Environ. Sci. Technol. 2002, 36, 3550-3555
Kinetics and Uptake Mechanisms for Monomethylmercury between Freshwater Algae and Water H . A N S O N M O Y E , * ,† C A R L J . M I L E S , †,‡ EDWARD J. PHLIPS,§ BETHANY SARGENT,§ AND KRISTEN K. MERRITT† Food and Environmental Toxicology Laboratory, Food Science and Human Nutrition Department, P.O. Box 110720, University of Florida, Gainesville, Florida 32611, and Fisheries and Aquatic Sciences Department, P.O. Box 110600, University of Florida, Gainesville, Florida 32611
Uptake kinetics of monomethylmercury chloride (MeHgCl) were measured for two species of green algae (Selenastrum capricornutum and Cosmarium botrytis), one blue-green algae (Schizothrix calcicola), and one diatom (Thalassiosira weissflogii), algal species that are commonly found in natural surface waters. Species differences were found with the two green algae giving the highest uptake rates, and one of them (Cosmarium) showing differences between cultures having widely different cell age (exponential versus stationary), where increases in uptake rate for cells 30 days old were about 25 times greater than cells only 3 days old when weights of cells were considered. Both Schizothrix and Thalassiosira exhibited nearly the same lower uptake rates, approximately 20 times lower than the two green algal species. Experiments with photosystem inhibitors, uncouplers, γ-radiation, light deprivation, and extended range uptake all point to an active transport mechanism for MeHgCl.
Introduction Bioaccumulation of monomethylmercury (MeHg) has been well-documented at all trophic levels of aquatic food webs. This observation has raised concerns of environmental interests around the world, including the Florida Everglades (1) and areas of Sweden and Finland. As primary producers, algae are key elements of the structure and function of aquatic food webs and are one of the first and most pronounced steps in the biomagnification of MeHg (2). Among the most important aspects of bioaccumulation and biomagnification are (i) the equilibrium distribution of MeHg between algae and water (3), (ii) the kinetics of uptake, and (iii) the mechanism of uptake. These parameters play central roles in the now widely used computer model, the Everglades Mercury Cycling Model (E-MCM),which addresses mercury uptake by algae in both canal and marshland ecosystems (4). This model and the related Dynamic Mercury Cycling Model (D-MCM) both suggest that MeHg dynamics in the lower parts of the aquatic food web are critically important but inadequately understood. The MCM uses a kinetic * Corresponding author phone: (352)392-1978; fax: (352)392-1988; e-mail:
[email protected]. † Food Science and Human Nutrition Department. ‡ Deceased. § Fisheries and Aquatic Sciences Department. 3550
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approach to algal MeHg uptake, but the approach needs validation with field and laboratory studies. In previous research on four species of freshwater algae, the three eucaryotic algae tested had similar MeHg partition constants, Kpl, while the single procaryotic algae examined, Schizothrix calcicola, had lower values; the log Kpl for the three eucaryotic algae range from 6.5 to 6.7, while that for Schizothrix calcicola was 6.3 (3). It was hypothesized that these observations could be interpreted in terms of fundamental differences in the structure and function of procaryotes and eucaryotes (3). In that study, phosphorus-limited cultures of the green algae Selenastrum capricornutum exhibited lower Kpl values than cells grown under nutrient sufficient conditions. These differences were hypothetically attributed to the changes in cellular composition of proteins, carbohydrates, and lipids undergone by cells grown under nutrient limiting conditions (5). There is also a need to resolve whether MeHg uptake by algae is governed by passive or facilitated transport and whether the facilitated transport is of the diffusion or active type. The proper function of the MCM is in part dependent on the resolution of this issue. Transport rates of chemicals across cell membranes are largely determined by the transport mechanism, which can be classified in several ways. Jain classifies them as passive diffusion, facilitated diffusion, or active transport (6). Grogan adds another classification by modifying the facilitated diffusion one to include a chemical binding phenomenon in the cytoplasm of the cell (7). He calls this “group translocation”, which occurs primarily for neutral species and results in a neutral product within the cell, which is not able to utilize the transporter to then exit the cell. Facilitated diffusion across cell membranes is characterized by several features: (i) it is driven only by the concentration gradient of the solute (permeant); (ii) the transporters across the membrane are saturable, giving hyperbolic plots typical of the Michaelis-Menten relationship; (iii) temperature coefficients for most transporters (Q10) are typically 2-3; and (iv) transporters are almost always some type of protein (6). Active transport in plant membranes in the plasmalemma is driven by proton pumps that hydrolyze ATP to power the transport of protons out of the cytosol, establishing electrochemical potentials across these membranes. Such transmembrane proton potentials are then used to power the transport of other ions and solutes across the membranes. Recent model simulations suggest that facilitated uptake by algae is more likely than passive diffusion for the range of freshwater conditions tested (8). This conclusion differs from another recent study, which suggests that passive diffusion is the major mechanism of MeHg uptake, a conclusion from experiments with T. weissflogii which showed a direct correlation between permeability coefficients for the various species of MeHg typically found in marine environments and the log Kow values of those species. These experiments were cited to support the conclusions about passive diffusion being the transport mechanism into phytoplankton drawn from MeHg uptake/octanol-water partitioning experiments (9). The studies reported here inescapably bring these interpretations and conclusions into question, an issue we leave to the reader for resolution. The goals of this study were to determine the kinetics of MeHg uptake by four selected algal species and to examine key aspects of the uptake mechanisms. In the latter case, we focused attention on whether uptake involved passive or active transport. The four algal species were selected to 10.1021/es011421z CCC: $22.00
2002 American Chemical Society Published on Web 07/11/2002
represent a range of structural and functional characteristics and include two species common to algal mats found in the Everglades. The major Everglades mat-forming blue-green alga (aka cyanobacterium) Sc. calcicola was the single procaryotic species included in the study. The other species common to the Everglades chosen for the study was the eucaryotic green alga C. botrytis. Also included among the eucaryotic algae tested were the green alga S. capricornutum, widely used in algal bioassays, and the diatom (Heterokontophyta) T. weisflogii, which has been the subject of several metal uptake studies. Structurally, all algae species share the presence of a thin cell membrane (i.e., plasma lemma); however, there are major distinctions in the structure of the cell wall between the three divisions included in this study (10). Schizothrix, like all cyanophytes, has a cell wall structure similar to Gramnegative bacteria. It is composed primarily of peptidoglycan, a series of polysaccharide chains linked by short chain peptides. In the Chlorophytes Cosmarium and Selenastrum, the major structural constituent of the cell wall is primarily made of cellulose. In the diatom Thalassiosira, the cell wall is encapsulated in a silica frustule containing pores to allow for chemical exchange with the water column. In addition to the silica layer, a wall of sulfated polysaccharides, protein, and lipid can be present (10). In addition to differences in the characteristics of the cell wall, the four species included in this study exhibit major differences in internal cell structure. The most profound distinction is that the procaryotic alga (Schizothrix) has no cellular organelles, such as mitochondria, chloroplasts, and nuclei. There are also differences between the divisions in a wide range of biochemical characteristics, including pigment composition and storage products. For example, blue-green algae contain large quantities of the protein-based pigments phycobiliproteins, absent in the Chlorophyta and Heterokontophyta. Conversely, the Heterokontophyta are characterized by the presence of large quantities of the lipidbased carotenoid pigment fucoxanthin, which is not found in the other two divisions. As for storage products, Cyanophytes most commonly store glycogen; Chlorophytes typically store starch; and diatoms store chrysolaminarin, a glucose-based polymer. In our effort to determine whether MeHg uptake involves passive or active transport, various metabolic inhibitors and cell treatments were employed. The tests included treatment with chemical uncouplers of phosphorylation, radiation treatment, temperature sensitivity analysis, and light/dark sensitivity analysis. The use of inhibitors of phosphorylation was based on the hypothesis that active transport is dependent on a supply of ATP. Some ionophores specific for protons, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP), disconnect the electron transport from ATP synthesis, thereby inhibiting photophosphorylation. These ionophores, known as uncouplers, prevent the formation of ATP and thereby disrupt the energetics of the cell (11). Another ATP/proton uncoupler, 2,4-dinitrophenol, has been shown to inhibit oxidative phosphorylation (12). The other class of inhibitors used were compounds that effect noncyclic photophosphorylation by shutting down the photosynthetic electron transport chain. One of the most widely used is the herbicide diuron (DCMU), 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks the flow of electrons from photosystem II to photosystem I. A second class of inhibitors acts at the reducing end of photosystem I, inhibiting the reduction of ferredoxin. The latter includes the herbicide paraquat (methyl viologen; 11). Another approach to determining the relative importance of active transport in MeHg uptake was to slow or affect the rate of metabolism in general. Alteration of light/dark regime
was used with the idea that extended periods of darkness slow the production of ATP through photophosphorylation, thereby reducing internal energy levels that are necessary for active transport mechanisms. Temperature sensitivity analysis was used as an indicator of the impact changing temperature has on transporters of enzyme-like function involved in active transport. Finally, in the most extreme case, cells were killed without disrupting membrane structures. This was done by exposing cells to γ-radiation at levels that have been shown not to cause structural damage to the cell walls but still bring about lethality. High energy radiation, such as γ-radiation, has been used to effect many chemical changes within cells, and it has been shown to greatly affect cellular membrane permeabilities. Cell wall membranes generally increase in permeability when exposed to nonlethal γ-radiation. This has been observed for postmitotic rhabdomyocytes (13), endothelial cells (14), B lymphocytes (15), and rat thymocytes (16). Changes in the chloroplasts of Eutetramorus planctonicus, a Chlorophyceae, after irradiation have been observed (17), and a slowing of the incorporation of uracil in irradiated Chlamydomanas reinhardii has been noted (18). MeHg has been shown to form strong complexes with humic substances which then could affect the rate at which it is absorbed by algae (19, 20), and humics in excess have been shown to directly affect the permeability of S. capricornutum for the transport of neutral species, such as sulforhodamine-B (21). We exposed that alga to various concentrations of Suwanee River natural organic matter (NOM) to determine whether such complexes would affect MeHgCl uptake.
Materials and Methods Container Preparation. All containers used in this study were made of borosilicate glass, which were washed with conventional laboratory detergent, rinsed with DI water (Barnstead Nanopure Infinity), and dried at 190 °C before use. Periodically, all glassware was soaked in 6 N nitric acid before rinsing and drying. Frequent “glassware blanks” demonstrated to us that contamination was never problematic and that losses to glassware surfaces did not occur. Algae Culturing. Two green algae species, S. capricornutum and C. botrytis, one blue-green alga, Sc. calcicola, and one diatom, T. weisflogii. were obtained from the University of Texas algal culture cell repository. Green and blue-green algae cultures were grown in Allen’s freshwater media at pH 7 (22) under Redfield ratio concentrations and were maintained at 20-22 °C and light levels of 20-30 µEinstein/m2 14/10 light/dark cycle in 250-mL flasks. Thallasiosira cells were cultured in 1 ppt Guillard media (23). Once sufficient biomass levels were reached, the cells were transferred to 8-L aerated carboys containing 6 L of sterile-filtered media solution for mercury experiments. Cells were harvested in the log phase (approximately 4 days) or stationary phase (>6 days) of growth and were transferred from the Allen’s media into 5 mM phosphate buffer for experiments. This was accomplished by centrifuging S. capricornutum cells for 15 min at 2000 rpm (550g) and 15 °C, decanting the supernatant, replacing it with an equal volume of phosphate buffer, and repeating the centrifugation and decantation process. Sc. calcicola cells were isolated identically except for centrifugation at 5000 rpm (3420g). C. botrytis and Thallasiosira spp. cells were removed from the culturing media with an 8-µm Nitex filter. The washed algae solutions were resuspended in a 5 mM phosphate buffer at the desired cell dilution and mixed, and triplicate samples were removed for cell counts. The phosphate buffer was pH adjusted first and then checked and adjusted if necessary with KOH or H2SO4 just prior to experimental initiation. VOL. 36, NO. 16, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Uptake of MeHg by Algal Speciesa species
cell type
cell vol (µm3/cell)
Selenastrum capricornutum Thalassiosira spp. Schizothrix calcicola Cosmarium botrytisc 3 days 4 days 14 days 30 days
green diatom blue-green green
11.96 965 212 113 098
n
amol/cell
SD
amol ng-1 nM-1 d
SD
amol g-1 h-1 d (×1010)
SD (×1010)
14 10 5
0.440 0.394 b
0.038 0.027 b
0.028 1.24 0.934
0.002 0.08 0.058
0.528 3.17 2.13
0.04 0.22 0.13
3 3 6 5
38.1 41.0 202 759
2.9 6.0 18 9
24.1 25.9 127 480
1.8 3.8 12 6
4.58 4.92 24.2 91.1
0.35 0.73 2.2 1.1
a Cells in stationary phase except for Cosmarium, 3 and 4 days (exponential phase). To assist the reader in better understand the column labels the following explanations are added. amol/cell ) attomoles of MeHgCl taken up by each cell; SD ) standard deviation; attmol ng-1 h-1 nM-1 ) attomoles of MeHgCl taken up per nanogram of algae per hour per nanomolar concentration of MeHgCl in solution; amol g-1 h-1 ) attomoles of MeHgCl taken up per gram of algae per hour; amol/g ) attomoles of MeHgCl taken up per gram of algae. b Filamentous algae; not able to calculate per cell value. c Data reported by cell age. d Cell weights expressed as dry weights.
Methods for Uptake Rate Determination. All uptake rate determinations, unless specified otherwise, were made using 14C-labeled monomethylmercury iodide (Amersham, 99.1% pure, 54 mCi/mmol), at a final concentration of 1.9 nM. Buffer pH was set to exactly 7.0, as described above, and the buffer contained 200 mg/L of chloride ion as KCl (5.6 mM), unless otherwise noted. Such high chloride concentrations are typical of what is found in the canals of the Everglades region of Florida where MeHg contamination has been problematic to large fish. Calculations by others and ourselves using MINEQL show that the MeHg is 85% MeHgCl under the pH and buffer strength conditions used here (9). Concentrations of cells were adjusted to give weights of approximately 5 × 10-6 g/mL; for Selenastrum, this was typically 5 × 105 cells/ mL, although some experiments were as low as 2.5 × 105 and as high as 1.5 × 106. Experiments were begun by measuring exactly 100 mL of stirred cells into a 250-mL beaker on a stirring plate, adding 1 mL of 1.9 × 10-7 M 14C-labeled MeHg, and stirring vigorously for exactly 5 min. The stirred cells were quickly filtered by vacuum through a 4.7-cm multigrade glass fiber disk (Whatman GF/C), followed by a rinse of 50 mL of EDTA (1 mM) in the 5 mM phosphate buffer. Although formation constants for a MeHg-EDTA complex have not been determined, we felt that an EDTA rinse may very well be helpful in removing MeHg sorbed to the algae cell exteriors. Air was pulled through the disks for about 1 min; the disks were removed with forceps, folded over into quarters, and placed into 20-mL scintillation vials containing 15 mL of the water-compatible liquid scintillation cocktail ScintiSafe Plus 50% (Fisher Scientific). The vials were capped, shaken vigorously, allowed to sit in the dark overnight, and counted in a liquid scintillation counter (LKB-Wallace model 1214). Quenching was calculated by additions of internal standards of 14C-labeled MeHg iodide. Cell concentrations were determined both by counting in a Coulter counter, a hemacytometer, and by weight after filtering through the Whatman GF/C filter disks and then drying at 85 °C for 24 h for each experiment. Methods for Inhibitor Effects. Those inhibitors studied were (i) CCCP, (ii) DCMU, (iii) 2,4-dinitrophenol (DNP), and (iv) methyl viologen (paraquat). All inhibitor stock solutions were made up in 5 mM phosphate buffer, except for DCMU, which was made up in methanol. Methanol was removed from subsequent dilutions with buffer by rotary evaporation. Less than 1-mL volumes were added to individual 100-mL quantities of cells exposed to laboratory fluorescent lights exactly 2 h before uptake experiments were done by the addition of labeled MeHg. Uptake was measured after a 5-min incubation, as done for the above-described uptake experiments. Q10 Determinations: Effect of Temperature on Uptake. Uptake measurements on exponential stage Selenastrum 3552
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were done at 20, 25, 30, 35, and 40 °C by stirring 100 mL of cells for 5 min in a refrigerated water bath (Fisher Scientific, model 900), capable of statting the temperature to (0.1 °C. Other aspects of this determination were done identically to those described above for the uptake experiments. Effect of MeHg Concentration on Uptake Rate: Extended Range Uptake Curve. Uptake rates for exponential stage Selenastrum were measured for MeHg concentrations ranging from 0.19 to 500 nM, using the same procedures described above. Effect of Dark on Uptake Rate. The effect of light on the uptake rate by exponential Selenastrum was measured. Two sets of light-deprived cells were used: denied light for 24 and 48 h. Control cells were continuously exposed to laboratory fluorescent lights over those periods. Uptake was measured in the dark, by covering the stirred beaker with foil before the addition of MeHg was done. Fortification was done at the 1.9 nM level. Effect of Humics on Uptake Rate. Suwannee River NOM (natural organic matter, catalogue no. 1N101; International Humic Substances Society) was used at concentrations of 2.4, 9.1, and 33 mg/L with exponential growth phase Selenastrum to determine the effect of humics on the uptake rate of MeHg. Cells were stirred with the NOM for 2 h before the addition of MeHg at the regular concentration of 1.9 nM. Uptake rates were then determined as described above. Effect of γ-Radiation on Uptake. Algae (Selenastrum, Cosmarium, Thalassiosira) were irradiated at 110 krad (kilorads) with a 137Cs γ-irradiator (Nordian 1000 blood irradiator) to determine the effect of cell virility on the uptake rate of MeHgCl. Preliminary experiments were performed on Selenastrum cells irradiated at 60, 110, 200, and 400 krad to determine the effect of radiation dosage on cell respiration, along with light microscopic examination, and total organic carbon (TOC) exudation measurements of the surrounding buffer. Irradiation at 110 krad produced approximately 80% lethality, as measured by oxygen evolution upon intense white light irradiation (1000 W xenon lamp), while at the same time causing no visible cell damage as viewed under a light microscope and no measurable increases in TOC in the irradiated samples as compared to the controls (2.22 mg of C/L; Dorhmann DC-190, Rosemount Analytical). Radiation at 200 krad and above produced puckers and lesions in the exterior of the cells, particularly the Selenastrum and produced approximately 95% lethality.
Results and Discussion MeHg Uptake Rates. Among the four species of algae included in this study, the two green algae, S. capricornutum and C. botrytis, had higher MeHg uptake rates than either the blue-green alga Sc. calcicola or the diatom T. weissflogii (Table 1). Sc. calcicola exhibited the lowest uptake rates,
TABLE 2. Effects of Metabolic Inhibitors, Light/Dark Periods, and Dissolved Humics on MeHg Uptake by Selenastrum concn
n
growth status
% inhibition
SD
Metabolic 3 stationary 3 stationary 6 stationary 6 stationary 6 stationary 6 stationary 6 stationary 6 stationary 3 stationary 3 stationary 3 stationary 3 stationary 3 exponential 3 exponential
-1.4 57.9 80.9 86.2 90.0 92.6 59.4 57.3 -18.9 7.5 -17.7 22.4 -13.2 -27.6
13.6 13.9 10.5 9.8 23.1 19.0 5.0 9.1 5.6 1.3 2.2 4.7 13.2 7.7
Light/Dark Effects 4 exponential 3 stationary
16.7 37.1
5.6 9.4
Humicsc exponential exponential exponential stationary
29.3 31.3 86.2 66.8
9.7 8.8 14.2 7.9
Inhibitorsa,b
diuron CCCP 2,4-DNP
paraquat
1 25 5 25 50 100 100 500 10 25 50 75 75 150
24 h 48 h 2.44 9.09 33.3 33.3
3 3 3 4
a Inhibitor concentrations in µM. b Cells incubated with inhibitor 2 h before addition of MeHg. c Humic concentrations in mg/L.
consistent with previous observations for partition constants (3). Consistent with the observations of others (19, 20), exposing MeHg to the humics in the “natural organic river matter” sample produced complexes that consequently reduced the uptake rate of MeHg by 86% at the highest concentration studied. Less of an inhibition effect (29%) was seen at humic levels typically found in marshland and canal environments (Table 2). Of the four species studied, Schizothrix is the only species that is procaryotic, not having organelles within the cell. The differences in MeHg uptake rate observed for this species could, in part, be explained by this distinction. Along with Schizothrix, Thalassiosira also showed significantly lower uptake rates than the other two species, Cosmarium and Selenastrum. One of the distinguishing physiological features of the latter species of algae is that they share the same type of chloroplast membrane structure, characterized by a double layer of lipoprotein-based plasmalemma (25). In contrast, Schizothrix does not contain chloroplasts and the chloroplasts of Thalassiosira are surrounded by two additional envelopes, including the endoplasmic reticulum that encompasses both the chloroplast and the nucleus. It may be hypothesized that chloroplasts enhance mercury bioaccumulation, particularly if they exist in the physiological configuration found in green algae. Chloroplast membranes may be the site of active and/ or passive transport of MeHg. While little is known about the mercury bioaccumulation potential of chloroplasts, it is possible that they are predisposed to exceptional bioaccumulation potential, thereby enhancing the overall uptake capability of green algae. The absence of such membranes in Schizothrix and the extra barrier represented by the endoplasmic reticulum envelope around the chloroplasts in Thalassiosira may lower the uptake rate significantly for those two species of alga. Clear differences in MeHg uptake rates were also observed for different culture ages of Cosmarium (Table 1), a phenomenon not observed for the other three species of algae. As Cosmarium cultures proceeded from exponential to stationary phases of growth, the uptake rate increased substantially. The increase of MeHg uptake associated with
the increasing culture age of Cosmarium provides further evidence that changes in cell physiology and function may affect MeHg bioaccumulation. As cultures age, the percentage of dividing cells drop dramatically. By the time stationary phase growth is reached, many algae species expend a larger percentage of energy in the accumulation of storage products, including lipids, than in the elements required for cell growth and division, particularly the production of proteins. Increases in the relative importance of lipids in older algal cells may contribute to the observed increases in mercury uptake with culture age, especially in light of recent preliminary evidence from this laboratory that lipids may be an important site for MeHg in certain algal species. Active versus Passive Uptake. The relationship between MeHg concentration (0.3-333 nM) and the rate of uptake showed a distinct bi-model character (Figure 2). The presence of two linear portions of differing slope can be clearly seen. The fact that the lower concentration portion exhibits a much greater slope (4.8) than the higher concentration portion (0.49) suggests the presence of differing modes of uptake, with the lower concentration portion representing a form of “active” uptake. The potential for active transport of MeHg is further supported by other results involving inhibitors, radiation, and light/dark exposure. It is interesting to note that the largest species studied, Cosmarium, exhibits the highest uptake rate (91 × 109 amol g-1 h-1), although its cellular surface area to biomass ratio is the lowest. If passive diffusion were the only mechanism of transport for algae, and membrane permeability were similar among species, then the rate of uptake would be expected to be the lowest in Cosmarium based on simple surface to volume relationships, indicating that passive diffusion is not the dominant mechanism of uptake here. In contrast, it may be argued that variability in uptake rate is primarily related to interspecies differences in membrane permeability. Since there is very little information on the species-specific membrane permeability of MeHg, the answer to this question will require additional research. As seen in Table 1, the two green algae (Cosmarium and Selenastrum) have the greatest uptake rates, when calculated on a mass basis, with Thalassiosira being slightly more efficient than Schizothrix. Of the four inhibitors studied, two of them were uncouplers of phosphorylation (CCCP and DNP), one was a photosystem I inhibitor (paraquat), and one was a photosystem II inhibitor (diuron; Table 2). Of these, the uncoupler CCCP, which disrupts photophosphorylation, had the most profound effect, even at the lowest concentration studied (5 µM). CCCP-treated cells were inhibited by 81-95%. The other uncoupler, 2,4-DNP, which disrupts oxidative phosphorylation, was significantly less potent, producing only a 27% inhibition at 50 µM concentrations but increasing to 59% at 100 µM. The lone PS II inhibitor, diuron, showed a strong effect on uptake rate at the 25 µM level; no effect was seen at the 1 µM level. The PS I inhibitor, paraquat, showed no consistent effect at any of the levels studied, even though the experiments were repeated several times with that inhibitor. The other inhibitors gave consistent and reproducible results. The results of the inhibitor experiments indicate that MeHg uptake is sensitive to the inhibition of phosphorylation, particularly photophosphorylation. This conclusion is further supported by the observation that prolonged dark periods (i.e., 48 h) depressed MeHg uptake by 37% (Table 2). These results suggest that ATP is important for MeHg uptake and is therefore at least in part “active” in character. A more general indication that MeHg uptake is dependent on active cellular metabolism was the response of uptake to γ-radiation. The exposure of cells to γ-radiation at the 110 krad greatly reduced uptake rates for all three algae tested (Table 3), with the effects being equal in magnitude to those observed for CCCP. This level of radiation produced no visual VOL. 36, NO. 16, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Extended and limited range curves for the uptake of MeHgCl by exponential phase Selenastrum. Uptake rate in amol cell-1 h-1 (Y axis) vs MeHgCl concentration in solution (X axis).
FIGURE 2. Effect of temperature on the uptake of MeHg by exponential phase Selenastrum. MeHg concentration of 1.9 × 10-9 M. Uptake rate in amol cell-1 h-1 nM-1 (Y axis) vs temperature of algae (X axis). structural damage to cell membranes, at least from the standpoint of microscopic observation. There was also no increase in total organic carbon observed in the surrounding buffer of irradiated cells, indicating that the cell membranes were left intact, although the possibility of loss of membrane permeability due to a “fusing effect” of the radiation was not tested. Irradiation of Selenastrum at 200 krad produced slight puckering of the cell walls, as seen by light microscopy, and gave an even more pronounced inhibition of MeHg uptake for the 5-min experiment, nearly eliminating uptake at the 3554
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1.9 nM level (97%). For most mammalian cells of various types, γ-radiation has been shown to increase transport of chemicals into the cells, ostensibly by increasing cell wall permeability (14-16). In contrast to the aforementioned indicators of active transport of MeHg, the temperature dependence study showed Q10 levels more consistent with passive transport. The relationship between temperature and MeHg uptake was linear from 20 to 40 °C, with an average Q10 of 0.7 (Figure 1). This value is lower than that observed for most transport-
ment for discussions on membrane transport, and Reed Harris at Tetra Tech for discussions on the Everglades Mercury Cycling Model. Thanks go to Susan Hillier who performed several of the inhibitor experiments.
TABLE 3. Effects of Irradiation on MeHg Uptake (1.9 nM MeHg) irradiation power (krad) n 110 110 110 200
3 5 6 3
cells/growth status
% inhibition
SD
Thalassiosira spp., exp Cosmarium botrytis, exp S. capricornutum, exp S. capricornutum, exp
64.7 76.0 82.9 96.6
17.8 18.8 24.8 28.9
ers, which range from 2 to 3, and corresponds to about 2.8 kcal/mol (6). Processes limited by diffusion in the aqueous phase are typically less than 5.0 kcal/mol, suggesting that transporters might be only partially involved in the uptake of MeHg by algae (6). In summary, there are several lines of evidence that suggest the involvement of active transport mechanisms in the uptake of MeHg by algae, including (i) the fact that the rate of MeHg uptake did not go up with increasing surface area to biovolume ratio, (ii) the strong inhibition of MeHg uptake by uncouplers of phosphorylation, (iii) the strong inhibition of MeHg uptake rates from exposure of cells to γ-radiation, (iv) the partial inhibition of MeHg uptake rates by inhibitors of photosynthetic electron transport, (v) the partial inhibition of MeHg uptake rates by prolonged periods of dark exposure, and (vi) the bimodal character of the relationship between MeHg concentration and MeHg uptake rate. The only counter-indicative observation in this study was the low Q10 value for temperature dependence of MeHg uptake. Overall, the weight of the evidence appears to point toward the involvement of active transport in MeHg uptake. This does not, of course, rule out the possibility that MeHg uptake involves more than one mechanism and depends on the species and environmental conditions in question. Recent research with prokaryotic models (e.g., E. coli and Vibrio) indicate the involvement of facilitated transport in Hg(II) uptake (26). While there is a clear need to be cautious in extrapolating these observations with bacteria to algae, it is also important to point out that the blue-green algae Schizothrix is a prokaryote (i.e., cyanobacterium) and shares in many of the physiological characteristics of bacteria. By the same token, it is well-known that the evolution of eucaryotic algae involved symbiotic relationships with bacteria. Clearly, the results of this study are only the beginning for the full examination of this issue.
Acknowledgments We thank Don Axelrad and Tom Atkeson at the Florida Department of Environmental Protection for financial support through Contract SP584 and Deans Bill Brown and Richard Jones of the UF/IFAS Dean for Research Office for salary support. We thank Matt Booth of the Department of Environmental Engineering Sciences for the total organic carbon measurements, Burrell Smittle of the Florida Linear Accelerator Research Center for the γ-radiation service, Henry Aldrich and Jim Preston of the Microbiology and Cell Science Department as well as George Bowes of the Botany Depart-
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Received for review November 15, 2001. Revised manuscript received May 14, 2002. Accepted May 17, 2002. ES011421Z
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