INTRODUCTION

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Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitous in the global environment which have been shown to have adverse effects on the health of many aquatic organisms (5, 16, 17, 20, 24). Limited biotransformations result in efficient transfer of many chlorinated biphenyls between trophic levels and accumulation in lipid-rich tissues (9, 19). Diffusion through cellular membranes is the only chlorobiphenyl (CB) uptake pathway available in nonphagocytotic unicellular organisms such as phytoplankton. For large organisms such as fish and marine mammals, PCBs accumulate through ingestion (6, 9, 12). Uptake via gill and dermal exposure has been suggested to play a minor role in the overall uptake of PCBs by fish and other large marine organisms (9, 19, 22).

Previous studies have explored the kinetics of CB equilibration with phytoplankton (25, 28), but to date, no work has been done concerning the accumulation of PCBs in organisms that can ingest contaminated prey. Heterotrophic protozoa offer a unique opportunity to study the relative rates and contributions of diffusive and ingestive uptake because they are capable of ingesting small particulate material yet are the same size as nonphagocytotic organisms already studied.

Within a bulk solution, CB speciation is determined by the nature and concentration of organic carbon in both dissolved and particulate pools (23). Studies have shown that PCBs associated with organic material (dissolved and/or particulate) are relatively unavailable for biological uptake in comparison to PCBs truly dissolved in the aqueous phase (for reviews, see references 10 and 18). Truly dissolved PCBs can enter a cell by diffusing through outer membranes. Ingestion of prey, on the other hand, involves the encapsulation of a parcel of water containing both free and complexed PCBs via invagination of the cellular membrane. Prey CB concentration is an important parameter in determining the CB uptake via ingestion and is affected by both prey size (i.e., surface-area-to-volume ratio) and cellular composition.

The steady-state CB concentration within the protozoan cell is the equilibrium value predicted by the CB congener K_ow (the n-octanol-water partition coefficient) and the relative size and composition of all organic carbon pools in the system. The uptake pathway does not affect the equilibrium concentration in the protozoan cell, simply the time needed to achieve this value (8, 9). Swackhamer and Skoglund (28) predicted that full equilibration of aquatic organisms (15 to 20 µm) with aqueous CB concentrations occurs on day time scales. Generation times of many aquatic organisms are shorter than days, and thus, the rate of uptake is an important factor in determining the accumulation of PCBs within the food web. Organisms such as phagotrophic protozoa can potentially access an additional pool of PCBs and achieve equilibrium faster than nonphagocytotic cells.

Initial calculation. An initial calculation for 3,3',4,4'-tetrachlorobiphenyl (IUPAC no. 77), however, showed that the diffusion uptake pathway should be faster than ingestion of contaminated prey. The calculation assumed that the rate-limiting step of diffusive uptake was transfer across the lipid membrane. The other slow step in this process, diffusion across the unstirred water boundary layer, was not considered to be due to the difficulty of estimating the width of this layer. Constant movement of surface cilia associated with filter feeding and swimming will lower the thickness of the unstirred water boundary layer.

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	MATERIALS AND METHODS

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Growth of organisms. Vineyard Sound seawater (VSW), used in all growth media, was collected by using a Masterflex pump during an incoming tide at Woods Hole, Mass. Seawater was stored in polycarbonate carboys in the dark at room temperature. Before media preparation, seawater was filtered through a 1.2-µm-pore-size in-line Versapor filter (Gelman) and a 0.2-µm-pore-size in-line nylon filter (Whatman). The water filtered through the 0.2-µm pores was autoclaved for at least 30 min and was stored at room temperature. All glassware used for culturing was washed with Citranox detergent (Fisher Scientific), was soaked in 10% ethanol-HCl in Milli-Q water overnight, and was rinsed with Milli-Q water and sterilized.

Experimental protocol. The PCBs used in this experiment were purchased (catalog no. C-CCSEC-R, lot no. 124-269; AccuStandard, New Haven, Conn.) as a mixture of 21 congeners (approximately 100 µg of each congener per ml of acetone), spanning a range of hydrophobicities (Table 2). All experimental cultures contained approximately 0.4 ng (total) of each CB congener per ml. For each congener, the aqueous concentration in each flask at the beginning of the experiment was significantly lower than the aqueous solubility (<10%), with the exception of three congeners: IUPAC no. 195 (11%), IUPAC no. 206 (44%), and IUPAC no. 209 (52%).

CB analyses. Congeners IUPAC no. 14 (3,5-dichlorobiphenyl) and IUPAC no. 198 (2,2',3,3',4,5,5',6-octachlorobiphenyl) were used as surrogate recovery standards in all samples. Congener IUPAC no. 103 (2,2',4,5',6-pentachlorobiphenyl) was used as the gas chromatography (GC) external quantitation standard. Individual congeners were purchased from AccuStandard (lot no. 024-212 [14], 081-186 [103], and 085-005 [198]), all at a concentration of 35 µg/ml in isooctane). Prior to use, anhydrous Na₂SO₄ (Fisher Scientific) was combusted for at least 4 h at 450°C and was stored in a desiccator. All solvents (hexane and acetone) were Ultra Resi-Analyzed grade (J. T. Baker, Phillipsburg, N.J.).

Ancillary analyses. Ag filters were dried overnight in a 60°C oven and were then cut into quarters, weighed, folded, and wrapped in a Sn boat (Microanalysis, Manchester, Mass.). Three of the four quarters were combusted and analyzed on a Fisons Instruments EA 1108 elemental analyzer. The average of the three quarters is presented in Table 3.

Radioactive experiments. Short (15-min) radioactive experiments were conducted with ¹⁴C-labeled 3,3',4,4'-tetrachlorobiphenyl ([¹⁴C]TCB) (IUPAC no. 77) (specific activity, 52.1 µCi/µmol [courtesy of J. Stegeman, Woods Hole Oceanographic Institution, Mass]) to better determine the protozoan uptake rate constant. This experiment was performed by using protozoan cultures with low concentrations of bacteria and was not repeated with high concentrations of bacteria. Filtrates (<5.0 µm) of the same protozoan culture were used to test retention of dissolved PCBs by Ag filters. In each experiment, an aliquot (600 ml) of either culture or filtrate was inoculated with [¹⁴C]TCB (in an acetone carrier) to a final concentration of 0.25 ng/ml (approximately 100 dpm/ml). While the concentration of radiolabel was low relative to typical radiofractionation studies, higher activities would have required higher CB mass concentrations, and the results would have not been comparable to the earlier study. No loss of sensitivity in measurements was observed, since 5 to 10 ml of solution and filters were analyzed on the scintillation counter (counts ranged from 700 to 1,400 dpm per sample). Blanks averaged 55 ± 7 dpm and were subtracted from all samples.

Numerical model. A four-box model was written to compare protozoan uptake and loss rate constants with an estimated bacterial loss rate constant. In addition, we compared the model results to the data from the bioaccumulation experiments. Initial values for the bacterial, DOC, and aqueous phases were assumed to be equal to the equilibrium values predicted by K_oc. The protozoan size class contained no PCBs at zero time. The model was run with 0.1-min time steps for 60 min. The following equations described the fluxes between pools:

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B_n, W_n, Aq_n, D_n, and P_n refer to the mass of CB congener in the bacterial, water (aqueous and DOC combined), aqueous, DOC, and protozoan pools, respectively. Superscripts refer to the case study (diff for diffusion and ing for ingestion), whereas subscripts refer to the time step number n. The other parameters are as follows: k_dep, bacterial depuration rate constant (per minute); IR, ingestion rate (cells per minute); k_for, protozoan uptake rate constant (per minute); k_rev, protozoan loss rate constant (per gram of organic carbon per minute); [P], protozoan concentration (cells per milliliter); and [OC]_P, organic carbon per protozoan (grams of organic carbon per cell). Equations 15 and 22 are used for grazing protozoa only (ingestion case study).

	RESULTS

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Bioaccumulation experiments. In the prey-limited, or diffusion, flask, bacterial cell numbers remained below the protozoan grazing threshold (10⁶ cells/ml) until the end of the experiment. In the prey-replete, or ingestion, flask, however, the prey concentration remained above the grazing threshold during the entire experiment. The protozoan population in each flask did not change significantly over the time course of the experiment (Table 3). Qualitatively, however, the health of the protozoa in the two flasks was different. It was observed microscopically that cells in the diffusion flask were very thin and contained few (<5) food vacuoles, whereas protozoa in the ingestion flask were robust with many (15 to 20) food vacuoles.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Mass of three selected congeners (18, 128, and 195) retained on a 5.0-µm-pore-size filter versus time. (A) Diffusion flask replicate 1; (B) ingestion flask replicate 1. Open symbols are used for samples in which one of the recovery standards in the filtrate was too low and only one recovery standard could be used to estimate CB content.

Organic-carbon-normalized CB concentrations. CB concentrations in the protozoan size class were normalized to organic carbon content by dividing PCBs caught on the 5.0-µm-pore-size filter (at 6 h) by the >5.0-µm particulate organic carbon (POC) concentration. Organic-carbon-normalized CB concentrations were calculated for each congener in each experimental flask and were plotted against log K_ow (Fig. 2). Data from the two treatments (diffusion and ingestion) were compared by using a Student's t test (95% confidence level) and two-way analysis of variance ( = 0.05; F = 3.58; F_crit = 4.35). For both tests, the organic-carbon-normalized concentrations for each congener were statistically indistinguishable in the two flasks, suggesting that PCBs had accumulated in the protozoan size class of each experimental flask according to organic carbon content.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Organic-carbon (OC)-normalized CB concentrations in the protozoan size class for each congener in the experimental flasks. The log of OC-normalized CB concentrations for the diffusion and ingestion flasks were plotted against the log Kow of the appropriate congener. OC-normalized concentrations were calculated by the following formula: (number of PCBs caught on a 5.0-µm-pore-size filter)/(POC > 5.0 µm). Errors were propagated from errors on both CB and POC measurements.

Rate constants for protozoan uptake and bacterial loss of PCBs. The Ag filters adsorbed a small fraction of the [¹⁴C]TCB (on average, 15.5 ± 2.0 dpm/ml filtered; n = 34, roughly 15% of the total [¹⁴C]TCB added). Background filter-associated [¹⁴C]TCBs were subtracted from the 5.0-µm-pore-size filters in the protozoan culture experiment to determine the amount associated with the protozoa (Fig. 3). The data from the short-term radioactive diffusion experiment was assumed to exhibit pseudo-first-order uptake of [¹⁴C]TCB by the protozoa (>5.0-µm size class).

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Radioactive bioaccumulation experiment. Fifty milliliters of a protozoan culture was filtered through a 5.0-µm-pore-size filter for each time point (solid lines). Three replicate experiments are shown. Aqueous CB concentrations are also shown as a function of time for three replicates (dashed lines) and are corrected for CBs associated with DOC. Organic carbon concentrations used for each replicate were as follows: experiment no. 1, 5.62 mg per liter; experiment no. 2, 4.63 mg per liter; and experiment no. 3, 9.16 mg per liter.

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View larger version (15K): [in this window] [in a new window]   FIG. 4.   Nonlinear regression fits for data from radioactive experiment. Data points are [14C]TCB aqueous concentrations as calculated from DOC concentrations and [14C]TCB (>5.0 µm). Average concentration of DOC was 5 × 106 g/ml. The aqueous pool refers to the concentration of truly dissolved CB. The analytical solution derived in the text was fit to the radioactive data by using the Levenberg-Marquardt method of nonlinear regression.

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Comparison of protozoan uptake rate and bacterial depuration rate. The numerical model was run with the protozoan rate constants derived from the regression analysis of the radioactive uptake experiment. The total activity of [¹⁴C]TCB in the model system was 100 dpm (in a 1-ml system). As written, the model did not include uptake into the bacteria. If alternate values were used for a bacterial uptake rate constant (10 × k_dep, 2 × k_dep, and 0.1 × k_dep), the relative amounts of PCBs in bacterial and protozoan pools changed, but the time required for equilibration did not (<15 min). The addition of ingestive uptake of PCBs did not change the model results for mass of PCBs within the protozoan pool or for time to equilibration (Table 4).

	DISCUSSION

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This data set demonstrates that the ingestion pathway does not contribute additional PCBs to the protozoan cell above those assimilated through diffusion. If ingestion was contributing PCBs over those accumulated via diffusion, a significant increase in organic-carbon-normalized concentration in the ingestion flasks would be observed. The observations in this study are consistent with the hypothesis that CB uptake is driven by diffusion and the steady-state cellular CB concentration is determined by the hydrophobicity of the CB congener.

The change in slope of log organic-carbon-normalized concentration versus log K_ow (log K_ow > 6.5 [Fig. 2]) has been observed by other investigators (26). They assumed that this plateau in particulate CB concentrations indicated the presence of a short-term surface adsorption constant that was independent of congener hydrophobicity (25). They hypothesized subsequent slow secondary uptake into internal cellular pools. Yet, the similarity of organic-carbon-normalized concentrations suggests that PCBs have been assimilated into all organic-carbon-containing cellular compartments (Fig. 2). Due to increased hydrophobicity, a greater percentage of higher-chlorinated congeners is associated with DOC. Low aqueous concentrations and high CB-DOC complex concentrations can potentially lead to decreased availability for diffusive uptake. DOC concentrations of approximately 7 mg per liter would be sufficient to explain lower concentration of organic-carbon-normalized CB in this system (13). This value is within the range of DOC concentrations observed in cultures of this organism (2 to 15 mg per liter).

The equivalence of organic-carbon-normalized CB concentrations in both diffusion and ingestion flasks (Fig. 2) is compelling evidence in this study that diffusion is the primary method of CB uptake for the ciliate studied here. This conclusion was further bolstered by subsequent radioactive experiments in which three replicate trials of CB no. 77 uptake into the ciliate exhibited pseudo-first-order kinetics with respect to aqueous CB concentration. In our study, equilibrium among different organic carbon pools was achieved quickly, and diffusion dominated CB uptake. Results of the numerical model supported our hypothesis in that equilibration occurred quickly (<10 min) and ingestion played a minor role in overall CB uptake.

These results could be extended to other prey species such as cyanobacteria or phytoplankton. Differences in cell composition among these various species would affect prey CB concentrations. However, the ratio of diffusive to ingestion uptake rates is so large that small variations in prey CB concentrations should not affect the general conclusions of this study. Changes in prey species will also influence protozoan clearance rates. Unless the increase in clearance rate is a factor of 10 or higher, diffusion will still out-compete ingestion and the conclusions of this study will remain unchanged.

The conclusion of this study has implications for the prediction of uptake pathways in other organisms. We have shown that diffusion dominates uptake in protozoa less than 15 µm in size. Trophic transfer studies have shown that ingestion dominates CB uptake in macroscopic organisms (22). Therefore, there must be a transition in the size spectrum of organisms between diffusion- and ingestion-dominated CB uptake. We can estimate this transitional size by comparing the ratios of diffusion and ingestion rates between species within a phylum. We assumed that cellular membrane characteristics did not change within this group of organisms, and thus, all parameters in equation 3 were held constant except those relating to cell size and ingestion and/or clearance rates.

The transitional size where uptake via diffusion and ingestion are equivalent was estimated in two ways: first, by varying feeding rates in a series of ciliate species and second, by varying clearance rates and optimal prey concentrations for the same series of species. We used feeding rates for a number of ciliate species ranging from 4 to 400 µm in diameter from Fenchel (11) and substituted them into the relationship between maximum ingestion rate and cell size from Fig. 2 of Fenchel (11): IR_max = 2.78 × 10⁴ × Vol^0.85, where IR is ingestion rate (in cubic meters per second) and Vol is cell volume (in cubic meters). After maximum ingestion rates for CR × BC × BV are substituted in equation 3, the ratio between diffusion and ingestion reduces to a function of cell radius (0.00834r^0.55). From this relationship, the cell radius at which diffusion and ingestion are equal is approximately 166 µm, corresponding to a cell diameter of 332 µm.

To obtain an independent estimate, maximum clearance rates (CR) and optimal prey concentrations (BC × BV) for each species studied by Fenchel (11) can be employed in a manner similar to that shown above. In this estimate, diffusion and ingestion were equivalent at approximately 50-µm cell radius or 100-µm cell diameter. This second estimate was consistent with field data of Axelman et al. (2), who observed that particles greater than 20 µm had lower CB concentrations than predicted from equilibrium calculations.

Both calculations presented above probably overestimate the importance of diffusion, because certain factors were not considered. After PCBs are incorporated into the cellular membrane, they are transported to other cellular compartments by diffusion and/or internal mixing. In equation 1, the cellular mixing rate was assumed to be practically instantaneous so that the rate-limiting step for cellular CB uptake was transport through the phospholipid membrane. As cell size increases, mixing within the cell will play a larger role in the overall accumulation of PCBs by lengthening the time for diffusive equilibration.

Full equilibration with internal cellular compartments will be further inhibited by cellular growth and addition of biomass, most noticeably in larger cells. This phenomenon was not observed in our cultures, but it is possible that the large surface-area-to-volume ratio of the ciliate obscured this effect. However, biomass dilution was observed in algal cultures by Swackhamer and Skoglund (28). Since algae (20 to 30 µm) were not capable of ingesting CB-laden particles, they were dependent on diffusion as an uptake mechanism and thus were potentially affected by the surface-area-to-volume dependence of diffusive equilibration. While the calculations above predicted diffusive equilibrium for organisms within this size range, it is possible that these organisms were large enough to be affected by internal equilibration barriers.

Lastly, there was no attempt in the above calculation to address the effect of cellular surface composition or increased surface area due to the presence of frustules (e.g., diatoms) or reticulopodia (e.g., foraminifera and radiolaria). These morphological features are composed of materials that are lipid poor and thus should have much lower affinity for PCBs than phospholipid bilayers. However, increases in surface area should increase the diffusive flux to the cell surface. The overall effect of these counterbalancing parameters will be species dependent. Being mindful of the limitations of these calculations, the best estimate at this time for the transitional size where diffusion is approximately equal to ingestion is a 50- to 170-µm cell radius (or a 100 to 340-µm cell diameter).