Environ. Sci. Technol. 2006, 40, 6341-6347
The Impact of Particle-Bound Cadmium on Bioavailability and Bioaccumulation: A Pragmatic Approach M A R IÄ A N . P I O L , † A N A G . L O Ä PEZ,† LELIA A. MIN ˜ O,† M A R IÄ A D O S S A N T O S A F O N S O , ‡ A N D N O E M IÄ R . V E R R E N G I A G U E R R E R O * , † Toxicologı´a y Quı´mica Legal, Departamento de Quı´mica Biolo´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. 4° Piso, Pab. II, Ciudad Universitaria. C1428EHA, Buenos Aires, Argentina. INQUIMAE y Departamento de Quı´mica Inorga´nica, Analı´tica y Quı´mica Fı´sica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. 3° Piso, Pab. II, Ciudad Universitaria. C1428EHA, Buenos Aires, Argentina
Studying the bioavailability of sediment-bound contaminants is complicated by many reasons, such as the variable composition of the particles, their temporal variations, the low levels of contaminant concentrations, their partitioning between diverse aqueous and particulate phases, and the variety of uptake routes that may involved with the biota. Therefore, simple and innovative methodologies should be tested as analogues for natural sediments. Among them, a diverse selection of artificial particles with welldefined surface properties, in the presence and absence of commercially available humic acids, has been proposed and used to investigate the bioavailability of several organic pollutants. For this work, this model was applied to investigate the uptake and accumulation of cadmium by the freshwater oligochaete Lumbriculus variegatus. The results showed that the uptake of the metal depended on the free dissolved Cd(II) species, while the contribution from the particles was negligible. Thus, the extent of cadmium bioaccumulated from each test system could be predicted as a function of the rate of absorption of the free dissolved Cd(II) species. These species were calculated either from the particle-water partition coefficients, or by using the MINEQL+ computer program. In general, the estimated accumulation levels were in good agreement with the experimental results.
Introduction Most contaminants that are discharged to aquatic environments have the tendency to bind to sediment particles at levels that largely exceed those present in the water column (1, 2). However, they can still pose a risk for the ecosystem in the case that they are released from the particles to the surrounding water, including pore water (3). Alternatively, they can be ingested by benthic organisms and released into the gastrointestinal tract of the animal (3). The assessment * Corresponding author phone: 54 114 576 3342; e-mail: noev@ qb.fcen.uba.ar. † Departamento de Quı ´mica Biolo´gica. ‡ INQUIMAE y Departamento de Quı ´mica Inorga´nica. 10.1021/es061135t CCC: $33.50 Published on Web 09/07/2006
2006 American Chemical Society
of sediment-bound contaminants is an issue of great concern, but it raises many difficulties due to the complex and variable composition of the natural particles. Sediment particles may be described as a heterogeneous mixture of particles from different sources. Basically, the particles are composed of an inorganic or biological matrix surrounded by a wide variety of inorganic and organic compounds that can act as substrates for binding or sorbing chemicals (4, 5). As a consequence of these interactions, the bioavailability of the chemicals may be markedly modified. Most of the works have tended to relate the interactions between the natural particles and contaminants in terms of variations in particle size, clay type and content, composition and amount of organic matter, acid volatile fraction, cation exchange capacity, redox potential, pH, and anaerobic profile (6). In certain types of sediments, metals are present as insoluble sulfides resulting virtually unavailable by biota (7, 8). However, there are also sediments with a very low acid volatile sulfide fraction where most of the metals interact with the natural organic matter present in the particles. In these cases, it is still difficult to predict the proportion that is bioavailable (9). Several approaches have been proposed to simplify these problems. The use of control sediments has become an important tool to assess and predict the bioavailability of particle-bound contaminants under laboratory conditions (10). Several types of formulated sediments have been proposed consisting of different kinds of sand, silt, and clay (11-13). In addition to the natural materials, the use of artificial particles as analogues for aquatic sediments has also been suggested (14, 15). However, this approach has not been extensively investigated yet. In particular, commercial materials commonly used as chromatographical resins have been selected as artificial particles because their size, backbone structure, and active groups on their surfaces are well characterized and standardized by the manufacturers (16-19). However, this model may be considered too simple, since it may not reflect fully the complexity of natural sediments. Thus artificial particles will clearly not show the full range of ligands that are present in natural organic matter (13, 15). It is well-known that humic acids constitute the most important natural source of organic matter in sediments from aquatic systems (20). Additionally, they can be obtained in a consistent form as a commercial product. For these reasons we have developed an experimental model consisting of artificial particles and humic acids for modeling the uptake and bioaccumulation of organic pollutants (21-23). For this work, we have used the same experimental device consisting of artificial particles with and without humic acid to investigate the bioavailability and bioaccumulation of cadmium. Lumbriculus variegatus was selected as the standard organism for the bioassays. This species of freshwater oligochaete has been recommended and widely used as an experimental animal model for sediment toxicity tests (24-26). However, we are aware that this experimental approach could not be applied to test all the chemicals of potential environmental relevance. For this reason, bioaccumulation levels were also predicted by using a curve of metal uptake versus cadmium concentrations in the dissolved phase. These species were estimated from two approaches: (a) the particle-water partition coefficients calculated for the different test systems, and (b) the MINEQL+ computer program. The final goal was, therefore, to compare the experimental results with those estimated by the both approaches. VOL. 40, NO. 20, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6341
TABLE 1. Some Characteristics of the Particles Selecteda
a
Reprinted from ref 45 with permission from Elsevier.
Materials and Methods All the glassware was prewashed with 5% nitric acid for 24 h, thoroughly rinsed with double distilled water several times, and dried before using. All solutions were prepared using reagent-grade chemicals and double distilled water. Cadmium dilutions for bioassays were prepared from a stock solution containing 1.0 mg Cd L-1, prepared from CdCl2.21/2H2O (M&B Laboratory Chemicals, United Kingdom). Acid prewashed marine sand, calcined at 900 °C, was obtained from Merck Company, Buenos Aires, Argentina. Commercial humic acids (HA) were obtained from Fluka Chemie AG, Switzerland, and were used as provided. The selected concentration (20 mg L-1 of humic acid) was prepared by dissolving the humic material in dechlorinated tap water treated as described below. Then, solutions were filtered through a 0.45 µm membrane filter to remove particles (27). The following resins were selected as artificial particles: Dowex 1 × 8400, an anionic exchanger (Sigma-Aldrich Company, Poole, Dorset, United Kingdom); Toyopearl SP650M, a cationic exchanger, and two neutral materials, Toyopearl Butyl 650M and Toyopearl Phenyl 650M. The last two resins are designed for studying hydrophobic interactions. All the Toyopearl particles were obtained from Fisher, Loughborough, United Kingdom. The backbone of Dowex particles was formed from a cross-linked copolymer of styrene and divinylbenzene. All the Toyopearl particles had the same backbone structure consisting of polymers of ethylene glycol and methyl methacrylate. Functional groups and particle sizes are presented in Table 1. Although some of the functional groups are not likely to occur in the natural sediments, the artificial particles selected may reflect some of the possible interactions that occur in the natural environment, such as charge effects and noncharged interactions. The particle sizes of the resins were all in the 40-60 µm range, ensuring that the animals could ingest them. Tap water was used for all the bioassays. It had been dechlorinated for at least 24 h and then filtered through a carbon column to eliminate any dissolved organic material. The following physicochemical parameters were recorded: total hardness ) 67 ( 3 mg CaCO3 L-1; alkalinity ) 29 ( 2 mg CaCO3 L-1; pH ) 7.0 ( 0.2 and conductivity ) 250 ( 17 µS. A culture of Lumbriculus variegatus was received from Prof. Simkiss, Ecotoxicology Research Group, School of Animal and Microbial Sciences, University of Reading, UK. The cultures were then maintained in our laboratory under static conditions in 12 L aquaria, at a temperature of 21 ( 2 °C, and a photoperiod of 16:8 h light/dark. The base of the aquaria was lined with shredded non-bleached paper towels 6342
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 20, 2006
(depth of 3-6 cm) and contained approximately 8 L of dechlorinated tap water, constantly aerated. The overlying water was changed every 7 days. Each culture was fed three times a week with suspensions of approximately 0.5 g finely ground TetraFin (TetraWerke, Melle, Germany) in 25 mL of dechlorinated water. Adult organisms of 2.5 ( 0.5 cm of length were used. All bioassays were performed for 48 h in static conditions at a temperature of 21 ( 2 °C. During the treatments, animals were not fed. To investigate the uptake of cadmium from the aqueous phase by L. variegatus, ten organisms were placed in glass vials, containing 20 mL of varying nominal concentrations of cadmium solutions (0.05-0.3 mg Cd L-1) prepared either in dechlorinated tap water or in the 20 mg L-1 of humic acid solution. After the treatments, animals were allowed to depurate for 6 h in clean water (28). For depuration studies, several sets of ten worms were exposed to a given level of the metal (0.1 mg Cd L-1) for 48 h. After these treatments one set was immediately processed for metal analysis (t ) 0), while the others sets were transferred to clean water for t ) 6, 24, and 48 prior to metal determinations. To evaluate the uptake of cadmium from the particles, ten organisms were placed in glass vials containing 20 mL of 0.1 mg Cd L-1 and 2 g of solid phase. The solid phase consisted of either 2 g of sand particles or 0.5 g of each resin plus 1.5 g of sand. In the absence of humic material, each test system had been equilibrated by shaking for 4 h before performing the bioassays. For the systems containing humic material, the particles were suspended in humic acid solution and equilibrated by stirring for 20 h. Then, an aliquot of the cadmium stock solution was added to obtain a final concentration of 0.1 mg Cd L-1 and the systems were allowed to equilibrate for another 4 h. After the treatments animals were transferred to clean water for 6 h prior to metal analyses. All results were based on the means of five replicates for absorption and depuration studies, and of ten replicates for the rest of the experiments. There was no mortality in any of the experimental procedures. After the treatments, animals were cooled on ice, during 6-8 min. Each pool of ten worms was placed on a watch glass, dried with filter paper, and weighed. The pools were transferred to borosilicate tubes and digested with concentrated analytical grade nitric acid (about 2-3 mL each 100 mg wet weight) at a temperature of 100 °C (29). Each 10 samples, a blank was performed and processed simultaneously. Then, the samples were diluted with 1% (v/v) nitric acid solution. Metal concentrations were measured by flame atomic absorption spectrophotometry (atomic absorption spectrophotometer varian 575 AA) (λ ) 228.8 nm), using a deuterium lamp for back-ground correction. To check the accuracy of the analyses, standard addition methods were used to overcome matrix effects. Blank values were negligible. In all cases metal concentrations were expressed as µg Cd g-1 wet tissue. Values of cadmium determined in tissues from control organisms were below 0.2 µg g-1. Batch adsorption experiments were conducted to determine the particle-water partition coefficients (Kd). Six different concentrations of cadmium (range 0.05-10.0 mg Cd L-1) were prepared in the same dechlorinated tap water and cadmium stock solution used for the bioassays. Experiments were performed in triplicate at a total solid concentration of 0.1 g mL-1 in borosilicate glass tubes that were stirred continually. The different particle systems were equilibrated using the same procedure as described for the bioassays. Previous experiments had shown that these periods were enough to reach the equilibrium times for all the particle systems. Then, the tube contents were filtered (filter paper Schleicher & Schu ¨ ll 5892, white ribbon) and aliquots of 3-4 mL of the filtrate were transferred to a 10-mL tube, adding
a drop of concentrated nitric acid to avoid adsorption onto the wall surfaces of the tubes. The residue on the filter paper was air-dried and then digested with concentrated acid nitric for 6-8 h at 100 °C (29). After this treatment, the digested material was diluted with 1% nitric acid solution. The samples were centrifuged for 15 min at 3000 rpm, and the aqueous phase transferred to another tube. Cadmium concentrations were determined in the solution (Ce ) equilibrium concentration) and in the solid phase (Cs) by atomic absorption spectrophotometry. The data showed that the equilibrium concentration plus the concentration in the solid phases were almost equal to the nominal cadmium level (g96%). The chemical speciation of cadmium in the different test systems was estimated using the MINEQL+ chemical equilibrium modeling system for water chemistry calculations, MINEQL+ software version 3.01 (30). The relative molecular mass of humic acids was in the range 600-1000, as reported by the manufacturers. Humic material was considered with two different adsorption sites responsible for binding metal ions (31, 32). The distribution constant between cadmium and humic acids was Kd ) 104, as reported by Ferna´ndez et al. (33). Regression analyses were carried out using the Excel software package (Microsoft, U.S.). The data were statistically evaluated applying analysis of variance tests (ANOVA) to study the differences among treatments (34). Analyses of the sample populations were performed by comparing cadmium levels in the organisms maintained in systems containing the metal with and without the different types of particles in the presence or absence of humic material. To investigate differences within the treatments, the Scheffe´ method was used (34). All the results were considered significant at p e 0.05.
Results Uptake and Elimination of Cd by L. variegatus. The uptake of cadmium by L. variegatus from the aqueous phase was linearly related with the exposure level within the range of concentrations tested, as shown in Figure 1A. Similarly, a significant regression line was obtained when the animals were exposed to the metal dissolved in the 20 mg L-1 humic acid solution. However, in this case, a lower slope was observed. Depuration studies were conducted to assess the rate of cadmium elimination. The results, presented in Figure 1B, showed that once the animals absorbed the metal, there was not a significant elimination over the period studied (p > 0.05). Bioaccumulation of Cd in the Different Test Systems. Figure 2 shows the levels of cadmium accumulated by L. variegatus exposed to 0.1 mg Cd L-1 from systems containing either water alone, a solution of 20 mg L-1 of humic acid, or the different particles both with and without the humic acid solution. The largest uptake of the metal was observed when the oligochaetes were exposed to the system containing water only or sand particles. The simultaneous presence of the humic acid solution induced significant decreases (p < 0.05) in the cadmium bioaccumulation from both systems. All the artificial particles tested, either with or without humic acids, induced lower levels of accumulation. In the absence of humic acids, the values followed the order Toyopearl Phenyl > Dowex ≈ Toyopearl Butyl > Toyopearl SP. These values were not significantly modified by the presence of humic acids (p > 0.05), excepting for the Toyopearl Phenyl particles that promoted a slight but significant lower accumulation than the value obtained when the particles were suspended in water alone (p < 0.05). Particle-Water Partition Coefficients (Kd). The values of the particle-water partition coefficients (Kd) found for
FIGURE 1. (A) Values of cadmium incorporated by L. variegatus (mean values ( SD) exposed for 48 h to varying levels of metal concentrations. (B) Values of cadmium concentrations in L. variegatus exposed for 48 h at 0.1 mg Cd L-1 and depurated in clean water for different periods. Data are expressed as µg Cd g-1 wet tissue. Ten organisms were treated at each condition by five replicates.
FIGURE 2. Values of cadmium incorporated by L. variegatus (mean values ( SD) exposed to 0.1 mg Cd L-1 for 48 h in the different test systems. Data are expressed as µg Cd g-1 wet tissue. T-Butyl, Toyopearl Butyl; T-Phenyl, Toyopearl Phenyl; T-SP, Toyopearl SP. Ten organisms were treated at each condition by ten replicates. The asterisk indicates significant differences from organisms exposed to water only. Significant differences between the same test systems with and without humic acids are indicated by different letters. the metal and the different particles both in the presence and in the absence of the humic acid solution are presented in Table 2. These values were obtained from highly significant regression coefficients (range of r2 ) 0.869 - 0.987), ensuring that any of the resins was saturated over the working range of cadmium concentrations. VOL. 40, NO. 20, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6343
TABLE 2. Values of Particle-Water Partition Coefficients (Kd) and pH for the Different Test Systems without HA
sand Dowex Toyopearl Phenyl Toyopearl Butyl Toyopearl SP
with HA
Kd
pH
Kd
pH
3.19 0.35 13.0 149 1430
7.0 ( 0.1 6.5 ( 0.2 7.0 ( 0.2 7.0 ( 0.2 6.9 ( 0.1
1.12 1.67 8.59 21.1 709
7.0 ( 0.1 6.5 ( 0.2 7.0 ( 0.2 7.0 ( 0.2 6.9 ( 0.1
The amount of metal sorbed onto the particles without humic acids followed the order Dowex < sand < Toyopearl Phenyl , Toyopearl Butyl , Toyopearl SP. In the presence of the humic acid solution the order was sand < Dowex < Toyopearl Phenyl < Toyopearl Butyl , Toyopearl SP. The values of pH for the different test systems are also presented in Table 2. Excepting for the system containing Dowex particles, the pH values were not significantly modified in comparison with the system containing only water (p > 0.05). Instead, the anionic exchanger induced a slight but significant decrease in the pH of the test solution (p < 0.05). The Kd Approach. The concentrations of dissolved Cd(II) species present at equilibrium can be calculated as a first approximation using the particle-water partition coefficients (Kd). In the case that the uptake of cadmium by L. variegatus had depended solely on the amount of Cd(II) dissolved species the equation obtained from Figure 1A without humic acids, may be applied to estimate the resulting metal bioaccumulation by the animals. The ratios between experimental and estimated bioaccumulation were calculated to check the goodness of the estimations. Data are presented in Table 3. Excepting for the Dowex resin, in all the systems containing the particles without humic acids the ratios were very close to the unity. However, in the presence of the humic acid solution, the ratios were, in all the cases, lower than the unity, varying from 0.38 to 0.76, indicating that the estimated values have overestimated the resulting experimental bioaccumulation. It may be argued that the equation of Figure 1A without humic acids could not be valid to properly predict the bioaccumulation from the particle systems when they contained the humic material. In order to improve the approach, the following steps were followed: (1) cadmium concentrations at equilibrium were calculated from the corresponding Kd values, (2) the MINEQL+ program was used to estimate the concentrations of the total inorganic cadmium species after complexation with the humic material, and (3) the equation obtained in Figure 1A without humic acids was applied to estimate the resulting metal bioaccumulation. Data are presented in Table 4. For sand and Toyopearl Phenyl, the ratios were now practically equal to the unity. Instead, for the rest of the particles they were still below 0.80. The MINEQL+ Computer Program Approach. In addition, the MINEQL+ program was used directly to predict the total levels of dissolved cadmium species in the systems containing particles both with and without humic acids. When introducing the data in the program it was shown that all the systems fitted the absorption model of Langmuir, excepting for the Dowex resin in the absence of humic material. Finally, the equation obtained in Figure 1A without humic acids was used, since they had been already introduced in the program. These results are presented in Table 5. When using this approach, the ratios varied between 1.15 and 0.77 with the only exception of the Dowex resin.
Discussion Uptake and Elimination of Cd by L. variegatus. The uptake of cadmium by Lumbriculus variegatus was linearly related to the nominal metal concentration dissolved either in 6344
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 20, 2006
dechlorinated water or in the 20 mg HA L-1 solution, over the range of exposure tested. However, a lower slope was found in the presence of the humic acid solution. This is a consequence of the complexation of cadmium with the dissolved organic matter, rendering the metal less available for uptake and bioaccumulation (29, 35, 36). It is worth noting that the influence of humic acids was more important at higher cadmium concentrations. Some aspects of the biology and feeding activity in L. variegatus have been well studied. It is generally accepted that the throughput time for ingested food is approximately 6 h (28, 37). Therefore, depuration periods of this length remove sediment particles from the alimentary tract, ensuring that metal concentrations are due to the amount of cadmium actually absorbed by the animals and not to the transitorily presence of particles in the alimentary tract. In addition, depuration studies showed that the rate of elimination of cadmium by L. variegatus was practically negligible for at least 48 h after the 48 h treatments. Experimental Bioaccumulation Results. For all of the bioassays using particles in the presence or absence of humic acids, the animals were exposed to the same concentration of 0.1 mg Cd L-1. This selected value corresponds to the maximum level of cadmium that can be released to natural freshwater courses according to the Argentine Law of Hazardous Substances (38). In addition, the experimental device, where there is a fixed mass balance, allows direct comparisons of accumulation by the animals among the treatments. In this way it is possible to investigate the relative importance of the aqueous phase (both pore water and overlying water) and ingested sediments as routes of uptake of metals. It is worth noting that, after the 48 h treatments, a steady state was not reached. But even in field conditions it is not always clear whether natural aquatic systems are actually at equilibrium conditions (39). Thus even if they are at equilibrium, intermittent or unexpected discharges of effluents, and several environmental factors (e.g., changes in nutrients loads, amount of suspended matter, salinity, etc.) will induce departures from the steady state. Therefore, for these bioassays we have adopted the same criterion as Burgess and McKinney (40) who considered that after such short time exposures, where an “early stage of bioaccumulation” occurs, it allows to investigate the principal routes of uptake. Furthermore, to reach this goal, great care must be taken to prevent oligochaetes from division. Fragmentation (architomy) is the typical reproduction strategy for L. variegatus. During division, the feeding activity and egestion rate of the animals is severely affected (41). Therefore, longterm bioassays may introduce further complications as a consequence of changes in the exposure pathways of contaminants. Understanding the bioaccumulation patterns requires of both the physicochemical characteristics of the systems and also of biological factors. In particular for metal contaminants it is generally accepted that: (1) the toxicity is related to the activity of the free ions, (2) the complexation of metals with both organic and inorganic ligands decreases the toxicity by decreasing the free ion activities, and (3) the water quality parameters affects metal toxicity either by affecting the organisms or by modifying metal speciation (42-44). In comparison with the systems containing water only, sand particles did not introduce any change in bioaccumulation by Lumbriculus variegatus either in the presence or absence of the humic acid solution, respectively. In the bioassays containing sand, a certain proportion of cadmium was adsorbed onto the particles decreasing the levels of metal in solution. However, due to the particle size of the sand (100-300 µm) they were too large to be ingested by the oligochaetes. Therefore, the uptake of cadmium could only occur from the Cd(II) species remaining in solution. The Kd
TABLE 3. Estimated Values of Cadmium Concentrations in the Test Systems and Accumulation by L. variegatus Using the Kd Approach equilibrium Cd concentration (mg L-1)
estimated Cd bioaccumulation (µg g-1)
ratio experimental and estimated Cd accumulation (Kd)
without HA sand Dowex Toyopearl Phenyl Toyopearl Butyl Toyopearl SP
0.0758 0.0966 0.0435 0.0063 0.0007
3.54 4.29 2.37 1.02 0.61
1.11 0.33 1.00 1.03 1.08
with HA sand Dowex Toyopearl Phenyl Toyopearl Butyl Toyopearl SP
0.0899 0.0857 0.0538 0.0322 0.0014
4.05 3.90 2.74 1.96 0.84
0.73 0.38 0.70 0.49 0.76
TABLE 4. Estimated Values of Cadmium Concentrations in the Test Systems Containing Particles Plus Humic Acids and Accumulation by L. variegatus Using the Kd Approach and the Mineql+ Program
sand Dowex Toyopearl Phenyl Toyopearl Butyl Toyopearl SP
inorganic Cd species MINEQL+ (mg L-1)
estimated Cd accumulation (µg g-1)
ratio experimental and estimated Cd accumulation (Kd + MINEQL+)
0.0593 0.0561 0.0353 0.0212 0.0009
2.94 2.83 2.07 1.56 0.82
1.01 0.53 0.93 0.62 0.78
TABLE 5. Estimated Values of Cadmium Concentrations in the Test Systems and Accumulation by L. variegatus Using the Mineql+ Program
without HA sand Dowex Toyopearl Phenyl Toyopearl Butyl Toyopearl SP with HA sand Dowex Toyopearl Phenyl Toyopearl Butyl Toyopearl SP
inorganic Cd species (mg L-1) Langmuir approximation
estimated Cd accumulation (µg g-1)
ratio experimental and estimated Cd accumulation (Langmuir)
0.0818
3.76
1.05
0.0383 0.0032 0.0007
2.18 0.91 0.61
1.09 1.15 0.92
0.0595 0.0592 0.0401 0.0032 0.0011
2.95 2.94 2.24 0.91 0.83
1.00 0.51 0.86 1.07 0.77
value for the system containing only sand was almost 3 times higher than the value obtained when the sand was pretreated with the humic acid solution. It could be hypothesized that, on one hand, as a consequence of the humic acid interactions with the sand particles, lower levels of cadmium could be adsorbed onto these particles. On the other hand, it could be possible that the complexation of the metal with the levels of humic acids remaining in solution induced a decrease in the amount of cadmium available for sorption to the particles. In comparison with the system containing water only, all the artificial particles selected induced marked decreases in cadmium accumulation by L. variegatus. This fact is clear evidence that the uptake from the artificial particles was practically negligible and ratifies the relevance of the dissolved Cd(II) species on metal uptake. A similar result was observed for lead (45). However, in contrast with these findings, these particles were able to increase the uptake and accumulation
of several hydrophobic organic contaminants in L. variegatus (18). The system containing the Dowex resin also induced a significant decrease in the pH of the test solutions. This resin is an anionic exchanger so it is expected that shows a very low affinity for the Cd(II) cations. As a consequence, most of the metal remained in solution and very low Kd values were found, especially for the system containing this resin in the absence of humic acids. However, values of accumulation were less than half of those observed for the systems containing water only. Some authors have reported that the proton ions could offer some protection for metal uptake and accumulation by competing with the elements for binding sites in the animals (46, 47). In addition, changes in pH values may modify the conformation of metal transport proteins. In agreement with this, it was observed a highly significant decrease (83%) in the cadmium internalization VOL. 40, NO. 20, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6345
flux in algae as the pH was decreased from 7.0 to 5.2, a pH range where the metal speciation was constant (48). The three Toyopearl resins have the same pore size and backbone composition, differing from each other only by their functional groups. The organisms exhibited a similar burrowing behavior in these test systems. This was in contrast to what had been observed in the system containing Dowex particles, where the animals showed an avoidance response in contact with this resin. By comparing the Toyopearl resins in systems without humic acids, the highest uptake and accumulation of cadmium in the oligochaetes was observed in the presence of Toyopearl Phenyl, followed by Toyopearl Butyl and finally Toyopearl SP. The Kd values followed exactly the inverse order. Therefore, in these systems, the metal bioavailability and bioaccumulation was directly related with the amount of Cd(II) that remained in solution. In the case of the cationic exchanger Toyopearl SP, the highest affinity for the Cd(II) species was expected and a large proportion of the metal was adsorbed onto the particles by strong ionic interactions, rendering the metal unavailable for uptake. As Toyopearl Phenyl, the Toyopearl Butyl is also a neutral resin designed for hydrophobic interactions. However, the Toyopearl Butyl particles presented a comparatively much higher Kd value than that of Toyopearl Phenyl particles. It is worth noting that, in both resins, the functional groups are attached to the backbone through an oxygen atom (Table 1). Therefore, it could be suggested that the dissolved Cd(II) species might be able to establish some kind of electrostatic interactions with the oxygen atom from the Toyopearl Butyl but not from the Toyopearl Phenyl resin as it was reported previously for lead (45). As for sand, when the systems contained the Toyopearl resins in the presence of humic acids, all the Kd values were lower than the values obtained for each respective resin free of organic matter. Consequently, higher cadmium concentrations remained in solution. However, the levels of metal incorporated by L. variegatus were similar to or even lower than (in the case of Toyopearl Phenyl) those obtained when the animals were exposed to each test system without humic acids. This is because of a certain proportion of cadmium present in solution was associated to the humic materials becoming less bioavailable. In summary, by using artificial particles with known composition, it is possible to understand the availability and uptake of cadmium by L. variegatus. But how far are these artificial particles from the real world? Natural particle surfaces are coated with organic films of biological origin, derived mainly from bacterial exopolymers, cell walls, and plant products. A great proportion of this organic carbon content is constituted by humic acids that have many carboxylic and phenolic moieties as principal functional groups and confer a net negative charge to the particles. Therefore, the Toyopearl SP resin, especially when coated with humic acids, could be suggested as the best analogue for a natural sediment. Previous results with organic chemicals support this hypothesis (21-23). Both neutral Toyopearl resins have simple hydrophobic surfaces without charges. In particular, Toyopearl Phenyl showed to be also a good analogue for a sample of a natural sediment for nonpolar organic contaminants when the particle-chemical associations occur principally by hydrophobic interactions (18, 19). On the other hand, Dowex particles, having a positive charge, are a very unrealistic model for a natural sediment and they were tested only for an experimental purpose. Estimated Bioaccumulation Results. The experimental accumulation results clearly showed that the amount of dissolved Cd(II) without complexation was the most available fraction for these oligochaetes at a neutral pH value. Therefore, cadmium incorporation in L. variegatus at a fixed 6346
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 20, 2006
point time (48 h) could be predicted from the values of metal concentration in the aqueous phase and the regression line obtained in Figure 1A. Following this hypothesis, metal concentrations were determined: (a) from the particle-water partition coefficients (Kd), and (b) by using the MINEQL+ computer program. Excepting for the Dowex resin, when using the Kd approach for all the particles tested without humic acids, the ratios were very close to the unity, showing the goodness of this approach (Table 3). Thus we can conclude that, in the absence of organic matter, Kd could be a good predictor of cadmium uptake. Instead, for the systems that contained the particles in the presence of humic acids, all the ratios were below the unity. In these cases, higher ratios were obtained when improving the Kd approach by considering also the interactions between the metal and the humic acids remaining in solution using the MINEQL+ program (Table 4). By using the computer program to modeling simultaneously the interactions between cadmium and sand, both in systems with and without humic acids, very significant ratios were obtained (Table 5). As a consequence of the anomalous uptake, due to the increase in acidity of the system, any of the approaches we tried were unable to accurately predict the resulting bioaccumulation from the Dowex resin since all of them overestimated it by far. It is worth noting that departures from the experimental bioaccumulation data were by overestimation. Therefore, both approaches might be useful for regulatory purposes.
Acknowledgments This work was partially supported by grants X-147 from the University of Buenos Aires, and PICTR 2002-00203 from the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANCPyT). We thank Valot S.A. and Whirlpool Argentina for providing some materials.
Literature Cited (1) Baudo, R.; Muntau, H. Lesser Known In-Place Pollutants and Diffuse Sources Problems. In Sediments: Chemistry and Toxicity of In-Place Pollutants; Baudo, R., Giesy, J.P., Muntau, H., Eds.; Lewis Publishers: Michigan, 1990; pp 1-14. (2) Burton, G. A. Assessing the toxicity of freshwater sediments. Environ. Toxicol. Chem. 1991, 10, 1585. (3) Chapman, P. M.; Ho, K. T.; Munns, W. R. Jr; Solomon, K; Weinstein, M.P. Issues in sediment toxicity and ecological risk assessment. Mar. Pollut. Bull. 2002, 44, 271-278. (4) Bradford, W. L.; Horowitz, A. J. The Role of Sediments in the Chemistry of Aquatic Systems, Circular 969; United States Geological Survey: Denver, 1988. (5) Fo¨rstner, U. Inorganic Sediment Chemistry and Elemental Speciation. In Sediments: Chemistry and Toxicity of in Place Pollutants; Baudo, R., Giesy, J.P., Muntau, H., Eds.; Lewis Publishers: Michigan:, 1990; pp 61-105. (6) Lee, H. Models, Muddles, and Mud: Predicting Bioaccumulation of Sediment-Associated Pollutants. In Sediment Toxicity Assessment; Burton GA, Ed.; Lewis Publishers: Chelsea, 1992; pp 267-293. (7) Di Toro, D. M.; Mahony, J. D.; Hansen, D. J.; Scott, K. J.; Hicks, M. B.; Mayr, S. M.; Redmond, M. S. Toxicity of cadmium in sediments: The role of acid volatile sulfide. Environ. Toxicol. Chem. 1990, 9, 1487-1502. (8) Warren, L. A.; Tessier, A.; Hare, L. Modelling cadmium accumulation by benthic invertebrates in situ: The relative constributions of sediment and overlying water reservoirs to organism cadmium concentrations. Limnol. Oceanogr. 1998, 43, 1442-1454. (9) Di Toro, D. M.; McGrath, J. H.; Berry, W. J.; Paquin, P. R.; Mathew, R.; Wu, K. B.; Santore, R. C. Predicting sediment metal toxicity using a sediment biotic ligand model: Methodology and initial application. Environ. Toxicol. Chem. 2005, 24, 2410-2427. (10) Ingersoll, C.G. Sediment Tests. In Fundamentals of Aquatic Toxicology, 2nd ed.; Rand, G.M., Ed.; Francis & Taylor: London, UK., 1995; pp 231-255.
(11) Suedel, B. C.; Rogers Jr.; J.H. Development of formulated reference sediments for use in freshwater and estuarine sediment tests. Environ. Toxicol. Chem. 1994, 13, 1163-1175. (12) Naylor, C.; Rodrigues, C. Development of a test method for Chironomus riparius using a formulated sediment. Chemosphere. 1995, 31, 3291-3303. (13) Kemble, N. E.; Dwyer, F. J.; Ingersoll, C. G.; Dawson, T. D.; Norberg-King, T. J. Tolerance of freshwater test organisms to formulated sediments for use as control materials in wholesediment toxicity tests. Environ. Toxicol. Chem. 1999, 18, 222230. (14) Simkiss, K. The application of controlled release and QSAR technology to sediments. Mar. Pollut. Bull. 1995, 3, 28-31. (15) Fleming, R. J.; Holmes, D.; Nixon, S. J. Toxicity of permethrin to Chironomus riparius in artificial and natural sediments. Environ. Toxicol. Chem. 1998, 17, 1332-1337. (16) Davies, N. A.; Edwards, P. A.; Lawrence, M. A. M.; Simkiss, K.; Taylor, M. G. Biocide testing using particles with controlled surface properties (artificial sediments). Environ. Toxicol. Chem. 1999, 18, 2337-2342. (17) Davies, N. A.; Edwards, P. A.; Lawrence, M. A. M.; Taylor, M. G.; Simkiss, K. Influence of particle surfaces on the bioavailability to different species of 2,4-dichlorophenol and pentachlorophenol. Environ. Sci. Technol. 1999, 33, 2465-2468. (18) Simkiss, K.; Edwards, P. A.; Lawrence, M. A. M.; Davies, N. A.; Taylor, M. G. The use of hydrophobic resins as analogues for sediment testing. Environ. Sci. Technol. 2000, 34, 2388-2392. (19) Simkiss, K.; Davies, N. A.; Edwards, P. A.; Lawrence, M. A. M.; Taylor, M. G. The use of sediment analogues to study the uptake of pollutants by chironomid larvae. Environ. Pollut. 2001, 115, 89-96. (20) Rand, G. M.; Wells, P. G.; McCarty, L. S. Introduction to Aquatic Toxicology. In Fundamentals of Aquatic Toxicology, 2nd ed.; Rand, G.M. Ed.; Francis & Taylor: London, UK, 1995; pp 231255. (21) Verrengia Guerrero, N. R.; Taylor, M. G.; Wider, E. A.; Simkiss, K. Modeling pentachlorophenol (PCP) bioavailability and bioaccumulation by the freshwater fingernail clam Sphaerium corneum using artificial particles and humic acids. Environ. Toxicol. Chem. 2001, 20, 2910-2915. (22) Verrengia Guerrero, N. R.; Taylor, M. G.; Wider, E. A.; Simkiss, K. Influence of particle characteristics and organic matter content on the bioavailability and bioaccumulation of pyrene by clams. Environ. Pollut. 2003, 121, 115-122. (23) Verrengia Guerrero, N. R; Taylor, M. G; Simkiss, K. Modelling 2, 4-dichlorophenol bioavailability and bioaccumulation by the freshwater fingernail clam Sphaerium corneum using artificial particles and humic acids. Environ. Pollut. in press. (24) Phipps, G. L.; Ankley, G. T.; Benoit, D. A.; Mattson, V. R. Use of aquatic oligochaete Lumbriculus variegatus for assessing the toxicity and bioaccumulation of sediment-associated contaminants. Environ. Toxicol. Chem. 1993, 12, 269-279. (25) American Society for Testing and Materials. Annual Book of ASTM Standards; American Society for Testing and Materials: Philadelphia, 1995; Vol 11.05, E 1393-94×bb. (26) United States Environmental Protection Agency. Methods for Measuring the Toxicity and Bioaccumulation of SedimentAssociated Contaminants with Freshwater Invertebrates, 600/ R-99/064; United States Environmental Protection Agency: Washington, DC, 2000. (27) Verrengia Guerrero, N. R.; Nahabedian, D. E; Wider, E. A. Analysis of some factors that may modify bioavailability of cadmium and lead by Biomphalaria glabrata. Environ. Toxicol. Chem. 2000, 19, 2762-2768. (28) Mount, D. R.; Dawson, T. D.; Burkhar, L. P. Implications of gut purging for tissue residues determined in bioaccumulation testing of sediment with Lumbriculus variegatus. Environ. Toxicol. Chem. 1999, 18, 1244-1249. (29) Verrengia Guerrero, N. R. Contaminantes Meta´licos en el Rı´o de La Plataqq: Monitoreo del Sistema Acua´tico y Estudio de Algunos Efectos To´xicos en Moluscos Bivalvos por Medio de Bioensayos. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Airesqq, Buenos Aires, Argentina, Ph.D. thesis. 1995. (30) Schecher, W. D.; Mcavoy, D. C. MINEQL+: A Chemical Equilibrium Program for Personal Computers, version 3.0; Environmental Research Software: Hallowell, Maine, 1994.
(31) Pandey, A. K.; Pandey, S. D.; Misra, V. Stability constants of metal-humic acid complexes and its role in environmental detoxification. Ecotoxicol. Environ. Saf. 2000, 47, 195-200. (32) Zhou, P.; Yan, H.; Gu; B. Competitive complexation of metal ions with humic substances. Chemosphere. 2005, 58, 13271337. (33) Ferna´ndez, F. M.; Tudino, M. B.; Troccoli, O. E. Automatic online ultratrace determination of Cd species of environmental significance in natural waters by FI-ETAAS. J. Anal. At. Spectrom. 2000, 15, 687-695. (34) Sokal, R. R.; Rohlf, F. J. Biometry. The Principles and Practice of Statistic in Biological Research; W.H. Freeman & Company: New York, 1997. (35) Ladonin, D. V.; Margolina, S. E. Interaction between humic acids and heavy metals. Eurasion Soil Sci. 1997, 30, 710-715. (36) van Ginneken, L.; Bervoets, L.; Blust, R. Bioavailability of Cd to the common carp, Cyprinus carpio, in the presence of humic acid. Aquat. Toxicol. 2001 52, 13-27. (37) Dawson, T. D.; Lott, K. G.; Leonard, E. N.; Mount, D. R. Time course of metal loss in Lumbriculus variegatus following sediment exposure. Environ. Toxicol. Chem. 2003, 22, 886889. (38) Ley N° 24051. Ley Nacional de Residuos Peligrosos N° 24051; Anales de la Legislacio´n Argentina LIIA: Buenos Aires, Argentina. 1992; pp 52-63. (39) Landrum, P. F.; Robbins, J. A. Bioavailability of SedimentAssociated Contaminants to Benthic Invertebrates. In Sediments: Chemistry and Toxicity of in Place Pollutants; Baudo, R., Giesy, J.P., Muntau, H., Eds.; Lewis Publishers: Michigan, 1990, 237-263. (40) Burgess, R. M.; McKinney, R. A. Importance of interstitial, overlying water and whole sediment exposures to bioaccumulation by marine bivalves. Environ. Pollut. 1999, 104, 373-382. (41) Leppa¨nen, M. T.; Kukkonen, J. V. K. Relationship between reproduction, sediment type, and feeding activity of Lumbriculus variegatus (Mu ¨ ller): Implications for sediment toxicity testing. Environ. Toxicol. Chem. 1998, 17, 2196-2202. (42) Campbell, P. G. 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 and Sons: New York, 1995, 45102. (43) Paquin, P. R.; Gorsuch, J. W.; Apte, S.; Batley, G. E.; Bowles, K. C.; Campbell, P. G. C.; Delos, C. G.; Di Toro, D. M.; Dwyer, R. L.; Galvez, F.; Gensemer, R. W.; Goss, G. G.; Hogstrand, C.; Janssen, C. R.; Mcgeer, J. C.; Naddy, R. B.; Playle, R. C.; Santore, R. C.; Schneider, U.; Stubblefield, W. A.; Wood, C. M.; Wu, K.; B. The biotic ligand model: A historical overview. Comp. Biochem. Physiol., Part C: Pharmacol., Toxicol. Endocrinol. 2002, 133, 3-35. (44) Niyogi, S.; Wood, C. M. Biotic ligand model, a flexible tool for developing site-specific water quality guidelines for metals. Environ. Sci. Technol. 2004, 38, 6177-6190. (45) Min ˜ o, L. A.; Folco, S.; Peche´n de D’Angelo, A. M.; Verrengia Guerrero, N. R. Modeling lead bioavailability and bioaccumulation by Lumbriculus variegatus using artificial particles. Potential use in chemical remediation processes. Chemosphere. 2006, 63, 261-268. (46) Campbell, P. G. C.; Stokes, P. M. Acidification and toxicity of metals to aquatic biota. Can. J. Fish. Aquat. Sci. 1985, 42, 20342049. (47) McDonald, D. G.; Reader, J. P.; Dalzier, T. R. K. Society for Experimental Biology, Seminar Series 34. In Acid Toxicity and Aquatic Animals; Morris, R., Taylor, E. W., Brown, D. J. A. Eds.; Cambridge University Press: Cambridge, 1989; pp 221-242. (48) Kola, H.; Wilkinson, K. J. Cadmium uptake by a green alga can be predicted by equilibrium modeling. Environ. Sci. Technol. 2005, 39, 3040-3047.
Received for review May 11, 2006. Revised manuscript received August 6, 2006. Accepted August 9, 2006. ES061135T
VOL. 40, NO. 20, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6347
