Estimation of polycyclic aromatic hydrocarbons and their conjugates in soils
その他（別言語等）
のタイトル
農用地および国道路肩土壌に分布する多環芳香族炭 化水素およその抱合体の同定・定量
著者（英） Fukushima Michihiro, Nakano Masuo, Negishi Takashi
journal or
publication title
Research bulletin of Obihiro Zootechnical University. Series I
volume 16
number 3
page range 199‑204
year 1989‑11‑30
URL http://id.nii.ac.jp/1588/00002042/
兄紺．軌JJ．伽上山ro（加fu．J，Jβけガタユ・Jタタへゼ朗． 199
Estimation of Polycyclic Aromatic Hydrocarbons
and their Conjugates in Soils
MichihiroFL：Kl｝S＝M＾■，MasuoNJ、K＾N＝】，TakashiNトニ（；［S‖l
（Rcei叩【】＝h′Iay31．1989）
AbsLract
I亡stimation of polycyclic arornatic hydrocarboLIS （PAT＝ and thcir gIutathio帆glucuroni（le and su】fate con」岬ateSiれ SOils of agrlCultul・alland
and road sideis described．
Each E，Oilwas extracted with mcthnnolWater（10：9．v／v）．rIlhe extract
WaS puri［ied byDEAE▼Sephadcx A−25coLumTIChromatography ar）d thin1aycr ChromaLographytoobtainP＾flan〔JtheiT〔OnJugateS． 1、helattertwoconjugates
WeTe treatCd further wjth enzymes and hydriodic acid to yield rcduced parent
PAH（8kinds〕・The products wcrehnalyzed by gas chronatographymaSS
SpeCtr（）met「Ⅴ．
Thc concentration of P＾Hin the agrlCulturalland w且S5g・29ppb andin thcl・Oad side soilwas馴一】5ppb・The parent PA＝based on thcir conJugateS
fr｛）m agrlCulturallzlnd wasl・08ppb forglutathioneconJugateS and6・48ppb for mixture of glucuroTLjde and sulfate conJug且Le＝ITeSPeCtively・l）AlIconstituting
COnJugatCS Were anthracenc，phcnarlthrerle．9methylanLhracene，fluoranthcnc，
pyrenc，Chrysene．benzo（且〕pyrene and dibenzo（且．h）anthracene．
COn】ugateS7・J
This papcr desL・T−ibes thc esLjmation。r PAH and け帽ir con」11gaLesin soils or agriculturalland and of road sides with
血eavy trarric．Materials alldルIethods Allorganic solvents werc use（t after djstillatio】1．R且ny nicket was ob一札ined rrom Kanto Chem．Co．；ary】sulfatase（fromIIe】jx l）（】matia，EC3・1・6・1・）from Boehrj71ger Mannheim Co・； βglucuronidas巳（from Imtroduction
PoJycyclicaromatichydrocarbons（PA上i），
SuCh as benzo（a）pyr即ほ（虻ap），hellZ（〉（a）
anthracene■ CtC・are abun血山1y presentln the environmer・t〜り．of Lhese PAH．a con Siderable rLUmber havc〔泊rCiT10genicity．c・gL airbo［Zle particulates b）r COmbustion．Or］
the othcr haTld，theseI，＾H are o7くidized t。
related hydroxyated com［】｛）Unds by microoganisms in soils and protluced their gluta仙one， glucuro山de and sulJale
l nepartmentoE＾gT（トEnviTlOnmentalScien（：e，
Me（1jcineIObihiro，IIokk月jdo，O8O．JapaLl．
ObihiroUniversILyofAgricultureaEl−1Veterinary
M．FtJK【1SIl【M人．M．NAI仏NU．T．NEGIS川
20￠
platcs developed wiLh ehloroEormrmethanol−
water（90：10：1，Y／v）．PAH conj11g8te SpOtS were detモCted t〉yirr且di且tlnn of UVl且mp且t 254nm 且nd colour reactions sprayl咽 With 50％ 6ulfuric acid（stero18），dnaphtol
（sugars）andninbydrinreagent（8minobase）
in the us11且Imanner。
Glu暮ath血Ie COnjl堰ateS：COnj岬ateS（10．0 曙）purified byTl．Cinmeth肌01（1−OmJ）we躇 reduced with aqueous slurry of rany nicl【el
（about2g）且t60℃for2hour日．The prod11etS
wereextractedwithetllylacetate and benzeJ−8 twotimesandsubjectedte・GLCanalys1810）・
Gl11CurOnide conjugates＝ the stopper豆d co山calcenLrifug8tube、C（〉ntaining con】ugateS
（5．0喝），0．2M sodium acぬte bu侮r 〈1・0皿J，
pI瓜5）且ndβglucuronidase（川00units）
wa眉incubated at 37℃in th8【hrk for 24 hoursin a water batl】inc11bator op打atぢd如 100 strokes／min．The tube was extracted with3．OmJof etbylacetate・
Sulfa捷COnJugat8S＝ the tube eo山且＝ling eonjug且teS（5．0曙），0．1M phosphateburfer、（1・D
nl，plT7．5），DLS且CCharjc acidrl．4lactone
（12．5moles）and且rylsulfat鋸婚（10u山値）wa6 incub且ted and extracted as described ab8ヤe・
The mixture of PAH 81eohoIB（3．3礪）
produced from upper2enzyme reactiom wa8 rerluxed withlmノof57％hydriodie acid（7．5 mm。】）8¶−1川．Om／of aceticacid solution（99 軋v／v），for15hけurS atl試ドC to obtaim paremt pAH of alc血01自・The hot solution wa写Lhen pouredintol％ aqu帥uS SOdium bit；ulifitc solutioTl（1DOm／）．The PAH were extractt）d with20mL of ethylacetate and the upper Layer was washed with water, and air dried．The crude extract waB diss｛〉lvedin sm且11volume of bcnzen8 and purified by pas姐gethrougbashortcolumnofsilicagel
〔5g）eluted with benzene．
PAH and parent PAIipr印ared from conjugates were analyzed by Shimad乙u QP lOO8gas chrom且tOgraphym8日S Spe￠trOmetry Ilelix ppm駐ti且，EC3．2．1．須．）from Miles
Lab．Co．；anthrac8叫 ehrysene，benzo（8）
pyrene乱nd dib8n印（a．h）anth一見cen￠from TokyoIくa8eilndustry Co■；pllenantbrene，9 methylarLtllraCe71e and pyrcne from Wako Pure Ch￠m．Co．；rluoranthene rroml∃且Stm且n Rodak Co．；D−SaCCharic acid−1．4lacton￠
from Sigma Chem．Co．；Other chemiJCals were obtaiTled rrom w且ko Pure Cbem．Co．
Soil組mples w8r8 COllected rrom agricul血ralland amd ro且d sides〔28，000 vehi亡I色盲／day，R（）ute38at Obihiro）．The soil
（5kg，dry soil）wa日SOnicatedwith3volumes
（w／v）of methanoトwater（10：9．v．／v）且nd
extracted thre￠ tjmes．Thc extracts were
combiTled 8nd conc8ntrated（〕n 且 rOtary evaporaもor f几UαC昆の．The residue（300喝）
wa5tak抑upinchlor（〉form−metbanol（1‥1，
v／v，10mL）and suhjectedto DEAESeph且dex A25column￠hro爪atOgraphy（20g）（The SOlid pllaSe WaSimmersed for 2 daysin
。hlorofprmmetbanoト1Msodiumac扇Ate（30＝
68：8．Ⅴ／v）sol11tion aTld w且Shed exhan緑Ⅴ￠ly withchloroform−methanol−Wat8r（訓＝68二8．Ⅴ
／v）to remove sodium 且Cetate and 乱fter d曙aSSing for15min，thF gelsuBp￠ruion was pack8dinto a column）・The eluting solv8dts werechloroform−mCthanoトwater（30：60：観
／v，160mJ）．methanol（70mり and加Ⅹt a gradient madc usi喝0・05・0・15amd O−45M amm。nium乱Cet8．t￠inmethanol（8伽／each），）
The el、1tion rat￠W且S adjusted to O，3n／／min and fractions oflOM／were co11那加d．To sh叩 the elutiollt汀Ofile，absorban亡ies of fra亡ti。nS Were meaSured by ultTaViolel（UV）
spe℃trOm￠try（A 254nm）and fluoresenee spectrometry（Åex365¶町 Åem407nm）・
Ammo−1ium acetate in the elu8teS WaS removed by cation−eXChange column 止romatogT叩hy（Dowex50W一Ⅹ8）・
Furth訂 p11rification of fractioIIS WaS ca汀ied（）uL by thin−1且yer Chrom且tOgrapby げLC）on Ml王RCK precoated Bilic8 ge160
−52
Polycyclic且rOmatic hydroc且rbonsin so上1s
201（EI，GCMS）on col11mn（0，33mmX25m）of CBP−1．Opcrating conditions weTe aS folloⅥ作＝1nJeぐti仰temperature，300CC：COlumn temperature，180270Uc（5℃／miLl）；flow rate of carrier gas（helium）．1．5kg／cd；i（】n SOurCe and molecular separator tempertature．
250℃andiomizationvoltage，70eV．
Results andl）iscussioII
Elution profile plotled 注S Op11c乳1 absorbancy of soilextract by 工）EAE Sephadex A 25colum†lChromatography was Shownin Fig．1．Fractions．chloroform−
meth且nOl−Water（Fr＿ A，highest peak），0．15
︵∈仁卜専岳ペす□頴琶ペl︻01×︶85qLまqイ巴口む警○⊇E
O り C C O ．〇 〇 6 月 2
0 乱 0 8 6 ィ 2． U O D．
︵∈く苫N︶むUu雲lh〇宍l亘．PD
EluLi（）n So】venL System
Fig・1・EluLion Profile of SoilExtract byDEAESephdex A25 Cvlumn Chromatography
【ニト→○：UV Absorbance；壬 こ：Fl110reSenCe Absorbancc．
Soilswereextract8dwjthmethanol−Water（10＝9，V／v）．Extractusedw且S30Dng
（Fr．B）and O．45M ammonium acct且tein metlはnOl（1叶．C）were collected．IL was COnSidered thatnonionic6ubstanees，S11Ch as pyrene，BaP and other PAH，Were eluLedin Fr．A a爪d polar substamc8S SuCh as C ｝nJug且teS W（）re eluLedin Fr．B and Fr．C．
The results or rll⊥C of each fraction
Were Shownin Fig．2．Rr v且1ues of Fr．A and correspon［ling PAH stand且rds（8kinds）
Were Shown to be about C−83 by UV
irrLldiation・ AIso， SpOtS Lemporarily identified as glutathionc conjugates（Fr，B），
glucuronidcJlnd sulfate conjugates（Fr．C）
w（汀e Shown to be Rf O．〔帽 for Fr．B
（ninhydrin reaction positiYe），0．12（馳％
S111fl⊥ric acid reactiorL POSitive）and O．21（α naphtolreaction positiv（））ror Fr．C．
The results or analysis of Fr．A（PAH Eraction）by GCMS were shownin Tablel，
The composition was agreed 且1most COInP］etelywith already reported reEerencesGl
18．11）
The fraction（8．060f Rf valuein Fr＿R）
COrreSpOndi岬tQ glutathione conjugaLes was
M．FuKし細則A．M．NAK／＼N（）．T．Nll：GjS川
Tablel．Conee‡ltration8f PAIIE曳tr8Cted
（rom S￠ils ofAgrictllt11ralLand and Road Side
CoTICemtr盆ti⊂ln（′ppb）
く⊃ ⊂⊃
Compound AgruralRoad畠i血
7 1 だU 9 1 7 6
1 g n︶ nV 負U ra l nn 幻q Rり 爪U 7 6 点V別 14 67 凋粥別 別 l 1 6 ▲uJ 1 3 ・d一 1 2
Anthraじ8¶e Pheれanthr帥や 9・Methylanthracene
Fluoranth、en8 Pyrene
Chrysell￠鮎mzo（a）pyrenぢ
Dib8n乙0（a，も）一札nthr且￠en￠
Tot且1
1▲25
4．3859＿29 64．15
Concentratior10f eaeh ℃OmpO11nd w年S calculated fromion peak aナia ofGCMS
Table2．Concentratio【10f Par8nt PAH ObtainedConjugatesinÅgr
iculturalI．乳nd Fig．2 TLC ofFractions prep且rCd by
DEAESephadex A 25ColumrL ChroTTlatOgraphy of SoilExtract
PAH standards：anthracene，phcnanthrene．g−
m8thylant11r且Cene， fl110Tar・th帥e． pyrene，
chrysene，BaPanddib8n芹0（且．h）且nthr且Cene；
Fr．A：eluate with ebloTOrOrm−metb且nOl−
wat8T（30：60二8 v／v）；Fr．B：elu8t8 Wltb q．15M ammoniumacetatein methanol；Fr▲C：
eluate with〔〉．45M ammonium acetate in meth且nOl；developing soIvent：Chlorororm一 血甜旭nol−Wat町（90：10：1v／Y）＜：＝〕：UV r伊88ti（）n；⑳：ninhydrin re且Ction；●＝ α一 n呵）htolreadion・
treated wilh rany nickel reaction whieh
catalyz8S hydrolysIS Of C−S bond spb−
eifically and the product was 良naly2巳d by GCMS＿The res111ts w8re Bh【｝Wnin Table2．
M札止y parent pAH wereidentified afld
quantitated．including anLhrac8ne（0．03ppb），
pム細孔nthrene（0，01ppb）．9m雨hylanthracene
（0．20ppb），pyrene（0．26ppb）andchry8en8（0▲58 ppb）．The composition of PAIipr叩ar￠d from glutathione eonユugateS Clo3ely resembled Fr．A （）f agrlCulturalland d￠SCri如d above．
Tlle mixed8ⅩtraCtS8fter treatmellt With
−54
C（）nC8苅tr乱ti ）n（ppb）
Compound
Gl11CurO山de＋Sulfate
（；1111且ull由ne
駆陀肌 飢∴拍感 60 20 nU l 八U l l V爪U ︿U
田口l如一鉢58 0 爪U ∧U O ハU
Antbraく†ene
Iうh8n且nthrene 9湖ethyla山ムrA既舶
FlⅥαraTltIlenQ Pyr￠n8
Cbrys印e
Ben耳0（a）pyrene DJ心enzo（a，h）
色nLhr且Ce皿e Tol且1
○，37
1．08 6．48
CoれCmtration（〉f PAII：S（∋e T8blel
＊：Mi）止ure of pr（）如ct富Of th8tWO erほyme readions
βglucuro扇dase and aryl童ul臨地se of
temporarilyidentiried gl11￠ur（】nid￠且nd gul転地
Polycyclic aromatic hydrocarbons in soils
203conj11gate6 （Fr，C）were reduced with
hydriodic acid and analyzed by GC MS，
Unfortunately，the e3（traCt from the enzyme reaction was ofins11fficient quantity，and had to be used as the mixture of products of the two enzyme reactions．The resulkako
Were Shownin Table2．Ågai11，many pAH Wereider］tified and quantitated，including
anthracene（0．96pph）．phenanthrene（l．56ppb），
9−methylanLhraceれe（0．31p、pb），fluoranthene
（1・15ppb），pyrene〔1．33pr）b），Chrysene（O．60
ppb），BaP（0．20ppb）Elnd dibenzo（a．h）
anthracene（0．37ppb）．The data also agreed
With reEerenct，S already de＄eribcdL乙▲ユ）It has he巳El
山ngus 仇花〝上作g力αmeJJα eJgg〔1几ぶ prOduce epoxyBaP from B且P by the cytochrome P 450 monooxygennse fiystems Rnd then
hydroxylaLe Lo trarlS BaP−7．8一1ihydrodiol．
formlng the ultimate carcinogenic，7，8 dihydroxy9，10 epoxy7．8．9．10 tetrahydro−BaPiTlanimals．In thiF3 rrLanner We COnrirmed tllat many art汀1cialPAH dirruse（lon the soilsurrace mainly formed gllltathione． glucuronide and sulrate
COn］ugatCS．by soj】microbe actiYity via hydroxy−PAT7 and dihydroxyLPATI．The COTl］ugateSidentificd from the soil，preSenLly，
Seem tO be detoxification prod11CtS Or SOil microorgaTLjsms・The PAflprcpared and ideLltiEied from Fr．A of soiIs，Showed abouL the same pattern as rro口1re†erences reporLe（l alreadyl引・This means，Or】glnS Of PA上1are
且rtlricja】sources．Rererene（・e
l）′1、．NlくG上川‖，M．N几K．へNr）．K，YAN叫 C．1I K爪1and 入4．Fl射」SlUMA，Isolation amd ideれtirication of β nitrostyl・ene rrom SrnOked chicken，Environ．PolluL．．弧
279283ぐ1988）．2）T・R山山肌川．G．Bi】CI汀1ft and A．B岬溝口・llト」，
NitraL即1polycyclicaromatic hydrocarbo11
in urban air partic）es，Environ．Sci．
Technoし18．861−865（1982）．
3）T．RAMl〕皿Ⅰ一．Polycyclic aromatic ketones
in environment且1samp】es，Environ．Sci．
Technol，】17，666670（1983）．
d）T・D・B柑Ⅶ王ERandR．A．H耶S，Simil且rity Of some org且nic compoundsin spark 1gnilion and dieselenglne p且rticul且te extracts．Environ．Sci．「11echれ0し18，202
2（）6（1981）．5）G・R・DしD＾Y and R．A．H…／S，Cyano−
arenes produced by combu＄tion of nitro
COntainlng fueJs，thviron．Sci．Teclm01．，
12，956 968（1978）．
6）T，R仙11）A川．and G＿ BI℃l丑：It，Characteri Zation of polynuc】ear aromatic hydro−
carbons derivatives im emission from wood and cerealstr＆W（｛）rrLb11Stion．Anal▼
Chem．Acta．．144，8391（1982）．
7）′11．N佑ISIU，M．NノⅦ価n，S Kr）】う．＼YA5tlland
C．H，l（LM，IsolaLion and determin且tion Of benzo（a）pyrene glucuronide 且nd SulraLe coれJugateSin soybean】eaves，Bu11．
Ⅵnviron．Contam．′roxicol．，39，294−298
（1987）．
8）C・E・C■く上川（；l．1八 andl〕．T．G‖−SON．
Oxidation of benzo（a）pyrene by Lhe filamentous fungus Cu乃几fg九αme〃α eJ堵α花S，J．Biol．Chem．，245，1217412180
（1979）．
9）M．1w＾VO7LLand Y．N＾GN，Anewchromato＿
graphic approach Lo the re501utiol10f individual ga叩1iosi【】es，Biochim．eL．
月iophs．Acta．．52月，257267〔1978）．
10）lT．K．S軋Kl帆 C＿ しI．Moofu】and M．C＿
Mノ＼【‥Lト：（川． Analysis of waterSOlut）1e eonJugateS PrOduc￠d by haTnSter embryo Ce】1t，eXpOSed to po）ynuclear aromatic hydrocarbons．1n一−Polynuclear aromatic hydrocarbons ，ed−L】y A．BJPTび昭T‖且nd A・rl＼D小爪町 Batte11e Press，Columbus，
1980，PP．923．
11）M．Kr〉Y川rヱNY and R．G．HAl‡ⅢY．Efficient
M．FuKUSHエMA，M．N八代AW㌧T．NⅨ11別】l
204red11Ction or polycyclic quinones，hydro quInOneS．a−−d phenoIs to polycyclic aromatic hydrocarboTIS with hydriodic
acid．一．Org．Chem＿，亜，亜13−4816（1979）・
12）M．ZA＼ly：T‡．Polycyclic arom8tic and hetero−arO一口atic hydroc且rbons，In Th金 丸andbo（〕k of environm8ntalehemistry ， Vol．3．part A．ed by O・Hu71ZING班，
Springer−Verlag，N8WYork▲1980・pp・1〔貯
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1ne．1983，pp．67．
農用地および国道路肩l二壌に分布する
多環芳香族炭化水素およぴ その抱合体の同定・定量
福島 道広・中野 益勇・根岸 孝
摘 要
農相地および交通量の多い国道路肩土壌（国道3折 線，帯広）に分布する多環芳香族横化水素（PAII）
とそのダルクオチン抱合体，グルタロン醒抱貪体およ
び硫醒抱合体の同定・定量を行った。
採取した土壌をメタ／−′レ水（10：g，V／v）で 抽出し，次いでその抽出物からPÅHと抱合体を分離●
精製するために．DEÅE セファデックスA班カ
ラムクロマトグラフィーおよこ博層クロマトグラフィー を待った。ダフレタチオン抱合体比 ラネー・ニッケル 処理によりPAHとした。ダルクロン観抱合伴および
硫醒抱合体ほ．それぞれβダルクロユダゼおよが ァリルスルプァターゼ処理で加水分解し，PAHめ水 酸化物を調整Lてから，ヲウ化水嚢鱒で還元し本来の PAHとした。得られたPAHはガスクロマトグラフィー
ー質量分析により同定・定蟄を行った。
PAliは，農用地土壌中および路肩土壌中にそれぞ れ乳29ppbおよび阻15ppb分布していた。また，農 用地土壌中に時グルダチオシ抱合体は1．08ppb，ダル
クロン醍および硫酸抱合体は合わせて6．48pノpb換出さ れた。抱合体構成PAIiとLてはアンもラセン．フよ ナントレン．9メチルアントラセン，フルオランチ ン，ビレン．クリセン，ペソゾ（鱒）ビレンおよぴジ ペンゾ（払h）アン1・ラセンなど8種頬を検出したb 以上の成績より，農用地土壌中に蓄積されているPAH は，抱合体として代謝をれていくことが確認された。
帯広研報1．16（1g約）：199〜204．
56
