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Influence of synthetic surfactants on the uptake of Pd, Cd and Pb by the marine macroalga, Ulva lactuca
Environmental Pollution 156 (2008) 897–904
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Influence of synthetic surfactants on the uptake of Pd, Cd and Pb by the marine macroalga, Ulva lactuca Kanaji Masakorala a,1, Andrew Turner a, *, Murray T. Brown b a b
School of Earth, Ocean and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK
Synthetic surfactants exert a significant impact on the uptake and internalisation of metals by a marine macroalga.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2008 Received in revised form 30 April 2008 Accepted 14 May 2008
Uptake of Pd, Cd and Pb by the marine macroalga, Ulva lactuca, has been studied in the presence of an anionic (sodium dodecyl sulphate, SDS), cationic (hexadecyltrimethylammonium bromide; HDTMA) and non-ionic (Triton X-100; TX) surfactant. Compared with the surfactant-free system, metal sorption was reduced in the presence of SDS or TX. Neither surfactant, however, had any measurable impact on cell membrane permeability, determined by leakage of dissolved free amino acids (DFAA), or on metal internalisation. We attribute these observations to the stabilisation of aqueous Cd and Pb by SDS and the shielding of otherwise amenable sorption sites by TX. Presence of HDTMA resulted in a reduction in the extent of both sorption and internalisation of all metals and a significant increase in the leakage of DFAA. Thus, by enhancing membrane permeability, HDTMA exerts the greatest influence on metal behaviour in the presence of U. lactuca. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Trace metals Surfactants Macroalgae Sorption Speciation
1. Introduction Marine macroalgae play an important role in the coastal environment as primary producers and in maintaining ecosystem stability. Macroalgae are also useful bioindicators since they are responsive to environmental change and accumulate many contaminants (Boisson et al., 1997; Haritonidis and Malea, 1999; Wang and Dei, 1999; Costanzo et al., 2001). Uptake of trace metal ions by algae is particularly favourable because of the high density of functional groups, including hydroxyls, carboxylates, amines, imidazoles and thiols, on the cell surface (Majidi et al., 1990; Sheng et al., 2004). Consequently, algal concentrations of some metals can exceed corresponding concentrations in ambient sea water by more than three orders of magnitude (Conti and Cecchetti, 2003; Muse et al., 2006). Among the environmental factors that affect the rate and extent of metal uptake by marine macroalgae are pH, temperature, light, salinity and algal concentration (Hu et al., 1996; Boisson et al., 1997; Vasconcelos and Leal, 2001; Turner et al., 2007). Less well defined,
* Corresponding author. Tel.: þ44 1752 233041; fax: þ44 1752 232406. E-mail address: [email protected] (A. Turner). 1 Present address: Department of Botany, University of Ruhuna, Matara, Sri Lanka. 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.05.030
however, are the effects of other, co-contaminants on metal uptake; this is perhaps surprising because many species are tolerant of moderately, but generally polluted conditions, such as those arising from the discharge of domestic sewage (Ho, 1990; Hu et al., 1996; Costanzo et al., 2001). Significant in this respect, therefore, are the studies of Lee and Wang (2001) and Ramelow et al. (1992). In the former, it was shown that macronutrients exerted a significant impact on the rate of accumulation of Cd, Cr, Zn and Se by Ulva fasciata due to a variety of chemical and physiological effects. In the latter, a reduction in the uptake of various metals by different (dried) algal species was demonstrated in the presence of EDTA, presumably through the complexation and stabilisation of metal ions in solution. In the present study, we examine the effects of commercially important surfactants on the uptake and internalisation of trace metals by a marine macroalga. Surfactants may be generated naturally by microbial and algal activity (Scoullos et al., 2004), but are more locally significant as contaminants in domestic and industrial effluent discharges (Mariani et al., 2006; Ying, 2006) and in applications used to control oil spills (Edwards et al., 2003) and, potentially, harmful algal blooms (Sun et al., 2004). Given that surfactants are known to (i) influence the sorption of other contaminants by soils and sediments (Jones-Hughes and Turner, 2005; Cruz-Guzma`n et al., 2006), and (ii) affect the structure and integrity of lipid membranes (Gustafsson et al., 1997; Groot and Rabone,
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2001), we hypothesise that they are able to modify interactions between trace metals and algae. Ulva lactuca was selected as the test species because of its wide distribution, relatively simple geometric form, high sorptive capacity and ability to thrive under polluted conditions (Ho, 1990; Wang and Dei, 1999). We selected Pd, Cd and Pb as trace metals which have high anthropic signals but different aquatic chemistries (Baes and Mesmer, 1976). In particular, they display significant differences in the nature and extent of their binding to natural organic substances (Muller, 1996; Tipping, 1998; Turner, 2007) and it is reasonable to assume, therefore, that they exhibit differences in their uptake by and effects on marine macroalgae. The commercially important surfactants we employ are sodium dodecyl sulphate (SDS), an anionic alkyl sulphate, hexadecyltrimethylammonium bromide (HDTMA), a cationic quaternary ammonium compound, and Triton X-100 (TX), a non-ionic octylphenol polyethoxylate with an average of 9.5 ethoxylate units. The chemical and environmental properties of these amphiphiles that are relevant to the current study are given in Table 1. 2. Materials and methods Unless otherwise stated, all plasticware and glassware were soaked in 0.5 M HCl for at least 24 h and rinsed twice with distilled water before being used. Reagents employed were purchased from Sigma, Fisher or BDH/Merck and were of analytical grade or better. Sea water used for culturing and experimental work was available on tap in the laboratory. Water is routinely collected from Plymouth Sound (UK) at high water (salinity w33; DOC w250 mM; Turner et al., 2007) and is stored in darkened fibreglass tanks at the university before being filtered through 5 mm extruded carbon filters on line. 2.1. Sampling and culturing Samples of U. lactuca were collected from intertidal rock pools at Wembury, a protected beach 7 km to the SE of Plymouth, as required during September and October of 2006. Samples were transported in sealed plastic bags containing locally collected sea water back to the laboratory. Here, particulate matter and epiphytes were carefully removed during rinsing with laboratory sea water. Ulva samples were acclimatised for about 3 days in sea water in aerated 10 L polyethylene tanks at 15 C and under fluorescent lighting (250 mmol m2 s1 photosynthetic active radiation for 12 h per day). As required, discs of 14 mm in diameter were cut from the central portions of the thalli with the sharpened end of a polyethylene cylinder. Discs were then maintained for a further 24 h under the conditions described above before being used in the experiments.
a nominal surfactant concentration of 3 mg L1 (or w5 mM of TX and 10 mM of HDTMA or SDS), and varied the concentration of metals (in combination) from 5 to 30 mg L1 (or w25–300 nM). Thus, the uptake of metals over a range of concentrations was examined in the presence of a fixed concentration of individual surfactant. Working concentrations of SDS, HDTMA and TX are well below their respective critical micelle concentrations (Table 1), and are representative of net surfactant concentrations encountered in highly contaminated environments (Ying, 2006). Significantly, previous research has indicated that concentrations of this order afford a good indication of the likely environmental impacts of surfactants on the sorption of co-contaminants to sediment particles (Jones-Hughes and Turner, 2005; Turner and Xu, 2008). Although working concentrations of metals are in excess of environmental levels, they did not appear to exert any toxic effects on U. lactuca, as evaluated in independent chlorophyll fluorescence studies, and are sufficiently low compared with concentrations of binding ligands, like Cl and undefined, heterogeneous organics (as DOC), to maintain realistic aqueous speciation in sea water.
2.3. Experimental Short-term, static experiments were undertaken in triplicate in 150 mL polyethylene terephthalate beakers containing 50 mL of sea water, two algal discs, metal and surfactant. Controls were performed likewise but in the absence of either surfactant, metal, or algal discs. Following the addition of the reactants, beakers, were loosely covered with their lids and the contents continuously but gently agitated on an orbital shaker under the experimental conditions described above for a period of 24 h. These conditions do not allow appreciable degradation of the surfactants (see Table 1) and caused an increase in the original pH (about 8.0) of no more than 0.3 units. Subsequently, 3 mL aliquots of sea water were pipetted into 20 mL screwcapped polyethylene test tubes and stored at 20 C awaiting amino acid determination. Meanwhile, 1 mL aliquots were pipetted into 15 mL polypropylene conical tubes containing 9 mL of 1 M HCl ready for metal analysis. Algal discs were carefully retrieved using plastic tweezers, gently shaken to remove excess water and stored individually at 20 C in clear specimen bags. Before freezing, and in order to discriminate adsorbed and internalised metal, single discs from selected experiments were transferred to 50 mL polyethylene beakers containing 30 mL of 0.003 M EDTA in 0.6 M NaCl and gently agitated for 15 min (Vasconcelos and Leal, 2001). Although an operational measure, results of this approach appear to be consistent with thermodynamic considerations, at least for Cd and Pb (Hassler et al., 2004). Frozen discs were freeze-dried and re-weighed before undergoing complete digestion in 2 mL of concentrated HNO3 in covered 50 mL Pyrex beakers at about 60 C on a hot plate. Digestion proceeded until brown fumes ceased (typically after about 2 h). The cooled contents plus Milli-Q water rinsings were transferred to 10 mL Pyrex volumetric flasks and diluted to mark with Milli-Q water. To evaluate the accuracy of this approach, a certified reference alga (U. lactuca; BCR279) was digested in triplicate likewise. Internalised metal was calculated from the concentration difference between untreated and EDTA-extracted discs derived from the same experimental incubation.
2.4. Metal analysis 2.2. Working solutions Individual working surfactant solutions of 1000 mg L1 were prepared by dilution (TX) or dissolution (HDTMA and SDS) of original reagents in Millipore Milli-Q water in 50 mL screw-capped polyethylene tubes. Solutions were stored at 4 C and in the dark until required (within a few days of preparation) but were briefly warmed to 40 C immediately before use to eliminate any precipitates. A mixed working metal solution containing 50 mg L1 of Pd, Cd and Pb was prepared in 0.3 M HNO3 by appropriate dilution of plasma emission standard solutions in a polyethylene volumetric flask. Clearly, the number of contaminants selected for study present an extensive number of possible combinations and permutations. In this study, however, we used
Algal digests and diluted, acidified sea water samples were analysed for 108Pd, Cd and 208Pb by inductively coupled plasma-mass spectrometry (ICP-MS) using a PlasmaQuad PQ2þ (Thermo Elemental, Winsford, UK). The instrument was calibrated with multi-element standards in either 0.3 M HNO3 (digests) or 1 M HCl (sea water) over the range 0.5–10 mg L1 (addition of 3 mg L1 of the individual surfactants to the standards did not affect the calibrations). Both 115In and 193Ir were added to all samples and standards to a concentration of 100 mg L1 for internal standardisation across the working atomic mass range. Measured concentrations of Cd and Pb in the reference U. lactuca (2.41 0.27 and 63.5 0.63 nmol g1, respectively) were within the range of certified values (2.51 0.18 and 64.8 1.0 nmol g1, respectively). 111
Table 1 Chemical and environmental properties of the surfactants studied (Okpokwasili and Olisa, 1991; Garcia et al., 2001; George, 2002; Jones-Hughes and Turner, 2005; Syracuse Research Corporation Database, 2007) surfactant
Molecular formula
Sodium dodecyl sulphate (SDS) Hexadecyltrimethylammonium bromide (HDTMA) Triton X-100 (TX)
CH3(CH2)11OSO3Na
a b c d e f
CH3(CH2)15N(CH3)3Br C8H17-C6H4-(OCH2-CH2)9.5OH
Aqueous solubility at 25 C. Critical micelle concentration at 25 C. Calculated octanol water partition coefficient. Indicative degradation half-life in natural waters at 10–20 C. SDS typically undergoes a period of acclimation before degradation. Miscible in all proportions.
Monomer mass (g mol1) 288.4 363.9 w625
Aqueous solubilitya (M)
CMCb (mM)
0.35
12
0.10 misc.f
0.93 0.26
Kowc
t1/2d (h)
50
<50e
1500 72000
40 150
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3.2. Metal sorption isotherms
2.5. Dissolved free amino acid analysis In order to evaluate cell membrane damage incurred by the surfactants, leakage of dissolved free amino acids (DFAA) was determined fluorometrically, according to the method outlined by Parsons et al. (1984), in samples without added metal. Thus, 3 mL of borate-buffered solution, containing o-phthaldialdehyde and 2-mercaptoethanol, were added to the thawed 3 mL sea water aliquots and the contents allowed to stand for 2 min. Fluorescence was then measured in a 1 cm cell using a Perkin Elmer LS50B spectrophotometer at an optimal excitation wavelength of 342 nm and an optimal emission wavelength of 452 nm. The standard curve was obtained using glycine solutions (0–1.0 mM) and the results were calculated as glycine equivalents.
3. Results and discussion 3.1. DFAA leakage The toxicity of surfactants to U. lactuca is a component of ongoing research by the authors. Here, we have elected to present results of dissolved free amino acid (DFAA) release, which are consistent with results from other toxicity tests, but are more relevant to our interpretation of metal uptake. Concentrations of DFAA in sea water containing U. lactuca, with and without the different surfactants, are shown in Fig. 1. In the absence of surfactant, concentrations are about 4 nM (as glycine equivalents) and are relatively low for coastal sea water (Tada et al., 1998). There is a small increase in DFAA concentration in the presence of the non-ionic surfactant, TX, and an increase of an order of magnitude in the presence of the cationic surfactant, HDTMA. Further experiments revealed that the effects of HDTMA were concentration-dependent, while additional toxicological tests (e.g. chlorophyll fluorescence) indicated that U. lactuca did not recover after being transferred to surfactant-free sea water for a period of 24 h. In contrast, no increase in DFAA concentration (or indeed any other toxicological marker) was observed in the presence of the anionic surfactant, SDS. The toxicity of HDTMA results from its relatively strong, electrostatic interaction with the cell surface, and its biocidal properties (Lewis, 1990; Li et al., 1998). Specifically, cell leakage appears to be associated with the ability of cationic surfactants to defect cell curvature and to form small, transient holes in lipid membranes (Gustafsson et al., 1997). Although non-ionic surfactants are able to affect membrane permeability (De Souza et al., 1996; Groot and Rabone, 2001), the present results suggest that the concentrations of TX employed were not sufficient to exert a significant impact in this respect. 60 50 40
DFAA, nM
30 20 10
TX
HDTMA
SDS
0 control
899
Fig. 1. DFAA concentration in sea water containing U. lactuca in the absence and presence of 3 mg L1 of individual surfactants. Error bars represent the standard deviation about the mean of three independent experimental determinations.
In Figs. 2–4, sorption isotherms defining the uptake of Pd, Cd and Pb by U. lactuca in the presence and absence of individual surfactants are shown. (For the purposes of the present discussion, sorption is defined as an overall process encompassing a variety of mechanisms by which metals are removed from the aqueous phase by the alga.) Metal concentrations taken up by U. lactuca, [Me–S] (w/w, on a dry mass basis), and in sea water, [Me], have been corrected for concentrations in the corresponding controls in which no metal had been added. Ambient, environmental concentrations of Pd are too low to be detected by ICP-MS (Ravindra et al., 2004; Turner et al., 2007) but measurable signals in the sea water and U. lactuca controls, reflecting molecular ion interferences during analysis, were subtracted from signals determined in the presence of added metal. Concentrations of Cd and Pb were detected in controls of U. lactuca but not sea water. Control concentrations of Cd were small (<3 nmol g1) compared with those in U. lactuca arising from metal addition, but concentrations of Pb were significant (about 15 nmol g1). For the purposes of this study, the latter concentration was subtracted from the measured Pb value in the algae in each case. Thus, we assume that Pb that has been accumulated by U. lactuca in situ is effectively independent of Pb that has been taken up during the relatively short period of experimental incubation. The Freundlich model was applied to each data set:
½Me—S ¼ KF ½Men where KF is the Freundlich constant and n is a term defining the curvature of the non-linear fit. Model fits are annotated on Figs. 2–4 and constants, derived from power regression analysis, are shown in Table 2. With respect to Pd, fits were significant (p < 0.05) in all cases with values of n close to 1. Regarding Cd, data are more scattered and, although the Freundlich model produced significant fits, presence of TX resulted in a relatively invariant concentration of Me–S and a high degree of isotherm curvature (n ¼ 0.17). Freundlich fits to the Pb data were significant in three cases but, as with Cd, presence of the non-ionic surfactant resulted in a relatively invariant concentration of Me–S which could not be modelled successfully. 3.3. Metal speciation and sorption mechanisms An understanding of the sorption mechanisms involved in the present experiments requires an evaluation of the aqueous speciation of Pd, Cd and Pb. Specifically, since the free ion is generally assumed to represent the most (or only) species able to interact with algae (Xue et al., 1988; Sheng et al., 2004), an indication of its concentration is desirable. The inorganic speciation of the metals in sea water was calculated using the Windermere Humic Aqueous Model (WHAM, v6) and either default (Cd and Pb) or published (Pd) constants after ion activity coefficients had been corrected for using the Debye–Hu¨ckel equation. (Further details of the thermodynamic constants used and the modelling approach adopted are given in Turner et al., 2008.) The results of the present calculations, exemplified in Table 3, were similar over the range of aqueous metal concentrations employed and the pH of the experiments. Organic complexation with pre-existent and exuded ligands is likely to be important for at least Pd and Pb under the present experimental conditions (Li and Byrne, 1990; Vasconcelos and Leal, 2001) but is difficult to evaluate precisely. For instance, incorporating a polyelectrolyte, modelled as default humic substances and at a concentration equal to the measured DOC concentration (Turner et al., 2008), resulted in the same relative proportions of the inorganic components of each metal but
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Fig. 2. Isotherms defining the sorption of Pd by U. lactuca in sea water (salinity w33; pH w8.1–8.4) in the absence and presence of 3 mg L1 of individual surfactants. Error bars represent the standard deviation about the mean of three independent experimental determinations. Lines represent Freundlich fits to mean data points; isotherm constants are given in Table 2.
Fig. 3. Isotherms defining the sorption of Cd by U. lactuca in sea water. See caption to Fig. 2 for further details. Note that in some cases Cd was not detected in U. lactuca or sea water.
K. Masakorala et al. / Environmental Pollution 156 (2008) 897–904
901
Fig. 4. Isotherms defining the sorption of Pb by U. lactuca in sea water. See caption to Fig. 2 for further details.
abundances of organically complexed Pd, Cd and Pb of about 99%, 0.05% and 85%, respectively. Moreover, the degree of computed organic complexation was sensitive to metal concentration and the presence of competing metals (e.g. Fe and Cu). Clearly, therefore, estimates of the absolute concentrations of metal species in the experiments (and particularly those of Pd and Pb) are subject to considerable uncertainty. Based on the results of inorganic speciation calculations (Table 3), the Freundlich constants in Table 2 are also shown normalised to the concentration of the free ion (i.e. KF/fMe 2þ, where fMe 2þ represents the fraction of aqueous metal as the free ion). By neglecting aqueous organic complexants, normalised sorption constants should be considered as lower estimates. Nevertheless, they
illustrate that binding (and/or internalisation) by U. lactuca is significantly greater for the Pd ion than either Cd2þ or Pb2þ. With respect to isotherm shape, convexity observed in most cases suggests either a shift in aqueous speciation or a reduction in site availability on the algal surface with increasing metal concentration. Given that inorganic speciation is rather insensitive to changes in metal concentration and assuming that the availability of organic binding ligands remains constant in each experiment, we suspect that isotherm convexity results largely from the progressive utilisation of binding sites on the algal surface or increasing competition among the metals for such sites (Webster et al., 1997; Lee and Wang, 2001). On this basis, therefore, and Table 3 Aqueous, inorganic speciation of 100 nM of Pd, Cd and Pb in sea water (salinity ¼ 33; pH ¼ 8.2), as computed by WHAM
Table 2 Freundlich constants defining the sorption of Pd, Cd and Pb by U. lactuca, derived from power regression analysis of the data shown in Figs. 2–4 (ns ¼ not significant; p < 0.05). KF/fMe 2þ represents the Freundlich constant normalised to the computed fraction of the free ion (Table 3) r2
KF/fMe 2þ
0.825 1.020 0.924 0.930
0.866 0.944 0.984 0.961
4.85 1014 1.93 1014 3.74 1014 2.56 1014
220 204 197 731
0.940 0.854 0.678 0.170
0.924 0.799 0.854 0.926
5450 5050 4880 18,100
3610 2250 826 4690
0.411 0.545 0.800 0.105
Added reactants
KF
Pd Pd þ HDTMA Pd þ SDS Pd þ TX
6650 2650 5120 3510
Cd Cd þ HDTMA Cd þ SDS Cd þ TX Pb Pb þ HDTMA Pb þ SDS Pb þ TX
n
0.964 0.917 0.944 0.147 (ns)
108,000 67,400 24,700 140,000
Species
Concentration (nM)
Pd2þ Pd(OH)2 PdCl3(OH)2 PdCl2 PdCl 3 PdCl2 4
1.37 109 42.3 48.8 6.32 103 1.29 7.51
Cd2þ CdSO4 CdClþ CdCl2 CdCl3
4.04 1.13 47.3 35.3 12.1
Pb2þ PbOHþ PbSO4 PbCO3 Pb(CO3)2 2 PbClþ PbCl2
3.34 1.87 2.09 72.0 3.31 12.5 4.89
K. Masakorala et al. / Environmental Pollution 156 (2008) 897–904
Pd2þ þ 2Cl þ G 4PdCl2 G ;
metabolic-dependent uptake, against the total concentration of metal taken up, [Me–S]. Results are presented for three added metal concentrations and for experiments undertaken in the presence and absence of HDTMA (results in the presence of SDS and TX showed no systematic or significant difference to those in the surfactant-free system). In the absence of HDTMA, the degree of internalisation was greatest for Pd (up to about 65%), and for all metals internalisation appeared to decline with increasing [Me–S]. These observations are consistent with the earlier assertion of increasing occupation of specific, and perhaps physiologically active sites (as precursors for transport across cationic channels), with increasing metal concentration, and the greatest affinity for these
400
[Me-S] int, nmol g-1
Pd
200
100
Fig. 5 shows the concentration of metal that was internalised by U. lactuca, [Me–S]int, as operationally defined by its resistance to mobilisation by EDTA and representing passive (diffusive) and
200
400
600
20
Cd 15
10
5
0
0
20
60
40
[Me-S], nmol g-1 50 Pb 40 30 20 10 0
3.4. Metal internalisation
0
[Me-S], nmol g-1
log K ¼ 19:7
In the absence of additional organic ligands, up to 12% of Pd was computed to be complexed by glycine over the working concentration range of metal; however, in the presence of aqueous polyelectrolyte, modelled as above, less than 1% was predicted to be complexed by the amino acid. We may conclude, therefore, that DFAA exert a small impact on extracellular metal speciation in the present experiments. Accordingly, competitive sorption between metals and the amphiphile (see above) and enhanced membrane permeability and mobilisation of internalised metal are more likely responsible for reduced uptake of Pd, Cd and Pb in the presence of HDTMA.
300
0
[Me-S] int, nmol g-1
consistent with its affinity for the algal surface, we predict that Pd, exhibiting quasi-linear sorption, is able to outcompete Pb (and perhaps Cd) for available binding sites. Examination of the sorption constants reveals that metal uptake is sensitive to the presence of the individual surfactants, and that this sensitivity is metal-dependent. Specifically, Pd sorption is considerably reduced in the presence of either HDTMA or TX, but little affected by SDS. Regarding Cd and Pb, however, sorption is reduced in the presence of all surfactants, but to the greatest extents by SDS and TX. These effects can be explained by surfactantinduced modification of both aqueous metal speciation and the algal surface. The cationic surfactant, HDTMA, is predicted to undergo effective sorption through both electrostatic and, in particular, hydrophobic interactions (Chen et al., 1998; Cruz-Guzma`n et al., 2006). The former interactions may compete directly with those involving metal and negatively charged algal surface sites, although mutually amenable sites are likely to be non-coordinating (for example, sulphate and unprotonated carboxyl oxygen; Rosen et al., 2001). That the reduction in metal sorption in the presence of HDTMA was greatest for Pd suggests there is some specificity or competition among the metals for such sites (as inferred above). Since the non-ionic surfactant, TX, is predicted to interact with algae non-specifically (e.g. hydrophobically or via hydrogen-bonding), we surmise that its influence on Cd and Pb sorption is related to the shielding of otherwise available (charged) sites rather than to any competitive effect. SDS is predicted to interact least with the algal surface because of the negative charge of the hydrophile and the relatively low Kow of the molecule (Table 1). A reduction in the uptake of Cd and Pb in the presence of SDS cannot, therefore, be explained in terms of algal surface modification or competitive sorption. Accordingly, we propose that these metals are partially stabilised in sea water by SDS through either complexation or counter ion exchange with the surfactant (Friedel et al., 1994; Juang et al., 2003). Although interactions (or complexes) between HDTMA and aqueous metal ions are unlikely, the cationic surfactant may also exert an indirect impact on the speciation and/or uptake of metals by mobilising intracellular amino acids as complexants in the extracellular medium (Fig. 1) or by enhancing cell membrane permeability. We do not know the precise makeup of DFAA mobilised by U. lactuca, but their potential impact on metal speciation was evaluated by repeating WHAM simulations for Pd, the metal having the greatest affinity for nitrogeneous ligands, in the presence of 10 nM of unprotonated glycine, G, by encoding the following reaction into the software (Li and Byrne, 1990):
[Me-S] int, nmol g-1
902
0
20
40
60
80
[Me-S], nmol g-1 Fig. 5. Internalised versus total concentration of metal in U. lactuca in the absence (-) and presence (,) of 3 mg L1 (w10 mM) of HDTMA. Error bars represent the standard deviation about the mean of three independent experimental determinations.
K. Masakorala et al. / Environmental Pollution 156 (2008) 897–904
sites displayed by Pd. In the presence of HDTMA, the fraction of internalised metal is reduced in all cases, presumably because of enhanced cell permeability and metal leakage incurred by the cationic surfactant. 3.5. General implications The results of this study have demonstrated that, while all surfactant types inhibit the uptake of trace metals by U. lactuca, only the cationic surfactant, HDTMA, exerts a measurable (and negative) impact on internalisation because of its ability to enhance cell membrane permeability. Although the precise effects observed in this study are specific to the particular reactants, the mechanisms involved and the biocidal nature of most cationic surfactants suggest that the broad findings are likely to apply to a wider range of metals, surfactants and macroalgae. Thus, the observations have important, general implications for the uptake, toxicity and trophic transfer of metals in environments that are concurrently impacted by both metals and surfactants. Such conditions may arise during the control of oil spills (Edwards et al., 2003) or harmful algal blooms (Sun et al., 2004), and in coastal areas that are moderately impacted by domestic or industrial effluents. Trace metal concentration factors or sorption constants based on biomonitoring programmes conducted in pristine environments are not, therefore, necessarily applicable where other types of contaminant co-exist. 4. Conclusions Common, synthetic surfactants exert a significant impact on the uptake of trace metals (Pd, Cd and Pb) by the marine macroalga, U. lactuca. Within the concentration range of the reactants studied, the anionic and neutral surfactants, sodium dodecyl sulphate and Triton X-100, respectively, inhibit the overall uptake of metals, but do not appear to alter cell membrane permeability or affect metal internalisation. In contrast, the cationic surfactant, hexadecyltrimethylammonium bromide, inhibits both the uptake of metals and their internalisation, effects attributed to the ability of this amphiphile to induce significant membrane damage. Acknowledgements KM was supported by an Erasmus Mundus studentship to undertake a Joint European Masters in Water and Coastal Management. We thank Miss Angela Watson and Dr. Andy Fisher for assistance with algal culturing and metal analysis, respectively. The insightful comments of two anonymous reviewers were greatly appreciated. References Baes, C.F., Mesmer, R.E., 1976. The Hydrolysis of Cations. Wiley and Sons, New York, 489 pp. Boisson, F., Hutchins, D.A., Fowler, S.W., Fisher, N.S., Teyssie, J.-L., 1997. Influence of temperature on the accumulation and retention of 11 radionuclides by the marine alga Fucus vesiculosus (L.). Marine Pollution Bulletin 35, 313–321. Chen, Y.M., Liu, J.C., Ju, Y.H., 1998. Flotation removal of algae from water. Colloids and Surfaces B 12, 49–55. Conti, M.E., Cecchetti, G., 2003. A biomonitoring study: trace metals in algae and molluscs from Tyrrhenian coastal areas. Environmental Research 93, 99–112. Costanzo, S.D., O’Donohue, M.J., Dennison, W.C., Loneragan, N.R., Thomas, M., 2001. A new approach for detecting and mapping sewage impacts. Marine Pollution Bulletin 42, 149–156. Cruz-Guzma`n, M., Celis, R., Hermonsin, M.C., Koskinen, W.C., Nater, E.A., Cornejo, J., 2006. Heavy metal adsorption by montmorillonites modified with natural organic cations. Soil Science Society of America Journal 70, 215–221. De Souza, M.P., Chen, Y.P., Yoch, D.C., 1996. Dimethylsulfoniopropionate lyase from the marine macroalgae Ulva curvata: purification and characterization of the enzyme. Planta 199, 433–438. Edwards, K.R., Lepo, J.E., Lewis, M.A., 2003. Toxicity comparison of biosurfactants and synthetic surfactants used in oil spill remediation to two estuarine species. Marine Pollution Bulletin 46, 1309–1316.
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Bioaccumulation of metals by the green alga Ulva rigida from Thermaikos Gulf, Greece. Environmental Pollution 104, 365–372. Hassler, C.S., Slaveykova, V.I., Wilkinson, K.J., 2004. Discriminating between intraand extracellular metals using chemical extractions. Limnology and Oceanography: 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. Hu, S., Tang, C.H., Wu, M., 1996. Cadmium accumulation by several seaweeds. Science of the Total Environment 187, 65–71. Jones-Hughes, T.L., Turner, A., 2005. Sorption of ionic surfactants to estuarine sediment and their influence on the sequestration of phenanthrene. Environmental Science and Technology 39, 1688–1697. Juang, R.-S., Xu, Y.-Y., Chen, C.-L., 2003. Separation and removal of metal ions from dilute solutions using micellar-enhanced ultrafiltration. Journal of Membrane Science 218, 257–267. Lee, W.-Y., Wang, W.-X., 2001. Metal accumulation in the green macroalga Ulva fasciata: effects of nitrate, ammonium and phosphate. Science of the Total Environment 278, 11–22. Lewis, M.A., 1990. Chronic toxicities of surfactants and detergent builders to algae: a review and risk assessment. Ecotoxicology and Environmental Safety 20, 123–140. Li, J.-H., Byrne, R.H., 1990. Amino acid complexation of palladium in seawater. Environmental Science and Technology 24, 1038–1041. Li, Z., Roy, S.J., Zou, Y., Bowman, R.S., 1998. Long-term chemical and biological stability of surfactant-modified zeolite. Environmental Science and Technology 32, 2628–2632. Majidi, V., Laude, D.A., Holcombe, J.A., 1990. Investigation of the metal-algae binding site with 113Cd nuclear magnetic resonance. Environmental Science and Technology 24, 1309–1312. Mariani, L., De Pascale, D., Faraponova, O., Tornambe, A., Sarni, A., Giuliani, S., Ruggiero, G., Onorati, F., Magaletti, E., 2006. The use of a test battery in marine ecotoxicology: the acute toxicity of sodium dodecyl sulfate. Environmetnal Toxicology 21, 373–379. Muller, F.L.L., 1996. Interactions of copper, lead and cadmium with the dissolved, colloidal and particulate components of estuarine and coastal waters. Marine Chemistry 52, 245–268. Muse, J.O., Carducci, C.N., Stripeikis, J.D., Tudino, M.B., Fernandez, F.M., 2006. A link between lead and cadmium kinetic speciation in seawater and accumulation by the green alga Ulva lactuca. Environmental Pollution 141, 126–130. Okpokwasili, G.C., Olisa, A.O., 1991. River water biodegradation of surfactants in liquid detergents and shampoos. Water Research 25, 1425–1429. Parsons, T.R., Maita, Y., Lalli, C.M., 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon, Oxford. Ramelow, G.J., Fralick, D., Zhao, Y., 1992. Factors affecting the uptake of aqueous metal ions by dried seaweed biomass. Microbios 72, 81–93. Ravindra, K., Bencs, L., van Grieken, R.V., 2004. Platinum group elements in the environment and their health risk. Science of the Total Environment 318, 1–43. Rosen, M.J., Li, F., Morrall, S.W., Versteeg, D.J., 2001. The relationship between the interfacial properties of surfactants and their toxicity to aquatic organisms. Environmental Science and Technology 35, 954–959. Scoullos, M., Plavsic, M., Karavoltsos, S., 2004. Speciation studies of copper in the Gulf of Elefsis: the role of the macroalgae Ulva rigida. Marine Chemistry 86, 51–63. Sheng, P.X., Ting, Y.-P., Chen, P.J., Hong, L., 2004. Sorption of lead, copper, cadmium, zinc and nickel by marine algal biomass: characterization of biosorptive capacity and investigation of mechanisms. Journal of Colloid and Interface Science 275, 131–141. Sun, X.-X., Han, K.-N., Choi, J.-K., Kim, E.-K., 2004. Screening of surfactants for harmful algal bloom mitigation. Marine Pollution Bulletin 48, 937–945. Syracuse Research Corporation Database. Available from: http://www.syrres.com/ esc/est_kowdemo.htm (accessed 5/2007). Tada, K., Tada, M., Maita, Y., 1998. Dissolved free amino acids in coastal seawater using a modified fluorometric method. Journal of Oceanography 54, 313–321. Tipping, E., 1998. Humic ion-binding model VI: an improved description of the interactions of protons and metal ions with humic substances. Aquatic Geochemistry 4, 3–48. Turner, A., 2007. Particle–water interactions of the platinum group elements under estuarine conditions. Marine Chemistry 103, 103–111. Turner, A., Lewis, M.S., Shams, L., Brown, M.T., 2007. Uptake of platinum group elements by the marine macroalga, Ulva lactuca. Marine Chemistry 105, 271–280. Turner, A., Xu, J., 2008. Influence of ionic surfactants on the flocculation and sorption of palladium and mercury in the aquatic environment. Water Research 42, 318–326.
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Turner, A., Pedroso, S.S., Brown, M.T., 2008. 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, 176–184. Vasconcelos, M.T.S.D., Leal, M.F.C., 2001. Seasonal variability in the kinetics of Cu, Pb, Cd and Hg accumulation by macroalgae. Marine Chemistry 74, 65–85. Wang, W.-X., Dei, R.C.H., 1999. Kinetic measurements of metal accumulation in two marine macroalgae. Marine Biology 135, 11–23.
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Influence of synthetic surfactants on the uptake of Pd, Cd and Pb by the marine macroalga, Ulva lactuca Kanaji Masakorala a,1, Andrew Turner a, *, Murray T. Brown b a b
School of Earth, Ocean and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK
Synthetic surfactants exert a significant impact on the uptake and internalisation of metals by a marine macroalga.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2008 Received in revised form 30 April 2008 Accepted 14 May 2008
Uptake of Pd, Cd and Pb by the marine macroalga, Ulva lactuca, has been studied in the presence of an anionic (sodium dodecyl sulphate, SDS), cationic (hexadecyltrimethylammonium bromide; HDTMA) and non-ionic (Triton X-100; TX) surfactant. Compared with the surfactant-free system, metal sorption was reduced in the presence of SDS or TX. Neither surfactant, however, had any measurable impact on cell membrane permeability, determined by leakage of dissolved free amino acids (DFAA), or on metal internalisation. We attribute these observations to the stabilisation of aqueous Cd and Pb by SDS and the shielding of otherwise amenable sorption sites by TX. Presence of HDTMA resulted in a reduction in the extent of both sorption and internalisation of all metals and a significant increase in the leakage of DFAA. Thus, by enhancing membrane permeability, HDTMA exerts the greatest influence on metal behaviour in the presence of U. lactuca. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Trace metals Surfactants Macroalgae Sorption Speciation
1. Introduction Marine macroalgae play an important role in the coastal environment as primary producers and in maintaining ecosystem stability. Macroalgae are also useful bioindicators since they are responsive to environmental change and accumulate many contaminants (Boisson et al., 1997; Haritonidis and Malea, 1999; Wang and Dei, 1999; Costanzo et al., 2001). Uptake of trace metal ions by algae is particularly favourable because of the high density of functional groups, including hydroxyls, carboxylates, amines, imidazoles and thiols, on the cell surface (Majidi et al., 1990; Sheng et al., 2004). Consequently, algal concentrations of some metals can exceed corresponding concentrations in ambient sea water by more than three orders of magnitude (Conti and Cecchetti, 2003; Muse et al., 2006). Among the environmental factors that affect the rate and extent of metal uptake by marine macroalgae are pH, temperature, light, salinity and algal concentration (Hu et al., 1996; Boisson et al., 1997; Vasconcelos and Leal, 2001; Turner et al., 2007). Less well defined,
* Corresponding author. Tel.: þ44 1752 233041; fax: þ44 1752 232406. E-mail address: [email protected] (A. Turner). 1 Present address: Department of Botany, University of Ruhuna, Matara, Sri Lanka. 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.05.030
however, are the effects of other, co-contaminants on metal uptake; this is perhaps surprising because many species are tolerant of moderately, but generally polluted conditions, such as those arising from the discharge of domestic sewage (Ho, 1990; Hu et al., 1996; Costanzo et al., 2001). Significant in this respect, therefore, are the studies of Lee and Wang (2001) and Ramelow et al. (1992). In the former, it was shown that macronutrients exerted a significant impact on the rate of accumulation of Cd, Cr, Zn and Se by Ulva fasciata due to a variety of chemical and physiological effects. In the latter, a reduction in the uptake of various metals by different (dried) algal species was demonstrated in the presence of EDTA, presumably through the complexation and stabilisation of metal ions in solution. In the present study, we examine the effects of commercially important surfactants on the uptake and internalisation of trace metals by a marine macroalga. Surfactants may be generated naturally by microbial and algal activity (Scoullos et al., 2004), but are more locally significant as contaminants in domestic and industrial effluent discharges (Mariani et al., 2006; Ying, 2006) and in applications used to control oil spills (Edwards et al., 2003) and, potentially, harmful algal blooms (Sun et al., 2004). Given that surfactants are known to (i) influence the sorption of other contaminants by soils and sediments (Jones-Hughes and Turner, 2005; Cruz-Guzma`n et al., 2006), and (ii) affect the structure and integrity of lipid membranes (Gustafsson et al., 1997; Groot and Rabone,
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2001), we hypothesise that they are able to modify interactions between trace metals and algae. Ulva lactuca was selected as the test species because of its wide distribution, relatively simple geometric form, high sorptive capacity and ability to thrive under polluted conditions (Ho, 1990; Wang and Dei, 1999). We selected Pd, Cd and Pb as trace metals which have high anthropic signals but different aquatic chemistries (Baes and Mesmer, 1976). In particular, they display significant differences in the nature and extent of their binding to natural organic substances (Muller, 1996; Tipping, 1998; Turner, 2007) and it is reasonable to assume, therefore, that they exhibit differences in their uptake by and effects on marine macroalgae. The commercially important surfactants we employ are sodium dodecyl sulphate (SDS), an anionic alkyl sulphate, hexadecyltrimethylammonium bromide (HDTMA), a cationic quaternary ammonium compound, and Triton X-100 (TX), a non-ionic octylphenol polyethoxylate with an average of 9.5 ethoxylate units. The chemical and environmental properties of these amphiphiles that are relevant to the current study are given in Table 1. 2. Materials and methods Unless otherwise stated, all plasticware and glassware were soaked in 0.5 M HCl for at least 24 h and rinsed twice with distilled water before being used. Reagents employed were purchased from Sigma, Fisher or BDH/Merck and were of analytical grade or better. Sea water used for culturing and experimental work was available on tap in the laboratory. Water is routinely collected from Plymouth Sound (UK) at high water (salinity w33; DOC w250 mM; Turner et al., 2007) and is stored in darkened fibreglass tanks at the university before being filtered through 5 mm extruded carbon filters on line. 2.1. Sampling and culturing Samples of U. lactuca were collected from intertidal rock pools at Wembury, a protected beach 7 km to the SE of Plymouth, as required during September and October of 2006. Samples were transported in sealed plastic bags containing locally collected sea water back to the laboratory. Here, particulate matter and epiphytes were carefully removed during rinsing with laboratory sea water. Ulva samples were acclimatised for about 3 days in sea water in aerated 10 L polyethylene tanks at 15 C and under fluorescent lighting (250 mmol m2 s1 photosynthetic active radiation for 12 h per day). As required, discs of 14 mm in diameter were cut from the central portions of the thalli with the sharpened end of a polyethylene cylinder. Discs were then maintained for a further 24 h under the conditions described above before being used in the experiments.
a nominal surfactant concentration of 3 mg L1 (or w5 mM of TX and 10 mM of HDTMA or SDS), and varied the concentration of metals (in combination) from 5 to 30 mg L1 (or w25–300 nM). Thus, the uptake of metals over a range of concentrations was examined in the presence of a fixed concentration of individual surfactant. Working concentrations of SDS, HDTMA and TX are well below their respective critical micelle concentrations (Table 1), and are representative of net surfactant concentrations encountered in highly contaminated environments (Ying, 2006). Significantly, previous research has indicated that concentrations of this order afford a good indication of the likely environmental impacts of surfactants on the sorption of co-contaminants to sediment particles (Jones-Hughes and Turner, 2005; Turner and Xu, 2008). Although working concentrations of metals are in excess of environmental levels, they did not appear to exert any toxic effects on U. lactuca, as evaluated in independent chlorophyll fluorescence studies, and are sufficiently low compared with concentrations of binding ligands, like Cl and undefined, heterogeneous organics (as DOC), to maintain realistic aqueous speciation in sea water.
2.3. Experimental Short-term, static experiments were undertaken in triplicate in 150 mL polyethylene terephthalate beakers containing 50 mL of sea water, two algal discs, metal and surfactant. Controls were performed likewise but in the absence of either surfactant, metal, or algal discs. Following the addition of the reactants, beakers, were loosely covered with their lids and the contents continuously but gently agitated on an orbital shaker under the experimental conditions described above for a period of 24 h. These conditions do not allow appreciable degradation of the surfactants (see Table 1) and caused an increase in the original pH (about 8.0) of no more than 0.3 units. Subsequently, 3 mL aliquots of sea water were pipetted into 20 mL screwcapped polyethylene test tubes and stored at 20 C awaiting amino acid determination. Meanwhile, 1 mL aliquots were pipetted into 15 mL polypropylene conical tubes containing 9 mL of 1 M HCl ready for metal analysis. Algal discs were carefully retrieved using plastic tweezers, gently shaken to remove excess water and stored individually at 20 C in clear specimen bags. Before freezing, and in order to discriminate adsorbed and internalised metal, single discs from selected experiments were transferred to 50 mL polyethylene beakers containing 30 mL of 0.003 M EDTA in 0.6 M NaCl and gently agitated for 15 min (Vasconcelos and Leal, 2001). Although an operational measure, results of this approach appear to be consistent with thermodynamic considerations, at least for Cd and Pb (Hassler et al., 2004). Frozen discs were freeze-dried and re-weighed before undergoing complete digestion in 2 mL of concentrated HNO3 in covered 50 mL Pyrex beakers at about 60 C on a hot plate. Digestion proceeded until brown fumes ceased (typically after about 2 h). The cooled contents plus Milli-Q water rinsings were transferred to 10 mL Pyrex volumetric flasks and diluted to mark with Milli-Q water. To evaluate the accuracy of this approach, a certified reference alga (U. lactuca; BCR279) was digested in triplicate likewise. Internalised metal was calculated from the concentration difference between untreated and EDTA-extracted discs derived from the same experimental incubation.
2.4. Metal analysis 2.2. Working solutions Individual working surfactant solutions of 1000 mg L1 were prepared by dilution (TX) or dissolution (HDTMA and SDS) of original reagents in Millipore Milli-Q water in 50 mL screw-capped polyethylene tubes. Solutions were stored at 4 C and in the dark until required (within a few days of preparation) but were briefly warmed to 40 C immediately before use to eliminate any precipitates. A mixed working metal solution containing 50 mg L1 of Pd, Cd and Pb was prepared in 0.3 M HNO3 by appropriate dilution of plasma emission standard solutions in a polyethylene volumetric flask. Clearly, the number of contaminants selected for study present an extensive number of possible combinations and permutations. In this study, however, we used
Algal digests and diluted, acidified sea water samples were analysed for 108Pd, Cd and 208Pb by inductively coupled plasma-mass spectrometry (ICP-MS) using a PlasmaQuad PQ2þ (Thermo Elemental, Winsford, UK). The instrument was calibrated with multi-element standards in either 0.3 M HNO3 (digests) or 1 M HCl (sea water) over the range 0.5–10 mg L1 (addition of 3 mg L1 of the individual surfactants to the standards did not affect the calibrations). Both 115In and 193Ir were added to all samples and standards to a concentration of 100 mg L1 for internal standardisation across the working atomic mass range. Measured concentrations of Cd and Pb in the reference U. lactuca (2.41 0.27 and 63.5 0.63 nmol g1, respectively) were within the range of certified values (2.51 0.18 and 64.8 1.0 nmol g1, respectively). 111
Table 1 Chemical and environmental properties of the surfactants studied (Okpokwasili and Olisa, 1991; Garcia et al., 2001; George, 2002; Jones-Hughes and Turner, 2005; Syracuse Research Corporation Database, 2007) surfactant
Molecular formula
Sodium dodecyl sulphate (SDS) Hexadecyltrimethylammonium bromide (HDTMA) Triton X-100 (TX)
CH3(CH2)11OSO3Na
a b c d e f
CH3(CH2)15N(CH3)3Br C8H17-C6H4-(OCH2-CH2)9.5OH
Aqueous solubility at 25 C. Critical micelle concentration at 25 C. Calculated octanol water partition coefficient. Indicative degradation half-life in natural waters at 10–20 C. SDS typically undergoes a period of acclimation before degradation. Miscible in all proportions.
Monomer mass (g mol1) 288.4 363.9 w625
Aqueous solubilitya (M)
CMCb (mM)
0.35
12
0.10 misc.f
0.93 0.26
Kowc
t1/2d (h)
50
<50e
1500 72000
40 150
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3.2. Metal sorption isotherms
2.5. Dissolved free amino acid analysis In order to evaluate cell membrane damage incurred by the surfactants, leakage of dissolved free amino acids (DFAA) was determined fluorometrically, according to the method outlined by Parsons et al. (1984), in samples without added metal. Thus, 3 mL of borate-buffered solution, containing o-phthaldialdehyde and 2-mercaptoethanol, were added to the thawed 3 mL sea water aliquots and the contents allowed to stand for 2 min. Fluorescence was then measured in a 1 cm cell using a Perkin Elmer LS50B spectrophotometer at an optimal excitation wavelength of 342 nm and an optimal emission wavelength of 452 nm. The standard curve was obtained using glycine solutions (0–1.0 mM) and the results were calculated as glycine equivalents.
3. Results and discussion 3.1. DFAA leakage The toxicity of surfactants to U. lactuca is a component of ongoing research by the authors. Here, we have elected to present results of dissolved free amino acid (DFAA) release, which are consistent with results from other toxicity tests, but are more relevant to our interpretation of metal uptake. Concentrations of DFAA in sea water containing U. lactuca, with and without the different surfactants, are shown in Fig. 1. In the absence of surfactant, concentrations are about 4 nM (as glycine equivalents) and are relatively low for coastal sea water (Tada et al., 1998). There is a small increase in DFAA concentration in the presence of the non-ionic surfactant, TX, and an increase of an order of magnitude in the presence of the cationic surfactant, HDTMA. Further experiments revealed that the effects of HDTMA were concentration-dependent, while additional toxicological tests (e.g. chlorophyll fluorescence) indicated that U. lactuca did not recover after being transferred to surfactant-free sea water for a period of 24 h. In contrast, no increase in DFAA concentration (or indeed any other toxicological marker) was observed in the presence of the anionic surfactant, SDS. The toxicity of HDTMA results from its relatively strong, electrostatic interaction with the cell surface, and its biocidal properties (Lewis, 1990; Li et al., 1998). Specifically, cell leakage appears to be associated with the ability of cationic surfactants to defect cell curvature and to form small, transient holes in lipid membranes (Gustafsson et al., 1997). Although non-ionic surfactants are able to affect membrane permeability (De Souza et al., 1996; Groot and Rabone, 2001), the present results suggest that the concentrations of TX employed were not sufficient to exert a significant impact in this respect. 60 50 40
DFAA, nM
30 20 10
TX
HDTMA
SDS
0 control
899
Fig. 1. DFAA concentration in sea water containing U. lactuca in the absence and presence of 3 mg L1 of individual surfactants. Error bars represent the standard deviation about the mean of three independent experimental determinations.
In Figs. 2–4, sorption isotherms defining the uptake of Pd, Cd and Pb by U. lactuca in the presence and absence of individual surfactants are shown. (For the purposes of the present discussion, sorption is defined as an overall process encompassing a variety of mechanisms by which metals are removed from the aqueous phase by the alga.) Metal concentrations taken up by U. lactuca, [Me–S] (w/w, on a dry mass basis), and in sea water, [Me], have been corrected for concentrations in the corresponding controls in which no metal had been added. Ambient, environmental concentrations of Pd are too low to be detected by ICP-MS (Ravindra et al., 2004; Turner et al., 2007) but measurable signals in the sea water and U. lactuca controls, reflecting molecular ion interferences during analysis, were subtracted from signals determined in the presence of added metal. Concentrations of Cd and Pb were detected in controls of U. lactuca but not sea water. Control concentrations of Cd were small (<3 nmol g1) compared with those in U. lactuca arising from metal addition, but concentrations of Pb were significant (about 15 nmol g1). For the purposes of this study, the latter concentration was subtracted from the measured Pb value in the algae in each case. Thus, we assume that Pb that has been accumulated by U. lactuca in situ is effectively independent of Pb that has been taken up during the relatively short period of experimental incubation. The Freundlich model was applied to each data set:
½Me—S ¼ KF ½Men where KF is the Freundlich constant and n is a term defining the curvature of the non-linear fit. Model fits are annotated on Figs. 2–4 and constants, derived from power regression analysis, are shown in Table 2. With respect to Pd, fits were significant (p < 0.05) in all cases with values of n close to 1. Regarding Cd, data are more scattered and, although the Freundlich model produced significant fits, presence of TX resulted in a relatively invariant concentration of Me–S and a high degree of isotherm curvature (n ¼ 0.17). Freundlich fits to the Pb data were significant in three cases but, as with Cd, presence of the non-ionic surfactant resulted in a relatively invariant concentration of Me–S which could not be modelled successfully. 3.3. Metal speciation and sorption mechanisms An understanding of the sorption mechanisms involved in the present experiments requires an evaluation of the aqueous speciation of Pd, Cd and Pb. Specifically, since the free ion is generally assumed to represent the most (or only) species able to interact with algae (Xue et al., 1988; Sheng et al., 2004), an indication of its concentration is desirable. The inorganic speciation of the metals in sea water was calculated using the Windermere Humic Aqueous Model (WHAM, v6) and either default (Cd and Pb) or published (Pd) constants after ion activity coefficients had been corrected for using the Debye–Hu¨ckel equation. (Further details of the thermodynamic constants used and the modelling approach adopted are given in Turner et al., 2008.) The results of the present calculations, exemplified in Table 3, were similar over the range of aqueous metal concentrations employed and the pH of the experiments. Organic complexation with pre-existent and exuded ligands is likely to be important for at least Pd and Pb under the present experimental conditions (Li and Byrne, 1990; Vasconcelos and Leal, 2001) but is difficult to evaluate precisely. For instance, incorporating a polyelectrolyte, modelled as default humic substances and at a concentration equal to the measured DOC concentration (Turner et al., 2008), resulted in the same relative proportions of the inorganic components of each metal but
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Fig. 2. Isotherms defining the sorption of Pd by U. lactuca in sea water (salinity w33; pH w8.1–8.4) in the absence and presence of 3 mg L1 of individual surfactants. Error bars represent the standard deviation about the mean of three independent experimental determinations. Lines represent Freundlich fits to mean data points; isotherm constants are given in Table 2.
Fig. 3. Isotherms defining the sorption of Cd by U. lactuca in sea water. See caption to Fig. 2 for further details. Note that in some cases Cd was not detected in U. lactuca or sea water.
K. Masakorala et al. / Environmental Pollution 156 (2008) 897–904
901
Fig. 4. Isotherms defining the sorption of Pb by U. lactuca in sea water. See caption to Fig. 2 for further details.
abundances of organically complexed Pd, Cd and Pb of about 99%, 0.05% and 85%, respectively. Moreover, the degree of computed organic complexation was sensitive to metal concentration and the presence of competing metals (e.g. Fe and Cu). Clearly, therefore, estimates of the absolute concentrations of metal species in the experiments (and particularly those of Pd and Pb) are subject to considerable uncertainty. Based on the results of inorganic speciation calculations (Table 3), the Freundlich constants in Table 2 are also shown normalised to the concentration of the free ion (i.e. KF/fMe 2þ, where fMe 2þ represents the fraction of aqueous metal as the free ion). By neglecting aqueous organic complexants, normalised sorption constants should be considered as lower estimates. Nevertheless, they
illustrate that binding (and/or internalisation) by U. lactuca is significantly greater for the Pd ion than either Cd2þ or Pb2þ. With respect to isotherm shape, convexity observed in most cases suggests either a shift in aqueous speciation or a reduction in site availability on the algal surface with increasing metal concentration. Given that inorganic speciation is rather insensitive to changes in metal concentration and assuming that the availability of organic binding ligands remains constant in each experiment, we suspect that isotherm convexity results largely from the progressive utilisation of binding sites on the algal surface or increasing competition among the metals for such sites (Webster et al., 1997; Lee and Wang, 2001). On this basis, therefore, and Table 3 Aqueous, inorganic speciation of 100 nM of Pd, Cd and Pb in sea water (salinity ¼ 33; pH ¼ 8.2), as computed by WHAM
Table 2 Freundlich constants defining the sorption of Pd, Cd and Pb by U. lactuca, derived from power regression analysis of the data shown in Figs. 2–4 (ns ¼ not significant; p < 0.05). KF/fMe 2þ represents the Freundlich constant normalised to the computed fraction of the free ion (Table 3) r2
KF/fMe 2þ
0.825 1.020 0.924 0.930
0.866 0.944 0.984 0.961
4.85 1014 1.93 1014 3.74 1014 2.56 1014
220 204 197 731
0.940 0.854 0.678 0.170
0.924 0.799 0.854 0.926
5450 5050 4880 18,100
3610 2250 826 4690
0.411 0.545 0.800 0.105
Added reactants
KF
Pd Pd þ HDTMA Pd þ SDS Pd þ TX
6650 2650 5120 3510
Cd Cd þ HDTMA Cd þ SDS Cd þ TX Pb Pb þ HDTMA Pb þ SDS Pb þ TX
n
0.964 0.917 0.944 0.147 (ns)
108,000 67,400 24,700 140,000
Species
Concentration (nM)
Pd2þ Pd(OH)2 PdCl3(OH)2 PdCl2 PdCl 3 PdCl2 4
1.37 109 42.3 48.8 6.32 103 1.29 7.51
Cd2þ CdSO4 CdClþ CdCl2 CdCl3
4.04 1.13 47.3 35.3 12.1
Pb2þ PbOHþ PbSO4 PbCO3 Pb(CO3)2 2 PbClþ PbCl2
3.34 1.87 2.09 72.0 3.31 12.5 4.89
K. Masakorala et al. / Environmental Pollution 156 (2008) 897–904
Pd2þ þ 2Cl þ G 4PdCl2 G ;
metabolic-dependent uptake, against the total concentration of metal taken up, [Me–S]. Results are presented for three added metal concentrations and for experiments undertaken in the presence and absence of HDTMA (results in the presence of SDS and TX showed no systematic or significant difference to those in the surfactant-free system). In the absence of HDTMA, the degree of internalisation was greatest for Pd (up to about 65%), and for all metals internalisation appeared to decline with increasing [Me–S]. These observations are consistent with the earlier assertion of increasing occupation of specific, and perhaps physiologically active sites (as precursors for transport across cationic channels), with increasing metal concentration, and the greatest affinity for these
400
[Me-S] int, nmol g-1
Pd
200
100
Fig. 5 shows the concentration of metal that was internalised by U. lactuca, [Me–S]int, as operationally defined by its resistance to mobilisation by EDTA and representing passive (diffusive) and
200
400
600
20
Cd 15
10
5
0
0
20
60
40
[Me-S], nmol g-1 50 Pb 40 30 20 10 0
3.4. Metal internalisation
0
[Me-S], nmol g-1
log K ¼ 19:7
In the absence of additional organic ligands, up to 12% of Pd was computed to be complexed by glycine over the working concentration range of metal; however, in the presence of aqueous polyelectrolyte, modelled as above, less than 1% was predicted to be complexed by the amino acid. We may conclude, therefore, that DFAA exert a small impact on extracellular metal speciation in the present experiments. Accordingly, competitive sorption between metals and the amphiphile (see above) and enhanced membrane permeability and mobilisation of internalised metal are more likely responsible for reduced uptake of Pd, Cd and Pb in the presence of HDTMA.
300
0
[Me-S] int, nmol g-1
consistent with its affinity for the algal surface, we predict that Pd, exhibiting quasi-linear sorption, is able to outcompete Pb (and perhaps Cd) for available binding sites. Examination of the sorption constants reveals that metal uptake is sensitive to the presence of the individual surfactants, and that this sensitivity is metal-dependent. Specifically, Pd sorption is considerably reduced in the presence of either HDTMA or TX, but little affected by SDS. Regarding Cd and Pb, however, sorption is reduced in the presence of all surfactants, but to the greatest extents by SDS and TX. These effects can be explained by surfactantinduced modification of both aqueous metal speciation and the algal surface. The cationic surfactant, HDTMA, is predicted to undergo effective sorption through both electrostatic and, in particular, hydrophobic interactions (Chen et al., 1998; Cruz-Guzma`n et al., 2006). The former interactions may compete directly with those involving metal and negatively charged algal surface sites, although mutually amenable sites are likely to be non-coordinating (for example, sulphate and unprotonated carboxyl oxygen; Rosen et al., 2001). That the reduction in metal sorption in the presence of HDTMA was greatest for Pd suggests there is some specificity or competition among the metals for such sites (as inferred above). Since the non-ionic surfactant, TX, is predicted to interact with algae non-specifically (e.g. hydrophobically or via hydrogen-bonding), we surmise that its influence on Cd and Pb sorption is related to the shielding of otherwise available (charged) sites rather than to any competitive effect. SDS is predicted to interact least with the algal surface because of the negative charge of the hydrophile and the relatively low Kow of the molecule (Table 1). A reduction in the uptake of Cd and Pb in the presence of SDS cannot, therefore, be explained in terms of algal surface modification or competitive sorption. Accordingly, we propose that these metals are partially stabilised in sea water by SDS through either complexation or counter ion exchange with the surfactant (Friedel et al., 1994; Juang et al., 2003). Although interactions (or complexes) between HDTMA and aqueous metal ions are unlikely, the cationic surfactant may also exert an indirect impact on the speciation and/or uptake of metals by mobilising intracellular amino acids as complexants in the extracellular medium (Fig. 1) or by enhancing cell membrane permeability. We do not know the precise makeup of DFAA mobilised by U. lactuca, but their potential impact on metal speciation was evaluated by repeating WHAM simulations for Pd, the metal having the greatest affinity for nitrogeneous ligands, in the presence of 10 nM of unprotonated glycine, G, by encoding the following reaction into the software (Li and Byrne, 1990):
[Me-S] int, nmol g-1
902
0
20
40
60
80
[Me-S], nmol g-1 Fig. 5. Internalised versus total concentration of metal in U. lactuca in the absence (-) and presence (,) of 3 mg L1 (w10 mM) of HDTMA. Error bars represent the standard deviation about the mean of three independent experimental determinations.
K. Masakorala et al. / Environmental Pollution 156 (2008) 897–904
sites displayed by Pd. In the presence of HDTMA, the fraction of internalised metal is reduced in all cases, presumably because of enhanced cell permeability and metal leakage incurred by the cationic surfactant. 3.5. General implications The results of this study have demonstrated that, while all surfactant types inhibit the uptake of trace metals by U. lactuca, only the cationic surfactant, HDTMA, exerts a measurable (and negative) impact on internalisation because of its ability to enhance cell membrane permeability. Although the precise effects observed in this study are specific to the particular reactants, the mechanisms involved and the biocidal nature of most cationic surfactants suggest that the broad findings are likely to apply to a wider range of metals, surfactants and macroalgae. Thus, the observations have important, general implications for the uptake, toxicity and trophic transfer of metals in environments that are concurrently impacted by both metals and surfactants. Such conditions may arise during the control of oil spills (Edwards et al., 2003) or harmful algal blooms (Sun et al., 2004), and in coastal areas that are moderately impacted by domestic or industrial effluents. Trace metal concentration factors or sorption constants based on biomonitoring programmes conducted in pristine environments are not, therefore, necessarily applicable where other types of contaminant co-exist. 4. Conclusions Common, synthetic surfactants exert a significant impact on the uptake of trace metals (Pd, Cd and Pb) by the marine macroalga, U. lactuca. Within the concentration range of the reactants studied, the anionic and neutral surfactants, sodium dodecyl sulphate and Triton X-100, respectively, inhibit the overall uptake of metals, but do not appear to alter cell membrane permeability or affect metal internalisation. In contrast, the cationic surfactant, hexadecyltrimethylammonium bromide, inhibits both the uptake of metals and their internalisation, effects attributed to the ability of this amphiphile to induce significant membrane damage. Acknowledgements KM was supported by an Erasmus Mundus studentship to undertake a Joint European Masters in Water and Coastal Management. We thank Miss Angela Watson and Dr. Andy Fisher for assistance with algal culturing and metal analysis, respectively. The insightful comments of two anonymous reviewers were greatly appreciated. References Baes, C.F., Mesmer, R.E., 1976. The Hydrolysis of Cations. Wiley and Sons, New York, 489 pp. Boisson, F., Hutchins, D.A., Fowler, S.W., Fisher, N.S., Teyssie, J.-L., 1997. Influence of temperature on the accumulation and retention of 11 radionuclides by the marine alga Fucus vesiculosus (L.). Marine Pollution Bulletin 35, 313–321. Chen, Y.M., Liu, J.C., Ju, Y.H., 1998. Flotation removal of algae from water. Colloids and Surfaces B 12, 49–55. Conti, M.E., Cecchetti, G., 2003. A biomonitoring study: trace metals in algae and molluscs from Tyrrhenian coastal areas. Environmental Research 93, 99–112. Costanzo, S.D., O’Donohue, M.J., Dennison, W.C., Loneragan, N.R., Thomas, M., 2001. A new approach for detecting and mapping sewage impacts. Marine Pollution Bulletin 42, 149–156. Cruz-Guzma`n, M., Celis, R., Hermonsin, M.C., Koskinen, W.C., Nater, E.A., Cornejo, J., 2006. Heavy metal adsorption by montmorillonites modified with natural organic cations. Soil Science Society of America Journal 70, 215–221. De Souza, M.P., Chen, Y.P., Yoch, D.C., 1996. Dimethylsulfoniopropionate lyase from the marine macroalgae Ulva curvata: purification and characterization of the enzyme. Planta 199, 433–438. Edwards, K.R., Lepo, J.E., Lewis, M.A., 2003. Toxicity comparison of biosurfactants and synthetic surfactants used in oil spill remediation to two estuarine species. Marine Pollution Bulletin 46, 1309–1316.
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