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Copper complexing capacity of phytoplanktonic cell exudates
Marine Chemistry, 18 (1986) 351--357 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
351
COPPER COMPLEXING CAPACITY OF PHYTOPLANKTONIC CELL EXUDATES* A L F R E D O SERITTI, DAVID PELLEGRINI, ELISABETTA MORELLI, CORRADO BARGHIGIANI and ROMANO F E R R A R A C.N.R. Istituto di Biofisica, Via S. Lorenzo, 26 -- 5 6 1 0 0 Pisa (Italy)
ABSTRACT Seritti, A., Pellegrini, D., Morelli, E., Barghigiani, C. and Ferrara, R., 1986. Copper complexing capacity o f Phytoplanktonic cell exu dates. Mar. Chem., 18: 351--357. Copper complexing capacity of cell exudates of Dunaliella salina in natural seawater culture medium was investigated in order to evaluate the influence of this organism on speciation of trace metals in seawater. Seawater samples were collected at 200 m and 2 miles off the coast and immediately filtered. Copper complexing capacity (CCGu) and stability constants (K') of related cupric complexes were then measured. They were, respectively, 27.1 × 10 - s tool 1-1 and 0.56 × 1071 mo1-1 for the samples collected at 2 0 0 m and 12.8 × 1 0 - S t o o l 1-1 and 6.10 x 1071 m o l - 1 for those collected 2 miles off the coast. A stock culture (20 ml, 106 cells ml- 1 ) in log-phase was inoculated in 21 of each sample of filtered natural seawater. The trend of cell influence was estimated on filtered culture medium by measuring CCcu and K ' after i h, 3 and 7 days. From the results it appears that CCcu increased with respect to time and this was related to the growth rate, indicating a certain relationship with cell metabolic activity. It can be concluded that a comparison between the culture referring to 200 m and 2 miles, respectively, shows that the former presents a CCcu two times higher than the latter while the K' is ten times higher at 2 miles than that at 200 m.
INTRODUCTION
The influence of organisms on speciation of trace metals in the aquatic environment is well known and is also the object of several studies (Davey et al., 1973; Sunda and Hanson, 1979; Gachter et al., 1978). Nevertheless, data on amounts and quality of natural organic ligands are scarce and difficult to gather. In marine life, phytoplankton represents the first link of the food chains and because of the large a m o u n t of these organisms in the oceans, the products of their metabolism play a role of primary importance in speciation problems. On the other hand the physico-chemical form of metals is of great interest regarding the toxicity of these elements to aquatic organisms. In fact, the response of algae to trace metals seems to be related to the free ion activity of the elements (Sunda and Guillard, 1976). Among the ligands, the most diffuse and important seem to be polysaccharides, * Presented at the VIII International Symposium on the Chemistry of the Mediterranean, Primosten, Yugoslavia, May 1984.
0304-4203/86/$03.50
© 1986 Elsevier Science Publishers B.V.
352 uronates, polyphenols, amino acids, polypeptides and proteins (Langston and Bryan, 1984). These ligands can represent a protective mechanism to the organisms as regards toxic effects of the metals (McKnight and Morel, 1980). Florence (1982) in a recent study has pointed out that copper, lead, cadmium and zinc in natural waters are largely b o u n d with highly stable complexes by unidentified organic ligands. Van den Berg et ai. (1979) have studied the influence of p h y t o p l a n k t o n media on the copper complexing capacity of lake water by anodic stripping voltammetry. Gnassia-Barelli et al. (1984) have estimated the copper and cadmium speciation in different p h y t o p l a n k t o n culture media by ultrafiltration techniques and atomic absorption spectrometry. In this paper the influence of the planktonic alga Dunaliella salina on the speciation of copper in seawater is reported by measuring the copper complexing capacity evaluated by DPASV. METHODS
Cultures Dunaliella salina was cultured in filtered natural seawater collected 200 m and 2 miles off the Tyrrhenian coast at the latitude of the city of Tirrenia (Tuscany, Italy) which, being in proximity to the Arno estuary ( 1 0 k m south) presents nearshore (200 m) a strong amount of organic pollutants. Two miles away from the coast the pollution decreases and this is more pronounced to the south of the estuary since the sea currents in this area point to the north. The batch cultures were prepared by inoculum of 20 ml of a 106 cells ml- 1 of a stock culture in log-phase in 21 of natural seawater medium. The stirred cultures were maintained in a thermostatic cell at 22°C under continuous light at 2500 + 200 lux (Philips fluorescent tubes mod. Th. 30 W/33 RS). Preparation of samples for D P A S V measurements After I h, 3 and 7 days from the inoculum, 500 ml of the cell suspension were filtered on 0.45-pm Sartorius membrane filters. Aliquots (30ml) of the filtrate were distributed in ten vessels of 50 ml each, to which increasing amounts of ionic copper were added. Another vessel was, in addition, acidified at pH 2 with hydrochloric acid. The solutions were prepared at least 12 h before the measurements in order to attain equilibrium. In order to avoid contamination all the vessels were pre-conditioned with HC1 (Merck Suprapur) at decreasing concentrations and finally with clean seawater, as reported by Mart (1979).
353
D P A S V measurements The DPASV measurements were performed with a PAR 174 A polarographic analyser connected to a Linseis 1800 XY recorder. The voltammetric cell was made from PTFE with a useful volume of ca. 50 ml. The electrode system consisted of a rotating thin mercury film electrode as working electrode, an Ag/AgC1 reference electrode, and a platinum wire as counter electrode. The formation of the appropriate mercury film on the glassy carbon surface, all the instrumental settings, and the analytical procedure were performed as reported by Plavsic et al. (1982). Calculation The titration equation for a 1 : 1 complex can easily be obtained as follows: x--y
K=
Y [Lt -- (x - - y ) ]
where x is the total concentration of the metal (i.e., the metal already present in the original sample plus the metal added), y the concentration of the free form and Lt the total a m o u n t of ligand. Solving with respect to y gives: Ky 2 +
[K(L t -- x) +
1] y -- x = 0
and finally KX
y
=
p(x)
--
KL t -- 1 + [(KL t + 1 -- Kx) 2 --
=
4Kx]
1/2
2K
For each experiment a set of N pairs of values (x~, yi) are obtained and, assuming a small error in x, it is possible to determine the values of Ki and Li as the best fit to the experimental data using the m e t h o d of least squares. This operation has been done by a computer program and a more detailed account is the object of a paper in preparation (Barbi et al., 1986). RESULTS AND DISCUSSION
In Fig. 1 the curves of complexing capacity (CCcu) corresponding to different control times for the culture referring to seawater collected 200 m off the coast are compared. The trend of CCcu with respect to time is in full agreement with the growth rate of the cells. In fact, as can be seen from the growth curve reported in Fig. 2, the log phase of the cells is between 3 and 7 days. In Fig. 3 the same comparison is made for the culture referring to 2 miles. Also in this case an increase of CCc~ with time can be observed. Furthermore, the decrease of the ionic copper at pH 2 in both cultures has
354
4 x I0-7
~
o
J/
2x,o-7 /
10-7
2 xlO-7 5xlO-7 4x 10-7 Cuodded(mol/I)
Fig. 1. Culture A (200 m). Curves representing the titration for the determination of CCcu. Comparison among the curves corresponding to different control times: t 1 (o, o), t2 (m, •) and t3 (A, ~). Closed symbols, pH 2; open symbols, natural pH.
106 '
, / ~ ~
~'~ 105 0
, jz • Z 104
Lo3
Days Fig. 2. Growth curve o f Dunaliella salina cultured in natural seawater. ( u ) ( A - - - A) 2 miles.
200 m ;
to be related to the u p t a k e o f metal by the cells as well as to adsorption o n the cell wall. However, metal adsorption o n t h e vessel walls c a n n o t be excluded. In Table I the values o f CCcu and t h e calculated stability constants, K', are summarized for b o t h cultures. The first observation w h i c h can be made is t h a t CCcu as well as K' for b o t h c.ultures, at the beginning and 1 h after the i n o c u l u m , remain practically u n c h a n g e d . This seems to s h o w t h a t at the
355 el-x 10-7
5xi0-7 fi 2 x 10-7
5 I0 -7
I
10- 7
2x10-7
5 x l O -7
I
4 x I0 -7
Cu added (moll1)
Fig. 3. Culture B (2 miles). Curves representing the titration for the d e t e r m i n a t i o n o f CCcu. Comparison a m o n g the curves corresponding to different control times: tl (o, ©), t2 (m, o) and t3 (4, ~). Closed symbols, pH 2 ; o p e n symbols, natural pH. TABLE I CCcu and K ' versus t i m e o f Dunaliella salina exudates. Culture A and B are referred to natural seawater m e d i u m collected 200 m and 2 miles off the coast, respectively, to = natural sea water before the i n o c u l u m ; t l , t2 and t 3 are referred to 1 h, 3 and 7 days after the inoculum, respectively.
CCcu (tooll-i)x 10 -8
K' (I mol-1) X 10 ~
Culture A to tl t2 t3
27.1 27.0 28.4 33.0
0.56 0.58 0.69 0.75
Culture B to tl t2 t3
12.8 13.0 16.0 21.0
6.10 6.39 7.00 7.00
beginning of the cultures, the presence of cells does not affect the value of CCcu. Furthermore, although the initial values of CCcu at 200 m are two times higher than that at 2 miles, the K' values are about ten times higher for the culture referring to 2 miles than t h a t for 200 m. This seems to suggest that independent of the amount, the ligands are stronger off-shore than in coastal waters. This trend is confirmed by several measurements made in other areas (Seritti et al., 1985).
356 The CCcu values increase with time for both cultures although the variations are not so pronounced. K ' , on the contrary, remains practically the same. This has also been observed by other authors in experiments done with synthetic ligands, such as EDTA, added to natural seawater (Plavsic et al., 1982). Furthermore the presence of surfactants produced by the cells could affect the response of the DPASV technique, in particular the K' values (Brezonic et al., 1976; Krznaric, 1984). On the other hand our results are comparable to those reported by Stolzberg and Rosin (1977) for the marine diatom Skeletonema and those by Swallow et al. (1978) for the freshwater alga Gloeocystis gigas. CONCLUSIONS
From these preliminary results the presence of cells in a seawater medium seems to have more effect on the value of CCcu than on that of K'. This seems to suggest that the contribution of p h y t o p l a n k t o n to dissolved organic matter (DOM) in the sea is not negligible and that their exudates form electrochemically non-labile copper complexes with a high affinity. In this direction, studies should be intensified to concentrate, isolate, and characterize these organic ligands in order to make decisive contributions to the knowledge of the speciation of trace metals, which is a crucial problem of inorganic and organic marine chemistry. REFERENCES Barbi, M., Morelli, E., Orsini, F. and Seritti, A., 1986. Voltammetric measurements of complexation capacity of seawater. Data analysis. Environ. Technol. Lett., submitted. Brezonic, P.L., Brauner, P.A. and Stumm, W., 1976. Trace metal analysis by anodic stripping voltammetry: effect of sorption by natural and model organic compounds. Water Res., 10: 605--612. Davey, E.M., Morgan, M.J. and Erickson, S. J., 1979. A biological measurement of copper complexation capacity of seawater. Limnol. Oceanogr., 18: 993--997. Florence, T.M., 1982. Development of physico-chemical speciation procedures to investigate the toxicity of copper, lead, cadmium and zinc towards aquatic biota. Anal. Chim. Acta, 141: 73--94. Gachter, R., Davis, J.S. and Mares, A., 1978. Regulation of copper availability to phytoplankton by macromolecules in lake water. Environ. Sci. Technol., 12: 1416--1422. Gnassia-Barelli, M., Romeo, M.H. and Nicolas, E., 1984. Copper and cadmium speciation in different phytoplankton culture media. In: C.J.M. Kramer and J.C. Duinker (Editors), Complexation of Trace Metals in Natural Waters. Martinus Nijhoff/Junk Publishers, The Hague, pp. 425--428. Krznaric, D., 1984. The influence of surfactants on the measurements of copper and cadmium speciation in model seawater by differential pulse anodic stripping voltammerry. Mar. Chem., 15: 117--132. Langston, W.J. and Bryan, G.W., 1984. T h e relationship between metal speciation in the environment and bioaccumulation in aquatic organism. In: C.J.M. Kramer and J.C. Duinker (Editors), Complexation of Trace Metals in Natural Waters. Martinus Nijhoff/ Junk Publishers, The Hague, pp. 375--392. Mart, L., 1979. Prevention of contamination and other accuracy risk in voltammetric trace metal analysis of natural waters. Fresenius Z. Anal. Chem., 296: 350--357.-
357 McKnight, D.M. and Morel, F.M.M., 1980. Copper complexation by siderophores from filamentous blue-green algae. Limnol. Oceanogr., 25: 62--71. Plavsic, M., Krznaric, D. and Branica, M., 1982. Determination of the apparent copper complexing capacity of seawater by anodic stripping voltammetry. Mar. Chem., 11: 17--31. Seritti, A., Pellegrini, D., Barghigiani, C., Ferrara, R., Morelli, E. and Orsini, F., 1985. Influence of phytoplankton on complexing capacity of seawater. Toxicol. Environ. Chem., in press. Stolzberg, R.J. and Rosin, D., 1977. Chromatographic measurement of submicromolar strong complexing capacity in phytoplankton media. Anal. Chem., 4~9: 226--230. Sunda, W. and Guillard, R.R.L., 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res., 34: 511--529. Sunda, W.G. and Hanson, P.J., 1979, Chemical speciation of copper in river water: effect of total copper, pH, carbonate and dissolved organic matter. In: E.A. Jenne (Editor), Chemical Modelling in Aqueous Systems. American Chemical Society, Syrup. Ser. 93, Washington, DC, 941 pp. Swallow, K.C., Westall, J.C., McKnight, D.M., Morel, N.M.L. and Morel, F.M.M., 1978. Potentiometric determination of copper complexation by phytoplankton exudates. Limnol. Oceanogr., 23: 538--542. Van den Berg, C.M.G., Wong, P.T.S. and Chau, Y.K., 1979. Measurement of complexing materials excreted from algae and their ability to ameliorate copper toxicity. J. Fish. Res. Board Can., 36: 901--905.
351
COPPER COMPLEXING CAPACITY OF PHYTOPLANKTONIC CELL EXUDATES* A L F R E D O SERITTI, DAVID PELLEGRINI, ELISABETTA MORELLI, CORRADO BARGHIGIANI and ROMANO F E R R A R A C.N.R. Istituto di Biofisica, Via S. Lorenzo, 26 -- 5 6 1 0 0 Pisa (Italy)
ABSTRACT Seritti, A., Pellegrini, D., Morelli, E., Barghigiani, C. and Ferrara, R., 1986. Copper complexing capacity o f Phytoplanktonic cell exu dates. Mar. Chem., 18: 351--357. Copper complexing capacity of cell exudates of Dunaliella salina in natural seawater culture medium was investigated in order to evaluate the influence of this organism on speciation of trace metals in seawater. Seawater samples were collected at 200 m and 2 miles off the coast and immediately filtered. Copper complexing capacity (CCGu) and stability constants (K') of related cupric complexes were then measured. They were, respectively, 27.1 × 10 - s tool 1-1 and 0.56 × 1071 mo1-1 for the samples collected at 2 0 0 m and 12.8 × 1 0 - S t o o l 1-1 and 6.10 x 1071 m o l - 1 for those collected 2 miles off the coast. A stock culture (20 ml, 106 cells ml- 1 ) in log-phase was inoculated in 21 of each sample of filtered natural seawater. The trend of cell influence was estimated on filtered culture medium by measuring CCcu and K ' after i h, 3 and 7 days. From the results it appears that CCcu increased with respect to time and this was related to the growth rate, indicating a certain relationship with cell metabolic activity. It can be concluded that a comparison between the culture referring to 200 m and 2 miles, respectively, shows that the former presents a CCcu two times higher than the latter while the K' is ten times higher at 2 miles than that at 200 m.
INTRODUCTION
The influence of organisms on speciation of trace metals in the aquatic environment is well known and is also the object of several studies (Davey et al., 1973; Sunda and Hanson, 1979; Gachter et al., 1978). Nevertheless, data on amounts and quality of natural organic ligands are scarce and difficult to gather. In marine life, phytoplankton represents the first link of the food chains and because of the large a m o u n t of these organisms in the oceans, the products of their metabolism play a role of primary importance in speciation problems. On the other hand the physico-chemical form of metals is of great interest regarding the toxicity of these elements to aquatic organisms. In fact, the response of algae to trace metals seems to be related to the free ion activity of the elements (Sunda and Guillard, 1976). Among the ligands, the most diffuse and important seem to be polysaccharides, * Presented at the VIII International Symposium on the Chemistry of the Mediterranean, Primosten, Yugoslavia, May 1984.
0304-4203/86/$03.50
© 1986 Elsevier Science Publishers B.V.
352 uronates, polyphenols, amino acids, polypeptides and proteins (Langston and Bryan, 1984). These ligands can represent a protective mechanism to the organisms as regards toxic effects of the metals (McKnight and Morel, 1980). Florence (1982) in a recent study has pointed out that copper, lead, cadmium and zinc in natural waters are largely b o u n d with highly stable complexes by unidentified organic ligands. Van den Berg et ai. (1979) have studied the influence of p h y t o p l a n k t o n media on the copper complexing capacity of lake water by anodic stripping voltammetry. Gnassia-Barelli et al. (1984) have estimated the copper and cadmium speciation in different p h y t o p l a n k t o n culture media by ultrafiltration techniques and atomic absorption spectrometry. In this paper the influence of the planktonic alga Dunaliella salina on the speciation of copper in seawater is reported by measuring the copper complexing capacity evaluated by DPASV. METHODS
Cultures Dunaliella salina was cultured in filtered natural seawater collected 200 m and 2 miles off the Tyrrhenian coast at the latitude of the city of Tirrenia (Tuscany, Italy) which, being in proximity to the Arno estuary ( 1 0 k m south) presents nearshore (200 m) a strong amount of organic pollutants. Two miles away from the coast the pollution decreases and this is more pronounced to the south of the estuary since the sea currents in this area point to the north. The batch cultures were prepared by inoculum of 20 ml of a 106 cells ml- 1 of a stock culture in log-phase in 21 of natural seawater medium. The stirred cultures were maintained in a thermostatic cell at 22°C under continuous light at 2500 + 200 lux (Philips fluorescent tubes mod. Th. 30 W/33 RS). Preparation of samples for D P A S V measurements After I h, 3 and 7 days from the inoculum, 500 ml of the cell suspension were filtered on 0.45-pm Sartorius membrane filters. Aliquots (30ml) of the filtrate were distributed in ten vessels of 50 ml each, to which increasing amounts of ionic copper were added. Another vessel was, in addition, acidified at pH 2 with hydrochloric acid. The solutions were prepared at least 12 h before the measurements in order to attain equilibrium. In order to avoid contamination all the vessels were pre-conditioned with HC1 (Merck Suprapur) at decreasing concentrations and finally with clean seawater, as reported by Mart (1979).
353
D P A S V measurements The DPASV measurements were performed with a PAR 174 A polarographic analyser connected to a Linseis 1800 XY recorder. The voltammetric cell was made from PTFE with a useful volume of ca. 50 ml. The electrode system consisted of a rotating thin mercury film electrode as working electrode, an Ag/AgC1 reference electrode, and a platinum wire as counter electrode. The formation of the appropriate mercury film on the glassy carbon surface, all the instrumental settings, and the analytical procedure were performed as reported by Plavsic et al. (1982). Calculation The titration equation for a 1 : 1 complex can easily be obtained as follows: x--y
K=
Y [Lt -- (x - - y ) ]
where x is the total concentration of the metal (i.e., the metal already present in the original sample plus the metal added), y the concentration of the free form and Lt the total a m o u n t of ligand. Solving with respect to y gives: Ky 2 +
[K(L t -- x) +
1] y -- x = 0
and finally KX
y
=
p(x)
--
KL t -- 1 + [(KL t + 1 -- Kx) 2 --
=
4Kx]
1/2
2K
For each experiment a set of N pairs of values (x~, yi) are obtained and, assuming a small error in x, it is possible to determine the values of Ki and Li as the best fit to the experimental data using the m e t h o d of least squares. This operation has been done by a computer program and a more detailed account is the object of a paper in preparation (Barbi et al., 1986). RESULTS AND DISCUSSION
In Fig. 1 the curves of complexing capacity (CCcu) corresponding to different control times for the culture referring to seawater collected 200 m off the coast are compared. The trend of CCcu with respect to time is in full agreement with the growth rate of the cells. In fact, as can be seen from the growth curve reported in Fig. 2, the log phase of the cells is between 3 and 7 days. In Fig. 3 the same comparison is made for the culture referring to 2 miles. Also in this case an increase of CCc~ with time can be observed. Furthermore, the decrease of the ionic copper at pH 2 in both cultures has
354
4 x I0-7
~
o
J/
2x,o-7 /
10-7
2 xlO-7 5xlO-7 4x 10-7 Cuodded(mol/I)
Fig. 1. Culture A (200 m). Curves representing the titration for the determination of CCcu. Comparison among the curves corresponding to different control times: t 1 (o, o), t2 (m, •) and t3 (A, ~). Closed symbols, pH 2; open symbols, natural pH.
106 '
, / ~ ~
~'~ 105 0
, jz • Z 104
Lo3
Days Fig. 2. Growth curve o f Dunaliella salina cultured in natural seawater. ( u ) ( A - - - A) 2 miles.
200 m ;
to be related to the u p t a k e o f metal by the cells as well as to adsorption o n the cell wall. However, metal adsorption o n t h e vessel walls c a n n o t be excluded. In Table I the values o f CCcu and t h e calculated stability constants, K', are summarized for b o t h cultures. The first observation w h i c h can be made is t h a t CCcu as well as K' for b o t h c.ultures, at the beginning and 1 h after the i n o c u l u m , remain practically u n c h a n g e d . This seems to s h o w t h a t at the
355 el-x 10-7
5xi0-7 fi 2 x 10-7
5 I0 -7
I
10- 7
2x10-7
5 x l O -7
I
4 x I0 -7
Cu added (moll1)
Fig. 3. Culture B (2 miles). Curves representing the titration for the d e t e r m i n a t i o n o f CCcu. Comparison a m o n g the curves corresponding to different control times: tl (o, ©), t2 (m, o) and t3 (4, ~). Closed symbols, pH 2 ; o p e n symbols, natural pH. TABLE I CCcu and K ' versus t i m e o f Dunaliella salina exudates. Culture A and B are referred to natural seawater m e d i u m collected 200 m and 2 miles off the coast, respectively, to = natural sea water before the i n o c u l u m ; t l , t2 and t 3 are referred to 1 h, 3 and 7 days after the inoculum, respectively.
CCcu (tooll-i)x 10 -8
K' (I mol-1) X 10 ~
Culture A to tl t2 t3
27.1 27.0 28.4 33.0
0.56 0.58 0.69 0.75
Culture B to tl t2 t3
12.8 13.0 16.0 21.0
6.10 6.39 7.00 7.00
beginning of the cultures, the presence of cells does not affect the value of CCcu. Furthermore, although the initial values of CCcu at 200 m are two times higher than that at 2 miles, the K' values are about ten times higher for the culture referring to 2 miles than t h a t for 200 m. This seems to suggest that independent of the amount, the ligands are stronger off-shore than in coastal waters. This trend is confirmed by several measurements made in other areas (Seritti et al., 1985).
356 The CCcu values increase with time for both cultures although the variations are not so pronounced. K ' , on the contrary, remains practically the same. This has also been observed by other authors in experiments done with synthetic ligands, such as EDTA, added to natural seawater (Plavsic et al., 1982). Furthermore the presence of surfactants produced by the cells could affect the response of the DPASV technique, in particular the K' values (Brezonic et al., 1976; Krznaric, 1984). On the other hand our results are comparable to those reported by Stolzberg and Rosin (1977) for the marine diatom Skeletonema and those by Swallow et al. (1978) for the freshwater alga Gloeocystis gigas. CONCLUSIONS
From these preliminary results the presence of cells in a seawater medium seems to have more effect on the value of CCcu than on that of K'. This seems to suggest that the contribution of p h y t o p l a n k t o n to dissolved organic matter (DOM) in the sea is not negligible and that their exudates form electrochemically non-labile copper complexes with a high affinity. In this direction, studies should be intensified to concentrate, isolate, and characterize these organic ligands in order to make decisive contributions to the knowledge of the speciation of trace metals, which is a crucial problem of inorganic and organic marine chemistry. REFERENCES Barbi, M., Morelli, E., Orsini, F. and Seritti, A., 1986. Voltammetric measurements of complexation capacity of seawater. Data analysis. Environ. Technol. Lett., submitted. Brezonic, P.L., Brauner, P.A. and Stumm, W., 1976. Trace metal analysis by anodic stripping voltammetry: effect of sorption by natural and model organic compounds. Water Res., 10: 605--612. Davey, E.M., Morgan, M.J. and Erickson, S. J., 1979. A biological measurement of copper complexation capacity of seawater. Limnol. Oceanogr., 18: 993--997. Florence, T.M., 1982. Development of physico-chemical speciation procedures to investigate the toxicity of copper, lead, cadmium and zinc towards aquatic biota. Anal. Chim. Acta, 141: 73--94. Gachter, R., Davis, J.S. and Mares, A., 1978. Regulation of copper availability to phytoplankton by macromolecules in lake water. Environ. Sci. Technol., 12: 1416--1422. Gnassia-Barelli, M., Romeo, M.H. and Nicolas, E., 1984. Copper and cadmium speciation in different phytoplankton culture media. In: C.J.M. Kramer and J.C. Duinker (Editors), Complexation of Trace Metals in Natural Waters. Martinus Nijhoff/Junk Publishers, The Hague, pp. 425--428. Krznaric, D., 1984. The influence of surfactants on the measurements of copper and cadmium speciation in model seawater by differential pulse anodic stripping voltammerry. Mar. Chem., 15: 117--132. Langston, W.J. and Bryan, G.W., 1984. T h e relationship between metal speciation in the environment and bioaccumulation in aquatic organism. In: C.J.M. Kramer and J.C. Duinker (Editors), Complexation of Trace Metals in Natural Waters. Martinus Nijhoff/ Junk Publishers, The Hague, pp. 375--392. Mart, L., 1979. Prevention of contamination and other accuracy risk in voltammetric trace metal analysis of natural waters. Fresenius Z. Anal. Chem., 296: 350--357.-
357 McKnight, D.M. and Morel, F.M.M., 1980. Copper complexation by siderophores from filamentous blue-green algae. Limnol. Oceanogr., 25: 62--71. Plavsic, M., Krznaric, D. and Branica, M., 1982. Determination of the apparent copper complexing capacity of seawater by anodic stripping voltammetry. Mar. Chem., 11: 17--31. Seritti, A., Pellegrini, D., Barghigiani, C., Ferrara, R., Morelli, E. and Orsini, F., 1985. Influence of phytoplankton on complexing capacity of seawater. Toxicol. Environ. Chem., in press. Stolzberg, R.J. and Rosin, D., 1977. Chromatographic measurement of submicromolar strong complexing capacity in phytoplankton media. Anal. Chem., 4~9: 226--230. Sunda, W. and Guillard, R.R.L., 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res., 34: 511--529. Sunda, W.G. and Hanson, P.J., 1979, Chemical speciation of copper in river water: effect of total copper, pH, carbonate and dissolved organic matter. In: E.A. Jenne (Editor), Chemical Modelling in Aqueous Systems. American Chemical Society, Syrup. Ser. 93, Washington, DC, 941 pp. Swallow, K.C., Westall, J.C., McKnight, D.M., Morel, N.M.L. and Morel, F.M.M., 1978. Potentiometric determination of copper complexation by phytoplankton exudates. Limnol. Oceanogr., 23: 538--542. Van den Berg, C.M.G., Wong, P.T.S. and Chau, Y.K., 1979. Measurement of complexing materials excreted from algae and their ability to ameliorate copper toxicity. J. Fish. Res. Board Can., 36: 901--905.