Document 4pG7kq45Z2Og2LpeYq73d0bG
Polychloi"'
Interferciiee
Introduction
XX XX
The RCD's X
X (X indicating
XX
Xx
possible chlorine positions) were studied as early as 1 ESI (1)
and by 1930 (3) were in vide use. They are known to be: quitc-
toxic, especially to livc-r cells, As early as 1S3G, Jones and
Aldon (3) repotted that men employed in the production of PCi's
developed r.cnc-type skin eruptions. Three years later, Gr<:cntu:g
and cot,`oi hers (4i) reported that FC5's and polychlorinated na.pl.-
thalcnos were resposible for the deaths of three workers.
Residue chemists, especially in Europe, have recently become
interested in these PCB's as well as the polychlorinated triphen
yls, naphthalenes, tqrpenes, and other related compounds, since
NOTE: This paper was presented at the Eastern Canada Sem inar oil Pesticide Residue Analysis, November 16-19, 1963, at' Guelph, Ontario, Canada.
ACKjJOnLKDGMvn.TS: This research was supported by funds from the Pesticide Section, Canadian V.'ildlife Service, Ottawa, and from the Province of Ontario through the Pcpnrtnent of Trade and Development.
' The technical assistance of Mary Coleman, Terry Cooper, find
other members of the ORF Festicide Laboratory is gratefully
acknowledged,
m
hull l:n of Ko.iror'rr-rrlal CoMjr'irMion & T`jtirlo;y, Vo). 4, .No. 3, 1 p:'t!i.licii L> Sprin^or-Vcrlj,- New York Inc.
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Jensen (5) in Sweden reported their presence in wildlife tissues
about two years ago.
The 1'CB's and related compounds (although in the rest of this
paper reference will be made to the I'CB's only, the other related
compounds eve also quite important) have very numerous and ir.ooc
tant industrial uses, but are not used as pesticides. Because of
their similarities in structure and properties to the DDT pesti
cide group, the PCB's, if present, are carried through the usual
pesticide extraction end screening procedures, and since they
possess electron absorbing properties, will interfere with gas
liquid chromatographic electron capture (GI.C-KC) analysis of '.lie
organochlori r.e compounds.
Before any discussion of the type of interference encountered
it would be appropriate to mention briefly so::.-:- cf the prei-T.
and uses of the PCB's,
They arc produced and marketed under a number of ce.mmorcial
trade names e,g 'Aroclor', 'Clophcn A50*, etc. The rcii'r are
available as liquids, resins, or solids; insoluble in water;
thermoplastic; non-drying;' stable on long heating at 150*C.;
electrically non-conducting; not affected by boiling with NaCn-i
solution; do not support combusticn when alone above JbO'C.j are
easily soluble in most common organic solvents and drying oils.
They are used in protective coatings, as plasticizers and
extenders, as sealers in water-proofing compounds ar.d putty, in
asphaltic materials, printing inks, waxes, and s>nthetic
'
J
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adhesive**.
.
Liquid FCh's arc used as dielectrics, as hydraulic fluids, in
thermostats, in cutting oils, as extreme pressure lubricants, as
Grinding fluids, and as heat transfer media.
Solid LCD's are used to impregnate carbon resistors, as
sealers or impregnating agents for electrical apparatus,
Obviously, the stability of thcSo compounds makes them ex
tremely useful and versatile for a great number of applications.
Considering their stability - not affected by boiling with b'cOii
or nitric acid, not metabolized in living organisms, and nonflam
mable if containing more than four chlorine groups, it is as
Jensen (C) pointed out, difficult to explain how those compounds
find their way into living, organism:..
However, with t.he name rues applications, it is not incon
ceivable that fish and other wildlife could be polluted as a
result of the flushing of wastes into rivers, lakes, etc. It is
also possible that contamination could proceed via the atmosphere
when wastes containing these compounds are burnt'i
.However, a third and nore likely source is the possibility
that some companies might he using PCB's in pesticide formula
tion to increase the kill-life of insecticides. The Monsanto
Company, which manufactures the Aroclors, stated back in 1 DC-5 (7)
that the Aroclors can "trap" and hold more volatile ingredients
making volatile insecticides, and repellents last longer in resi
dual activity. The most pronounced effect for increasing the
130
.
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kill-life of insecticides vac obtained with lindane, chlordar.e,
ami bennenc haxachiori.de (D1IC), A ten-fcld cffcctivoncfs for
lindane was reported by the U.S.D.A. by including 5-2bx DCB's in
th.e formulation. Attempts to determine whether this idee, had been put into practice by some companies have so fat been unsuc
cessful. Bet there is no doubt that, if t_hc DCB's arc being used
ir. pesticide formulation, then this voul d certainly explain their
presence in wildlife tissues and other samples,
.
j#
Jensen (i ) has used a nitration procedure in order to jlit:cr
ept i ate the KB's from the pesticide residues. He treated the
^i
cl car.cd-up extract vrith a mixture of concentrated lillO and ca'u
ceutratod H7, i`0i,* (1:1) for i> tain. at 0C. After the addition of crushed iec, he extracted the reaction mixture with hexane and
reinjected the extract. He states that the method should leave
ITB's, lindane, and BHC unaffected. Our nttc-r.pts to repeat thi?
reaction have not been fully successful. Thorc appears to be
some loss of the more volatile (early emerging) PCB's, hopt uchiov
epoxide is not affected, and peaks with longer retention tines
appear.
Although Jensen, did not elaborate as to the fate cf the pes
ticides, we have demonstrated that apparently, nitration does
occur. This was shown for DDT when a large peak (probably due to
the* tetranitr'o derivative) appeared on the chromatogram about 7
hours after injection of the nitrated extract.
-
' Of course, this reaction is a modification of the old
Ml
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Schceh'er-hal l.cr (8) DDT method in which more drastic conditions
(flirting flNOj and concentrated ll^SO^ with heating on steam bath)
were used to ensure oxidation and removal of interfering biolog
ical materials. The nitrated pesticides were extracted with ether
and a colorimetric method was used in the final determinative
stop.
F.rro et al . used this technique to determine toxaphene in
the presence of DDT, on the basis that the chromatographic pattern
of tox.-.phoue is not affected by nitration while the nitrated DL'T
docs not chromatograph under the specified conditions.
Obviously, nitration docs not appear to be the ansvoi Tor
complex mixture.' of pesticides end lots's sir.ee seme pc: tic ids r-
(lindane, rHC, l.oxaphene, 'Strobar.e-', etc.) apparently will not
nitrate while some of the FCh's night nitrate. Although we har e
not used Jensen's column packing (the liquid phase SF-96 is a
methyl silicone), it is in.possihl e to avoid complication, and in
terference from the nitro derivatives fornsd, especially when the
pesticides are present in large amounts.
There arc three main reasons why vt prefer an approach dif
ferent from Jensen'st.
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 complicate the inter
pretation of the chromatograms, since the nitro derivatives 132
'
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possess treater electron absorbing power and with their longer
retention times, should emerge and interfere with subsequent
injections.
.
3. We have been unable to repeat Jensen's cleav-cuy dif
ferentiation, apparently partly because of the nitration of sor.c
of the FCB's.
Interference of TCP's
We have attempted a more ideal approach to differentiate the
two troupe by separation followed by the separate analysis of
each troup.
The GLC work was carried out under the followirt conditiers;
Gar Ciiro;.:a.tcf,roph; V.trian Model 1200, fitted with triLivr.-
cleetror, capture detector; column. glass, spiral, C :< 1
O.M.,
packed with 6 QF-1 and 4" SK-20 on Ci.ror.osor\> V (AW), bo. cf
theoretical plates for DDT = 2227.
Operating Condi liens; Co1u-ji temperature 190'C.; injector
temperature 245"C.; detector (base) temperature 240''C.; f*^ .flow
rate, approximately 40 nl./min.; volume injected, Spl. Recorder:
Varian Aerotraph Model 20, 1 mV, full scale deflection. Chart
speed; 2/3" per min.
FiG* 1 indicates the degree of separation of pesticides
in a standard mixture. The excellent separation obtained for
DDE and dleldrin in. this-column which was first used by McCully
and McKinley (10) should be r.otod,
.
las
.
'*
.
' *' DSW 030061
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f I G . l CHROWATCGRAM CP STANOAftO MIXTOHE OP OnCANOCHLO-i.'NE ^tr.T IC iO E S .
m
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H*
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Ficurc 2 shows the number of peaks and the reparation ob
tained for a sample of FCB's ('Aroelbr' 1254) vb.ile Figure 3
demonstrates the decree of. interference encountered when the
pesticides are nixed with the ICD's.
.
It ir. interesting to note chat the. peaks of the cotamonly
fennel pesticides all have a corrcr.pendins PCB peak that would
interfere if present in the same extract. This is in agreement
with Jensen's work.
Separation of FCB's fro:.. Post ici >.h:s_b_y thc_ Use cf Florici1
Kith thin layer clu cratogt c.phy (Tl.C) it was observed that:
the rCC t (V.iochr1 12M) ter.de.J to i.en towirdr Che solvent
front on the 7L plates. Bearing this ir. rind and the fact Civ..:.
our cleanup procedures for pesticide residues in ar.i:.:atissue.;
usually involve a final Florisil step, we experimented to see if
the BCD's could be eluted front the Florisil col inn idth n-hexane
knowing th;<t most of the pesticides are r.ot eluted under the*.e
specific conditions.
\
..
Four preliminary experiments were carried out to test the
feasibility of this separation on Florisil, In Fxpt. I, 5 r.l.
of standard PCD preparation was added to the s'ass column (50 cm,
x 2.5 cm. O.D.) packed with 40 nl. (cn. 19 gra, or 10 cm. in
height) Florisil (60-100 mesh, Floridin Co., stored at 130*C.
until ready for use) and topped with n:i 1/2" layer of anhydrous
Na.SO, . Elution was carried out with 100 nl. n-hoxane, an;'- the 24
percentage recoveries were determined. This t->:pcrir.ent was
l.V.
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repeated but the elution was effected with 200 nil. hexane (Expt.
II). The sn.T.o experiments were carried out with the standard pes
ticide mixture eluting with 100 (Expt. Ill) and 200 nl. (Expt. IV)
hexane respectively.
TABLE-I .
Percent recovery of PCb's and Pesticides from Florisil columns by elution with hexene (a)
PC!', peak ! Expt. 1 sxpt Yf :Pes ticircCOExpt. IJ1 : Err TV
. (GI.C) 100
hex 200 t:l us:! peak
100 r.l h-v 200 r.l "r.ov
1
0.1
92.2
j Lindane
hone
None
2
86.7
103.1
ilr-pt acV.lor
None
62.7
3
63.6
100.0
Aldrin
62. S
S'-.1
4
9$ .2
101,0
'kept. epex. None
Non e
5
62.1
100.0
j DDE
' 20.5
97.5
(>
44.9
98.7
jDuel drin
None
None
'
C'i.0
101.2
| ODD
None
None
1b
96.3
105.2
|P,p'-DDT
Fore
I-o:'0
0
60.4
105.8
10
72.6
IC-3.3
i
11
76.9
57.9
i
12
57.2
100.0
l13
100.0
100.0
j
1 ft 71.6 100.0
Here.vc r i>- are based c-n perk height comparisons and each value represent; the average of duplicate detnrr.ir-.cticns.
b. Unde.'- the cxrir.r ir.ental conditions, 250 ul. cf 20.'.' ethyl ether in hexane is used corm-.liy to elute the pesticides
. although 200 ml, can quantitatively remove then.
The experimental results which are shown in Table I indicate tlu.t separation on a Florisil column is feasible. Almost quanti tative removal of the FCB's i s effected with 200 mI , hexane, \ r.i! a
under the same conditions qi.ly three of the S pesticides trird shoved evidence of elution (heptachlor 92,72, aldrin 94.12, and DOE 97.52). It is interesting to note that these three pcstl-
1.T7
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-cidcs showing some elui.ion fro:-; Florisil with hexane, are, like the FCB's, quite mobile under our TLC conditions.
Two further experiments were carried out to see if the sep aration was still effective when FCB's and pesticides were mixed (L'xpt. V) and when they were present in the extract from an ani mal tissue (Expt. VI), The first elution was made with 2C0 ml. hexane, the receiver was chang'd, and the second elution was carried out with 250 ml. of 20*- ethyl ether in hexane to remove the pesticides.
The results of the two experiments ate shown in Table II, and confirm our earlier finding that with the exception of DDK, aldrin, and heptachlcr, a clcar-cut separation of the FCB's and pesticide; can he rad-.' by the use of a florist: column.
The fact that DPT is elutvd with the FCB's by pure hexere can be used to advantage in the confirmation ar.d quant if hear, ion of Dl'T by dehydrccl.lorination. The estimation of small amounts of DDT in Die presence of interference (for example, a I'CB) is enhanced if DDE is previously removed. The DDE produced by dchydrocMorinatior. can then be used to estimate che amount of DDT originally present. In the presence oT comparatively large amounts of DDE, tfiis approach is not very dependable.
- Discussion The results of the above experiments coupled with the work of Jensen indicate that there are serious problems confronting residue analysts, however, as far as the writer is aware, there
138 '
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TAP.LF II
Percent IlccovtT5.es of f'CB's and Pesticides from a Nixture after Separation on Florisil (Expts, V L VI)*
Fluted with 200 ml
PCI) r.nd/or , * ;>est. peak Kccov,
hexane
V.'ith 2 50 r.iT . 70:1 at her in hexane
^ fh') KCCOV,
I't-sticide pec.K
r 'jcov. (a)
f,lecov. (b)
Hoptachlor FCB1 FCB2 + Aid 5
TCP 3
FCB4 PC PS + I)!)KC
POP 6
92.7 1 04 . 0 102.1 100.0 10A .2
97.0 101.3
FCP7 PC PS
POP 9 !`i~ 1> 10 i-r.bl l PCK12 PC 1)13 )'C 1) 1 r.
97.8 100.0 91.6 104.7 100.0 100.0 100.0
96.2
93.1 101.7
96.6 10C.O 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
lieptr.chlor None
Aldrin
1.3
ilept. op ox. 96.4 IfD** Kono
Dieldrin 109.0
DDD . 102.3
DDT 99.3
9S.5 None
4.0
102.2 None
100.0
95.9
92.5
* The peak? are arranged ir, cider of their e.r.orpc-nce (increas ing retention tir.e) from the C.LC column, and whore a FCf. and pesticide peak appear in the sar.e line (horizontally) they have similar retention times.
a. A standard mixture of.PCS's and pesticides in pure hexane was pieced on the Florisil column; first elution was made with 200 ml. hexane, receiver was changed, and the column elxitcd with 250 ml. 20'i ethyl ether in hexane.
b. Same as in (a) except that the ICS's and pesticides were first nixed with an extract from an anir.al tissue which was known to bo essentially free of pesticides.
C. Since a single peak was obtained, the recovery was calcu lated by a comparison of the peak hoichr against that in
' the combined standard mixture of Fell and pesticide. In all other cases the peak heipht was compared to that in the standard injected separately.
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has been no positive confirmation of the presence of TCB's in
wildlife tissues by techniques other than chromatography. This
leaves doubts that the presence of the unidentified peaks (Uir's)
is actually due to TCB's. There is the possibility that some or
all of the peaks are due to condensation products of the netabo-
litcs of pesticides like DDT. For ewar.pl c, 4,0'-dichlcrobenro-
phenone (DCD)
is known
to be a metabolite of the DPT group.
The'presence of the keto troop nakos it quite feasible for
condensation to lake place.
There arc at least two points that lend support to this pos
sibility.
1. The DIF's (being called TCP's) are usually observed
oe.ly when 1 ar(,fi amounts of the DDT group .ire present.
2, Jensen cheeked eagle feathers collected since l?30 aid
first detected FC5 (not confirmed) ii; an =,i[,l<; from 19V';. It
ini gbit be a coincidence, but this is approximately the time that
I DDT use came into prominence. It should be noted also that the
I PCB's were in wide use as early as 1930 (2). Thus until positive
I confirmation (e.g. with pass spectra) is obtained, there will
remain some doubt that, these HIT'S are due to TCP's - especially
I if FCB's are not used in pesticide formulations.
It is certainly true, however, that whether or not the DIF's
are TCP's their presence leads to difficulties. The results
obtained for some samples of fat recently .analyzed In our labora-
140
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--tc>ry arc typical of the problem. Tho sample containing the
highest levels of residues contained the following pesticides in
p.p.m.: Dl>E 1.42, di el dr in - 2.13, DDI) - 5.61, and p,p'-DDI -
2.60.
Even prior to subjection to TEC confirmation, the 1)1)1) value
appeared unusually high when it is considered that its presence in
tissues is usually accounted for by three main routes;
a) It. fs used cs a pesticide, but not extensively,
h) It is one of the metabolites of I'PT - however, the DPT--
pathway is much nore prevalent than DDT -->DDD, vith the latter
usually occurt5ng in the liver, hence the fat tissue is an unlike
ly location for largo ascent s of DDI),
t:
c) It is a frerjucr.t Cuntnnina.nt. of technical PbT use--1 in spray
programs.
hTien confirmation of the pesticides was attempted, the TEC
plates showed no PCD, although tic apparent amount present
should have given a distinct spot o.o the plate. However, a spot
was observed running near the solvent front, a considerable dis
tance from DDt). When this spot, was scraped off the TLC plate, .
eluted, and reinjected into the gas chromatograph, a peak having
retention time identical to PDD was observed. Although this in
terfering material has not been identified - it could be a PCB
since its retention time, coincides with one of the ICB's - it is
obvious how easily one could report false results, especially if
use is made of the GLC-EC results without further confirmation.
141
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TI.C continues to le our main confivaj.itory method, but thorp avo times, especially with smaller (tut significant) amounts cf pesticides, wh.cn it is impassible to make a positive confirmation with this technique alone.
The determination of CLC retention times on two or more sta tionary phases is quite useful in some cases, but as F.obinron (li)
i has pointed out, it cannot be regarded as an independent parame ter of identity since it ray be shown that, various orgaaochlo: i:.e pesticides on different stationsy phases are sign!ficantly cori elated.
Tearing in mind these problems and the difficulty of apply ing infrared, toss sped: a, and other spcc-troscopi c methods for confirmation of small amounts of pesticide residues, moiu .-.r.nhacis and reliance should bo given to chemical modification cf the pesticides end reinjection into the gas chromatograph, using the retention timer, of the products as r.ee.ns of confirmation.
Kith our SMI technique (S = Separation of PCB's on Fieri.5 il, M = Modification of the pesticide by chemical moans, I = Injec tion of the extract containing the product into the CLC apparatus) wc have observed some cases where a single CLC peak indicating one pesticide was in fact a mixture consisting of the pesticide plus some other FCE-type unknown having the sene retention tine. With TLC as the sole confirmatory method, one could quantify the whole as being due to the pesticide and be out by many factors depending on the ratios of the two compounds giving rise to the
142 `
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single C.EC peak
Ref crenr.cs
1. If. SCHMIDT and C. SCHULTZ, Ann. 207, 333 (1881)
2. C.h\ i'LNiU'NG, lnd. Eng. CUr.u 22, 1180-2 (1930)
3. J.E'. JOKE'S and H.S. ALDLK, Arch. Dern.al. Sypliilol. 33, 1022-
103'i (19 35)
4. h. C!:?-TNT.I,1:C, M.H. MAYERS and A, P.. SMITH, J. lnd. Hyg. Tc.:< in.
2J_, 29-3T (1939)
8. S, jn.'SEN, Me-,: Scientist, p. 612 (15 Dcccnljcr 1566)
6. 5. JrSSE.'i, Private corai'micatic>n (1967)
7. 'The /.l oci o' Ccr.ponnd.s *, Monsanto Clio,ai cal Co.Tpm.y Bulletin, p. 1 7 (1 965)
5. M.S. SHLCIITLK, S.!i. SOLOKAY, I;.A. HAYES, and II.L. IIALUR, lnd. Kr.g. Chon., Anal. Ed., _J_7, 704 (1945)
9. F.
A. REVEl.Tn and II. pr.CKK.AL, Pull. Fnvivon. Coni. an.
and Tox. 2, 372 (1967)
10. K.a. KeCUTLY and W.T-. KcKIKU-1, JA0AC 67, 452 (1964)
11. J. ROSINSOH, CLtr.istrry and Industry, p. 1974 (23 Novo:.:her 1967)
t 1U
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