Copper Complexation Capacity of Marine Water Samples from Southern California Richard F. Srna," Kenn S. Garrett, Sandra M. Miller, and Alan B. Thum Lockheed Center for Marine Research, 6350 Yarrow Drive, Carlsbad. California 92008 The ability of a variety of natural water samples from southern California to complex cupric ion was measured by using anodic stripping voltammetry (ASV) and a technique employing algal bioassay. Samples investigated ranged from oceanic waters to a freshwater lake which drained into a coastal lagoon. According to ASV results, complexation capacity ranged from undetectable to 0.93 pM Cu2+/L. The difference between ECjo values obtained from bioassays using a 0.45-pm filtered and ultrafiltered ( G O O M,) water was used to measure the reduction in free cupric ion by high-molecular-weight compounds. Cupric ion EC50 values differed by up to 2.3 pM between filtered and ultrafiltered samples. The correlation between results using the titration method and the bioassay technique was 0.993. Quantitative differences between chemical measures of complexation and biological effect indicated the importance of verifying the accuracy of chemical techniques used to measure complexation capacity. Results of the survey suggested that coastal lagoons provide a useful test site for study of the dynamics of complexing compounds in the marine environment. Introduction Certain organic compounds present in natural environments can alter both the mobility ( 1 , Z ) and the toxicity ( 3 , 4 ) of dissolved trace metals such as copper. Phytoplankton are particularly sensitive to toxic effects of cupric ion. I t has been suggested that naturally occurring concentrations of copper may sometimes be toxic to marine phytoplankton (5) and that observed variations in copper toxicity could be due to differences in complexation capacity of natural waters (6, 7). Significant efforts have been directed toward evaluation of cupric ion toxicity to phytoplankton in the presence of complexing organic compounds. Recent work has been successful in demonstrating a correlation between reduced phytoplankton growth and cupric ion activity (3, 7, 8).Similar toxicity data are available for some higher organisms (9).Many of these studies involved use of strong artificial chelators and natural water samples with limited definition of organic content. At present, it is not possible to describe exactly the chemical structure of classes of natural compounds which are capable of strong interactions with metal ions. However, a number of biological and chemical techniques have been suggested as methods for quantifying the ability of natural water to mitigate cupric ion toxicity. Davey et al. (6) suggested the use of the algal species Thalassiosira pseudonana 13-1 as a biological probe to indicate end points of titration of samples of natural water with copper. Sunda and Lewis (8) and Bresnahan et al. ( 4 ) were able to use an ionspecific electrode for titrations with cupric ion in low-salinity water. Mantoura et al. (IO)used gel-filtration techniques to determine stability constants of complexes formed with copper. Shuman and Woodward ( I I ) , Smith ( 1 2 ) ,and Sugai and Healy ( 1 ) used anodic stripping voltammetry (ASV) to measure the complexation capacity of natural waters. However, the accuracy of anodic stripping techniques in natural waters has been questioned. Shuman and Michael (13)suggested that electrode processes a t the mercury droplet may obscure the true nature of interactions of cupric ion with natural organic ligands. In addition, Gachter et al. (14) suggested that a chemical measurement of natural complexation capacity of a water sample does not by itself guarantee that 1482
Environmental Science & Technology
an equivalent amount of copper can be tolerated without an adverse effect on phytoplankton production. The purpose of this study was to obtain information regarding the importance, the quantity, and the range of concentrations of complexing compounds present in southern California coastal environments. Because of uncertainties associated with assigning a level of environmental significance to results from chemical measures of complexation capacity, samples of water were characterized by using both chemical and biological techniques. Chemical characterization involved the determination of complexation capacity and conditional association constants of cupric ion with natural ligands by using anodic stripping voltammetry (ASV). Biological characterization utilized a new phytoplankton bioassay technique for measuring reduction in cupric ion concentration. The results from the chemical and biological determinations were tested for a correlation. The data were then used to provide insight into the importance and the possible sources of complexing compounds in a coastal marine environment. Materials and Methods Sample Collection. Samples were obtained from 10 nearshore locations between San Diego and Oceanside, CA. Sources of water are shown in Figure 1. Water was collected in 4-L glass or plastic carboys which had been washed with 30% nitric acid and 10%hydrochloric acid and then rinsed with doubly distilled water five times. Upon arrival in the laboratory, particles were removed by filtration through a 0.45-m Millipore filter. Water was stored a t 5 "C before analysis. Analysis took place within 48 h after collection of the samples. Sunda and Lewis (8) reported that no change in complexation capacity occurred when samples were stored in this manner. Dissolved Organic Carbon. The organic-carbon content of the water samples was determined by using an Oceanography International total carbon system (Model 0524B). This technique for analysis of naturally occurring dissolved organic compounds used a mixture of potassium persulfate and phosphoric acid to oxidize carbon to carbon dioxide. Standards for carbon analysis consisted of oven-dried reagentgrade potassium biphthalate dissolved in water which had been distilled from alkaline permanganate solution. Copper Content of Water Samples. A Jarrell-Ash Atomsorb atomic absorption spectrophotometer with a Varian carbon rod atomizer (Model 63) was used for determination of the natural copper content of the samples. Copper was extracted and concentrated from seawater by using APDC/ MIBK according to methods outlined by Mulford ( 1 5 ) . Salinity. Values for the salinity of water were determined by the argentometric method (16). Ultrafiltration. An Amicon ultrafiltration cell (Model 402) containing a UM05 filter was used to remove molecules with sizes equivalent to -500 M, or larger. Filters were extensively washed before use to reduce contamination of water samples by leached organics. The performance of these filters was monitored to ensure that blanks contained less than 0.5 ppm C contamination from the filter. Complexometric Titrations. Titrations of water with cupric ion were followed by using a Princeton Applied Research polarographic analyzer (Model 174A) coupled to an automated electroanalyzer controller (Model 315A). The temperature was 23 "C. The polarograph was operated in the differential pulse anodic stripping volammetry mode
0013-936X/80/0914-1482$01.OO/O
@ 1980 American Chemical Society
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A
Oceanside 28 26
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I-
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24 C
MAP KEY STATION 1
I1 111 IV V VI VI1 VI11 IX X
LOCATION Lake Val Sereno San Elijo Lagoon Batlquitor Lagoon Mission Bay San Diego Bay Oceanride Harbor Cardiff aurf Agua Hcdionda Lagoon Del Mar Burf Open ocean
bx
0
01 02
0 3 0 4 0 5 06 0 7 0 8 0 9 1 0 1 1 1 2 1 3 1 4
15 16
1 7 18
COPPER CONCENTRATION l p M I L l
Figure 2. Example of ASV titration of filtered (0)and ultrafiltered (0) water from San Elijo Lagoon. End point was determined graphically from linear regression of data before and after end point.
P a c i f i c Ocean
Figure 1. Location of sampling stations along southern California coast. Samples were obtained from nearshore lagoon and a freshwater lake at locations from San Diego to Oceanside, CA.
(DPASV). Ten milliliters of a sample to be titrated was placed in a three-electrode hanging mercury droplet cell supplied by the manufacturer. Aliquots of standard C u ( N 0 3 ) ~solution were sequentially added to the cell by using an Eppendorf micropipette. Oxygen was purged from the cell by bubbling COz through the solution. A mercury droplet was conditioned a t -1.20 V before deposition. The deposition of copper took place for 80 s a t -0.70 V. The copper concentration was determined by monitoring the current while scanning the potential between -0.70 and +0.10 V a t a rate of 5 mV/s. End points of titrations were determined by regression analysis of linear portions of the titration curves before and after the end points. Complexation capacity and stability constants were determined by using methods of Shuman and Woodward (11, 17). Complexation Capacity Using Algal Bioassay. A portion of the 0.45-pm filtered water obtained from the field was filtered again with an Amicon UM05 ultrafilter. A modified formulation of F/2 nutrients ( 1 8 )containing no NazEDTA was used to enrich water which had been filtered or ultrafiltered. The p H of all samples was then adjusted t o between 7.9 and 8.1. Fifty-milliliter aliquots of water were dosed with Cu(NO& to obtain a suitable range of concentrations. These aliquots were inoculated with algal cells by using sterile techniques -30 min after dosing. The inoculum consisted of unialgal cultures of the marine diatom Thalassiosira pseudonana 3H which was in early log growth phase. Portions of dosed, inoculated solution were placed in 10-mL test tubes to provide a t least three replicates of each concentration. Algal cells were cultured a t 19.0 OC. “Cool White” fluorescent lights provided 400 f t cd of illumination. A Turner fluorometer (Model 111) was used to monitor
growth. Every 24 h, readings were obtained for individual cultures by placing test tubes directly into the fluorometer for measurement of in vivo fluorescence. The data were used to establish the concentration of added copper which reduced the growth of algal cultures by 50% (ECjo) when compared to undosed cultures. ECjo values for filtered and ultrafiltered water were determined by probit analysis (16).The difference between these two ECjo values was used as a biological measure of the complexation of copper.
Results The data from analysis of samples from locations in southern California coastal waters are listed in Table I. The salinity ranged from 0.5 ppt for Lake Val Sereno to 32.4 ppt for coastal seawater samples. The quantity of dissolved organic carbon contained in the water samples varied considerably from less than 1ppm for water from a coastal seawater source to nearly 90 ppm for one of the lagoon samples. Natural concentrations of trace metals in water from all locations were low. All samples contained less than 2 ppb Cu2+ as determined by atomic absorption analysis. ASV scans for Cd, Pb, and Zn yielded similar low concentrations for these metals. The complexation capacity of dissolved matter was determined by titration with cupric ion. Figure 2 is an example of the titration of filtered and ultrafiltered water from San Elijo Lagoon. Water which had been filtered through a 0.45-pm filter showed a distinct break in the titration curve, whereas water which had been passed through an Amicon filter gave no evidence of a break in the titration curve. Titration data were obtained by adding microliter aliquots of cupric ion to a IO-mL sample. Approximately 20 min after each addition of titrant, copper was deposited on a mercury droplet and subsequently oxidized into solution, yielding a current peak whose value (in arbitrary units) was recorded on the abscissa of a graph. The detection limit using this technique was -5 ppb. The precision for five replicate determinations of the San Elijo Lagoon samples was &8%.Slopes and end points obtained from the titration curves were used to calculate conditional stability constants. Errors associated with this technique were discussed by Shuman and Woodward ( 17). The precision for conditional stability constants estimated from five titrations of the San Elijo sample was &l5Y,. Conditional stability constants reported in Table I are the Volume 14, Number 12, December 1980
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Table 1. Characteristics of Water Samples Obtained from Stations in Southern California Coastal Zone dlssolved organic carbon, ppm
complex capaclty DPASV, p M Cu*+/L
10’ Kassoc’
equlv/ dissolved carbon, pWmg C
(F) Lake Val Sereno
0.5
5.90
0.52
4.5
0.08
(S) San Elijo Lagoon (F) San Elijo Lagoon
2.5 23.9
15.30 14.70
0.52 0.66
3.7 3.6
0.03 0.04
(S) Batiquitos Lagoon
14.5
9.40
0.63
3.7
0.06
(F) Batiquitos Lagoon
24.1
89.75
0.93
2.3
0.01
(S) Mission Bay
29.2
2.91
0
0
(S) San Diego Bay
29.7
2.52
0
0
sallnlty, PPt
water samplea
ioxlcn b pM C U ~ ~ / L
complexn blologlcal, pM C”2+ /L
0.330 f0.006
0.20
2.14 f0.06 1.874 f0.005 3.1 f0.2 0.519 f0.005 0.63
1.45 1.45 2.27
0 0
fO.O1 (S) Oceanside Harbor
31.5
2.72
0.11
0.04
(S) Cardiff Surf (S) Aqua Hedionda Lagoon
31.8 32.0
2.10 1.10
