Environ. Sci. Technol. 2003, 37, 3405-3412
Phenanthrene Accumulation Kinetics in Marine Diatoms CHENG-WEI FAN† AND JOHN R. REINFELDER* Department of Environmental Sciences, Rutgers University, 14 College Farm Road, New Brunswick, New Jersey 08901
Cell surface and intracellular accumulation kinetics of phenanthrene were studied in two coastal marine diatoms. Cell surface uptake and depuration rate constants were two to three times greater in the smaller Thalassiosira pseudonana than in T. weissflogii, reflecting the 2.8-fold difference in surface-area-to-volume ratio (A/V) between the two species. However, cell surface accumulation was faster than most other environmental and biological process, supporting the assumption that cell surfaces are in equilibrium with the water. Intracellular depuration rate constants of phenanthrene were similar (0.56 d-1, 0.62 d-1) for both diatoms and for other hydrophobic organic contaminants (ca 1 d-1) with a range of hydrophobicities and chemical structures in other microalgae. Biodilution could be a significant factor in the internal accumulation of phenanthrene, as phenanthrene loss rate constants were on the order of phytoplankton growth rates in the spring and summer (1-3 d-1). Organic carbon-normalized phenanthrene bioconcentration factors were not significantly different for the two diatom species. The fraction of phenanthrene in the cell surface compartment at steadystate (x1) was directly related to the A/V of the two diatoms and a Chlorophyte microalga, showing that intracellular partitioning, which may affect phenanthrene trophic transfer, depends on phytoplankton cell size.
Introduction Phytoplankton play an important role in the fate and transport of hydrophobic organic contaminants (HOCs) in aquatic environments (1). Once accumulated in phytoplankton, HOCs can be carried to bottom sediments with settling cells or ingested by higher organisms, such as zooplankton (2), in the first step of bioconcentration up the food web. A significant portion of HOCs found in aquatic food webs may be accumulated through trophic transfer, rather than directly from water. Therefore, understanding the accumulation mechanisms of HOCs in phytoplankton is necessary for developing predictive models of HOC bioaccumulation (3-5). The partitioning of HOCs between phytoplankton and the dissolved aqueous phase has often been modeled as a thermodynamically controlled process which is driven by the concentration or fugacity gradients between phases. Observations of slow accumulation, which contradict the assumption of rapid equilibrium, demonstrate the need for a kinetic evaluation and long-term (days to weeks) * Corresponding author phone: (732)-932-8013; fax: (732)-9328644; e-mail:
[email protected]. † Current address: Environmental Change Research Center, Academia Sinica, No. 128, Academia Road, Sec. 2, Taipei 115, Taiwan. 10.1021/es026367g CCC: $25.00 Published on Web 06/26/2003
2003 American Chemical Society
accumulation experiments (4,6). Long-term studies of HOC bioaccumulation in microalgae support a two-compartment accumulation model in which the compound first rapidly partitions in the cell surface and subsequently diffuses into the cell’s interior (7-10). The extent and rates of HOC accumulation in either compartment may be influenced by dissolved organic carbon (6,11-14), temperature (15), and phytoplankton growth rate (8,9), cell size (8,16), or biochemical composition (5,6,17). A theoretical framework for evaluating the effects of many of these parameters on the accumulation of PCBs, polycyclic aromatic hydrocarbons (PAHs), and other HOCs by phytoplankton has recently been developed by Del Vento and Dachs (32). PAHs are of environmental interest because of health concerns related to their carcinogenicity and their ubiquity in aquatic environments. The steady-state bioaccumulation of PAHs in phytoplankton has been examined (5,12,13,1820), but the kinetics of PAH accumulation have not been quantified. The objectives of this study were to determine the accumulation kinetics of phenanthrene in two coastal diatom species and thus evaluate empirically the two-compartment HOC accumulation mechanism (cell surface and cell interior). Phenanthrene is one of the most abundant PAHs found in coastal aquatic environments (21) and is on the EPA’s Priority Pollutant list (USEPA water quality criteria). A secondary objective was to evaluate the importance of cell surfacearea-to-volume ratio (A/V) on the phenanthrene uptake kinetics and intracellular partitioning. Thus, the two study diatoms differed in A/V by a factor of 2. The impact of environmental factors such as water temperature and dissolved organic carbon (DOC), and biological factors such as growth rate and cell lipid content, on phenanthrene accumulation were also examined.
Materials and Methods Cultures. Coastal diatoms Thalassiosira weissflogii (clone ACTIN) and Thalassiosira pseudonana (3H) were cultured in acid-cleaned polycarbonate bottles containing synthetic seawater medium (Aquil; 22) at 18 °C, under constant illumination (200 µmol m-2 s-1). Growth was monitored by measurements of in vivo chlorophyll-a fluorescence (ex. 440 nm) in 2-mL samples of cultures using a Turner fluorometer (model 450) with seawater as a blank. Diatom cells were harvested in late exponential growth stage by filtration on 3-µm polycarbonate filters (Poretics), and then resuspended in experimental media. Chemicals. Radiolabeled PAH (9-14C-Phenanthrene, chemical purity >98%) with a specific activity of 12.4 mCi mmol-1 was purchased from Sigma. A phenanthrene stock solution of 10-4 M was prepared in 100% methanol (Fisher Scientific). Phenanthrene Accumulation. The short- and long-term accumulation of phenanthrene was measured in marine diatoms exposed to radiolabeled phenanthrene for up to 11 days. Diatoms were resuspended in 250 mL of synthetic ocean water (SOW, without addition of macronutrients, vitamins, and trace metals) and incubated at 18-20 °C in 300-mL glass BOD bottles with glass stoppers. Short-term accumulation was measured in bottles exposed to low light (PAR 60 µmol photon m-2 s-1) or no light (foil-wrapped bottles) with no observed light effect (Table 2). Long-term accumulation was measured in bottles exposed to light (PAR 200 µmol m-2 s-1) to maintain cell vitality. Light was provided by 40-W fluorescent bulbs (Philips F40T12, UV emission ) 3.5% of visible emission). Initial cell densities ranged from 4 × 104 VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Characteristics of Algal Culturesa species
dry weight (10-12 g cell-1)
T. weissflogii D. tertiolecta T. pseudonana
300 ( 30 163 ( 78d 30 ( 2
total lipid 10-12 g cell-1
organic carbon 10-12 g cell-1 wt %
wt %
30 ( 2b nd 3.5 ( 0.2c
250 ( 50 24d 15.7 ( 3.2
10 12
83 15 52
cell dimensions (µm)
A/V (µm-1)
4.5 R; 13 H 2.6 R 1.5 R; 6 H
0.59 1.16 1.66
Values are means ( 1 standard deviation (n ) 3). Cell dimensions are radii (R), and, for diatoms, cylinder heights (H). A/V is the ratio of cell surface area to cell volume. nd: not determined. b Data were determined by charring method, phosphatidyl choline as standard (23). c Data were determined gravimetrically. d A. Quigg (personal commun.). a
TABLE 2. Surface Accumulation Rate Constants and Dry-Weight-Normalized Partition Coefficientsa culture number
CT b (10-9 M)
1 2 3 5
34.6 34.6 34.9 31.5 42.0 24.9 44.5 90.8 25.8
T. weissflogii (20 ( 2 °C) 5652 807 7111 761 9262 884 4727 532 4489 504 3409 384 3450 361 2862 311 4762 633 5080 ( 2027 575 ( 208
7.01 9.34 10.48 8.88 8.90 8.87 9.55 9.21 7.53 8.86 ( 1.04
45.3c 25.1 34.1 48.7
T. weissflogii (5 ( 1°C) 2041 171 1810 163 1793 172 1690 153 1834 ( 148 165 ( 9
11.94 11.10 10.42 11.08 11.13 ( 0.62
44.6 90.0 20.7 39.0 80.8 20.0c 39.0c 78.2c
T. pseudonana (20 ( 2 °C) 11000 1406 8971 1122 11335 1436 11374 1261 11186 1257 10187 1254 9767 1175 10779 1275 10575 ( 861 1273 ( 105
7.83 7.99 7.89 9.02 8.90 8.12 8.31 8.45 8.31 ( 0.45
45.0 93.2 21.0c 41.8c 79.1c
T. pseudonana (5 ( 1 °C) 6420 517 6717 547 3824 310 5612 453 4257 315 5366 ( 1285 429 ( 111
12.41 12.28 12.31 12.40 13.50 12.58 ( 0.52
6 7 average 5 7 average 1 2
average 1 2 average
kad (m3 kg-1 d-1)
kdes (d-1)
Ks (m3 kg-1)
a Means and standard deviations also shown. b C is the dissolved T phenanthrene concentration at the start of each experiment. c Conducted in complete darkness.
to 6 × 104 and 7 × 105 to 9 × 105 cells mL-1 for T. weissflogii and T. pseudonana, respectively (corresponding biomass of 12-18 mg L-1 and 20-27 mg L-1 for T. weissflogii and T. pseudonana, respectively). Uptake experiments were begun with the addition of 50-200 µL of 14C-phenanthrenemethanol stock solution, yielding final phenanthrene concentrations of 2 × 10-8 to 9 × 10-8 M. Control bottles receiving methanol only indicated that the carrier of methanol has no negative effect on the growth of experimental cultures. Sampling times ranged from 1 min to 11 d. Short-term uptake sampling was conducted intensively in the first 4 - 5 h of exposure and long-term uptake sampling was conducted daily after the short-term sampling. At each sampling time, cell samples were collected by filtering 5-10 mL of cells on glass-fiber filters (Whatman, GF/F), and rinsed with 5 mL of SOW. Filtering time was approximately 5 s. Unfiltered samples (1 mL) were collected simultaneously for total radioisotope 3406
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TABLE 3. Intracellular Accumulation and Depuration Rate Constants, Dry-Weight-Normalized Intracellular Partition Coefficients (Ki), and Overall Dry-Weight-Normalized Bioconcentration Factors (BCF) of Phenanthrene in Marine Diatomsa CT a ku (10-9M) (m3 kg-1 d-1)
kd (d-1)
Ki (m3 kg-1)
BCF (m3 kg-1)
T. weissflogii 6
24.9 44.5 90.8
17.1 0.69 24.6 33.5 17.3 0.64 27.0 36.6 19.1 0.54 35.6 44.8 17.8 ( 1.10 0.62 ( 0.08 29.1 ( 5.8 38.3 ( 5.9
20.7 39.0 80.8
5.66 0.68 8.3 16.2 4.55 0.58 7.9 16.9 3.72 0.41 9.1 17.9 4.64 ( 0.97 0.56 ( 0.14 8.4 ( 0.6 17.0 ( 0.9
average
T. pseudonana 2
average
a Long-term phenanthrene accumulation experiments were conducted in continuous light at 18 °C. b CT is the dissolved phenanthrene concentration at the start of each experiment.
analysis. Collected samples were put into 7-mL scintillation vials, and 5 mL of scintillation fluor (Scintisafe 30%, Fisher Scientific) was added. All vials were held at room temperature for more than 4 h before quantifying radioactivity by scintillation counting using a Beckman LS 6500 liquid scintillation counter with external standard ratio correction of quenching. The concentrations of 14C-phenanthrene were calculated using the specific activity of the chemical and measured dpm. Short-term phenanthrene accumulation was measured in cells from seven different parent cultures for T. weissflogii and from two different parent cultures for T. pseudonana (Table 2). Different parent cultures were grown up to months apart. Within cultures, short-term uptake replicate experiments (n ) 2-6) were performed simultaneously. Long-term phenanthrene accumulation was measured in triplicate simultaneous experiments from one parent culture for each diatom species (Table 3). Parent cultures are designated by number in Tables 2, 3, and 5. Short-Term Treatments. The effects of DOC concentrations and sources and temperature on short-term phenanthrene accumulation were examined in T. weissflogii and T. pseudonana. Three DOC sources were used, including natural seawater, humic acid (Aldrich), and diatom exudate. The natural seawater was obtained from Cheesequake Creek (Raritan Bay, New Jersey) and Sandy Hook (New Jersey). Seawater from the two locations was filtered through 0.2-µm polycarbonate filters (Poretics) to remove suspended particles. Cheesequake seawater was diluted 1:1 with SOW. Various concentrations of humic acid (10-40 mg mL-1) were prepared by dissolving humic acid into SOW followed by GF/F filtration. Diatom exudate was obtained from the 0.2µm filtrate (polycarbonate filters) of late exponential growth stage (>3 d) T. weissflogii cultures. Short-term phenanthrene accumulation was measured in the presence and absence of the 0.2-µm filtrate. DOC was analyzed with a Shimadzu 5000
total organic carbon analyzer. Temperature-effect experiments were performed at 20 ( 2 °C and 5 ( 1 °C. Control bottles were defined as the SOW experiment at 20 °C. Algal Characteristics Measured. Dry weights were determined by weighing dried cells (60 °C) on preweighed 3-µm polycarbonate filters (Poretics). Total lipid and particulate organic carbon (POC) samples were collected on precombusted GF/F filters (400 °C, 2 h). Total lipid samples were determined gravimetrically or by the charring method (23). Particulate organic carbon samples were analyzed at the Chesapeake Biological Laboratory using an Exeter Analytical CE-440 elemental analyzer. Diatom cell volumes and surface areas were calculated on the basis of 20 measurements of cell dimensions with a compound microscope assuming a cylindrical cell shape. The cell density of each sampling time was determined by counting Lugol’s solution-preserved cell samples with a Fuchs-Rosenthal counting chamber. Bioaccumulation Model. The bioaccumulation of phenanthrene in phytoplankton is conceptually modeled as accumulation into two compartments: cell surface and cell interior. The accumulation of phenanthrene by diatom cells can be described with a first-order, two-compartment kinetic model, using separate surface and interior uptake and depuration rate constants. Thus, the experimental rate of phenanthrene accumulation in diatom cells is given by
dCP ) (ku + kad)D × Wd × Cw - (kdes + kG) × dt Cp,s - (kd + kG)Cp,i (1) where Cp is the total activity of radiotracer in cells (dpm mL-1, Cp ) Cp,s + Cp,i), Cp,s is the activity of radiotracer in the surface compartment (dpm mL-1), Cp,i is the activity of radiotracer in the interior compartment (dpm mL-1), Cw is the dissolved activity of radiotracer (dpm mL-1), D is the cell density (cell m-3, D is a function of time), Wd is the dry weight of diatom cells (kg cell-1), ku is the uptake rate constant to the interior (m3 kg-1 d-1), kd is the depuration rate constant from the interior (d-1), kad is the adsorption constant to the surface (m3 kg-1 d-1), kdes is the desorption constant from the surface (d-1), kG is growth rate constant (d-1), and t is the exposure time (d). At steady-state, the partition coefficients in the cell surface and cell interior, Ks and Ki, and the overall kinetic bioconcentration factor (BCF), a quantitative measure of bioaccumulation, are calculated using the following equations on a dry weight basis:
Ks ) Ki )
kad kdes
(2)
ku kd
(3)
BCF ) Ks + Ki )
Cp∞ Cw∞
(4)
where the partition coefficients and bioconcentration factor are in units of m3 kg-1, Cp∞ is the activity of radiotracer in the cells at steady-state (dpm mL-1), and Cw∞ is the dissolved activity of radiotracer at steady-state (dpm mL-1). The fraction of total steady-state phenanthrene in the cell surface compartment, x1, was calculated using the following equation:
x1 )
Ks Ks + K i
(5)
Determination of kad and kdes. Given the assumptions that during the short-term exposure (1) the sum of Cp and
FIGURE 1. Short-term 14C-phenanthrene accumulation in T. weissflogii at (A) 20 °C with 3.46 × 10-8 M initial dissolved phenanthrene and (B) 5 °C with 4.53 × 10-8 M initial dissolved phenanthrene. Cw is constant, (2) the accumulation in the cell interior is negligible, (3) the initial Cp is zero, and (4) the cell density is constant (the kG is zero), eq 1 can be simplified and solved as
Cp ) C t ×
D × Wd × kad + (1 - e-(kad‚D‚Wd kdes)t) D × Wd × kad + kdes
(6)
where Ct is the total activity of radiotracer (Ct ) Cp + Cw, dpm mL-1). Short-term phenanthrene accumulation data were fit to eq 6 in the form of y ) a(1 - e-bt) using SigmaPlot to estimate the rate constants kad and kdes. Determination of ku and kd. Long-term phenanthrene accumulation results were used to estimate the rate constants, ku and kd, by solving eq 1 using a numerical method. In this method, it was assumed that long-term steady state was reached after 7 days and therefore that the overall BCF can be estimated using cellular phenanthrene concentrations measured at 11 d according to eq 4. The overall BCF was used to calculate Ki (see eq 4). Using Ki and eq 3, kd was eliminated from eq 1. Equation 1 was then iterated using measured cellular radioactivities (Cp), cell densities (D), and growth rates (kG) for each time point and previously determined adsorption and desorption rate constants (kad and kdes) to determine the long-term uptake rate constant, ku. The long-term depuration rate constant kd was then calculated with eq 3.
Results and Discussion Diatom Characteristics and Growth. The dry weights and lipid and organic carbon contents of Thalassiosira weissflogii and T. pseudonana are shown in Table 1. Because these cellular parameters did not vary during long-term phenanthrene uptake experiments (data not shown), cell number densities were used to monitor diatom biomass. Cell densities were constant during short-term experiments, but increased during the long-term accumulation experiments (Figures 3 and 4). Growth rate constants derived from cell density measurements in long-term accumulation experiments were 0.14-0.22 d-1 and 0.08-0.36 d-1 for T. weissflogii and T. pseudonana, respectively, during the first 3 d and gradually decreased to less than 0.01 d-1 thereafter. VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Short-term 14C-phenanthrene accumulation in T. pseudonana at (A) 20 °C with 3.90 × 10-8 M initial dissolved phenanthrene and (B) 5 °C with 4.18 × 10-8 M initial dissolved phenanthrene.
FIGURE 4. Long-term 14C-phenanthrene accumulation in T. pseudonana at 18 °C with 2.07 × 10-8 M initial dissolved phenanthrene.
TABLE 4. Phenanthrene Bioconcentration Factors and Percent Surface Accumulation (x1) in T. pseudonana, T. weissflogii, and D. tertiolectaa species
x1 (%)
BCF (m3 kg-1)
BCFoc (m3 kg-1)
BCFlipid (m3 kg-1)
T. weissflogii D. tertiolecta T. pseudonana
23 36 49
38.3 ( 5.9 4.6 ( 1.1 17.0 ( 0.9
46.1 ( 12 32.2 ( 16 32.7 ( 7
383 ( 74 nd 141 ( 14
a BCF values are dry-weight-normalized partition coefficients. BCF oc and BCFlipid are normalized to cellular organic carbon or lipid content, respectively. Values are means ( 1 standard deviation (n ) 3). nd: not determined.
FIGURE 3. Long-term 14C-phenanthrene accumulation in T. weissflogii at 18 °C with 2.49 × 10-8 M initial dissolved phenanthrene. Phenanthrene Accumulation Kinetics. Representative phenanthrene accumulation curves are shown in Figures 1-4. Total radioactivity (phytoplankton plus dissolved phase) in the media was constant during the short- and long-term exposures (Figures 1A-4A), and chemical analysis by HPLC (24) of phenanthrene in long-term (11 d) incubations with T. weissflogii in SOW showed no degradation of this PAH. Phenanthrene accumulation in both T. weissflogii and T. pseudonana cells reached a short-term steady state within 10 min at 20 °C (Figures 1A and 2A), and within approximately 30 min at 5 °C (Figures 1B and 2B). The short-term steady state, which was stable for several hours, was followed by a period of slower uptake of phenanthrene (Figures 3 and 4), leading to a long-term steady state in 5 to 7 days. 3408
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Partition Coefficients and Overall Bioconcentration Factors. The observation of two phases of phenanthrene accumulation in T. weissflogii and T. pseudonana (Figures 1-4) is consistent with a two-compartment uptake mechanism in which phenanthrene initially accumulates in the cell surface and subsequently accumulates in the cell interior. The cell surface phenanthrene partition coefficients (Ks) of the two diatoms measured at 20 °C were not significantly different (t test, p > 0.1; Table 2), but the cell interior partition coefficient (Ki) was significantly higher (p < 0.01) in T. weissflogii than in T. pseudonana (Table 3). As a result, the average overall, dry weight-normalized BCF in T. weissflogii was twice that in T. pseudonana (significant difference, p < 0.01; Table 3). Organic carbon normalization reduced much of the difference in bioconcentration factors between the two diatoms, but lipid normalization resulted in BCFs that differed by a factor of 2.7 (Table 4). Cell surface partition coefficients, Ks, were 3% to 17% lower in the presence of humic acid, estuarine DOC, or diatom exudate than those measured in DOC-free controls (Table 5). Although inter-culture variability in the value of Ks for T. weissflogii measured in SOW was observed, the trends in the effects of DOC on surface accumulation were consistent across cultures. Only the Ks values measured at the highest humic acid concentration and those with 100% Cheesequake Creek or Sandy Hook seawater were significantly different from those of the SOW controls at the 1 sigma level. Natural
TABLE 5. Cell Surface Accumulation Rate Constants and Dry-Weight-Normalized Partition Coefficients of Phenanthrene in T. weissflogii in the Presence of Humic Acid (HA), Estuarine Dissolved Organic Carbon from Cheesequake Creek or Sandy Hook, NJ, or Dissolved (0.2-µm Filtrate) Exudates from a 3-Day-Old Diatom Culturea experimental conditions culture
media
1
HA-40 mg L-1 HA-20 mg L-1 HA-10 mg L-1 SOW Cheesequake Sandy Hook SOW Cheesequake 50% Cheesequake SOW 100% exudate SOW
2 3 4
CT b kad kdes Ks (10-9 M) (m3 kg-1 d-1) (d-1) (m3 kg-1) 40 36 37 35 34 33 35 37 39 35 37 34
5320 5347 5676 5652 4489 6314 7111 5492 6991 9262 nd nd
840 824 882 807 576 773 761 617 689 884 nd nd
6.34* 6.49 6.43 7.01 7.79* 8.17* 9.34 8.90* 10.1 10.5 10.9c 11.3c
a Short-term phenanthrene accumulation experiments were conducted at 20 °C. nd: not determined. Ks values that were significantly (1 sigma) lower than those of the SOW controls are indicated with asterisks. b CT is the dissolved phenanthrene concentration at the start of each experiment. c Ks values were calculated as the measured cellular phenanthrene divided by dissolved phenanthrene.
estuarine DOC from Cheesequake Creek had the largest effect on cell surface phenanthrene accumulation, causing a 1.6 to 1.7-fold drop in kad, whereas diatom exudates had very little effect (Table 5). The fractions of steady-state phenanthrene accumulated in the cell surface compartment (x1) were estimated using the average Ks and Ki values according to eq 5 (Table 4). The x1 value in T. weissflogii (23%) was about half that in T. pseudonana (49%). Kinetic Parameters and Model Results. The average phenanthrene desorption rate constants, kdes, in T. weissflogii and T. pseudonana (Table 2) reflect the rapid cell surface exchange kinetics observed in these experiments (Figures 1 and 2). Biomass-normalized, cell surface exchange rate constants (kad, kdes) were two times greater in T. pseudonana than in T. weissflogii at 20 °C and nearly three times greater at 5 °C (both significant differences, p < 0.01; Table 2). Phenanthrene desorption rate constants decreased by factors of 3.5 and 3 in T. weissflogii and T. pseudonana, respectively, with a drop in temperature from 20 to 5 °C (Table 2). Over this temperature range, phenanthrene adsorption rate constants, kad, in the two diatoms did not decrease as much as kdes. As a result, the average cell surface partition coefficients, Ks, increased by factors of 1.25 and 1.50 for T. weissflogii and T. pseudonana, respectively. For T. weissflogii, short-term kinetic constant results showed greater interculture than intra-culture variability. For example, kad had a coefficient of variation (CV ) SD/mean) of 4-10% for within-culture replicate experiments, but a CV of 37% among different parent cultures. This variability likely reflects physiological differences among the algal cultures. However, because the effect of these differences was an increase or decrease in both kad and kdes, Ks was less affected (Table 2). Phenanthrene depuration rate constants for the cell interior compartment, kd (Table 3), were much lower than those for the cell surface and were not significantly different (p > 0.5) in the two diatoms (0.62 d-1 and 0.56 d-1 for T. weissflogii and T. pseudonana, respectively). As a result of the higher phenanthrene Ki in T. weissflogii than T. pseudonana, the internal compartment uptake rate constant (ku) of the former was nearly four times that of the latter (Table 3). Long-term uptake and depuration rate constants varied by
FIGURE 5. Measured (b) and modeled (lines) cellular phenanthrene concentrations in T. weissflogii over (A) 1 h and (B) 11 d of exposure at 18 °C with an initial dissolved phenanthrene concentration of 9.08 × 10-8 M. between 6 and 25%. This intraculture variability reflects accumulated error associated with measuring dry weight (10%), cell density (5%), and radiological counting (
