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PolycIiIoroMphcnyli-, (PCS's) unci In civ Interference Kith Pesticide Residue Analysis
by Llvcoi.n M. JIkynolds
Oiitnrie Jlesiwrh
Shci idatt
(V.fu'io, Ccnur!u
(X indicative t-i'.j
possible cblorins positions) verc studied ns early as 16S1 (1)
and by 19X) (2) verc In vide use. They :.ro known to be quite
toxic, especially to liver cells. As early as 1935, Jct.oj miJ
Alden O) repot rad that nen omply/ed In the production of ['CD's
developed ecnc-type Skin eruptions. Throe years later, Cr.-cntur^
and cowtV.crs (4) reported that FCVs and polychlorinated
thairnet were resposiLle for the deaths of three workers.
Eosidue chcniste, especially in Europe, have recently beer::?
interested in these PCS's aa veil as the polychlorinated triphen-
yl6, naphthalenes, terpenes, and other related compounds, since y
EOTEi This paper was presented at the Eastern Canada Sc:>ituir ott Pesticide Residue Analysis, Xoveabcr 16-19, 1963, at Cuelply Ontario, Canada. Atj?!OWLEhGHrTfTSi Thia research vas supported by funds fre.-n
the Pesticide Section, Canadian Wildlife Service, Ottawa, and free the Province of Ontario through the nrpartr.cnt of Trad.- and Development.
V The technical assistance of Mary Colc.-aan, Terry Cooper, and ' ether members of the 03F Pesticide Laboratory la gratefully
actoovl edged*
us
EalhlhtW T*I , Jf* S. l.
.
CvvuMnilimi t Tn*4in. by Spfir.^t.VtrU: N**r Y* Ik. 1
-
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Jensen (5) in Sweden reported their presence in wildlife tissues
about two years ago.
The FCB's and related compounds (although in the reel of this
paper reference will be made to the PCB's only, the other reicted
compounds are also quite important) have very nu: erous and impor
tant industrial uses, but arc not used as pesticides. Because of
their simi.l arities in structure end properties to the bbT pc.-ti.-
cido croup, the FCB's, if present, are carried thrush the \ v.:al
pesticide extraction and screening procedures, and since they
possess electron absorbing properties, will interfere with
liquid chromatographic electron capture (GLC-KC) analysis of the
orgonochlorire compounds.
Before any discussion of the typo of interference e..c<
r^-i
It would be appropriate to nc*ntion brief?/ scr. of the - r- ;:
and usc3 of the PCB'r,.
They are produced and marketed under a mimbrr of ccr. :u: r i; 1
trade names e.g. 'Aroclor', 'Clopb.in A50', etc. The 1CL>' r nre
available as liquids, resins, or sol ids; insoluble in water;
thermoplastic; non-drying; stable on Iong heating at 150'C.;
electrically non-conductLng; not affected by boiling with NrC'l
solution; do not support cor.busticn when alone above 2fO*C,; arc
easily soluble In mo3t corraon organic solvents and drying oils.
They are used in protective coatings, as plasticizers ar.d
ex tend eis, as sealers in water-proofing compounds ar.d putty, in
asphaltic materials, printing inks, waxes, and synthetic
129
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adhesive* liquid FCU's arc used as dielectrics, as hydraulic fluids, in
thermostats, in cutting oils, as ext rente pressure lubricants, as Grinding fluids, and as heat transfer media.
Solid PCB's are used to Lu-pregnatc cart-on resistors, as sealers or impregnating agpnta for electrical apparatus.
Obviously, the stability of these ecsipcuods nak.es thMi cxtrencly yseful ai.d versatile for a great number of applications. Cousiderin thilr stability - not affected by boilLr.g with f'tO:! or nitric acid, not metabolized in living organises, and nor.fl mel-lv if containing more than four chlorine groups, it is as Jensen (6) point ed out, dii ficult to explain how thevc to.-.p.vjpds find their way into living organisr.s.
However, with the runvri/JS appl icalionr., it is not incon ceivable that fish and othr-r wildlife could be pil luted as a result of the flushing of wastes into rivers, le'rcs, etc. It is *al so possible that contaniiiati on could proceed via the atr.uspber? when wastes containing these compounds are burnt''.
However, a third and nore likely source is Che possibility that some companies might he using rCB's in pesticide formula tion to increase the kill-life of insecticides. The Monsanto Company, which manufactures the Aroclcrs, seated back in 19C5 (7) that the Aroclors can "trap" and hold more volatile lngrcdients making volatile insecticides and repellents last longer in resi dual activity. The most pronounced effect for increasing the-
IJO
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! ' ,. t "M ' : *1 * ,
. '_
kill-life of insecticides was obtained with lindane, chlordar.c,
and benxenc ImxacM oride (CMC). A ten-fold cf fcctivenef S for
-.au. . .. I ... t . ... w . . .
.
Undent wus reported by the U.S.D.A. by Including S-2ST! rcB'; i:.
the formulation. Attempts to determine whether this idem had been put into practice by soir:c companies have so fat been enrve-
ccasful. Bet there is -no doubt titrt., if tfic ICS's ate beinp, urcJ.
ir. pesticide formulation, then this would certainly explain t!-.v
presence iri wildlife tissues and other samples.
Jensen (t) has used a nitration procedure in order to differ
entiate the roll's from the pesticide ret 5 dues. Ke treated the
clcar.cd-vp extract with a kiixturc of concentrated lil-0^ ;.nd co--
c ecntratrd
(111 > for i> tcin. at 0*C. After the tddltice. er
crushed ice, he extracted the reaction mixture with hexane and
reinjected the extract. He stales that the method thcj'd l.-avi
rCB's, lindane, and BHC unaffected. Our- arterpts to repeat '' i
reaction have not been fully successful. There appears to be
some loss of the more volatile (early emerging) FlA's, hept itb.i or
epoxide is not affected, and peaks with longer retention tine:
appear.
Although Jensen did not elaborate as to the fate cf the pes
ticides, we have demonstrated that apparently, nitration docs
occur. This was ahovn for DDT when a l.rge peak (probably due to
the tetranitro derivative) appeared on the chromatogram about 2
hours after injection of the nitrated extract.
.
` Of course, this reaction is a itodif icaticn o' the old
ill
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(
Schcehtcr-Nal1cr (8) PDT method In which rsrc drastic cc-ndi ti ons
(funing HN'O J and concentrated IIt. SO*4 with h-iating on stcr.-i b.'.th) were used to ensure oxidation and removal of interferinc biolog
ical mtcrials. The nitrated pesticides w.ic extracted with ether
ond a colorimetric method was used in the fivr.l dot erninot ive
step. Erro ct al. used this tcc'm.i.yje to deUTmir.e toxa. phene in
the presence of PbT, on the basis that the chromatographic pattern
oT toxr.phene is not affected by nitration while the niti.it cJ DL'T
docs not chromatograph under the specif led conditions.
Obviously, nitration docs not appor.r to he th.o answer for
cor.pl ex mixture.? of pesticides rad F'.B's since son? pc' ? 5 cf.-. r.
(lindane, F-fC, inxaphene, 'Strobane', etc.) .-.ppat entiy wi 1 / net
nitrate while some of the TCP's r.ii jht nitrate. Although we
not used Jensen's column paeMng (the liquid j on sc SF-SS i-'- a
methyl silicone), it Is impossible to avoid cor.pZicaiio.i .-.-.d h -
tcrfcrcnce front the nitro derivatives for:.: especially when the
pesticides ere present in large ar.ounts.
There arc three main reasons why vt prefer an approach dif
ferent froa Jensen1si.
1. It is preferable to separate the two groups rather than
destroying one, especially when it is the pesticides that are
being destroyed.
.
2. The nitration approach tends to cor,plicate the inter
pretation of the chronwCogratr.s, since the nitro derivatives
131
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possess treater electron absorbing power and with their longer
retention tie-.es, should emerge and interfere vith subsequent
injections.
.
3. We have been unable to repeat Jensen's elect--cut dif
ferentiation, apparently partly because of the nitration of r.o-.e
of the FCB's.
Interference of PCT's
We have attempted a more ideal approach to dif f cre.-.t inf o the
two groups by separation followed by the separate analysis of
each group.
The GT,C work vas carried out under the follovir.f, cor.di i irr.<:
Car- Chro.v.at cr.raph: V.rrian Model 1200, fitted vi;h triliv.-
clectron capture detector; colu:.\r.. glass, spircl, f' x l
packed with 6? QJ'-l ar.d 4" SK-20 on C0orosorb V (A"). bo. of
theoretical pistes for DOT c 2221.
Operating Conditions: Column terrpcrcture 190*C.; injector
temperature 245'C.; detector (base) temperature 240*C.;
flow
rate, approximately 40 al./r.in.; volufse injected, 5pl, P.rc:-rda::
Varian Aerograph Model 20, 1 mV, full scale deflection. Chatt
speedi 2/3" per min.
Fig. I Indicate* the dogrP~ of separation of F pesticides
In a standard mixture. The excellent separation obtained for
DDE end dieldrln in. this- column which was first used by KcCully
and McKinley CIO) should be noted.
.
ui
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I
o
134
DSW 032011 STLCOPCB4015973
t .1.
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O
Figure 2 shows the number of peaks and the separation ob
tained for a sample of ICB's ('Arcelor' 1254) while Figure 3
demonstrates the degree of. interference encountered when the
pesticides are nixed with the FtB's.
.
It is interesting to note that the peaks of the coivnonl y
found pesticides all have a corTcr.pc.'niing PCB peak that would
interfere if present in the sane extract. This is In agreement
with Jensen's work.
Separation of FCB's f re:.. Pestici ds_tn the_ Use of Fieri1
With thin layer chi ctr atogt e.phy (Tl.C) it was observed tier
the Fee's ('/.rodor* 1254) tended to ice. tewTd: the rolvon:
front on the TL plates. Bearing tM.< ir. rind and the ir.rt .'o.:
our cleanup procedures for pesticide residue.; in a:;Lis.:.:..'
usually involve a final Florisil step, ve experimented to sc? L!
the FCE's could be clutrJ fron the Florisil column inch n-l.'n.inc
knowing that most of the pesticides are not cld c.' unde: the*
specific conditions.
.
Four preliminary experiments were carried out to test the
feasibility of this separation on Florisil. In Expl. I, 3 ml.
of standard PCB preporation was added to the glass column (~9 c.t.
X 2.5 ex. O.D.) packed with 40 ml. (ca, 19 era* or 10 cm. in
height) Florisil (60-100 mesh, Flcridin Co-, stored at 130'C.
until ready (or use) and topped with a.i 1/2" layer of anhydrous
Na^SO^. Elution was carried out with 100 r.l. n-hexane, and the
percentage recoveries veto determined. This experiment wis
IX.
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. f i
'
,i
` '`
.
*
repeated but the elution was effected with 200 ml. hexane (Ey.pr..
II). The same experiments were carried out`with the sttndarri p--s-
tlcide mixture eluting with
1............................................
----------------------
hexane respectively.
100
|
|
(Expt.
Ill) and 200 ml.
(Expt.
IV)
TABLE -1 .
Percent recovery of PCb's and Pesticides frem Florisil columns by elution with hexene (*)
JO B peak Expt. 1 Expt. II jrertiri':'clw:'\pt. Ill f - ; -. >.v
no. (CLC) 100 r! hex 200 ml he: peak
* ICO r.l h-x 200 r \ r.e'
1
E0.1
92.2
! Lindane
Non e
N'oc
2
86 * *
103.1
fl'-pi achlor
None
12.7
3
65.6
lOO.O
Ald.rin
62. S
S'-.l
(>
9S.2
101 .0
iliept. epox. None
None
5
42.1
100.0
j n:>?;
20.5
97.5
6
44.9
98.7
jDLeldrin
None
None
4
Ci.O
101.2
J 01)0
None
None
6
96.3
105.2
,p,p`-L'ff
Kore
IV.eo
9
60.4
105.8
1
I
10
72.6
1C 3.5
1
11
76.r
SJ.9
I
`
J12
57.2
100.0
13
100.0
100.0
14
71.6
100.'!
.L
.
, Recovcrler *re bastd on per'.-, height comparisons and tv.h value represents th? aviraqe of duplicate dctorr.iracl r:.s.
b. Under the experimental corditiur.s , 2V.- i.l. cf 2u\' etl 1 ether in hexane is used norm-iliy to elute the pent cid'.r.
. although 200 .-1. can quantitatively remove then.
The experimental results which are shown in Table I indie a:e
that separation or, a Florisil column is f east hie. Almost quanti
tative removal of the FOB'S is effected with 2 CO i-d .
c, v'-.:. ??
. under the s-n.x* conditions <ji,ly three of the S pesticides trird
shoved evidence of elution (heptachlor 92.?,'.', aldriti 94.12, anJ
DDE 97.52), It is interesting to note that these three pcs*, i-
m
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-cldc* showing *om elution Iron Florisli with hexane, arc, like
the FCVs, quite mobile under our TLC conditions.
Two further expcrir.ents were carried out to see if the sep
aration was still effective when PCB'a and pesticides were nixed
(Expt, V) and when they were present in the extract from an ani
mal tissue (Expt. VI). The first elution was ratio v.'ith 2C0 i.il.
hexane, the receiver was chang'd, and the second elution was
carried out with 230 til, of 20- ethyl ether in hexane to remove
the pesticides.
I The results of the two experiments an* -shown in
e II,
and confirm our earlier finding that with the-exception of DDE, I1
a'.drin, end heptachlcr, a clcar-ent scpaiv.* io.i of the FCf-"ar.d ,i
pesticide; can be rad-.1 ly ti e jse of a rior-ir.''. col vc-in. I_
The f?.ct that DDE is elutrd with the Fall's by p>:v< !. nc. c
can be used to advarta^e in che cotifirmtiun ir..T quaiit'.fc.,m Ion 1 i
of DDT r>y ,v1^,nM j 4>- ochl or inat ior.. The es t. i::a t ior. of f^.all .t .o- l':tS ,- i'
of DDT in the pr osoi'.ce of inter! e? "rxe (tor cxj-nplc? a ITD) Is '
enhanced if DDE is previously to moved. The DDE produced hy dchy-
drochlorination can then be used to estir.utv the a;..our,v of DDT
originally present. In the presence of co"par?.tivi*ly larcc
.
amounts of DUE, this approach is not very dependable.
' Discussion
,
1 .
The results of th-? above experiments coupled ttritli the worV.
,V ,,
of Jensen indicate that there are serious problems*confronting residue analysts, hewevtr, as far as the writer is avarc, tljere
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TABLE II
Percent Kccovc-ri.es of I'CB's and Pesticides from a Mixture after Separation on riorisil (Bxpts. V ( VI)-
LluLcd villi 200 ml. h c r a; i **
With 250 ml , 7G.i et her i r. r.
l*Cii and/or pest, peak
, * CO Kecov, 7
Kecov/b^
Pesticide peek
F t,nv.(i)
liecov.
Hcptachlor
PCB1 PCK2 + Ald.C
rcB3 \X U4 PCB5 D!>LC
POPS
92.7 104.0 102.1 100.0 104.2
97. E 101.3
PCB7 1'CBS PC 59 POLIO PCB11 PCB12 PCB13 PC HI A
97.8 100.0
91.6 104.7 100.0 1GO.O 100.0
96.2
98.1 101.7
96.6 10C.0 102.6 102.4 100.0
105.1 100.0
97.0 104.3 105.5 100.0 100.0 100.0
Lindane
93.6
Heptr.chl or None
Aldrin
1.3
liopt. c-pox. 94 .4 dd;: Kone
Dicldrin LOO .0
DDD 102.3
DDT 99i. S
1
95.5 None
4.0
107.2 None
100.0
9-,.5
5?. 5
i * The* peaks arc artaer.ed ir. order oi their ^.r.cvccnce (ire:rir-
Inc retention tir.e) [:oe the- OLC colu-r., and-whore a rcr. and a pesticide peak appear in the sar*.- line (hovi? .-"tally) they have sir.ilar retention tirv?s.
A standard mixture of TCh's and pesticides in pure hr/v.-ie
va3 pieced on the Florisil column; first elution v-.s :.*tdc
with 200 nl. hexane, receiver was changed, and the col urn
eluted with 250 nl. 20- ethyl ether in hey.ane, I`
'
b. Sane as in (a) except that the ICR'k ^nd >fcticldes were
first nixed with an extract Iron an ar^ir.a tissue which
was known to be essentially free of pettiitfdes.
Since a single peak was obtained, the Ver )kcry was calcu
lated by a comparison of the peak height ipainsc that in the combined standard mixture of PCD and pesticide. In all other case* the pea!; here*11 w.is co.*,voapMl to th^i in the standard Injected separately.
is*
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>1 .1
has been no positive confirmation of the presence of PCB's in wildlWoj1 Hs/ucj| by techniques other than ehro.T.atOEraphy, This
leaves doubts tlyic the presence of the unidentified peaks (UIT's) to rC^'*. There is the possibility that so-ii or
is actual 1)' due
til of the pcaksj are diie to condensation products of the notabo-
lltcs
pesticides ;li|te DDT.
9 4,4' --dichl cL'ol'tn 7 (.)-
phcrvolnj j^DCD)
V/ i\
/7~\
. Clfl'
y >Cl
1 Vrr/ 6 \"y
is known
to he a metabolite of the t>DT croup.
j
' The1preserve of Inc koto group makes it quite feasible for
;i
'
, .-
h
condjcnration to take place.
1 Thlre arc at least two points that 1 end 'support to this pos
sibility.
.
f 1. live DIP'S (being called TCh's) are usually observ-'d
.only1 when large (mounts of live D'JT firowp arc present.
2. Jensen cheeked eagle feathers collected sir.ee lv'i'J cud
first del ected PCS (not confivr.cd) i>; an eagle from 19V';. It
aright be a coincidence, but this is approximately the tir.ic that
DDT use ca:ie into pro.*Iner.ee. It should to noted also that th-?
PCB *s wore in vide use as early as 1930 (2). Thus until positivv
confirmation (e.g. with Mass spectra) in obtained, there will
remain sor.c doubt that these HIT'S are due to PCB's - especially
. j it
1`
I lf'PCB** nre rot used in pesticide formulations.
I 'I
'
It Is .certainly true, however, tint whether nr r.ot the
.
. '
,
are !thoir presence leads to difficulties. The results
, ,t
*
obtained for sooaLsamples cf fat .recently analysed in our labora-
Itt
* tclfj , 1- i
> `,
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%i / .
, \\r '!
'
-tory ara typical af the problem. The sample containinj the highest levels of residues contained the followlnfc pesticide* in
p.p.u.i DlfE - 1.42* dleldrin - 2.13, DDD - 5.61, and p.p'-DDT -
7.40.
'
Even prior to subjection to TLC confirmation, the D:>D value appeared unusually hlh vhen it is considered that its presence in tissues is usually accounted for hy three main routes: a) ijt is used cs a pesticide, hut not extensively.
h) It is one of the metabolites oT DDT - however, the D!;T''>DbK #'
pathway is much more prevalent than DDT'->31)0, with the la: ter
usif-lly occultinj in the liver, hence the fat tissue is ;w. unlike
ly location for ltr<- amounts ef DDD. t
c) It Is a ,fr r'v.'cr.t Cont.vnintnt of toe!..-iie;:l F>I>7 use* i . spray
procrons.
1
When confirmation of the pesticides was attempted, toe ~]r
plates showed r.o DDD, although the apparent amount prose:;!
I'
Should have \vcn a distLnet spot on the plate. However, a spot
war observed running near the solvent front, a considerable dis
tance from DOI). When this spot was scrapwd off the The plate,
eluted, and reinjected into the f,as chromatograph, a peak havin',
retention time"identLeal to HDD was observed. Although this in
: tetf crime.riaterlal has not been identified - it could lw a PCB
1; v
i
.
since its retention time coincides with one of the ICB's - it is
. 1 i.
i,
otrious how easily one could report false results, especially if
| use Is made of the CLC-EC results without further confirmation.
Ml
,I
'*
.
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i
I
I
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l
I \ Mr l '
' < . , ] I: -i iii i
If
I1 I-
Tl.C continue* Co be our nain confirmatory method, but thoro are tines, especially with smaller {hut si.cnif ic?r.t) amounts cf
pesticide#, w! icn it i# l;; possible to Make a positive conf ir-..at ion
vith this technique alone.' i
Tiie determination of CLC retention limes on two or note sta
tionary phases is quite useful in some cases, bet as F.obi n: on (1 i )
has pointed out, it cann;.: be recar,ltd as an independent p;\ra".e-
. = *
'
,
ter of. identity since it ray be she..".! that varic-us orjja.-.oohl o'3 :.o
pesticides on different stationary phases arc r j cni fica'it I > ccr-
related.
'
Bearing in Mind these problems an.1 the difficulty of epp'y-
inp, infrared, mtss spcet:.<, and other spcctroscopic i..\'.th;'d ^ for
tenfiraat ion >f r:al 1
:.s of pcr-ticids residues, moi. -..`.j-
""cis and reliance should be iven to eh mica l modification cf the
pesticides and reinjection into the ,ns chror.itc.~i nnh, usir,;
retention tiros of the products as r.eans of corif irr.at 5 on.
With our SMI technique (S = Separation of Pub's on fieri:l1,
H = Modification of the pesticide by chemical reins, I = Ir. Uc-
tion of the extract conf r, ioin the product into the CLC a;. j ntut)
wc have observed sor.e cases where a single CLC peak indicating
c&k pesticide ves in fact a isixture consisting of the pesticide
^lus some other FCB-typc unknown having the sane retention tin:.
, jf
,,
' f"'
.
, J! ,
Hith TLC as1 th Mlt confirr^ntory method, one could quantify the
whole as beinc'du* to the pesticide and be out by many factors
on t^he .ratios
the tvo compounds fcivire rise to the
Ml
i. ! r'j, .i:.- J
-'
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single GLC peak. Rof crcnces
1. IJ, SCi:;-:iDT and G. SCHULTZ, Ann. 207, 333 (186L)
.2 C.H. i'LNNINC, lnd. EnC. CH-;?. 22, 11 SO-2 (1930)
3. J.W. JOKES end H.S. ALDLN, Aich. Derr.'.jl.. SypHilol. JJ3, 1021030 (1935)
.4 I,. G!!i-ENr.t:r,, N.H. MWF.RS and A.fc. SMITH, J. J.nd. `(ye. Toy:-.
N, ?9-33 (1939) 5. S. .TIJSEN, Net: Scientist, p. 612 (15 Deccnber 1SC6)
.6 S. JF:.SEN, Private corjacnication (19C7)
7. 'Thc/.ioclor Coriponnds', lions on to Clicrncal Company Pul let ; p. 17 (1965)
6. H.S. StUXHTER, S.5. S0Lc;:.\V, I:.A. HAVT'S, and H.L. Il'.UJ.!:, lnd. Kr.|j. Chon,, Anal. Ed., 17, 7C0 (19j5)
9. V. El:?;0, A. BLVLT.rC and H. BECKHAM, null, fnvivon. Cer t an. and Tox. 2, 172 (1907)
.10 K. A. H._c:-LLY and W. ?. Y.,KlfM.Ki, J.\n\r >. 5 52 (1
.11 J. K051NSON', CT.er.istr> and Industry, p. 197 4 (2 5 N. v ->e: 1967)
Iti
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