- Email: [email protected]
Biosynthesis of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) from red alga—Bangia atropurpurea
ARTICLE IN PRESS
Water Research 38 (2004) 1014–1018
Biosynthesis of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) from red alga—Bangia atropurpurea Chih Yu Chen* Department of Tourism, Hsing Wu College, No. 11-2, Fen-Liao Road, Lin-Kou, Taipei County, Taiwan 244-41, Republic of China Received 17 September 2002; received in revised form 30 October 2003; accepted 10 November 2003
Abstract The contents of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) of red alga, Bangia atropurpurea, filaments cultured in artificial sea water medium were similar to those cultured in natural sea water medium. In the culture experiment, B. atropurpurea filaments were found to synthesize de novo phthalate esters. Additionally, DEHP and DBP contents in different species of algae grown in the same environment were different significantly, suggesting that it was due to the intrinsic nature of algae. r 2003 Elsevier Ltd. All rights reserved. Keywords: DEHP; DBP; Bangia atropurpurea; De novo synthesis
1. Introduction Phthalate esters have been widely applied as plasticizers or solvents in chemical industry [1,2]. Among them, di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) are most commonly used [3]. DEHP and DBP are pollutants released from plastic wares to the environment or in the bag-stored blood [4,5]. Many findings of phthalate esters have been reported in nature, such as sediments, natural waters, soils, plants, and aquatic organisms [6–11]. Phthalate esters pose analytical challenges since phthalate esters are present in many laboratory products, tools, reagents, solvents and supplies [12]. What remains unclear, however, is whether some of these substances are naturally occurring or originate from some commercial processes and subsequently, enter the ecosystem. In a screen of survey of the lipid-soluble components of seaweed commonly found on the northern coast of *Tel.: +886-2-2601-53-10X430; fax: +886-2-2601-5310X439. E-mail address: [email protected] (C.Y. Chen).
Taiwan, the two compounds mentioned above were found in relatively large amount in all the seaweed samples studied [13]. Furthermore, the occurrence of phthalate esters in algae has been reported previously [14–16]. In order to understand the origin of phthalate esters, we cultured red alga, Bangia atropurpurea, filaments in an artificial sea water (ASW) medium (free from the possible solvent transferred contamination and environment impurities accumulation) and a natural sea water (NSW) medium and analyzed the contents of DEHP and DBP in both the plants. Furthermore, B. atropurpurea filaments were cultured in a medium containing NaH14CO3. After two weeks, the radioactivity of DEHP and DBP fractionated by HPLC from cultured filaments was analyzed. However, in order to know whether environmental factor or the intrinsic nature of algae will affect the phthalate esters level in algae, DEHP and DBP contents of seaweed collected from different coasts were analyzed, and we also cultured the filaments of three different species of algae in the same laboratory condition to determine the DEHP and DBP contents in them.
0043-1354/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2003.11.029
ARTICLE IN PRESS C.Y. Chen / Water Research 38 (2004) 1014–1018
2. Materials and methods 2.1. Algal specimens and culture condition Stock culture of the filamentous phase of B. atropurpurea (Roth) C. Agardh was kindly given by Dr Y.M. Chiang, Institute of Oceanography, National Taiwan University. Filaments were propagated by fragmentation and grew into colonies of filamentous clusters in SWMIII medium [17]. The filamentous plants (‘Conchocelis’) were maintained at 20 C under fluorescent light and illuminated with continuous light of 60 mE m2 s1. 2.2. DEHP and DBP standard DEHP and DBP standards were purchased from Fluka (Switzerland). Serially diluted chloroform solutions were prepared. 0.5 ml of each solution was injected into the GC-MS column to determine the system sensitivity and linear range. Measurements were taken in triplicate and values of the peak areas were recorded as GC-MS responses to the different amounts of authentic DEHP and DBP injected. A linear regression line was plotted to show the correlation between peak areas and injected amounts of authentic standards. 2.3. De novo synthesis of DEHP and DBP 2.3.1. The comparison of NSW and ASW media NSW Medium cultured group: harvested filaments were cultured in SWM-III medium, and algae were collected with a plankton net. Removal of excess water was done by filtration on a Buchner funnel using suction. The wet weights were measured (1.20 g; 0.84 g)ðn ¼ 2Þ and then lyophilized overnight to determine the dry weights (0.1940 g; 0.1457 g). ASW Medium cultured group: The wet weight of harvested filaments cultured in SWM-III medium were measured (1.22 g; 1.25 g)ðn ¼ 2Þ; and the filaments were washed several times with ASW-III [18]. The ASW medium contained deionized water and inorganic salts. After a month of incubation, plants were collected and the wet weights were measured (3.05 g; 3.10 g), then lyophilized overnight to determine the dry weight (0.5524 g; 0.5534 g). The dry filaments of the alga cultured in NSW and ASW media were extracted by supercritical fluid extraction (SFE) method [13]. The extract was dissolved in a small volume of chloroform and the contents of DEHP and DBP were analyzed by GC-MS. 2.3.2. Culture the filaments with radioactive carbon source First, the filaments cultured in SWM-III medium were harvested, wet weight was measured (1.80 g) and then inoculated into fresh SWM-III medium. Second, 3.2 mg
1015
of NaH14CO3 (250 mCi, 6.8 mCi/mmole, Sigma, Paris, France) was added into the medium. The filaments were cultured for two weeks (without aeration). When collected, the wet and dry weights of the filaments were 2.22 and 0.3443 g, respectively. The algae were extracted by a high pressure Soxhlet extractor (SFE), and then DEHP and DBP were purified by HPLC system. 2.4. SFE and HPLC, GC-MS analysis condition SFE was carried out using high pressure Soxhlet extractor (J & W Scientific Comp., NJ, USA) under 500 psi at 55 C for 3 h. The condenser temperature was 15 C, and the supercritical solvent used was CO2. HPLC was run by two Hitachi L-6000 pumps and one L-5000 LC controller. DEHP and DBP were detected by a Hitachi L-4200 UV-VIS detector (Tokyo, Japan). The column used was a 5 mm, 4.6 250 mm2 Econosil column at a flow rate of 1 ml/min. Mobile phase was isocratic CHCl3/hexane=3:7. Elutes were monitored at 280 nm and the DEHP and DBP fractions were collected from the comparative retention time of DEHP and DBP standards. The radioactive DEHP and DBP were counted by liquid scintillation counter (Beckman LS5801, IL, USA) and further identified by GC-MS. Analysis of DEHP and DBP were carried out using HP 5890 series II GC and HP 5971 MSD. The GC-MS column was an Ultra-2 fused silica capillary column (25 m 0.2 id). Column temperature range was programmed from 160 C to 240 C. The carrier gas (helium) flow rate was 31 cm/s, injector and detector temperatures were 250 C and 280 C, respectively. The electron impact mass spectrum (70 eV) of DEHP showed the molecular ion at M=z 390, other important ions were detected at M=z 279, 167, and 149 (base peak); DBP showed the molecular ion at M=z 278, other important ions were detected at M=z 223, 205, 167, and 149 (base peak). 2.5. DEHP and DBP contents of algae collected from different polluted area Three species of algae—Ulva fasciata, Enteromorpha intestinalis and Porphyra angusta, which grew in three different neighbor coasts (A, B, C) of northern Taiwan were collected. All species of the algae were collected twice as duplicate. Collected algae were lyophilized, extracted by a Soxhlet extractor (SFE), the supercritical solvent was CO2, and the contents of DEHP and DBP were analyzed by GC-MS. 2.6. DEHP and DBP contents of algae cultured at the same environment Stock cultures of red algae B. atropurpurea, P. angusta and P. dentata filaments were blended into fragments
ARTICLE IN PRESS 1016
C.Y. Chen / Water Research 38 (2004) 1014–1018
and then inoculated into fresh flasks. The filaments were cultured under no aeration, cultured conditions were the same as previous, but temperature was 23 C. After three weeks of incubation, the filaments were harvested and lyophilized, extracted by Soxhlet extractor (SFE). The contents of DEHP and DBP were analyzed by GC-MS.
3. Results and discussion Quantitative analysis of DEHP and DBP showed good linear relationship between the GC-MS response (peak area on chromatograms) and the amount of injected authentic standard. A regression line of DEHP was plotted as Y ¼ 7:766X þ 112:667 ðr2 ¼ 0:964Þ and DBP was plotted as Y ¼ 20:322X 69:16 ðr2 ¼ 0:981Þ; where X represents the amount of the standard in nanograms, and Y represents the peak area ð104 Þ: The comparison of DEHP and DBP contents of B. atropurpurea filaments cultured in NSW and ASW media is shown in Table 1. The contents of DEHP and DBP in NSW cultured filaments were similar as those of ASW cultured filaments (at time zero). After one month incubation, the biomass of the algae cultured in ASW medium increased 2.5 fold (ASW 1) and 2.48 fold (ASW 2), respectively. The data showed that DEHP and DBP contents were similar in ASW and NSW medium cultured groups. At the beginning of the test, we found the concentrations of DEHP in NSW and ASW media were less than 0.4% DEHP of total algae DEHP. However, DBP was not found in both the media. The result proves that DEHP and DBP were not impurities taken by the algae from the environment. In the radioactive compound labeled experiment, we used NaH14CO3 as the carbon source of B. atropurpurea
filaments. Fig. 1 showed the HPLC chromatogram of DEHP and DBP. The first peak fraction of which yellow color quenched easily, showed no radioactivity. We collected two peak fractions of DEHP and DBP, then analyzed by a scintillation counter and GC-MS. The average radioactivity showed that single peak fractions of DEHP (160.00 cpm) and DBP (4786.67 cpm) have significant higher radioactivities than the background (28.00 cpm). We also analyzed the radioactivities of other compounds whose retention times were earlier than DEHP. We found the radioactivity range was from
Fig. 1. HPLC chromatograms and radioactivities of DEHP and DBP purified from B. atropurpurea.
Table 1 The DEHP and DBP contents of B. atropurpurea filaments cultured in ASW and NSW media ASW medium cultured
DEHP experiment Algae dry weight (before cultivation) (g) Algae dry weight (after cultivation) (g) SFE crude extract (g) Calibrated weight (ng)
1
2
1
2
0.2186 0.5524 0.0042 95.3871.30
0.2220 0.5534 0.0044 97.0271.43
— 0.1940 0.0016 33.8470.83
— 0.1457 0.0014 25.2770.41
Av. content (mg/g) DBP experiment Algae dry weight (before cultivation) (g) Algae dry weight (after cultivation) (g) SFE crude extract (g) Calibrated weight (ng) Av. content (mg/g)
NSW medium cultured
69.6070.76
0.2186 0.5524 0.0042 98.0072.36 70.6070.52
0.2220 0.5534 0.0044 97.1670.44
69.5870.28
— 0.1940 0.0016 34.2671.07
— 0.1457 0.0014 25.3970.66
70.1670.66
ARTICLE IN PRESS C.Y. Chen / Water Research 38 (2004) 1014–1018
65.35 to 1350.45 cpm, which showed that radioactive 14C also labeled other algal organic compounds. Furthermore, the HPLC fractions of DEHP and DBP were subjected to GC-MS, each fragment of the single compound mass spectrum matches DEHP and DBP, respectively. Thus the possibility that radioactivities originated from other compounds was eliminated. The results indicate that algae incorporate 14C as the carbon source to de novo synthesize phthalate esters. This is also an evidence that DEHP and DBP were biosynthesized by algae. The concentrations of DEHP in the water from where the samples were collected (northern Taiwan coasts) were 0.2570.02% of total algae DEHP content. However, no DBP was found in water samples of the three locations. The contents of DEHP and DBP varied in the same species of algae collected from different polluted coasts (Table 2). One explanation was possible based on the environment effect, namely, the algae grew in a more polluted coast might accumulate higher phthalate esters than those that grew in a cleaner coast. However, in our results, we did not find higher DEHP and DBP contents in more polluted area (location A). The other explanation was that growth conditions, such as light, water temperature and nutrients might affect the phthalate ester accumulation rate and metabolic mechanism in algae [11,19]. The purpose of culturing different species of algal filaments in the same laboratory condition was to prevent the possible phthalate esters contamination coming from different polluted areas. The contents of DEHP and DBP in the stock cultures of three species of algae (at time zero) were similar to those of three weeks incubation. The results showed that the three different species of red algae filaments had different DEHP and DBP contents even when grown in the same environments and these results were owing to the intrinsic nature of algae (Table 3). If the phthalate esters originate only from the environment impurities, the DEHP and DBP contents among algae species should be similar. Sastry and Rao [20] mentioned that cross contamination of phthalate esters can arise from many sources. To avoid cross contamination, we used glass container and
1017
avoided using plastics, which eliminated the possibility of phthalate ester contamination from containers. Chloroform and hexane were the organic solvents used in our studies. Furthermore, we also analyzed all the water samples, chloroform and hexane used in our experiments, it was found that DEHP concentration coming from water and organic solvents were less than 0.4% DEHP of total algae. However, DBP was not found in all of the analyzed water samples. Although the effects of phthalate esters on biological systems have been learned more than 40 years, the biogenic origin has not been proved. These results showed the production of phthalate esters in the algae, but their physiological functions are not known. In animals, Nazir et al. [21] found DEHP in the heart muscle of bovine; they suggested that the DEHP might play a role in bioenergetics. DEHP and DBP are lipid soluble compounds, which suggest that phthalate esters might be stored in cell membrane and maintain the flexibility of algal cells. Some papers suggest that bacteria or plants can biosynthesize phthalate esters [22,23], but they did not use analytical chemistry methods to prove their results, whether bacteria could synthesize phthalate esters. In our experiments, we used 0.45 mm membrane to filter the algal culture medium, thus excluded the possibility of contamination resulting from attached or coexistent bacteria. Sastry and Rao [16] collected many species of algae and isolated high concentrations of dioctyl phthalate only in brown alga Sargassum wightii. They suggested that phthalate ester was not an impurity taken up by the algae from the environment. In this paper, we
Table 3 The DEHP and DBP contents of three species of red algae filaments Compound Species (mg/g dry weight) B. atropurpurea P. angusta DBP DEHP
62.1470.48 34.7471.20
P. dentata
22.1670.52 33.4770.05 6.3570.91 18.5370.18
The algae were cultured in the same laboratory conditions.
Table 2 The DEHP and DBP contents of three species of algae collected from three different neighbor coasts (A, B, C) of northern Taiwan Sampling sites
A B C
DEHP (mg/g dry weight)
DBP (mg/g dry weight)
E. intestinalis
U. fasciata
P. angusta
E. intestinalis
U. fasciata
P. angusta
16.0271.81 26.2771.18 5.0370.37
14.4370.32 9.5870.14 10.7770.62
3.9870.83 0.8070.46 15.7971.10
16.90711.12 16.2373.45 21.8371.64
14.5376.94 19.8379.32 18.3370.66
8.1770.51 8.9073.39 17.9573.62
A: harbor near the effluent from wastewater treatment plant; B: intertidal zone which is 0.5 km off the coast; C: 2 km off the coast.
ARTICLE IN PRESS 1018
C.Y. Chen / Water Research 38 (2004) 1014–1018
proved that algae could synthesize phthalate esters. The production process and physiological role of phthalate esters in algae still needs further investigation.
4. Conclusion
[9]
[10]
The results obtained in the present study prove that red algae Bangia atropurpurea can de novo synthesize di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP). Three species of algae which were cultured under the same laboratory condition showed different DEHP and DBP contents, such results were due to the intrinsic nature of algae.
[11]
Acknowledgements
[14]
We express our thanks to Dr. Y. M. Chiang for his gift of Bangia atropurpurea ‘conchocelis’ stock culture. This work was supported by grant from the National Science Council.
References [1] Fishbein L, Albro PW. Chromatographic and biological aspects of the phthalate esters. J Chromatogr 1972;70: 365–412. [2] Thomas JA, Thomas DD, Richard FW, Paul JG, Leo M. A review of the biological effects of di-(2-ethylhexyl) phthalate. Toxicol Appl Pharmacol 1978;45:1–27. [3] Giam CS, Chan HS, Neff GS, Atlas EL. Phthalate ester plasticizers: a new class of marine pollutant. Science 1978;199:419–21. [4] Jaeger RJ, Rubin RJ. Plasticizers from plastic devices: extraction, metabolism, and accumulation by biological systems. Science 1970;170:460–2. [5] Daniel JW. Toxicity and metabolism of phthalate esters. Clin Toxicol 1978;13:257–68. [6] Hunneman DH. A cautionary note on phthalate esters of Cryptotaenia canadensis. Tetrahedron Lett 1968;14:1743. [7] Atlas E, Giam CS. Global transport of organic pollutants: ambient concentrations in the remote marine atmosphere. Science 1980;211:163–5. [8] Sodergren A. Significance of interfaces in the distribution and metabolism of di-2-ethylhexyl phthalate in an aquatic
[12]
[13]
[15]
[16]
[17] [18]
[19]
[20]
[21]
[22]
[23]
laboratory model ecosystem. Environ Pollut Ser A 1982; 27:263–74. Giam CS, Atlas E, Powers MA, Leonard JE. Phthalic acid esters. In: Hutzinger O, editor. Anthropogenic compounds, vol. 3, Part C. Berlin: Springer; 1984. p. 67–140. Thuren A. Determination of phthalates in aquatic environments. Bull Environ Contam Toxicol 1986;36: 33–40. Staples CA, Peterson DR, Parkerton TF, Adams WJ. The environmental fate of phthalate esters: a literature review. Chemosphere 1997;35:667–749. Staples CA, Parkerton TF, Peterson DR. A risk assessment of selected phthalate esters in North American and Western European surface waters. Chemosphere 2000;40: 885–91. Chen CY. Studies on the essential oils in some Formosan seaweeds. Master thesis, National Taiwan University, Taipei, 1993. p.16–24. Noguchi T, Ikawa M, Uebel JJ, Andersen KK. Lipid constituents of the red algae Ceramium rubrum. A search for antimicrobial and chemical defense substances. In: Hoppe HA, Levring T, Tanaka Y, editors. Marine algae in pharmaceutical science. New York: Walter de Gruyter & Co.; 1979. p. 711–8. Stefanov K, Konaklieva M, Brechany EY, Christie WW. Fatty acid composition of some algae from the black sea. Phytochemistry 1988;27:3495–7. Sastry VMVS, Rao GRK. Dioctyl phthalate, and antibacterial compound from the marine brown alga— Sargassum wightii. J Appl Phycol 1995;7:185–6. Chen LCM, Edelstein T, McLachlan J. Bonnemaisonia hamifera in nature and in culture. J Phycol 1969;5:211–20. Provasoli L, McLaughlin JJA, Droop MR. The development of artificial media for marine algae. Arch Microbiol 1957;25:392–428. Casserly DM, Davis EM, Down TD, Guthrine RK. Sorption of organics by Selenastrum capricornutum. Water Res 1983;17:1591–4. Sastry VMVS, Rao GRK. Antibacterial substances from marine algae: successive extraction using benzene, chloroform and methanol. Bot Mar 1994;37:357–60. Nazir DJ, Alcaraz AP, Bierl BA, Beroza M, Nazir PP. Isolation, identification, and specific localization of di-2ethylhexyl phthalate in bovine heart muscle mitochondria. Biochemistry 1971;10:4228–32. Hayashi S, Asakawa Y, Ishida T, Matsuura T. Phthalate esters of Cryptotaenia canadensis DC .var. japonica Makino. Tetrahedron Lett 1967;50:5061–3. Marx JL. Phthalic acid esters: biological impact uncertain. Science 1972;178:46–7.
Water Research 38 (2004) 1014–1018
Biosynthesis of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) from red alga—Bangia atropurpurea Chih Yu Chen* Department of Tourism, Hsing Wu College, No. 11-2, Fen-Liao Road, Lin-Kou, Taipei County, Taiwan 244-41, Republic of China Received 17 September 2002; received in revised form 30 October 2003; accepted 10 November 2003
Abstract The contents of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) of red alga, Bangia atropurpurea, filaments cultured in artificial sea water medium were similar to those cultured in natural sea water medium. In the culture experiment, B. atropurpurea filaments were found to synthesize de novo phthalate esters. Additionally, DEHP and DBP contents in different species of algae grown in the same environment were different significantly, suggesting that it was due to the intrinsic nature of algae. r 2003 Elsevier Ltd. All rights reserved. Keywords: DEHP; DBP; Bangia atropurpurea; De novo synthesis
1. Introduction Phthalate esters have been widely applied as plasticizers or solvents in chemical industry [1,2]. Among them, di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) are most commonly used [3]. DEHP and DBP are pollutants released from plastic wares to the environment or in the bag-stored blood [4,5]. Many findings of phthalate esters have been reported in nature, such as sediments, natural waters, soils, plants, and aquatic organisms [6–11]. Phthalate esters pose analytical challenges since phthalate esters are present in many laboratory products, tools, reagents, solvents and supplies [12]. What remains unclear, however, is whether some of these substances are naturally occurring or originate from some commercial processes and subsequently, enter the ecosystem. In a screen of survey of the lipid-soluble components of seaweed commonly found on the northern coast of *Tel.: +886-2-2601-53-10X430; fax: +886-2-2601-5310X439. E-mail address: [email protected] (C.Y. Chen).
Taiwan, the two compounds mentioned above were found in relatively large amount in all the seaweed samples studied [13]. Furthermore, the occurrence of phthalate esters in algae has been reported previously [14–16]. In order to understand the origin of phthalate esters, we cultured red alga, Bangia atropurpurea, filaments in an artificial sea water (ASW) medium (free from the possible solvent transferred contamination and environment impurities accumulation) and a natural sea water (NSW) medium and analyzed the contents of DEHP and DBP in both the plants. Furthermore, B. atropurpurea filaments were cultured in a medium containing NaH14CO3. After two weeks, the radioactivity of DEHP and DBP fractionated by HPLC from cultured filaments was analyzed. However, in order to know whether environmental factor or the intrinsic nature of algae will affect the phthalate esters level in algae, DEHP and DBP contents of seaweed collected from different coasts were analyzed, and we also cultured the filaments of three different species of algae in the same laboratory condition to determine the DEHP and DBP contents in them.
0043-1354/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2003.11.029
ARTICLE IN PRESS C.Y. Chen / Water Research 38 (2004) 1014–1018
2. Materials and methods 2.1. Algal specimens and culture condition Stock culture of the filamentous phase of B. atropurpurea (Roth) C. Agardh was kindly given by Dr Y.M. Chiang, Institute of Oceanography, National Taiwan University. Filaments were propagated by fragmentation and grew into colonies of filamentous clusters in SWMIII medium [17]. The filamentous plants (‘Conchocelis’) were maintained at 20 C under fluorescent light and illuminated with continuous light of 60 mE m2 s1. 2.2. DEHP and DBP standard DEHP and DBP standards were purchased from Fluka (Switzerland). Serially diluted chloroform solutions were prepared. 0.5 ml of each solution was injected into the GC-MS column to determine the system sensitivity and linear range. Measurements were taken in triplicate and values of the peak areas were recorded as GC-MS responses to the different amounts of authentic DEHP and DBP injected. A linear regression line was plotted to show the correlation between peak areas and injected amounts of authentic standards. 2.3. De novo synthesis of DEHP and DBP 2.3.1. The comparison of NSW and ASW media NSW Medium cultured group: harvested filaments were cultured in SWM-III medium, and algae were collected with a plankton net. Removal of excess water was done by filtration on a Buchner funnel using suction. The wet weights were measured (1.20 g; 0.84 g)ðn ¼ 2Þ and then lyophilized overnight to determine the dry weights (0.1940 g; 0.1457 g). ASW Medium cultured group: The wet weight of harvested filaments cultured in SWM-III medium were measured (1.22 g; 1.25 g)ðn ¼ 2Þ; and the filaments were washed several times with ASW-III [18]. The ASW medium contained deionized water and inorganic salts. After a month of incubation, plants were collected and the wet weights were measured (3.05 g; 3.10 g), then lyophilized overnight to determine the dry weight (0.5524 g; 0.5534 g). The dry filaments of the alga cultured in NSW and ASW media were extracted by supercritical fluid extraction (SFE) method [13]. The extract was dissolved in a small volume of chloroform and the contents of DEHP and DBP were analyzed by GC-MS. 2.3.2. Culture the filaments with radioactive carbon source First, the filaments cultured in SWM-III medium were harvested, wet weight was measured (1.80 g) and then inoculated into fresh SWM-III medium. Second, 3.2 mg
1015
of NaH14CO3 (250 mCi, 6.8 mCi/mmole, Sigma, Paris, France) was added into the medium. The filaments were cultured for two weeks (without aeration). When collected, the wet and dry weights of the filaments were 2.22 and 0.3443 g, respectively. The algae were extracted by a high pressure Soxhlet extractor (SFE), and then DEHP and DBP were purified by HPLC system. 2.4. SFE and HPLC, GC-MS analysis condition SFE was carried out using high pressure Soxhlet extractor (J & W Scientific Comp., NJ, USA) under 500 psi at 55 C for 3 h. The condenser temperature was 15 C, and the supercritical solvent used was CO2. HPLC was run by two Hitachi L-6000 pumps and one L-5000 LC controller. DEHP and DBP were detected by a Hitachi L-4200 UV-VIS detector (Tokyo, Japan). The column used was a 5 mm, 4.6 250 mm2 Econosil column at a flow rate of 1 ml/min. Mobile phase was isocratic CHCl3/hexane=3:7. Elutes were monitored at 280 nm and the DEHP and DBP fractions were collected from the comparative retention time of DEHP and DBP standards. The radioactive DEHP and DBP were counted by liquid scintillation counter (Beckman LS5801, IL, USA) and further identified by GC-MS. Analysis of DEHP and DBP were carried out using HP 5890 series II GC and HP 5971 MSD. The GC-MS column was an Ultra-2 fused silica capillary column (25 m 0.2 id). Column temperature range was programmed from 160 C to 240 C. The carrier gas (helium) flow rate was 31 cm/s, injector and detector temperatures were 250 C and 280 C, respectively. The electron impact mass spectrum (70 eV) of DEHP showed the molecular ion at M=z 390, other important ions were detected at M=z 279, 167, and 149 (base peak); DBP showed the molecular ion at M=z 278, other important ions were detected at M=z 223, 205, 167, and 149 (base peak). 2.5. DEHP and DBP contents of algae collected from different polluted area Three species of algae—Ulva fasciata, Enteromorpha intestinalis and Porphyra angusta, which grew in three different neighbor coasts (A, B, C) of northern Taiwan were collected. All species of the algae were collected twice as duplicate. Collected algae were lyophilized, extracted by a Soxhlet extractor (SFE), the supercritical solvent was CO2, and the contents of DEHP and DBP were analyzed by GC-MS. 2.6. DEHP and DBP contents of algae cultured at the same environment Stock cultures of red algae B. atropurpurea, P. angusta and P. dentata filaments were blended into fragments
ARTICLE IN PRESS 1016
C.Y. Chen / Water Research 38 (2004) 1014–1018
and then inoculated into fresh flasks. The filaments were cultured under no aeration, cultured conditions were the same as previous, but temperature was 23 C. After three weeks of incubation, the filaments were harvested and lyophilized, extracted by Soxhlet extractor (SFE). The contents of DEHP and DBP were analyzed by GC-MS.
3. Results and discussion Quantitative analysis of DEHP and DBP showed good linear relationship between the GC-MS response (peak area on chromatograms) and the amount of injected authentic standard. A regression line of DEHP was plotted as Y ¼ 7:766X þ 112:667 ðr2 ¼ 0:964Þ and DBP was plotted as Y ¼ 20:322X 69:16 ðr2 ¼ 0:981Þ; where X represents the amount of the standard in nanograms, and Y represents the peak area ð104 Þ: The comparison of DEHP and DBP contents of B. atropurpurea filaments cultured in NSW and ASW media is shown in Table 1. The contents of DEHP and DBP in NSW cultured filaments were similar as those of ASW cultured filaments (at time zero). After one month incubation, the biomass of the algae cultured in ASW medium increased 2.5 fold (ASW 1) and 2.48 fold (ASW 2), respectively. The data showed that DEHP and DBP contents were similar in ASW and NSW medium cultured groups. At the beginning of the test, we found the concentrations of DEHP in NSW and ASW media were less than 0.4% DEHP of total algae DEHP. However, DBP was not found in both the media. The result proves that DEHP and DBP were not impurities taken by the algae from the environment. In the radioactive compound labeled experiment, we used NaH14CO3 as the carbon source of B. atropurpurea
filaments. Fig. 1 showed the HPLC chromatogram of DEHP and DBP. The first peak fraction of which yellow color quenched easily, showed no radioactivity. We collected two peak fractions of DEHP and DBP, then analyzed by a scintillation counter and GC-MS. The average radioactivity showed that single peak fractions of DEHP (160.00 cpm) and DBP (4786.67 cpm) have significant higher radioactivities than the background (28.00 cpm). We also analyzed the radioactivities of other compounds whose retention times were earlier than DEHP. We found the radioactivity range was from
Fig. 1. HPLC chromatograms and radioactivities of DEHP and DBP purified from B. atropurpurea.
Table 1 The DEHP and DBP contents of B. atropurpurea filaments cultured in ASW and NSW media ASW medium cultured
DEHP experiment Algae dry weight (before cultivation) (g) Algae dry weight (after cultivation) (g) SFE crude extract (g) Calibrated weight (ng)
1
2
1
2
0.2186 0.5524 0.0042 95.3871.30
0.2220 0.5534 0.0044 97.0271.43
— 0.1940 0.0016 33.8470.83
— 0.1457 0.0014 25.2770.41
Av. content (mg/g) DBP experiment Algae dry weight (before cultivation) (g) Algae dry weight (after cultivation) (g) SFE crude extract (g) Calibrated weight (ng) Av. content (mg/g)
NSW medium cultured
69.6070.76
0.2186 0.5524 0.0042 98.0072.36 70.6070.52
0.2220 0.5534 0.0044 97.1670.44
69.5870.28
— 0.1940 0.0016 34.2671.07
— 0.1457 0.0014 25.3970.66
70.1670.66
ARTICLE IN PRESS C.Y. Chen / Water Research 38 (2004) 1014–1018
65.35 to 1350.45 cpm, which showed that radioactive 14C also labeled other algal organic compounds. Furthermore, the HPLC fractions of DEHP and DBP were subjected to GC-MS, each fragment of the single compound mass spectrum matches DEHP and DBP, respectively. Thus the possibility that radioactivities originated from other compounds was eliminated. The results indicate that algae incorporate 14C as the carbon source to de novo synthesize phthalate esters. This is also an evidence that DEHP and DBP were biosynthesized by algae. The concentrations of DEHP in the water from where the samples were collected (northern Taiwan coasts) were 0.2570.02% of total algae DEHP content. However, no DBP was found in water samples of the three locations. The contents of DEHP and DBP varied in the same species of algae collected from different polluted coasts (Table 2). One explanation was possible based on the environment effect, namely, the algae grew in a more polluted coast might accumulate higher phthalate esters than those that grew in a cleaner coast. However, in our results, we did not find higher DEHP and DBP contents in more polluted area (location A). The other explanation was that growth conditions, such as light, water temperature and nutrients might affect the phthalate ester accumulation rate and metabolic mechanism in algae [11,19]. The purpose of culturing different species of algal filaments in the same laboratory condition was to prevent the possible phthalate esters contamination coming from different polluted areas. The contents of DEHP and DBP in the stock cultures of three species of algae (at time zero) were similar to those of three weeks incubation. The results showed that the three different species of red algae filaments had different DEHP and DBP contents even when grown in the same environments and these results were owing to the intrinsic nature of algae (Table 3). If the phthalate esters originate only from the environment impurities, the DEHP and DBP contents among algae species should be similar. Sastry and Rao [20] mentioned that cross contamination of phthalate esters can arise from many sources. To avoid cross contamination, we used glass container and
1017
avoided using plastics, which eliminated the possibility of phthalate ester contamination from containers. Chloroform and hexane were the organic solvents used in our studies. Furthermore, we also analyzed all the water samples, chloroform and hexane used in our experiments, it was found that DEHP concentration coming from water and organic solvents were less than 0.4% DEHP of total algae. However, DBP was not found in all of the analyzed water samples. Although the effects of phthalate esters on biological systems have been learned more than 40 years, the biogenic origin has not been proved. These results showed the production of phthalate esters in the algae, but their physiological functions are not known. In animals, Nazir et al. [21] found DEHP in the heart muscle of bovine; they suggested that the DEHP might play a role in bioenergetics. DEHP and DBP are lipid soluble compounds, which suggest that phthalate esters might be stored in cell membrane and maintain the flexibility of algal cells. Some papers suggest that bacteria or plants can biosynthesize phthalate esters [22,23], but they did not use analytical chemistry methods to prove their results, whether bacteria could synthesize phthalate esters. In our experiments, we used 0.45 mm membrane to filter the algal culture medium, thus excluded the possibility of contamination resulting from attached or coexistent bacteria. Sastry and Rao [16] collected many species of algae and isolated high concentrations of dioctyl phthalate only in brown alga Sargassum wightii. They suggested that phthalate ester was not an impurity taken up by the algae from the environment. In this paper, we
Table 3 The DEHP and DBP contents of three species of red algae filaments Compound Species (mg/g dry weight) B. atropurpurea P. angusta DBP DEHP
62.1470.48 34.7471.20
P. dentata
22.1670.52 33.4770.05 6.3570.91 18.5370.18
The algae were cultured in the same laboratory conditions.
Table 2 The DEHP and DBP contents of three species of algae collected from three different neighbor coasts (A, B, C) of northern Taiwan Sampling sites
A B C
DEHP (mg/g dry weight)
DBP (mg/g dry weight)
E. intestinalis
U. fasciata
P. angusta
E. intestinalis
U. fasciata
P. angusta
16.0271.81 26.2771.18 5.0370.37
14.4370.32 9.5870.14 10.7770.62
3.9870.83 0.8070.46 15.7971.10
16.90711.12 16.2373.45 21.8371.64
14.5376.94 19.8379.32 18.3370.66
8.1770.51 8.9073.39 17.9573.62
A: harbor near the effluent from wastewater treatment plant; B: intertidal zone which is 0.5 km off the coast; C: 2 km off the coast.
ARTICLE IN PRESS 1018
C.Y. Chen / Water Research 38 (2004) 1014–1018
proved that algae could synthesize phthalate esters. The production process and physiological role of phthalate esters in algae still needs further investigation.
4. Conclusion
[9]
[10]
The results obtained in the present study prove that red algae Bangia atropurpurea can de novo synthesize di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP). Three species of algae which were cultured under the same laboratory condition showed different DEHP and DBP contents, such results were due to the intrinsic nature of algae.
[11]
Acknowledgements
[14]
We express our thanks to Dr. Y. M. Chiang for his gift of Bangia atropurpurea ‘conchocelis’ stock culture. This work was supported by grant from the National Science Council.
References [1] Fishbein L, Albro PW. Chromatographic and biological aspects of the phthalate esters. J Chromatogr 1972;70: 365–412. [2] Thomas JA, Thomas DD, Richard FW, Paul JG, Leo M. A review of the biological effects of di-(2-ethylhexyl) phthalate. Toxicol Appl Pharmacol 1978;45:1–27. [3] Giam CS, Chan HS, Neff GS, Atlas EL. Phthalate ester plasticizers: a new class of marine pollutant. Science 1978;199:419–21. [4] Jaeger RJ, Rubin RJ. Plasticizers from plastic devices: extraction, metabolism, and accumulation by biological systems. Science 1970;170:460–2. [5] Daniel JW. Toxicity and metabolism of phthalate esters. Clin Toxicol 1978;13:257–68. [6] Hunneman DH. A cautionary note on phthalate esters of Cryptotaenia canadensis. Tetrahedron Lett 1968;14:1743. [7] Atlas E, Giam CS. Global transport of organic pollutants: ambient concentrations in the remote marine atmosphere. Science 1980;211:163–5. [8] Sodergren A. Significance of interfaces in the distribution and metabolism of di-2-ethylhexyl phthalate in an aquatic
[12]
[13]
[15]
[16]
[17] [18]
[19]
[20]
[21]
[22]
[23]
laboratory model ecosystem. Environ Pollut Ser A 1982; 27:263–74. Giam CS, Atlas E, Powers MA, Leonard JE. Phthalic acid esters. In: Hutzinger O, editor. Anthropogenic compounds, vol. 3, Part C. Berlin: Springer; 1984. p. 67–140. Thuren A. Determination of phthalates in aquatic environments. Bull Environ Contam Toxicol 1986;36: 33–40. Staples CA, Peterson DR, Parkerton TF, Adams WJ. The environmental fate of phthalate esters: a literature review. Chemosphere 1997;35:667–749. Staples CA, Parkerton TF, Peterson DR. A risk assessment of selected phthalate esters in North American and Western European surface waters. Chemosphere 2000;40: 885–91. Chen CY. Studies on the essential oils in some Formosan seaweeds. Master thesis, National Taiwan University, Taipei, 1993. p.16–24. Noguchi T, Ikawa M, Uebel JJ, Andersen KK. Lipid constituents of the red algae Ceramium rubrum. A search for antimicrobial and chemical defense substances. In: Hoppe HA, Levring T, Tanaka Y, editors. Marine algae in pharmaceutical science. New York: Walter de Gruyter & Co.; 1979. p. 711–8. Stefanov K, Konaklieva M, Brechany EY, Christie WW. Fatty acid composition of some algae from the black sea. Phytochemistry 1988;27:3495–7. Sastry VMVS, Rao GRK. Dioctyl phthalate, and antibacterial compound from the marine brown alga— Sargassum wightii. J Appl Phycol 1995;7:185–6. Chen LCM, Edelstein T, McLachlan J. Bonnemaisonia hamifera in nature and in culture. J Phycol 1969;5:211–20. Provasoli L, McLaughlin JJA, Droop MR. The development of artificial media for marine algae. Arch Microbiol 1957;25:392–428. Casserly DM, Davis EM, Down TD, Guthrine RK. Sorption of organics by Selenastrum capricornutum. Water Res 1983;17:1591–4. Sastry VMVS, Rao GRK. Antibacterial substances from marine algae: successive extraction using benzene, chloroform and methanol. Bot Mar 1994;37:357–60. Nazir DJ, Alcaraz AP, Bierl BA, Beroza M, Nazir PP. Isolation, identification, and specific localization of di-2ethylhexyl phthalate in bovine heart muscle mitochondria. Biochemistry 1971;10:4228–32. Hayashi S, Asakawa Y, Ishida T, Matsuura T. Phthalate esters of Cryptotaenia canadensis DC .var. japonica Makino. Tetrahedron Lett 1967;50:5061–3. Marx JL. Phthalic acid esters: biological impact uncertain. Science 1972;178:46–7.