Environ. Sci. Technol. 2001, 35, 4277-4282
Partitioning of Monomethylmercury between Freshwater Algae and Water C A R L J . M I L E S , †,‡ H . A N S O N M O Y E , * ,† EDWARD J. PHLIPS,§ AND BETHANY SARGENT§ 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
Phytoplankton-water monomethylmercury (MeHg) partition constants (Kpl) have been determined in the laboratory for two green algae Selenastrum capricornutum and Cosmarium botrytis, the blue-green algae Schizothrix calcicola, and the diatom Thallasiosira spp., algal species that are commonly found in natural surface waters. Two methods were used to determine Kpl, the Freundlich isotherm method and the flow-through/dialysis bag method. Both methods yielded Kpl values of about 106.6 for S. capricornutum and were not significantly different. The Kpl for the four algae studied were similar except for Schizothrix, which was significantly lower than S. capricornutum. The Kpl for MeHg and S. capricornutum (exponential growth) was not significantly different in systems with predominantly MeHgOH or MeHgCl species. This is consistent with other studies that show metal speciation controls uptake kinetics, but the reactivity with intracellular components controls steady-state concentrations. Partitioning constants determined with exponential and stationary phase S. capricornutum cells at the same conditions were not significantly different, while the partitioning constant for exponential phase, phosphorus-limited cells was significantly lower, suggesting that P-limitation alters the ecophysiology of S. capricornutum sufficiently to impact partitioning, which may then ultimately affect mercury levels in higher trophic species.
Introduction Bioaccumulation of methylmercury (MeHg) in high trophic levels of aquatic food webs is an important environmental contamination issue, especially in the Florida Everglades as well as in other parts of the world, including Sweden and Finland (1). Algae are important at the base of aquatic food webs; therefore, understanding the dynamics and extent of MeHg uptake by algae is fundamental to the bioaccumulation and biomagnification processes (2). The range of partitioning constants estimated from field measurements is fairly restricted, and there is evidence suggesting that certain water quality conditions (i.e., pH, DOC) affect MeHg accumulation (2, 3). * Corresponding author phone: (352)392-1978, ext 401; fax: (352)392-1988. † Food Science and Human Nutrition Department. ‡ Deceased. § Fisheries and Aquatic Sciences Department. 10.1021/es010792c CCC: $20.00 Published on Web 10/03/2001
2001 American Chemical Society
Values for Kpl (in liters per kilogram) determined from field measurements and models range from about 105 to 107 for periphyton and microseston. In one study, the distribution constant (Kd) for MeHg in periphyton and water in the central Everglades ranged from about 104 to 105 wet weight (4). In another study, the Kd ranged from 102.0 to 103.6 wet weight (5). Assuming that this periphyton was 90% moisture, the Kd dry weight values increase an order of magnitude. Morrison and Watras (6) reported a Kpl ) 106.3-106.8 for microseston/ MeHg in Lake Barco in north-central Florida. Watras and Bloom (7) reported a Kd of about 105 for phytoplankton/ MeHg in Little Rock Lake, WI, although it was on a wet weight basis. Maximum uptake of mercury can be calculated from cell geometry and measured physical constants, as was done by Hudson et al., for a lake with pH 6 and DOC 3 mg/L, which showed a Kpl of about 106 (2). Having well-validated partitioning constants as well as uptake rate constants are requirements for proper employment of one or more versions of the several mercury cycling models prepared by Tetra Tech Inc., which describe how mercury in many forms distributes itself through the various compartments of the model, including the water column, sediments, and food chain. One of these models, the Regional Mercury Cycling Model, predicts the cycling and fate of the major forms of mercury in lakes (8). It is designed to consider the various factors affecting fish mercury concentrations in lakes. Another version, the Everglades Mercury Cycling Model, has components specific to that type of canal/ marshland system (9). It is for this model that we have compiled our data on the four species of algae discussed here. The lack of such literature values for laboratory-derived phytoplankton-water MeHg Kpl values promoted this study. Laboratory-derived Kpl values have advantages over those derived in natural systems in that individual algae species can be singled out; there is control over the stage of growth of the algae and, therefore, cell characteristics; and the elimination of competing sorbers, such as detritus and other types of particulates. We used two independent methods to determine Kpl in order to increase our confidence in the results. We also studied MeHg partitioning into S. capricornutum as a function of algae growth phase, phosphorus limitation, and MeHg speciation (MeHgCl vs MeHgOH) to determine the effects of those parameters on such partitioning. In addition, we examined MeHg partitioning into several species of algae including the two green algae S. capricornutum and Cosmarium botrytis, the blue-green algae Schizothrix calcicola, and the diatom Thallasiosira spp. to determine if Kpl is dependent upon algae type. Cosmarium (10) and Schizothrix (11) have been identified in previous Everglades’s surveys. After we observed that the Kpl values were identical for exponential and stationary phase S. capricornutum, it was decided that we would focus our subsequent efforts on exponentially growing cells of the other three species since in that stage of growth there would be more consistency in cell size, virility, and partitioning characteristics. We would also focus on the Freundlich sorption isotherm method since it was seen to be rapid, reliable, and consumed less laboratory resources. Confirmation of the suitability of this method was accomplished by replicating the experiments using a flowthrough method and a radiolabeled method (see below). Since aquatic phosphorus has been an issue in south Florida for several years, we also performed a set of experiments designed to determine whether algae exposed to varying concentrations of that nutrient might exhibit atypical partitioning behavior. VOL. 35, NO. 21, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Methods and Materials Container Preparation. All containers used in this study were made of borosilicate glass, which after use 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. The analytical method used for MeHg distinguished that species from all other species, including inorganic mercury, which made glassware cleanliness a simple issue to deal with. Frequent “glassware blanks” demonstrated to us that contamination was never problematic. Algae Culturing. Algal species were selected on the basis of their being indigenous to the freshwater marshlands of the Everglade regions of Florida, their ability to be manipulated in the laboratory without clumping or adsorption to containers, and their ready availability from cultures free from bacteria. Two green species (S. capricornutum and C. botrytis), one blue-green species (S. calcicola), and one diatom (Thallasiosira spp.) 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 (12) under Redfield ratio concentrations and were maintained at 20-22 °C and light levels of 20-30 µEinstein/m2 with a 14/ 10 light/dark cycle in 250-mL flasks. Thallasiosira spp. cells were cultured in 1 ppt Guillard media (13). Once sufficient biomass levels were reached, the cells were then transferred to 8-L aerated carboys containing 6 L of sterile-filtered media solution for mercury experiments. Two nutrient levels were considered: phosphorus limited (50 µg of P/L, 3500 µg of N/L) and the Redfield ratio of nutrients (500 µg of P/L, 3500 µg of N/L). The Redfield ratio condition represents a balanced addition of nutrients so that growth is not preferentially limited by either nutrient. P-limited experiments were done individually because the cell size/volumes were unique for each batch. 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 a 5 mM phosphate buffer for experiments. This was accomplished by centrifuging S. capricornutum cells for 15 min at 2000 rpm (550 RCF) and 15 °C, decanting the supernatant, replacing it with an equal volume of phosphate buffer, and repeating the centrifugation and decantation process. S. calcicola cells were isolated identically except for centrifugation at 5000 rpm (3420 RCF). 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, then checked, and adjusted if necessary with KOH or H2SO4 just prior to experimental initiation. Methods for Kpl Determination. Freundlich Sorption Isotherm Method. Six 100-mL aliquots of control solution and six 100-mL aliquots of algae solution were placed into clear, acid-washed, glass bottles and fortified with amounts of MeHg ranging from 0.05 to 8 ng/L. After 20-24 h at 25 ( 1 °C and a 12/12 light/dark cycle (10 µEinstein m-2 s-1), the solutions were filtered (0.45 µm Teflon), and the filtrate [MeHg] was measured. The acid-washed bottles were examined for surface adsorption by taking solutions of MeHg through the experiment without the presence of algae. Algaesorbed MeHg was determined by difference, after the measurement of supernatant concentrations was determined. No significant adsorption of MeHg to container walls was observed at the working concentrations employed. In selected experiments, samples were filtered through GF/C glass fiber filters, and the amount of MeHg on the algae was 4278
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measured directly by digestion with methanolic KOH and nitrogen-assisted distillation. The amount of MeHg on the algae was also assayed by liquid scintillation counting after fortification with14C-labeled methylmercury at similar concentrations. Cells were enumerated with both a hemocytometer and a Coulter counter, which gave comparable results. For all S. capricornutum experiments, cell counts were converted to dry weight using the experimentally determined cell mass of 1.9 × 10-14 kg dry wt/cell. For C. botrytis, S. calcicola, and Thallsiosira spp. experiments, dry weights were determined for the individual experiments. For calculation of the partition constant, units were in liters per kilogram dry weight, as determined by
Kpl ) (ng of MeHg kg-1)cell/(ng of MeHg L-1)water Flow-Through, Dialysis Bag Method. Dialysis bag experiments were conducted in a flow-through mode so as to provide an environment where the aqueous concentration of MeHg did not diminish during the partitioning process. A 20-L batch of 5 mM phosphate buffer (pH 7.0) was prepared and pumped into a 1-L beaker with magnetic stirrer at 12.4 mL/min. A 2-L batch of MeHg stock (ca 45 ng/L) in phosphate buffer was pumped into the same beaker at 1.2 mL/min. The target concentration in the beaker was 5 ng/L, and the solution volumes were sufficient for approximately 24-h operation. Algae were prepared as described above, loaded into two 3.1 × 17 cm cellulose ester dialysis tubes (500 MWCO), and placed inside the stirred beaker. At the end of the experiment (24 h), the dialysis bag contents were filtered through a glass fiber filter, digested, and distilled before GC/ AFS analysis along with the supernatant solutions. The beaker solution was measured periodically and at the end of the experiment (24 h), the dialysis bag contents were filtered through a glass fiber filter. The filters were then digested and distilled for MeHg analysis as described above. The supernatant solutions were also analyzed. MeHg Speciation Experiments. To test if MeHg speciation was a significant factor in partitioning of MeHg and S. capricornutum, we varied the pH (7-8) and chloride concentration (20-200 mg/L) over a range that would produce MeHgCl or MeHgOH as the dominant species. This range was nearly comparable to be one that could exist under natural Everglades marsh conditions and not significantly stress the algae during the course of the experiment (14). Short-Term Uptake Experiments with EDTA Rinse. In an effort to determine whether MeHgCl is first loosely sorbed to the algae cells and then stays in that environment or penetrates into the interior of the cell with time, we performed experiments on the two green algae, S. capricornutum and C. botrytis. The former algae has a cell wall comprised only of a thin cellulose structure and a phospholipid bilayer, while the latter has a much more rigid cell wall. These experiments were done with 14C-labeled MeHgCl, but incubations were limited to only 5 min, followed by a 50-mL rinse with 50 mL of 1 mM EDTA in the pH phosphate buffer described above. Controls were run by rinsing only with 50 mL of water. Filtration, solubilization of algae, and scintillation counting were done as described above. Sample Preparation for Methylmercury Analysis. The filtered sample solution, 0.2 mL of a 2 M citrate buffer, 50 µL of the internal standard (7.9 µg/L propylmercury), and 50 µL of sodium tetraethylborate mixture (1% w/v in 1%w/v KOH) were combined in a 44-mL volatile organic analysis vial, filled to volume (no headspace) with filtered sample, capped, and mixed. Solutions were analyzed within 12 h. Methods for MeHg Analysis. Gas Chromatography/Atomic Fluorescence Detector System (GC/AFS). A 10-mL aliquot of sample mixture was loaded (Dynatech PTA-30 autosampler)
TABLE 1. Shapes and Dimensions of Algae Cells Used in This Study
a
cell
type
shape used
dimensions (µm)a
vol (µm3/cell)
S. capricornutum S. capricornutum, P-lim, rep 1 S. capricornutum, P-lim, rep 2 S. capricornutum, P-lim, rep 3 Cosmarium botrytis Schizothrix calcicola Thallasiosira spp.
green green green green green blue-green diatom
prolate spheroid prolate spheroid prolate spheroid prolate spheroid sphere cylinder cylinder
a ) 4, b ) 0.875 a ) 4.53, b ) 1.07 a ) 5.90, b ) 1.04 a ) 5.32, b ) 1.04 r ) 30 r ) 0.75, h ) 120 r ) 5, h ) 12.17
11.96 21.7 48.4 43.7 113 098 212 965
a ) major semi-axis; b ) minor semi-axis.
into a purge-and-trap concentrator/injector (Tekmar LSC2). After a 5-min purge onto a Tenax column (T < 30 °C), the sample was desorbed for 1 min at 180 °C into a splitless injector (1 mm bore liner, Silcosteel liner seal, 200 °C), with the purge off from 0 to 1 min. The Tenax column was then baked at 190 °C for 2 min before cool-down for the next cycle. The injector was connected to a wide-bore capillary column (15 m × 0.53 mm; 1.5 µm DB-1 J&W) inside a gas chromatograph (HP 5890) with 14.5 mL/min of He carrier gas. After an initial hold at 40 °C for 2 min, the oven was heated at 10 °C/min for 5 min (total run time 7 min). The GC effluent was pyrolyzed in a NiCAT tube (OI Analytical) heated in a furnace at 630 °C, combined with argon (18 mL/ min) via a PEEK mixing tee, and swept into a Tekran (model 2500) cold vapor atomic fluorescence spectrophotometer mercury detector. The detector signal was processed by ClassVP software (Shimadzu Corp). Methylethylmercury, ethylethylmercury (ethylated inorganic mercury), and propylethylmercury (internal standard) eluted at 2.8, 4.8, and 6.0 min, respectively. Kpl Determinations Using 14C-Labeled MeHg. Methylmercury (TCI-GR, MeHgCl, 98%) and 14C-labeled MeHg (54 mCi/mmol; Amersham) were used without purification. Primary stock solutions were prepared annually in ethanol, secondary stock solutions were prepared weekly in water, and all other standards were prepared daily. Primary and stock solutions were stored at 4 °C when not in use. Scintillation counting was performed on a model 1214 LKBWallace scintillation counter at 3 min per sample. Incubations and filtrations were done as for the Freundlich method, except counts were done on the filtered cells with proper quench corrections employed (see above for the Freundlich method). DOC was measured on a Dohrmann DC-100 TOC analyzer. Samples were filtered, acidified, and purged with helium. MINEQL+ (15) was used for all equilibrium calculations. Volume-volume concentrations factors (VCF) were determined according to
VCF ) (mol of metal µm-3)cell/(mol of metal µm-3)water using cell dimensions and shapes summarized in Table 1. Such VCF values factor in differences in cell versus water densities since they are volume-based calculations. Biovolumes were calculated using the program BIOVOL (16). Attempts at measuring cell volume with the Coulter were futile due to large measures of variability for unknown reasons.
Results and Discussion The Freundlich sorption isotherm method examines how MeHg partitions between algae and water by observing the concentration of MeHg on the solid, Cs, as a function of MeHg in water, Cw. The data are fitted to the Freundlich equation (Cs ) KCwn) where K is the Freundlich or equilibrium constant and n is a measure of the nonlinearity. In dilute solutions of MeHg, the plot of Cs vs Cw is linear and n ) 1. A plot of the
FIGURE 1. Example Freundlich isotherm. S. capricornutum, pH 7.0, [Cl-] 200 mg/L, intercept ln K ) 14.95 ) log K 6.49, slope n ) 0.86. natural log of the amount of MeHg sorbed to algae (ln Cs) vs the amount of MeHg in solution (ln Cw) yields a slope ) n and an intercept of ln K. Figure 1 shows an example from an experiment with S. capricornutum. The Kpl for MeHg algae/MeHg water for all algae tested ranged from 106.0 to 106.7 (Table 2). If we compare the Kpl for four freshwater algae species in exponential growth, the P-limited S. capricornutum (106.0-6.1) and the blue-green algae, Schizothrix (106.3), are significantly lower (one-way ANOVA, p 0.05) (see Figure 2 also). Figure 2 shows this graphically where the three, P-lim S. capricornutum sets of cells had significantly larger volumes than the controls, and the Kpl values were also significantly smaller. However, even though there were huge differences in cell sizes for those cells that were not phosphorus limited, only Schizothrix exhibited a significantly different Kpl, it being lower than for the other three. Obviously, factors other than cell size are involved in determining Kpl. Using EDTA to rinse the algae cells rather than water did not diminish the amount of MeHg remaining in or on the cells after 5-min incubations, whether the cells were S. capricornutum or C. botrytis. Consequently, sorption of MeHg appears to be via a mechanism other than by superficial surface adsorption. The mechanism of transport across the cell wall is addressed in another manuscript now in preparation (17). The sorption isotherms were linear (r2 mean ) 0.96) with slopes that were near unity. Control solutions showed quantitative recovery of MeHg. Partitioning constants determined with exponential and stationary phase cells (all at pH 7, [Cl-] ) 200 mg/L) were not significantly different. To verify that the amount of MeHg associated with the algae could be accurately determined by the difference between the initial amount and the filtrate amount, we measured the MeHg concentrations in algae by digestion and distillation in some experiments. In addition, we verified the accuracy of MeHg concentrations associated with the VOL. 35, NO. 21, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Partition Constants (K) and Volume-Volume Concentrations Factors (VCF) for MeHg and Algae Using Various Determination Methods method
cells/growth status
Freundlich Freundlich Freundlich Freundlich Freundlich Freundlich Freundlich Freundlich flow-through
S. capricornutum, exp S. capricornutum, stat S. capricornutum, exp, P-lim, rep 1 S. capricornutum, exp, P-lim, rep 2 S. capricornutum, exp, P-lim, rep 3 Cosmarium botrytis, exp Schizothrix calcicola, exp Thallasiosira spp., exp S. capricornutum, exp
a A C C C A,B B,C A,B
log K ( SD
log VCF ( SD
n
slopeb
6.66 ( 0.19 6.72 ( 0.39 5.85 ( 0.01 5.95 6.07 6.74 ( 0.25 6.26 ( 0.25 6.72 ( 0.21 6.54 ( 0.16
6.81 ( 0.20 6.91 ( 0.58 6.00 ( 0.08 6.61 6.72 5.94 ( 0.69 5.60 ( 0.21 5.37( 0.04 6.67 ( 0.13
6 4 2 1 1 4 4 4 4
1.05 0.98 0.78 0.94 1.11 0.92 0.89 1.08
a Treatments with similar letters are not significantly different at the 0.05 level (Tukey’s test). in this table were taken at pH 7 and 200 mg/L chloride.
FIGURE 2. Partition constant vs cell volume for the four cell types in exponential growth phase. Cell volume error assumed to be 10% or that measured. Kpl error from Table 2. algae by fortification with 14C-[MeHg] and liquid scintillation counting. There was no significant difference between the three methods of determining MeHg on algae (one-way ANOVA, p ) 0.05). We also used a flow-through, dialysis bag method for determination of the MeHg/S. capricornutum partition constant. Media and MeHg stock solution were combined to yield the target concentration (5 ng/L) and approximately 1 turnover/h. The partition constant measured by this method (106.54 ; n ) 4) was not significantly different from the isotherm method determined at the same pH and chloride concentration (Table 2). Results showed that the beaker solution maintained the target MeHg concentration (4.5 vs 5 ng/L) throughout the experiment. We believe that this method could be used at lower MeHg concentrations, but it was not tested. An objective in the determination of partition constants was to work at typical environmental concentrations. We initiated our Kpl determinations using the Freundlich isotherm since it is commonly used in environmental studies. Using algae cell densities of around 105 cells/mL, which were used in other similar studies, we found that MeHg fortification concentrations above 10 ng/L (40 pM) were needed to yield final filterable MeHg concentrations that were reliably measured (i.e., >0.2 ng/L, which was the practical method limit of quantitation for the GC method). Method limit of detection was that MeHg concentration in the filtrate that gave a peak approximately 10 times baseline noise. To keep the depletion of MeHg in lower concentrations of solutions from occurring, MeHg can be continuously added at a lower concentration, as was done with the flow-through method. The flow-through method has the advantage of using low and relatively constant MeHg concentrations. However, low 4280
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b
Only applicable to Freundlich method. All data
concentrations of MeHg and algae allow potentially significant interactions with the reaction bottle surfaces or the dialysis bag material. Also, it is difficult to maintain suspended algae in the dialysis bag. The Freundlich isotherm method suffers from the high initial MeHg concentrations used, although in many cases, the MeHg concentrations in the filtrate were in the upper range observed in the natural environment after equilibrium was reached (i.e., 0.2-0.5 ng/ L). This method was much more user-friendly and less timeconsuming than the flow-through method and is to be recommended for those reasons. MeHg forms inorganic complexes with chloride and hydroxide, and these two species predominate in natural waters devoid of organic material and reduced sulfur. How a chemical species distributes itself between immiscible octanol and water is a measure of the “hydrophobicity” of that species. When multiple species are present, such as MeHgOH and MeHgCl as found in the systems we used, the more descriptive term is the Dow, which is defined by
Dow,Hg ) (fMeHgCl)(Kow,MeHgCl) + (fMeHgOH)(Kow,MeHgOH) + (fMeHg+)(Kow,MeHg+) where f is the fraction of any particular species in the aqueous phase and Kow is the octanol/water partition coefficient for any particular species (13). Thus, Dow can be a measure of how all species of MeHg might partition into algae since the interior of the cells are a “hydrophobic” environment. As one would expect, the numerical value of this term is highly pH dependent and also dependent upon the anionic species and concentrations present. For example, the Dow of the test solutions used in our studies varied from 0.13 (pH 8, [Cl-] ) 20 mg/L) where MeHgOH predominates (96%) to 1.36 (pH 7, [Cl-] ) 200 mg/L) where MeHgCl is the major species (79%). The stability constants are such that at slightly basic conditions and above, MeHgOH is dominant, while at slightly acidic conditions and below and/or with moderate chloride concentrations, MeHgCl is dominant (12). In addition, MeHgCl has a significantly higher octanol/water partition constant (Kow ) 1.7) than MeHgOH (Kow ) 0.3; 18). Realizing the significance of this situation, Mason et al. (19) theorized that MeHg uptake into alga cells is governed by MeHg speciation. They reasoned that the more lipophilic MeHgCl should partition through the cell membrane much faster than the more polar MeHgOH. They found a correlation (r ) 0.75) between MeHg uptake rate into the saltwater diatom, T. wiessflogii, and the Dow of MeHg. In the studies we report here, the partition constant for MeHg and S. capricornutum (exponential growth) was relatively constant over the range of Dow tested (Figure 3). This suggests that the amount of MeHg that partitions into these algae was not significantly affected by MeHg speciation. Reinfelder and Chang (20) found that silver uptake kinetics
FIGURE 3. Partition constant of S. capricornutum (exp growth phase) and MeHg as a function of Dow. by the marine diatom, T. weissfolgii, is controlled by silver speciation, similar to that observed by Mason et al. (19) for inorganic mercury and methylmercury in the same diatom and also found that the volume-volume concentration factors were relatively constant over the range tested. They noted that while the hydrophobicity of AgCl controlled Ag uptake kinetics, the reactivity with intracellular components controlled steady-state concentrations. Our observation that Kpl for S. capricornutum was insensitive to MeHg species is consistent with these findings. In addition, partitioning constants determined with exponential and stationary phase cells at the same conditions were not significantly different, while the partitioning constant for exponential phase, phosphorus-limited cells was significantly lower. This suggests that these intracellular components were decreased in S. capricornutum by phosphorus limitation (by simple dilution); however, we have no cellular chemistry data to support this theory. Our experiments indicate that Plimitation alters the ecophysiology of S. capricornutum sufficiently to impact partitioning. For our experiments, we assumed that MeHg speciation was controlled by chloride and hydroxide since sulfide and dissolved organic carbon (strong complexers of MeHg) were not added. However, algal exudates are known to occur and be significant sources of DOC. Algal exudates are poorly defined mixtures of metabolic intermediates (glycollic acids, polysaccharides) and metabolic end products (carbohydrates, peptides, enzymes, vitamins, and toxins; 21). In our experiments with S. capricornutum, we measured DOC concentrations of 0.9 mg/L before the equilibration period and slightly higher (1.4 mg/L) after equilibration, a concentration not uncommon in natural surface waters. Koelmans and Heugens (22) found similar DOC concentrations for S. capricornutum exudates. The binding constant for MeHg with these algal exudates is unknown so their effect on MeHg speciation is unknown. However, these levels of DOC are extremely low as compared to natural waters. Assuming that these exudates bind MeHg like humics and using the MeHg-humic stability constants determined by Hintlemann et al. (23) in MINEQL, these levels of DOC will not result in a significant fraction of the MeHg-DOC species. The similarity of MeHg partition constants, Kpl, observed for the three eucaryotic algae reported in this paper and the distinct value observed for the procaryotic alga provides several interesting insights into the nature of mercury bioaccumulation by algae. Some of the physiological features of these four taxa are different enough to shed some light on what may and may not affect the partitioning coefficient for methyl mercury. The four species of algae presented in this paper are members of three major algal divisions: the
procaryotic division Cyanophyta (i.e., cyanobacteria) and two eucaryotic divisions, Chlorophyta (i.e., green algae) and Heterokontophyta (i.e., in this case diatoms or Bacillariophyceae, a class within the division; 24). While all of the algal divisions share the presence of a thin cell membrane, or plasmalemma, there are major distinctions between the three divisions is the structure of the cell wall. 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 Chlorophytes, Cosmarium and Selenastrum, the major structural constituent of the cell wall is cellulose. In the diatom Thalassiosira, the outer cell wall is composed primarily of silica deposits with a system of pores to allow for chemical exchange with the water column. In addition to the silica layer, a secondary wall of sulfated polysaccharides, protein, and lipid can be present. Within the cell wall, the three algal divisions represented in this study exhibit many fundamental differences in structure and chemical composition (24). The most structurally profound distinction involves the lack of cellular organelles (e.g., mitochondria, chloroplasts, and nuclei) in the procaryotic algae. There are also differences between the divisions in a wide range of biochemical characteristics, including pigment composition and storage products. For example, the Cyanophyta contain large quantities of the protein-based pigments phycobiliproteins absent in the Chlorophyta and Heterokontophyta. By the same token, the Heterokontophyta are characterized by the presence of large quantities of the lipid-based carotenoid pigment fucoxanthin not found in the other two divisions. In terms of principal carbon storage products, cyanophytes most commonly store glycogen, Chlorophytes typically store starch, while diatoms store chrysolaminarin, a glucose-based polymer. The apparent lack of significant differences in the partition constants for methyl mercury among the three species of eucaryotic algae included in this study, despite the aforementioned structural and chemical differences between the two algal divisions represented by these species, suggests that mercury bioaccumulation capacity is controlled by more conservative features shared by eucaryotic algae (e.g., the presence of cellular organelles). The most obvious structural distinction between procaryotic and eucaryotic algae that may have a bearing on the lower partition coefficient observed for Schizothrix is the lack of true organelles in the former. Since our preliminary experiments with the distribution of MeHg in algal cells indicate a preference for association with membrane rather than cytoplasmic fractions (25), it may be hypothesized that membranes of organelles or endoplasmic reticulum of eucaryotes enhance the availability of binding sites for MeHg. In contrast, binding sites in cyanophytes may be more limited, e.g., thylakoid membranes. Another observation pertinent to this issue is the decrease in the MeHg partition constant with phosphorus limitation in Selenastrum. It is well-known that nutrient limitation can have profound effects on the structure and chemistry of algae (26, 27). Physically, these effects include changes in cell size and the number and size of organelles within cells. Phosphorus limitation has also been shown to alter the biochemical composition and relative concentrations of proteins, carbohydrates, and lipids within algal cells (28, 29). It is therefore not surprising that phosphorus limitation has a significant impact on the nature of mercury bioaccumulation.
Acknowledgments We thank Tom Atkeson and Don Axelrad at the Florida Department of Environmental Protection for financial support through Contract 454 and Deans Bill Brown and Richard Jones of the UF/IFAS/Office of Research for salary support. We thank Tim Fitzpatrick. Kerry Tate, and T. M. Chandrasekar VOL. 35, NO. 21, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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also with FDEP central laboratories for equipment support. We thank Curt Pollman and Reed Harris at Tetra Tech, Cindy Gilmour of the Academy of Natural Sciences Benedict Laboratory, and Rob Mason of the University of Maryland for helpful discussions during the course of this work.
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Received for review March 27, 2001. Revised manuscript received August 23, 2001. Accepted August 24, 2001. ES010792C
