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I~NVIRONILIENl~.lL RESE.\RCII2, 22-29 (1968)

Experimental Studies on the Formation of PolycyclicAromatic Hydrocarbons in Plants

J. BORNEFF, F. SELENICA, H. KTNTE, ASD A. MAXIMOSInstitute of Hygiene, University of fliluixz, Germnily

llew imi Dewnllrf~r~ 14, 1nhY

Algn c (Cl~lor~elln vrtlgaris) grown in YLxcc1 ate medium synthesized bcneo [al-IIyrcnc and other pol~c~clic aromatic h~drocarhons with 10 times higher spe cific:activity than algar from a cont,rol batch without the isot,ope . Th is obserxxtion con-firms earlier results and sccm~ to show unrquivoc~nllg the l)ioch(,micsl synthesis ofrarcinogrns in plants . Thr ~s ulis of t III, esp crimc nls ofkr an esp lana iion for thf)occurrence of lmizp)-rcnc- in \qot:~bl(~r, vrgctabk f:lls and oils, in the soil, and the

ground watrr. Purpose of thr synthe sis may br the, formaiion of promotcra of plantgrowth as sugg ested by GrSf and Nowak (1966).

Mallet et al. (1960) demonstrated in marine fauna and flora (English ChanneltAtlantic Ocean) the consistent presenw of traces of benzo [a] pyrcne. Malletand H&OS (1962) reported additional results on the occurrence of polycyclicaromatic compounds in oak Ieavcs. These findings were explained as being due tocontamination of the leaves by carcinogens present in the air aerosol. This in-terpret,ation was prompted by the observations of Goulden and Tipler (1949))Waller (1952)) Commins et al. (1957), Kotin and Falk (19631, Cooper (1954))Hoffman et al. (1965), Hueper and Payne (1960), Bonnet (1962)) and others whohad confirmed the presence of these compounds in soot and other emissions. Rc-suits similar to those of Mallet and H&OS were obtained in our analytical work,but led to different conclusions.

Initially Borneff and Fischer (1962a) found polycyclic aromatic hydrocarbons(PAH) in the filter sludge of a mat8cr work at Lake Constance. The material

contained profusely silicious algae which suggestecl he investig:lt8ion of phyto-plankton. The analysis of Asterionella fomosa from Lake Constance showed thepresence of 1 mg of fluorescent hydrocarbons per kilogram dry substance. Theorigin of the carcinogens from lubricant,s and fuel of ships, from t hc exhaust ofcombustion cngincs, road dust, soot, and other combustion waste products wasconsidered. However, there existed the possibility that the formation of thecarcinogens was correlated with the mctaboliam of plants, since according toBIumer (1961) hcnzo[a]pyrene can be found in soil samples from nonindustrialarcas. Pyrolytic processes caused by forest fires could not be accepted as a solesatisfactory esplanntion.

Controlled laboratory esperimcnts were required to exclude the role of ex-ogenous factors. Borneff and Fischer (1962~) stud&xl first, asparagus and cauli-

22

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FORMATION OF BEXZO-a-PYRESE IN PLANTS 23

liower, i.c., vegetables which were certainly r free of scclimcnted Soot, :%lld fila-nlclltous algae grown in llutricrlt mcdin; YAH \V~W plwent in ~~11SamplCs:,

although in very small amounts. Tllcrefore it ~viis stntcc:l tll:lt. tJlCW S~lbSt:lll~Woriginated in the plants possibly by fault,y synthesis.

Further invostig:~tions confirmctl the finding;-: of Blumcr ( 1961 ,I :~ntl Mallet:knd H&s (lg62j 011 t,l le presence of bcnzl)yrcne in Joi1 ~;anll~L~ Th~c rc*ultscould be reconciled with the assumption of the bioqntlicsi~ of PAH. The corn-pout~tls are formed in the plants, arc tlanaftrred to the soil by hid plants, andare finally carried by seeping precipitation to the ground water ~h~rc thcv can beconsist.cntly demonstrated.

The mark of Grimmer (1966), GrSf (1961)) and Gr5f and Die111 (1966) madeimportant cont,ributions to the observat.ions on the occurrence of PAH in plant.

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24 BORNEFF ET AL.

MATIJXIALS AND MlilTHODS

Algul Cultures

Of the various plankton genera we sclc clrd CJJo rrllu and used the strain C hlorella vulguris211-llh which was recommended to us on account of its growth property utilizing acetateas the sole C-source. Cultivation with aeration i.e., in presence of COa was not possible in

this type of espcriment. The nutrient medium had the following compos ition:SoIutio?~ I: 1,O g CH,.COONa.3HsO, 0,24 g Na2HPO I.12H,0, 0,7 g NaH2P 04.H20, and

10,O ml distille d water.Solutio n II: l,l, g KNO,, 0,34 g MgSO,.7H,O, and 10,O ml distille d water.Solutio n III: 0,02 g CaC12.2Hz0 and 10,O ml dist illed water.Solutior~ IV: 0,028g Titrip lcs, 0,014g FeS01*7Hz0; 0,02 mg MnSOI.HzO, 0,03 mg ZnSo,.

7H,O, trace CuSOI.5H,0, trace (NH&MorOzr.4H20; 0,006 mg H&O,, and 10,O ml dist illed

water.Solutions I, II, III, and IV added to 960 ml distilled water. pH adjusted to 7.0.For inoculation of mass cultures, seeding cultures grown on the same medium in lOO- ml

flasks were used. They were derived from single Chlorella colonies. The single colonies haddcvelopcd a.fter several w recks on a solid medium consisting of the above-described liquidmedium with 2@ /b agar added. T ests for absence of bacteria were carried out by subculture onnutrient agar, blood agar, beer wort agar, Mouton agar (nutrient broth agar, conta ining Ylostrength of nutrient broth), and liquid media. Prevention of microb ial conta mination was

one of t,he greatest difficulties of the mass cultures and made the culture in large batchesof 50 liters impo ssible in spite of various attempts of thermal and chem ical sterilization.Finally, the followin g procedure was adopted:

White glass bottles of 5-liter capacity were filled with 4 liters of medium (Solutions I, II,IV), closed with rubber stoppers and ahuninum foil, and sterilized by autoclaving at 120Cfor 2 hours. After coo ling, Solutio n III wa s added in a sterile inocu lation hood under u ltra-

violet light and inoculated with 50 ml of a well-grown culture of the algae. The bottles werekept at room temperature at the laboratory window s without add itional exposure to light orncration. Twice daily the cultures were briefly shaken. Under these conditions maximalgrowth was obtained after approximately 16 days. The important correction of the pH topH 7.0 was carried out on day 11.

After maximal growth was obtained the algae were separated from the medium by mea nsof a continuous-flow centrifuge and dried to constant weight at 50C in a drying oven. The

yield of every 10 X 5-liter batch was about 8g. In 11$ years we had colle cted the 200 g ofmaterial required for our experiments.

C-Lnbelivg

In preliminary rspcrimen ts it was found that the algae tolerated an activity up to 2 &/mlwithout growth inhib ition and that 40%, of the nvailable*C-acetate was ultilized in the(oursc of 3 weeks. According to our calculation a single culture period with *C-acetate(using 20 mC) in 34 liters of mcGum should be adequate for measurement of spec ific activityin the main expcrimcnt. Howcvcr, earlier cspcricnc c had shown that the dry algal substance

that shou ld be obtained by a sing le culture was insuffic irnt for thin-layer chromatography.At leas t 1OOg wPre rrquircd for yhp sicoc hcm ical analyse s. We, therrforc, chos e the followingprocedure :

Approsima tply 1OOg of algae cultured wit,hout, radioartivc isotoprs served as carrier for

:I sma ll amount of algae grown in a single culture with C. We used 20 mC sodium acctate-l-14C in 34 liters of medium. The nuclide was obtained from the Radiochemiral Crnterrlmershnm (England) and containe d a spe cific activity of 29.0 mC/mM. Acco unting for theacetate in the medium a theoretical initial impulse rate of 7,358 impulses per minute (Ipm)pcxr 1 fig C was calculated. The algae assimilat.ed 35.7% of the offered carbon in a 19-dayculture. ilft,c,r prolongrd culturing no furt,her utilization of thr nutrient cou ld be espectrdns shown by t,hc phy siolog ical cond ition of the algae. Thercxfore, we trrminattxd the experi-

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FORMATION OF BEKZO-Cl-PYREKE IN PLANTS 25

merits at this tinle a nd cOll(xctLbd the algae by filtration thruughIJalK 'l' filters that had h'llestracte tl \vil,)l bcnzcn,, f0r the rrmoral of pos$h ly intc,rfcring impu rities. TlI c &W wm

xVasl~c Li twicr with d istilled \\-atcr and dried in an incubator at50C. The process gave a yieldof 9.5 g dricxd algal substance which was :~Idcd 1.0 104.0 p radioinactivt:algac~, Idcnticnl amountr;0f 113.5 g radioinartive algw wcw prtl,arcd in para llel control bat chcs .

Extractions ~~~~r~ g.arried out lvith p urified bcnzenc at 100111 tcmperalurt~ ; the algae wtr(:

thoroughly sllakerl with b,,nzvne and eollwted by filtration after a fewhoura scdimentatiun.r],is llroccss \~:1s Iy,j,,aa ttd 11 tiiilpsduring 4 rltlys. Prc,sh kic~llZ:l,Ilc W:15 l:Ill~)lO~L Y~, dtllOU Zll,v, uscx cl occ;l*io nally the, solv,.nt after it 11x1 ))c%c>n r(~cov(,r~:11 from tllc cstract by distillativn .

The total amount of bcnzcnc used wxs2.5 litcrs. The cstrncts wcrc concentrated to 10 ml

:~lltl nm through z colum ns of 26 nnu tliaow tc~r cl~.rg(~1 wyI(h SO g,21,0, (basic, r:~tionotrollic,;lctivily lc\~l 1) alltl rlutr,l with 2000 1111bc~ncm~. Since tlw calun~(,s had :L strong ~~110~ ~0101

:kffc>r c,on(:cntration :mcI a~ sdtlitio nal ~~~wifkation wrrktl 0rlt in a colum n with 60 g AII~O:;(lit1 not r,xmove tlicx imlnuity, it was finally cliniin atc,d l)y further p\irification hJ- clnwnal.og -~,:tphy on acety1atc.d pa1~r. l+:\-(~ntunlly the c~lukd extra

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26 BORNEFF ET AL.

algae was also prepared. Under these experimental conditions 1.0 pg fluoranthrne in 20

ml chloroform/m ct,hanol was offcrcd a pos sibility of exchange with onp tenth of the benzeno-solu hlc fraction cxtractcd from the algae. After 25 days r>sposure the chloroform-methan olmixture was dist illed off and replaced by benzene. Fluoranthrnc was separated by chroma-tography o n an a.luminium oxide colum n, followed by two-dime nsional thin-layer chromatog-raphy. Sftcr qualitative and quan titntivc determina tion by fluorrsccn ce photometry theimpulse rates were measured.

Chemical Results

RESULTS

Seven polycyclic aromatic hydrocarbons (Table I) were found and quantita-tively determined by chromatography and spectroscopy.

TABLE IPAH CON TENT OF INACTIVE AND W-LABELED ALGAE

PAH (pga) found in:

Compound Inactive algae W-labeled algae

Fluorant,hene 5.SO 6.20Benz[n]anthracene 0.78 0.65Benzo[b]fluoranthene 0.39 0.42Benzo[a]pyrene 0.07 0.08Benzo[ghi]perylene 0 1 0.23Benzo[k]fluoranthene 0.14 0.15Indeno[1,2,3-cdlpyrene 0 1s 0.17

tZ The data refer to total extracied amc~ur~t of algae disreg artling loss es due IO c,hrorllatogr:rphyand elutioll.

Physical Results

The impulse rates of the individual PAH are given in Table II.The investigations on the occurrence of exchange reactions showed impulse

rates consistently lower than twice the background (Table III).

TABLE IIIYPULSE RliTES OF PAH F~OJIlQ2-LABELED AND NONLABELED ALGAL CULTURES

PAH compound

lGlabeletl

IvIpm (corr.)

Ipm (c0rr.a) per pg C

Nonlabeled

Ipm ratio non-Ipm (corr.) labeled :

Ipm (corr.) per rg C labele d

FluorantheneBenz[n]anthracene

Benzo[b]fluorantheneBenzo[a]pyreneBenzo[ghi]perylene

Be~~zo[k]fluorant~herleIildeno[l,2,3-ctl]I)yrelltl

1975 1973 334 144 126 23 1: 15.7427 408 663 50 30 41 1:13.6

151 133 333 x2 12 32 1:ll.l137 118 1540 31 11 165 7:10.7034 934 4247 76 57 271 1:16.41% 15s 1 I ( I6 42 2 165 I:7.27 252 7650 40 I ) 1 IO 1 : 1%:;

u Corrertetl for background and esterlutl star~tlardizatioll; Ipm = irnplks per rnillute.

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FORXIATIOS OF BEXZO-Cl-PYREKE IX ILAKTS 27

I~n]~lsc I&& of 11~4ive Fluomnthene Kept 2s daysill l&C-Co nt:tining and %-Free dolut ion

Aqueous*Chluroform/methallolBackground

a (Jjrrecte(l for t,ackground :md rsterunl st;tndnrdizatiou.I) A(*1 ivc or inactive nut,rientj medium .

(jr1 tllc basis of the absolute weights given in Table I the impulse rate forI ,,,g C can be calculated. The expected value should hc 485 Ipm//.~g C. Thisfigure is obtained from the amount of C added to the culture medium (~7358I~n?/,~g Cj, a counting yield of 78.85% (=5798 Imp/pug C) and the fact that9.5 g. lahcled dry alga l substance was added to 104 g inactive material.

The calculated value was lower in the case of fluoranthene and benzo [blfluor-anthene, namely 333 Ipm./,,,g C. Considering the extremely small amounk ofmaterial, this discrepancy may lie in the range of the cxpcrimental error. Ont8he other hand the values for hcnzo [a] pyrene, benzo [g/G] perylene, benzo [k] fluo-Innthene do not agree with the theoretical impulse rate, being considerablyhigher. We assume that, this is cnuecd by the presence of nonfluoresccnt P,4Hwhich :w located at the s:mw spots of the thin-layer chromntogram as the flu-orcswllt8 ~oml~ounds. Both t ypes of substances are mlc-nsurcd by the radiot~hcmicaltechnique, whereas only the fluorescenC cornpound.. q arc demonstrated by fluores-wncc photometry. This is particularly evident, in the cast of benzo [ghi]pcryIene;it is our cxlwricwce that thc compouncl is always contaminnted with a compara-t,ivclly large amount, of we:~kly fluorescent PAH, at least if twtc(l with the mcthotlof t,lrin-layer cllroni:ttography used in our cxperinwnts.

Tlrc significance of the rndiorhcmical results is, however, not influenced bythese obpcrvat8ions; the parallel expcrimcnts with nonlnbcled meclia rcsultctl inal l instances in markeclly lower impulse rates.

DISCUSSION

The results of our analytical work are unequivocal: in algal cult,urcs grown inthe presence of nonlabeled acetate as sole C-source the specific activity of al lisolated PAH (except fluoranthene1 was found witllin the range of tmice thebackground. In presrncc of C acetate the COurlt of inipulsrs was more than 10times hgcr, indicating that t,llc lal~c~lcd C-atoms had j)~(ln incorl,orated in the

PAI I-nloleculcs. T%c occurmire of csch:tnge reactions was esclu(led by controlrslwrimcnts. Thw:e findings demonstrntc the l)iosyntllcsis of PAH in plants.

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28 BORNEFF ET AL.

total amount can be larger than 100 ,,,g/kg. In plant-bearing soil approximately10 p,g benzo[n]pyrene is found; 1.0 pg/rn is dissolved in the water. Humans and

animals have been always subjected to a certain exposure to carcinogenic PAH.The compounds contained in food amount to 10 mg per year (Borneff and Fabian,1966) ; additional traces arc inhaled as soil dust and lodged in the lungs.

The health risk caused by orally introduced PAH has not yet been completelyelucidated. There a nutritional influence certainly exists since the caloric intakecan have an effcrt on tumor de\-clopmcnt,, although ndequntc epidemiologicalstudies on the possible cffcct of certain food constituents arc still missing. How-ever, the studies of Dungal (1959, 1961a,b) on smoked food stuffs and the viewsof Wyndcr et nl. (1963) regarding the correlation of vcgetnrian rlict and gastriccancer indicate that the consistent uptake of PAN with vegctablcs may exert aninfluence on intest,iiial neoplastic diseases. Similar conclusions can be reachedon the hasis of the findings of Hakama and Saxen (1967) with regard to thesignificant correlation of t hr consumption of cereals with gastric cancer. Itshould bc pointed out that according to Wynder et nl. (1963) starchy food stuffsplay an important role, nlthough their P,4H content is lower than that of greenvegctahles. We believe that, this llroblcm should be cluridated by an internation-ally coordinated epidemiological st udy.

The evidcncc of PAH synthesis in plants raises the question of the biologicalinterpretation of this process. Our first assumption of a faulty synthesis has to

be corrected ; GrKf and Sowak ( 1966) succeeded recently in demonstrating de-finite growth-promoting cfft,cts of PBH in higher :ind lower plants. Hydrocarbonswith high carcinogenic potency in animal tests were also particularly cffect,ivegrowth factors. The aut hors assume that PAH arc plant-growth hormones ortheir precursors. Consequently PAH should be included in the group of Lnaturalproducts with definahle biological functions in the T-egetablc kingdom. The con-ccntrations are, however, SO small that rarcinogenesis in humans and animalsdoes not occur nornially. Ncverthc>lcss, it is our opinion that. al l possibilities forelimination of carcinogens should bc conai(lcred, bccausc t,lie natural level ofcarcinogenic compounds will unavoiclal~ly incrcn:dc owing t,o the marlifol(l py-rolytic product,ion of these substances.

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