Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
lL
2, 22-29
RESE.\RCII
Experimental
(1968)
Studies
Aromatic
on the Formation
Hydrocarbons
in Plants
of Hygiene,
llewimi
of Polycyclic
ASD A. MAXIMOS
University
of fliluixz,
Dewnllrf~r~
14, 1nhY
Germnily
Algnc
(Cl~lor~elln
vrtlgaris)
grown
in YLxcc1 ate medium
synthesized
bcneo [alIIyrcnc
and other
pol~c~clic
aromatic
h~drocarhons
with
10 times
higher
specific:
activity
than algar from a cont,rol
batch without
the isot,ope. This obserxxtion
confirms earlier
results
and sccm~ to show unrquivoc~nllg
the l)ioch(,micsl
synthesis
of
rarcinogrns
in plants.
Thr
~sulis
of t III, espcrimcnls
ofkr
an esplanaiion
for thf)
occurrence
of lmizp)-rcncin \qot:~bl(~r,
vrgctabk
f:lls and oils, in the soil, and the
ground
watrr.
Purpose
of thr synthesis
may br the, formaiion
of promotcra
of plant
growth
as suggested
by GrSf and Nowak
(1966).
Mallet et al. (1960) demonstrated in marine fauna and flora (English Channelt
Atlantic Ocean) the consistent presenw of traces of benzo [a] pyrcne. Mallet
and H&OS (1962) reported additional results on the occurrence of polycyclic
aromatic compounds in oak Ieavcs. These findings were explained as being due to
contamination of the leaves by carcinogens present in the air aerosol. This interpret,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 who
had confirmed the presence of these compounds in soot and other emissions. Rcsuits 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 suggesteclthe investig:lt8ion of phytoplankton. The analysis of Asterionella fomosa from Lake Constance showed the
presence of 1 mg of fluorescent hydrocarbons per kilogram dry substance. The
origin of the carcinogens from lubricant,s and fuel of ships, from t#hc exhaust of
combustion cngincs, road dust, soot, and other combustion waste products was
considered. However, there existed the possibility that the formation of the
carcinogens was correlated with the mctaboliam of plants, since according to
BIumer (1961) hcnzo[a]pyrene can be found in soil samples from nonindustrial
arcas. Pyrolytic processescaused by forest fires could not be accepted as a sole
satisfactory esplanntion.
Controlled laboratory esperimcnts were required to exclude the role of exogenous factors. Borneff and Fischer (1962~) stud&xl first, asparagus and cauli22
FORMATION
OF
BEXZO-a-PYRESE
IN
PLANTS
23
:%lld filaliower, i.c., vegetables which were certainly r free of scclimcnted Soot,
nlclltous algae grown in llutricrlt
mcdin; YAH \V~W plwent
in ~~11SamplCs:,
it ~viis
stntcc:l
tll:lt.
tJlCW
S~lbSt:lll~W
although in very small amounts. Tllcrefore
originated 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,lle presence of bcnzl)yrcne in Joi1 ~;anll~L~ Th~c rc*ults
could be reconciled with the assumption of the bioqntlicsi~
of PAH. The cornpout~tls are formed in the plants, arc tlanaftrred
to the soil by hid
plants, and
are finally carried by seeping precipitation to the ground water ~h~rc thcv can be
consist.cntly demonstrated.
The mark of Grimmer (1966), GrSf (1961)) and Gr5f and Die111 (1966) made
important cont,ributions to the observat.ions on the occurrence of PAH in plant.<.
It, was shown that vegetables, lettuce, and cereals cont,ain considerable amounts
of hydrocarbons.
Analysts
by Grimmer and Hildebrandt
(1965) indicated that
grain s:amples of the Ruhr district contained ten times more carcinogens than
and other nonindustrial
arcas. This
specimens from lower Saxonia, Holstein,
emphasizes the causal significtlnce of air pollution. GrBf and Diehl, on the othck
hand, brought forth evidence that the foliage of various trees as well as vegetables
level of PAH indcpcndctlt from that
for human coneumpt,ion contain a normal
locale and even if grown under protective plastic sheets. The range of benzo[n]pyrene is 10 t,o 20 pg/kg dry substance. Thrct~ to fivcb times higher values are
obtainetl from milting and yellowing parts of tlw pl:ll1ts. Scc~l~, f1,uit.S. and tuhcrs
with comparatively
high starch content containecl only 1 to lO(;/ of the PAT-I
concentrations found in green plant,s.
In an extension of our own studies in algae n-c 11nw pow11 C,h/orc/ln in thcl
laboratory
under controlled contlit,ion; wc found in t.lw bcnzcne cstract of 100 g
dry material bcnzo [CL]pyrenr, hcne [al anthracenc, bellzo [[I] fluorant,hene, indenopyrene, benzo [Jfluoranthene,
benzo [k] fluoranthene,
benzo [ghi] peryIenc, Auornnthene, and pyrene. The nutrient media and the chen~icals wed did not contain
dcmonstrnhle
amounts of PAH.
Griif and Diehl (1966) delnonstrated biochemical synthesis of PAH iI1 l~igll~~l
plants. hft.cr having determinccl the amounts of PAN in r;vc gr;tins, jvll(qt,, :Lncl
lentils they induced germination of the same numlwr of grains in aqupous medium. The synthetic nut,rient solution was prepared wit11 chemicals of highest
purity and W:M tested for absence of PAH. Tllc~ young plants were analyzed
nftcr 10 clays when they wre about IO cm long. Colnparcd to the grailIs the
content of benzo [crlpyrene and other hydrocarbons
had incrcascrl 10-100 fold.
In further experiments
rridcnce
was brought forth that el~(log~~l~o~~~spnth(Lsis
of P-AH took plncr indrpcndcnt
of photos~nth&s.
In principle, the biosynthwis
of carcinogenic
and noncarcinogenic
PAH in
lo~cr and higher plants had brcn cst~ablishetl by these studies. Tl~c unprctlictal)le
implications
of three results prompted us to study the synthesis not only by
cwntitntive
comparison, but :-tlso by means of carbon assimilation. In this report
experiments
are clcscrihed in which algae mcrc offered isotope-labeled
carbon ;
PAH thus formed wore extracted , Wpal%tPd,
Fill4 the
specific act,ivity of the
purified compounds determined.
24
BORNEFF
MATIJXIALS
Algul
ET
AND
AL.
MlilTHODS
Cultures
Of the various
plankton
genera we sclcclrd
CJJorrllu
and used the strain Chlorella
vulguris
211-llh
which
was recommended
to us on account
of its growth
property
utilizing
acetate
as 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
composition:
SoIutio?~
I: 1,O g CH,.COONa.3HsO,
0,24 g Na2HPOI.12H,0,
0,7 g NaH2P04.H20,
and
10,O ml distilled
water.
Solution
II: l,l, g KNO,,
0,34 g MgSO,.7H,O,
and 10,O ml distilled
water.
Solution
III:
0,02 g CaC12.2Hz0
and 10,O ml distilled
water.
Solutior~
IV:
0,028g
Titriplcs,
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 distilled
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
had
dcvelopcd
a.fter several
wrecks on a solid medium
consisting
of the above-described
liquid
medium
with 2@/b agar added. Tests for absence of bacteria
were carried
out by subculture
on
nutrient
agar, blood
agar, beer wort agar, Mouton
agar (nutrient
broth
agar, containing
Ylo
strength
of nutrient
broth),
and liquid
media.
Prevention
of microbial
contamination
was
one of t,he greatest
difficulties
of the mass cultures
and made the culture
in large batches
of 50 liters
impossible
in spite of various
attempts
of thermal
and chemical
sterilization.
Finally,
the following
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 120C
for 2 hours. After
cooling,
Solution
III was added in a sterile inoculation
hood under ultraviolet
light and inoculated
with 50 ml of a well-grown
culture
of the algae. The bottles
were
kept at room temperature
at the laboratory
windows
without
additional
exposure
to light or
ncration.
Twice
daily
the cultures
were briefly
shaken.
Under
these conditions
maximal
growth
was obtained
after
approximately
16 days. The important
correction
of the pH to
pH 7.0 was carried
out on day 11.
After
maximal
growth
was obtained
the algae were separated
from the medium
by means
of 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 collected
the 200 g of
material
required
for our experiments.
C-Lnbelivg
In preliminary
rspcriments
it was found that the algae tolerated
an activity
up to 2 &/ml
without
growth
inhibition
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 specific activity
in the main expcrimcnt.
Howcvcr,
earlier
cspcricncc
had shown that the dry algal substance
that should
be obtained
by a single culture
was insufficirnt
for thin-layer
chromatography.
At least 1OOg wPre rrquircd
for yhpsicochcmical
analyses.
We, therrforc,
chose the following
procedure
:
Approsimatply
1OOg of algae cultured
wit,hout,
radioartivc
isotoprs
served
as carrier
for
:I small amount
of algae grown
in a single culture
with C. We used 20 mC sodium
acctate-l14C in 34 liters
of medium.
The
nuclide
was obtained
from
the Radiochemiral
Crnter
rlmershnm
(England)
and contained
a specific
activity
of 29.0 mC/mM.
Accounting
for the
acetate
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-day
culture.
ilft,c,r
prolongrd
culturing
no furt,her
utilization
of thr nutrient
could
be espectrd
ns shown
by t,hc physiological
condition
of the algae. Thercxfore,
we trrminattxd
the experi-
FORMATION
OF
BEKZO-Cl-PYREKE
IN
PLANTS
25
merits
at this tinle and cOll(xctLbd the algae by filtration
thruugh
IJalK'l'
filters
that
had
h'll
estractetl
\vil,)l bcnzcn,,
f0r the rrmoral
of pos$hly
intc,rfcring
impurities.
TlIc &W
wm
xVasl~cLi twicr with distilled
\\-atcr and dried in an incubator
at 50C. The process gave a yield
algac~, Idcnticnl
amountr;
of 9.5 g dricxd algal substance
which was :~Idcd 1.0 104.0 p radioinactivt:
0f 113.5 g radioinartive
algw wcw prtl,arcd
in parallel
control
batchcs.
Extractions
thoroughly
~~~~r~ g.arried
out
sllakerl
with b,,nzvne
lvith
and
purified
bcnzenc
at 100111 tcmperalurt~
; the algae wtr(:
eollwted
by filtration
after a few houra
scdimentatiun.
r],is
llroccss
\~:1s Iy,j,,aattd
11 tiiilps
during
4 rltlys.
Prc,sh kic~llZ:l,Ilc
W:15 l:Ill~)lO~LY~,
dtllOU!Zll
,v,! uscxcl occ;l*ionally
the, solv,.nt
after it 11x1 ))c%c>n r(~cov(,r~:11 from tllc cstract
by distillativn.
The cstrncts
wcrc concentrated
to 10 ml
The total
amount
of bcnzcnc
used wxs 2.5 litcrs.
:~lltl
nm
through
z columns
of 26 nnu tliaowtc~r
cl~.rg(~1
wyI(h SO g ,21,0, (basic, r:~tionotrollic,
;lctivily
lc\~l
1) alltl rlutr,l
with 2000 1111 bc~ncm~.
Since tlw calun~(,s had :L strong ~~110~ ~0101
:kffc>r c,on(:cntration
:mcI a~ sdtlitional
~~~wifkation
wrrktl
0rlt in a column
with 60 g AII~O:;
(lit1 not r,xmove
tlicx imlnuity,
it was finally
cliniinatc,d
l)y further
p\irification
hJ- clnwnal.og~,:tphy on acety1atc.d
pa1~r.
l+:\-(~ntunlly
the c~lukd extra<.&
w(rc separated
by two-dimensional
196i),
thtl sllots lo~~tctl
by Auorcs1bin layer
chromntogral~hy
(Kiihlcr
~1 (II.. 1964 ; li~mtt~,
ctrncc under
the UV-an:alylical
lamp
and
the collected
lwnder
lxycr
clutcd
with
benzcw,.
Absorption
and fluorcwt~ncc
CIII'VW
of thtx solutions
were ;,lott~l
for clualitativc
and quantitativc dctrrmination
of the subetanws.
The
cluates
of the individual
substancw
wvere concentrated
t,o 0.2-0.3
ml, adjusted
to
0.5 ml, mtl
trnnsfcrrrll
t,o 10 ml of wintilla.(or
fluid c:onsisting
of equal parts of tolwne
amI
dioxanc,
anci 0.15$;
PPU
(2,j-,lil)llc,nvlosazolt~).
The m?nwring
instrument
was n Trienrb
LScintillation
Spcctrornc~tc~r
3003 with cw)lwl
ssmplc
than, cp>r :md cstcrnal
standardization.
1111,
xljlwt~mcnt
sc~l~tcd
(gain 15%, wintlow
50-1000)
nllowc~d a ? ield of 78.5% of C. lhc background
of li-20
Ilm
was sntisfn~torily
low. Tlw
counting
period
was 100 minutes.
Ewr)
batch
of wm11lca
IV:~S twtcatl
for possit)lc
inlc~rf~~rt,n(~c, crf cllrc~ii~~lling cffwts
by m(wx
of
~wxrn:tl
stand:trtlizntion.
Exchange,
wactions
I)clwcen
radiowti\-cly
labrlcd
;ml
unlabeled
substances
can caux
cwors.
~111hough
an cwhangc
in llC-con~lwunds
should
be minimal,
the 1%day
duration
of
11~ culturing
0i tilt, algae made a control
c~spwirncnt
in aquc~~~~x
;LIKI
organic
medium
atlrisablr~.
Thwc
cslwrim,n(s
were undcrtsken
to indicate
whrthcr
undckctc~d
traws
of P-kH
arc able to incorporntc
C radiochemically
(i.e., without
biosynthetic
cellular
function)
and
\vlut iml)ulw
ratw codd
he obtained
by this llromss.
The W~II~OLIS
medium
consisted
of 3.4 liters of the used algal medium
and still contained
64.3~ 0) tllc C c~onccntration
oripinnlly
llrcscnt
in 3.4 lit,eN. As coml,cnsation
for the rcduccd
tOt:l~l ncalivity
tllc, duration
of thcx c~sllcrinwnt
was prolongfd
to 2s days. One microgram
of
fiuor:mt.hc~nc~.
whirl1 \~as di;;solved
in 1.0 ml mcthan01,
was added t0 the medium
to participntc
in f~~hangc
reactions.
In a furthr,r
control
batch
1.0 pg flLloranthcn(:
Tras added to 3.4 liters
of unlabeled
mrdium.
Fluoranthenc
was recovered
at the termination
of the cspcrimcnt
t)Y cstraction
with 600 ml bcnzcne,
concentrated,
and separated
by mrans
of two-dimension:ll
thin-lawr
chromatography.
The qualitative
and quantitative
determination
of flu0ranthcncs
nxs
carried
out
u&g
th,* :I ) ,ove-lileni.ionl~~l
l~roc(xl11rw
of fiuowsw1wf:
~~llotornctry
an,1
lnc:wllr~ment
of radionct,ivity.
26
BORNEFF
ET
AL.
Chemical
Results
TABLE
OF INACTIVE
CONTENT
I
AND W-LABELED
PAH
Compound
Inactive
Fluorant,hene
Benz[n]anthracene
Benzo[b]fluoranthene
Benzo[a]pyrene
Benzo[ghi]perylene
Benzo[k]fluoranthene
Indeno[1,2,3-cdlpyrene
tZ The data
and elutioll.
Physical
(pga)
ALGAE
found
in:
algae
W-labeled
5.SO
0.78
0.39
0.07
0 11
0.14
0 1s
refer
to total
extracied
amc~ur~t
algae
6.20
0.65
0.42
0.08
0.23
0.15
0.17
of algae
disregartling
Results
The impulse rates of the individual PAH are given in Table II.
The investigations
on the occurrence of exchange reactions showed
rates consistently
lower than twice the background
(Table III).
IYPULSE
RliTES
OF PAH
F~OJI
TABLE
lQ2-LABELED
II
AND NONLABELED
ALGAL
lGlabeletl
PAH
compound
Fluoranthene
Benz[n]anthracene
Benzo[b]fluoranthene
Benzo[a]pyrene
Benzo[ghi]perylene
Be~~zo[k]fluorant~herle
Iildeno[l,2,3-ctl]I)yrelltl
u Corrertetl
for background
Ipm
1975
427
151
137
034
1%
27
Ipm
(c0rr.a)
1973
408
133
118
934
15s
252
and esterlutl
impulse
CULTURES
Nonlabeled
Iv
(corr.)
per pg C
334
663
333
1540
4247
1 I ( I6
7650
star~tlardizatioll;
Ipm
(corr.)
per rg C
ratio nonlabeled :
labeled
126
30
23
41
1: 15.7
1:13.6
31
12
11
32
165
76
42
40
57
2
I!)
271
165
1 IO
Ipm
144
50
x2
Ipm
Ipm
(corr.)
= irnplks
per rnillute.
1:ll.l
7:10.7
1:16.4
I:7.
1 : 1%:;
FORXIATIOS
I~n]~lsc
I&&
of 11~4ive
OF
Fluomnthene
BEXZO-Cl-PYREKE
Kept
2s days
IX
ill
27
ILAKTS
l&C-Cont:tining
and
%-Free
dolut ion
Aqueous*
Chluroform/methallol
Background
a (Jjrrecte(l
for t,ackground
I) A(*1 ivc or inactive
nut,rientj
:md rsterunl
medium.
st;tndnrdizatiou.
(jr1 tllc basis of the absolute weights given in Table I the impulse rate for
I ,,,g C can be calculated. The expected value should hc 485 Ipm//.~g C. This
figure is obtained from the amount of C added to the culture medium (~7358
I~n?/,~g Cj, a counting yield of 78.85% (=5798 Imp/pug C) and the fact that
9.5 g. lahcled dry algal substance was added to 104 g inactive material.
The calculated value was lower in the case of fluoranthene and benzo [blfluoranthene, namely 333 Ipm./,,,g C. Considering
the extremely small amounk of
material, this discrepancy
may lie in the range of the cxpcrimental
error. On
t8he other hand the values for hcnzo [a] pyrene, benzo [g/G] perylene, benzo [k] fluoInnthene do not agree with the theoretical
impulse rate, being considerably
higher. We assume that, this is cnuecd by the presence of nonfluoresccnt
P,4H
which :w located at the s:mw spots of the thin-layer
chromntogram
as the fluorcswllt8 ~oml~ounds. Both t#ypes of substances are mlc-nsurcd by the radiot~hcmical
technique, whereas only the fluorescenC cornpound.. q arc demonstrated
by fluoreswncc photometry. This is particularly
evident, in the cast of benzo [ghi]pcryIene;
it is our cxlwricwce that thc compouncl is always contaminnted with a comparat,ivclly large amount, of we:~kly fluorescent PAH, at least if twtc(l with the mcthotl
of t,lrin-layer cllroni:ttography
used in our cxperinwnts.
Tlrc significance of the rndiorhcmical
results is, however, not influenced by
these obpcrvat8ions; the parallel expcrimcnts
with nonlnbcled meclia rcsultctl in
all instances in markeclly lower impulse rates.
DISCUSSION
28
BORNEFF
ET
AL.