Influence of Humic Acid on Algal Uptake and Toxicity of Ionic Silver

Article pubs.acs.org/est

Influence of Humic Acid on Algal Uptake and Toxicity of Ionic Silver Zhongzhi Chen, Céline Porcher,† Peter G. C. Campbell, and Claude Fortin* Institut National de la Recherche Scientifique, Centre Eau Terre Environnement (INRS-ETE), 490 rue de la Couronne, Québec (QC), Canada G1K 9A9 S Supporting Information *

ABSTRACT: The biogeochemical cycle of silver has been profoundly disturbed by various anthropogenic activities. To better understand the relationship among silver speciation, bioavailability, and toxicity in freshwaters, we have studied the short-term uptake of silver by two species of green algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata, in the presence or absence of a well-characterized humic acid (Suwannee River Humic Acid, SRHA). The free Ag+ concentrations in the exposure solutions were determined using an equilibrium ion-exchange technique. According to the biotic ligand model, for a given free metal ion concentration, metal uptake should remain the same in the presence or absence of humic acid. However, short-term silver uptake in the presence of SRHA was greater than anticipated on the basis of free Ag+ concentration. Subsequent determination of silver subcellular distribution revealed that significantly more silver was present in the “cell debris” fraction (known to contain the cell wall and fragmented membranes) in the presence of SRHA than in its absence. Finally, this increase in silver uptake in the presence of humic acid did not result in decreased algal growth. These results suggest that the increase in silver uptake observed in the presence of SRHA is surface-bound, not truly internalized.

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INTRODUCTION Silver (Ag) is naturally present at very low concentrations in surface waters, and is considered to be a nonessential metal. However, it has been widely used by humans, with usages ranging from photographic processes to new applications as nanoparticulate silver, resulting in increased inputs into the aquatic environment.1,2 Generally, cationic metals bind to natural humic substances in aquatic environments, and these complexes may dominate the metal’s speciation in the water column. Humic substances are ubiquitous in natural waters, and have been implicated in many direct and indirect interactions with freshwater organisms: not only complexing metal cations, but also inducing oxidative stress, interfering with photosynthesis and growth, and providing nutrients.3 However, the influence of humic substances on silver−algae interactions has not, to our knowledge, been explored so far. In principle, complexation of a cationic metal by humic acid should lead to a decrease in its bioavailability,4 and indeed there are several reports in the literature that are in qualitative agreement with this expectation for silver.5,6 However, humic substances might also affect silver uptake by interacting with the algal cell surface and altering chemical and physical processes at the cell−solution interface.7 For example, Slaveykova et al. reported that in the presence of fulvic acid, lead uptake by Chlorella kesslerii was much greater than predicted on the basis of the free Pb2+ concentration.8 Sánchez-Marı ́n and Beiras also reported that humic acid (HA) enhanced Pb internalization by the marine phytoplankter Isochrysis galbana, and attributed this enhancement to HA adsorption on the cell membrane surface.9 The biotic ligand model (BLM) provides a useful construct to study interactions between metals and aquatic organisms.10 The BLM assumes that hydrophilic metal complexes cannot pass across biological membranes. If the BLM applies to silver− © 2013 American Chemical Society

algae interactions, complexation of silver by humic substances should reduce its bioavailability, and uptake should be directly proportional to the free Ag+ concentration (or activity), as long as Ag+ concentrations are well below the level needed to saturate the algal transport sites. To determine if the BLM correctly predicts the bioavailability of silver in the presence of humic substances, it is also essential to distinguish between free and complexed silver in solution. An ion-exchange technique (IET) was used to determine the free Ag+ concentrations in our exposure media.11 The present study focused on experimental media containing a unicellular alga (two different species), ionic silver and a standard humic acid (Suwannee River Humic Acid, SRHA). Our objective was to determine if the effect of humic acid on silver uptake and toxicity is limited to the complexation of Ag+ in solution, that is, to evaluate if Ag−SRHA complexes are indeed biologically unavailable as predicted by the BLM, and if not, to examine potential explanations. Initial efforts focused on the relationship between silver speciation and biological uptake. Short-term algal uptake of silver was followed in the presence or absence of SRHA (≤ 10 mg C·L−1). The subcellular distribution of silver was also determined to differentiate between metal adsorption to the surface of the cells and metal binding to cytosolic ligands or intracellular organelles.12 Finally, algal growth inhibition experiments were performed in the presence or absence of humic acid to determine if the uptake of silver could be used to predict its toxicity in the presence of SRHA. Received: Revised: Accepted: Published: 8835

March 11, 2013 May 31, 2013 June 21, 2013 June 21, 2013 dx.doi.org/10.1021/es401085n | Environ. Sci. Technol. 2013, 47, 8835−8842

Environmental Science & Technology

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Determination of Silver Uptake. To prepare a batch of algal cells for uptake experiments, algae were initially inoculated into fresh growth medium at a density of ∼40 000 cells·mL−1, and then gently harvested after 48 h (C. reinhardtii) or 72 h (P. subcapitata) of growth, corresponding to the midexponential growth phase. A single algal culture was used for comparisons between each treatment to avoid interbatch variations. Cells were gently harvested on polycarbonate filter membranes (2 μm pore size, Millipore), using a low vacuum pressure (< 10 cm Hg). Harvested cells were rinsed with 4 × 10 mL of sterile simplified culture medium (MHSM-R, see section 1 of the SI) without phosphate or trace metals. The algal cells were then resuspended in 10 mL MHSM-R. Cell density, size distribution and average surface area were determined rapidly with a particle counter (Multisizer 3; Coulter Electronics, 70 μm aperture). Aliquots of concentrated cell suspensions were then swiftly distributed into all exposure flasks. The exposure solution volume and number of algal cells were selected to provide sufficient signal from the γ-counter, while minimizing the depletion of dissolved AgT in exposure solutions; cell numbers never exceeded 80 000 cells·mL−1. Exposures were carried out for short periods (≤ 60 min) to minimize the influence of the algae on their exposure medium (metal-binding exudates from the algal cells could affect silver speciation). Algae were exposed under ambient laboratory conditions (22 °C) and were manually shaken at the beginning and end of the exposure period. Silver uptake over time (0−60 min) at fixed AgT concentrations (35−85 nM) was determined in the presence or absence of SRHA (0, 5, and 10 mg C·L−1) to confirm linearity of uptake. Algal cells were then exposed to increasing concentrations of Ag+ (up to 160 nM Ag+) with or without SRHA at pH 7.0, for up to 60 min. Finally, silver uptake was determined at pH 5.5 (50 nM Ag+; 60 min) in the presence or absence of SRHA (0 and 5 mg C·L−1) and compared to uptake observed at pH 7.0. Extra silver was added to the media containing SRHA in order to obtain a free Ag+ concentration in the desired range. Thus, total [AgT] in the media with SRHA was higher than in the inorganic media (e.g., at 5 mg C·L−1 and total dissolved [AgT] ∼75 nM, [Ag+] ∼ 50 nM). Prior to all the experiments testing the effect of SRHA, the algal cells were pre-exposed for 90 min to a simplified culture solution (MHSM-E, see section 1 of the SI) with or without SRHA, in the absence of silver, in order to condition the cells to the exposure media. Exposure times were calculated from the time of addition of the cells to the exposure solution until all cells were removed from the exposure medium by filtration. After exposure to Ag, the cells were harvested on a 2 μm filter membrane. An aliquot was collected from each filtrate to measure the total “dissolved” silver for mass balance calculations and the remaining solution was analyzed using the IET to determine the free Ag+ concentration. The cells were then rinsed with 4 × 10 mL of rinse MHSM-R medium supplemented with nonradioactive silver (100 nM). This step was designed to remove any radioactive silver that had bound to the algal surface, through isotopic exchange.13 An aliquot of this filtrate was also collected to determine the amount of radiosilver initially adsorbed on the surface of the cell. The algae were initially collected on two superimposed polycarbonate filter membranes, the lower of which was used to correct for passive retention of silver. Initial results showed that the amount of radioactive silver on the second filter was always less than 3% of the signal from the upper filter, so that only one

MATERIALS AND METHODS Test Organisms and Culture Conditions. The two green algae, Chlamydomonas reinhardtii (CPCC 11) and Pseudokirchneriella subcapitata (CPCC 37), were obtained from the Canadian Phycological Culture Centre (CPCC, Waterloo, ON, Canada). Batch cell cultures were regularly maintained by transferring 2 mL aliquots into 100 mL of fresh sterile modified high salt medium (MHSM-1, see section 1 of the Supporting Information (SI)) in 250 mL polycarbonate Erlenmeyer flasks. All stock solutions were filtered through polycarbonate membranes (0.2 μm pore size, Millipore) and kept in previously autoclaved polypropylene bottles (121 °C, 15 min) at 4 °C. The axenic batch cultures were maintained at 22 °C in an environmental chamber (Conviron CMP4030) with 50 rpm rotary agitation, under constant illumination (∼100 μE·m−2·s−1). Reagents and Labware. All exposure solutions were left to equilibrate overnight under ambient laboratory conditions before use and a small aliquot was removed to determine initial total silver (AgT) concentration. All solutions were buffered with 10 mM MOPS (N-morpholino-3-propanesulfonic acid, Sigma Aldrich; pH 7.0) or 10 mM MES (2-(Nmorpholino)ethanesulfonic acid, Sigma Aldrich; pH 5.5). The ionic strength was adjusted in the range from 0.022 to 0.024 eq·L−1 by adding the required volume of a 1 M NaNO3 (ACS, EMD Chemicals) solution. Radiolabeled silver was used to facilitate quantification of silver using a gamma counter (Wallac 2480 Wizard2; Perkin-Elmer). The radioactive silver (110mAg; 45 mCi·mmol−1) was purchased from Eckert & Ziegler Isotope Products (Atlanta, GA). Stock solutions of SRHA (batch #1S101H; International Humic Substances Society, St. Paul, MN) were prepared by dissolution in 0.01 M NaOH followed by 24 h equilibrium and filtration (0.4 μm), and were kept at 4 °C in the dark. All experiments were performed in triplicate (minimum) in polycarbonate ware to minimize adsorptive losses of silver to the container walls during the incubation period. Silver Speciation. To study the effects of SRHA on silver uptake by the test algae, we needed to determine the speciation of silver in the exposure media. For the solutions without SRHA, equilibrium speciation calculations indicated that there was minimal complexation of Ag+ in the exposure medium (i.e., [Ag+]/[Ag]T > 98%; estimated using visual MINTEQ 3.0). For experiments in the presence of SRHA (5 mg C·L−1) at pH 7.0, the target initial Ag+ concentrations were estimated based on a previously determined silver titration curve11 and then determined experimentally at the end of the exposure period. At pH 5.5, the binding of Ag+ by SRHA at three different concentrations of total silver (20, 50, and 100 nM) was probed by measuring the free Ag+ concentration using the IET (brief description and distribution coefficients are provided in section 2 of the SI).11 Electrophoretic Mobility (EPM). The electrophoretic mobility (EPM) of the algal cells was determined using a Zetaphoremeter IV (ZetaCompact, Z8000, CAD Instrumentation) following a 20-min exposure to the experimental solution with or without SRHA at pH 7.0. Triplicate cell samples of C. reinhardtii were first deflagellated using an Ultra-Turrax homogenizer (T25, Inter Science) at 30 000 rpm for 10 min. Cellular integrity was verified afterward by microscopy. The Zeta potential (ζ) was calculated using the ZetaCompact software (version 4.31) based on the Smoluchowski equation. 8836

dx.doi.org/10.1021/es401085n | Environ. Sci. Technol. 2013, 47, 8835−8842


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filter was used for the subsequent experiments. Finally, the filters were individually counted for radioactivity to determine the amount of silver accumulated by the algal cells. Subcellular Distribution Experiments. Subcellular fractionation protocols have been successfully used in the past with C. reinhardtii.12,14 Briefly, to determine the subcellular location of silver in algal cells, a suspension of C. reinhardtii cells (∼150 000 cells·mL−1) was exposed to 110mAg with or without SRHA for 30 min, as done earlier in the uptake experiments. At the end of each exposure, 0.2 mL of a 0.1 M Na2S2O3 solution (to reach a final concentration of 200 μM Na2S2O3) was added to remove adsorbed Ag+ ions from the cell surface. The cells were then separated from their 100 mL exposure solutions by centrifugation (20 000g, 15 min, 4 °C), followed by four successive resuspension and centrifugation steps (20 000g, 5 min, 4 °C), each time with the addition of 10 mL MHSM-R. After the final centrifugation, the algae were resuspended in 2.5 mL MHSM-R. This washing process was designed to remove dissolved silver and thiosulfate, which might affect silver partitioning during the subsequent manipulations. A small aliquot of the algal suspension was taken to determine the final cell densities. At the same time, to determine the total accumulated silver (Agcellular), the 2 mL concentrated algal samples were transferred into glass scintillation vials and analyzed with the γ-counter. After radioassay, the 2 mL algal samples were transferred into 7 mL polypropylene tubes. To rupture the cells, samples were sonicated (sonicator, Branson 250, Danbury, CT) for 4 min (power = 22 W; pulse = 0.2 s·s−1).12 The samples were kept at 4 °C during and after sonication. The ruptured cells were then transferred into 3 mL ultracentrifugation tubes and were separated into five subcellular fractions by a differential centrifugation procedure: debris; NaOH-resistant or granule-like compounds; organelles; heat-stable proteins (HSP); heat-denaturable proteins (HDP or enzymes).15 The relative proportions of silver in each fraction were obtained by dividing the subcellular silver content of each fraction by the total cellular silver (Agcellular). All results are presented as means ± standard deviation (n ≥ 3) unless otherwise mentioned. The relative proportions (percentages) were transformed (arcsine) before the statistical tests. Toxicity Tests. These experiments were designed to determine the growth response of C. reinhardtii in batch cultures in MHSM-1 medium during chronic exposures to Ag+ (24 h) at an initial density of 10 000 cells·mL−1. Cells were exposed to free Ag+ (0−60 nM) with or without SRHA (5 mg C·L−1). Total AgT concentrations were selected in order to reach targeted range of initial free Ag+ concentrations based on the previously determined titration curve.11 At the end of the exposure period, duplicate subsamples (2 mL) were collected and the cell density and AgT concentration in the filtrate were determined as described above. Growth rates were calculated according to the following equation: μ=

if a decrease in cell density was recorded (e.g., due to cell lysis), growth was considered to be null.

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RESULTS Humic Binding. In preliminary experiments, we titrated SRHA (5 mg C·L−1) with Ag+ at pH 7.0 and compared IETdetermined Ag+ concentrations with values measured by equilibrium dialysis (EqD).11 We performed a similar titration using the same humic acid (5 mg C·L−1 SRHA) but at pH 5.5. However, Ag+ was not significantly bound to SRHA at pH 5.5 (average free Ag+ proportion of 101 ± 8%). Humic acid may sorb to surfaces, including biological membranes thereby altering the net surface charge.16 We thus determined the electrophoretic mobility of C. reinhardtii cells in the presence or absence of SRHA. The EPM measurements of the algal cells reflect the overall surface potential of the algae. Only a slight increase in mobility (t test, p < 0.05) was observed between EPM measurements made on C. reinhardtii suspensions in the absence (−2.16 ± 0.10 × 10−8 m2 V−1 s−1) or presence of 5 mg C·L−1 SRHA (−2.44 ± 0.31 × 10−8 m2 V−1 s−1). Effects of Humic Acid on Silver Uptake. Time Series Kinetics of Silver Uptake. The cellular accumulation of silver over time was studied at two Ag+ concentrations in the absence of SRHA using C. reinhardtii. Linear uptake was observed over the 60 min period with average fluxes (±standard error) of 13.0 ± 1.5 and 27.1 ± 3.5 nmol·m−2·min−1 at 40 and 85 nM Ag+, respectively (see S1 Figure SI). The Y-intercepts of the cellular silver uptake curves were not significantly different from zero (p > 0.05), suggesting that the rinsing step with nonradioactive silver was effective in removing 110mAg adsorbed at the algal surface. Dissolved AgT concentrations decreased slightly during the first minutes of the exposure and then remained almost constant; the maximum variation from the beginning to the end of the experiment was ∼15%, indicating that there was little depletion of ambient silver during the uptake period. Mass balance analysis showed that the recovery was generally between 80% and 110%, the slight decrease in AgT being attributable to absorption/adsorption by the algae. Silver Uptake in the Absence or Presence of SRHA. Algal cells (C. reinhardtii) were preconditioned to the MHSM-E exposure medium with or without SRHA, in the absence of silver, for 90 min prior to all experiments. This pre-exposure of the algal cells did not affect subsequent silver uptake (compared to cells that had not been pre-exposed; p > 0.05). In this comparison of pretreatments, all the silver exposures were carried out under the same conditions (50 nM Ag+ in the absence of SRHA). Based on the results of the time-course experiments, we studied silver uptake for a fixed time period (60 min), and exposed the algae to different concentrations of Ag+ at pH 7.0 in the absence or presence of SRHA in parallel exposure experiments. Cellular silver was measured over a broad range of free Ag+ concentrations (5−200 nM). Measurements of free Ag+ in the filtrates collected at the end of the exposure period using the IET showed that significant decreases of free Ag+ had occurred (between 26 and 52% reduction in Ag+), despite the relative constancy of the AgT concentration, most likely due to binding to cellular exudates. To take into account these lower free Ag+ values, we have used the mean free Ag+ concentration, calculated as the arithmetic mean of (a) the [Ag+] expected from the titration curve of SRHA and the AgT concentration

(ln Nt − ln Nt0) (t − t0)

(1)

where μ is the growth rate, t − t0 is the silver exposure period, and Nt and Nt0 are the cell densities at time t and at the beginning of test, respectively. Growth relative to control cells (both in presence and absence of SRHA; n = 2) was plotted as a function of the silver cell quota and fitted using SigmaPlot 11.0 software and its built-in nonlinear “four parameter logistic curve” regression tool. For the purpose of fitting the algorithm, 8837



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Figure 1. Ag uptake by C. reinhardtii for a fixed exposure period (60 min) with (○) and without SRHA (●) for 10 000 (A) or 80 000 cells·mL−1 (B) at different free Ag+ concentrations. Note that the X-axis refers to average free Ag+ concentrations; the mean free silver concentrations were calculated as the arithmetic mean of the [Ag+] that was predicted by the titration curve of SRHA based on the [AgT] before the experiment, and the final [Ag+] in the filtrate after exposure as measured by the IET.

Silver Subcellular Distribution. We compared the subcellular distribution of silver (as percentages of total Agcellular content) in C. reinhardtii cells that had been exposed to ∼50 nM Ag+ in the absence (50 nM AgT) or presence of SRHA (75 nM AgT). The relative silver distribution was similar in both exposures except for the debris fraction, which includes membranes, cell walls and nuclei,12 and which was significantly higher in the presence of SRHA (t test, p < 0.05, Figure 3A). The subcellular partitioning experiment was then repeated with twice as much SRHA (10 mg C·L−1) at ∼35 nM Ag+ (90 nM AgT). Silver uptake was higher in the presence (0.83 ± 0.07 μmol·m−2) than that in the absence of SRHA (0.39 ± 0.12 μmol·m−2), but again only the contribution of the cell debris fraction was significantly higher (t test, p < 0.05), and this difference was more important than that observed at 5 mg C·L−1 SRHA (Figure 3B). Mass balance calculations showed 85 ± 5% mean recovery (total Agcellular compared to the sum of the silver contents of the five subcellular fractions). The loss was considered to be due to adsorption on tubes and vials. Silver Toxicity. Cell growth was progressively inhibited with the increase in average free Ag+ concentration, regardless of the presence or absence of SRHA (Figure 4A). When silver toxicity was expressed as a function of silver cell quotas, the response curves in the presence and absence of SRHA also overlapped (Figure 4B). In both cases, no significant differences between the EC50 values in the absence (21.2 ± 1.8 nM; 2.26 ± 1.14 fmol·cell−1) or presence of SRHA (21.2 ± 0.7 nM; 1.76 ± 0.36 fmol·cell−1) were observed (EC50 = mean free Ag+ concentration or cellular silver quota at which cell growth is inhibited by 50%; four parameter logistic curve regression, p > 0.05). If all the data are treated together, the overall EC50 values are 21.2 ± 1.8 nM and 1.96 ± 0.46 fmol·cell−1.

added at the beginning of the experiment, and (b) the final [Ag+] in the filtrate after exposure, as measured by the IET. The uptake results, as expected, showed an increase in Agcellular in both algal species with increasing [Ag+] (Figures 1 and 2). In the presence of SRHA however, silver uptake was

Figure 2. Ag uptake by P. subcapitata after a fixed exposure period (25 min) with (○) or without SRHA (●) for 20 000 cell·mL−1 at different free Ag+ concentrations. Note that the X-axis refers to average free Ag+ concentrations; the mean free silver concentrations were calculated as the arithmetic mean of the [Ag+] that was predicted by the titration curve of SRHA based on the [AgT] before experiment, and the final [Ag+] in the filtrate after exposure as measured by the IET.

greater than anticipated based on the mean free Ag + concentration (linear regression, Extra sum-of-squares F test, p < 0.05; Figures 1 and 2). The data presented in Figure 1A and B were generated in separate experiments, with different algal batch cultures and different algal cell densities but with overlapping Ag+ concentration ranges. In parallel, we determined silver uptake by C. reinhardtii at pH 5.5 in the presence or absence of SRHA. In contrast to the results obtained at pH 7.0, no significant difference (t test, p > 0.05) in Ag+ uptake was detected in the absence (1.16 ± 0.11 μmol·m−2) or presence of SRHA (1.04 ± 0.02 μmol·m−2) at pH 5.5 ([Ag+] ∼ 50 nM in each exposure solution, t = 60 min).

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DISCUSSION

For constant free-metal ion concentrations, the BLM predicts that metal uptake should remain the same, regardless of the presence or absence of a ligand.4 However, our results suggest an increase in silver uptake in the presence of SRHA at neutral pH. In this context, we have examined several possible explanations for the enhanced bioaccumulation of silver in the presence of SRHA: (a) diffusion limitation is alleviated by the presence of the Ag−SRHA complex; (b) the SRHA affects 8838



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Figure 3. Subcellular silver distributions within C. reinhardtii cells in percentages, after 30 min exposure to ∼50 nM (A) or ∼35 nM (B) [Ag+] in the absence (blank) or presence (hatched) of 5 mg C·L−1 (A) or 10 mg C·L−1 SRHA (B). Values are given as means ± standard deviation (n ≥ 3). Significant differences between mean relative silver distributions with or without SRHA are indicated by the asterisks (p < 0.05); only the relative silver distribution in the cell debris fraction was significantly different in the presence of SRHA.

Figure 4. Silver toxicity to C. reinhardtii as a function of the mean free Ag+ (A) and as a function of the silver cell quota (B) in the absence (●) or presence (○) of SRHA (5 mg C·L−1) after 24 h. The curve represents a nonlinear regression through all the data points (four parameter logistic curve, R2 = 0.89 and 0.96, respectively).

limitation by Ag−SRHA complexes seems untenable in this case. Considering the imprecision inherent in such flux calculations,18 another algal species (P. subcapitata) was used to repeat the silver uptake experiment in the absence or presence of SRHA. This species was chosen since its silver uptake is slower than for C. reinhardtii, and we had previously shown that diffusion limitation of silver uptake does not occur with this alga in Ag−Cl systems.19 Our present result demonstrates that silver uptake by P. subcapitata in the presence of SRHA is also greater than anticipated from the BLM (linear regression, Extra sum-of-squares F test, p < 0.05, Figure 2). Since both algal species behave similarly, we can rule out alleviation of diffusion limitation as a possible explanation for the enhanced uptake. (b). SRHA Affects the Algal Surface Charge and Increases the Effective Concentration of Ag+ Close to the Algal Surface. It has been hypothesized in the past that sorption of humic substances at an algal surface could increase the net negative surface charge, and thus increase the concentration of metal cations near the membrane, leading to enhanced metal uptake.8,20 To verify this possible explanation, we determined the electrophoretic mobility of C. reinhardtii cells in the presence or absence of SRHA. The EPM measurements of the algal cells reflect the overall surface potential of the algae. Indeed, a small but significant increase (t test, p < 0.05) in the negative surface charge was observed when the SRHA concentration is increased from 0 to 5 mg

the algal surface charge and increases the effective concentration of Ag+ close to the algal surface; (c) the adsorption of SRHA at the membrane surface alters the rate of ion transport; and (d) our estimates of Agcellular at pH 7.0 in the presence of SRHA include a contribution from a tightly bound silver surface complex. (a). Diffusion Limitation Alleviated by the Ag−SRHA Complex. For the BLM to be applicable, metal internalization should be rate-limiting.17 However, previous work showed that for silver uptake by C. reinhardtii, mass transport of Ag+ (and its labile complexes) from the bulk solution toward the algal surface (biological interface) could be the rate-limiting step under certain conditions.13 Indeed, Fortin and Campbell observed a partial diffusion limitation at low (8 nM Ag+) but not at high (100 nM Ag+) free silver concentrations.13 In the present study, increasing the concentration of labile silver species (Ag−SRHA) in the bulk solution would lead to a greater concentration gradient across the phycosphere (the diffusive boundary layer), and increase the diffusive supply of Ag+ to the algal surface. We first examined the computed maximum diffusional flux for Ag+ from the bulk solution to the cell surface at pH 7.0 (see section 4 of the SI). The computed maximum diffusional flux for 50 nM Ag+ exposures is ∼3 pmol·cm−2·s−1, whereas the observed uptake fluxes of Ag+ are ∼0.03 pmol·cm−2·s−1. The computed maximum diffusion flux from the bulk solution to the algal surface is thus much higher than the internalization flux, and alleviation of diffusive 8839



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C·L−1 SRHA. However, we calculated that the silver ion enrichment at the cell surface due to this increased negative charge would only represent a 16% increase (see section 5 of the SI). We conclude that this slight increase in surface charge cannot fully explain the enhancement of silver uptake observed in the presence of SRHA. Moreover, earlier work with C. reinhardtii and the same humic acid, but with Cd2+ as the cation rather than Ag+, did not show any enhancement of Cd uptake.21 Since Cd2+ is divalent, it would have been more influenced by the proposed electrostatic attraction than the monovalent Ag+ ion (assuming that the attraction is only electrostatic in nature, it would be directly dependent on the charge of the ion). (c). Adsorption of SRHA at the Membrane Surface May Alter the Rate of Trans-Membrane Ion Transport. Humic substances are natural surfactants with amphiphilic character, and tend to adsorb and accumulate in the “phycosphere” of a phytoplankton cell.7 Boullemant et al. showed that the presence of SRHA could affect algal uptake of metal lipophilic complexes by increasing the permeability of the algal membrane.22 The adsorption of humic substances at the cell surface is influenced by pH: since at neutral pH, the deprotonation of the cell surface increases electrostatic repulsion between the SRHA and the algal surface, the amount of humic substances adsorbed at the biological surface tends to increase with decreasing pH.7,23 In addition, Vigneault et al. found that natural organic matter could adsorb to phytoplankton and lead to enhanced permeability to neutral lipophilic membrane probes at acidic pH.24 If the enhanced uptake of silver at pH 7.0 in the presence of SRHA were due to the adsorption of SRHA at the membrane surface and its influence on the rate of trans-membrane ion transport, then increased silver uptake in the presence of SRHA should be even more obvious under acidic pH conditions. In fact, for a given [Ag+], the presence or absence of SRHA gave no significant difference in silver uptake observed at pH 5.5 and we can thus discard the possibility of an increase in the Ag+ ion transport rate due to adsorbed SRHA. (d). Cellular Silver at pH 7.0 in the Presence of SRHA Includes a Contribution from a Tightly Bound Silver Surface Complex. Our estimate of cellular silver was operationally defined, based on the use of an excess of nonradioactive Ag+ to remove all surface-bound 110mAg+ at the end of the exposure period.13 We first considered the possibility that SRHA could coat the algal surface and hinder the removal of radiolabeled 110mAg+ during the exchange step with nonradioactive Ag+. If this were the case, longer contact times for the final rinsing step might be able to reduce the coating effect. Silver uptake and adsorption in the absence or presence of SRHA ([Ag+] ∼ 50 nM) were determined as a function of the contact time with the rinsing solution (MHSMR), up to 60 min. Increasing the rinsing time up to 1 h did not change the observed silver uptake (see section 6 of the SI), indicating that the cellular silver was quasi-irremovable. We then considered the possibility that, in the presence of SRHA, the Ag−SRHA complex may bind to the algal surface, either as a ternary surface complex where the silver acts as a bridge between the algal surface and the humic acid (alga−Ag− SRHA), or as silver−humic acid complex that binds to the algal surface without involving the silver ion (e.g., hydrophobically, as alga−SRHA−Ag); in both cases, the binding of the Ag− SRHA complex might contribute to the apparent increase in silver uptake (provided that the silver was not readily

exchangeable). Preliminary tests with algal cells exposed to Ag+ in the absence of SRHA showed that treatment with excess nonradioactive Ag+ did remove surface-bound silver effectively (SI Figure S1; zero Y-intercept). However, when uptake experiments were performed at AgT concentrations of 75 nM and 90 nM, in the presence of 5 or 10 mg C·L−1 SRHA, respectively, positive Y-intercepts were detected that were significantly different from zero (SI Figure S2, p < 0.05) and increased with SRHA concentration. Despite the different Yintercepts, the observed fluxes normalized for the exposure Ag+ concentration were identical (0.41 ± 0.03 and 0.42 ± 0.04 nmol·m−2·min−1·nM−1 at 5 and 10 mg C·L−1 SRHA, respectively; SI Figure S2). These observations are consistent with the formation of a surface complex, since both the binding of Ag+ to SRHA and the amount of SRHA adsorbed onto the cells would be expected to increase with a doubling of the SHRA concentration in solution without affecting the Ag+ internalization flux. Binding of SRHA to the cell surface would also be expected to increase at acidic pH (5.5), but since the IET results indicated no Ag+ binding to SRHA at pH 5.5, the additional presence of SRHA at the algal surface at this pH would not increase the apparent Agcellular concentration. Preliminary results indicated that SRHA alone should be removed by the MHSM-R rinsing step when there is no silver present (see section 7 of the SI). To test if the “extra” radioactive silver that accumulates in the algae in the presence of SRHA is truly intracellular, we compared the subcellular distribution of silver in algal cells that had been exposed to ∼50 nM free Ag+ in the absence (50 nM AgT) or presence of SRHA (75 nM AgT). The relative contribution of the debris fraction was significantly higher in the presence of SRHA than in its absence (Figure 3A). This result suggests that the increase in silver uptake in the presence of SRHA at pH 7.0 may be due to silver binding to the cell surface. To test this explanation, the subcellular partitioning experiment was repeated with twice as much SRHA (10 mg C·L−1) at ∼35 nM free Ag+ (90 nM AgT). As anticipated, silver uptake was enhanced in the presence of the humic acid, but again only the contribution of the cell debris fraction was significantly higher than in the media without humic acid, this difference being more important than that observed at 5 mg C·L−1 SRHA (Figure 3B; p < 0.05). This result supports the idea that the Ag−SRHA complex binds to the cell surface and that the silver resists exchange with nonradioactive silver. The suggestion that adsorbed Ag−SRHA contributes to the enhanced silver uptake observed in the presence of SRHA is not without precedent. Lamelas et al. suggested that Pb−SRHA complexes were bound to the surface of the green alga Chlorella kessleri, contributing to an increase in cellular Pb.25 In subsequent work on the same alga,20 this same research group compared the behavior of Cd, Cu, and Pb in the presence of SRHA, and concluded that only in the case of Pb did this mechanism lead to an increase in the concentration of cellular metal. The relatively poor affinity of Ag+ for SRHA suggests that the simple association of Ag−SRHA complexes with the algal surface, as envisaged for Pb, is not likely to contribute significantly to the apparent silver uptake (see section 8 of the SI). On the other hand, Bell and Kramer suggested that Ag+ could bridge thiol functional groups, and that when Ag− thiolate complexes come into contact with a cell or cellreceptor containing free thiol groups, silver could transfer from the aqueous-phase to the cellular mercaptan.26 Jacobson et al. 110m

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of Canada as a strategic project grant. Some of the preliminary work was supported by Kodak Canada. C.F. and P.G.C.C. are supported by the Canada Research Chair Program.

reported that in solutions containing humic acids and the mineral acanthite, thiols could complex to Ag+ on the surface of the mineral, and silver could form ternary complexes by acting as a bridge between the S in the mineral and the dissolved thiols (i.e., −S−Ag−S−).27 Algal cell membranes contain a number of functional groups,28 including membrane-bound enzymes that can effect metal reduction,29 for example, copper and ferric reductase activity occurring at the surface of C. reinhardtii.30 In addition, it has recently been suggested that SRHA can reduce surface-bound Ag+ ions.31 We thus speculate that the tightly bound silver at the algal surface in the presence of humic acid may include reduced Ag(0). Finally, 24 h exposures showed no increase in silver toxicity in the presence of SRHA, despite the enhancement in silver uptake observed in the short term (1 h) experiments. Indeed, EC50 values expressed in terms of [Ag+] were identical in the presence or absence of SRHA. Similarly, growth inhibition of C. reinhardtii could be predicted on the basis of its silver cellular quota. If we compare the amount of silver uptake increase in the presence of SRHA at ∼50 nM Ag+ (intercept in SI Figure S2, 0.3 μmol·m−2 × 80 μm2·cell−1 = 0.024 fmol·cell−1) with the amount required to induce growth inhibition (EC50 = 1.96 ± 0.46 fmol·cell−1), the contribution of the SRHA to the silver algal pool becomes nonsignificant. We conclude that ternary metal complexes with humic acids at the surface of aquatic organisms may increase the apparent uptake of metals, leading to an apparent deviation from BLM predictions. In the present case, the greater surface accumulation of silver does not lead to increased toxicity, although the increased uptake could be transferred to higher-level consumers through trophic pathways. Caution in interpreting uptake results and relating metal accumulation to toxicity is thus warranted.

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(1) Gottschalk, F.; Nowack, B. The release of engineered nanomaterials to the environment. J. Environ. Monit. 2011, 13 (5), 1145−1155. (2) Nowack, B.; Krug, H. F.; Height, M. 120 years of nanosilver history: Implications for policy makers. Environ. Sci. Technol. 2011, 45 (4), 1177−1183. (3) Steinberg, C. E. W.; Kamara, S.; Prokhotskaya, V. Y.; Manusadzianas, L.; Karasyova, T. A.; Timofeyev, M. A.; Jie, Z.; Paul, A.; Meinelt, T.; Farjalla, V. F.; Matsuo, A. Y. O.; Burnison, B. K.; Menzel, R. Dissolved humic substancesEcological driving forces from the individual to the ecosystem level? Freshwater Biol. 2006, 51 (7), 1189−1210. (4) Campbell, P. G. C., Interactions between trace metals and aquatic organisms: A critique of the free-ion activity model. In Metal Speciation and Bioavailability in Aquatic Systems; Tessier, A., Turner, D. R., Eds.; John Wiley & Sons: New York, NY, 1995; pp 45−102. (5) Glover, C. N.; Wood, C. M. Physiological interactions of silver and humic substances in Daphnia magna: Effects on reproduction and silver accumulation following an acute silver challenge. Comp. Biochem. Physiol. C 2004, 139 (4), 273−280. (6) Bury, N. R.; McGeer, J. C.; Wood, C. M. Effects of altering freshwater chemistry on physiological responses of rainbow trout to silver exposure. Environ. Toxicol. Chem. 1999, 18 (1), 49−55. (7) Campbell, P. G. C.; Twiss, M. R.; Wilkinson, K. J. Accumulation of natural organic matter on the surfaces of living cells: Implications for the interaction of toxic solutes with aquatic biota. Can. J. Fish. Aquat. Sci. 1997, 54 (11), 2543−2554. (8) Slaveykova, V. I.; Wilkinson, K. J.; Ceresa, A.; Pretsch, E. Role of fulvic acid on lead bioaccumulation by Chlorella kesslerii. Environ. Sci. Technol. 2003, 37 (6), 1114−1121. (9) Sánchez-Marín, P.; Beiras, R. Adsorption of different types of dissolved organic matter to marine phytoplankton and implications for phytoplankton growth and Pb bioavailability. J. Plankton Res. 2011, 33 (9), 1396−1409. (10) Di Toro, D. M.; Allen, H. E.; Bergman, H. L.; Meyer, J. S.; Paquin, P. R.; Santore, R. C. Biotic ligand model of the acute toxicity of metals. 1. Technical basis. Environ. Toxicol. Chem. 2001, 20 (10), 2383−2396. (11) Chen, Z.; Campbell, P. G. C.; Fortin, C. Silver binding by humic acid as determined by equilibrium ion-exchange and dialysis. J. Phys. Chem. A 2012, 116 (25), 6532−6539. (12) Lavoie, M.; Bernier, J.; Fortin, C.; Campbell, P. G. C. Cell homogenization and subcellular fractionation in two phytoplanktonic algae: Implications for the assessment of metal subcellular distributions. Limnol. Oceanogr. Methods 2009, 7, 277−286. (13) Fortin, C.; Campbell, P. G. C. Silver uptake by the green alga Chlamydomonas reinhardtii in relation to chemical speciation: Influence of chloride. Environ. Toxicol. Chem. 2000, 19 (11), 2769− 2778. (14) Lavoie, M.; Le Faucheur, S.; Boullemant, A.; Fortin, C.; Campbell, P. G. C. The influence of pH on algal cell membrane permeability and its implications for the uptake of lipophilic metal complexes. J. Phycol. 2012, 48 (2), 293−302. (15) Lavoie, M.; Le Faucheur, S.; Fortin, C.; Campbell, P. G. C. Cadmium detoxification strategies in two phytoplankton species: Metal binding by newly synthesized thiolated peptides and metal sequestration in granules. Aquat. Toxicol. 2009, 92 (2), 65−75. (16) Campbell, P. G. C.; Twiss, M. R.; Wilkinson, K. J. Accumulation of natural organic matter on the surfaces of living cells: Implications for the interaction of toxic solutes with aquatic biota. Can. J. Fish. Aquat. Sci. 1997, 54 (11), 2543−2554.

ASSOCIATED CONTENT

* Supporting Information S

Details on the composition of MHSM media, a brief description of the IET and the resin Ag+ distribution coefficient at pH 5.5, the kinetics of silver uptake, the time-course of silver uptake, the calculation of maximal diffusive fluxes of Ag+, the calculation of surface enrichment factors for the silver ion, the cellular silver concentration as a function of rinse time, the removal of SRHA in the absence of silver and the extent of SRHA adsorption at the cell surface are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +1 418 654 3770; fax: +1 418 654 2600; e-mail: [email protected]. Present Address †

C.P.: Department of National Defense, Valcartier Garrison, PO Box 1000, Station Forces, Courcelette (QC), Canada G0A 4Z0 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Comments provided by P. Sánchez-Marı ́n an earlier version of the manuscript were greatly appreciated. Funding of this research was provided by the Fonds de Recherche du Québec Nature et Technologies (FRQNT) as a team grant and by the Natural Sciences and Engineering Research Council (NSERC) 8841



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(17) Slaveykova, V. I.; Wilkinson, K. J. Predicting the bioavailability of metals and metal complexes: Critical review of the biotic ligand model. Environ. Chem. 2005, 2 (1), 9−24. (18) Whitfield, M.; Turner, D. R., Critical assessment of the relationship between biological thermodynamic and electrochemical availability. In Chemical Modeling in Aqueous Systems, ACS Symposium Series No. 93; Jenne, E. A., Ed.; American Chemical Society: Washington, DC, 1979; pp 657−680. (19) Lee, D. Y.; Fortin, C.; Campbell, P. G. C. Influence of chloride on silver uptake by two green algae, Pseudokirchneriella subcapitata and Chlorella pyrenoidosa. Environ. Toxicol. Chem. 2004, 23 (4), 1012− 1018. (20) Lamelas, C.; Slaveykova, V. I. Comparison of Cd(II), Cu(II), and Pb(II) biouptake by green algae in the presence of humic acid. Environ. Sci. Technol. 2007, 41 (11), 4172−4178. (21) Vigneault, B.; Campbell, P. G. C. Uptake of cadmium by freshwater green algae: Effects of pH and aquatic humic substances. J. Phycol. 2005, 41 (1), 55−61. (22) Boullemant, A.; Le Faucheur, S.; Fortin, C.; Campbell, P. G. C. Uptake of lipophilic cadmium complexes by three green algae: Influence of humic acid and its pH dependence. J. Phycol. 2011, 47 (4), 784−791. (23) Parent, L.; Twiss, M. R.; Campbell, P. G. C. Influences of natural dissolved organic matter on the interaction of aluminum with the microalga Chlorella: A test of the free-ion model of trace metal toxicity. Environ. Sci. Technol. 1996, 30 (5), 1713−1720. (24) Vigneault, B.; Percot, A.; Lafleur, M.; Campbell, P. G. C. Permeability changes in model and phytoplankton membranes in the presence of aquatic humic substances. Environ. Sci. Technol. 2000, 34 (18), 3907−3913. (25) Lamelas, C.; Wilkinson, K. J.; Slaveykova, V. I. Influence of the composition of natural organic matter on Pb bioavailability to microalgae. Environ. Sci. Technol. 2005, 39 (16), 6109−6116. (26) Bell, R. A.; Kramer, J. R. Structural chemistry and geochemistry of silver-sulfur compounds: Critical review. Environ. Toxicol. Chem. 1999, 18 (1), 9−22. (27) Jacobson, A. R.; Martinez, C. E.; Spagnuolo, M.; McBride, M. B.; Baveye, P. Reduction of silver solubility by humic acid and thiol ligands during acanthite (β-Ag2S) dissolution. Environ. Pollut. 2005, 135 (1), 1−9. (28) Xue, H. B.; Stumm, W.; Sigg, L. The binding of heavy metals to algal surfaces. Water Res. 1988, 22 (7), 917−926. (29) Hill, K. L.; Hassett, R.; Kosman, D.; Merchant, S. Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability. Plant Physiol. 1996, 112 (2), 697−704. (30) Weger, H. G. Ferric and cupric reductase activities in the green alga Chlamydomonas reinhardtii: Experiments using iron-limited chemostats. Planta 1999, 207 (3), 377−384. (31) Adegboyega, N. F.; Sharma, V. K.; Siskova, K.; Zbořil, R.; Sohn, M.; Schultz, B. J.; Banerjee, S. Interactions of aqueous Ag+ with fulvic acids: Mechanisms of silver nanoparticle formation and investigation of stability. Environ. Sci. Technol. 2012, 47 (2), 757−764.

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Influence of Humic Acid on Algal Uptake and Toxicity of Ionic Silver

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