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A link between lead and cadmium kinetic speciation in seawater and accumulation by the green alga Ulva lactuca
Environmental Pollution 141 (2006) 126e130 www.elsevier.com/locate/envpol
A link between lead and cadmium kinetic speciation in seawater and accumulation by the green alga Ulva lactuca J.O. Muse a,*, C.N. Carducci a, J.D. Stripeikis b, M.B. Tudino b, F.M. Ferna´ndez b,1 a
Departamento de Quı´mica Analı´tica y Fisicoquı´mica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Qulmica Analitica, 3er. Piso, Junin 956-1113, Buenos Aires, Argentina b Departamento de Quı´mica Inorga´nica, Analı´tica y Quı´mica Fı´sica/INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, 3er. Piso, Pabello´n 2, Ciudad Universitavia, Buenos Aires, Argentina Received 17 September 2005; accepted 2 August 2005
Metal kinetic speciation can overcome CF limitations for the evaluation of marine pollution. Abstract In this work, studies on the bioaccumulation of Cd and Pb by Ulva lactuca at different sites of Gulf San Jorge (Patagonia, Argentina) are presented. Higher values of bioaccumulated Cd were found in Punta Maqueda e a site believed to serve as a control e in comparison to those in Punta Borja, a place highly exposed to urban and industrial activities. Consequently; the labile fractions of Cd and Pb in seawater were determined with a flow injection-preconcentration manifold interfaced to a graphite furnace-atomic absorption spectrometer (FI-GFAAS). The results obtained by kinetic speciation showed that the variable that correctly explains heavy metals accumulation in the alga, is the labile metal fraction in seawater. We propose to use an enhancement ratio e on the basis of the kinetically labile metal fraction e for calculation of the metal accumulated by the alga relative to its environment. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cd and Pb; Seawater; Kinetic speciation; Ulva lactuca; Concentration factor
1. Introduction Benthic marine algae are useful in marine pollution assessment and biomonitoring studies (Phillips, 1977, 1990; Volterra and Conti, 2000) since they are able to accumulate metals in concentrations several thousand times higher than those in the surrounding seawater (Foster, 1976; Bryan and Langston, 1982). The Concentration Factor (CF) (Foster, 1976) is defined as the ratio between the content of a given metal in an organism and its concentration in any compartment of the environment such as the total dissolved metal fraction in seawater (Morel, * Corresponding author. Tel.: C54 114 964 8262; fax: C54 114 964 8263. E-mail address: [email protected] (J.O. Muse). 1 Present address. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA. 0269-7491/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2005.08.021
1983) or the acid extractable fraction in sediment (Luoma and Bryan, 1978). CF is often used to estimate evolution of contamination over time (Melhuus et al., 1978) and, as proposed by Conti and co-workers (Conti et al., 2002), it is also useful to compare pollution levels among different sites. However, inter-site comparison of metal pollution through CF values is affected by sampling design, by temporal and spatial variability, and also by biological variables inherent to the organism under study (Langston and Spence, 1995). Consequently, studies on bioaccumulation in each place show that the link between CF and the actual water pollution is poor if sites under different geochemical, hydrodynamic and environmental conditions need to be compared (Moriarty, 1999). According to the Free Ion Activity Model (FIAM) proposed by Morel (1983), the interaction between metals and living organisms should be explained through the chemical reaction of free and weakly complexed ionic species with biological
J.O. Muse et al. / Environmental Pollution 141 (2006) 126e130
membranes (Morel, 1983; Xue et al., 1988). For example, the metal uptake in algae (Sunda and Guillard, 1976) under controlled, static conditions has been shown to be proportional to the concentration of free ionic and weakly-bound metal species, and not to the total metal concentrations. FIAM assumes that the transport of the metal in the solution bulk towards the cell membrane is fast in comparison to the actual biological uptake rate, and thus, the activities of all the metal species in the bulk medium and at the cell surface are approximately the same. A second assumption is that the cell surface is in ‘‘pre-equilibrium’’ with the metal species in solution and that the uptake rate is only limited by the membrane transport processes. In this case, the complexation of metal ions with ligands that form highly stable complexes decreases the bioavailability to metals (Tessier et al., 1994). However, if the metal ions are present as kinetically inert complexes, the surface ligand exchange reactions are conditioned by the rate of release of the metal from the complex. In this case, chemical kinetic considerations must be accounted for and the concept of dynamic speciation needs to be introduced. Dynamic speciation takes into account both the equilibrium distribution of the different species and the kinetics of their interconversion (Templeton et al., 2000). The development of an analytical method capable of performing kinetic speciation analysis involves to mimic the surface complexation reactions taking place at the cell membranes with a laboratory-based setup. Competitive ligand displacement assays between the metal complex under investigation and a chelating resin (Figura and McDuffie, 1980; Chakrabarti et al., 1994) are useful to perform kinetic speciation analysis. Figura and McDuffie (1980) reported that iminodiacetate-based resins such as Chelex-100 can modify their metal retention ability by operating under different pH and sample flow rate conditions. In fact, under suitable conditions, the resin is able to selectively retain the most reactive fraction of metal species which is directly related to toxicity effects on aquatic biota (Florence et al., 1983; Buckley et al., 1985). In a previous work, we have shown the potential of iminodiacetate-based resins to selectively retain the bioavailable fraction of the total dissolved metal content in seawater (Ferna´ndez et al., 1997, 2000). The aim of this study is to establish a relationship between the kinetically-labile fraction of Cd and Pb in coastal S. Atlantic seawater and the accumulation of both analytes in Ulva lactuca growing in the area of Gulf San Jorge (Patagonia, Argentina). Obtained results will be employed to show that CF can achieve a better environmental significance if the sum of the concentrations of labile metal species in seawater instead of the total metal contents is taken into account. 2. Materials and methods 2.1. Sampling design Four sites undergoing similar oceanographic conditions in a restricted area of the Gulf San Jorge were chosen to compare the distribution of Cd and Pb in Ulva lactuca exposed to pollutants derived from the petroleum extraction
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industry. The sampling sites are: Caleta Cordova (45 43# S 67 20# W), Punta Borja (45 52# S 67 29# W), Rada Tilly Beach (46 00# S 67 33# W) and Punta Maqueda (46 01# S 67 34# W) chosen as the control site. Caleta Cordova is a sandy beach with pebbles and stones with an underdeveloped harbor but with important petroleum extraction activity. Punta Borja is sited in an open area, plenty of rocks and tide pools with dark spots spread over the beach, highly exposed to industrial, urban and harbor activities and absence of waste-treatment facilities. Rada Tilly is a fine sandy beach with mild contamination derived from a small touristic village. Punta Maqueda is a naturally protected sandy beach, surrounded by shoals and far away from any source of contamination. The evaluation of the degree of exposure to different sources of contamination is qualitative and has taken into account the number of inhabitants, the types of activities and the presence of waste-treatment facilities. A description of the geology and oceanographic conditions along the S. Atlantic coasts has been given by Commendatore et al. (1996). The materials under study were collected following the records of geographical distribution of seaweeds along the Patagonian coasts reported by Ku¨hneman (1972).
2.2. Sampling procedure and sample conditioning Composite and pooled samples of Ulva lactuca plants were collected at low tide at each of the aforementioned sites during August 2001 according to the current methodology (Keith, 1991). The material (500e1000 g, fresh weight) was gathered from the intertidal area, rinsed to remove solid debris and associated organisms, washed with few portions of deionized water to avoid leaching of metals, and finally stored in plastic bags under freezing. Dried samples were ground to pass a 212-mm sieve and stored in a dessicator prior to analysis. Surface seawater samples were collected at low tide and at the same time and place of algal collection. The mean Cd and Pb concentrations were used for calculation of CF values. A second set of seawater samples was also collected under the calm weather conditions of the autumn-winter period (between March and August 2001). In all occasions natural perturbing phenomena such as solid resuspension (Whitfield and Turner, 1987) and phytoplankton blooms (Wallace, 1982) were avoided. An estimation of the concentration means of total dissolved and labile Cd and Pb in the aquatic environment prior to algal sampling was obtained. Seawater samples conditioning for total trace metal analysis was performed by the addition of nitric acid (SuprapurÒ grade, Merck, Darmstadt, Germany) until the sample pH was between 4 and 5. Samples were then filtered through acid cleaned 0.22-mm membrane filters, stored in pre-cleaned 1000-ml polypropylene bottles and kept frozen at ÿ20 C. Samples for speciation studies were kept at their natural pH, filtered and frozen in PFTEÒ bottles (Keith, 1991).
2.3. Reagents All reagents were of analytical grade or better. Standards were prepared on a laminar flow clean bench. Doubly deionized water (DIW, 18 MU cm) was obtained from a MilliQ water system (Millipore, Beldford, MA, USA). Chelex-100 chelating resin (100e200 mesh, Bio-Rad, Richmond, CA, USA) was used for the retention of the labile Cd and Pb fractions following the procedure described in our previous works (Ferna´ndez et al., 1997, 2000). Seawater samples were preconditioned using a 0.1 M acetic acid-ammonium acetate buffer which was purified through an auxiliary Chelex-100 column before its use, following the procedure described below. Working standard solutions of cadmium (50e200 ng lÿ1) and lead (50e 300 ng lÿ1) were prepared by appropriate dilution of stock standard solutions containing 1000 g lÿ1 Cd and 1000 g lÿ1 Pb (Merck), respectively. Dilutions were performed in Nalgene (Nalge, Rochester, NY, USA) volumetric flasks. All glassware was thoroughly cleaned with nitric acid (1 C 1) rinsed with DIW, further cleaned with hot aqueous 0.05% APDC solution and rinsed again. Once in use, only DIW rinsing of the flasks was performed. CASS-3 Seawater Reference Material from the National Research Council of Canada (NRCC) and Sea lettuce (Ulva lactuca) Reference Material (BCR 279) from the Community Bureau of Reference were used for analytical validation purposes.
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2.4. Analytical determinations in seawater
3. Results and discussion
Temperature, pH and salinity were determined in situ with an Orion model 250A pHmeter (Boston, MA, USA) and a Hach model sensIon 5 conductivity/ salinity meter (Loveland, CO, USA), respectively. Soluble reactive phosphorus (SRP molybdate-ascorbic) was determined following the method described by Strickland and Parsons (1972). Total dissolved Cd and Pb concentrations were determined according to Sturgeon et al. (1979). An on-line solid-phase chelation method already reported (Ferna´ndez et al., 1997) was employed for the determination of kinetically labile fractions of Cd and Pb in seawater. On-line conditioned water samples (pH Z 5.2) were allowed to flow through a micro-column packed with Chelex-100. The micro-column was placed in the autosampler arm of a graphite-furnace atomic absorption spectrometer (model 6701, Shimadzu, Kyoto, Japan). In most cases, 4 ml of sample were preconcentrated on this material. The retained heavy metals were eluted by aspiration of 80 ml of 2 M solution of nitric acid (SuprapurÒ grade, Merck, Darmstadt, Germany) and subsequent injection of the whole volume into the graphite furnace atomizer. After elution, the heating program of the atomizer was started: the sample was dried at 80 and 130 C for 5 and 20 s respectively, ashed for 16 s at 280 C, and atomized with a 2 s-temperature pulse reaching 1500 C for Cd and 1700 C for Pb analyses. The hollow cathode lamp emission lines used were 228.8 nm for Cd and 283.3 for Pb. The background absorption was corrected using a D2 lamp. An external calibration curve was generated using synthetic Cd and Pb standards in the range of 50e300 ng lÿ1, and the Cd and Pb contents in the samples were calculated by interpolation. The values obtained for Cd and Pb concentrations in the analysis of seawater reference material (CASS-3) were in good agreement (95% confidence interval) with the certified values.
Results obtained for the mean concentrations of total and labile cadmium and lead species in seawater from the four locations under study are shown in Table 1. Table 2 shows the total contents of these metals in algal tissues, the percentage of labile fraction of Cd and Pb in each site and calculated ER (enhancement ratio) values. CF was calculated as the ratio between the metal content in algae and the Total Dissolved Metal (TDM) concentration. Cd CF values were 1100; 580; 2200 and 8500 for Caleta Cordova, Punta Borja, Rada Tilly Beach and Punta Maqueda, respectively. Values of Pb CF were 730, 1180, 600 and 690 for the four locations mentioned before. According to Phillips (1977, 1990) one of the requisites for selection of a biological indicator is that CF should be similar at all sites under study for a correct comparison of levels of contaminants over time. In the case that this feature is not accomplished, CF may be used to identify factors that influence availability to the organism (Langston and Spence, 1995). However, a lower CF value in Punta Borja, the most heavily impacted site with the highest total cadmium concentration in seawater, than in Punta Maqueda (the control site) seemed unreasonable and this finding deserved further investigation. As a matter of fact, Cd CF values are quite far from being similar between places and differences of more than one order of magnitude are noticed in Punta Borja and Punta Maqueda. Metal speciation is the keypoint to better understand metal availability to biota in natural aquatic systems (Campbell, 1995). Free and weakly complexed ionic species in seawater are likely to be in the labile fraction. Cadmium chloro species and lead carbonate and chloride species are the major components in seawater in the absence of dissolved organic matter (DOM). In the presence of DOM from contaminated environments different Cd and Pb species may exist in solution as organic complexes (Fergusson, 1990). Hydrological parameters such as temperature, pH, salinity and reactive phosphate and environmental factors such as
2.5. Determination of Cd and Pb in digested algae Dried algal powders (400e500 mg) were digested in closed PFTEÒ vessels with 70% (w/w) nitric acid using a microwave digestion system, MDS 2000 (CEM Co, Matthews, NC, USA). The pressure conditions inside the vessels were automatically controlled by the microwave unit. The digestion procedure was programmed in four 8-min-long steps at 500 W each. The pressure limits in the vessel were set to: 40, 85, 135 and 175 psi, respectively. After the cycle was completed, the PTFE vessels were allowed to cool down to room temperature and the final clear solution was made up to 25 ml with distilled water. Duplicate digestion blanks were also prepared. Analysis of the certified reference material (BCR 279) showed results in good agreement with the certified values (Muse et al., 1999).
Table 1 Concentrations of cadmium and lead in surface seawater from the Southern Atlantic coasts Site
Cd in seawater (mg/litre) TDM Mean a
Pb in seawater (mg/litre) RLDM
Range
Mean a
TDM Range
Mean a
RLDM Range
Mean a
Range
Caleta Cordova
0.40 0.50b
(0.15e0.55)
0.100 0.110b
(0.090e0.120)
2.30 2.20b
(2.20e2.50)
1.15 1.20b
(0.60e1.30)
Punta Borja
0.48 0.40
(0.28e0.68)
0.090 0.080
(0.070e0.100)
3.00 3.25
(2.65e3.45)
2.80 2.70
(2.40e3.20)
Playa Rada Tilly
0.30 0.35
(0.20e0.40)
0.110 0.100
(0.100e0.150)
2.00 2.00
(1.95e2.15)
0.80 0.75
(0.60e0.90)
Punta Maqueda
0.20 0.25
(0.10e0.30)
0.180 0.175
(0.110e0.210)
2.40 2.45
(2.30e2.50)
1.30 1.20
(0.90e1.40)
TDM: Total Dissolved Metal. RLDM: Resin Labile Dissolved Metal. a Seawater samples collected in August, 2001. Mean values of five determinations. b Seawater samples monthly collected from March to August 2001.
J.O. Muse et al. / Environmental Pollution 141 (2006) 126e130 Table 2 Labile Cd and Pb species in seawater and total metal accumulated in Ulva lactuca from the area of Gulf San Jorge Sampling site
Caleta Cordova
Punta Borja
Rada Tilly Beach
Punta Maqueda
37
90
0.68 G 0.07
1.70 G 0.03
6000
10,000
40
54
1.68 G 0.30 3.55 G 0.26
1.21 G 0.29
1.65 G 0.31
1500
1500
1300
Cadmium % labile Cd in 25 19 seawatera Amount of Cd in 0.45 G 0.04 0.28 G 0.03 algaeb,c 3000 Cd enhancement 4500 ratiod Lead % labile Pb in seawatere Amount of Pb in algaef,c Pb enhancement ratiog
50
93
1300
a
The percentage for Cd of RLDM in TDM fraction (see line a in Table 2). Total Cd determined in digested algal powder as mg Cd/g dry algae. c Population means (X) and standard deviation of pooled samples (SN) calculated for each collection site in August, 2001 (n Z 6). d The ratio for Cd concentration enhancement was calculated as mg Cd/g dry algae divided by mg labile Cd/ml filtered seawater !1000. e The percentage for Pb of RLDM in TDM fraction (see line a in Table 2). f Total Pb determined in digested algal powder as mg Pb/g dry algae. g The ratio for Pb concentration enhancement was calculated as mg Pb/g dry algae divided by mg labile Pb/ml filtered seawater !1000. b
nature and concentrations of competitive metal ions, colloidal particles and natural DOM contribute to change the heavy metals speciation in seawater (Van den Berg, 1984) and thus, the metal availability to algae and other marine organisms. The sampling sites located along the Gulf San Jorge are close to each other and are exposed to similar oceanographic and climatic conditions. The values of temperature (12e15 C), pH (8.0e8.5), salinity (32.0e33.9 g Lÿ1) and soluble reactive phosphate (0.20e0.25 mM) in seawater were in the range of natural variability, and thus, differences in CF cannot be attributed to these parameters. Regarding the influence of other metals such as Zn e a well known competitor of Cd uptake at the surface of the algal membrane-, no depletion in seawater was observed. So, the reduction of Cd availability in the exposed site should not be attributed to Zn antagonism. Studies performed with the macroalgae Enteromorpha prolifera (chlorophyceae) and Porphyra columbina (rhodophyceae), collected together with U. lactuca, showed similar spatial trends in the bioaccumulation of Cd. These findings suggest that it is not the organism the responsible for the anomalous behavior. On the contrary, bioaccumulation of other metals such as Pb, Cu, Zn and Cr in Ulva lactuca (Muse et al., 1999) followed the expected trends and the contents of these heavy metals accumulated in P. Borja were significantly higher (P O 0.10) than those accumulated in P. Maqueda. The anomalous response to Cd (Tables 1 and 2) shows that its chemical speciation in P. Borja is different from that in P. Maqueda. As a matter a fact, 90% of total soluble Cd in P. Maqueda corresponds to the labile fraction and
129
just 19% of total soluble Cd comprises this fraction in P. Borja. Probably, the human activities developed in P. Borja (urban discharges and petroleum extraction) drastically influence its chemical environment through the increase of DOM, reducing the concentrations of bioavailable Cd and hence, its accumulation in Ulva spp. The percentages of labile Cd found in Caleta Cordova and Rada Tilly beach were close to that of P. Borja and deserve a similar explanation. In brief, considering that Cd CF is referred to the total soluble Cd concentration in seawater, the CF approach might not be useful to evaluate the state of the environment with respect to cadmium in the area of study (Moriarty, 1999). Instead, Pb contamination could be assessed through the CF approach since there is a positive correlation between total and labile Pb concentrations in seawater (see Table 1). Pb CF values are close between places and evolution of Pb contamination with time can be followed in this case. In order to find a parameter able to perform a better description of the state of the environment for cadmium, we propose to recalculate CF as the enhancement ratio (ER) of the metal in algae relative to its kinetically labile forms in seawater (see Table 2). Values of Cd ER range between 0.3 and 1.0 ! 104, showing practically the same order of magnitude in the four locations. These ER values fulfill the criterium established by Phillips (1977, 1990) at the time of defining an accumulation factor. Now, if ER is employed instead of CF, the values of accumulated Cd in Ulva spp. become unsurprising. Calculated ER values allow us to know about the degree of pollution of the environment with respect to Cd. Now, these values are in good agreement with those found by Vasconcelos and Leal (2001) and they correspond to low polluted areas. Regarding Pb accumulation, any of both parameters (CF or ER) can be used for comparison between places. 4. Conclusions From an environmental point of view, when the contents of total dissolved metal in the aquatic medium and that accumulated in the bioindicator are not directly related CF lacks of significance. In such case, ER values can provide baseline data, qualify the state of the environment and help to evaluate those factors affecting bioavailability in an environmental assessment. Therefore, the ER approach based on metal kinetic speciation in seawater can overcome CF limitations for the evaluation of marine pollution.
Acknowledgements The authors thank Dr. A. Boraso, National University of Patagonia San Juan Bosco, Comodoro Rivadavia, Chubut, for her assistance in selection of algal species and Dr. M.A. Fajardo (NUPSJB) for supplying algal and seawater samples. Participants in a collaborative program on Marine Pollution Research developed by National University of Buenos Aires
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and NUPSJB, and also Dr. J. Lopez Gappa, Museum of Natural Sciences, Buenos Aires, are gratefully acknowledged for their collaboration. Buenos Aires University, Science and Technology (UBACYT), National Research Council, Science and Technology (CONICET) and Institute of Chemistry and Environment (INQUIMAE) granted financial support.
References Bryan, G.W., Langston, W.J., 1982. Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review. Environ. Pollut. 76, 89e131. Buckley, J.A., Yoshida, G.A., Wells, N.R., Aquino, R.T., 1985. Toxicities of total and chelex-labile cadmium to salmon in solutions of natural waters and diluted sewage with potentially different cadmium complexing capacities. Water Res. 19, 1549e1554. Campbell, P.G.C., 1995. Interactions between trace metals and aquatic organisms: a critique of the free-ion activity model. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. John Wiley and Son, West Sussex, UK, pp. 45e102. Chakrabarti, C.L., Lu, Y.J., Gregoire, D.C., Back, M.H., Schroeder, W.H., 1994. Kinetic studies of metal speciation using chelex cation e exchange resin e application to cadmium, copper and lead speciation in River Water and Snow. Environ. Sci. Technol. 28, 1957e1967. Commendatore, M., Gil, M., Harvey, M.A., Colombo, J.C., Esteves, J.L., 1996. Evaluacio´n de la contaminacio´n por hidrocarburos y metales en la costera patago´nica. Informes Te´cnicos del Plan de Manejo Integrado de la Zona Costera Patago´nica. Fundacio´n Patagonia Natural (Puerto Madryn, Argentina) 21, 1e47. Conti, M.E., Tudino, M.B., Muse, J.O., Cecchetti, F., 2002. Biomonitoring of heavy metals in the marine environment: the contribution of atomic absorption spectrometry and inductively coupled plasma spectrometry. Res. Trends Appl. Spectros. 4, 295e324. Fergusson, J.E., 1990. Heavy Elements, Chemistry, Environmental Impact and Health Effects. Pergamon Press, Oxford UK. Ferna´ndez, F.M., Stripeikis, J.D., Tudino, M.B., Troccoli, O.E., 1997. Fully automatic on-line separation preconcentration system for electrothermal atomic absorption spectrometry: determination of cadmium and lead in seawater. Analyst Lond 122, 679e684. Ferna´ndez, F.M., Tudino, M.B., Troccoli, O.E., 2000. Automatic on-line ultrtrace determination of Cd species of environmental significance in natural waters by FI-ETAAS.J. Anal. At. Spectrom. 15, 687e695. Figura, P., McDuffie, B., 1980. Determination of labilities of soluble trace metal species in aqueous environmental samples by anodic stripping voltammmetry and Chelex column and batch methods. Anal. Chem. 52, 1433e1439. Florence, T.M., Lumsden, B.G., Fardy, J.J., 1983. Evaluation of some physicochemical techniques for the determination of the fraction of dissolved copper toxic to the marine diatom, Nitzchia closterium. Anal. Chim. Acta 151, 281e295. Foster, P., 1976. Concentrations and concentration factors of heavy metals in brown algae. Environ. Pollut. 10, 45e53. Keith, L.H., 1991. Environmental Sampling and Analysis A Practical Guide. Lewis Publishers, Chelsea, UK.
Ku¨hneman, O., 1972. Bosquejo fitogeogra´fico de la vegetacio´n marina del litoral argentino. Contr, CIBIMA, Argentina, Te´c. No. 66, pp.1e32. Langston, W.J., Spence, S.K., 1995. Biological factors involved in metal concentrations observed in aquatic organisms. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. John Wiley and Son, West Sussex, UK, pp. 407e478. Luoma, S.N., Bryan, J.W., 1978. A statistical assessment of the form of trace metals in oxidized estuarine sediments employing chemical extractants. Sci. Tot. Environ. 17, 65e196. Melhuus, A., Seip, K.L., Seip, H.M., Myklestad, S., 1978. A preliminary study of the use of benthic algae as biological indicators of heavy metal pollution in Sorfjorden. Norway. Environ. Pollut. 15, 101e107. Morel, F.M.M., 1983. Principles of Aquatic Chemistry. Wiley-Interscience, New York. Moriarty, F., 1999. Ecotoxicology. The Study of Pollutants in Ecosystems. Academic Press, London. Muse, J.O., Stripeikis, J.D., Ferna´ndez, F.M., d’Huicque, L., Tudino, M.B., Carducci, C.N., Troccoli, O.E., 1999. Seaweeds in the assessment of heavy metal pollution in the Gulf San Jorge, Argentina. Environ. Pollut. 104, 315e322. Phillips, D.J.H., 1977. The use of biological indicator organism to monitor trace metal pollution in marine and estuarine environmentsea review. Environ. Pollut. 13, 281e317. Phillips, D.J.H., 1990. Use of macroalgae and invertebrates as monitors of metal levels in estuaries and coastal waters. In: Furness, R.W., Rainbow, P.S. (Eds.), Heavy Metals in the Marine Environment. CRC Press, Boca Raton, FL, pp. 81e99. Sturgeon, R.E., Berman, S.S., Desaulniers, A., Russell, D.S., 1979. Preconcentration of trace metals from seawater for determination by graphite furnace atomic absorption spectrometry. Talanta 27, 85e94. Sunda, W.G., Guillard, R.R.L., 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res. 34, 511e529. Templeton, D.M., Ariese, F., Cornelis, R., Danielsson, L.-G., Muntau, H., Van Leeuwen, H.P., Lobinski, R., 2000. Guidelines for terms related to chemical speciation and fractionation of element. Definitions, structural aspects and methodological approach. Pure Appl. Chem. 72, 1453e1470. Tessier, A., Buffle, S., Campbell, P.G.C., 1994. Uptake of trace metals by aquatic organisms. In: Buffle, J., De Vitre, R. (Eds.), Chemical and Biological Regulation of Aquatic Systems. Lewis, Boca Raton, FL (Chapter 6). Van den Berg, C.M.G., 1984. Organic and inorganic speciation of copper in the Irish Sea. Mar. Chem. 14, 201e212. Vasconcelos, M.T.S.D., Leal, M.F.C., 2001. Seasonal variability in the kinetics of Cu, Pb, Cd and Hg accumulation by macroalgae. Mar. Chem. 74, 65e85. Volterra, L., Conti, M.E., 2000. Algae as biomarkers, bioaccumulators and toxin producers. Int. J. Environ. Pollut. 13, 92e125. Wallace, G.T., 1982. The association of copper, mercury and lead with surface active organic matter in coastal seawater. Mar. Chem. 11, 379e394. Whitfield, M., Turner, D.R., 1987. The Role of Particles in Regulating the Composition of Seawater. In: Stumm, W. (Ed.), Wiley, New York, pp. 457e493. Xue, H.-B., Stumm, W., Sigg, L., 1988. The binding of heavy metals to algal surfaces. Water Res. 22, 917e926.
A link between lead and cadmium kinetic speciation in seawater and accumulation by the green alga Ulva lactuca J.O. Muse a,*, C.N. Carducci a, J.D. Stripeikis b, M.B. Tudino b, F.M. Ferna´ndez b,1 a
Departamento de Quı´mica Analı´tica y Fisicoquı´mica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Qulmica Analitica, 3er. Piso, Junin 956-1113, Buenos Aires, Argentina b Departamento de Quı´mica Inorga´nica, Analı´tica y Quı´mica Fı´sica/INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, 3er. Piso, Pabello´n 2, Ciudad Universitavia, Buenos Aires, Argentina Received 17 September 2005; accepted 2 August 2005
Metal kinetic speciation can overcome CF limitations for the evaluation of marine pollution. Abstract In this work, studies on the bioaccumulation of Cd and Pb by Ulva lactuca at different sites of Gulf San Jorge (Patagonia, Argentina) are presented. Higher values of bioaccumulated Cd were found in Punta Maqueda e a site believed to serve as a control e in comparison to those in Punta Borja, a place highly exposed to urban and industrial activities. Consequently; the labile fractions of Cd and Pb in seawater were determined with a flow injection-preconcentration manifold interfaced to a graphite furnace-atomic absorption spectrometer (FI-GFAAS). The results obtained by kinetic speciation showed that the variable that correctly explains heavy metals accumulation in the alga, is the labile metal fraction in seawater. We propose to use an enhancement ratio e on the basis of the kinetically labile metal fraction e for calculation of the metal accumulated by the alga relative to its environment. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cd and Pb; Seawater; Kinetic speciation; Ulva lactuca; Concentration factor
1. Introduction Benthic marine algae are useful in marine pollution assessment and biomonitoring studies (Phillips, 1977, 1990; Volterra and Conti, 2000) since they are able to accumulate metals in concentrations several thousand times higher than those in the surrounding seawater (Foster, 1976; Bryan and Langston, 1982). The Concentration Factor (CF) (Foster, 1976) is defined as the ratio between the content of a given metal in an organism and its concentration in any compartment of the environment such as the total dissolved metal fraction in seawater (Morel, * Corresponding author. Tel.: C54 114 964 8262; fax: C54 114 964 8263. E-mail address: [email protected] (J.O. Muse). 1 Present address. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA. 0269-7491/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2005.08.021
1983) or the acid extractable fraction in sediment (Luoma and Bryan, 1978). CF is often used to estimate evolution of contamination over time (Melhuus et al., 1978) and, as proposed by Conti and co-workers (Conti et al., 2002), it is also useful to compare pollution levels among different sites. However, inter-site comparison of metal pollution through CF values is affected by sampling design, by temporal and spatial variability, and also by biological variables inherent to the organism under study (Langston and Spence, 1995). Consequently, studies on bioaccumulation in each place show that the link between CF and the actual water pollution is poor if sites under different geochemical, hydrodynamic and environmental conditions need to be compared (Moriarty, 1999). According to the Free Ion Activity Model (FIAM) proposed by Morel (1983), the interaction between metals and living organisms should be explained through the chemical reaction of free and weakly complexed ionic species with biological
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membranes (Morel, 1983; Xue et al., 1988). For example, the metal uptake in algae (Sunda and Guillard, 1976) under controlled, static conditions has been shown to be proportional to the concentration of free ionic and weakly-bound metal species, and not to the total metal concentrations. FIAM assumes that the transport of the metal in the solution bulk towards the cell membrane is fast in comparison to the actual biological uptake rate, and thus, the activities of all the metal species in the bulk medium and at the cell surface are approximately the same. A second assumption is that the cell surface is in ‘‘pre-equilibrium’’ with the metal species in solution and that the uptake rate is only limited by the membrane transport processes. In this case, the complexation of metal ions with ligands that form highly stable complexes decreases the bioavailability to metals (Tessier et al., 1994). However, if the metal ions are present as kinetically inert complexes, the surface ligand exchange reactions are conditioned by the rate of release of the metal from the complex. In this case, chemical kinetic considerations must be accounted for and the concept of dynamic speciation needs to be introduced. Dynamic speciation takes into account both the equilibrium distribution of the different species and the kinetics of their interconversion (Templeton et al., 2000). The development of an analytical method capable of performing kinetic speciation analysis involves to mimic the surface complexation reactions taking place at the cell membranes with a laboratory-based setup. Competitive ligand displacement assays between the metal complex under investigation and a chelating resin (Figura and McDuffie, 1980; Chakrabarti et al., 1994) are useful to perform kinetic speciation analysis. Figura and McDuffie (1980) reported that iminodiacetate-based resins such as Chelex-100 can modify their metal retention ability by operating under different pH and sample flow rate conditions. In fact, under suitable conditions, the resin is able to selectively retain the most reactive fraction of metal species which is directly related to toxicity effects on aquatic biota (Florence et al., 1983; Buckley et al., 1985). In a previous work, we have shown the potential of iminodiacetate-based resins to selectively retain the bioavailable fraction of the total dissolved metal content in seawater (Ferna´ndez et al., 1997, 2000). The aim of this study is to establish a relationship between the kinetically-labile fraction of Cd and Pb in coastal S. Atlantic seawater and the accumulation of both analytes in Ulva lactuca growing in the area of Gulf San Jorge (Patagonia, Argentina). Obtained results will be employed to show that CF can achieve a better environmental significance if the sum of the concentrations of labile metal species in seawater instead of the total metal contents is taken into account. 2. Materials and methods 2.1. Sampling design Four sites undergoing similar oceanographic conditions in a restricted area of the Gulf San Jorge were chosen to compare the distribution of Cd and Pb in Ulva lactuca exposed to pollutants derived from the petroleum extraction
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industry. The sampling sites are: Caleta Cordova (45 43# S 67 20# W), Punta Borja (45 52# S 67 29# W), Rada Tilly Beach (46 00# S 67 33# W) and Punta Maqueda (46 01# S 67 34# W) chosen as the control site. Caleta Cordova is a sandy beach with pebbles and stones with an underdeveloped harbor but with important petroleum extraction activity. Punta Borja is sited in an open area, plenty of rocks and tide pools with dark spots spread over the beach, highly exposed to industrial, urban and harbor activities and absence of waste-treatment facilities. Rada Tilly is a fine sandy beach with mild contamination derived from a small touristic village. Punta Maqueda is a naturally protected sandy beach, surrounded by shoals and far away from any source of contamination. The evaluation of the degree of exposure to different sources of contamination is qualitative and has taken into account the number of inhabitants, the types of activities and the presence of waste-treatment facilities. A description of the geology and oceanographic conditions along the S. Atlantic coasts has been given by Commendatore et al. (1996). The materials under study were collected following the records of geographical distribution of seaweeds along the Patagonian coasts reported by Ku¨hneman (1972).
2.2. Sampling procedure and sample conditioning Composite and pooled samples of Ulva lactuca plants were collected at low tide at each of the aforementioned sites during August 2001 according to the current methodology (Keith, 1991). The material (500e1000 g, fresh weight) was gathered from the intertidal area, rinsed to remove solid debris and associated organisms, washed with few portions of deionized water to avoid leaching of metals, and finally stored in plastic bags under freezing. Dried samples were ground to pass a 212-mm sieve and stored in a dessicator prior to analysis. Surface seawater samples were collected at low tide and at the same time and place of algal collection. The mean Cd and Pb concentrations were used for calculation of CF values. A second set of seawater samples was also collected under the calm weather conditions of the autumn-winter period (between March and August 2001). In all occasions natural perturbing phenomena such as solid resuspension (Whitfield and Turner, 1987) and phytoplankton blooms (Wallace, 1982) were avoided. An estimation of the concentration means of total dissolved and labile Cd and Pb in the aquatic environment prior to algal sampling was obtained. Seawater samples conditioning for total trace metal analysis was performed by the addition of nitric acid (SuprapurÒ grade, Merck, Darmstadt, Germany) until the sample pH was between 4 and 5. Samples were then filtered through acid cleaned 0.22-mm membrane filters, stored in pre-cleaned 1000-ml polypropylene bottles and kept frozen at ÿ20 C. Samples for speciation studies were kept at their natural pH, filtered and frozen in PFTEÒ bottles (Keith, 1991).
2.3. Reagents All reagents were of analytical grade or better. Standards were prepared on a laminar flow clean bench. Doubly deionized water (DIW, 18 MU cm) was obtained from a MilliQ water system (Millipore, Beldford, MA, USA). Chelex-100 chelating resin (100e200 mesh, Bio-Rad, Richmond, CA, USA) was used for the retention of the labile Cd and Pb fractions following the procedure described in our previous works (Ferna´ndez et al., 1997, 2000). Seawater samples were preconditioned using a 0.1 M acetic acid-ammonium acetate buffer which was purified through an auxiliary Chelex-100 column before its use, following the procedure described below. Working standard solutions of cadmium (50e200 ng lÿ1) and lead (50e 300 ng lÿ1) were prepared by appropriate dilution of stock standard solutions containing 1000 g lÿ1 Cd and 1000 g lÿ1 Pb (Merck), respectively. Dilutions were performed in Nalgene (Nalge, Rochester, NY, USA) volumetric flasks. All glassware was thoroughly cleaned with nitric acid (1 C 1) rinsed with DIW, further cleaned with hot aqueous 0.05% APDC solution and rinsed again. Once in use, only DIW rinsing of the flasks was performed. CASS-3 Seawater Reference Material from the National Research Council of Canada (NRCC) and Sea lettuce (Ulva lactuca) Reference Material (BCR 279) from the Community Bureau of Reference were used for analytical validation purposes.
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2.4. Analytical determinations in seawater
3. Results and discussion
Temperature, pH and salinity were determined in situ with an Orion model 250A pHmeter (Boston, MA, USA) and a Hach model sensIon 5 conductivity/ salinity meter (Loveland, CO, USA), respectively. Soluble reactive phosphorus (SRP molybdate-ascorbic) was determined following the method described by Strickland and Parsons (1972). Total dissolved Cd and Pb concentrations were determined according to Sturgeon et al. (1979). An on-line solid-phase chelation method already reported (Ferna´ndez et al., 1997) was employed for the determination of kinetically labile fractions of Cd and Pb in seawater. On-line conditioned water samples (pH Z 5.2) were allowed to flow through a micro-column packed with Chelex-100. The micro-column was placed in the autosampler arm of a graphite-furnace atomic absorption spectrometer (model 6701, Shimadzu, Kyoto, Japan). In most cases, 4 ml of sample were preconcentrated on this material. The retained heavy metals were eluted by aspiration of 80 ml of 2 M solution of nitric acid (SuprapurÒ grade, Merck, Darmstadt, Germany) and subsequent injection of the whole volume into the graphite furnace atomizer. After elution, the heating program of the atomizer was started: the sample was dried at 80 and 130 C for 5 and 20 s respectively, ashed for 16 s at 280 C, and atomized with a 2 s-temperature pulse reaching 1500 C for Cd and 1700 C for Pb analyses. The hollow cathode lamp emission lines used were 228.8 nm for Cd and 283.3 for Pb. The background absorption was corrected using a D2 lamp. An external calibration curve was generated using synthetic Cd and Pb standards in the range of 50e300 ng lÿ1, and the Cd and Pb contents in the samples were calculated by interpolation. The values obtained for Cd and Pb concentrations in the analysis of seawater reference material (CASS-3) were in good agreement (95% confidence interval) with the certified values.
Results obtained for the mean concentrations of total and labile cadmium and lead species in seawater from the four locations under study are shown in Table 1. Table 2 shows the total contents of these metals in algal tissues, the percentage of labile fraction of Cd and Pb in each site and calculated ER (enhancement ratio) values. CF was calculated as the ratio between the metal content in algae and the Total Dissolved Metal (TDM) concentration. Cd CF values were 1100; 580; 2200 and 8500 for Caleta Cordova, Punta Borja, Rada Tilly Beach and Punta Maqueda, respectively. Values of Pb CF were 730, 1180, 600 and 690 for the four locations mentioned before. According to Phillips (1977, 1990) one of the requisites for selection of a biological indicator is that CF should be similar at all sites under study for a correct comparison of levels of contaminants over time. In the case that this feature is not accomplished, CF may be used to identify factors that influence availability to the organism (Langston and Spence, 1995). However, a lower CF value in Punta Borja, the most heavily impacted site with the highest total cadmium concentration in seawater, than in Punta Maqueda (the control site) seemed unreasonable and this finding deserved further investigation. As a matter of fact, Cd CF values are quite far from being similar between places and differences of more than one order of magnitude are noticed in Punta Borja and Punta Maqueda. Metal speciation is the keypoint to better understand metal availability to biota in natural aquatic systems (Campbell, 1995). Free and weakly complexed ionic species in seawater are likely to be in the labile fraction. Cadmium chloro species and lead carbonate and chloride species are the major components in seawater in the absence of dissolved organic matter (DOM). In the presence of DOM from contaminated environments different Cd and Pb species may exist in solution as organic complexes (Fergusson, 1990). Hydrological parameters such as temperature, pH, salinity and reactive phosphate and environmental factors such as
2.5. Determination of Cd and Pb in digested algae Dried algal powders (400e500 mg) were digested in closed PFTEÒ vessels with 70% (w/w) nitric acid using a microwave digestion system, MDS 2000 (CEM Co, Matthews, NC, USA). The pressure conditions inside the vessels were automatically controlled by the microwave unit. The digestion procedure was programmed in four 8-min-long steps at 500 W each. The pressure limits in the vessel were set to: 40, 85, 135 and 175 psi, respectively. After the cycle was completed, the PTFE vessels were allowed to cool down to room temperature and the final clear solution was made up to 25 ml with distilled water. Duplicate digestion blanks were also prepared. Analysis of the certified reference material (BCR 279) showed results in good agreement with the certified values (Muse et al., 1999).
Table 1 Concentrations of cadmium and lead in surface seawater from the Southern Atlantic coasts Site
Cd in seawater (mg/litre) TDM Mean a
Pb in seawater (mg/litre) RLDM
Range
Mean a
TDM Range
Mean a
RLDM Range
Mean a
Range
Caleta Cordova
0.40 0.50b
(0.15e0.55)
0.100 0.110b
(0.090e0.120)
2.30 2.20b
(2.20e2.50)
1.15 1.20b
(0.60e1.30)
Punta Borja
0.48 0.40
(0.28e0.68)
0.090 0.080
(0.070e0.100)
3.00 3.25
(2.65e3.45)
2.80 2.70
(2.40e3.20)
Playa Rada Tilly
0.30 0.35
(0.20e0.40)
0.110 0.100
(0.100e0.150)
2.00 2.00
(1.95e2.15)
0.80 0.75
(0.60e0.90)
Punta Maqueda
0.20 0.25
(0.10e0.30)
0.180 0.175
(0.110e0.210)
2.40 2.45
(2.30e2.50)
1.30 1.20
(0.90e1.40)
TDM: Total Dissolved Metal. RLDM: Resin Labile Dissolved Metal. a Seawater samples collected in August, 2001. Mean values of five determinations. b Seawater samples monthly collected from March to August 2001.
J.O. Muse et al. / Environmental Pollution 141 (2006) 126e130 Table 2 Labile Cd and Pb species in seawater and total metal accumulated in Ulva lactuca from the area of Gulf San Jorge Sampling site
Caleta Cordova
Punta Borja
Rada Tilly Beach
Punta Maqueda
37
90
0.68 G 0.07
1.70 G 0.03
6000
10,000
40
54
1.68 G 0.30 3.55 G 0.26
1.21 G 0.29
1.65 G 0.31
1500
1500
1300
Cadmium % labile Cd in 25 19 seawatera Amount of Cd in 0.45 G 0.04 0.28 G 0.03 algaeb,c 3000 Cd enhancement 4500 ratiod Lead % labile Pb in seawatere Amount of Pb in algaef,c Pb enhancement ratiog
50
93
1300
a
The percentage for Cd of RLDM in TDM fraction (see line a in Table 2). Total Cd determined in digested algal powder as mg Cd/g dry algae. c Population means (X) and standard deviation of pooled samples (SN) calculated for each collection site in August, 2001 (n Z 6). d The ratio for Cd concentration enhancement was calculated as mg Cd/g dry algae divided by mg labile Cd/ml filtered seawater !1000. e The percentage for Pb of RLDM in TDM fraction (see line a in Table 2). f Total Pb determined in digested algal powder as mg Pb/g dry algae. g The ratio for Pb concentration enhancement was calculated as mg Pb/g dry algae divided by mg labile Pb/ml filtered seawater !1000. b
nature and concentrations of competitive metal ions, colloidal particles and natural DOM contribute to change the heavy metals speciation in seawater (Van den Berg, 1984) and thus, the metal availability to algae and other marine organisms. The sampling sites located along the Gulf San Jorge are close to each other and are exposed to similar oceanographic and climatic conditions. The values of temperature (12e15 C), pH (8.0e8.5), salinity (32.0e33.9 g Lÿ1) and soluble reactive phosphate (0.20e0.25 mM) in seawater were in the range of natural variability, and thus, differences in CF cannot be attributed to these parameters. Regarding the influence of other metals such as Zn e a well known competitor of Cd uptake at the surface of the algal membrane-, no depletion in seawater was observed. So, the reduction of Cd availability in the exposed site should not be attributed to Zn antagonism. Studies performed with the macroalgae Enteromorpha prolifera (chlorophyceae) and Porphyra columbina (rhodophyceae), collected together with U. lactuca, showed similar spatial trends in the bioaccumulation of Cd. These findings suggest that it is not the organism the responsible for the anomalous behavior. On the contrary, bioaccumulation of other metals such as Pb, Cu, Zn and Cr in Ulva lactuca (Muse et al., 1999) followed the expected trends and the contents of these heavy metals accumulated in P. Borja were significantly higher (P O 0.10) than those accumulated in P. Maqueda. The anomalous response to Cd (Tables 1 and 2) shows that its chemical speciation in P. Borja is different from that in P. Maqueda. As a matter a fact, 90% of total soluble Cd in P. Maqueda corresponds to the labile fraction and
129
just 19% of total soluble Cd comprises this fraction in P. Borja. Probably, the human activities developed in P. Borja (urban discharges and petroleum extraction) drastically influence its chemical environment through the increase of DOM, reducing the concentrations of bioavailable Cd and hence, its accumulation in Ulva spp. The percentages of labile Cd found in Caleta Cordova and Rada Tilly beach were close to that of P. Borja and deserve a similar explanation. In brief, considering that Cd CF is referred to the total soluble Cd concentration in seawater, the CF approach might not be useful to evaluate the state of the environment with respect to cadmium in the area of study (Moriarty, 1999). Instead, Pb contamination could be assessed through the CF approach since there is a positive correlation between total and labile Pb concentrations in seawater (see Table 1). Pb CF values are close between places and evolution of Pb contamination with time can be followed in this case. In order to find a parameter able to perform a better description of the state of the environment for cadmium, we propose to recalculate CF as the enhancement ratio (ER) of the metal in algae relative to its kinetically labile forms in seawater (see Table 2). Values of Cd ER range between 0.3 and 1.0 ! 104, showing practically the same order of magnitude in the four locations. These ER values fulfill the criterium established by Phillips (1977, 1990) at the time of defining an accumulation factor. Now, if ER is employed instead of CF, the values of accumulated Cd in Ulva spp. become unsurprising. Calculated ER values allow us to know about the degree of pollution of the environment with respect to Cd. Now, these values are in good agreement with those found by Vasconcelos and Leal (2001) and they correspond to low polluted areas. Regarding Pb accumulation, any of both parameters (CF or ER) can be used for comparison between places. 4. Conclusions From an environmental point of view, when the contents of total dissolved metal in the aquatic medium and that accumulated in the bioindicator are not directly related CF lacks of significance. In such case, ER values can provide baseline data, qualify the state of the environment and help to evaluate those factors affecting bioavailability in an environmental assessment. Therefore, the ER approach based on metal kinetic speciation in seawater can overcome CF limitations for the evaluation of marine pollution.
Acknowledgements The authors thank Dr. A. Boraso, National University of Patagonia San Juan Bosco, Comodoro Rivadavia, Chubut, for her assistance in selection of algal species and Dr. M.A. Fajardo (NUPSJB) for supplying algal and seawater samples. Participants in a collaborative program on Marine Pollution Research developed by National University of Buenos Aires
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and NUPSJB, and also Dr. J. Lopez Gappa, Museum of Natural Sciences, Buenos Aires, are gratefully acknowledged for their collaboration. Buenos Aires University, Science and Technology (UBACYT), National Research Council, Science and Technology (CONICET) and Institute of Chemistry and Environment (INQUIMAE) granted financial support.
References Bryan, G.W., Langston, W.J., 1982. Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review. Environ. Pollut. 76, 89e131. Buckley, J.A., Yoshida, G.A., Wells, N.R., Aquino, R.T., 1985. Toxicities of total and chelex-labile cadmium to salmon in solutions of natural waters and diluted sewage with potentially different cadmium complexing capacities. Water Res. 19, 1549e1554. Campbell, P.G.C., 1995. Interactions between trace metals and aquatic organisms: a critique of the free-ion activity model. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. John Wiley and Son, West Sussex, UK, pp. 45e102. Chakrabarti, C.L., Lu, Y.J., Gregoire, D.C., Back, M.H., Schroeder, W.H., 1994. Kinetic studies of metal speciation using chelex cation e exchange resin e application to cadmium, copper and lead speciation in River Water and Snow. Environ. Sci. Technol. 28, 1957e1967. Commendatore, M., Gil, M., Harvey, M.A., Colombo, J.C., Esteves, J.L., 1996. Evaluacio´n de la contaminacio´n por hidrocarburos y metales en la costera patago´nica. Informes Te´cnicos del Plan de Manejo Integrado de la Zona Costera Patago´nica. Fundacio´n Patagonia Natural (Puerto Madryn, Argentina) 21, 1e47. Conti, M.E., Tudino, M.B., Muse, J.O., Cecchetti, F., 2002. Biomonitoring of heavy metals in the marine environment: the contribution of atomic absorption spectrometry and inductively coupled plasma spectrometry. Res. Trends Appl. Spectros. 4, 295e324. Fergusson, J.E., 1990. Heavy Elements, Chemistry, Environmental Impact and Health Effects. Pergamon Press, Oxford UK. Ferna´ndez, F.M., Stripeikis, J.D., Tudino, M.B., Troccoli, O.E., 1997. Fully automatic on-line separation preconcentration system for electrothermal atomic absorption spectrometry: determination of cadmium and lead in seawater. Analyst Lond 122, 679e684. Ferna´ndez, F.M., Tudino, M.B., Troccoli, O.E., 2000. Automatic on-line ultrtrace determination of Cd species of environmental significance in natural waters by FI-ETAAS.J. Anal. At. Spectrom. 15, 687e695. Figura, P., McDuffie, B., 1980. Determination of labilities of soluble trace metal species in aqueous environmental samples by anodic stripping voltammmetry and Chelex column and batch methods. Anal. Chem. 52, 1433e1439. Florence, T.M., Lumsden, B.G., Fardy, J.J., 1983. Evaluation of some physicochemical techniques for the determination of the fraction of dissolved copper toxic to the marine diatom, Nitzchia closterium. Anal. Chim. Acta 151, 281e295. Foster, P., 1976. Concentrations and concentration factors of heavy metals in brown algae. Environ. Pollut. 10, 45e53. Keith, L.H., 1991. Environmental Sampling and Analysis A Practical Guide. Lewis Publishers, Chelsea, UK.
Ku¨hneman, O., 1972. Bosquejo fitogeogra´fico de la vegetacio´n marina del litoral argentino. Contr, CIBIMA, Argentina, Te´c. No. 66, pp.1e32. Langston, W.J., Spence, S.K., 1995. Biological factors involved in metal concentrations observed in aquatic organisms. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. John Wiley and Son, West Sussex, UK, pp. 407e478. Luoma, S.N., Bryan, J.W., 1978. A statistical assessment of the form of trace metals in oxidized estuarine sediments employing chemical extractants. Sci. Tot. Environ. 17, 65e196. Melhuus, A., Seip, K.L., Seip, H.M., Myklestad, S., 1978. A preliminary study of the use of benthic algae as biological indicators of heavy metal pollution in Sorfjorden. Norway. Environ. Pollut. 15, 101e107. Morel, F.M.M., 1983. Principles of Aquatic Chemistry. Wiley-Interscience, New York. Moriarty, F., 1999. Ecotoxicology. The Study of Pollutants in Ecosystems. Academic Press, London. Muse, J.O., Stripeikis, J.D., Ferna´ndez, F.M., d’Huicque, L., Tudino, M.B., Carducci, C.N., Troccoli, O.E., 1999. Seaweeds in the assessment of heavy metal pollution in the Gulf San Jorge, Argentina. Environ. Pollut. 104, 315e322. Phillips, D.J.H., 1977. The use of biological indicator organism to monitor trace metal pollution in marine and estuarine environmentsea review. Environ. Pollut. 13, 281e317. Phillips, D.J.H., 1990. Use of macroalgae and invertebrates as monitors of metal levels in estuaries and coastal waters. In: Furness, R.W., Rainbow, P.S. (Eds.), Heavy Metals in the Marine Environment. CRC Press, Boca Raton, FL, pp. 81e99. Sturgeon, R.E., Berman, S.S., Desaulniers, A., Russell, D.S., 1979. Preconcentration of trace metals from seawater for determination by graphite furnace atomic absorption spectrometry. Talanta 27, 85e94. Sunda, W.G., Guillard, R.R.L., 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res. 34, 511e529. Templeton, D.M., Ariese, F., Cornelis, R., Danielsson, L.-G., Muntau, H., Van Leeuwen, H.P., Lobinski, R., 2000. Guidelines for terms related to chemical speciation and fractionation of element. Definitions, structural aspects and methodological approach. Pure Appl. Chem. 72, 1453e1470. Tessier, A., Buffle, S., Campbell, P.G.C., 1994. Uptake of trace metals by aquatic organisms. In: Buffle, J., De Vitre, R. (Eds.), Chemical and Biological Regulation of Aquatic Systems. Lewis, Boca Raton, FL (Chapter 6). Van den Berg, C.M.G., 1984. Organic and inorganic speciation of copper in the Irish Sea. Mar. Chem. 14, 201e212. Vasconcelos, M.T.S.D., Leal, M.F.C., 2001. Seasonal variability in the kinetics of Cu, Pb, Cd and Hg accumulation by macroalgae. Mar. Chem. 74, 65e85. Volterra, L., Conti, M.E., 2000. Algae as biomarkers, bioaccumulators and toxin producers. Int. J. Environ. Pollut. 13, 92e125. Wallace, G.T., 1982. The association of copper, mercury and lead with surface active organic matter in coastal seawater. Mar. Chem. 11, 379e394. Whitfield, M., Turner, D.R., 1987. The Role of Particles in Regulating the Composition of Seawater. In: Stumm, W. (Ed.), Wiley, New York, pp. 457e493. Xue, H.-B., Stumm, W., Sigg, L., 1988. The binding of heavy metals to algal surfaces. Water Res. 22, 917e926.