11. of sample to 25 ml by the CoAPDC method provides a net concentration factor of 40. By adjustment of sample volume and the nitric acid solution volume, concentration factors as high as 400 can be obtained.
ACKNOWLEDGMENT The authors thank J. M. Edmond of Massachusetts Institute of Technology and Michael Orren of Woods Hole Oceanographic Institute for the discussion of their experimental results prior to publication. We appreciate the critical review of the manuscript by G. W. Fuhs of the New York State Department of Health. We gratefully acknowledge the technical assistance of C. P. Snyder and Kaiwen Wang in performing anodic stripping voltammetry. LITERATURE CITED (1)D. W. Spencer and P. W. Brewer, Geochim. Cosmochim. Acta, 33,325 (1969). (2)P. G. Brewer, D. W. Spencer, and C. L. Smith, Am. Soc. Test. Mater., Spec. Tech. pub/., 443,70-77 (1969). (3)American Public Health Association, New York, "Standard Methods for the Examination of Water and Wastewater", 14th Ed., 1976,pp 144162. (4)J. F. Slowey and D. W. Hood, Geochim. Cosmochim. Acta, 35, 121 (1971). (5) C.L. Luke, Anal. Chem., 36,318 (1964). (6)W. J. Campbell, E. F. Spano, and T. S. Green, Anal. Chem., 38, 987 (1966). (7)C.W. Blount, D. E. Leyden, T. L. Thomas, and S.M. Guili, Anal. Chem., 45, 1045 (1973). (8)J. P. Riley and D. Taylor, Anal. Chim. Acta, 40,479 (1968). (9)H. L. Windom and R. G. Smith, Deep-sea Res., 19, 727 (1972). (10)R. Chester and J. H. Stoner, Mar. Chem., 2, 17 (1974). (11)D. E. Leyden, T. A. Patterson, and J. J. Alberts, Anal. Chem., 47, 733 (1975).
(12)D. E. Leyden and G. H. Luttreil, Anal. Chem., 47, 1612 (1975). (13)J. E. Going, G. Wesenberg, and A. Andryat, Anal. Chim. Acta, 81, 349 (1976). (14)M. M. Reddy, unpublished results, 1976. (15)E. Rona, D. W. Hood, L. Mase, and B. Baglio, Limnol, Oceanogr., 7,201 (1962). (16)H. R. Fleck, Analyst(London), 62,378 (1937). (17)R. L. Mitchell and R. 0. Scott, Spectrochim. Acta, 3,368 (1947). (18)G. E. Heggen and L. W. Strock, Anal. Chem., 25, 859 (1953). (19)J. A Buono, J. C. Buono, and J. L. Fasching. Anai. Chem., 47, 1926 (1975). (20)T. N. Tweeten and J. W. Knoech, Anal. Chem., 48,64 (1976). (21)R. A. Nadkarni and G. H. Morrison, Anal. Chem., 46,232 (1974). (22)R. A. Nadkarni and G. H. Morrison, Anal. Chem., 47,2285 (1975). (23)E. A. Boyle and J. M. Edmond, Adv. Chem. Ser., 147,44-55 (1975). (24)J. F. Elder, S. K. Perry, and F. P. Brady, Environ. Sci. Techno/., 9, 1039 (1975). (25)A. Hulanicki, Talanta, 14, 137 (1967). (26)J. Stary and K. Krantzer, Anal. Chim. Acta, 40,93 (1968). (27)A. Wyttenbach and S. Bajo. Anal. Chem., 47,2 (1975). (28)K. Gleu and R . Schwab, Angew. Chem., 62,320(1950). (29)H. Malissa and E. Schoffmann, Mikrochim. Acta, 22, 187 (1955). (30)C.R. Parker, "Water Analysis by Atomic Absorption Spectroscopy", Varian Techtron, Palo Alto, Calif., 1972. (31)H. Siegerman and G. O'Dom, Am. Lab., 4,59 (1972). (32)K. V . Krishnamurty, E. Shpirt, and M. M. Reddy, At. Absorpt. News/., 15, 68 (1976). (33)K. V. Krishnamurty, M. M. Reddy, and G. Kartha, unpublished results, 1976.
RECEIVEDfor review July 19, 1976. Accepted October 26, 1976. This study was conducted as part of the Task C activities. of the Pollution from Land Use Activities Reference Group, International Joint Commission, and is funded through the United States Environmental Protection Agency and the State of New York. Presented at the Symposium on Chemical Separations during the 171st National Meeting, American Chemical Society, New York, N.Y., April 4-9,1976.
Chromatographic Measurement of Submicromolar Strong Complexing Capacity in Phytoplankton Media Richard J. Stoizberg" and Diane Rosin Harold Edgerton Research Laboratory, New England Aquarium, Central Wharf, Boston, Mass. 02 1 10
A technique for measurlng strong complexing capaclty in seawater has been developed and applied to a number of matrices, lncludlng phytoplankton media used In continuous culture experiments. Samples are spiked wlth an excess of copper and passed through a column of Chelex 100. The copper that is not strongly complexed Is removed from solution at the head of the column, but copper assoclated wlth large, strong ligands elutes from the column. The effects of flow rate, column length, and pH were studied. The precision of the entire technique is indistinguishablefrom the precision of the copper measuring technique when laboratory solutions and artificial phytoplankton media are analyzed. When an Echelle grating plasma emission spectrometer is used, the standard deviation of the technique is 2 X M when measuring submicromolar complexing capacity.
The importance of the speciation of trace metals in natural waters has been recognized only in the past few years. It is clear now that metal speciation determines bioavailability to primary producers (1-4) and can regulate transport and sorption mechanisms (5-7). This is not to say that the mechanisms responsible for changes in bioactivity or transport due to complexation of the metal are well understood. 226
ANALYTICAL CHEMISTRY, VOL.
Two methods have been used in attempts to elucidate the speciation of metals in natural waters-direct measurement (8-10) and theoretical modeling (11-13). In addition, measurements of total strong complexing capacity have been made using titrimetric techniques (14-16) and a column chromatographic method (17) using both the sodium and the copper forms of the chelating resin Chelex 100, a specially purified form of Dowex A-1. We are particularly interested in the biochemical response of marine phytoplankton to trace metal stress. One response, production of extracellular metal binding organic matter (EMBO), could be triggered by a number of stimuli. These include the presence of excess uncomplexed toxic metal and the presence of insufficient micronutrient metal available to the organism. The production of strong organic complexing agents by diatoms in response to the presence of high concentrations of uncomplexed or weakly complexed copper has been hypothesized (18),but to our knowledge this hypothesis has never been proven. Recently, blue-green algae in fresh water have been observed to excrete iron selective chelators during periods of iron deprivation (19),and it is not unlikely that the same effect may be observed with copper deficient algae since copper has been shown to be an essential micronutrient ( 4 ) . Measurement of submicromolar quantities of total com-
49, NO. 2, FEBRUARY 1977
plexing capacity in seawater has been accomplished only by Davey (15) using a bioassay technique. While Chau’s ( 1 4 ) titrimetric technique may be useful in seawater, it is slow, requires a relatively large volume of sample, and could produce erroneous results due to surface or kinetic phenomena (20,211, Jones’ technique, which is perhaps applicable to seawater samples, is not well suited for large numbers of samples and is insufficiently sensitive for our application. T h e success of Jones’ technique (17) implies that ligand exchange chromatography (22) will not work as a preconcentrating technique for ethylenediaminetetraacetic acid (EDTA) and possibly other large or strong ligands. Ligand exchange chromatography has successfully been used t o concentrate metal binding substances from tobacco smoke condensate (23) and t o separate amino acids from lake water ( 2 4 ) ,seawater (25), and other dilute solutions (26). Results obtained by Loewenschuss and Schmuckler (27) indicate that small ligands such as glycine, glutamate, and iminodiacetate bind to the metal which is in turn bound to the iminodiacetate moiety on the Dowex A-1 resin. However, t h e large polydentate ligand N-hydroxyethylenediaminetriaceticacid does not enter the resin phase. It seemed likely that column chromatography using Chelex 100 could be used t o separate copper bound t o large or strong ligands from uncomplexed or weakly complexed copper. The uncomplexed copper is sorbed at the top of the column. Strongly complexed copper should elute from the column unretarded and nearly quantitatively. The rationale for spiking samples with copper is t o associate all of the ligands present with a single metal. Copper complexes of organic ligands are among the strongest formed when competing inorganic reactions are taken into consideration, and copper can therefore readily displace most other metals from ligands of interest. Metals that might form stronger complexes than copper hydrolyze extensively at the p H of seawater, interact strongly with chloride, or react slowly. Copper reacts rapidly and does not form strong chloride complexes. In addition, determination of copper is done easily and rapidly with high sensitivity using either absorption or emission spectrometry.
EXPERIMENTAL Reagents. All chemicals used were of reagent grade. Stock ligand solutions were prepared by dissolving an accurately weighed quantity of ligand and diluting to volume. Distilled, deionized water was used throughout. The sodium form of the chelating resin Chelex 100 (Bio-Rad Laboratories), 100-200 mesh, was washed repeatedly with distilled water to remove the fines. The pH of the suspension was then adjusted to near 8 with HC1, and sufficient pH 8 tris(hydroxymethy1)aminomethane (Tris) buffer was added to give a 0.1 M solution. Apparatus. Copper determinations were done using both atomic absorption (AA) and atomic emission (AE) spectrometry. AA analysis was done a t 324.7 nm with an air-acetylene flame, hydrogen continuum lamp background correction, and a 10-cm Boling burner (Instrumentation Laboratory Model 151). AE analysis was done with a relatively new commercial instrument, a Spectrametrics Echelle grating plasma emission spectrometer (Spectraspan III), at 324.7 and 327.4 nm. Statistical analyses of data were performed using a Wang 600 programmable calculator. Procedures. Bulk samples were filtered or centrifuged to remove particulates, if necessary, and the pH was adjusted to 7 to 8 with HCl or NaOH. Tris buffer is used to ensure pH stability. Subsamples, typically 10 ml, were transferred to polypropylene snap-cap tubes (Falcon 2059) and spiked with an excess of copper. For most natural water samples and our phytoplankton media, an addition of 15 pM copper was sufficientto ensure complexation of most, if not all, strong ligands by copper. After the addition of copper, the sample was equilibrated with the indicator metal. A period of 1h was sufficient to establish equilibrium in all samples we have examined. Columns were rinsed with distilled water, an appropriate buffer, or synthetic seawater prior to use. When synthetic seawater was used to equilibrate
the column, the bed volume decreased to approximately 60% of its original value as the resin was converted from the sodium form to the calcium form. Column flow rate was adjusted to approximately 30 ml min-1 cm-2 in a 1-cm i.d. column for natural water samples or to 10 ml min-1 cm-2 in a 0.6-cm i.d. column for phytoplankton media; the spiked, equilibrated sample was chromatographedon a 2- to 3-cm long column of Chelex 100; and the effluent was collected in polypropylene tubes. Blanks and standards were chromatographed with samples. Many samples, standards, and blanks could be run on the same column as long as they all had similar major ion composition and care was taken to collect effluent that is representative of individual samples. Development of a blue band of the copper form of Chelex 100 was observed at the head of the column, and the size of the band was useful for determining when the column should be replaced. The effluent was analyzed for copper and the complexing capacity was calculated from comparison with standards or by assuming stoichiometric passage of copper associated with strong ligands. Application of the technique to algal growth medium involved a similar procedure. Duplicate 10-mlsamples were taken from both the culture and the medium reservoir, spiked with copper, left for approximately 1 h to allow metal and ligands to equilibrate, and chromatographed. EDTA standards in synthetic seawater were chromatographed on the same column, and quantification was made by means of a calibration curve, both for unused and for used medium. Skeletonemu costutun, a marine diatom, was grown in the synthetic medium Aquil (28) (nominal concentration of EDTA = 5 X M) in continuous culture in all experiments reported here. This procedure is not applicable to all ligands and all media. Ligands present as inert complexes will not associate with the copper spike and consequently will not be measured. In some phytoplankton media, trace metals are present with total concentration greater than 10-5 M, and calculations should be made to calculate the concentration of copper necessary to ensure near complete association of ligands with copper. These same media contain greater than M added ligand, and the copper spike should be increased to maintain a moderate uncomplexed copper concentration. These media do not lend themselves to measurement of the production of EMBO because of the difficulty of measuring small increases in an initially high total complexing capacity.
RESULTS A N D DISCUSSION Preliminary results in synthetic seawater (SSW) demonstrated the utility of the technique with EDTA as the model ligand. A set of EDTA standards (0 to 6 pM, in 1 pM increments) was prepared. Duplicate samples at each EDTA level were taken, spiked with copper, chromatographed, and the effluents were analyzed by AA for copper. The resulting plot of copper concentration of the effluent vs. EDTA concentration of the influent was straight (standard deviation of the slope 0.03) an the slope of the plot was within 4%of the theoretical value of 1.00. The standard deviation of the analysis based on the duplicate analyses was 0.26 pM and was indistinguishable from the standard deviation of the copper analysis itself (0.2 pM). Further experiments demonstrated that the linear range extends to a t least M if the effluent is diluted prior t o the copper determination. The effects of flow rate and column length on copper concentration in the effluent were investigated for both EDTA and NTA in 10-2 M Tris buffer a t p H 8. As shown in Table I and Figure 1,the effects in the presence of EDTA are minimal. When NTA is the ligand, the copper concentration in the effluent decreased markedly as the column was lengthened or the flow rate was decreased. Naturally-occurring ligands may be weaker than EDTA or NTA. In a medium like Aquil, the quantity of EMBO produced by algae is likely to be only a fraction of the synthetic ligand already present, and precise measurements must be made t o measure EMBO produced. Short (2 cm) columns have been used successfully, and this minimizes variability in copper content of effluent from samples containing unknown natural ligands in addition to EDTA. The use of a peristaltic pump to obtain high flow rates has not proven successful when small diameter (3 or 6 mm) columns were used because of channeling in the resin bed. T h e pump-induced channeling
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
227
1.o I
h
cC" 5igand
0.5
0 I
I
0.0
I
10 20 flow rate, rnt rnin-1 crn-2
0
30
Figure 1. Effect of flow rate on copper !n effluent (0)2 and 10 pM EDTA. (0) 80 and 100 pM NTA
Table I. Effect of Column Length on Effluent Copper Concentrationa Column length, cm
2 5 10
CCdCligend
NTA
EDTA 2.5pM
10pM
60pM
100pM
0.91 0.90 0.88
0.83 0.85 0.84
0.86 0.51 0.22
0.91 0.72 0.54
Flow rate = 10 ml min-l cm-2.
Table 11. Complexing Capacity Measurements in Chemostatsa
Date Feb 18 19 20 25 26
Chemo- Difference, Chemo- Difference, Inflow, stat, # 3 - feed; stat, #4 - feed, pM #3,pM pM #4,pM pM
4.45 4.42 4.44 4.68 4.68 28 4.48 29 4.84 Mar 1 4.70 2 4.78 3 b 4.71 4 4.75 5 4.88 6 4.94 7 4.71 8 4.57 Mean 4.67 Std dev 0.16
4.88 4.83 4.66 5.00 4.98 5.22 5.34 4.82 5.12 5.26 5.40 5.15
5.30 5.36 5.04 5.09 0.23
0.43 0.41 0.22 0.32 0.30 0.74 0.50 0.12 0.34 0.55 0.65 0.27 0.36 0.65 0.47 0.42 0.17
5.06 4.80 4.50 5.04 4.99 4.81 5.21 4.68
0.61 0.38 0.06 0.36 0.31 0.33 0.37 -0.02
4.89 0.23
0.30 0.20
Each figure is the mean of two complexing capacity measurements. Medium in reservoir changed.
is not apparent in 1-cm i.d. columns, and the larger columns are quite useful where large numbers of samples must be analyzed and 10 to 15 ml of sample is available. Complexing capacity measurements were made to investigate the stability of Aquil and to determine if Skeletonema costaturn produces a measurable quantity of EMBO when grown in continuous culture. Table I1 presents results of measurements made over a three-week period. The excellent 228
day-to-day repeatability of the technique is attested to by the standard deviation of measurements made for the inflow medium. The slight decrease in precision of measurements made for chemostat complexing capacity reflects favorably on the applicability of the technique to more complex and less well defined systems. We have observed a consistent excess complexing capacity of 3 to 4 X M in continuous cultures not stressed by excess copper. Although we have not completely ruled out artifacts due to cell rupture, the evidence points to the production of the EMBO as being a real phenomenon in the chemostat. T o our knowledge, this is the first direct measurement of copper binding EMBO produced by marine phytoplankton. Determination of less than M EDTA complexing capacity in SSW has proven feasible using the 1-cm i.d. columns. ' Table I11 presents results of calibration curve experiments performed first with a 3-mm i.d. column and then with a 1-cm i.d. column. The data obtained with the small diameter column reflect leakage of uncomplexed copper. Results obtained with the large diameter column are comparable to those observed with 5 MMEDTA in SSW in smaller columns. The slope of the calibration curve is 0.92 to 0.95 and the line extends to close to the origin. Our experience has shown that complexing capacity measurements below lom6M are adversely affected by uncomplexed copper leakage, and for that reason large diameter columns should be used. The ultimate sensitivity of the technique for ligands like EDTA appears to be limited by the sensitivity of the copper measurement technique if leakage of uncomplexed copper does not occur. With the technique described here, the detection limit is certainly less than 5 X M. Throughout the entire developmental portion of this study, we have observed that the precision of the entire technique is indistinguishable from that of the copper measurement step. This fact implies that the chromatographic step is reproducible and reliable with a wide variety of samples. Atomic absorption measurements of copper in seawater are limited by flame noise and nonatomic absorbance, and the average M with the standard deviation is approximately 2 X non-optimum system described previously. The emission instrument has a comparable standard deviation (1.6 X 10-7 M) when measuring 5 X M copper, but it is better suited than the AA for measuring copper in seawater. The standard deviation of measurements of M copper in SSW is 7 X M, and the emission system has been used to take advantage of its higher precision. The overall standard deviation of duplicate complexing capacity measurements of 45 samples of Aquil, used Aquil, and EDTA standards in SSW during the three-week period that the data in Table I1 were collected was 1.3 X lod7M with plasma emission detection. The day-to-day repeatability of the technique can be seen in Table 11, as noted previously. The standard deviation of the average of feed measurements over the entire test period is indistinguishable from that calculated for the technique based on duplicate analyses on a single day. The standard deviation of the technique based on the duplicate analyses presented in Table I11 is 2.0 X M. The precision of the technique for the samples described above and for Boston Harbor water samples is tabulated in Table IV. Analysis of synthetic seawater samples is less demanding than that of natural samples, because of the heterogeneous physical and chemical nature of the latter. Sixteen Boston Harbor samples were analyzed for complexing capacity in early June. Four of the samples were spiked with 0.9 FmM EDTA, and all were analyzed for total complexing capacity. Results presented in Table IV show that recovery of EDTA added to Harbor water was only 55% complete. It is possible that irreversible sorption of the ligand to particulates is responsible for the incomplete recovery because the sample was
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1077
Table 111. Sub-Micromolar EDTA Complexing Capacity Determinations in SSW
WMCopper in effluent Day l a
Day 2 a
Day 3a
gM EDTA
Series Ab
Bb
A
B
A
B
0.00 0.09 0.18 0.36 0.54 0.72 0.90
0.07 0.16 0.30 0.47 0.72 0.92 1.03
0.34 0.32 0.54 0.72 0.83 0.98 1.16
0.02 0.15 0.25 0.38 0.57 0.60 0.81
0.02 0.14 0.20 0.40 0.52 0.64 0.78
C
0.00
0.08 0.21 0.34 0.48 0.68 0.85
0.09 0.18 0.35 0.52 0.67 0.84
Day 1: 0.3-cm i.d. X 3-cm column; 21 ml minc-1 cm-2. Day 2 and 3: 1.0-cm i.d. X 3-cm column; 12 ml min-l cm-*. Two separate calibration curve sets run through the same column, one complete set at a time. Contaminated.
Table IV. Precision of the Technique Sample otogx10-7~ EDTA in SSW 0 to 5 X 10-6 M EDTA in Aquil, used Aquil, and SSW Boston Harbor “A” (found 6.0 X 10-7 M) Boston Harbor “B” (found 4.6 X M) Boston Harbor “B” + 9 x 10-7 M EDTA (found 9.6 X 10-7)
Number
Std dev
7 duplicates 2.4 X lob8 M (day 2) 1.6 X M (day 3) 45 duplicates 1.3 X M 8
0.3 x 10-7 M
4
0.2 x 10-7 M
4
0.4 x 10-7 M
neither filtered not centrifuged. We have seen analogous behavior in Charles River water samples concentrated sixfold with a rotary vacuum evaporator. Spikes of 1WMEDTA added to the concentrate increased the copper concentration of the column effluent from 4.25 to 4.80 pM. We have shown that the uptake of complexed copper is virtually 100%efficient. In the presence of EDTA, >90% of the copper associated with the ligand elutes from the column over a wide range of flow rates, column lengths, and ligand concentration. This is not the case with the weaker ligand NTA. The strong dependence on flow rate and column length on the effectiveness of complexed copper uptake by the resin suggests that thermodynamic and kinetic factors may regulate the uptake of complexed metals on Chelex 100. Simple competition between the ligand in solution and the iminodiacetate on the resin can determine whether or not the ligand elutes from the column nearly quantitatively associated with copper. Kinetic factors, particularly involving dissociation of the metal-ligand complex in solution and mass transport a t the resin-aqueous interface, could also be important. Lowenschuss (27) suggested that ligand size may be important. A fourth possibility that is probably intimately involved with the other three is that of the number of basic sites on the ligand. Association of a metal with Chelex 100 reduces the number of empty d orbitals available to accept metal donated electrons. A ligand that is unidentate or bidentate is liable to become immobilized on the resin as a mixed complex of the type Chelex-M-L. Larger polydentate ligands would be less likely to do so, because of the limited number of d orbitals available. Competition between the Chelex and
the mobile ligand for metal in solution occurs, and strong polydentate ligands carry a stoichiometric quantity of metal through the column. One must recognize that the data obtained using this technique represent operational complexing capacities only. As with other techniques of this sort (14-17), it is biased against complete detection of polydentate ligands forming complexes with the added indicator metal if the stability constant is less than a certain critical value. Ligands forming complexes with the indicator metal, having a stability constant greater than that critical value, pass through the column with a stoichiometric quantity of indicator metal. The value of the critical stability constants is determined by column length, flow rate, sample pH, sample cationic makeup, and ligand concentration. For samples with similar ionic composition, direct comparison of calculated apparent complexing capacity can be made with confidence. This is the approach taken in measuring the production of EMBO in the continuous culture experiments. Changes in sample pH affect column behavior as the result of two opposing protonation reactions. Lowering the pH of the solution tends to protonate the ligands in solution and the Chelex 100. Metal affinity is decreased both for Chelex 100 and for the soluble ligand. The relative degree of protonation determines whether more or less indicator metal elutes from the column associated with soluble ligand as the pH of the solution is changed. The concentration of copper in the column effluent decreases for EDTA but is unchanged for NTA as the pH of the influent decreases from eight to four. The protonation side reaction coefficient for both NTA and Chelex 100 increases by lo4, whereas the value for EDTA increases by The EDTA may be weakened sufficiently compared to the Chelex so that it cannot carry a stoichiometric quantity of copper through the resin bed. Passage of seawater through a column of NaChelex 100 undoubtedly changes a large portion of the resin to the calcium form. The identity of the counterion. on the Chelex 100 will determine the relative affinity of the resin an?. ligand for the metal. Substitution of calcium for sodium will decrease the resin affinity for copper, thereby decreasing the critical stability constant. However, an opposing effect is observed in solution. The calcium competes with the added copper for the ligand, decreasing the conditional stability constant for the copper-ligand complex. As in the case of the pH effect, the direction and magnitude of this effect depends on the relative values of stability or distribution constants. To optimize the system with respect to stability, sensitivity, and response to moderately strong ligands, we have chosen
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
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to use a very short column, For some samples, high flow rates are employed. In this way variations in copper throughput are minimized for ligands that exhibit nonstoichiometric behavior, In addition, the critical stability constant defining a very strong ligand is probably reduced. The resin has worked ideally to sorb uncomplexed and weakly complexed indicator metal. Errors due to leakage of uncomplexed metal have been recognized and larger diameter columns are used when necessary. We hypothesize that another factor may cause erroneously high results. It is possible that indicator metal sorbed onto organic or inorganic colloidal particles will not be retained by the resin (29) and will contribute to the calculated complexing capacity. Since adsorbed metals are not truly complexed, and since they can exhibit different behavior regarding bioactivity and transport, care must be taken in interpreting results. The same caveat must be made for complexing capacity measurements based on other techniques. Electrochemistry, molecular filtration, and bioassay all give operational complexing capacities where sorbed metal can be mistaken for complexed metal. Finally, it should be clear that kinetic factors exist and can affect results. Ligands that are slow to exchange their original metal for the copper spike will not be detected using this technique unless sufficient time is allowed for equilibrium to be established between the added copper spike and the metal bound ligand. This technique is rapid, sensitive, and quite precise when applied to phytoplankton medium, Boston Harbor water, and concentrated Charles River water. Its accuracy is excellent for EDTA in phytoplankton medium, although it is less so in unfiltered natural water samples spiked with EDTA. It is possible that the effective strength of ligands in natural waters could be estimated using a modification of the chromatographic step. A plot of copper concentration in the effluent vs. flow rate or column length could be compared to a plot obtained with ligands of known metal binding strength. In fresh water there would be a much wider latitude of metal which might be used as indicator metal (Fe, Hg, Ni) to further characterize the metal specificity of the strong complexing species present.
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LITERATURE CITED ( I ) R. T. Barber In "Trace Metals and Metal-Organic Interaction In Natural
Waters", P. C.Singer, Ed., Ann Arbor Sclence Publlshere, Inc., Ann Arbor, Mlch., 1973, p 321. (2) R. Johnston, J. Mar. Biol. Assoc. U.K., 44, 87 (1964). (3) J. Lewln and C. H. Chen, Limnol. Oceanogr., 16,670 (1971). (4) S. E. Manahan and M. J. Smith, Environ. Sci. Technol., 7, 629 (1973). (5) A. Lerrnan and C. W. Chllds in "Trace Metals and Metal-Organic lnteractlon in Natural Waters", P. C. Singer, Ed., Ann Arbor Science Publishers, Inc., Ann Arbor, Mlch., 1973, p 201. (6) K. L. Jewett, F. E. Brinckman,and J. M. Beiiarna in ACS Symposium Series No. 18, "Marine Chemistry in the Coastal Environment", R. E. Baier, Ed., 1975, p 304. (7) T, P. O'Connor and D. R. Kester, Geochim. Cosmochim. Acta. 30. 1531 (1975). M. J. Stiff, WaterRes., 5, 565 (1971). G. E. Batley and T. M. Florence, Anal. Lett., 0, 379 (1976). Y. K. Chau and K. L. S. Chan, Water Res., 8,383 (1974). C. W. Chiids, Proc. 14th Conf. Great Lakes Res., 1971, p 198. L. R. Gardner, Geochim. Cosmochim. Acta, 38, 1297 (1974). F. Morel and J. Morgan, fnviron. Sci. Technol., 6, 58 (1972). Y. K. Chau, R. Gachter, andK. L. S. Chan, J. Fish. Res. Boardcan., 31, 1515 (1974). E. W. Davey, M. J. Morgan, and S.J. Erickson, Limnol. Oceanogr., 18,993 (1973). R. G. Smith, Anal. Chem., 48, 74 (1976). D. R. Jones and S.E. Manahan, Anal. Lett., 8,421 (1975). E. Steemann Nieisen and S. Wiurn-Andersen, Physiol. Plant., 24, 480 (1971). T. P. Murphy, D. R. S. Lean, and C. Nalewajko, Sclence, 102, 900 (1976). D. N. Hume and J. N. Carter, Chem. Anal. (Warsaw), 17, 747 (1972). M. S.Shuman and G. P. Woodward, Anal. Chem., 45, 2032 (1973). F. Heiferich, J. Am. Chem. Soc., 84, 3237 (1962). V. N. Fineili, E. Menden, and H. Petering, Environ. Scl. Techno/., 8 , 740 (1972). W. Gardner and G. F. Lee, Environ Scl. Technol., 7, 719 (1973). A. Siege1 and E. T. Degens, Science, 15, 1098 (1966). 6 . Hemmasi and E. Bayer, J. Chromatogr., 100, 43 (1975). H. Loewenschussand G. Schmuckler, Talanta, 11, 1399 (1969). F. M. M. Morel, J. C. Westall, J. 0. Rueter, and J. P. Chapllck, Descrlption of the Algal Growth Media "Aqull" and "Fraqull", Technical Note No. 16, Ralph M. Parsons Laboratory, M.I.T., Cambridge, Mass., Sept. 1974. T. M. Florence and G. E. Battey, Talanta, 23, 179 (1976).
RECEIVEDfor review September 20, 1976. Accepted November 15,1976.This research was supported by the Oceanography Section, National Science Foundation, Grant DES74-21642. Portions of this paper were presented a t the 172d National Meeting, American Chemical Society, San Francisco, Calif., September 1, 1976.
