Environ. Sci. Technol. 2007, 41, 2255-2260
Impact of pH on Cu Accumulation Kinetics in Earthworm Cytosol M A R T I N A G . V I J V E R , †,‡ MARIJKE KOSTER,† AND W I L L I E J . G . M . P E I J N E N B U R G * ,† Laboratory for Ecological Risk Assessment, National Institute for Public Health and the Environment, Bilthoven, The Netherlands, and Institute of Environmental Sciences (CML), University of Leiden, Leiden, The Netherlands
We studied the interaction between toxic stress and accumulation in the earthworm Aporrectodea caliginosa, as induced by different soil copper pools and soil constituents (especially pH). Earthworms were exposed in quartz sand, spiked soils, and field soils with different Cu concentrations and varying soil composition. The copper content in the earthworms was determined in the following: the cytosolic fraction, a granular fraction and a fraction consisting of tissue fragments, cell membranes and intact cells. The highest amount of Cu was found in the cytosolic fraction. The other fractions varied only slightly in response to changes in any of the copper pools in soil. Cytosolic copper was the best predictor of Cu availability to earthworms collected from soils at constant pH, as statistically significant correlations were obtained with pore water pCu at constant pH in earthworms exposed in quartz sand. This correlation was lost for cytosolic Cu concentrations in earthworms exposed to spiked soils and field soils at differing pHs. Instead, cytosolic copper correlated well to Cu in either pore water or solid phase. Soil pH not only plays an important role in the availability of metals and therefore on their uptake fluxes, but internal competition of Cu2+ and H+ at physiologically active binding sites also explained these apparent contradictions and increased the predictability of body burdens significantly.
Introduction Physical, chemical, and biological processes operate in a delicate interplay. The challenge in ecotoxicology is to visualize and quantify the variety of ingenious interactions. A clear shift in metals risk assessment is becoming visible, moving from the use of total metal concentrations for assessing adverse effects on ecosystems toward dissolved concentrations. The most recent knowledge is to express risks in terms of the activity of the free metal ion within the solution, explicitly taking into account physicochemical interactions in the abiotic environment and at the biological interface. This approach is the basis for the development of Biotic Ligand Models (BLMs). Whereas a number of BLMs have been formulated for aquatic organisms (see for instance (1) for a recent overview), similar approaches are virtually lacking for the terrestrial compartment. Given the similarities in metal metabolism between aquatic and terrestrial species, * Corresponding author phone: +31-30-2743129; fax: +31-302744413; e-mail:
[email protected]. † National Institute for Public Health and the Environment. ‡ University of Leiden. 10.1021/es061212k CCC: $37.00 Published on Web 02/21/2007
2007 American Chemical Society
the principles underlying the BLMs are likely to be valid for terrestrial species too (2, 3). Another approach to identifying the toxic pressure of metals to soil organisms is by determining uptake and elimination fluxes, also known as kinetic modeling. As demonstrated for isopods by Van Straalen et al. (4), the use of fluxes increases the predictability of toxic effects as compared to total body burdens. This is due to the ability of isopods of metal sequestration. Like isopods, earthworms are capable of sequestering metals at high internal levels, and different strategies may be used for this purpose. Specific strategies of metal handling can also be expected by essential metals. Essential metals may be subject to regulation either by limiting metal uptake at the level of the total body content, or by involving organism-specific accumulation strategies with active excretion from the metal excess pool, storage in an inert form, and/or excretion of stored (detoxified) metal (5). An internal metal pool required for normal metabolism can be distinguished from a metal pool above the metabolic requirements. Nevertheless, tissue- and organ-specific metal accumulation are ultimately determined by cellular mechanisms. At the cellular level, biota have evolved control mechanisms to minimize accumulation of reactive metal species and to facilitate optimal utilization of essential metals. The excess of metal ions are potentially toxic and must be removed from the vicinity of important biological molecules. This is achieved by the various chemical forms in which metals can be present, including binding in the active center of functional proteins and enzymes, binding in the active center of enzymes, binding to metallothionein (MT), transport proteins, or other sequestration proteins, and precipitation in extracellular granules, mineral deposits, residual bodies, and exoskeletons (6, 7). A pragmatic approach toward determining metal sequestration can be used to get insight in how organisms cope with exposure to polluted soil. Wallace et al. (8) isolated cytosolic fractions from a homogenate of the entire organism by separating this fraction from granules and from cell membranes, and subsequently analyzed each fraction for its metal content. Up to now some researchers have applied this approach of fractionation to investigate the capabilities of organisms to respond to metal exposure (9-13). In the research reported here we studied uptake of copper in the earthworm Aporrectodea caliginosa following exposure under a series of exposure regimes. Copper distribution was determined in (1) cytosolic fraction, the proteins, and the microsomal fraction, (2) granular fraction, and (3) tissue fragments, cell membranes, and intact cells. The amount of copper within these fractions was related to a few copper pools in the soil. It is postulated that one or more of the subcellular fractions thus identified may serve as an improved indicator of copper-induced stress. In line with the principles underlying the BLM approach we state that metal uptake by earthworms takes place predominantly via the aqueous phase (14-16). The aim of our study is to assess the interaction between stress induced by the presence of copper in the pore water, and soil-related stresses as affected by pH. Copper pools considered were the total copper concentration in the solid phase, the dissolved copper pool in the pore water, and the copper activity (expressed as pCu), whereas competition with cations was explicitly accounted for. Especially pH is an important parameter in this respect. The impact of soil constituents was first studied by using inert quartz sand as the exposure medium. Quartz sand provides an inert matrix that does not bind any copper from solution, thus allowing for univariate modification of the VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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composition of the water phase (adjustment of pCu) and minimizing gut uptake of Cu bound to ingested particles. Copper-spiked soils were used as a means of modifying the magnitude of copper exposure while maintaining similar soil properties. Finally, copper-contaminated and subsequently aged field soils (for at least one decade) were used to allow the study of copper uptake over a range of soils and soil properties.
Materials and Methods Earthworms. Adult Aporrectodea caliginosa species were collected from an uncontaminated sandy soil in The Netherlands and kept in the laboratory for at least two weeks before the start of the experiments. Wet weight of individual worms was on average 381 mg (SE 7 mg). Earthworms were kept at 15 ( 2 °C during all tests, and continuous illumination (1500 Lux) was employed to avoid animals escaping from the containers. Additional food was not provided. Experimental Design. Exposure in Quartz Sand. Worms were exposed in containers filled with 300 g of quartz sand that was percolated with an aqueous Steiner nutrient solution (17) containing a range of Cu2+-concentrations while keeping all other parameters as constant as possible. The inert quartz sand was rinsed with demineralized water. To reach equilibrium, the sand was kept in the test solution for at least 1 day and the solution was circulated through the equipment for another day prior to the start of the bioassays. The pH of the test solution was set at 5.5. A maximum variation of 0.15 pH units was allowed. The flow rate of the modified nutrient solution was 1 L per day. The free Cu activity (reported as pCu) and the pH of the Steiner solution were checked daily and adjusted if necessary. The pCu was adjusted by adding Cu(NO3)2 to the Steiner solution. The pH was adjusted using KOH or HNO3. Representative samples of the (percolated) Steiner solution were taken to measure the total dissolved Cu concentration. Each container held 4 worms. After 7 days of exposure, the earthworms were collected, placed on moist filter paper for 48 h to void their gut, and frozen at -18 °C. Exposure in Cu-Spiked Soils. Five Dutch soils were spiked with different concentrations of CuCl2 and allowed to equilibrate for at least 1 month at 15 °C. The five soils can be characterized as follows: one sandy soil with a high pH, generated artificially by addition of ground mollusc shells and allowed to age outdoors for around 10 years (VU-100), two clay soils with high pH from Oosterhout and Epen, one loamy sandy soil with low pH from Boxtel, and one sandy soil with low pH from Lepelstraat. The soil composition is described in more detail by Koster et al. (18). For reasons of consistency the same soil codes are used in this contribution as were used in ref 18. Twelve worms were used at each exposure concentration. The individual jars were filled with 500 g of spiked soil. Four worms per exposure duration were examined, namely after 7, 14, and 28 days of exposure. The pH, pCu, and Cu concentration in pore water were measured at the start and end of the experiments. The composition of the pore water in terms of pH and Cu concentrations is given in Table A-1 of the Supporting Information (SI). Exposure in Field Soils. The three field soils used originated from Canada, Denmark, and The Netherlands. Nine Canadian soils were collected at various distances near an abandoned copper mine in the Rouyn Noranda region (ROU). Five Danish soils were collected along a gradient of copper contamination from a site near Hygum (HYG), contaminated with Cu(SO4)2 during some decades. Emission stopped about 70 years ago (19). Eight soils from The Netherlands were contaminated in situ with CuSO4 and aged for over 20 years. These were sandy soils from Wageningen (WA) in which two pH ranges were created; a low pH of 4.5 obtained by addition of sulfur and a higher pH of about 6.5 obtained by addition of calcium 2256
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carbonate. The composition of pore water in terms of pH and copper concentrations is given in Table A-2 in the SI. The soil composition is described in more detail by Koster et al. (18). For the earthworm exposures, the same experimental design was used as for the spiked soils. Measurement of Soil and Pore Water Characteristics. Pore water was collected by suction over a 0.45 µm acetate filter of about 250 g of soil stored for at least one week at 15 °C at 80% of its maximum water holding capacity. Cation concentrations in the pore water were determined by ICPAES (Spectro Analytical Instruments, Kleve, Germany). Samples for quality standard control were within certified ranges, recovery was between 85 and 105%, and no systematic correction of analytical results was therefore performed. The pH was measured with a pH glass electrode Sentix stored in a buffer solution, employing an electronic voltmeter against a saturated solution of KCl. A copper selective electrode in combination with a voltmeter with 0.1 mV resolution (Cole-Palmer Copper Electrode) was used to measure the pCu in pore water. Standard solutions of Cu(NO3)2 in the concentration range of pCu 5 to 9 were used to generate calibration curves for measuring pCu according to the Nernst equation. All measurements were carried out at 20 ( 2 °C. Fractionation of Earthworms. Earthworms were thawed and homogenized using an Omni TH115 tissue homogenizer fitted with a 7 mm sawtooth blade in 5 mL of ice-cold 0.01 M Tris-HCl buffer (pH 7.5, Fisher Scientific, Houston, TX). Homogenates were centrifuged at 10 000g for 30 min at 5 °C. Supernatants were decanted and contained the metal present in cytosolic fractions including proteins and the microsomal fractions. The pellet fractions were boiled at 100 °C for 2 min and hydrolyzed at 60-70 °C for 1 h in 0.5 M NaOH (Merck, Darmstadt, Germany). The granules (pellet fraction) were subsequently separated from tissue fragments, cell membranes, and intact cells (supernatant) by centrifugation at 10 000g for 10 min at 11 °C. All fractions were dried at 100 °C and digested with HNO3 (ultrapure, Sigma-Aldrich, Seelze, Germany). The dried residues were dissolved in 0.5 M HNO3 (ultrapure, SigmaAldrich, Seelze, Germany). Metal quantification was performed by ICP-AES (Spectro Analytical Instruments, Kleve, Germany). Data Analysis. The amount of Cu accumulated in the different internal fractions was correlated to external metal pools in the soil by univariate regression analysis: log Y ) a*log X + b, in which Y ) amount of Cu in each of the internal fractions distinguished in this study, or total Cu amount in the earthworms, X ) either the pCu (no further transformation), the soluble copper concentrations, or the total Cu concentration. Statistics were expressed as: R 2adj ) correlation coefficient after correction for the number of degrees of freedom, n ) number of data points, F ) value of F-test, p ) significance level, a cutoff level of 0.005 was selected.
Results Quartz Sand Experiment. Earthworms exposed in quartz sand were collected after 7 days of exposure. The amount of Cu in the cytosolic fraction, in the granules, and in the tissue fragments, cell membranes, and intact cells was measured in pooled earthworm samples of each exposure duration. The Cu content in these internal fractions is plotted in Figure 1 as a function of pCu and the Cu concentration in the pore water, respectively. As can be seen from Figure 1, about 60% of the Cu that is taken up by the earthworms is typically present in the cytosolic fraction of earthworms. Only minor amounts of Cu (on average 30%) were present in the fraction containing tissue fragments, cell membranes, and intact cells. The Cu content of the granular fraction did not vary significantly,
FIGURE 1. Distribution of Cu in earthworms after 7 days of exposure to quartz sand as a function of pCu in pore water (left) and the soluble Cu concentration. C indicates the cytosolic fraction, D indicates the granules fraction, and E is the fraction containing the tissue fragments, cell membranes, and intact cells.
TABLE 1. Relationship Between the (log-transformed) Amount of Cu Present in Three Operationally Defined Subcellular Fractions of A. caliginosa as Well as the Total Amount of Cu in the Earthworms after Exposure for 7 Days in Quartz Sand, and Two (Log-transformed) Copper Pools in the Exposure Solution internal pool
external pool
C-fraction D-fraction E-fraction total
pCu pCu pCu pCu
C-fraction D-fraction E-fraction total
[Cu]pw [Cu]pw [Cu]pw [Cu]pw
a
b
R 2adj
N
F
P
-0.23 0.70 0.98 6 297.96
