dialkyl phthalates , such as di-(2-ethylhexyl ) phthalate ( dehp ) , diisononyl phthalate ( dinp ) , diisodecyl phthalate ( didp ) and dibutyl phthalate ( dbp ) , are petrochemicals used as plasticizers or solvents in a variety of industrial products . these chemicals have been thought to be environmental pollutants and detected in soils , sediments , terrestrial and marine waters , and also living organisms . dehp has been isolated from terrestrial and marine organisms including plants , marine algae [ 25 ] , and fungal and bacterial culture broths . however , it is difficult to determine whether dehp was produced by these organisms , the sample was contaminated during the separation process , or dehp accumulated in the organisms , because dehp has been used abundantly in petrochemical phthalate esters . more than 65% of all the phthalate esters used in japan in 2003 were dehps followed by dinps ( > 26% ) , didps ( about 3% ) , and dbps ( about 1% ) , and this relative ratio has changed little in recent years . interestingly , there has been no report of the isolation of dinp and didp from organisms , whereas dbp has been isolated from plants , marine algae , bacteria , and fungi [ 2 , 811 ] . diethyl phthalate ( dep ) , a less common petrochemical product , was obtained as a chemotactic factor from the culture medium of helicobacter pylori and also detected in the broth of streptomyces sp . moreover , di-(2-methylheptyl ) phthalate , which is not a petrochemical used in the chemical industry , was isolated from a plant hypericum hyssopifolium ( guttiferae ) . chen reported the incorporation of c into dbp and dehp by culturing the filamentous phase of a red alga , bangia atropurpurea , with c - enriched nahco3 . dbp was enriched by about 171 times the background level and dehp , by 5.7 times . recently , teuten et al . demonstrated that polybrominated diphenyl esters isolated from a true s beaked whale ( mesoplodon mirus ) were naturally produced compounds and not derived from artificial products by analyzing the natural abundance c content of the isolated compounds and industrial products . therefore , we intended to investigate if dbp and dehp obtained from marine algae are natural products or contaminants of petrochemical products using a similar method . in this report , we suggest that dbp isolated from the ch2cl2-methanol extracts of undaria pinnatifida ( harvey ) suringar ( alariaceae , phaeophyceae ) , laminaria japonica areschoug ( laminariaceae , phaeophyceae ) and ulva sp . ( ulvaceae , chlorophyceae ) was a natural product based on an analysis of the natural abundance c content of the isolates . industrial products of dbp and dehp contain very small amounts of c which can not be detected even by the most sensitive method , accelerator mass spectrometry ( ams ) , because these compounds are made from fossil oils . if these phthalate esters are produced by living organisms , natural abundance c will be detected in the same amounts as modern carbon . the isolation of dbp and dehp was first examined using ulva sp . collected in may 2005 to determine solvents suitable for the extraction process and procedures for concentrating lipophilic compounds . the conditions for hplc and gc were established using authentic samples of dbp and dehp . the extraction and separation of u. pinnatifida were performed according to the procedures used for small amounts of ulva sp . the aqueous residue obtained after the evaporation of organic solvents from the ch2cl2-meoh extract of the alga , was dissolved in meoh and adsorbed on hyflo supercell . the adsorbent was placed in a glass column and lipophilic compounds were eluted with ch2cl2 . the lipophilic fraction was adsorbed on hyflo super - cell and applied to a silica gel column with hexane . the silica gel column was eluted successively with hexane and hexane - etoac ( gradient ) . the eluate was monitored by tlc ( hexane - etoac = 4:1 ) and three fractions containing dbp and/or dehp were obtained . the first fraction ( fr . dbp ( 1.24 mg ) was separated by hplc ( ods ) with 80% meoh - h2o from frs . 2 and 3 , and dehp since the particles of hyflo super - cell were too small , dispersion and formation of masses occurred during the evaporation of solvents when large amounts of extract or lipophilic fraction were adsorbed . therefore , silica gel was used in lieu of hyflo super - cell for the separation from l. japonica and ulva sp . the other separation procedures for these algae were the same as those for u. pinnatifida . dbp and dehp obtained from three algae were identified and analyzed the purity by h nmr and gc - mass spectra . the h nmr spectra of dbp and dehp from u. pinnatifida were clean and only a very few faint signals due to contaminants were detected . on the other hand , pigments and lipophilic compounds disturbed the separation of dbp and especially dehp from l. japonica and ulva sp . dehp fractions obtained by the first hplc separation from l. japonica and ulva sp . contained a large quantity of contaminants , which were most probably unsaturated fatty acids . the h nmr spectra of the final fraction of dbp and dehp from these two algae still showed the signals due to the contaminants . it is unclear if the difference in the amounts / species of contaminants between u. pinnatifida and the other two algae is due to the nature of these algae or the season when they were collected . l. japonica and ulva sp . were harvested in the winter ( cold water ) , when they usually contain more unsaturated fatty acids . the concentration ( w / w% ) of dbp and dehp in the final samples , analyzed by gc - ms utilizing the selected ion monitoring ( sim ) method , was 60% ( dbp ) and 70% ( dehp ) from l. japonica and 75% ( dbp ) and 60% ( dehp ) from ulva sp . it was not feasible to conduct further purification since the separation of contaminant(s ) is difficult and would significantly reduce the amounts of dehp . we thought that the measurement of c with these samples would give decipherable results because petrochemical compounds contain no measurable c and biogenetic products contain modern levels of c. therefore , we submitted dbp and dehp samples obtained from l. japonica and ulva sp . without further separation , together with dbp and dehp from u. pinnatifida . natural abundance c content was analyzed at the tandetron ams c dating laboratory , center for chronological research at nagoya university by the ams method . this unit represents a percentage value of the ratio of sample c concentration to the absolute c concentration , which is defined as corresponding to ad1950 . the industrial samples of dbp and dehp did not give measurable c concentration as they are petrochemical products . the dbp samples from three algae contained modern c , which indicated that dbp was synthesized naturally and not of industrial origin . the higher c content in dbp can be ascribed to the enrichment of the c level caused by the testing of nuclear bombs during the 1950s and 1960s . significant amounts of c were detected in the dehp samples from three algae . in the most reliable ( highly purified ) sample obtained from u. pinnatifida , the contaminant(s ) ( unsaturated fatty acid(s ) ) in the former two samples would raise the c concentration . it may be that remarkable amounts of industrial dehp were incorporated into the algae and/or contaminated the sample during the isolation procedures and that some amount of dehp was biosynthesized to give the significant c content . this study revealed that dbp obtained from two edible brown algae , u. pinnatifida and l. japonica , and a green alga , ulva sp . , was a natural product since modern c was detected in the isolated dbp samples . dehp may also be produced naturally , but the results of this study were not conclusive . therefore , further studies will be required to confirm whether this compound is biosynthesized by these algae . di-(2-methylheptyl ) phthalate isolated from h. hyssopifolium must be a biogenetic product of this plant , because it has not been synthesized . dbp and dep detected in the culture media of microorganisms would also be produced by microorganisms [ 813 ] . it may be true that they are stored in the cell membrane and affect the flexibility of the cell . h nmr ( 400 mhz ) and c nmr ( 100 mhz ) spectra were measured on a jeol al-400 spectrometer in cdcl3 . gc - ms data were obtained with a shimadzu gcms - qp5050a spectrometer using class-5000 software and a tc-70 column ( 0.25 m x 30 m ) : carrier gas , he ( 0.4 ml / min ) ; interface and ion source temperatures , 300 c . for a typical elution , the column temperature was first set at 100 c for 5 min , raised to 300 c at 2 c / min , and kept at 300 c for 20 min . hplc was carried out with a hitachi l-2130 pump and l-2400 uv detector ( 280 nm ) equipped with a d-2500 data processor using a senshu pack pegasil - ods column ( 10 mm x 250 mm ) at a flow rate of 3 ml / min . standard samples ( petrochemical products ) of dbp and dehp were provided by showa ether co. , ltd . and cg ester corporation , respectively . all other solvents and reagents were of the highest grade commercially available and used without further purification . the alga ( about 13.5 kg ) was cut into pieces and extracted four times with ch2cl2-meoh ( 2:1 , each 10.5 l ) . the alga ( about 9.3 kg ) was cut into pieces and extracted four times with ch2cl2-meoh ( 2:1 , each 12 l ) . was collected at yokohama marine park , yokohama , japan in may and december 2005 . the alga collected in december ( about 13.5 kg ) was cut into pieces and extracted four times with ch2cl2-meoh ( 2:1 , each 12 l ) . the aqueous residue obtained from the organic extract of u. pinnatifida was dissolved in meoh and the solution was mixed with hyflo super - cell ( 1 kg ) . meoh and water were evaporated and the dried adsorbent was placed in a glass column ( diameter , 70 mm ; length , 600 mm ) . the column was eluted with ch2cl2 ( 4 l ) to give a lipophilic fraction ( 32.5 g ) . the lipophilic fraction was dissolved in ch2cl2 , and the solution was mixed with 100 g of hyflo super - cell . after evaporation of the organic solvent , the dried adsorbent was mixed with hexane and poured on to a silica gel column ( 700 g ) made up with hexane . the column was eluted with 2 l of hexane followed by hexane - etoac = 9:1 ( 2 l ) and 4:1 ( 2 the eluates were monitored by tlc ( hexane - etoac = 4:1 ) and divided into three fractions ( frs . dbp and dehp were detected in two fractions ( frs . 2 and 3 ) . fr . 2 ( 120.2 mg ) was separated by hplc ( ods ) with 80% meoh - h2o to obtain a dbp fraction and then a dehp fraction was recovered with 100% meoh . fr . 3 ( 12.1 mg ) was subjected to a similar hplc separation to give dbp and dehp fractions . two dbp fractions were combined and separated by hplc ( 80% meoh - h2o ) to afford 1.24 mg of dbp . 1 , and dehp ( 2.14 mg ) was isolated by hplc separation ( 100% meoh ) . the aqueous residue from ulva sp . was dissolved in meoh and the solution was mixed with silica gel ( 1.22 kg ) . meoh and water were evaporated and the dried silica gel was placed in a glass column ( diameter , 70 mm ; length , 600 mm ) . the column was eluted with ch2cl2 ( 3 l ) to give a lipophilic fraction ( 216.8 g ) . the ch2cl2 solution of the lipophilic fraction was mixed with 250 g of silica gel , and , after evaporation of the organic solvent , the adsorbed silica gel was applied to a silica gel column ( 800 g ) with hexane . the column was eluted successively with 1 l each of hexane and hexane - ch2cl2 = 9:1 , 7:3 , 1:1 , 3:7 , and 1:4 . the eluates were monitored by tlc ( hexane - etoac = 4:1 ) and divided into five fractions . dbp and dehp were contained into two fractions ( frs . 3 and 4 ) . fr . 3 ( 127.1 mg ) was separated by hplc ( ods ) with 80% meoh - h2o to obtain a dbp fraction and then a dehp fraction was recovered with 100% meoh . fr . 4 ( 240.7 mg ) was subjected to a similar hplc separation to give dbp and dehp fractions . the combined dbp fraction ( 12.8 mg ) was further separated by repeated hplc ( 80% meoh - h2o ) to afford 1.78 mg of dbp . dehp ( 3.87 mg ) was isolated from the dehp fraction ( 135.7 mg ) by repeated hplc separation ( 100% meoh ) . dbp ( 1.93 mg ) and dehp ( 2.47 mg ) were isolated from the aqueous residue obtained from the organic extract of l. japonica by similar procedures . dbp and dehp isolated from these algae were identified based on the h nmr and gc - mass spectra with the standard samples of dbp and dehp . were analyzed by gc - ms using the selected ion monitor ( sim ) method . a calibration table was created by injecting standard solutions containing 10 , 5 , 2 , 1 , 0.5 , and 0.1 g of the authentic dbp or dehp . the samples in the same solvent were injected and the chromatogram was integrated in terms of concentration . the gc column temperature used for the analysis of dbp was first set at 100 c for 6 min , raised to 300 c at 2 c / min , and held at 300 c for 20 min . the amounts of dbp isolated from l. japonica and ulva sp . were about 60 and 75 ( w / w)% , respectively . the column temperature for dehp was set at 100 c for 6 min and raised to 300 c at 4 c / min , and dehp was eluted at around 38 min . natural abundance c content was measured by the following three steps at the tandetron ams c dating laboratory , center for chronological research , nagoya university utilizing a model 4130- ams system : ( 1 ) each organic compound was first burned into co2 , ( 2 ) the co2 was reduced to graphite , and ( 3 ) the graphite was analyzed by accelerator mass spectrometry ( ams ) ( kitagawa , 1993 ; nakamura et al . , 2004 ) . the results ( table 1 ) were expressed as percent modern carbon ( pmc ) , a percentage value of the ratio of sample c concentrations to the absolute c concentration defined as corresponding to ad 1950 . h nmr ( 400 mhz ) and c nmr ( 100 mhz ) spectra were measured on a jeol al-400 spectrometer in cdcl3 . gc - ms data were obtained with a shimadzu gcms - qp5050a spectrometer using class-5000 software and a tc-70 column ( 0.25 m x 30 m ) : carrier gas , he ( 0.4 ml / min ) ; interface and ion source temperatures , 300 c . for a typical elution , the column temperature was first set at 100 c for 5 min , raised to 300 c at 2 c / min , and kept at 300 c for 20 min . hplc was carried out with a hitachi l-2130 pump and l-2400 uv detector ( 280 nm ) equipped with a d-2500 data processor using a senshu pack pegasil - ods column ( 10 mm x 250 mm ) at a flow rate of 3 ml / min . standard samples ( petrochemical products ) of dbp and dehp were provided by showa ether co. , ltd . and cg ester corporation , respectively . all other solvents and reagents were of the highest grade commercially available and used without further purification . the alga ( about 13.5 kg ) was cut into pieces and extracted four times with ch2cl2-meoh ( 2:1 , each 10.5 l ) . the alga ( about 9.3 kg ) was cut into pieces and extracted four times with ch2cl2-meoh ( 2:1 , each 12 l ) . was collected at yokohama marine park , yokohama , japan in may and december 2005 . the alga collected in december ( about 13.5 kg ) was cut into pieces and extracted four times with ch2cl2-meoh ( 2:1 , each 12 l ) . the aqueous residue obtained from the organic extract of u. pinnatifida was dissolved in meoh and the solution was mixed with hyflo super - cell ( 1 kg ) . meoh and water were evaporated and the dried adsorbent was placed in a glass column ( diameter , 70 mm ; length , 600 mm ) . the column was eluted with ch2cl2 ( 4 l ) to give a lipophilic fraction ( 32.5 g ) . the lipophilic fraction was dissolved in ch2cl2 , and the solution was mixed with 100 g of hyflo super - cell . after evaporation of the organic solvent , the dried adsorbent was mixed with hexane and poured on to a silica gel column ( 700 g ) made up with hexane . the column was eluted with 2 l of hexane followed by hexane - etoac = 9:1 ( 2 l ) and 4:1 ( 2 the eluates were monitored by tlc ( hexane - etoac = 4:1 ) and divided into three fractions ( frs . dbp and dehp were detected in two fractions ( frs . 2 and 3 ) . fr . 2 ( 120.2 mg ) was separated by hplc ( ods ) with 80% meoh - h2o to obtain a dbp fraction and then a dehp fraction was recovered with 100% meoh . fr . 3 ( 12.1 mg ) was subjected to a similar hplc separation to give dbp and dehp fractions . two dbp fractions were combined and separated by hplc ( 80% meoh - h2o ) to afford 1.24 mg of dbp . 1 , and dehp ( 2.14 mg ) was isolated by hplc separation ( 100% meoh ) . the aqueous residue from ulva sp . was dissolved in meoh and the solution was mixed with silica gel ( 1.22 kg ) . meoh and water were evaporated and the dried silica gel was placed in a glass column ( diameter , 70 mm ; length , 600 mm ) . the column was eluted with ch2cl2 ( 3 l ) to give a lipophilic fraction ( 216.8 g ) . the ch2cl2 solution of the lipophilic fraction was mixed with 250 g of silica gel , and , after evaporation of the organic solvent , the adsorbed silica gel was applied to a silica gel column ( 800 g ) with hexane . the column was eluted successively with 1 l each of hexane and hexane - ch2cl2 = 9:1 , 7:3 , 1:1 , 3:7 , and 1:4 . the eluates were monitored by tlc ( hexane - etoac = 4:1 ) and divided into five fractions . dbp and dehp were contained into two fractions ( frs . 3 and 4 ) . fr . 3 ( 127.1 mg ) was separated by hplc ( ods ) with 80% meoh - h2o to obtain a dbp fraction and then a dehp fraction was recovered with 100% meoh . fr . 4 ( 240.7 mg ) was subjected to a similar hplc separation to give dbp and dehp fractions . the combined dbp fraction ( 12.8 mg ) was further separated by repeated hplc ( 80% meoh - h2o ) to afford 1.78 mg of dbp . dehp ( 3.87 mg ) was isolated from the dehp fraction ( 135.7 mg ) by repeated hplc separation ( 100% meoh ) . dbp ( 1.93 mg ) and dehp ( 2.47 mg ) were isolated from the aqueous residue obtained from the organic extract of l. japonica by similar procedures . dbp and dehp isolated from these algae were identified based on the h nmr and gc - mass spectra with the standard samples of dbp and dehp . dbp and dehp isolated from l. japonica and ulva sp . were analyzed by gc - ms using the selected ion monitor ( sim ) method . a calibration table was created by injecting standard solutions containing 10 , 5 , 2 , 1 , 0.5 , and 0.1 g of the authentic dbp or dehp . the samples in the same solvent were injected and the chromatogram was integrated in terms of concentration . the gc column temperature used for the analysis of dbp was first set at 100 c for 6 min , raised to 300 c at 2 c / min , and held at 300 c for 20 min . the amounts of dbp isolated from l. japonica and ulva sp . were about 60 and 75 ( w / w)% , respectively . the column temperature for dehp was set at 100 c for 6 min and raised to 300 c at 4 c / min , and dehp was eluted at around 38 min . the amounts of dehp isolated from l. japonica and ulva sp . were about 70 and 60 ( w / w)% , respectively . natural abundance c content was measured by the following three steps at the tandetron ams c dating laboratory , center for chronological research , nagoya university utilizing a model 4130- ams system : ( 1 ) each organic compound was first burned into co2 , ( 2 ) the co2 was reduced to graphite , and ( 3 ) the graphite was analyzed by accelerator mass spectrometry ( ams ) ( kitagawa , 1993 ; nakamura et al . , 2004 ) . the results ( table 1 ) were expressed as percent modern carbon ( pmc ) , a percentage value of the ratio of sample c concentrations to the absolute c concentration defined as corresponding to ad 1950 .