Bioaccumulation of PBDE Congener 2, 2′, 4, 4′-Tetrabromodiphenyl

Oceanological and Hydrobiological Studies International Journal of Oceanography and Hydrobiology

Volume 42, Issue 2

ISSN 1730-413X (139–148) eISSN 1897-3191 2013

DOI: 10.2478/s13545-013-0067-x Received: March 19, 2012 Original research paper Accepted: December 17, 2012

lipid content. log BAFlip for BDE-47 in H. akashiwo ranged from Bioaccumulation of PBDE congener 6.70 to 7.25. The results of this study indicate that variation in 2,2′,4,4′-tetrabromodiphenyl ether by BDE-47 accumulation by H. akashiwo corresponds to the change in cellular lipid content induced by different nitrate and Heterosigma akashiwo in response to phosphate concentrations. different nutrient concentrations INTRODUCTION

Wei Gea, Xundong Yinb, Chao Chaib,∗, Jiao With the rapid population growth, Zhangb industrialization and urbanization, coastal waters are receiving a variety of land-based pollutants, ranging from nutrients (nitrogen, phosphorus) to persistent a College of Life Sciences, Qingdao Agricultural University, organic pollutants (POPs) (Smith 2003, Chiuchiolo et Qingdao 266109, China al. 2004). Among the POPs, polybrominated b College of Resources and Environment, Qingdao diphenyl ethers (PBDEs) have caused serious public Agricultural University, Qingdao 266109, China concern due to their rapidly rising levels in organisms and their environments. PBDEs are organobromine compounds used for more than 30 years to retard Key words: Nutrient availability, POPs flame in many products, including plastics, electrical bioaccumulation, Microalgae, Lipid content and electronic equipment, textiles, building materials, and paint (Rahman et al. 2001). So far, PBDEs are found in fresh and marine water, sediment, shellfish, Abstract mammals, fish, bird eggs, and even in human blood, breast milk and tissues (Sellström et al. 1993, Ohta et Changes in the bioaccumulation of 2,2′,4,4′- al. 2002, Hale et al. 2003, Hites 2004, Oros et al. 2005, tetrabromodiphenyl ether (BDE-47) in the marine alga Suzuki et al. 2006, Law et al. 2006, Borghesi et al. Heterosigma akashiwo (Raphidophyceae) were examined for 2008, Luo et al. 2008). Furthermore, it should be -3 different concentrations of nitrate (0, 128, and 512 μmol dm ) noted that PBDE levels in these samples have and phosphate (0, 8, and 32 μmol dm-3) in the semi-continuous culture with 20% renewal rate. The BDE-47 content per cell and increased exponentially during the last 30 years (Hites per culture, as well as the accumulated percentage of available 2004). BDE-47, presented a significant decreasing trend with the Chinese coastal waters are exposed to the increase in nitrate and phosphate concentrations. The N-0 pollution by PBDEs and nutrients. In Laizhou Bay, (4.0 × 10-6 ng cell-1) and P-0 (5.8 × 10-6 ng cell-1) treatments had significantly higher BDE-47 content per cell than other the PBDE concentrations in plants and shellfish were treatments. In comparison, the difference in BDE-47 per algal in the range of (70 to 5900) ng g-1 lipid and (230 to culture and accumulated percentage between the nitrate 720) ng g-1 lipid, respectively (Jin et al. 2008). The treatments or phosphate treatments was not as obvious as the PBDE levels in sediment and shellfish in Jiaozhou BDE-47 content per cell. BDE-47 per cell presented significantly Bay were 5500 pg g-1 dw and 860 pg g-1 dw, negative correlation with nitrate and phosphate concentrations, and the accumulated BDE-47 was in positive correlation with respectively (Yang et al. 2003). In the Pearl River estuary, the PBDE concentrations in the water ranged from 0.344 to 68.0 ng dm-3, and the yearly ∗ Corresponding author: [email protected]

140 | Wei Ge, Xundong Yin, Chao Chai, Jiao Zhang input of PBDEs into this estuary continued to MATERIALS AND METHODS increase (Guan et al. 2011). In addition to PBDEs, these coastal waters are exposed to serious nitrogen Algal growth and phosphorus pollution (Huang et al. 2003, Liu et al. 2005, Ji et al. 2007). H. akashiwo was obtained from the Institute of Depending on the number and location of Oceanology, Chinese Academy of Sciences. The bromine atoms, PBDEs are formed by a family of axenic stock culture was maintained in log-phase 209 congeners. Lower brominated PBDEs, with less growth in F/2 medium. The stock and experimental than five bromine atoms per molecule, reach higher cultures were kept at 20 ±1°C with a 12:12 h concentrations in aquatic life (De Wit 2002, De Wit light:dark cycle. Illumination of 45 μmol (m2s)-1 was et al. 2006, Wang et al. 2007). Among the lower supplied by cool-white fluorescent tubes. brominated PBDEs, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) was the only substantial compound Experimental conditions in both fish (Meng et al. 2008) and non-fish seafood (Guo et al. 2007) in the coastal area of China, with a At the start of the experiment, H. akashiwo in the relative abundance of about 50% (Guan et al. 2009). log-phase was grown in sterilized N-free and P-free Also, Hites (2004) reported that the level of BDE-47 seawater for 48 h, thereby exhausting N and P in the in marine mammals in the Arctic was higher than media. Next, 2 dm-3 cultures were inoculated into that of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153). 3 dm-3 flasks with sterilized artificial seawater. For the Therefore, we used BDE-47 to observe the experiments on the response to nitrate, three nitrate bioaccumulation of PBDEs by microalgae. treatments were designed as follows: 0, 128, and Microalgae are at the base of the aquatic food 512 μmol dm-3 NaNO3, with an initial phosphate chain. Microalgae can easily accumulate POPs, which concentration for all nitrate treatments of 36 μmol -3 are then transferred to higher trophic levels through dm NaH2PO4. For the experiments on the response the food chain because of their high lipid content to phosphate, three phosphate treatments were and small size. The bioaccumulation of POPs by designed: 0, 8, and 32 μmol dm-3 NaH2PO4, with an microalgae is influenced by the properties of organic initial nitrate concentration for all phosphate compounds (such as molecular structure and treatments of 883 μmol dm-3 NaNO3. Trace metals, hydrophobicity), and by algal lipids, which are vitamins and other nutrients in all treatments were variable between species and influenced by added according to the F/2 medium. The algal environmental factors (Stange & Swackhamer 1994, cultures were semi-continuous with daily 20% Berglund et al. 2000, Carlson & Swackhamer 2006). changes in the medium. The 0.4 dm-3 algal cultures Several studies reported the bioconcentration of were renewed every 24 h with sterilized artificial POPs, such as PCBs, by microalgae in nutrient- seawater containing different nitrate (0, 128, and limited conditions (Halling-Sørensen et al. 2000, 512 μmol dm-3) and phosphate (0, 8, and 32 μmol Datta 2001, Lynn et al. 2007). However, few studies dm-3) concentrations. The experiments were done in have examined the nutrient concentration-induced triplicate for each treatment. change in the bioaccumulation of PBDE. Heterosigma akashiwo is a bi-flagellated, single- BDE-47 exposure celled, golden brown alga in the Raphidophyceae family. With the increasing nitrogen and phosphorus A 0.4 × 10-3 dm-3 stock solution of BDE-47 levels in coastal waters, H. akashiwo forms massive (50 mg dm-3, AccuStandard Inc) was diluted in 0.1 harmful algal blooms (Khan et al. 1997). The present dm-3 methanol, resulting in a 0.2 mg dm-3 working study focused on the relationship between the solution. After the daily media renewal on the fifth bioaccumulation of BDE-47 by H. akashiwo, and day of cultivation, each algal culture flask was concentrations of nitrogen (N) and phosphorus (P) inoculated with 0.002 dm-3 BDE-47 working solution in the media. The effects of different nitrate and (0.2 mg dm-3). The BDE-47 concentration in the phosphate concentrations on the growth, cellular algal cultures was 0.2 μg dm-3. The exposure of H. biochemical composition, and bioaccumulation of akashiwo to BDE-47 persisted up to 24 h. BDE-47 by H. akashiwo were investigated.

Bioaccumulation of PBDE congener 2,2′,4,4′-tetrabromodiphenyl ether by Heterosigma akashiwo| 141

Growth parameters and nutrients extracts through a multilayer silica/alumina column according to Luo et al. (2008). Extracts were eluted Before the daily media renewal, 0.02 dm-3 of the with 1:1 hexane:dichloromethane, and then were algal culture was obtained and fixed with Lugol's concentrated to 0.1 × 10-3 dm-3. To serve as an solution to determine the cell density. The growth internal standard, 0.010 × 10-3 dm-3 of 2,2',4,5',6- rate (μ, d-1) was estimated by the equation: pentachlorobiphenyl (PCB-103, 1 mg dm-3) was added to all extracts. Chromatographically pure grade ln solvents were used in BDE-47 extraction and analysis. = Samples were analyzed by a gas chromatograph 푁푡 � � �� (GC, Agilent 6890N), equipped with an electron- 푁0 휇 capture detector and HP-5 column (30 m × 0.25 mm where Nt and N0 are the final푡 and initial cell density, i.d., 0.25 µm film thickness). The oven temperature respectively; and t is the incubation time (d) (Landry program was operated as follows: initial temperature & Hassett 1982). Another ~0.1 dm-3 algal culture was of 150°C for 1 min, followed by a rate of 40°C min-1 filtered through a 0.45 μm micro-pore filter to 250°C, then by a rate of 10°C min-1 to 300°C, membrane, and the filtrate was used for measuring with a final temperature of 300°C for 7 min. The nitrate and phosphate concentrations. Nitrate injector temperature was 300°C. The GC was concentration was determined using the cadmium- operated in the splitless injection mode (Fontana et al. copper reduction method (Grasshoff 1976). 2009). Phosphate concentration was determined using Quantitation was done using the internal standard phosphomolybdenum blue (Strickland & Parsons method. For every 10 samples, a standard was 1972). Nutrient concentrations were analyzed after measured to confirm the calibration curve. daily sampling. Procedural blanks and surrogate standards were performed for quality assurance or quality control. Protein, carbohydrate, and lipid The levels of BDE-47 in procedural blanks corresponded closely to the limit of quantification, After 24 h exposure to BDE-47, 0.2 dm-3 of the and they were not subtracted from those in samples. algal culture was collected onto fiberglass Whatman The recoveries of surrogate standards were GF/F filters (pre-combusted at 450°C for 7 h). 83% ±9%. The BDE-47 results were reported with Samples were stored at -20°C before the analysis of no surrogate recovery correction. protein, carbohydrate, and lipid content. Protein was extracted according to Rausch (1981), and quantified Statistical analysis using the Coomassie Brilliant Blue dye-binding method, with bovine albumin as the standard The lipid-normalized bioaccumulation factor (Bradford 1976). Carbohydrate content was (BAFlip) was derived from Eq. (1) (Lynn et al. 2007). measured using the phenol-sulfuric acid method, with D-glucose as the standard (Kochert 1978). Lipid μg BDE-47/dm-3algal culture was extracted according to Bligh & Dyer (1959), and kg lipid/dm 3- algal culture BAF = (1) measured according to Pande et al. (1963), with lip μg BDE-47/dm 3- media palmitic acid as the standard. where the mass of BDE-47 in the media was BDE-47 analysis obtained by subtracting the mass of BDE-47 in the algal phase from the nominal whole-flask BDE-47 A 0.25 dm-3 aliquot of the algal culture was mass. filtered through fiberglass Whatman GF/F filters The differences were analyzed by ANOVA (LSD (pre-combusted at 450°C for 7 h). The filters were test), and P < 0.05 was regarded as significant. frozen before BDE-47 extraction and analysis. All Correlation analysis was performed with the samples received surrogate standards (50 ng Spearman correlation. The software SPSS 16.0 was 2,2',4,4',5,5'-hexabromodiphenyl ether) and were used for the statistical analyses. Soxhlet extracted for 48 h in hexane with activated copper added to a flask for desulphurization. Interferences were manually removed by passing the

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142 | Wei Ge, Xundong Yin, Chao Chai, Jiao Zhang

RESULTS 1000 40

(a) P-0 ) -3 ) -3 30 Nutrient concentrations 800 20 Concentrations of nitrate and phosphate in all 600 treatments decreased during the cultivation period 10 Nitrate (μmol dm (μmol Nitrate (Figs. 1 and 2). On the fifth day, nitrate 400 0 dm (μmol Phosphate concentrations in N-0, N-128, and N-512 treatments 1000 40 (b) P-8 ) -3 -3 ) were 0.4, 12.7, and 378.0 μmol dm , respectively; and -3 30 phosphate concentrations in all nitrate treatments 800 ranged from 15.1 μmol dm-3 to 24.8 μmol dm-3. 20 Justic et al. (1995) proposed that thresholds for 600 10 dissolved inorganic nitrogen (DIN) and phosphate of Nitrate (μmol dm (μmol Nitrate phytoplankton growth were 1 μmol dm-3 and 400 0 dm (μmol Phosphate 0.1 μmol dm-3, respectively. According to these 1000 40 (c) P-32 ) -3 ) thresholds, cultures in the N-0 treatment were -3 30 limited by nitrogen, but there was no phosphorus 800 limitation in all nitrate treatments. 20 600 On the fifth day of cultivation, phosphate 10 concentration in the P-0 treatment was undetectable, Nitrate Nitrate (μmol dm (μmol Nitrate and 1.5 μmol dm-3 and 8.2 μmol dm-3 in P-8 and P- 400 Phosphate 0 dm (μmol Phosphate 32 treatments, whereas the values for nitrate 0 1 2 3 4 5 concentration in all phosphate treatments ranged Time (d) from 726.4 μmol dm-3 to 766.0 μmol dm-3. According to Justic et al. (1995), the algae growth Fig. 2. Changes in nutrient concentrations in H. was severely limited by phosphorus in the P-0 akashiwo cultures in different phosphate conditions. treatment, and no nitrate limitation was found in all phosphate treatments. Algal growth

(a) N-0 40 ) There was no significant difference in the cell -3 ) 500

-3 400 30 density between different nitrate treatments on the

300 20 first day of cultivation (P > 0.05) (Fig. 3). On the 200 fifth day, the cell densities in the N-128 and N-512 10 treatments were 11.3 × 107 cells dm-3 and Nitrate (μmol dm (μmol Nitrate 100 0 dm (μmol Phosphate 7 -3 0 13.3 × 10 cells dm , respectively, and both were

(b) N-128 40 ) -3 ) 500 significantly higher than in the N-0 treatment -3 400 30 (P < 0.05). The growth rate of H. akashiwo in the N-0 -1 300 20 treatment was 0.05 d on the fifth day, which is -1 200 significantly lower than in the N-128 (0.20 d ) and 10 -1

Nitrate (μmol dm (μmol Nitrate 100 N-512 (0.25 d ) treatments (P < 0.05) (Fig. 4). 0 dm (μmol Phosphate 0 With the nitrate treatments, the algae growth in

(c) N-512 40 ) -3 ) 500 phosphate treatments presented the same trend. The -3 400 30 cell densities on the fifth day reached 9.7 × 107 cells -3 7 -3 300 20 dm and 12.0 × 10 cells dm in the P-8 and P-32 200 10 treatments, respectively, which are about three to Nitrate Nitrate (μmol dm (μmol Nitrate 100 four times as high as that in the P-0 treatment (Fig. Phosphate 0 dm (μmol Phosphate 0 3). The algal growth rate on the fifth day was 0.24 0 1 2 3 4 5 and 0.25 d-1 in the P-8 and P-32 treatments, Time(d) respectively, whereas in the P-0 treatment − 0.001 d-1

(Fig. 4). The average growth rate (μave) and the Fig. 1. Changes in nutrient concentrations in H. maximum growth rate (μmax) of H. akashiwo during akashiwo cultures in different nitrate conditions. the cultivation period increased with the rising nitrate

Bioaccumulation of PBDE congener 2,2′,4,4′-tetrabromodiphenyl ether by Heterosigma akashiwo| 143

and phosphate concentrations (Fig. 5). H. akashiwo

) -1 -3 15 N-0 reached μmax in the N-512 treatment (0.82 d ) and in N-128 the P-32 treatment (0.74 d-1). Spearman correlation 12 N-512 analysis showed that cell densities and growth rates cells dm 7 9 were significantly correlated with concentrations of nitrate and phosphate (P < 0.05). 6

3 1.2 μave Density (10 Density (a) Nitrate

) μmax in nitrate treatments 0 -1

) 0.9 μmax in phosphate treatments

-3 15 P-0 P-8 12 P-32 0.6 cellsdm

7 9 Growth rateGrowth (d 0.3 6 0.0 3 0 128 512 0 8 32

Density (10 Density (b) Phosphate Nitrate Phosphate 0 Nutrient concentration (μmol dm-3) 0 1 2 3 4 5

Time (d) Fig. 5. The average growth rate (μave) and the maximum growth rate (μmax) of H. akashiwo in different nitrate Fig. 3. The growth curve of H. akashiwo in different and phosphate conditions. nitrate and phosphate conditions.

Biochemical composition

(a) Nitrate 1.0 N-0 With the increasing nitrate concentrations, the

) N-128 total protein level per cell and per culture presented a -1 0.8 N-512 significantly increasing trend (P < 0.05) (Fig. 6a). The 0.6 total protein level per cell increased from 27.7 × 10-9 mg cell-1 in the N-0 treatment to 44.4 × 10-9 mg 0.4 cell-1 in the N-512 treatment. The total protein level -3

Growth rateGrowth (d 0.2 per culture increased from 1.2 mg dm to 5.9 mg dm-3, respectively. There was no significant 0.0 difference in the total protein level per cell between 1.0 (b) Phosphate P-0 different phosphate concentrations. In contrast, the

) P-8 total protein per culture in the P-32 treatment -1 0.8 P-32 (5.3 mg dm-3) was significantly higher than in the P-0 0.6 treatment (1.3 mg dm-3) (P < 0.01). 0.4 The cellular carbohydrate levels decreased with increasing nitrate concentration (Fig. 6b). The cellular -9 -1 Growth rateGrowth (d 0.2 carbohydrate level was 8.6 × 10 mg cell in the N-0 0.0 treatment, significantly higher than that in the N-128 (4.2 × 10-9 mg cell-1) and N-512 (3.4 × 10-9 mg cell-1) 1 2 3 4 5 treatments. The carbohydrate level per culture -3 Time (d) presented a different trend since it was 0.38 mg dm in the N-0 treatment, which is significantly lower Fig. 4. The growth rate of H. akashiwo in different compared to the N-128 (0.47 mg dm-3) and N-512 nitrate and phosphate conditions. (0.45 mg dm-3) treatments. Similar to the nitrate treatments, the cellular carbohydrate levels decreased with increasing phosphate concentration. The cellular www.oandhs.org

144 | Wei Ge, Xundong Yin, Chao Chai, Jiao Zhang

accumulated percentage of available BDE-47, (a) Protein per cell 8 60 per culutre presented a significantly decreasing trend with ) -1 increasing nitrate concentration (P < 0.05) (Fig. 7). 6 ) 40 -3 The BDE-47 content per cell was 4.0 × 10-6 ng cell-1

mg cell 4 -9 in the N-0 treatment, nearly three to four times (mg dm

(10 20 2 higher than in the N-128 and N-512 treatments. The Total protein per cell

Total protein per culture difference in the accumulated percentage between 0 0 (b) Carbohydrate per cell the nitrate treatments was not as obvious as the 12 0.8 ) per culutre -3 ) per cell

-1 BDE-47 content per cell. The accumulated 9 0.6 percentage of BDE-47 in the N-0 and N-128

mg cell 0.4

-9 6 treatments was 87.5% and 83.0%, respectively, higher carbohydrate (10

carbohydrate than the N-512 treatment (P < 0.05). BDE-47 per 3 0.2 Total per culture(mg dm algal culture presented a similar trend to the Total 0 0.0 accumulated percentage. (c) Lipid per cell 1.6 30 per culutre ) ) -1

-1 8 1.2 ) -3 (a) per cell 20 per culture

mg cell 0.8 lipid -9 ng cell lipid 6 (mg dm -6

(10 10 0.4 Total Total 4 0 0.0 0 128 512 0 8 32 Nitrate Phosphate 2 Nutrient concentration (μmol dm-3)

BDEcell per (10 0 Fig. 6. The biochemical composition of H. akashiwo in 100 (b) different nitrate and phosphate conditions. 80 60 carbohydrate level in the P-0 treatment was about two to three times higher than in the P-8 and P-32 40 treatments. 20 The total lipid content per cell presented a of available BDE (%) Accumulated percentage significantly declining trend with increasing nitrate 0 ) and phosphate concentrations (Fig. 6c). The total -3 0.20 (c) lipid content per cell was 22.5 × 10-9 mg cell-1 in the N-0 treatment, nearly three and sixteen times higher 0.15 than in the N-128 and N-512 treatments, respectively. 0.10 The P-0 treatment had the highest total lipid content with 25.1 × 10-9 mg cell-1, which is significantly 0.05 higher than in the P-8 (11.6 × 10-9 mg cell-1) and P-32

-9 -1 dm BDE per culture (μg 0.00 (1.4 × 10 mg cell ) treatments. As for the total lipid 0 128 512 0 8 32 -3 content per culture, values for the N-0 (0.99 mg dm ) Nitrate Phosphate -3 and N-128 (0.98 mg dm ) treatments were Nutrient concentration (μmol dm-3) significantly higher than in the N-512 treatment -3 (0.19 mg dm ). The P-8 treatment had the highest Fig. 7. The bioaccumulation of BDE-47 by H. akashiwo lipid content per culture compared to the three in different nitrate and phosphate conditions. phosphate treatments.

A similar trend was also observed in the Bioaccumulation of BDE-47 bioaccumulation of BDE-47 using different phosphate treatments (Fig. 7). The P-0 treatment had The bioaccumulation of BDE-47 varied with different nutrient concentrations. The BDE-47 significantly higher BDE-47 content per cell -6 -1 -6 -1 content per cell and per culture, as well as the (5.8 × 10 ng cell ) than the P-8 (1.9 × 10 ng cell ) and P-32 (1.3 × 10-6 ng cell-1) treatments. The P-0

Bioaccumulation of PBDE congener 2,2′,4,4′-tetrabromodiphenyl ether by Heterosigma akashiwo| 145 and P-8 treatments had the highest accumulated lipids per culture of H. akashiwo (Table 2). percentage of 92.1% and 93.5%, respectively, which log BAFslip for BDE-47 in H. akashiwo ranged are significantly higher than those of the P-32 from 6.70 to 7.25 in all treatments (Table 3) with the treatment (73.1%) (P < 0.01). highest value in the N-512 treatment and the lowest In nitrate treatments, BDE-47 per cell, per culture, in the N-128 treatment for nitrate concentrations, and accumulated percentage in H. akashiwo presented and the highest in the P-32 treatment and the lowest a significantly negative correlation with nitrate in the P-8 treatment for phosphate concentrations. concentration. BDE-47 per cell had a positive correlation with lipids per cell; whereas BDE-47 per DISCUSSION culture and accumulated percentage were positively correlated with lipids per culture of H. akashiwo The accumulated amount of POPs by microalgae (Table 1). In phosphate treatments, BDE-47 per cell varies with nutrient availability. The bioaccumulated was significantly negatively correlated with phosphate 2,2’,6,6’-tetrachlorobiphenyl (PCB-54) content per concentration and positively correlated with lipids Stephanodiscus minutulus cell (Lynn et al. 2007) and per cell. BDE-47 per culture and accumulated 2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB-101) per percentage were not correlated with phosphate Cyclotella meneghiniana cell in silica-, phosphorus-, and concentrations, but were positively correlated with nitrogen-limited media were higher than in non-

Table 1

Spearman correlation for the nitrate concentration, biochemical composition and BDE-47 content in H. akashiwo. Nitrate Protein per Protein per Carbohydrate Carbohydrate Lipid per BDE-47 per BDE-47 per Accumulated Lipid per cell concentration cell culture per cell per culture culture cell culture percentage Nitrate concentration 1.000 Protein per cell 0.949** 1.000 Protein per culture 0.949** 0.967** 1.000 Carbohydrate per cell -0.900** -0.812** -0.862** 1.000 Carbohydrate per culture 0.635 0.711* 0.611 -0.328 1.000 Lipid per cell -0.949** -0.867** -0.867** 0.795* -0.695* 1.000 Lipid per culture -0.738* -0.717* -0.650 0.494 -0.594 0.850** 1.000 BDE-47 per cell -0.949** -0.900** -0.967** 0.946** -0.460 0.867** 0.600 1.000 BDE-47 per culture -0.896** -0.900** -0.933** 0.879** -0.561 0.867** 0.683* 0.933** 1.000 Accumulated percentage -0.896** -0.900** -0.933** 0.879** -0.561 0.867** 0.683* 0.933** 1.000** 1.000 * Correlation is significant at the 0.05 level (2-tailed) ** Correlation is significant at the 0.01 level (2-tailed)

Table 2

Spearman correlation for the phosphate concentration, biochemical composition and BDE-47 content in H. akashiwo. Phosphate Protein per Protein per Carbohydrate Carbohydrate Lipid per BDE-47 per BDE-47 per Accumulated Lipid per cell concentration cell culture per cell per culture culture cell culture percentage Phosphate concentration 1.000 Protein per cell 0.422 1.000 Protein per culture 0.791* 0.517 1.000 Carbohydrate per cell -0.949** -0.300 -0.833** 1.000 Carbohydrate per culture 0.158 0.150 0.600 -0.167 1.000 Lipid per cell -0.953** -0.510 -0.812** 0.929** -0.192 1.000 Lipid per culture -0.476 -0.259 -0.025 0.444 0.728* 0.479 1.000 BDE-47 per cell -0.896** -0.417 -0.917** 0.883** -0.400 0.828** 0.226 1.000 BDE-47 per culture -0.527 -0.233 -0.150 0.467 0.517 0.485 0.854** 0.417 1.000 Accumulated percentage -0.527 -0.233 -0.150 0.467 0.517 0.485 0.854** 0.417 1.000** 1.000 * Correlation is significant at the 0.05 level (2-tailed) ** Correlation is significant at the 0.01 level (2-tailed)

Table 3

BAFlip and log BAFlip for BDE-47 in H. akashiwo in different nitrate and phosphate conditions. Concentration BAF log BAF Nutrients lip lip (μmol dm-3) (106(μg kg−1) (μg dm-3)-1) (μg kg−1) (μg dm-3)-1) 0 7.05 6.85 Nitrate 128 4.97 6.70 512 13.14 7.12 0 14.66 7.17 Phosphate 8 12.86 7.11 32 17.97 7.25

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146 | Wei Ge, Xundong Yin, Chao Chai, Jiao Zhang limited media (Datta 2001). Moreover, the amount of concentrations (0.20 d-1 to 0.25 d-1) (Fig. 4). tritiated polychlorinated dibenzofurans (PCDFs) Therefore, the effect of the growth status on the accumulated by Nitzschia sp. in silica-limited media bioaccumulated amount of BDE-47 per H. akashiwo was higher than in non-limited and phosphorus- cell may not be excluded. However, the growth rate limited conditions (Kilham 1998). In this study, H. of microalgae in previous studies was obtained by akashiwo limited by nitrogen and phosphorus in the different cultivation temperatures and not by N-0 and P-0 treatments exhibited the highest different nutrient concentrations, as in this study. In accumulated level of BDE-47 per cell (Fig. 7a), which addition, the microalgae in previous studies were is consistent with previous studies. The declining grown in the media with POPs for more than trend in the accumulated amount of BDE-47 in H. 20 days, whereas in this study, the exposure time of akashiwo cells was found with increasing nitrate and H. akashiwo to BDE-47 was only one day. Therefore, phosphate concentrations. Changes in the growth dilution could not be a major element in bioaccumulation through different nutrient the reduction of the bioaccumulated amount of treatments may result from the biochemical BDE-47 in H. akashiwo cells. composition of algae. The biochemical composition, Highly hydrophobic compounds (log Kow >6) including cellular protein, carbohydrate and lipid were transported to a lesser extent within plants levels, varied in microalgae with nutrient availability. compared to less hydrophobic compounds (log The declining levels of nitrogen and phosphorus in Kow<6) (Cunningham & Berti 1993). In general, the the media inhibited protein synthesis, but induced lipid-normalized BAF for microalgae was a linear the storage of carbohydrates and lipids in microalgae function of Kow with a slope close to 1 when log (Fogg 1959, Berdalet et al. 1994, Kilham et al. 1997, Kow < 6. Meanwhile, the lipid-normalized BAF was Lourenço et al. 1997, Li et al. 2005, Zhao et al. 2009, less than Kow when log Kow > 6 (Stange & Lai et al. 2011). The lipid content per H. akashiwo cell Swackhamer 1994, Gerofke et al. 2005). If there is exhibited a reduction with the increase of initial equilibrium between the hydrophobic organic nitrate and phosphate concentrations (Fig. 6c), which chemicals partitioning the water and cell lipids, the was consistent with other related research. However, linear relationship between BAF and Kow exists when as influenced by the cell density, the lipid content per log Kow is close to 7 (Swackhamer & Skoglund 1993). algal culture did not decease in phosphate treatments The values for BAFlip in this study ranged from 6.70 (Fig. 6c). Correlation analysis indicated that the to 7.25 (Table 3), corresponding with the reported BDE-47 level per H. akashiwo cell had a positive log Kow of BDE-47 (6.81). However, in one of our correlation with the lipid level per H. akashiwo cell, experiments on the BDE-47 bioaccumulation by a similar to the BDE-47 level per culture and lipid level dinoflagellate, Prorocentrum donghaiense, with the same per culture (Tables 1 and 2). The results in this study nitrate and phosphate treatments as in this study, the indicate that the accumulated amount of BDE-47 in values for log BAFlip ranged from 5.63 to 6.30, which H. akashiwo cells varies with changes in the quantity is lower than the currently reported log Kow of BDE- of lipids, which are induced by the nutrient 47. Also Swackhamer (1985) found in a field concentrations. experiment that BAFs for PCBs in diatom-dominant Besides the nutrients, the growth status of communities were lower than in non-diatoms. The microalgae also affected the bioaccumulation of difference may result from algae species, which differ POPs. Previous studies found that the quantity of in morphology, structure, and lipid composition. The lipids was a major factor in the bioaccumulation of absorption amount of PCBs in Nitzschia closterium was POPs by microalgae at low growth rates, whereas higher than in Chlorella sp. due to larger surface area bioaccumulation was reduced due to growth dilution of N. closterium cells (Fang 2006). H. akashiwo cells when microalgae were grown at active growth range in size from 18 to 34 μm in diameter, which is conditions (Swackhamer & Skoglund 1993). In this larger than P. donghaiense (10 μm × 10 μm), and so the study, the average and maximum growth rate of H. surface area of H. akashiwo is greater than that of P. akashiwo during the cultivation period increased with donghaiense. Furthermore, H. akashiwo had no cell wall, the increasing initial nitrate and phosphate which may facilitate the diffusion of hydrophobic concentrations (Fig. 5). The growth rate on the fifth compounds into the cell. Therefore, their day of algal cultivation was 0.05 d-1 and 0.001 d-1 in morphology and structure helped H. akashiwo cells to N-0 and P-0 treatments, respectively, obviously lower accumulate BDE-47. In addition, the lipid than that under higher nitrate and phosphate composition of algae also affected BAFs. Algal lipids

Bioaccumulation of PBDE congener 2,2′,4,4′-tetrabromodiphenyl ether by Heterosigma akashiwo| 147 consist of neutral lipids, glycolipids and Datta, S. (2001). Relationship between PCB uptake and nutrient phospholipids (Thompson 1996), which are different limitation in three algal species. Unpublished doctoral dissertation, Drexel University, Philadelphia, PA, USA. in different algae species and are affected by De Wit, C.A. (2002). An overview of brominated flame nutritional conditions (Khozin-Goldberg & Cohen retardants in the environment. Chemosphere, 46(5), 583-624. 2006, Merzlyak et al. 2007). There is a difference in De Wit, C.A., Alaee, M. & Muir, D.C.G. (2006) Levels and the total lipids, neutral lipids, glycolipids, and trends of brominated flame retardants in the Arctic. Chemosphere, 64(2), 209-233. phospholipids of normalized BAFs for PCBs by Fang K. (2006). Studies on the adsorption of PCBs to marine microalgae. microalgae (Halling-Sørensen et al. 2000). 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