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Influence of Nitrogen Status on the Bioconcentration of Hydrophobic Organic Compounds to Selenastrum capricornutum
Ecotoxicology and Environmental Safety 45, 33±42 (2000) Environmental Research, Section B Article ID eesa.1999.1818, available online at http://www/idealibrary.com on
In£uence of Nitrogen Status on the Bioconcentration of Hydrophobic Organic Compounds to Selenastrum capricornutum Bent Halling-Sùrensen,*,1 Niels Nyholm,{ Kresten Ole Kusk,{ and Eva Jacobsson{ *Royal Danish School of Pharmacy, Section of Environmental Chemistry, Universitetsparken, DK-2100 Copenhagen é, Denmark; {Institute of Environmental Science and Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark; and {Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Received July 29, 1998
the same species, in relation to nutritional status. Bioconcentration of HOCs by algae has been described in several publications (Rice and Sikka, 1973; Urey et al., 1976; Casserly et al., 1983; Richer and Peters, 1993; Swackhamer and Skoglund, 1993; Stange and Swackhamer, 1994; Koelmans and JimeÂnez, 1994), but the level of knowledge about algae is limited compared to that available for ®sh. Models predicting the transport and fate of HOCs in the environment rely on the HOC octanol/water partition coecient (Kow), a physicochemical property representing a chemical's lipophilicity. Bioaccumulation is quanti®ed in terms of the bioaccumulation factor (BAF), which is the ratio of HOC concentration in the organism's biomass to the HOC concentration dissolved in water, expressed in equivalent units. At true equilibrium, this ratio is called the biological concentration factor (BCF). Thus, when the system is at equilibrium, the BAF equals the BCF. A proportional relationship between BCF and Kow has been suggested for several aquatic organisms by several investigations (Veith et al., 1979; Mackay, 1982). Veith et al. (1979) showed that this correlation could be re®ned by lipid normalization of BCF values. However, Swackhamer and Skoglund (1993) found that for algae this linear correlation between BCF for HOCs and Kow did not function. Stange and Swackhamer (1994) found that normalization to total lipid worked for lower substituted PCB congeners (Kow 5 106.5). They also showed that by normalizing to phospholipids, BCF values converged for Kow 4 106.5. These ®ndings led to the hypothesis that PCBs with Kow 4 106.5 associate mainly with the cell membrane, where most of the phospholipids occur. Chessells et al. (1992) suggested that the curved relationship between the solubility of HOCs in biotic lipid (SL) and Kow probably accounted for the decreased bioconcentration of superhydrophobic compounds, due to the fact that
Changes in algal nitrogen status that increase algal lipid content also aect the bioconcentration of hydrophobic organic compounds (HOCs). Bioconcentration factors (BCFs) for several HOCs increased up to nine times as the total algal lipid content of the green algae Selenastrum carpricornutum increased from 17 to 44% of the algal dry weight as a consequence of nitrogen starvation. An increase in total lipid from 17 to 44% should theoretically increase the BCFs by a factor of 2.6. BCFs for PCB 31, PCB 49, PCB 153, and DDT increased with maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively, thus more than theoretically predicted from the lipid normalization of BCFs obtained at exponential growth phase (17% total lipid for S. carpricornutum), whereas BCFs for PCB 105, phenanthrene, and 4-chloroaniline increased at 44% lipid content, only by factors of 1.5, 1.5, and 2.5, respectively, and thus less than or equal to the theoretical prediction. Lipidclass normalization of BCFs did not reveal signi®cant information beyond that available from normalizing to total lipid. # 2000 Academic Press
Key Words: nutritional status; HOCs; algal bioconcentration; lipid normalization; PCBs; S. carpricornutum.
INTRODUCTION
Phytoplankton are signi®cant as primary producers that sustain the pelagial food chains in aquatic ecosystems and can in¯uence the speciation and transport of pollutants in natural waters (Thomann et al., 1992; Millard et al., 1993). More speci®cally the lipid content of algae is known to be the major factor in¯uencing the bioconcentration of HOCs (Geyer et al., 1984; Manthey et al., 1993) and algal lipid status may vary considerably, both between species and for 1
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34
HALLING-SéRENSEN ET AL.
superhydrophobic compounds are much more soluble in octanol than in lipids. In general, nitrogen-de®cient microalgae have larger cell volumes and contain more dry matter with a higher proportion of fat than actively growing algae (Fogg, 1959). Exponential growth ceases when the nitrogen supplied is exhausted but, when the growth rate decreases, assimilation of carbon continues at a reduced rate for some time. The net result is an increase in total biomass or dry weight of algal material, by up to a factor of two to three (Nyholm, 1976). While the nitrogen content declines, fats or carbohydrates accumulate in the cells. Several sets of data illustrating this phenomenon have been published (Spoehr and Milner, 1949; Ketchum and Red®eld, 1949; Collyer and Fogg, 1955), as has a mathematical description of nitrogen-limited algal growth (Nyholm, 1976). Nitrogen-starved Neochloris oleoabundus increased its lipid content to 35±54% of cell dry weight (Thornabene et al., 1983). The lipid levels of Scenedesmus obliquus and Chlorella vulgaris reaching as much as 45% of the biomass were found when growing slowly in a medium containing low nitrogen concentrations (Piorreck et al., 1984). N-de®cient algal cells can be prepared in the laboratory in several ways: (1) cultures can be grown with a limited amount of available N so they become nitrogen-de®cient after a period of growth (external growing on internal nitrogen); (2) N-sucient cells can be transferred to a Nfree medium thus forcing them to grow only through the use of internal N reserves; or (3) a chemostat culture with N limitation can be set up (Nyholm, 1976, 1977). Algal lipids may roughly be fractionated into three major classes: glycolipids, neutral lipids, and phospholipids (see Thompson, 1996). Glycolipids are found intracellularly, whereas neutral lipids are major constituents of membrane lipids. Phospholipids are associated mainly with the cell membrane (Stange and Swackhamer, 1994). A review of lipids in green algae is provided in Thompson (1996). The objective of this study was to determine if increases in algal total lipid content due to changes in nutritional status would aect the ability of algae to bioconcentrate HOCs. In our study bioaccumulation is predicted to equal bioconcentration because an equilibrium between the algal phase and the water phase was expected to be achieved quickly. So BAF is predicted to equal BCF. The term BCF is therefore used on all graphs. In the present study, the BCF of six HOCs ranging in lipophilicity from log Kow = 4.57 to log Kow = 6.90 was measured in the green algae Selenastrum capricornutum. The BCF measurements were made at dierent algal lipid contents achieved by manipulating the extent of N starvation. A compound with relatively lower lipophilicity, 4-chloroanilin (log Kow= 2.78), was used as negative control. The other six compounds were phenanthrene, 2,4',5-trichlorobiphenyl
(PCB 31), 2,2',4,5'-tetrachlorobiphenyl (PCB 49), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), 2,2',4,4',5,5'hexachlorobiphenyl (PCB 153), and 4,4'-DDT. THEORY
Algal bioconcentration of hydrophobic chemicals can be described by a ®rst-order one-compartment kinetic model, such as that for ®sh (Branson et al., 1975). Growth of the algae should be included, since algae are fast-growing organisms with a growth rate of days (Sijm et al., 1995): dCalg =dt k1 Cw ÿ k2 Calg :
1
where Calg is the concentration in the algae (mg/kg), k1 is the uptake rate constant (l/kg * d), Cw is the concentration of the freely dissolved chemical in water (mg/liter), k2 is the elimination rate constant (1/d ), and m is the growth rate constant (1/d ). At steady state, when dCalg/dt = 0, the BCF (l/kg) can be derived from Eq. [1]: BCF
k1 Calg : k2 Cw
2
The lipid-normalized bioconcentration factor, BCFlipid (l/ kg), can then be derived from Eq. (2), knowing the fraction (weight or percent) of total algal lipid content, a. BCFlipid
BCF
3
MATERIAL AND METHODS
Chemicals and Radiochemicals Information about the radiolabeled compounds used in this investigation is given in Table 1. [UL-14C]2,4',5Trichlorobiphenyl (PCB 31, 19 mCi/mol), [UL-14C]2,2', 4,5'-tetrachlorobiphenyl (PCB 49, 25 mCi/mol), [UL-14C] 2,3,3',4,4'-pentachlorobiphenyl (PCB 105, 26.5 mCi/mol), and [UL-14C]2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153, 12.6 mCi/mol) were obtained from the Department of Environmental Chemistry at the Wallenberg Institute, Stockholm University (Stockholm, Sweden), with a purity of 498%. The four PCBs were synthesized in accordance with methods described elsewhere (Sundstrùm, 1974; Bergman and Wachtmeister, 1977; Bergman et al., 1981). [UL-14C]4-Chloroanilin, [9-14C]phenanthrene, and [UL-14C]4,4'-DDT-Ring were obtained from Sigma Chemical Co. (St. Louis, MO) at a purity of 498%. All compounds were stored at 48C and diluted in Uvasol grade
35
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
TABLE 1 List of Substances Used in the Experimental Studies
[14C] Substancea 14
Speci®c activity (mCi/mmol)
[UL- C] 4-Chloroanilin [9-14C] Phenanthrene [UL-14C] 2,4',5-Trichlorobiphenyl (PCB 31)
25.5 8.3 19
[UL-14C] [UL-14C] [UL-14C] [UL-14C]
25 26.5 12.6 10
2,2',4,5'-Tetrachlorobiphenyl (PCB 49) 2,3,3',4,4'-Pentachlorobiphenyl (PCB 105) 2,2',4,4'5,5'-Hexachlorobiphenyl (PCB 153) 4,4'-DDT-Ring
Water Solubility (mg/liter)
Log Kow
b
2.78 4.57d 5.67 f
0.016g 0.009 0.001e 0.006h
5.85 f 6.65 f 6.9 f 6.19 i
3900 1.2d 0.31e
c
Source Sigma Chemical Co. (St. Louis, MO) Sigma Department of Environmental Chemistry, Wallenberg Institute, Stockholm University (Stockholm, Sweden) Wallenberg Institute Wallenberg Institute Sigma Sigma
Note. Data are obtained from the following sources; apurity 498%, based on manufacturer's standards and tests performed at the Department of Environmental Chemistry, Wallenberg Institute, Stockholm, Sweden; bKilzer et al. (1979); cGeyer et al. (1981); dMackay et al. (1993); eBurkhard et al. (1985); fHawker and Connell (1988); gShiu and Mackay (1986); hMackay (1991); iBiggar and Riggs (1974).
acetone (min 99.9%; Merck KGaA, Darmstadt, Germany) to a working concentration of about 1000 dpm/100 ml.
and drying to constant weight at 1058C and equilibration in an exsiccator. Con®rmation of N Limitation
Algal Cultures Unialgae cultures of S. capricornutum were obtained from the Norwegian Institute of Water Research culture collection. Nonaxenic populations of these cells were maintained in log-phase development, in ISO medium (ISO 8692, 1989) at 22+18C, under a light intensity of 6 klux supplied by cool-white ¯uorescent tubes. Cells in the culture were kept in suspension by use of magnetic stirrers. The algae were grown in six 10-liter glass ¯asks (one for each study day). An increase in algal lipids was obtained by allowing growth after N in the media had been depleted. Algal biomass from each 10-liter ¯ask (between 300 and 900 mg algal dry wt) was harvested and used for both lipid analysis and bioaccumulation studies, 1 to 24 days after the test medium had become N-de®cient. Day 0 of the experiment was de®ned as the last day of exponential growth, day 1 is the ®rst day after nitrogen exhaustion in the media, day 3 is the third day, and so forth. On days 1, 3, 7, 13, 19, and 24 after the start of the study, one 10-liter ¯ask each was used for experiments. The algal concentration was quanti®ed as total volume (mm3/ml) and cell numbers per liter of both unwashed and washed algal samples, by use of an electronic particle counter (Coulter Multisizer, 70-mm capillary tube) using IsotoneR as electrolyte. Triplicate measurements were made on each sample. The relationship between cell numbers and algal dry weight was determined for each of the six 10-liter ¯asks by ®ltering 100-ml washed algal samples in triplicate through a dried and pre-weighed GFC ®lter (Whatmann, 1.2 mm)
Nitrogen de®ciency in the media was con®rmed by analyzing the media for the level of Kjeldahl nitrogen during the exponential growth phase and during the study period (day 1 to day 24). As a positive control a culture incubated at the same time under the same conditions as the six other ¯asks, but with a higher N concentration (N=50 mg/liter), was used. Accumulation Pro®le Studies Before the lipid-depended bioaccumulation studies were started, accumulation pro®les of phenanthrene and the four PCBs were measured over time, in a separate experiment to allow us to estimate the time needed to achieve steady-state conditions. To minimize possible sorption by nonalgal particulate matter (e.g., bacteria), S. capricornutum cells (exponential growing from the 10liter ¯ask at day 0) were concentrated from the growth medium by gentle centrifugation. The spent medium overlying the cell pellet was discarded, and the algal cell pellet was resuspended in fresh N-free growth medium (to minimize cell growth). The process was repeated three times. For each compound triplicate samples of 100 ml washed algal suspension were exposed under a shaking procedure (100 rpm) in 250-ml open Erlenmeyer ¯asks. At various time intervals (0 min, 2 min, 5 min, 10 min, 30 min, 1 h, and 2 h) (except for PCB 31 data only up to 1 h) 10-ml suspensions of algal cells were separated by ®ltration with a GFC ®lter (Whatmann, 1.2 mm) into algal biomass and medium and both were analyzed for the content of
36
HALLING-SéRENSEN ET AL.
compound. Results from the uptake study were used to evaluate the study time to be used in the bioaccumulation study below to ensure a steady-state situation. Uptake experiments for DDT were not performed in this study as they were made by Rice and Sikka (1973). Bioaccumulation Studies at Dierent Algal Lipid Content For the bioaccumulation experiments S. capricornutum cells at an initial cell density of 106 cells per milliliter were used in order to avoid any intervention of changes in cell density with the BCF. The same algal washing procedure was used as described above. The bioaccumulation studies were performed by adding the radiolabeled test compound (1000 dpm per milliliter of algal sample) to 50 ml prewashed algal sample, as explained above on each of the indicated days. Triplicate measurements were made for all seven compounds each day. Prewashed algal suspension were shaken for 2 h with the test compound to permit phase preference. The uptake experiment described above showed that steady state was achieved within 30 to 60 min for all six compounds why a prolonged study period of 2 h was used in all bioaccumulation studies with N-de®cient algae to ensure steady-state situations. The phases (algae and media) were then separated by ®ltrating 10 ml of the suspension through a GFC ®lter (25 mm). Radioactivity was quanti®ed in both algae and water phases by liquid scintillation counting (Tri-Carb 2000 liquid scintillation counter, United Technologies Packard). The amount of toxicant associated with algal biomass was determined by dissolving the ®lter and biomass using 5 ml Ecoscint A (National Diagnostics). The radioactivity of the ®ltrate was determined using 10 ml Optiphase HiSafe and 10 ml sample. For background correction due to sorption of compounds retained in the GFC ®lter, blank samples (without algal culture) were spiked with the radiolabeled compounds and 10 ml was ®ltered through a GFC ®lter and analyzed in the same way as the algal samples.
One-dimensional polar-lipid separation was achieved using a solvent system consisting of hexane and isopropanol (8:2 ratio). Lipids were visualized by using a spray of 8anilino-1-naphtalene sulfonic acid (0.2% in methanol), followed by inspection under UV light. Lipids were identi®ed by comparison with authentic standards. The individual lipid classes, including glycolipids, neutral lipids, and phospholipids, were quanti®ed by scraping the lipid bands of each class from the TLC plate and resuspending them in chloroform. The chloroform was evaporated at low temperature from preweighed aluminum foil cups and the lipids were quanti®ed gravimetrically. All lipid analyses were accompanied by two blanks to correct (mean value was used) for nonlipid dry weight contribution. RESULTS
Con®rmation of N Limitation Before N depletion was evident S. carpricornutum grew at a mean rate of 1.6 day71 (based on cell numbers for the six 10-liter ¯asks) at 22+18C. After N depletion had become evident, the growth rate of alga decreased gradually to 50.1 day71. Nitrogen de®ciency in the media was also con®rmed by analyzing the media for the level of Kjeldahl nitrogen which was below 1 mg/liter N after day 1. The supposition of N limitation in the N-free cultures was con®rmed by the positive control at an N concentration of 50 mg/liter, which sustained high rates of cell division. Lipids Lipid content of S. capricornutum increased with the onset of N depletion by a factor of 2.6 (from 17% on day 0 to 44% on day 7, on a dry weight basis) (Fig. 1). After day 13, lipid content declined to about 10% (Fig. 1).
Extraction and Analysis of Algal Lipids Lipids were extracted using the method of Gardener et al., (1985) modi®ed to accommodate our experimental design and laboratory facilities. The modi®cations included use of isopropanol rather than methanol, to avoid lipid degradation by endogenous lipolysis. Non-lipid material was removed by twice mixing the extract with an equal volume of 0.9% NaCl in water; phase separation was then achieved using a 250-ml separation funnel. The chloroform phase was dried with Na2SO4 and the volume reduced to about 500 ml by evaporation. Analytical TLC was carried out using silica gel plates precoated with a layer of 0.5-mmthick silica gel G (E. Merck No. 5721, Darmstadt, Germany).
FIG. 1. Relationship of ( ) percentage total lipid of algal dry weight, (*) average algal cell volume, and (~) average algal cell dry weight during the study period.
37
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
TABLE 2 Distribution of Neutral, Glyco-, and Phospholipids (Given as Percentages of Total Lipid Content) for S. capricornutum during the Study Study day Ratio
0 ND
1 24:48:28
3 46:13:41
7 37:54:9
13 ND
19 50:27:23
24 38:24:38
Note: ND not determined.
These results, on day 0, are in accordance with ®ndings by Swackhamer and co-workers, who found the average total lipid content of S. capricornutum in exponential growth phase as 19.5%. Lipid-class distribution was measured on each sample except at day 13. Results are shown as ratios among the lipid classes in Table 2. The neutral lipids remained fairly constant during the incubation period. The percentage of glycolipids (the internal lipids) gained a maximum at day 7. During the incubation period with high glycolipids the level of phospholipids associating mainly with the cell membrane was low.
In accordance with Eq. (3), an increase in total lipid content from 17 to 44% should theoretically increase the BCFs by a factor of 2.6. Figure 3 shows that BCFs for PCB 31 (A), PCB 49 (B), PCB 153 (D), and DDT (E) increased at maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively,
Uptake and Partitioning of HOCs into Algal Phase For all compounds the uptake was fast and a steadystate level was reached within 60 min. Figures 2A to 2E show the bioaccumulation pro®les for the four PCBs and phenanthrene. Uptake experiments for DDT were not performed in this study because they were performed by Rice and Sikka (1973). Rice and Sikka found that a steady state for DDT was reached within 2 h as demonstrated for several algal species. Our results dier from these reported by Swackhamer and co-workers, but are in accordance with other ®ndings reported in the literature (e.g., Rice and Sikka, 1973; Urey et al., 1976; SoÈdergren and Gelin, 1973). Figures 2A to 2E show that depuration of the PCBs and phenanthrene occurred due to evaporation (open test vessels) from the aqueous phase after steady state was obtained. Calculations showed no changes in phase preference during depuration because BCF was constant. Bioaccumulation Factors All BCFs in the present study were calculated on a dry weight basis. Figures 3A to 3G show both the experimental and theoretical relationships between the algal lipid content and log BCF obtained in the present study. Table 3 shows all data (results from day 19 and day 24 were not presented in ®gures; see below) with corresponding 95% con®dence limits. Equation (3) was used to calculate theoretical predicted lipid-normalized BCFs (lines in Fig. 3) based on the lipid content at exponential growth phase (17% total lipid for S. capricornutum). Results were compared with the experimental values (dots in Fig. 3) obtained in the present study.
FIG. 2. Partitioning of substances into algal phase over the time course of experiments with S. capricornutum of (A) PCB 31, (B) PCB 49, (C) PCB 105, (D) PCB 153, and (E) phenanthrene.
FIG. 3. Relationship between the biological concentration factors (BCFs) and total lipid content of S. capricornutum related to algal dry weight for (A) PCB 31, (B) PCB 49, (C) PCB 105, (D) PCB 153, (E) DDT, (F) phenanthrene, and (G) 4-chloroaniline. (^) Experimental values with corresponding 95% con®dence limits. (Ð) Theoretical predicted lipidnormalized BCFs for increasing lipid content (17 to 44%).
thus more than theoretically anticipated, whereas BCFs for PCB 105 (C), phenanthrene (F), and 4-chloroaniline (G) increased at 44% lipid content only by factors of 1.5, 1.5, and 2.5, respectively, thus less or equal to the theoretical prediction.
Lipid Normalization
Figure 4A shows the BCFs for S. capricornutum at dierent N status, when total lipid content ranged from 17 to 44% based on dry algal weight. We found that up to
6.3
4.88 4.85 4.49 4.59 5.19 5.03 5.29
0.064 0.093 0.091 0.107 0.077 0.123 0.193
8.9
5.06 4.73 4.33 4.94 5.00 5.03 5.28
Note. All results are means of three measurements.
24 10 19 10.3 0 17.1 1 27.6 13 31.1 3 43.8 7 44.2 Factor of increase in BCF between exponential growth conditions (17% lipid) and highest algal lipid content
Study day 0.206 0.039 0.027 0.048 0.088 0.175 0.059
1.5
5.41 5.04 5.49 5.31 5.50 5.37 5.66
0.095 0.406 0.414 0.197 0.178 0.354 0.278
8.9
5.19 4.88 4.51 4.58 5.26 5.36 5.46
0.074 0.194 0.033 0.248 0.205 0.188 0.375
6.6
5.10 4.57 4.68 4.83 4.95 5.31 5.50
0.064 0.191 0.044 0.053 0.073 0.405 0.387
1.5
4.47 4.41 4.24 4.22 4.29 4.46 4.43
0.036 0.194 Ð 0.084 0.021 0.223 0.025
2.5
Ð Ð 2.45 Ð 2.27 2.85 Ð
Ð Ð 0.115 Ð 0.088 0.099 Ð
%total lipid content of 95% 95% 95% 95% 95% 95% 95% algal dry con®dence con®dence con®dence con®dence con®dence con®dence con®dence weight PCB 31 limit PCB 49 limit PCB 105 limit PCB 153 limit DDT limit Phenanthrene limit 4-Chloroaniline limit
TABLE 3 Log BCF in Selenastrum capricornutum for the Dierent HOC Model Compounds and Dierent Degree of Nitrogen Limitation Achieved by Harvesting Cells from Batch Cultures with Nitrogen as a Minimum Medium Constituent, Mean95% Con®dent Limit
38 HALLING-SéRENSEN ET AL.
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
39
DISCUSSION
FIG. 4. Relationship of total lipid and lipid-class normalized biological concentration factors (BCF) to Kow of S. capricornutum related to algal dry weight. (A) Normalization to total lipid content at dierent lipid contents. (B) Normalization to neutral lipids at dierent lipid contents. (C) Normalization to glycolipids at dierent lipid contents. (D) Normalization to phospholipids at dierent lipid contents.
about log Kow56 lipid-normalized BCFs were directly proportional to Kow. For chemicals having log Kow values 6, greater lipid contents improved the linearity of the relationship between lipid-normalized Kow and BCFs (see part of the graph in the box). The results of lipid normalization relative to neutral lipids, glycolipids, and phospholipids, respectively, are given in Figs. 4B to 4D. The ratio between these three lipid classes changed greatly during the study. Lipid-class normalization in all cases led to overestimation of the BCFs, especially when the lipid contents were low.
The experimental setup used to culture the algal biomass in these experiments has the disadvantage of introducing a transient growth period during the biomass development, because N declines in the media over time. Transient growth periods may produce an unde®ned biomass with respect to cell size, cell volume, cell shape, and lipids. Producing the algal biomass in a chemostat system with short dilution rate would have been more correct, but a chemostat system introduces other disadvantages. In a chemostat system it is impossible to keep the culture for more than 10 days without contaminating the algal biomass with bacteria. Therefore, we chose to use the 10-liter ¯asks as growth vessels. The ¯asks were inoculated with a low algal density (104 cells/ml), which allowed the biomass to develop in the media and therefore reduced the inhomogeneity. It was thus considered that a homogeneous algal biomass was obtained due to a long period of balanced growth after the transient growing period (until N depletion). This system allowed to maintain the culture for 24 days without bacteria contamination. As indicated by Fogg (1959), and also found in this study, one of the consequences of N-de®cient conditions is the development of a higher proportion of fat than in exponentially growing cells. Other consequences of N de®ciency are changes in algal shape, weight, and volume. Using the test procedures described in this study we tried to keep as many of these factors unchanged for as long as possible. As shown in Fig. 1, the average algal volume was found to be constant and equal to approximately 50 mm3 until day 19. Afterward, the average algal volume increased to 85 mm3. The average cell weight increased as shown in Fig. 1 from 261078 to 161077 mg dry wt/cell during the incubation under nitrogen de®ciency. It might thus be stressed that during most of this investigation (day 1 to day 19) the algal cell weight and volume were kept constant. The algal shape of S. capricornutum was controlled microscopically and found to be unchanged during the study period. Only the total lipid content increased. It is impossible to evaluate if a speci®c trend in change of lipid distribution took place (see Table 2). A decrease in phospholipid level may be due to degradation of algal membranes, whereas an increase in glycolipids could be explained by the fact that the fat accumulation observed during the study period is primarily ``stored'' as internal lipids. When cell volume, shape, and weight increased, at the end of the incubation period (day 19 and day 24), the amount of glycolipids again declined. Swackhamer and co-workers found in experiments with S. capricornutum that partitioning into the algal phase continued for several days, except for monochlorinated PCB homologs. Tri- and pentachlorinated homologs
40
HALLING-SéRENSEN ET AL.
appeared to reach steady-state partitioning after the ®rst week, whereas for decachlorobiphenyl the percentage associated with algae was still increasing at the end of the experiment at day 40. The major dierence between the uptake experiments in this study and those in the study performed by Swackhamer and co-workers is that we did short-term experiments in open test vessels, under a shaking procedure, while Swackhamer and co-workers carried out their experiments over a time period of nearly 40 days without disturbing their vessels. During this long time period, even at very low growth rates, the algal cells will continuously extrude dissolved and particulate exudates and therefore the total particle/algal surface in the algal culture will increase, resulting in increased apparent bioconcentration. In our study algal cells were washed just prior to the accumulation experiments so the total surface area could be attributed primarily to algal cells only. This may explain the dierence in uptake pro®le for HOC found in short-term experiments by (e.g.) Uray et al. (1976), Rice and Sikka (1973), and in this study and the pro®les found by Swackhamer and co-workers, respectively. Because a maximum accumulation of the HOCs was found in less than 1 h incubation time, a study duration of 2 h during the partitioning was applied in all experiments estimating BCFs at dierent algal nutritional status. The bioconcentration factor of PCB 31, PCB 49, and of the two superhydrophobic substances, PCB 153 and DDT, increased most, whereas the bioconcentration factor of the less hydrophobic substances was less aected by an increase in algal total lipid. Surprisingly PCB 105 (log Kow=6.65) at the highest lipid content only increased by a factor of 1.5. This result was obtained as the mean of three individual measurements with a narrow 95% con®dence limit. No explanation may be given for that. For phenanthrene (log Kow=4.57) and 4-chloroaniline (log Kow=2.78), greater levels of algal total lipid content were associated with only a minor increase in BCF. Consistent with a hypothesis introduced by Swackhamer and co-workers, one possible explanation for this result could be that the less hydrophobic compounds (log Kow56) adsorb primarily to the cell membrane, whereas the more hydrophobic substances (log Kow46) partition into the internal lipid pool of the cells. The BCFs found at day 24 (showed only in Table 2) were higher than might be expected, given the low algal total lipid content. Maybe this was due to the circumstance that the average algal volume increased by a factor of 2 before day 24. The contaminant transport can therefore increase signi®cantly, assuming no changes in algal loading, as a consequence of nitrogen starvation. An increase in total algal lipid content from 17 to 44% changed BCFs (dry weight basis) for PCB 153 from log 4.51 to log 5.46 (Table
3). This would result in an increase in transport from 3.2 6 1076 to 2.9 6 1075 mg contaminant liter71 assuming a constant algal loading of 10 mg dry wt liter71 and a contaminant concentration in water of 1075 mg liter71. Lipid-normalized BCFs for phytoplankton are often assumed to be directly proportional to Kow (Thomann and Connolly, 1984; Connolly and Pederson, 1988). Swackhamer and Skoglund (1993) showed that when bioconcentration is lipid normalized, BCFs may exceed octanol/water partition coecients by up to a factor of 100 or more. No explanation is given in the literature for such high lipidnormalized bioconcentration factors. One possible explanation is that dierent classes of lipids may have dierent binding anities and that some chemicals adsorb predominantly to the algal surface (which contains dierent speci®c types of lipids), whereas lipid as normally analyzed represents a summation of all extractable lipid types (Swackhamer and Skoglund, 1993; Stange and Swackhamer, 1994). A straight line with a slope equal to unity was thus not obtained for the superlipophilic compounds as theoretically expected, but as shown in Fig. 4A (see the enlargement of the ®gure between log Kow=5.5 and log Kow=6.8) BCFs found for algae having a total lipid content of 44% were much closer to unity as those having lipid contents of 31.1 and 27.6%, respectively. We hypothesize therefore that an increase in total algal lipid increases the fugacity dierence (or driving force) across the algal cell wall, thus counteracting diusion or other uptake barriers. To test the hypothesis that limited cell wall permeability reduces uptake rates of hydrophobic compounds, we inspected class-speci®c lipid data in relation to BCFs (Table 2). The results of lipid normalization relative to neutral lipids, glycolipids, and phospholipids, respectively, are given in Figs. 4B to 4D. The ratio between these three lipid classes changed greatly during the study. Lipid-class normalization in all cases leads to overestimation of the BCFs, especially when the lipid contents were low. Thus, we found no clear advantage to normalizing BCFs to lipid class compared to normalization to total algal lipids. Normalization with total lipid in fact appeared preferable, due to its simplicity. This ®nding is in agreement with observations in other studies (Swackhamer and Skoglund, 1993). CONCLUSION
Our results show that algal N status, by aecting the lipid content, may in¯uence the bioconcentration factor of superhydrophobic compounds by up to a factor of 8.9. BCFs for PCB 31, PCB 49, PCB 153, and DDT increased with maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively, thus more than theoretically predicted
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
from the lipid normalization of BCFs obtained at exponential growth phase (17% total lipid), whereas BCFs for PCB 105, phenanthrene, and 4-chloroaniline increased at 44% lipid content only by factors of 1.5, 1.5, and 2.5, respectively, thus less than or equal to the theoretical prediction. These ®ndings may perhaps account for the fact that bioconcentration factors for superhydrophobic compounds in algae are always found to be less than predicted from Kow due to diusional and other resistances across the cell wall/membrane, becoming important only when the fugacity dierences are high. No general advantages were found in lipid-class normalizing BCFs compared to a normalization to total algal lipid. ACKNOWLEDGMENTS Thanks are given to Philipp Mayer for many helpful comments on the manuscript. This research was funded by the Danish Centre for Ecotoxicological Research, Project 7.1, under the Danish Strategic Environmental Research Program 1993±1996.
REFERENCES Bergman, AÊ., and Wachtmeister, C. (1977). Synthesis of 14C-biphenyl and of some chlorinated 14C-biphenyls containing 4-chloro, 2,4-dichloroand 2,3,6-trichlorophenyl nuclei from the corresponding labeled anilines. Chemosphere 6, 759±767. Bergman, AÊ., Bramford, I., and Wachtmeister, C. (1981). Synthesis of 14Clabeled polychlorobiphenyls derived from the labeled 4-chloro-, 2,3dichloro-, 2,5-dichloro-, 3,4-dichloro-, 2,4,5-trichloro- and 2,3,6-trichloroanilines. J. Labeled Comp. Radiopharm. 18, 1023±1032. Biggar, J. W., and Riggs, I. R. (1974). Apparent solubility of organochlorine insecticide in water at various temperatures. Hilgardia 42, 383±391. Branson, D. R., Blau, G. E., Alexander, H. C., and Neely, W. B. (1975). Bioconcentration of 2,2',4,4'-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. Trans. Am. Fish Soc. 104, 785±792. Burkhard, L. P., Kuehl, D. W., and Veith, G. D. (1985). Evaluation of reversed phase LC/MS for estimation of n-octanol/water partition coecient of organic chemicals. Chemosphere 14, 1551±1560. Casserly, D. M., Davis, E. M., Downs, T. D., and Guthrie, R. K. (1983). Sorption of organics by Selenastrum capricornutum. Water Res. 17, 1591±1594. Chessells, M., Hawker, D. W., and Connell, D. W. (1992). In¯uence of solubility in lipid bioconcentration of hydrophobic compounds. Ecotoxicol. Environ. Saf. 23, 260±273. Collyer, D. M., and Fogg, C. G. (1955). Studies of fat accumulation by algae. J. Exp. Bot. 6, 256±275.
41
Geyer, H., Politzki, G., and Freitag, D. (1984). Prediction of ecotoxicological behaviour of chemicals: Relationship between n-octanol/ water partition coecient and bioaccumulation of organic chemicals by algae Chlorella. Chemosphere 13, 269±284. Hawker, D. W., and Connell, D. W. (1988). Octanol±water partitioning coecients of polychlorinated biphenyl congers. Environ. Sci. Technol. 22, 382±387. ISO 8692 (1989). Water Quality Ð Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus or Selenastrum capricornutum. International Organisation for Standardisation, Switzerland. Ketchum, B. H., and Red®eld, A. C. (1949). Some physical and chemical characteristics of algae growth in mass culture. J. Cell. Comp. Physiol. 33, 281±299. Kilzer, L., Scheunert, H., Klein., W., and Korte, F. (1979). Laboratory screening of the volatilization rates of organic chemicals from water. Chemosphere 8, 751±761. Koelmans, A. A., and JimeÂnez, C. S. (1994). Temperature dependency of chlorobenzene bioaccumulation in phytoplankton. Chemosphere 28, 2041±2048. Mackay, D. (1982). Correlation of bioconcentration factors. Environ. Sci. Technol. 16, 274±278. Mackay, D. (1991). Multimedia Environmental Models: The Fugacity Approach. Lewis, Chelsea, MI. Mackay, D., Shiu, W. Y., and Ma, K. C. (1993). Illustrated Handbook of Physical±Chemical Properties and Environmental Fate for Organic Chemicals, Vol. 2 Ð Polynuclear Aromatic Hydrocarbons (PAHs), Polychlorinated Dibenzo-p-dioxins and Dibenzofurans. Lewis, Chelsea, MI. Manthey, M., Faust, M., Smolka, S., and Horst, L. (1993). Herbicide bioconcentration in algae: Studies on lipohilicity±sorption±activity relationships (LSAR) with Chlorella fusca. Sci. Total Environ. Suppl., 453±459. Millard, E. S., Halfon, E., Minns, C. K., and Charlton, C. C. (1993). Eects of primary productivity and vertical mixing on PCB dynamics in planktonic model ecosystems. Environ. Toxicol. Chem. 12, 931±946. Nyholm, N. (1976). A mathematical model for microbial growth under limitation by conservative substrates. Biotechnol. Bioeng. 18, 1043±1056. Nyholm, N. (1977). Kinetics of nitrogen-limited algal growth. Prog. Water Technol. 8, 346±358. Piorreck, M., Baasch, K. H., and Pohl, P. (1984). Biomass production, total protein, chlorophylls, lipids and fatty acids of freshwater green and blue±green algae under dierent nitrogen regimes. Phytochemistry 23, 207±216. Rice, C. P., and Sikka, H. C. (1973). Uptake and metabolism of DDT by six species of marine algae. J. Agric. Food Chem. 21, 148±152. Richer, G., and Peters, R. H. (1993). Determinants of the short-term dynamics of PCB uptake by plankton. Environ. Toxicol. Chem. 12, 207±218.
Connolly, J. P., and Pederson, C. J. (1988). A thermodynamic-based evaluation of organic chemical accumulation in aquatic organisms. Environ. Sci. Technol. 22, 99±103. Fogg, G. E. (1959). Nitrogen nutrition and metabolic patterns in algae. Symp. Soc. Exp. Biol. 13, 106±125.
Shiu, W. Y., and Mackay, D. (1986). A critical review of aqueous solubilities, vapour pressures, Henry's law constant and octanol±water partition coecient of the polychlorinated biphenyls. J. Phys. Chem. Ref. Data 15, 911±929.
Gardner, W. S., Frez, W. A., Cichocki, E. A., and Parrich, C. C. (1985). Micromethod for lipid analysis in aquatic invertebrates. Limnol. Oceanogr. 30, 1099±1105. Geyer, H., Viswanathan, R., Freitag, D., and Korte, F. (1981). Relationship between water solubility of organic chemicals and their bioaccumulation by the alga Chlorella. Chemosphere 10, 1307±1313.
SoÈdergren, A., and Gelin, C. (1983). Eect of PCBs on the rate of carbon14 uptake in phytoplankton isolates from oligotrophic and eutrophic lakes. Bull. Environ. Contam. Toxicol. 30, 191±198.
Sijm, D. T. H. M., Middelkoop, J., and Vrisekoop, K. (1995). Algal density dependent bioconcentration factors of hydrophobic chemicals. Chemosphere. 31(9), 4001±4012.
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HALLING-SéRENSEN ET AL.
Spoehr, H. A., and Milner, H. W. (1949). The chemical composition of Chlorella: Eect of environmental conditions. Plant Physiol. 24, 120±149.
Thomann, R. V., Connolly, J. P., and Parkerton, T. F. (1992). An equilibrium model of organic chemical accumulation in aquatic food webs with sediment interaction. Environ. Toxicol. Chem. 11, 615±629.
Stange, K., and Swackhamer, D. L. (1994). Factors aecting phytoplankton species-speci®c dierences in accumulation of 40 polychlorinated biphenyls (PCBs). Environ. Toxicol. Chem. 13, 1849±1860.
Thompson, G. A., Jr. (1996). Lipids and membrane function in green algaeÐReview. Biochim. Biophys. Acta 1302, 17±45. Thornabene, T. G., Holzer, G., Lien, S., and Burris, N. (1983). Lipid composition of the nitrogen starved green alga Neochloris oleoabundans. Enzyme Microb. Technol. 5, 435±440.
Sundstrùm, G. (1974). Polychlorinated biphenyls V. Synthesis of 14Clabeled 2,2',4,4',5,5'-hexabiphenyl, a major component in PCB-mixtures. Bull. Environ. Contam. Toxicol. 11, 39. Swackhamer, D. L., and Skoglund, R. S. (1993). Bioaccumulation of PCBs by algae: Kinetics versus equilibrium. Environ. Toxicol. Chem. 12, 831±838. Thomann, R. V., and Connolly, J. P. (1984). Model of PCB in the Lake Michigan lake trout food chain. Environ. Sci. Technol. 18, 65±71.
Urey, J. C., Kricher, J. C., and Boylan, J. M. (1976). Bioconcentration of four pure PCB isomers by Chlorella pyrenoidosa. Bull. Environ. Contam. Toxicol. 16, 81±85. Veith, G. D., DeFoe, D. L., and Bergstedt, B. V. (1979). Measuring and estimating the bioconcentration factor of chemicals in ®sh. J. Fish. Res. Board Can. 36, 1040±1048.
In£uence of Nitrogen Status on the Bioconcentration of Hydrophobic Organic Compounds to Selenastrum capricornutum Bent Halling-Sùrensen,*,1 Niels Nyholm,{ Kresten Ole Kusk,{ and Eva Jacobsson{ *Royal Danish School of Pharmacy, Section of Environmental Chemistry, Universitetsparken, DK-2100 Copenhagen é, Denmark; {Institute of Environmental Science and Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark; and {Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Received July 29, 1998
the same species, in relation to nutritional status. Bioconcentration of HOCs by algae has been described in several publications (Rice and Sikka, 1973; Urey et al., 1976; Casserly et al., 1983; Richer and Peters, 1993; Swackhamer and Skoglund, 1993; Stange and Swackhamer, 1994; Koelmans and JimeÂnez, 1994), but the level of knowledge about algae is limited compared to that available for ®sh. Models predicting the transport and fate of HOCs in the environment rely on the HOC octanol/water partition coecient (Kow), a physicochemical property representing a chemical's lipophilicity. Bioaccumulation is quanti®ed in terms of the bioaccumulation factor (BAF), which is the ratio of HOC concentration in the organism's biomass to the HOC concentration dissolved in water, expressed in equivalent units. At true equilibrium, this ratio is called the biological concentration factor (BCF). Thus, when the system is at equilibrium, the BAF equals the BCF. A proportional relationship between BCF and Kow has been suggested for several aquatic organisms by several investigations (Veith et al., 1979; Mackay, 1982). Veith et al. (1979) showed that this correlation could be re®ned by lipid normalization of BCF values. However, Swackhamer and Skoglund (1993) found that for algae this linear correlation between BCF for HOCs and Kow did not function. Stange and Swackhamer (1994) found that normalization to total lipid worked for lower substituted PCB congeners (Kow 5 106.5). They also showed that by normalizing to phospholipids, BCF values converged for Kow 4 106.5. These ®ndings led to the hypothesis that PCBs with Kow 4 106.5 associate mainly with the cell membrane, where most of the phospholipids occur. Chessells et al. (1992) suggested that the curved relationship between the solubility of HOCs in biotic lipid (SL) and Kow probably accounted for the decreased bioconcentration of superhydrophobic compounds, due to the fact that
Changes in algal nitrogen status that increase algal lipid content also aect the bioconcentration of hydrophobic organic compounds (HOCs). Bioconcentration factors (BCFs) for several HOCs increased up to nine times as the total algal lipid content of the green algae Selenastrum carpricornutum increased from 17 to 44% of the algal dry weight as a consequence of nitrogen starvation. An increase in total lipid from 17 to 44% should theoretically increase the BCFs by a factor of 2.6. BCFs for PCB 31, PCB 49, PCB 153, and DDT increased with maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively, thus more than theoretically predicted from the lipid normalization of BCFs obtained at exponential growth phase (17% total lipid for S. carpricornutum), whereas BCFs for PCB 105, phenanthrene, and 4-chloroaniline increased at 44% lipid content, only by factors of 1.5, 1.5, and 2.5, respectively, and thus less than or equal to the theoretical prediction. Lipidclass normalization of BCFs did not reveal signi®cant information beyond that available from normalizing to total lipid. # 2000 Academic Press
Key Words: nutritional status; HOCs; algal bioconcentration; lipid normalization; PCBs; S. carpricornutum.
INTRODUCTION
Phytoplankton are signi®cant as primary producers that sustain the pelagial food chains in aquatic ecosystems and can in¯uence the speciation and transport of pollutants in natural waters (Thomann et al., 1992; Millard et al., 1993). More speci®cally the lipid content of algae is known to be the major factor in¯uencing the bioconcentration of HOCs (Geyer et al., 1984; Manthey et al., 1993) and algal lipid status may vary considerably, both between species and for 1
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0147-6513/00 $35.00 Copyright # 2000 by Academic Press All rights of reproduction in any form reserved.
34
HALLING-SéRENSEN ET AL.
superhydrophobic compounds are much more soluble in octanol than in lipids. In general, nitrogen-de®cient microalgae have larger cell volumes and contain more dry matter with a higher proportion of fat than actively growing algae (Fogg, 1959). Exponential growth ceases when the nitrogen supplied is exhausted but, when the growth rate decreases, assimilation of carbon continues at a reduced rate for some time. The net result is an increase in total biomass or dry weight of algal material, by up to a factor of two to three (Nyholm, 1976). While the nitrogen content declines, fats or carbohydrates accumulate in the cells. Several sets of data illustrating this phenomenon have been published (Spoehr and Milner, 1949; Ketchum and Red®eld, 1949; Collyer and Fogg, 1955), as has a mathematical description of nitrogen-limited algal growth (Nyholm, 1976). Nitrogen-starved Neochloris oleoabundus increased its lipid content to 35±54% of cell dry weight (Thornabene et al., 1983). The lipid levels of Scenedesmus obliquus and Chlorella vulgaris reaching as much as 45% of the biomass were found when growing slowly in a medium containing low nitrogen concentrations (Piorreck et al., 1984). N-de®cient algal cells can be prepared in the laboratory in several ways: (1) cultures can be grown with a limited amount of available N so they become nitrogen-de®cient after a period of growth (external growing on internal nitrogen); (2) N-sucient cells can be transferred to a Nfree medium thus forcing them to grow only through the use of internal N reserves; or (3) a chemostat culture with N limitation can be set up (Nyholm, 1976, 1977). Algal lipids may roughly be fractionated into three major classes: glycolipids, neutral lipids, and phospholipids (see Thompson, 1996). Glycolipids are found intracellularly, whereas neutral lipids are major constituents of membrane lipids. Phospholipids are associated mainly with the cell membrane (Stange and Swackhamer, 1994). A review of lipids in green algae is provided in Thompson (1996). The objective of this study was to determine if increases in algal total lipid content due to changes in nutritional status would aect the ability of algae to bioconcentrate HOCs. In our study bioaccumulation is predicted to equal bioconcentration because an equilibrium between the algal phase and the water phase was expected to be achieved quickly. So BAF is predicted to equal BCF. The term BCF is therefore used on all graphs. In the present study, the BCF of six HOCs ranging in lipophilicity from log Kow = 4.57 to log Kow = 6.90 was measured in the green algae Selenastrum capricornutum. The BCF measurements were made at dierent algal lipid contents achieved by manipulating the extent of N starvation. A compound with relatively lower lipophilicity, 4-chloroanilin (log Kow= 2.78), was used as negative control. The other six compounds were phenanthrene, 2,4',5-trichlorobiphenyl
(PCB 31), 2,2',4,5'-tetrachlorobiphenyl (PCB 49), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), 2,2',4,4',5,5'hexachlorobiphenyl (PCB 153), and 4,4'-DDT. THEORY
Algal bioconcentration of hydrophobic chemicals can be described by a ®rst-order one-compartment kinetic model, such as that for ®sh (Branson et al., 1975). Growth of the algae should be included, since algae are fast-growing organisms with a growth rate of days (Sijm et al., 1995): dCalg =dt k1 Cw ÿ k2 Calg :
1
where Calg is the concentration in the algae (mg/kg), k1 is the uptake rate constant (l/kg * d), Cw is the concentration of the freely dissolved chemical in water (mg/liter), k2 is the elimination rate constant (1/d ), and m is the growth rate constant (1/d ). At steady state, when dCalg/dt = 0, the BCF (l/kg) can be derived from Eq. [1]: BCF
k1 Calg : k2 Cw
2
The lipid-normalized bioconcentration factor, BCFlipid (l/ kg), can then be derived from Eq. (2), knowing the fraction (weight or percent) of total algal lipid content, a. BCFlipid
BCF
3
MATERIAL AND METHODS
Chemicals and Radiochemicals Information about the radiolabeled compounds used in this investigation is given in Table 1. [UL-14C]2,4',5Trichlorobiphenyl (PCB 31, 19 mCi/mol), [UL-14C]2,2', 4,5'-tetrachlorobiphenyl (PCB 49, 25 mCi/mol), [UL-14C] 2,3,3',4,4'-pentachlorobiphenyl (PCB 105, 26.5 mCi/mol), and [UL-14C]2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153, 12.6 mCi/mol) were obtained from the Department of Environmental Chemistry at the Wallenberg Institute, Stockholm University (Stockholm, Sweden), with a purity of 498%. The four PCBs were synthesized in accordance with methods described elsewhere (Sundstrùm, 1974; Bergman and Wachtmeister, 1977; Bergman et al., 1981). [UL-14C]4-Chloroanilin, [9-14C]phenanthrene, and [UL-14C]4,4'-DDT-Ring were obtained from Sigma Chemical Co. (St. Louis, MO) at a purity of 498%. All compounds were stored at 48C and diluted in Uvasol grade
35
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
TABLE 1 List of Substances Used in the Experimental Studies
[14C] Substancea 14
Speci®c activity (mCi/mmol)
[UL- C] 4-Chloroanilin [9-14C] Phenanthrene [UL-14C] 2,4',5-Trichlorobiphenyl (PCB 31)
25.5 8.3 19
[UL-14C] [UL-14C] [UL-14C] [UL-14C]
25 26.5 12.6 10
2,2',4,5'-Tetrachlorobiphenyl (PCB 49) 2,3,3',4,4'-Pentachlorobiphenyl (PCB 105) 2,2',4,4'5,5'-Hexachlorobiphenyl (PCB 153) 4,4'-DDT-Ring
Water Solubility (mg/liter)
Log Kow
b
2.78 4.57d 5.67 f
0.016g 0.009 0.001e 0.006h
5.85 f 6.65 f 6.9 f 6.19 i
3900 1.2d 0.31e
c
Source Sigma Chemical Co. (St. Louis, MO) Sigma Department of Environmental Chemistry, Wallenberg Institute, Stockholm University (Stockholm, Sweden) Wallenberg Institute Wallenberg Institute Sigma Sigma
Note. Data are obtained from the following sources; apurity 498%, based on manufacturer's standards and tests performed at the Department of Environmental Chemistry, Wallenberg Institute, Stockholm, Sweden; bKilzer et al. (1979); cGeyer et al. (1981); dMackay et al. (1993); eBurkhard et al. (1985); fHawker and Connell (1988); gShiu and Mackay (1986); hMackay (1991); iBiggar and Riggs (1974).
acetone (min 99.9%; Merck KGaA, Darmstadt, Germany) to a working concentration of about 1000 dpm/100 ml.
and drying to constant weight at 1058C and equilibration in an exsiccator. Con®rmation of N Limitation
Algal Cultures Unialgae cultures of S. capricornutum were obtained from the Norwegian Institute of Water Research culture collection. Nonaxenic populations of these cells were maintained in log-phase development, in ISO medium (ISO 8692, 1989) at 22+18C, under a light intensity of 6 klux supplied by cool-white ¯uorescent tubes. Cells in the culture were kept in suspension by use of magnetic stirrers. The algae were grown in six 10-liter glass ¯asks (one for each study day). An increase in algal lipids was obtained by allowing growth after N in the media had been depleted. Algal biomass from each 10-liter ¯ask (between 300 and 900 mg algal dry wt) was harvested and used for both lipid analysis and bioaccumulation studies, 1 to 24 days after the test medium had become N-de®cient. Day 0 of the experiment was de®ned as the last day of exponential growth, day 1 is the ®rst day after nitrogen exhaustion in the media, day 3 is the third day, and so forth. On days 1, 3, 7, 13, 19, and 24 after the start of the study, one 10-liter ¯ask each was used for experiments. The algal concentration was quanti®ed as total volume (mm3/ml) and cell numbers per liter of both unwashed and washed algal samples, by use of an electronic particle counter (Coulter Multisizer, 70-mm capillary tube) using IsotoneR as electrolyte. Triplicate measurements were made on each sample. The relationship between cell numbers and algal dry weight was determined for each of the six 10-liter ¯asks by ®ltering 100-ml washed algal samples in triplicate through a dried and pre-weighed GFC ®lter (Whatmann, 1.2 mm)
Nitrogen de®ciency in the media was con®rmed by analyzing the media for the level of Kjeldahl nitrogen during the exponential growth phase and during the study period (day 1 to day 24). As a positive control a culture incubated at the same time under the same conditions as the six other ¯asks, but with a higher N concentration (N=50 mg/liter), was used. Accumulation Pro®le Studies Before the lipid-depended bioaccumulation studies were started, accumulation pro®les of phenanthrene and the four PCBs were measured over time, in a separate experiment to allow us to estimate the time needed to achieve steady-state conditions. To minimize possible sorption by nonalgal particulate matter (e.g., bacteria), S. capricornutum cells (exponential growing from the 10liter ¯ask at day 0) were concentrated from the growth medium by gentle centrifugation. The spent medium overlying the cell pellet was discarded, and the algal cell pellet was resuspended in fresh N-free growth medium (to minimize cell growth). The process was repeated three times. For each compound triplicate samples of 100 ml washed algal suspension were exposed under a shaking procedure (100 rpm) in 250-ml open Erlenmeyer ¯asks. At various time intervals (0 min, 2 min, 5 min, 10 min, 30 min, 1 h, and 2 h) (except for PCB 31 data only up to 1 h) 10-ml suspensions of algal cells were separated by ®ltration with a GFC ®lter (Whatmann, 1.2 mm) into algal biomass and medium and both were analyzed for the content of
36
HALLING-SéRENSEN ET AL.
compound. Results from the uptake study were used to evaluate the study time to be used in the bioaccumulation study below to ensure a steady-state situation. Uptake experiments for DDT were not performed in this study as they were made by Rice and Sikka (1973). Bioaccumulation Studies at Dierent Algal Lipid Content For the bioaccumulation experiments S. capricornutum cells at an initial cell density of 106 cells per milliliter were used in order to avoid any intervention of changes in cell density with the BCF. The same algal washing procedure was used as described above. The bioaccumulation studies were performed by adding the radiolabeled test compound (1000 dpm per milliliter of algal sample) to 50 ml prewashed algal sample, as explained above on each of the indicated days. Triplicate measurements were made for all seven compounds each day. Prewashed algal suspension were shaken for 2 h with the test compound to permit phase preference. The uptake experiment described above showed that steady state was achieved within 30 to 60 min for all six compounds why a prolonged study period of 2 h was used in all bioaccumulation studies with N-de®cient algae to ensure steady-state situations. The phases (algae and media) were then separated by ®ltrating 10 ml of the suspension through a GFC ®lter (25 mm). Radioactivity was quanti®ed in both algae and water phases by liquid scintillation counting (Tri-Carb 2000 liquid scintillation counter, United Technologies Packard). The amount of toxicant associated with algal biomass was determined by dissolving the ®lter and biomass using 5 ml Ecoscint A (National Diagnostics). The radioactivity of the ®ltrate was determined using 10 ml Optiphase HiSafe and 10 ml sample. For background correction due to sorption of compounds retained in the GFC ®lter, blank samples (without algal culture) were spiked with the radiolabeled compounds and 10 ml was ®ltered through a GFC ®lter and analyzed in the same way as the algal samples.
One-dimensional polar-lipid separation was achieved using a solvent system consisting of hexane and isopropanol (8:2 ratio). Lipids were visualized by using a spray of 8anilino-1-naphtalene sulfonic acid (0.2% in methanol), followed by inspection under UV light. Lipids were identi®ed by comparison with authentic standards. The individual lipid classes, including glycolipids, neutral lipids, and phospholipids, were quanti®ed by scraping the lipid bands of each class from the TLC plate and resuspending them in chloroform. The chloroform was evaporated at low temperature from preweighed aluminum foil cups and the lipids were quanti®ed gravimetrically. All lipid analyses were accompanied by two blanks to correct (mean value was used) for nonlipid dry weight contribution. RESULTS
Con®rmation of N Limitation Before N depletion was evident S. carpricornutum grew at a mean rate of 1.6 day71 (based on cell numbers for the six 10-liter ¯asks) at 22+18C. After N depletion had become evident, the growth rate of alga decreased gradually to 50.1 day71. Nitrogen de®ciency in the media was also con®rmed by analyzing the media for the level of Kjeldahl nitrogen which was below 1 mg/liter N after day 1. The supposition of N limitation in the N-free cultures was con®rmed by the positive control at an N concentration of 50 mg/liter, which sustained high rates of cell division. Lipids Lipid content of S. capricornutum increased with the onset of N depletion by a factor of 2.6 (from 17% on day 0 to 44% on day 7, on a dry weight basis) (Fig. 1). After day 13, lipid content declined to about 10% (Fig. 1).
Extraction and Analysis of Algal Lipids Lipids were extracted using the method of Gardener et al., (1985) modi®ed to accommodate our experimental design and laboratory facilities. The modi®cations included use of isopropanol rather than methanol, to avoid lipid degradation by endogenous lipolysis. Non-lipid material was removed by twice mixing the extract with an equal volume of 0.9% NaCl in water; phase separation was then achieved using a 250-ml separation funnel. The chloroform phase was dried with Na2SO4 and the volume reduced to about 500 ml by evaporation. Analytical TLC was carried out using silica gel plates precoated with a layer of 0.5-mmthick silica gel G (E. Merck No. 5721, Darmstadt, Germany).
FIG. 1. Relationship of ( ) percentage total lipid of algal dry weight, (*) average algal cell volume, and (~) average algal cell dry weight during the study period.
37
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
TABLE 2 Distribution of Neutral, Glyco-, and Phospholipids (Given as Percentages of Total Lipid Content) for S. capricornutum during the Study Study day Ratio
0 ND
1 24:48:28
3 46:13:41
7 37:54:9
13 ND
19 50:27:23
24 38:24:38
Note: ND not determined.
These results, on day 0, are in accordance with ®ndings by Swackhamer and co-workers, who found the average total lipid content of S. capricornutum in exponential growth phase as 19.5%. Lipid-class distribution was measured on each sample except at day 13. Results are shown as ratios among the lipid classes in Table 2. The neutral lipids remained fairly constant during the incubation period. The percentage of glycolipids (the internal lipids) gained a maximum at day 7. During the incubation period with high glycolipids the level of phospholipids associating mainly with the cell membrane was low.
In accordance with Eq. (3), an increase in total lipid content from 17 to 44% should theoretically increase the BCFs by a factor of 2.6. Figure 3 shows that BCFs for PCB 31 (A), PCB 49 (B), PCB 153 (D), and DDT (E) increased at maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively,
Uptake and Partitioning of HOCs into Algal Phase For all compounds the uptake was fast and a steadystate level was reached within 60 min. Figures 2A to 2E show the bioaccumulation pro®les for the four PCBs and phenanthrene. Uptake experiments for DDT were not performed in this study because they were performed by Rice and Sikka (1973). Rice and Sikka found that a steady state for DDT was reached within 2 h as demonstrated for several algal species. Our results dier from these reported by Swackhamer and co-workers, but are in accordance with other ®ndings reported in the literature (e.g., Rice and Sikka, 1973; Urey et al., 1976; SoÈdergren and Gelin, 1973). Figures 2A to 2E show that depuration of the PCBs and phenanthrene occurred due to evaporation (open test vessels) from the aqueous phase after steady state was obtained. Calculations showed no changes in phase preference during depuration because BCF was constant. Bioaccumulation Factors All BCFs in the present study were calculated on a dry weight basis. Figures 3A to 3G show both the experimental and theoretical relationships between the algal lipid content and log BCF obtained in the present study. Table 3 shows all data (results from day 19 and day 24 were not presented in ®gures; see below) with corresponding 95% con®dence limits. Equation (3) was used to calculate theoretical predicted lipid-normalized BCFs (lines in Fig. 3) based on the lipid content at exponential growth phase (17% total lipid for S. capricornutum). Results were compared with the experimental values (dots in Fig. 3) obtained in the present study.
FIG. 2. Partitioning of substances into algal phase over the time course of experiments with S. capricornutum of (A) PCB 31, (B) PCB 49, (C) PCB 105, (D) PCB 153, and (E) phenanthrene.
FIG. 3. Relationship between the biological concentration factors (BCFs) and total lipid content of S. capricornutum related to algal dry weight for (A) PCB 31, (B) PCB 49, (C) PCB 105, (D) PCB 153, (E) DDT, (F) phenanthrene, and (G) 4-chloroaniline. (^) Experimental values with corresponding 95% con®dence limits. (Ð) Theoretical predicted lipidnormalized BCFs for increasing lipid content (17 to 44%).
thus more than theoretically anticipated, whereas BCFs for PCB 105 (C), phenanthrene (F), and 4-chloroaniline (G) increased at 44% lipid content only by factors of 1.5, 1.5, and 2.5, respectively, thus less or equal to the theoretical prediction.
Lipid Normalization
Figure 4A shows the BCFs for S. capricornutum at dierent N status, when total lipid content ranged from 17 to 44% based on dry algal weight. We found that up to
6.3
4.88 4.85 4.49 4.59 5.19 5.03 5.29
0.064 0.093 0.091 0.107 0.077 0.123 0.193
8.9
5.06 4.73 4.33 4.94 5.00 5.03 5.28
Note. All results are means of three measurements.
24 10 19 10.3 0 17.1 1 27.6 13 31.1 3 43.8 7 44.2 Factor of increase in BCF between exponential growth conditions (17% lipid) and highest algal lipid content
Study day 0.206 0.039 0.027 0.048 0.088 0.175 0.059
1.5
5.41 5.04 5.49 5.31 5.50 5.37 5.66
0.095 0.406 0.414 0.197 0.178 0.354 0.278
8.9
5.19 4.88 4.51 4.58 5.26 5.36 5.46
0.074 0.194 0.033 0.248 0.205 0.188 0.375
6.6
5.10 4.57 4.68 4.83 4.95 5.31 5.50
0.064 0.191 0.044 0.053 0.073 0.405 0.387
1.5
4.47 4.41 4.24 4.22 4.29 4.46 4.43
0.036 0.194 Ð 0.084 0.021 0.223 0.025
2.5
Ð Ð 2.45 Ð 2.27 2.85 Ð
Ð Ð 0.115 Ð 0.088 0.099 Ð
%total lipid content of 95% 95% 95% 95% 95% 95% 95% algal dry con®dence con®dence con®dence con®dence con®dence con®dence con®dence weight PCB 31 limit PCB 49 limit PCB 105 limit PCB 153 limit DDT limit Phenanthrene limit 4-Chloroaniline limit
TABLE 3 Log BCF in Selenastrum capricornutum for the Dierent HOC Model Compounds and Dierent Degree of Nitrogen Limitation Achieved by Harvesting Cells from Batch Cultures with Nitrogen as a Minimum Medium Constituent, Mean95% Con®dent Limit
38 HALLING-SéRENSEN ET AL.
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
39
DISCUSSION
FIG. 4. Relationship of total lipid and lipid-class normalized biological concentration factors (BCF) to Kow of S. capricornutum related to algal dry weight. (A) Normalization to total lipid content at dierent lipid contents. (B) Normalization to neutral lipids at dierent lipid contents. (C) Normalization to glycolipids at dierent lipid contents. (D) Normalization to phospholipids at dierent lipid contents.
about log Kow56 lipid-normalized BCFs were directly proportional to Kow. For chemicals having log Kow values 6, greater lipid contents improved the linearity of the relationship between lipid-normalized Kow and BCFs (see part of the graph in the box). The results of lipid normalization relative to neutral lipids, glycolipids, and phospholipids, respectively, are given in Figs. 4B to 4D. The ratio between these three lipid classes changed greatly during the study. Lipid-class normalization in all cases led to overestimation of the BCFs, especially when the lipid contents were low.
The experimental setup used to culture the algal biomass in these experiments has the disadvantage of introducing a transient growth period during the biomass development, because N declines in the media over time. Transient growth periods may produce an unde®ned biomass with respect to cell size, cell volume, cell shape, and lipids. Producing the algal biomass in a chemostat system with short dilution rate would have been more correct, but a chemostat system introduces other disadvantages. In a chemostat system it is impossible to keep the culture for more than 10 days without contaminating the algal biomass with bacteria. Therefore, we chose to use the 10-liter ¯asks as growth vessels. The ¯asks were inoculated with a low algal density (104 cells/ml), which allowed the biomass to develop in the media and therefore reduced the inhomogeneity. It was thus considered that a homogeneous algal biomass was obtained due to a long period of balanced growth after the transient growing period (until N depletion). This system allowed to maintain the culture for 24 days without bacteria contamination. As indicated by Fogg (1959), and also found in this study, one of the consequences of N-de®cient conditions is the development of a higher proportion of fat than in exponentially growing cells. Other consequences of N de®ciency are changes in algal shape, weight, and volume. Using the test procedures described in this study we tried to keep as many of these factors unchanged for as long as possible. As shown in Fig. 1, the average algal volume was found to be constant and equal to approximately 50 mm3 until day 19. Afterward, the average algal volume increased to 85 mm3. The average cell weight increased as shown in Fig. 1 from 261078 to 161077 mg dry wt/cell during the incubation under nitrogen de®ciency. It might thus be stressed that during most of this investigation (day 1 to day 19) the algal cell weight and volume were kept constant. The algal shape of S. capricornutum was controlled microscopically and found to be unchanged during the study period. Only the total lipid content increased. It is impossible to evaluate if a speci®c trend in change of lipid distribution took place (see Table 2). A decrease in phospholipid level may be due to degradation of algal membranes, whereas an increase in glycolipids could be explained by the fact that the fat accumulation observed during the study period is primarily ``stored'' as internal lipids. When cell volume, shape, and weight increased, at the end of the incubation period (day 19 and day 24), the amount of glycolipids again declined. Swackhamer and co-workers found in experiments with S. capricornutum that partitioning into the algal phase continued for several days, except for monochlorinated PCB homologs. Tri- and pentachlorinated homologs
40
HALLING-SéRENSEN ET AL.
appeared to reach steady-state partitioning after the ®rst week, whereas for decachlorobiphenyl the percentage associated with algae was still increasing at the end of the experiment at day 40. The major dierence between the uptake experiments in this study and those in the study performed by Swackhamer and co-workers is that we did short-term experiments in open test vessels, under a shaking procedure, while Swackhamer and co-workers carried out their experiments over a time period of nearly 40 days without disturbing their vessels. During this long time period, even at very low growth rates, the algal cells will continuously extrude dissolved and particulate exudates and therefore the total particle/algal surface in the algal culture will increase, resulting in increased apparent bioconcentration. In our study algal cells were washed just prior to the accumulation experiments so the total surface area could be attributed primarily to algal cells only. This may explain the dierence in uptake pro®le for HOC found in short-term experiments by (e.g.) Uray et al. (1976), Rice and Sikka (1973), and in this study and the pro®les found by Swackhamer and co-workers, respectively. Because a maximum accumulation of the HOCs was found in less than 1 h incubation time, a study duration of 2 h during the partitioning was applied in all experiments estimating BCFs at dierent algal nutritional status. The bioconcentration factor of PCB 31, PCB 49, and of the two superhydrophobic substances, PCB 153 and DDT, increased most, whereas the bioconcentration factor of the less hydrophobic substances was less aected by an increase in algal total lipid. Surprisingly PCB 105 (log Kow=6.65) at the highest lipid content only increased by a factor of 1.5. This result was obtained as the mean of three individual measurements with a narrow 95% con®dence limit. No explanation may be given for that. For phenanthrene (log Kow=4.57) and 4-chloroaniline (log Kow=2.78), greater levels of algal total lipid content were associated with only a minor increase in BCF. Consistent with a hypothesis introduced by Swackhamer and co-workers, one possible explanation for this result could be that the less hydrophobic compounds (log Kow56) adsorb primarily to the cell membrane, whereas the more hydrophobic substances (log Kow46) partition into the internal lipid pool of the cells. The BCFs found at day 24 (showed only in Table 2) were higher than might be expected, given the low algal total lipid content. Maybe this was due to the circumstance that the average algal volume increased by a factor of 2 before day 24. The contaminant transport can therefore increase signi®cantly, assuming no changes in algal loading, as a consequence of nitrogen starvation. An increase in total algal lipid content from 17 to 44% changed BCFs (dry weight basis) for PCB 153 from log 4.51 to log 5.46 (Table
3). This would result in an increase in transport from 3.2 6 1076 to 2.9 6 1075 mg contaminant liter71 assuming a constant algal loading of 10 mg dry wt liter71 and a contaminant concentration in water of 1075 mg liter71. Lipid-normalized BCFs for phytoplankton are often assumed to be directly proportional to Kow (Thomann and Connolly, 1984; Connolly and Pederson, 1988). Swackhamer and Skoglund (1993) showed that when bioconcentration is lipid normalized, BCFs may exceed octanol/water partition coecients by up to a factor of 100 or more. No explanation is given in the literature for such high lipidnormalized bioconcentration factors. One possible explanation is that dierent classes of lipids may have dierent binding anities and that some chemicals adsorb predominantly to the algal surface (which contains dierent speci®c types of lipids), whereas lipid as normally analyzed represents a summation of all extractable lipid types (Swackhamer and Skoglund, 1993; Stange and Swackhamer, 1994). A straight line with a slope equal to unity was thus not obtained for the superlipophilic compounds as theoretically expected, but as shown in Fig. 4A (see the enlargement of the ®gure between log Kow=5.5 and log Kow=6.8) BCFs found for algae having a total lipid content of 44% were much closer to unity as those having lipid contents of 31.1 and 27.6%, respectively. We hypothesize therefore that an increase in total algal lipid increases the fugacity dierence (or driving force) across the algal cell wall, thus counteracting diusion or other uptake barriers. To test the hypothesis that limited cell wall permeability reduces uptake rates of hydrophobic compounds, we inspected class-speci®c lipid data in relation to BCFs (Table 2). The results of lipid normalization relative to neutral lipids, glycolipids, and phospholipids, respectively, are given in Figs. 4B to 4D. The ratio between these three lipid classes changed greatly during the study. Lipid-class normalization in all cases leads to overestimation of the BCFs, especially when the lipid contents were low. Thus, we found no clear advantage to normalizing BCFs to lipid class compared to normalization to total algal lipids. Normalization with total lipid in fact appeared preferable, due to its simplicity. This ®nding is in agreement with observations in other studies (Swackhamer and Skoglund, 1993). CONCLUSION
Our results show that algal N status, by aecting the lipid content, may in¯uence the bioconcentration factor of superhydrophobic compounds by up to a factor of 8.9. BCFs for PCB 31, PCB 49, PCB 153, and DDT increased with maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively, thus more than theoretically predicted
BIOCONCENTRATION OF HOCs AT DIFFERENT ALGAL NITROGEN STATUS
from the lipid normalization of BCFs obtained at exponential growth phase (17% total lipid), whereas BCFs for PCB 105, phenanthrene, and 4-chloroaniline increased at 44% lipid content only by factors of 1.5, 1.5, and 2.5, respectively, thus less than or equal to the theoretical prediction. These ®ndings may perhaps account for the fact that bioconcentration factors for superhydrophobic compounds in algae are always found to be less than predicted from Kow due to diusional and other resistances across the cell wall/membrane, becoming important only when the fugacity dierences are high. No general advantages were found in lipid-class normalizing BCFs compared to a normalization to total algal lipid. ACKNOWLEDGMENTS Thanks are given to Philipp Mayer for many helpful comments on the manuscript. This research was funded by the Danish Centre for Ecotoxicological Research, Project 7.1, under the Danish Strategic Environmental Research Program 1993±1996.
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