Environ. Sci. Technol. 2007, 41, 5679-5684
Humic Acids Increase Dissolved Lead Bioavailability for Marine Invertebrates P A U L A S AÄ N C H E Z - M A R IÄ N , * , † J. IGNACIO LORENZO,† RONNY BLUST,‡ AND RICARDO BEIRAS† Departamento de Ecoloxı´a e Bioloxı´a Animal, Fac. Ciencias do Mar, Universidade de Vigo, Crta. Colexio Universitario s/n. E-36310, Vigo, Spain. Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium
The presence of dissolved organic matter (DOM), as humic acids (HA), in natural waters is assumed to decrease dissolved metal bioavailability by binding metal ions and, therefore, decreasing the free ion concentration in solution. In this study, Pb complexation by HA in artificial seawater was checked by means of square wave anodic stripping voltammetry (SWASV). Uptake and toxicity of this metal in the absence and presence of HA was tested using excised gills of Mytilus edulis and the Paracentrotus lividus embryo-larval bioassay respectively. Both Pb uptake by mussel gills and Pb toxicity to sea urchin larvae increased in the presence of HA, and this increase was higher at higher HA concentrations. Since it is shown that the presence of DOM can enhance the uptake and toxicity of lead in some important marine invertebrate species, these results challenge the general applicability of the free ion activity and related models used for deriving environmental water quality criteria for metals.
Introduction An important goal in aquatic ecotoxicology is to predict the bioavailability of dissolved metals as a function of their speciation in the environment. The widely used models FIAM (free ion activity model) (1) and BLM (biotic ligand model) (2, 3) were developed to address this challenge. The theoretical basis is that in a system in equilibrium, the free metal ion activity reflects the chemical reactivity of the metal, i.e., the extent to what the metal reacts with binding sites on the cell membrane surface, and hence its bioavailability (4). The presence of ligands, as dissolved organic matter (DOM), in the bulk solution plays an important role decreasing the free metal ion concentration. Although these models have been widely used in bioavailability studies, there are also a number of studies showing deviating results and questioning their general applicability to natural waters (reviewed in refs 4 and 5). Currently, the application of this model for copper is generally accepted, and even the U.S. Environmental Protection Agency has included the BLM into its regulatory framework for Cu (6). However, for other metals, the number of bioavailability studies including speciation measurements is still scarce, especially in seawater, given the difficulty of measuring metal * Corresponding author e-mail:
[email protected]. † Universidade de Vigo. ‡ University of Antwerp. 10.1021/es070088h CCC: $37.00 Published on Web 07/11/2007
2007 American Chemical Society
speciation with ion-selective electrodes, due to limited sensitivity and interferences caused by major ions. The present study aims at testing the applicability of FIAM to dissolved Pb in the presence of DOM in marine systems. After Hg and Cu, Pb is the next toxic metal whose speciation is significantly altered by DOM complexation. HAs are part of the pool of DOM in natural waters. They bind metals by means of their carboxylic and phenolic groups, and have been extensively used in bioavailability studies. Previous work by Lorenzo et al. (7-9) revealed that Cu complexation by humic acids decreased its toxicity and biouptake for Paracentrotus lividus larvae and excised mussel gills, respectively, and these results are in agreement with FIAM predictions for that metal. In the present study, the same biological responses (Pb uptake by excised mussel gills and sea urchin larval growth) were used to test the applicability of the FIAM to Pb in the presence of HA in seawater. Due to their bioaccumulation capacity and worldwide distribution, mussels are frequently used in pollution monitoring programs (10). The use of excised gills in metal uptake experiments provides a well-defined model system to study the uptake of metals by the gill epithelium. The exposure of whole mussels involves different sites of metal uptake including the digestive system, which may result in the break down of complexes and uptake of released metals, leading to higher uptakes than predicted by the FIAM (7, 11, 12). Sea urchin larval bioassays have been widely used to assess toxicity in seawater and marine sediments (13, 14). They are, together with bivalve embryo bioassays, the most frequently used bioassays in seawater, and their use is comparable to that of Daphnia in freshwater.
Materials and Methods Reagents. All experimental solutions were prepared in chemically defined artificial seawater (ASW) according to the formulation described in Lorenzo et al. (8). Deionized water purified by ion exchange (resistivity g18.2 MΩ/cm; MilliQ; Millipore, Mosheim, France) and analytical grade salts were used. The seawater was aerated with 0.22 µm filtered air overnight to establish CO2 equilibrium with the atmosphere. Before each experiment, pH and salinity were checked and found to be 8.14 ( 0.05 and 35 ( 0.5‰, respectively. A 1.000 g Pb/L standard solution (Pb(NO3)2 in 0.5N HNO3; Panreac Quı´mica SA; Barcelona, Spain) was used for preparing Pb2+ dilutions. Humic acid stock solutions of 1 g/L were prepared by dissolving the acid form of HAs (Fluka, Aldrich; Steinheim, Germany) in 7 × 10-3 M NaOH. In order to remove particulate forms of HAs, stock solutions were immersed for 30 min in an ultrasonic bath and filtered afterward through 0.45 µm polyethersulfone filters (PALL Gelman Laboratory; Ann Arbor, Michigan). HA solutions were stored at 4 °C in the dark to prevent photochemical aging. Before use, they were sonicated again for 10 min to redissolve HA aggregates. DOC content of the HA stock solutions was analyzed using a total carbon analyzer (Shimadzu TOC-5000, model ASI-5000-S, Japan) and was found to be 0.33 ( 0.01 g/L (n ) 2). Lead and HA solutions in ASW were let to equilibrate for 24 h at 15 °C in the case of the mussel gills uptake experiments and 20 °C in the case of the sea urchin larvae toxicity tests. It was verified that neither HA nor lead additions altered the pH of the solutions. All glassware and polypropylene labware were soaked for 24 h in 10% HNO3 and rinsed several times with ultrapure water before use. Estimation of Pb Complexation by HA. In order to check Pb complexation by HA in seawater and to choose appropriate VOL. 41, NO. 16, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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[HA] to be used in the experiments, Pb-HA complexation was checked by square wave anodic stripping voltammetry (SWASV). A series of increasing quantities of HA (13 additions ranging from 1 to 60 mg HA/L) were added to different sets of total Pb concentrations (0.25, 0.5, 1, and 2 µM) in ASW. Electrochemically labile lead was measured with a hanging mercury drop electrode (HMDE), a Ag/AgCl reference electrode and a Pt-rod auxiliary electrode held in a Metrohm 663 VA polarographic stand coupled to an Eco-Chemie AutoLab PGSTAT10 pontenciostat (Metrohm; Herisau, Switzerland). Measurements were performed in a Teflon cell thermostated at 20 °C. The cell was preconditioned with 10 mL of each sample for 5 min, then the solution was discarded and the remaining 10 mL of the sample were measured. The deposition potential was -0.65 V and deposition time varied depending on Pb concentration (30 s for solutions with less than 1 µM of Pb and 10 s for higher Pb concentrations). A equilibration time of 5 s was allowed before the voltage scan (from -0.65 to -0.25 V). Three voltammograms were recorded for each solution and the arithmetic mean of the peak current (Ip) was calculated. Quality Control of Experimental Solutions. To study the background Zn, Cd, Pb, and Cu content in the ASW a 20 mL sample was acidified to pH 2 with HNO3, and the four metals were measured by SWASV by standard additions. Pb Uptake by Mussel Gills. Mussels between 50 and 60 mm length were collected from an intertidal site at Wemeldinge (Oosterschelde, The Netherlands). They were transported to the laboratory on ice in a cooling box. They were cleaned from epibionts and transferred to an acclimatization tank filled with artificial seawater of 35‰ at 15 °C. The mussels were fed with microalgae cultures until 2 days before the experiment. Each experimental solution was prepared in a 2 L polypropylene bottle and distributed in six plastic vessels (replicates) of 300 mL. Total lead concentrations in exposure solutions ranged from 0.1 to 3 µM, and [HA] ranged from 5 to 40 mg/L. The gill uptake experiment was performed following the methodology described by Lorenzo et al. (7). Gills were cut from the mantle and incubated for an hour at 15 °C in the test solutions. After incubation, gills were washed twice for 15 s in ASW and once for 5 s in ultrapure water and then transferred into 4 mL polypropylene vials. Gill dry weight was determined after drying for 48 h at 65 °C. Dried gills were digested by a microwave assisted procedure adapted from De Wit and Blust (15). Blank vials and reference material (Mussel tissue; CRM 278R; European Community Bureau of Reference Materials, Geel, Belgium) were also prepared and digested together with the gill samples. Pb concentration was measured by inductively coupled plasma atomic-emission spectroscopy (ICP-AES) using a Varian Liberty Series II ICP-AES (Varian, Mulgrave, Victoria, Australia). Samples with [Pb] below 20 µg/L were measured by inductively coupled plasma mass spectroscopy using a Varian ICP-MS (Varian, Mulgrave, Victoria, Australia). Lead concentrations in gills were expressed on a dry-weight basis (µg/gdw). Measured Pb concentration in the reference material agreed well with the certified concentration, within (6%. Sea Urchin Embryogenesis Bioassay. Adult sea urchins were collected from a subtidal population at Canido (Rı´a of Vigo; NW Iberian Peninsula), immediately transported to the laboratory in a cooler, kept in a 200 L aquarium at 12-18 °C, and fed with Ulva lactuca and Mytilus galloprovincialis. Total lead concentrations in exposure solutions ranged from 0.1 to 3 µM and [HA] ranged from 2.5 to 30 mg/L. The sea urchin bioassay was conducted following the procedures of Ferna´ndez and Beiras (16). After in vitro fertilization, around 80 eggs (20 µL) were delivered into 4 mL polypropylene vials containing the test solutions to get a final density of 20 eggs per mL. Five replicates per treatment 5680
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and ten ASW blanks were assayed. The vials were incubated at 20 °C for 48 h in the dark. After the incubation period, larvae were fixed with a few drops of 40% formalin. The length of 35 individuals was recorded under inverted microscope as the endpoint of the bioassay. Data Analysis and Statistics. Linear and nonlinear regression analyses were performed using Sigma-Plot 2002 for Windows (SPSS Inc.). Parameters (slopes and EC50) obtained in different models were compared using the extra sum-of-squares F test by means of global fitting (17). Some parameters were fixed in multiple regression analysis to avoid under or overestimation of the variable’s influence produced by the effect of covariation (18). Fixed parameters (lead uptake rate for gills and Hill slope in the Pb toxicity curve) were previously determined experimentally. Goodness of fit of the models was reported by the coefficient of determination (R2).
Results Estimation of Pb Complexation by HA. The presence of increasing quantities of HA in the solutions caused the following polarographic responses: a decrease in Ip, an increase in peak width, a shift in peak potentials toward more negative values and an increase in the background current. The decrease in Ip is attributed to the formation of Pb-HA complexes. A higher decrease in Ip was observed with increasing HA concentrations. For instance, in the presence of 5 mg/L of HA, the Ip obtained ranged from 50% (0.25 µM of PbT) to a 70% (2 µM of PbT) as compared with the Ip obtained without HA; while for 30 mg/L of HA this ratio ranged from 20 to 30% (from 0.25 to 2 µM of PbT). Observed peak intensities in the presence of HA within the treatments used in the biological experiments ranged from 75 to 25% of the Ip observed for the same total lead concentration in the absence of HA. The other polarographic responses are related with the adsorption of HA on the mercury electrode and the electrochemical behavior of Pb-HA complexes. The effect of these phenomena in the measured Ip will be discussed afterward. Quality Control of Experimental Solutions. Total metal concentrations in the stocks of ASW were very low compared to the lead concentrations used in the experiments: [Zn] < 2 µg/L, [Pb] and [Cu] < 1 µg/L and Cd was below detection limit for the measurement conditions used (Cd < 0.05 µg/L). Actual dissolved lead concentrations in the only lead exposure solutions were measured and found to be the nominal concentrations within (4%. Measured Ip in the experimental solutions were consistent with the expected from the previous complexation measurements. Any significant organic complexation capacity was not detected in the artificial seawater at the range of Pb concentrations used in the experiments. Pb Uptake by Mussel Gills in the Presence of HAs. Pb Uptake by Mussel Gills. Pb concentrations in mussel gills exposed for 1 h to Pb increased linearly with Pb concentration in exposure solutions (Figure 1, black dots), and no evidence of saturation was apparent at tested concentrations. A general uptake model was obtained from gills exposed only to Pb. The initial lead concentration in gills ([Pb]ini) was fixed to the measured value (0.88 ( 0.23 µg/gdw) and the uptake rate obtained was 12.22 ( 0.45 µg/gdw per µM of Pb and per hour (Table 1, model 1). Effect of HA on Pb Uptake. The presence of HA increases Pb accumulation in gills (Figure 1, open dots) and this effect is higher at higher HA concentrations (Figure 2). Bars in Figure 2 represent Pb concentration in gills ([Pb]gills) grouped according to the total Pb concentration in the exposure solutions. Regression analysis showed that, at constant PbT concentrations, [HA] had a significant enhancing effect on
FIGURE 1. Mean Pb concentration ( stdv (n ) 6) in gills exposed for 1 h to different concentrations of Pb and Pb + HAs represented versus total Pb concentration in exposure solutions. B represent [Pb] in gills exposed only to Pb, and n correspond to gills exposed to Pb+HA. Solid and dashed line represent the regression line fitted to the data of Pb-only and Pb + HA solutions, respectively.
FIGURE 3. Mean percentage of larval growth ( stdv (n ) 5) plotted versus total Pb concentration in exposure solutions. B correspond to only Pb exposures and n correspond to Pb + HA exposures. Solid and dashed lines represent the fitting of Pb-only and Pb + HA exposures respectively to the toxicity model described in eq 1. variation observed (model 2; Figure 1, dashed line). When both [PbT] and [HA] were considered in the regression (model 3), the explained variability increased up to 95%. The c parameter obtained was significantly different from zero (p < 0.001), showing that the overall effect of HA is to increase Pb uptake at a rate of 0.41 ( 0.05 µg/gdw/h per mg/L of HA. Toxicity of Pb in the Presence of HA Evaluated by the Sea Urchin Embryogenesis Bioassay. Pb Effect on Larval Growth. Pb effect on P. lividus embryo-development was reported as the decrease in larval growth as compared to the control expressed in percentage (%LG). Black dots and the solid line in Figure 3 show the dose-response relationship for Pb exposure. Data were fitted to the following log-logistic model by least-squares regression analysis:
%LG )
FIGURE 2. Pb concentrations in gills exposed for 1 h to different PbT concentrations with increasing additions of HA. Error bars represent standard deviations (n ) 6). [Pb]gills, with slopes ranging between 0.2 and 0.6 (p < 0.01). This effect was found for all [PbT] except for 0.2 µM. At 0.2 µM of PbT, a slightly significant negative slope was found (-0.06, p ) 0.02), showing a small protective effect of HA. However, when the uptake at this [PbT] was expressed as a function of labile lead concentrations, the uptake was still higher than expected on the basis of FIAM (slope significantly higher; F test; p < 0.05). The overall effect of HA on Pb uptake was quantified by means of multiple regression analysis. The regression coefficients obtained are shown in Table 1. Model 1 describes the general uptake model obtained with the “Pb only” exposures; the uptake rate was 12.22 ( 0.45 µg/gdw/h. When considering gills exposed to Pb and HA, the uptake rate (16.05 ( 0.83 µg/gdw/h) was significantly higher (F test; p < 0.01) and total Pb concentrations could explain 87.5% of the
100 [Pb] 1+ EC50
( )
(1)
a
where a is the Hill slope of the toxicity curve and the EC50 is the median effective concentration. This model is equivalent to the sigmoid Emax model used in pharmacology and first proposed by Hill (19). The fitted parameters and their standard errors are EC50 ) 2.25 ( 0.25 µM of Pb and a ) 1.37 ( 0.21. This model explains 97% of the variation observed. Effect of HA on Pb Toxicity. Results of toxicity tests are shown in Figures 3 and 4. The addition of HA to Pb solutions causes a clear increase in toxicity (decrease of larval growth) for sea urchin larvae. This effect is higher at higher HA concentrations. Only at a total lead concentration of 10 µM, where larval growth is almost zero, toxicity is not significantly altered by HAs addition. In order to quantify the effect of HA on Pb toxicity, [HA] was included in the toxicity equation as shown in Table 2. The Hill slope (a) was fixed to the value obtained with the Pb-only exposures. Data from “Pb and HA” exposures were fitted to this model as function of [PbT] in solutions, and the EC50 obtained (0.97 ( 0.12 µM) was significantly lower (p
