ligands. The high molecular weight fractions which bind the metals more strongly and might be less likely to pass the cell membrane, showed a very slight effect. In both trials, a marker of vitamin Bi2was employed as a control o n the column used to isolate the ligand material to ensure that the effects seen would not be caused by uncomplexed B12in the sewage. Conclusions
Fractions isolated from secondary sewage effluent containing organic ligands for metal cations show a definite effect in increasing the growth of algae. Effort is being made a t present to rule out the possibility that other substances in the sewage fractions, such as vitamins, plant hormones, or organic phosphate, could be responsible for the stimulation of algal growth detected.
1107 677
1096 776
High mol. wt. ligands Fe (3 X 1 0 . m
g o . Fe
794 61 3
644 516
Achnowledgrnent
The authors gratefully acknowledge the assistance of the crew of the research vessel, Inland Seas, Great Lakes Research Division, University of Michigan. Literature Cited Allen, H. E., Matson, W. R., Mancy, K . H., J . Wafer Pollut. Contr. Fed. (in press). Hoover, S. R., Jasewicz, L. B., Porges, N., Science 144, 213 (195 1). Johnston, R., J . Mar. B i d . Ass. U . K . 44,87-109 (1964). Matson. W. R.. Roe. D. K.. Carritt. D. E.. Anal. Clrem. 37. 1594-5 (1965). Matson, W. R., Roe, D. K., Carritt, D. E. Analysis Instrumentation 7.19-22 (1967). Matson, W. -R., Ph.D. Thesis, MIT, Cambridge, Mass., 1968. McGauhey, P. H., Rohlich, G. A., Pearson, E. A., Progress Report, FWPCA Grant WPO-48, 1968. Prakash, A., Rashid, M. A,, Litnnol. Oceanngr. 13, 598-606 (1968). Shapiro, J., J . Amer. Water Works Ass. 56, 1062-82 (1964). Shelske, C. L., Science 136,45-6 (1962). Stewart, K. M., Rohlich, G. A., Eutrophication-A Review, State of California. Water Quality Control Board, Publication No. 34, 1967. Receiued,for review July 14, 1969. Accepted January 22, 1970. The work was partially supported b y USPHS-NIH Grant 5-Sol-FR-05447-07 to Wayne Matson and b y FWPCA Traineeship 1-FI- WP-26-294-01. Volume 4, Number 6 , June 1970 521
