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Influence of salinity and humic substances on the uptake of trace metals by the marine macroalga, Ulva lactuca: Experimental observations and modelling using WHAM
Marine Chemistry 110 (2008) 176–184
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Marine Chemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a r c h e m
Influence of salinity and humic substances on the uptake of trace metals by the marine macroalga, Ulva lactuca: Experimental observations and modelling using WHAM Andrew Turner a,⁎, Silvia S. Pedroso a, Murray T. Brown b a b
School of Earth, Ocean, and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA, UK School of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK
a r t i c l e
i n f o
Article history: Received 17 December 2007 Received in revised form 2 April 2008 Accepted 9 April 2008 Keywords: Macroalgae Trace metals Biouptake Salinity Humics Speciation modelling
a b s t r a c t Uptake of the trace metals, Pd, Cd, Hg and Pb, by the marine macroalga, Ulva lactuca, has been studied along a salinity gradient (S = 15–35; pH~ 8.3) created by batch mixing of synthetic sea water and pure water, both in the absence and presence of humic substances. Factors defining the concentration ratio of metal taken up (w/w) to metal remaining in solution ranged from about 102 mL g− 1 for Cd to 103 mL g− 1 for Pd and Hg. Within experimental error, only the biouptake of Cd appeared to exhibit a dependence on salinity, while the addition of 3 mg L− 1 of humics resulted in a small suppression of Pd and Hg uptake and a moderate enhancement of Pb uptake compared with the humic-free system. Metal internalisation, evaluated from an EDTA wash of the alga, followed the sequence: HgN Pd N Cd N Pb; and was notably inhibited in the presence of humics for Pb. Metal uptake (as adsorption and internalisation) was modelled using the Windermere Humic Aqueous Model (WHAM, v6) by encoding the macroalga as a polyelectrolytic binding phase whose properties were defaulted to those of aqueous humics in the software database. By setting the “activity” of the binding phase to about 0.1 and systematically reducing the default constants for metal binding, the magnitude of metal uptake by U. lactuca was reproduced. However, for all metals the model predicted a reduction in algal uptake as a function of salinity that was not always observed experimentally. Moreover, calculations performed in the presence of aqueous humic substances and using the earlier fitted constants significantly underestimated metal uptake by U. lactuca. Discrepancies between experimental observations and model calculations, which are attributed to the formation of ternary complexes at the algal surface, suggest that conventional equilibrium speciation considerations alone are not applicable for modelling metal interactions with marine macroalgae. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Marine macroalgae are important and abundant bioindicators of metal contamination in near shore coastal environments. Algae are able to concentrate essential and non-essential metals from ambient water by several orders of magnitude because of the density of surface functional sites and the binding capacity of intracellular ligands. For monitoring and, to some extent, predictive purposes, accumulation is often expressed in terms of a concentration factor, CF, defined as the w/w concentration ⁎ Corresponding author. Tel.: +44 1752 233041; fax: +44 1752 232406. E-mail address: [email protected] (A. Turner). 0304-4203/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2008.04.003
of metal in the organism divided by the w/v concentration of metal in the local water (Vasconcelos and Leal, 2001). From a chemical perspective, however, it is generally assumed that the free ionic form of aqueous metal is the most (or only) amenable form for accumulation by algae (Worms et al., 2006), rendering CF a rather conditional parameter. More suitable chemical models that consider the role of aqueous speciation on metal bioavailability include the free ion activity model (FIAM) and biotic ligand model (BLM), but these do not appear to have been applied to marine macroalgae. On the basis of equilibrium speciation, we would predict that the uptake of metal ions at the algal surface, hence their net accumulation, is inhibited in the presence of competing (aqueous)
A. Turner et al. / Marine Chemistry 110 (2008) 176–184
inorganic and organic ligands. Accordingly, we would expect that metal uptake by marine macroalgae exhibits a dependence on both salinity and the presence of natural, heterogeneous polyelectrolytes, like humic and fulvic substances. However, results of recent experiments employing fresh water microalgae and marine invertebrates suggest that equilibrium speciation alone does not always form the basis of a successful model because some metals appear to form ternary complexes at the cell surface, while certain ligands can alter the permeability of the cell membrane (Lamelas and Slaveykova, 2007; Sanchez-Marin et al., 2007). In the present study, we examine the uptake of a range of trace metals by the marine macroalga, Ulva lactuca. This species is often employed as a bioindicator of metals (e.g. El-Moselhy and Gabal, 2004; Daby, 2006; Chaudhuri et al., 2007) because it is widely distributed, able to withstand moderately polluted conditions and, being just two cells thick, has a relatively high surface area to volume ratio (Ho, 1990). The metals we study (Pd, Cd, Hg, Pb) have high anthropic signatures but no clearly established biological function in marine macroalgae. Experiments are performed in mixtures of synthetic sea water and pure water over a range of salinities representative of the habitat of U. lactuca (Wang and Dei, 1999), and in the presence and absence of a commercial humic substance. The aqueous speciation of the metals is computed using the Windermere Humic Aqueous Model (WHAM, v6), and adsorption and internalisation by the alga, configured as a polyelectrolyte, is modelled via surface complexation and a strong binding function, respectively. Experimental observations and speciation calculations allow us to evaluate whether conventional equilibrium considerations alone are appropriate for defining metal uptake by marine macroalgae. 2. Materials and methods 2.1. Reagents and experimental medium preparation
All plasticware used in the experiments and for the processing, storage and analysis of samples was soaked in 10% HCl for at least 24 h and subsequently rinsed with Milli-Q water (MQW). Unless otherwise stated, reagents employed were purchased from VWR International, Sigma-Aldrich or Fisher Scientific and were of VWR AnalaR or Aristar grade or equivalent. Filtered (b 5 μm) seawater collected from Plymouth Sound at high water was used for rinsing and acclimation of the macroalgae, and a synthetic medium based on Aquil (see below) was employed for all experimental work. A mixed working standard of 100 μM of each metal (or about 10 mg L− 1 of Cd and Pd and 20 mg L− 1 of Hg and Pb) was prepared by dilution of analytical standards in 0.1 M HCl, and a concentrated stock solution of humics was prepared by dissolving 500 mg of Acros Organics humic acid sodium salt (equivalent to about 300 mg of humic acid) in 1 L of MQW. Synthetic sea water (S=35), and nutrient and minor (trace) element solutions were prepared separately by diluting the appropriate salts in MQW. We followed the recipe given by Morel et al. (1979) in all cases but did not add EDTA to the minor element solution. The chelating agent was omitted because we required an inorganic medium in one experiment, and a medium with an aqueous polyelectrolyte (humic) as the sole organic complexant in a second experiment. A salinity range representative of the habitat of U. lactuca (S=15 to 35; Wang and Dei, 1999) was attained in both experiments by mixing synthetic sea water and MQW in different proportions. A constant concentration of the remaining components was obtained by the subsequent addition of fixed quantities of nutrient and minor element solutions to each mixture. 2.2. Sampling and sample preparation
U. lactuca was collected from the intertidal rock pools of a protected area of coastline (Wembury, SW England) during late spring (6/07).
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Samples were transported to the laboratory in zip-locked plastic bags and subsequently rinsed with filtered sea water to remove epibionts and debris. Samples were then transferred to 12 L clear plastic tanks filled with aerated sea water and maintained at 15 °C and under 250 μmol m− 2 s− 1 photosynthetic active radiation on a 12:12 h cycle for about 48 h. Discs of U. lactuca of 14 mm in diameter and about 6 mg in dry weight were cut, as required, from the central thallus using a sharpened, polypropylene cylinder, and were allowed to acclimatize for a further 24 h in clean sea water. Immediately before experimental use, discs were conditioned in full strength synthetic sea water for a few minutes. 2.3. Experimental
Short-term, static experiments were designed to investigate the effects of salinity on metal uptake by U. lactuca, both in an organic-free system and in the presence of 3 mg L− 1 of humic substances. The two experiments were performed at five salinities and in quadruplicate in a series of 150 mL polyethylene terephthalate beakers. Two discs of U. lactuca, a 50 μL spike of mixed metal standard and, where appropriate, a 500 μL spike of concentrated humic stock were added to 50 mL of water and the contents incubated on an orbital shaker at 100 rpm under the conditions described above for a period of 24 h. Controls, comprising water and algal discs without added metal, were performed under each set of conditions. The pH of each beaker was measured about 10 min after metal had been added and just before the termination of the incubations. The effects of metals on photosynthetic performance were assessed in both experiments by measuring the chlorophyll fluorescence of each disc before exposure and at the end of the incubation period using the Hansatech fluorescence monitoring system (FMS). Disks were initially dark-adapted for 15 min before readings of Fm, the maximum fluorescence yield, and Fo, the initial fluorescence yield, were recorded. The maximum quantum efficiency of PSII in the dark-adapted state is expressed as the ratio of variable to maximal chlorophyll fluorescence (Fv/Fm), derived from (Fm − Fo) / Fo (Maxwell and Johnson, 2000). At the end of the experiments, 1 mL of water from each beaker was pipetted into a polyethylene conical tube containing 9 mL of 0.1 M HCl. Both discs were carefully retrieved, from either the beaker or the FMS, using plastic tweezers. One disc from each beaker was immediately freeze-dried in a plastic specimen bag and subsequently weighed into a 60 mL screw-capped Teflon digestion vessel. The disc was then microwave-digested in 3 mL of concentrated HNO3 using a CEM-2000 microwave on medium power for 10 min, and the digested contents transferred to a polyethylene conical tube and diluted to 10 mL using MQW. The second disc from each beaker was rinsed in 10 mL of 3 mM EDTA in 0.6 M NaCl for 15 min to release adsorbed metal (Hassler et al., 2004) prior to its complete digestion in HNO3 as above. The efficacy of the HNO3 digestion and accuracy of subsequent analyses was evaluated by triplicate digestions of a certified reference material (U. lactuca; BCR279, IRMM). 2.4. Metal analysis
Palladium, Cd, Hg and Pb were analysed in all diluted aqueous samples and HNO3 digests by inductively coupled plasma-mass spectrometry using a Thermoelemental PQ2+. Ten μg L− 1 of both 115 In and 193Ir were added to all standards and samples to correct for variations in plasma conditions and drift in detector sensitivity, and the instrument was calibrated in the range 0.5 to 15 μg L− 1 for each metal using mixed standards prepared in 0.1 M HNO3. A standard was run as a check after every ten samples, and the instrument was flushed with 0.1 M HNO3 between samples and standards. Cadmium, Hg and Pb were not detected in diluted aqueous controls. However, background Cd and Pb in the alga and molecular ion interferences with Pd during analysis were corrected for in the samples by subtracting signals registered in the appropriate controls. Analysis of the digested reference alga revealed concentrations of Cd and Pb of 2.41 ± 0.27 and of 63.5 ± 0.63 nmol g− 1, respectively, compared with respective certified values of 2.51 ± 0.18 and 64.8 ± 1.0 nmol g− 1. Palladium and Hg are not certified for this material, and were not detectable from our analysis of the digests.
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2.5. Speciation modelling
Table 2 Binding constants used to model metal uptake by U. lactuca in WHAM
The aqueous equilibrium speciation of the metals in the experiments was computed using the Windermere Humic Aqueous Model (WHAM, v6). The chemical composition of synthetic sea water and MQW dilutions thereof was used to define the ionic composition of the samples. Fixed concentrations of the appropriate nutrient trace metals (e.g. Fe and Zn), as competitors for humic binding, were also included. A temperature of 288 K and a pH of 8.3, based on measurements at the end of the incubations (see below), were employed and equilibrium with the atmosphere was assumed (pCO2 = 3.5 × 10− 4 atm). For the inorganic speciation computations, default stability constants were adopted. However, we added a constant for the formation of CdCl−3, and encoded thermodynamic data relevant to Pd speciation in saline solutions reported by van Middlesworth and Wood (1999) (Table 1). In the absence of specific information regarding the physicochemical characteristics and binding properties of the humic phase, default-mode values, based on a number of observations in the literature (Tipping, 1998), were employed. For the binding of Cd, Hg and Pb and their first hydrolysis products, default constants defining overall complexation at a variety of carboxylic and weakly acidic sites, log KMeHS, and the distribution of “strong” binding sites, ΔLK2, are given in Table 2. Since no such constants exist for Pd, they were estimated from empirical correlations based on the binding of other metals. Thus, log KPdHS = 3.34 (with a standard error of ± 0.22) was derived from extrapolation of the relationship between first hydrolysis constants and overall binding constants with the default humic phase, while ΔLK2 = 5.58 (with a standard error of ± 0.74) was obtained by extrapolating the relationship between this parameter and binding constants for metal-NH3 complexes (Tipping, 1998), thereby assuming that “strong” Pd-binding sites are nitrogeneous. Both the extended Debye–Hückel equation and the Davies equation were used to calculate ion activity coefficients, and broadly similar speciation results were obtained with either approach. However, despite a smaller range in ionic strength application, the Debye–Hückel equation appeared to give estimates of free ion contributions that were more consistent with those from previous, independent metal speciation computations (e.g. Turner et al., 1981; Bourg, 1983; Paalman et al., 1994), and results based on this equation are, therefore, presented below. 3. Results and discussion 3.1. Reactants and reaction conditions The salinity-distributions of pH, measured at the beginning and end of the incubations, are shown in Fig. 1. In the humic-free experiment, mean values of pH were originally between about 7 and 7.5 and did not display any clear dependence on salinity. In the presence of 3 mg L− 1 of aqueous (or colloidal) humics, mean, original values of pH were higher (between about 7.5 and 8.0) and displayed a small reduction with increasing salinity. By the end of the experiments, and despite ready gaseous exchange between the head space of the conTable 1 Stability constants for Cd (Turner et al., 1981) and Pd (van Middlesworth and Wood, 1999) added to the WHAM database for the equilibrium speciation calculations Reaction
log K
Cd2+ + 3Cl− ⇌ CdCl−3 Pd2+ + OH− ⇌ PdOH+ Pd2+ + 2OH− ⇌ Pd(OH)2 Pd2+ + 3OH− ⇌ Pd(OH)−3 Pd2+ + 4OH− ⇌ Pd(OH)2− 4 Pd2+ + Cl− ⇌ PdCl+ 2+ − Pd + 2Cl ⇌ PdCl2 Pd2+ + 3Cl− ⇌ PdCl−3 Pd2+ + 4Cl− ⇌ PdCl2− 4 Pd2+ + 3Cl− + OH− ⇌ PdCl3OH2−
2.4 12.1 23.4 26.2 26.9 5.1 8.3 10.9 11.8 18.2
Default humic binding
Adsorption to U. lactuca surface
Total uptake by U. lactuca
Metal
log KMeHS
ΔLK2
log KMeS
ΔLK2
log KMeS
ΔLK2
Pd Cd Hg Pb
3.34 1.30 3.50 2.00
5.58 1.48 5.10 0.93
1.93 1.30 2.48 1.36
0 0 0 0
1.93 1.30 2.48 1.36
2.45 1.20 3.10 1.10
tainers and the ambient atmosphere, pH had risen by between a half and one and a half units such that values were between about 8.2 and 8.5 in all cases. For the purposes of the discussion and modelling, an invariant, “equilibrium” pH of 8.3 is assumed throughout. Photosynthetic activity, determined during the acclimation period in filtered sea water and at the end of the incubations in the synthetic mixtures, is shown for both experiments in Fig. 2. In the presence of humics, activity had not changed compared with that determined during acclimation. In the humic-free incubations, however, the rate of photosynthesis had measurably increased from the period of acclimation to the end of the experiment. We suspect that this is related to the gradual depletion of nutrients in sea water used to acclimatize the discs employed in the humic-free system. By the end of the incubations, photosynthetic activity was statistically indistinguishable (p N 0.05 according to one-way ANOVA) both within and between experiments. These observations suggest that varying salinity and the presence of 3 mg L− 1 of humic substances and/or 100 nM of Cd, Pd, Hg and Pb do not exert any adverse effects on the health of individual discs, at least according to the measure employed and within the timeframe of the experiments, or that any effects are offset by the nutrient amendment of the synthetic mixtures. 3.2. Aqueous metal speciation The aqueous equilibrium speciation of Cd, Pd, Hg and Pb in the experiments, as computed by WHAM, is shown in the absence and presence of aqueous humic substances in Figs. 3 and 4, respectively. The distributions of inorganic species of Cd and Hg are in good agreement with respective estuarine distributions computed using different software (Paalman et al., 1994; Leermakers et al., 1995). The inorganic speciation of Pb concords with distributions derived from defaulted for the Humber Estuary (UK) (Tipping et al., 1998). However, we note that there exist discrepancies in the relative abundances of carbonato- and chlorocomplexes of Pb as computed by WHAM and using other models (Turner et al., 1981; Bourg, 1983). With respect to Pd, the calculated inorganic speciation is in agreement with that presented by van Middlesworth and Wood (1999), whose constants were used in the present computations. However, it should be noted that there is some uncertainty about the relative magnitudes of the stability constants relevant to this metal in saline solutions. Specifically, other studies indicate that chloro-complexes are the dominant inorganic form in sea water rather than the neutral hydroxide and hydroxychloride, and that mixed chlorocarbanato-complexes may also be present (Cosden and Byrne, 2003). In the presence of a fixed concentration (3 mg L− 1) of humic substances, modelled from the default WHAM database, the speciation of Cd is largely unaffected since organic complexes were predicted to comprise less than one percent of total aqueous Cd. For the remaining metals, however, complexation by humics is highly significant, exceeding 65%, 95% and 99% throughout the salinity range for Pb, Pd and Hg, respectively. 3.3. Metal uptake by U. lactuca By the end of the experiments, metal concentrations in the aqueous phase, [Mew], ranged from about 30 to 80 nM for Pd, 70 to 100 nM for Cd, 40 to 60 nM for Hg and 50 to 80 nM for Pb. Respective concentrations in U. lactuca, [Mealga], normalised to the dry mass of solid, malga, ranged from about 10 to 50 nmol g− 1, b 5 to 30 nmol g− 1, 50 to 100 nmol g− 1 and 10 to 25 nmol g− 1. Results are shown in Fig. 5 as a
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Fig. 1. Initial (▲) and final (■) pH measured in (a) the humic-free incubations and (b) the presence of 3 mg L− 1 of aqueous humics. Error bars represent the standard deviation about the mean of four independent experimental measurements.
function of salinity and in the absence and presence of humics in terms of a concentration factor, CF: CF ¼
Mealga =malga ½Mew
ð1Þ
Mean values of CF range from about 102 mL g− 1 for Cd to in excess of 103 mL g− 1 for Hg, but in some cases replicate results exhibit considerable variation, with relative standard deviations of CF up to 40%. Presumably, such variations are a reflection of analytical errors, an inherent heterogeneity in the chemical and surface composition of the macroalga (and, with respect to Pb, background concentrations), and small variations in “equilibrium” pH. A reduction in CF for Cd with increasing salinity was observed in the present experiments, at least in the absence of humics, consistent with results reported by Wang and Dei (1999) using the same species of macroalga. A salinity dependence is also predicted for Pd, Hg and Pb on the basis of aqueous equilibrium speciation considerations (specifically, a reduction in both the concentration and activity of the respective free ions was computed with increasing salinity), and interactions with other, abiotic surfaces (Turner, 1996, 2007a). However, we suspect that the precision of the results has precluded detection of such a dependence in the present experiments. A reduction in the biouptake of Pd, Hg and Pb is also predicted in the presence of humics because of the competing effects of the aqueous complexant (Fig. 4). A statistically significant reduction (p b 0.05 according to a two sample t-test) was observed for Pd throughout the salinity range and for Hg in three cases. For Pb, however, an increase in uptake was observed at four out of five salinities. Metal taken up by U. lactuca may be adsorbed at the algal surface or internalised within the cytosol of component cells. Metal adsorption involves surface complexation, while internalisation may proceed subsequent to surface complexation, or independently as a passive
mechanism if metal complexes are sufficiently lipophilic. Particularly significant with respect to the latter is the neutral chloride of Hg, whose octanol-water partition coefficient is around 3 (Mason et al., 1996) and which makes a substantial contribution to the aqueous speciation of Hg in the absence of humics (Fig. 3). We operationally discriminated intracellular uptake and surface adsorption by washing one of the two discs from each incubation in 3 mM EDTA prior to its total digestion and analysis for metals. The results revealed no clear dependence of internalised metal (i.e. that resistant to EDTA washing) on salinity and are, therefore, pooled for each metal, both in the absence and presence of aqueous humics, in Fig. 6. Mean values of internalisation follow the sequence:HgN PdN CdN Pb; under both sets of conditions. The presence of humics exerted no significant influence on intracellular uptake of Pd, Cd and Hg, but appeared to greatly inhibit the internalisation of Pb. 3.4. Modelling metal adsorption by U. lactuca Since the algal surface bears many similarities with an aqueous or colloidal polyelectrolyte (e.g. humic substances), in particular in respect of its heterogeneous metal-binding properties (Xue et al., 1988; Koelmans et al., 1996; Kaulbach et al., 2005), we modelled metal adsorption by U. lactuca in WHAM by encoding the alga as a separate binding phase (or a biotic ligand). Complexation of a divalent metal ion, Me2+ (and its first hydrolysis product; MeOH+), at the algal surface, ≡S, was defined by the following general reaction (Xue et al., 1988): uSH þ Me2þ
K MeS
X
uSMeþ þ Hþ
ð2Þ
where the overall equilibrium constant, KMeS, encompasses binding at a variety of sites and is equivalent to the overall constant, KMeHS, defining metal binding by humic substances in WHAM. In the absence of additional information, we assumed that the surface properties of U.
Fig. 2. Photosynthetic activity of U. lactuca measured at the end of the incubations in (a) the absence of aqueous humics and (b) the presence of 3 mg L− 1 of humics. Error bars represent the standard deviation about the mean of four independent experimental measurements; broken lines define mean activities measured during the corresponding acclimation periods in filtered sea water.
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Fig. 3. Calculated salinity-dependent aqueous equilibrium speciation of Pd, Cd, Hg and Pb in the humic-free experiment.
lactuca (e.g. acidity constants, binding site density) were equivalent to those of the default humic substances contained in the WHAM database and based on averaged values in the literature (Tipping,1998). For a given metal, adsorption at the algal surface was modelled using a single, overall binding constant (Table 2), but the distributional term for strong surface binding, ΔLK2, was neglected (i.e. strong, specific binding is ignored). The concentration of metal adsorbed by U. lactuca in the experiments, [Meads], was evaluated from the chemical measure described
above (i.e. total metal taken up, [Mealga], minus internalised metal, [Meint]). Thus, neglecting internalised metal (see below), the percentage of metal adsorbed at equilibrium was calculated as follows: k adsorbed ¼
½Meads 100k ½Meads þ ½Mew
ð3Þ
and is plotted against salinity in Fig. 7. Initial fitting of adsorption data involved Cd because its uptake by phytoplankton in saline suspensions
Fig. 4. Calculated salinity-dependent aqueous equilibrium speciation of Pd, Cd, Hg and Pb in the experiment conducted in the presence of 3 mg L− 1 of aqueous humics.
A. Turner et al. / Marine Chemistry 110 (2008) 176–184
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Fig. 5. Concentration factors defining the uptake of Cd, Pd, Hg and Pb by U. lactuca as a function of salinity in the humic-free experiment (□) and in the presence of 3 mg L− 1 of aqueous humics (■). Error bars represent the standard deviation about the mean of four independent experimental measurements. Note that data for Cd in the humic system are incomplete because of detection problems in some algal samples.
has previously been modelled by surface complexation (Koelmans et al., 1996) and default binding constants for this metal with humic substances are based on a relatively large dataset that exhibits little dispersion (Tipping, 1998). Thus, log KCdHS was employed to define interactions of Cd with the algal surface, and the percentage of “active” U. lactuca was then adjusted in order to give the best fit to the observed adsorption. Activity is defined as the proportion of the total mass of a substance that participates in metal binding, and a value of 0.091 (equivalent to a net binding phase concentration of 22 mg L− 1) was able to reproduce both the extent (between about 1 and 4%) and salinity-dependence of Cd adsorption with reasonable success. Our estimate of activity is low
compared with that of natural dissolved organic matter (around 0.5; Tipping et al., 1998), presumably reflecting the relatively large mass contribution of the inert cellular matrix. For the remaining metals, the same surface properties and activity of the alga were employed, and default metal binding constants were subsequently manipulated individually in order to fit the data by eye. The results of this exercise are annotated on Fig. 7, and the constants used to generate such are given in Table 2. Thus, by “calibrating” metal binding with respect to the default constants for Cd, a reduction in the default binding constants for the remaining metals was necessary to attain fits of the correct order of magnitude. However, because of the effects of ionic
Fig. 6. Fraction of metal internalised by U. lactuca in the humic-free experiment (□) and in the presence of 3 mg L− 1 of aqueous humics (■). Error bars represent the standard deviation about the mean of 20 measurements.
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strength and complexation by seawater anions, a decrease in the uptake of all metals as a function of salinity was predicted by the WHAM software that was not observed experimentally (see above).
well with the measured, internalised fractions of metal, but are not proportional to the respective constants defining strong metal binding by the default humic substances (see also Table 2).
3.5. Modelling metal internalisation by U. lactuca
3.6. Modelling metal uptake by U. lactuca in the presence of aqueous humics
With the surface binding constants derived above, we then attempted to model total metal uptake by U. lactuca by incorporating an internalisation phase. The latter may be considered as a first-order process successive to surface complexation: uSH þ Me2þ
k
K MeS
uSMeþ þ Hþ Y Meint
X
ð4Þ
where Meint represents internalised metal and k is a first-order rate constant, or as an independent, passive mechanism: MeL
K int
X
ð5Þ
MeLint
where L represents a ligand forming a lipophilic complex with a metal ion and Kint is an equilibrium constant defining passive uptake. Using WHAM, we modelled total uptake of metal as an equilibrium process, and simulated internalisation as strong binding at the algal surface and parameterized by the distributional term, ΔLK 2. Although internalisation and strong surface binding are, mechanistically, quite different, the rationale for this approach is that (i) binding at specific sites on the algal surface is believed to precede internalisation (Galceran et al., 2006), and (ii) the two processes bear some mathematical similarity. The percentage of metal taken up by U. lactuca (i.e. adsorbed and internalised) was calculated as follows:
k uptake ¼
Mealga 100k Mealga þ ½Mew
ð6Þ
We used the fitted values derived above to define the binding characteristics of the alga, together with the properties and constants of aqueous humic substances contained in the WHAM default database (Table 2) to compute metal uptake by U. lactuca in the presence of the aqueous polyelectrolyte. The results of the calculations are not shown, but revealed the following. Cadmium uptake by U. lactuca was little affected by the addition of aqueous humics, consistent with experimental observations reported above. For the remaining metals, however, binding by aqueous humics was so intense that the computed uptake by the alga was considerably less than that determined experimentally. For example, bb1% of Pd and Hg was predicted to be taken up (adsorbed plus internalised) under these conditions, but up to 20% removal from the aqueous phase was observed. The broad findings of this exercise were not significantly altered by adjusting the properties and constants of the default aqueous humic phase within realistic limits (Tipping, 1998), nor by “calibrating” the surface characteristics of the alga with respect to a metal other than Cd (recent evidence suggests that, in some cases, humic binding by Cd is overestimated in the WHAM default mode; Turner et al., 2008). Thus, while the discrepancies between observed and modelled results (including salinity dependence) are partly attributable to experimental and analytical variability and the limitations and assumptions of the computations and fitting, more important appear to be factors related to the general problems inherent in modelling metal uptake by marine macroalgae. 3.7. Difficulties in modelling metal uptake by macroalgae
and is also plotted in Fig. 7. Best eye-fits to these data were obtained using the binding constants derived above to define the adsorption process, and the values of ΔLK2 given in Table 2. The latter correlate
The approach to modelling metal uptake by U. lactuca that we have adopted is based on complexation with relatively weak sites on the
Fig. 7. Percentage of metal taken up (■; calculated using Eq. (6)) and adsorbed (△; calculated using Eq. (3)) by U. lactuca in the humic-free experiment. Error bars represent the standard deviation about the mean of four independent experimental measurements. Solid lines represent best eye-fits to the data modelled using WHAM and the constants reported in Table 2.
A. Turner et al. / Marine Chemistry 110 (2008) 176–184
algal surface, whose chemical characteristics are assumed to bear some semblance to natural aqueous or colloidal polyelectrolytes, and strong complexation as an analogue of (or precursor to) internalisation. Calculations and data-fitting based on this approach would suggest that the algal surface is less efficient in metal binding than aqueous polyelectrolytes in terms of both the density (activity) and binding capacity of its functional groups. However, it is important to appreciate that the constants derived are conditional because of the potential confounding effects of slow coordination-dissociation kinetics (Turner, 2007b) and competition for metal ions from algal exudates. Intracellular molecules released to the external medium may serve as natural algal antifoulants (Harder et al., 2004) or be a proportional defensive response to an increase in contaminant loading in the external medium (Vasconcelos and Leal, 2001), but generally have some propensity to bind, often strongly, with trace metals (Scoullos et al., 2004). Although complexation by exudates may result in an underestimation of true algal binding constants for Pd and Hg, this effect alone cannot account for the magnitude of the discrepancies between observed and modelled results in the presence of aqueous humics (Masakorala et al., in press). Specifically, under otherwise identical conditions, why should the presence of a highly active aqueous polyelectrolyte not have a greater, negative impact on the uptake of the most strongly complexing metals, Pd and Hg, by U. lactuca? Moreover, why is the adsorption of Pb enhanced but its internalisation inhibited in the presence of aqueous humics? Results of recent research regarding the uptake of Pb by fresh water microalgae and marine invertebrates are significant in this respect (Lamelas and Slaveykova, 2007; Sanchez-Marin et al., 2007; Slaveykova, 2007). Thus, enhanced uptake in the presence of humic or fulvic substances has been attributed to (i) the formation of a ternary complex involving either the adsorption of Pb onto adsorbed humics or the adsorption of a Pb-humic complex, and (ii) an increase in cell permeability, hence propensity for passive diffusion of the metal, incurred by the surfactancy of the humic molecule itself. These effects appear to exhibit some specificity among the metals that have been studied to date, such that Cd and Cu are not regulated by the same controls (Vigneault and Campbell, 2005; Lamelas and Slaveykova, 2007). Regarding our experiments with U. lactuca, we surmise that the stabilising effects of aqueous humic substances are partly (Pd and Hg) or more than (Pb) offset by the uptake of ternary complexes at the algal surface, a mechanism that cannot be directly modelled using software such as WHAM. With respect to Pb at least, and in contrast to results obtained using freshwater algae (Vigneault and Campbell, 2005), a reduction in passive, diffusive uptake appears to be a consequence of this effect; in other words, ternary complexation involves metal that would otherwise be available for internalisation. Since these observations have important, general implications regarding our understanding of and ability to model metal uptake by marine macroalgae, further studies in this area are necessary.
4. Conclusions Of the trace metals examined in the present study, only Cd uptake appeared to be measurably and systematically affected by variations in salinity that are representative of the habitat of U. lactuca. Of more general importance among the remaining, more strongly binding metals (Pd, Hg and Pb), is the presence of polyelectrolytic aqueous organic matter (humics). However, the effects of humic substances are quantitatively inconsistent with equilibrium speciation calculations performed in WHAM; specifically, observed uptake of Pd, Hg and Pb by U. lactuca far exceeds that predicted from the competitive effects of the algal surface and aqueous polyelectrolyte for a given metal ion. Our observations concord with recent, independent experimental results derived in the presence of microalgae and invertebrates, and further support assertions that current models of metalorganism interactions (e.g. FIAM and BLM) may not be generally applicable where additional processes, such as ternary complexation, are significant.
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Acknowledgement We thank Miss Angela Watson for assistance with the medium preparation and incubations, and Dr. Andy Fisher for help with the ICP-MS analyses. The comments of Dr. Steve Lofts (CEH, Lancaster) and two anonymous reviewers were greatly appreciated. References Bourg, A.C.M., 1983. Role of fresh water/sea water mixing on trace metal adsorption phenomena. In: Wong, C.S., Burton, J.D., Boyle, E., Bruland, K., Goldberg, E.D. (Eds.), Trace Metals in Sea Water. Plenum, New York, pp. 195–208. Chaudhuri, A., Mitra, M., Havrilla, C., Waguespack, Y., Schwarz, J., 2007. Heavy metal biomonitoring by seaweeds on the Delmarva Peninsula, east coast of the USA. Bot. Mar. 50, 151–158. Cosden, J.M., Byrne, R.H., 2003. Comparative geochemistries of PdII and PtII: formation of mixed hydroxychloro and chlorocarbanato-complexes in seawater. Geochim. Cosmochim. Acta 67, 1331–1338. Daby, D., 2006. Coastal pollution and potential biomonitors of metals in Mauritius. Water Air Soil Pollut. 174, 63–91. El-Moselhy, K.M., Gabal, M.N., 2004. Trace metals in water, sediments and marine organisms from the northern part of the of the Gulf of Suez, Red Sea. J. Mar. Syst. 46, 39–46. Galceran, J., Monne, J., Puy, J., van Leeuwen, H.P., 2006. Transient biouptake flux and accumulation by microorganisms: the case of two types of sites with Langmuir adsorption. Mar. Chem. 99, 162–176. Harder, T., Dobretsov, S., Qian, P.Y., 2004. Waterborne polar macromolecules act as algal antifoulants in the seaweed Ulva reticulata. Mar. Ecol. Prog. Ser. 274, 133–141. Hassler, C.S., Slaveykova, V.I., Wilkinson, K.J., 2004. Discriminating between intra- and extracellular metals using chemical extractions. Limnol. Oceanogr. Methods 2, 237–247. Ho, Y.B., 1990. Ulva lactuca as bioindicator of metal contamination in intertidal waters in Hong Kong. Hydrobiologia 203, 73–81. Kaulbach, E.S., Szymanowski, J.E.S., Fein, J.B., 2005. Surface complexation modelling of proton and Cd adsorption onto an algal cell wall. Environ. Sci. Technol. 39, 4060–4065. Koelmans, A.A., Gillissen, F., Lijklema, L., 1996. Influence of salinity and mineralization on trace metal sorption to cyanobacteria in natural waters. Water Res. 30, 853–864. Lamelas, C., Slaveykova, V.I., 2007. Comparison of Cd(II), Cu(II), and Pb(II) biouptake by green algae in the presence of humic acid. Environ. Sci. Technol. 41, 4172–4178. Leermakers, M., Meuleman, C., Baeyens, W., 1995. Mercury speciation in the Scheldt estuary. Water Air Soil Pollut. 80, 641–652. Masakorala, K., Turner, A., Brown, M.T., in press. Influence of synthetic surfactants on the uptake of Pd, Cd and Pb by the marine macroalga, Ulva lactuca. Environ. Pollut. Mason, R.P., Reinfelder, J.R., Morel, F.M.M., 1996. Uptake, toxicity, and trophic transfer of mercury in a coastal diatom. Environ. Sci. Technol. 30, 1835–1845. Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence — a practical guide. J. Exp. Bot. 5, 659–668. Morel, F.M.M., Rueter, J.G., Anderson, D.M., Guillard, R.R.L., 1979. Aquil — chemically defined phytoplankton culture medium for trace metal studies. J. Phycol. 15, 135–141. Paalman, M.A.A., van der Weijden, C.H., Loch, P.G., 1994. Sorption of cadmium on suspended matter under estuarine conditions; competition and complexation with major sea water ions. Water Air Soil Pollut. 73, 49–60. Sanchez-Marin, P., Lorenzo, J.I., Blust, R., Beiras, R., 2007. Humic acids increase dissolved lead bioavailability for marine invertebrates. Environ. Sci. Technol. 41, 5679–5684. Scoullos, M., Plavsic, M., Karavoltsos, S., 2004. Speciation studies of copper in the Gulf of Elefsis: the role of the macroalgae Ulva rigida. Mar. Chem. 86, 51–63. Slaveykova, V.I., 2007. Predicting Pb bioavailability to freshwater microalgae in the presence of fulvic acid: algal cell density as a variable. Chemosphere 69, 1438–1445. Tipping, E., 1998. Humic ion-binding model VI: an improved description of the interactions of protons and metal ions with humic substances. Aquat. Geochem. 4, 3–48. Tipping, E., Lofts, S., Lawlor, A.J., 1998. Modelling the chemical speciation of trace metals in the surface waters of the Humber system. Sci. Total Environ. 210/211, 63–77.
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Turner, A., 1996. Trace-metal partitioning in estuaries: importance of salinity and particle concentration. Mar. Chem. 54, 27–39. Turner, A., 2007a. Particle–water interactions of platinum group elements under estuarine conditions. Mar. Chem. 103, 103–111. Turner, A., 2007b. A binary aqueous component model for the sediment– water partitioning of trace metals in natural waters. Environ. Sci. Technol. 41, 3977–3983. Turner, D.R., Whitfield, M., Dickson, A.G., 1981. The equilibrium speciation of dissolved components in freshwater and seawater at 25 °C and 1 atm pressure. Geochim. Cosmochim. Acta 45, 855–881. Turner, A., Le Roux, S.M., Millward, G.E., 2008. Adsorption of cadmium to iron and manganese oxides during estuarine mixing. Mar. Chem. 108, 77–84. van Middlesworth, J.M., Wood, S.A., 1999. The stability of palladium(II) hydroxide and hydroxy-chloride complexes: an experimental solubility study at 25–85 °C and 1 bar. Geochim. Cosmichim. Acta 63, 1751–1765.
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Marine Chemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a r c h e m
Influence of salinity and humic substances on the uptake of trace metals by the marine macroalga, Ulva lactuca: Experimental observations and modelling using WHAM Andrew Turner a,⁎, Silvia S. Pedroso a, Murray T. Brown b a b
School of Earth, Ocean, and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA, UK School of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK
a r t i c l e
i n f o
Article history: Received 17 December 2007 Received in revised form 2 April 2008 Accepted 9 April 2008 Keywords: Macroalgae Trace metals Biouptake Salinity Humics Speciation modelling
a b s t r a c t Uptake of the trace metals, Pd, Cd, Hg and Pb, by the marine macroalga, Ulva lactuca, has been studied along a salinity gradient (S = 15–35; pH~ 8.3) created by batch mixing of synthetic sea water and pure water, both in the absence and presence of humic substances. Factors defining the concentration ratio of metal taken up (w/w) to metal remaining in solution ranged from about 102 mL g− 1 for Cd to 103 mL g− 1 for Pd and Hg. Within experimental error, only the biouptake of Cd appeared to exhibit a dependence on salinity, while the addition of 3 mg L− 1 of humics resulted in a small suppression of Pd and Hg uptake and a moderate enhancement of Pb uptake compared with the humic-free system. Metal internalisation, evaluated from an EDTA wash of the alga, followed the sequence: HgN Pd N Cd N Pb; and was notably inhibited in the presence of humics for Pb. Metal uptake (as adsorption and internalisation) was modelled using the Windermere Humic Aqueous Model (WHAM, v6) by encoding the macroalga as a polyelectrolytic binding phase whose properties were defaulted to those of aqueous humics in the software database. By setting the “activity” of the binding phase to about 0.1 and systematically reducing the default constants for metal binding, the magnitude of metal uptake by U. lactuca was reproduced. However, for all metals the model predicted a reduction in algal uptake as a function of salinity that was not always observed experimentally. Moreover, calculations performed in the presence of aqueous humic substances and using the earlier fitted constants significantly underestimated metal uptake by U. lactuca. Discrepancies between experimental observations and model calculations, which are attributed to the formation of ternary complexes at the algal surface, suggest that conventional equilibrium speciation considerations alone are not applicable for modelling metal interactions with marine macroalgae. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Marine macroalgae are important and abundant bioindicators of metal contamination in near shore coastal environments. Algae are able to concentrate essential and non-essential metals from ambient water by several orders of magnitude because of the density of surface functional sites and the binding capacity of intracellular ligands. For monitoring and, to some extent, predictive purposes, accumulation is often expressed in terms of a concentration factor, CF, defined as the w/w concentration ⁎ Corresponding author. Tel.: +44 1752 233041; fax: +44 1752 232406. E-mail address: [email protected] (A. Turner). 0304-4203/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2008.04.003
of metal in the organism divided by the w/v concentration of metal in the local water (Vasconcelos and Leal, 2001). From a chemical perspective, however, it is generally assumed that the free ionic form of aqueous metal is the most (or only) amenable form for accumulation by algae (Worms et al., 2006), rendering CF a rather conditional parameter. More suitable chemical models that consider the role of aqueous speciation on metal bioavailability include the free ion activity model (FIAM) and biotic ligand model (BLM), but these do not appear to have been applied to marine macroalgae. On the basis of equilibrium speciation, we would predict that the uptake of metal ions at the algal surface, hence their net accumulation, is inhibited in the presence of competing (aqueous)
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inorganic and organic ligands. Accordingly, we would expect that metal uptake by marine macroalgae exhibits a dependence on both salinity and the presence of natural, heterogeneous polyelectrolytes, like humic and fulvic substances. However, results of recent experiments employing fresh water microalgae and marine invertebrates suggest that equilibrium speciation alone does not always form the basis of a successful model because some metals appear to form ternary complexes at the cell surface, while certain ligands can alter the permeability of the cell membrane (Lamelas and Slaveykova, 2007; Sanchez-Marin et al., 2007). In the present study, we examine the uptake of a range of trace metals by the marine macroalga, Ulva lactuca. This species is often employed as a bioindicator of metals (e.g. El-Moselhy and Gabal, 2004; Daby, 2006; Chaudhuri et al., 2007) because it is widely distributed, able to withstand moderately polluted conditions and, being just two cells thick, has a relatively high surface area to volume ratio (Ho, 1990). The metals we study (Pd, Cd, Hg, Pb) have high anthropic signatures but no clearly established biological function in marine macroalgae. Experiments are performed in mixtures of synthetic sea water and pure water over a range of salinities representative of the habitat of U. lactuca (Wang and Dei, 1999), and in the presence and absence of a commercial humic substance. The aqueous speciation of the metals is computed using the Windermere Humic Aqueous Model (WHAM, v6), and adsorption and internalisation by the alga, configured as a polyelectrolyte, is modelled via surface complexation and a strong binding function, respectively. Experimental observations and speciation calculations allow us to evaluate whether conventional equilibrium considerations alone are appropriate for defining metal uptake by marine macroalgae. 2. Materials and methods 2.1. Reagents and experimental medium preparation
All plasticware used in the experiments and for the processing, storage and analysis of samples was soaked in 10% HCl for at least 24 h and subsequently rinsed with Milli-Q water (MQW). Unless otherwise stated, reagents employed were purchased from VWR International, Sigma-Aldrich or Fisher Scientific and were of VWR AnalaR or Aristar grade or equivalent. Filtered (b 5 μm) seawater collected from Plymouth Sound at high water was used for rinsing and acclimation of the macroalgae, and a synthetic medium based on Aquil (see below) was employed for all experimental work. A mixed working standard of 100 μM of each metal (or about 10 mg L− 1 of Cd and Pd and 20 mg L− 1 of Hg and Pb) was prepared by dilution of analytical standards in 0.1 M HCl, and a concentrated stock solution of humics was prepared by dissolving 500 mg of Acros Organics humic acid sodium salt (equivalent to about 300 mg of humic acid) in 1 L of MQW. Synthetic sea water (S=35), and nutrient and minor (trace) element solutions were prepared separately by diluting the appropriate salts in MQW. We followed the recipe given by Morel et al. (1979) in all cases but did not add EDTA to the minor element solution. The chelating agent was omitted because we required an inorganic medium in one experiment, and a medium with an aqueous polyelectrolyte (humic) as the sole organic complexant in a second experiment. A salinity range representative of the habitat of U. lactuca (S=15 to 35; Wang and Dei, 1999) was attained in both experiments by mixing synthetic sea water and MQW in different proportions. A constant concentration of the remaining components was obtained by the subsequent addition of fixed quantities of nutrient and minor element solutions to each mixture. 2.2. Sampling and sample preparation
U. lactuca was collected from the intertidal rock pools of a protected area of coastline (Wembury, SW England) during late spring (6/07).
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Samples were transported to the laboratory in zip-locked plastic bags and subsequently rinsed with filtered sea water to remove epibionts and debris. Samples were then transferred to 12 L clear plastic tanks filled with aerated sea water and maintained at 15 °C and under 250 μmol m− 2 s− 1 photosynthetic active radiation on a 12:12 h cycle for about 48 h. Discs of U. lactuca of 14 mm in diameter and about 6 mg in dry weight were cut, as required, from the central thallus using a sharpened, polypropylene cylinder, and were allowed to acclimatize for a further 24 h in clean sea water. Immediately before experimental use, discs were conditioned in full strength synthetic sea water for a few minutes. 2.3. Experimental
Short-term, static experiments were designed to investigate the effects of salinity on metal uptake by U. lactuca, both in an organic-free system and in the presence of 3 mg L− 1 of humic substances. The two experiments were performed at five salinities and in quadruplicate in a series of 150 mL polyethylene terephthalate beakers. Two discs of U. lactuca, a 50 μL spike of mixed metal standard and, where appropriate, a 500 μL spike of concentrated humic stock were added to 50 mL of water and the contents incubated on an orbital shaker at 100 rpm under the conditions described above for a period of 24 h. Controls, comprising water and algal discs without added metal, were performed under each set of conditions. The pH of each beaker was measured about 10 min after metal had been added and just before the termination of the incubations. The effects of metals on photosynthetic performance were assessed in both experiments by measuring the chlorophyll fluorescence of each disc before exposure and at the end of the incubation period using the Hansatech fluorescence monitoring system (FMS). Disks were initially dark-adapted for 15 min before readings of Fm, the maximum fluorescence yield, and Fo, the initial fluorescence yield, were recorded. The maximum quantum efficiency of PSII in the dark-adapted state is expressed as the ratio of variable to maximal chlorophyll fluorescence (Fv/Fm), derived from (Fm − Fo) / Fo (Maxwell and Johnson, 2000). At the end of the experiments, 1 mL of water from each beaker was pipetted into a polyethylene conical tube containing 9 mL of 0.1 M HCl. Both discs were carefully retrieved, from either the beaker or the FMS, using plastic tweezers. One disc from each beaker was immediately freeze-dried in a plastic specimen bag and subsequently weighed into a 60 mL screw-capped Teflon digestion vessel. The disc was then microwave-digested in 3 mL of concentrated HNO3 using a CEM-2000 microwave on medium power for 10 min, and the digested contents transferred to a polyethylene conical tube and diluted to 10 mL using MQW. The second disc from each beaker was rinsed in 10 mL of 3 mM EDTA in 0.6 M NaCl for 15 min to release adsorbed metal (Hassler et al., 2004) prior to its complete digestion in HNO3 as above. The efficacy of the HNO3 digestion and accuracy of subsequent analyses was evaluated by triplicate digestions of a certified reference material (U. lactuca; BCR279, IRMM). 2.4. Metal analysis
Palladium, Cd, Hg and Pb were analysed in all diluted aqueous samples and HNO3 digests by inductively coupled plasma-mass spectrometry using a Thermoelemental PQ2+. Ten μg L− 1 of both 115 In and 193Ir were added to all standards and samples to correct for variations in plasma conditions and drift in detector sensitivity, and the instrument was calibrated in the range 0.5 to 15 μg L− 1 for each metal using mixed standards prepared in 0.1 M HNO3. A standard was run as a check after every ten samples, and the instrument was flushed with 0.1 M HNO3 between samples and standards. Cadmium, Hg and Pb were not detected in diluted aqueous controls. However, background Cd and Pb in the alga and molecular ion interferences with Pd during analysis were corrected for in the samples by subtracting signals registered in the appropriate controls. Analysis of the digested reference alga revealed concentrations of Cd and Pb of 2.41 ± 0.27 and of 63.5 ± 0.63 nmol g− 1, respectively, compared with respective certified values of 2.51 ± 0.18 and 64.8 ± 1.0 nmol g− 1. Palladium and Hg are not certified for this material, and were not detectable from our analysis of the digests.
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2.5. Speciation modelling
Table 2 Binding constants used to model metal uptake by U. lactuca in WHAM
The aqueous equilibrium speciation of the metals in the experiments was computed using the Windermere Humic Aqueous Model (WHAM, v6). The chemical composition of synthetic sea water and MQW dilutions thereof was used to define the ionic composition of the samples. Fixed concentrations of the appropriate nutrient trace metals (e.g. Fe and Zn), as competitors for humic binding, were also included. A temperature of 288 K and a pH of 8.3, based on measurements at the end of the incubations (see below), were employed and equilibrium with the atmosphere was assumed (pCO2 = 3.5 × 10− 4 atm). For the inorganic speciation computations, default stability constants were adopted. However, we added a constant for the formation of CdCl−3, and encoded thermodynamic data relevant to Pd speciation in saline solutions reported by van Middlesworth and Wood (1999) (Table 1). In the absence of specific information regarding the physicochemical characteristics and binding properties of the humic phase, default-mode values, based on a number of observations in the literature (Tipping, 1998), were employed. For the binding of Cd, Hg and Pb and their first hydrolysis products, default constants defining overall complexation at a variety of carboxylic and weakly acidic sites, log KMeHS, and the distribution of “strong” binding sites, ΔLK2, are given in Table 2. Since no such constants exist for Pd, they were estimated from empirical correlations based on the binding of other metals. Thus, log KPdHS = 3.34 (with a standard error of ± 0.22) was derived from extrapolation of the relationship between first hydrolysis constants and overall binding constants with the default humic phase, while ΔLK2 = 5.58 (with a standard error of ± 0.74) was obtained by extrapolating the relationship between this parameter and binding constants for metal-NH3 complexes (Tipping, 1998), thereby assuming that “strong” Pd-binding sites are nitrogeneous. Both the extended Debye–Hückel equation and the Davies equation were used to calculate ion activity coefficients, and broadly similar speciation results were obtained with either approach. However, despite a smaller range in ionic strength application, the Debye–Hückel equation appeared to give estimates of free ion contributions that were more consistent with those from previous, independent metal speciation computations (e.g. Turner et al., 1981; Bourg, 1983; Paalman et al., 1994), and results based on this equation are, therefore, presented below. 3. Results and discussion 3.1. Reactants and reaction conditions The salinity-distributions of pH, measured at the beginning and end of the incubations, are shown in Fig. 1. In the humic-free experiment, mean values of pH were originally between about 7 and 7.5 and did not display any clear dependence on salinity. In the presence of 3 mg L− 1 of aqueous (or colloidal) humics, mean, original values of pH were higher (between about 7.5 and 8.0) and displayed a small reduction with increasing salinity. By the end of the experiments, and despite ready gaseous exchange between the head space of the conTable 1 Stability constants for Cd (Turner et al., 1981) and Pd (van Middlesworth and Wood, 1999) added to the WHAM database for the equilibrium speciation calculations Reaction
log K
Cd2+ + 3Cl− ⇌ CdCl−3 Pd2+ + OH− ⇌ PdOH+ Pd2+ + 2OH− ⇌ Pd(OH)2 Pd2+ + 3OH− ⇌ Pd(OH)−3 Pd2+ + 4OH− ⇌ Pd(OH)2− 4 Pd2+ + Cl− ⇌ PdCl+ 2+ − Pd + 2Cl ⇌ PdCl2 Pd2+ + 3Cl− ⇌ PdCl−3 Pd2+ + 4Cl− ⇌ PdCl2− 4 Pd2+ + 3Cl− + OH− ⇌ PdCl3OH2−
2.4 12.1 23.4 26.2 26.9 5.1 8.3 10.9 11.8 18.2
Default humic binding
Adsorption to U. lactuca surface
Total uptake by U. lactuca
Metal
log KMeHS
ΔLK2
log KMeS
ΔLK2
log KMeS
ΔLK2
Pd Cd Hg Pb
3.34 1.30 3.50 2.00
5.58 1.48 5.10 0.93
1.93 1.30 2.48 1.36
0 0 0 0
1.93 1.30 2.48 1.36
2.45 1.20 3.10 1.10
tainers and the ambient atmosphere, pH had risen by between a half and one and a half units such that values were between about 8.2 and 8.5 in all cases. For the purposes of the discussion and modelling, an invariant, “equilibrium” pH of 8.3 is assumed throughout. Photosynthetic activity, determined during the acclimation period in filtered sea water and at the end of the incubations in the synthetic mixtures, is shown for both experiments in Fig. 2. In the presence of humics, activity had not changed compared with that determined during acclimation. In the humic-free incubations, however, the rate of photosynthesis had measurably increased from the period of acclimation to the end of the experiment. We suspect that this is related to the gradual depletion of nutrients in sea water used to acclimatize the discs employed in the humic-free system. By the end of the incubations, photosynthetic activity was statistically indistinguishable (p N 0.05 according to one-way ANOVA) both within and between experiments. These observations suggest that varying salinity and the presence of 3 mg L− 1 of humic substances and/or 100 nM of Cd, Pd, Hg and Pb do not exert any adverse effects on the health of individual discs, at least according to the measure employed and within the timeframe of the experiments, or that any effects are offset by the nutrient amendment of the synthetic mixtures. 3.2. Aqueous metal speciation The aqueous equilibrium speciation of Cd, Pd, Hg and Pb in the experiments, as computed by WHAM, is shown in the absence and presence of aqueous humic substances in Figs. 3 and 4, respectively. The distributions of inorganic species of Cd and Hg are in good agreement with respective estuarine distributions computed using different software (Paalman et al., 1994; Leermakers et al., 1995). The inorganic speciation of Pb concords with distributions derived from defaulted for the Humber Estuary (UK) (Tipping et al., 1998). However, we note that there exist discrepancies in the relative abundances of carbonato- and chlorocomplexes of Pb as computed by WHAM and using other models (Turner et al., 1981; Bourg, 1983). With respect to Pd, the calculated inorganic speciation is in agreement with that presented by van Middlesworth and Wood (1999), whose constants were used in the present computations. However, it should be noted that there is some uncertainty about the relative magnitudes of the stability constants relevant to this metal in saline solutions. Specifically, other studies indicate that chloro-complexes are the dominant inorganic form in sea water rather than the neutral hydroxide and hydroxychloride, and that mixed chlorocarbanato-complexes may also be present (Cosden and Byrne, 2003). In the presence of a fixed concentration (3 mg L− 1) of humic substances, modelled from the default WHAM database, the speciation of Cd is largely unaffected since organic complexes were predicted to comprise less than one percent of total aqueous Cd. For the remaining metals, however, complexation by humics is highly significant, exceeding 65%, 95% and 99% throughout the salinity range for Pb, Pd and Hg, respectively. 3.3. Metal uptake by U. lactuca By the end of the experiments, metal concentrations in the aqueous phase, [Mew], ranged from about 30 to 80 nM for Pd, 70 to 100 nM for Cd, 40 to 60 nM for Hg and 50 to 80 nM for Pb. Respective concentrations in U. lactuca, [Mealga], normalised to the dry mass of solid, malga, ranged from about 10 to 50 nmol g− 1, b 5 to 30 nmol g− 1, 50 to 100 nmol g− 1 and 10 to 25 nmol g− 1. Results are shown in Fig. 5 as a
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Fig. 1. Initial (▲) and final (■) pH measured in (a) the humic-free incubations and (b) the presence of 3 mg L− 1 of aqueous humics. Error bars represent the standard deviation about the mean of four independent experimental measurements.
function of salinity and in the absence and presence of humics in terms of a concentration factor, CF: CF ¼
Mealga =malga ½Mew
ð1Þ
Mean values of CF range from about 102 mL g− 1 for Cd to in excess of 103 mL g− 1 for Hg, but in some cases replicate results exhibit considerable variation, with relative standard deviations of CF up to 40%. Presumably, such variations are a reflection of analytical errors, an inherent heterogeneity in the chemical and surface composition of the macroalga (and, with respect to Pb, background concentrations), and small variations in “equilibrium” pH. A reduction in CF for Cd with increasing salinity was observed in the present experiments, at least in the absence of humics, consistent with results reported by Wang and Dei (1999) using the same species of macroalga. A salinity dependence is also predicted for Pd, Hg and Pb on the basis of aqueous equilibrium speciation considerations (specifically, a reduction in both the concentration and activity of the respective free ions was computed with increasing salinity), and interactions with other, abiotic surfaces (Turner, 1996, 2007a). However, we suspect that the precision of the results has precluded detection of such a dependence in the present experiments. A reduction in the biouptake of Pd, Hg and Pb is also predicted in the presence of humics because of the competing effects of the aqueous complexant (Fig. 4). A statistically significant reduction (p b 0.05 according to a two sample t-test) was observed for Pd throughout the salinity range and for Hg in three cases. For Pb, however, an increase in uptake was observed at four out of five salinities. Metal taken up by U. lactuca may be adsorbed at the algal surface or internalised within the cytosol of component cells. Metal adsorption involves surface complexation, while internalisation may proceed subsequent to surface complexation, or independently as a passive
mechanism if metal complexes are sufficiently lipophilic. Particularly significant with respect to the latter is the neutral chloride of Hg, whose octanol-water partition coefficient is around 3 (Mason et al., 1996) and which makes a substantial contribution to the aqueous speciation of Hg in the absence of humics (Fig. 3). We operationally discriminated intracellular uptake and surface adsorption by washing one of the two discs from each incubation in 3 mM EDTA prior to its total digestion and analysis for metals. The results revealed no clear dependence of internalised metal (i.e. that resistant to EDTA washing) on salinity and are, therefore, pooled for each metal, both in the absence and presence of aqueous humics, in Fig. 6. Mean values of internalisation follow the sequence:HgN PdN CdN Pb; under both sets of conditions. The presence of humics exerted no significant influence on intracellular uptake of Pd, Cd and Hg, but appeared to greatly inhibit the internalisation of Pb. 3.4. Modelling metal adsorption by U. lactuca Since the algal surface bears many similarities with an aqueous or colloidal polyelectrolyte (e.g. humic substances), in particular in respect of its heterogeneous metal-binding properties (Xue et al., 1988; Koelmans et al., 1996; Kaulbach et al., 2005), we modelled metal adsorption by U. lactuca in WHAM by encoding the alga as a separate binding phase (or a biotic ligand). Complexation of a divalent metal ion, Me2+ (and its first hydrolysis product; MeOH+), at the algal surface, ≡S, was defined by the following general reaction (Xue et al., 1988): uSH þ Me2þ
K MeS
X
uSMeþ þ Hþ
ð2Þ
where the overall equilibrium constant, KMeS, encompasses binding at a variety of sites and is equivalent to the overall constant, KMeHS, defining metal binding by humic substances in WHAM. In the absence of additional information, we assumed that the surface properties of U.
Fig. 2. Photosynthetic activity of U. lactuca measured at the end of the incubations in (a) the absence of aqueous humics and (b) the presence of 3 mg L− 1 of humics. Error bars represent the standard deviation about the mean of four independent experimental measurements; broken lines define mean activities measured during the corresponding acclimation periods in filtered sea water.
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Fig. 3. Calculated salinity-dependent aqueous equilibrium speciation of Pd, Cd, Hg and Pb in the humic-free experiment.
lactuca (e.g. acidity constants, binding site density) were equivalent to those of the default humic substances contained in the WHAM database and based on averaged values in the literature (Tipping,1998). For a given metal, adsorption at the algal surface was modelled using a single, overall binding constant (Table 2), but the distributional term for strong surface binding, ΔLK2, was neglected (i.e. strong, specific binding is ignored). The concentration of metal adsorbed by U. lactuca in the experiments, [Meads], was evaluated from the chemical measure described
above (i.e. total metal taken up, [Mealga], minus internalised metal, [Meint]). Thus, neglecting internalised metal (see below), the percentage of metal adsorbed at equilibrium was calculated as follows: k adsorbed ¼
½Meads 100k ½Meads þ ½Mew
ð3Þ
and is plotted against salinity in Fig. 7. Initial fitting of adsorption data involved Cd because its uptake by phytoplankton in saline suspensions
Fig. 4. Calculated salinity-dependent aqueous equilibrium speciation of Pd, Cd, Hg and Pb in the experiment conducted in the presence of 3 mg L− 1 of aqueous humics.
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Fig. 5. Concentration factors defining the uptake of Cd, Pd, Hg and Pb by U. lactuca as a function of salinity in the humic-free experiment (□) and in the presence of 3 mg L− 1 of aqueous humics (■). Error bars represent the standard deviation about the mean of four independent experimental measurements. Note that data for Cd in the humic system are incomplete because of detection problems in some algal samples.
has previously been modelled by surface complexation (Koelmans et al., 1996) and default binding constants for this metal with humic substances are based on a relatively large dataset that exhibits little dispersion (Tipping, 1998). Thus, log KCdHS was employed to define interactions of Cd with the algal surface, and the percentage of “active” U. lactuca was then adjusted in order to give the best fit to the observed adsorption. Activity is defined as the proportion of the total mass of a substance that participates in metal binding, and a value of 0.091 (equivalent to a net binding phase concentration of 22 mg L− 1) was able to reproduce both the extent (between about 1 and 4%) and salinity-dependence of Cd adsorption with reasonable success. Our estimate of activity is low
compared with that of natural dissolved organic matter (around 0.5; Tipping et al., 1998), presumably reflecting the relatively large mass contribution of the inert cellular matrix. For the remaining metals, the same surface properties and activity of the alga were employed, and default metal binding constants were subsequently manipulated individually in order to fit the data by eye. The results of this exercise are annotated on Fig. 7, and the constants used to generate such are given in Table 2. Thus, by “calibrating” metal binding with respect to the default constants for Cd, a reduction in the default binding constants for the remaining metals was necessary to attain fits of the correct order of magnitude. However, because of the effects of ionic
Fig. 6. Fraction of metal internalised by U. lactuca in the humic-free experiment (□) and in the presence of 3 mg L− 1 of aqueous humics (■). Error bars represent the standard deviation about the mean of 20 measurements.
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strength and complexation by seawater anions, a decrease in the uptake of all metals as a function of salinity was predicted by the WHAM software that was not observed experimentally (see above).
well with the measured, internalised fractions of metal, but are not proportional to the respective constants defining strong metal binding by the default humic substances (see also Table 2).
3.5. Modelling metal internalisation by U. lactuca
3.6. Modelling metal uptake by U. lactuca in the presence of aqueous humics
With the surface binding constants derived above, we then attempted to model total metal uptake by U. lactuca by incorporating an internalisation phase. The latter may be considered as a first-order process successive to surface complexation: uSH þ Me2þ
k
K MeS
uSMeþ þ Hþ Y Meint
X
ð4Þ
where Meint represents internalised metal and k is a first-order rate constant, or as an independent, passive mechanism: MeL
K int
X
ð5Þ
MeLint
where L represents a ligand forming a lipophilic complex with a metal ion and Kint is an equilibrium constant defining passive uptake. Using WHAM, we modelled total uptake of metal as an equilibrium process, and simulated internalisation as strong binding at the algal surface and parameterized by the distributional term, ΔLK 2. Although internalisation and strong surface binding are, mechanistically, quite different, the rationale for this approach is that (i) binding at specific sites on the algal surface is believed to precede internalisation (Galceran et al., 2006), and (ii) the two processes bear some mathematical similarity. The percentage of metal taken up by U. lactuca (i.e. adsorbed and internalised) was calculated as follows:
k uptake ¼
Mealga 100k Mealga þ ½Mew
ð6Þ
We used the fitted values derived above to define the binding characteristics of the alga, together with the properties and constants of aqueous humic substances contained in the WHAM default database (Table 2) to compute metal uptake by U. lactuca in the presence of the aqueous polyelectrolyte. The results of the calculations are not shown, but revealed the following. Cadmium uptake by U. lactuca was little affected by the addition of aqueous humics, consistent with experimental observations reported above. For the remaining metals, however, binding by aqueous humics was so intense that the computed uptake by the alga was considerably less than that determined experimentally. For example, bb1% of Pd and Hg was predicted to be taken up (adsorbed plus internalised) under these conditions, but up to 20% removal from the aqueous phase was observed. The broad findings of this exercise were not significantly altered by adjusting the properties and constants of the default aqueous humic phase within realistic limits (Tipping, 1998), nor by “calibrating” the surface characteristics of the alga with respect to a metal other than Cd (recent evidence suggests that, in some cases, humic binding by Cd is overestimated in the WHAM default mode; Turner et al., 2008). Thus, while the discrepancies between observed and modelled results (including salinity dependence) are partly attributable to experimental and analytical variability and the limitations and assumptions of the computations and fitting, more important appear to be factors related to the general problems inherent in modelling metal uptake by marine macroalgae. 3.7. Difficulties in modelling metal uptake by macroalgae
and is also plotted in Fig. 7. Best eye-fits to these data were obtained using the binding constants derived above to define the adsorption process, and the values of ΔLK2 given in Table 2. The latter correlate
The approach to modelling metal uptake by U. lactuca that we have adopted is based on complexation with relatively weak sites on the
Fig. 7. Percentage of metal taken up (■; calculated using Eq. (6)) and adsorbed (△; calculated using Eq. (3)) by U. lactuca in the humic-free experiment. Error bars represent the standard deviation about the mean of four independent experimental measurements. Solid lines represent best eye-fits to the data modelled using WHAM and the constants reported in Table 2.
A. Turner et al. / Marine Chemistry 110 (2008) 176–184
algal surface, whose chemical characteristics are assumed to bear some semblance to natural aqueous or colloidal polyelectrolytes, and strong complexation as an analogue of (or precursor to) internalisation. Calculations and data-fitting based on this approach would suggest that the algal surface is less efficient in metal binding than aqueous polyelectrolytes in terms of both the density (activity) and binding capacity of its functional groups. However, it is important to appreciate that the constants derived are conditional because of the potential confounding effects of slow coordination-dissociation kinetics (Turner, 2007b) and competition for metal ions from algal exudates. Intracellular molecules released to the external medium may serve as natural algal antifoulants (Harder et al., 2004) or be a proportional defensive response to an increase in contaminant loading in the external medium (Vasconcelos and Leal, 2001), but generally have some propensity to bind, often strongly, with trace metals (Scoullos et al., 2004). Although complexation by exudates may result in an underestimation of true algal binding constants for Pd and Hg, this effect alone cannot account for the magnitude of the discrepancies between observed and modelled results in the presence of aqueous humics (Masakorala et al., in press). Specifically, under otherwise identical conditions, why should the presence of a highly active aqueous polyelectrolyte not have a greater, negative impact on the uptake of the most strongly complexing metals, Pd and Hg, by U. lactuca? Moreover, why is the adsorption of Pb enhanced but its internalisation inhibited in the presence of aqueous humics? Results of recent research regarding the uptake of Pb by fresh water microalgae and marine invertebrates are significant in this respect (Lamelas and Slaveykova, 2007; Sanchez-Marin et al., 2007; Slaveykova, 2007). Thus, enhanced uptake in the presence of humic or fulvic substances has been attributed to (i) the formation of a ternary complex involving either the adsorption of Pb onto adsorbed humics or the adsorption of a Pb-humic complex, and (ii) an increase in cell permeability, hence propensity for passive diffusion of the metal, incurred by the surfactancy of the humic molecule itself. These effects appear to exhibit some specificity among the metals that have been studied to date, such that Cd and Cu are not regulated by the same controls (Vigneault and Campbell, 2005; Lamelas and Slaveykova, 2007). Regarding our experiments with U. lactuca, we surmise that the stabilising effects of aqueous humic substances are partly (Pd and Hg) or more than (Pb) offset by the uptake of ternary complexes at the algal surface, a mechanism that cannot be directly modelled using software such as WHAM. With respect to Pb at least, and in contrast to results obtained using freshwater algae (Vigneault and Campbell, 2005), a reduction in passive, diffusive uptake appears to be a consequence of this effect; in other words, ternary complexation involves metal that would otherwise be available for internalisation. Since these observations have important, general implications regarding our understanding of and ability to model metal uptake by marine macroalgae, further studies in this area are necessary.
4. Conclusions Of the trace metals examined in the present study, only Cd uptake appeared to be measurably and systematically affected by variations in salinity that are representative of the habitat of U. lactuca. Of more general importance among the remaining, more strongly binding metals (Pd, Hg and Pb), is the presence of polyelectrolytic aqueous organic matter (humics). However, the effects of humic substances are quantitatively inconsistent with equilibrium speciation calculations performed in WHAM; specifically, observed uptake of Pd, Hg and Pb by U. lactuca far exceeds that predicted from the competitive effects of the algal surface and aqueous polyelectrolyte for a given metal ion. Our observations concord with recent, independent experimental results derived in the presence of microalgae and invertebrates, and further support assertions that current models of metalorganism interactions (e.g. FIAM and BLM) may not be generally applicable where additional processes, such as ternary complexation, are significant.
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