Gas chromatographic and mass spectrometric identification of chlordane components in fish from Manoa stream, Hawaii

Chemosphere,Vol.12,No.9/IO,pp P r i n t e d in G r e a t B r i t a i n

1229-1242,1983

0045-6535/83 $3.00 + .OO ©1983 P e r g a m o n P r e s s Ltd.

GAS CHROMATOGRAPHIC AND MASS SPECTROMETRIC IDENTIFICATION OF CHLORDANE COMPONENTS IN FISH FROM MANOA STREAM, HAWAII

Michael A. Ribick!/ Jim Zajicek!/

I-]U.S. Department of the Interior Fish and Wildlife Service Columbia National Fisheries Research Laboratory Route 1 Columbia, Missouri

65201

ABSTRACT

A series of chlordane-related compounds derived from impurities in the hexachlorocyclopentadiene starting material used to produce chlordane, were detected in fish from Hawaii. Identities were assigned to these compounds by using a combination of gas chromatography and mass spectrometry in the electron impact and negative ionization modes. INTRODUCTION

Residues of technical chlordane have been detected in a variety of environmental and biological samples, notably in food productsl, 2 in fish 3,4, and in humans 5,6.

In most instances

the reported residues were a combination of the major components and metabolites of chlordane. These are cis- and trans-chlordane (chlordanes), cis- and trans-nonachlor, and the metabolites oxychlordane and heptachlor epoxide. the total technical mixtureT, 8. complicated by its complexity:

The first four compounds represent about 50% by weight of

Determination of total chlordane residues in the mixtures is it is composed of more than 45 components 9, and variations are

frequent in both the number and relative composition of these components in weathered chlordane. Other difficulties are encountered from analytical interferences such as PCB's and the chlorinated pesticide toxaphene.

Furthermore, the exact structure has not been determined for a number of the

compounds and the majority of them have not been isolated or synthesized for use as comparative standards. The U.S. Fish and Wildlife Service has periodically analyzed for these chlordane components and metabolites since 1967 in fish collected from a national network of stations as part of the National Pesticide Monitoring Program (NPMp)3,4.

For the compounds for which standards are

available, only the chlordanes and nonachlors have been detected as significant residues in NPMP fish samples over the years.

Cis____-chlordanewas usually the most abundant component, followed by

1229

1230

trans-nonachlor, trans-chlordane, and cis-nonachlor.

The two most abundant components were

detected in about 93% of the 330 NPMP samples in 1978 and 1979, and generally the residues were highest in Hawaii, the Great Lakes, and the Corn Belt 4. Recently, several of the minor components of technical chlordane have given unusually large responses relative to the major components in the gas chromatogrems of eXtracts of fish from Ranoa Stream, Hawaii.

Previous to this discovery, these components were either unresolved from the

peaks of major chlordane components and other pesticides, or not detected.

These unusual

chlordane compositions led us to investigate and confirm that these residues were indeed related to commercial technical chlordane, which is one of the major organochlorine insecticides used in Hawaiian pineapple culture and termite control 10. In the analysis for these compounds in the Hawaiian fish, a method for determining complex mixtures of organochlorine contaminants (pesticides and PCB's) was used in the extraction and cleanup.

After evaluating the complex residue compositions by packed column gas chromatography

(GC), we used additional column substrates and high resolution capillary columns and mass spectrometry (MS) to identify and confirm the chlordane and related components in these samples.

EXPERIMENTAL

Materials and Standards.

All analytical standards of technical chlordane components and

metabolites -- technical chlordane, heptachlor, cis-chlordane, trans-chlordane, trans-nonachlor, heptachlor epoxide, oxychlordane, and compound K --

cis.___-nonachlor,

were obtained from the UoS.

Environmental Protection Agency Pesticides Repository, Research Triangle Park, NC. All solvents were pesticide grade from Burdick and Jackson'/ and J.To Baker or equivalent. All other chemicals not mentioned in this section were ACS reagent grade or equivalent.

. ~ .

The samples c o n s i s t e d of composites of f i v e whole, a d u l t Cuban l i m i a (Limia

c u l e n s i s ) and Mozambique t i l a p i a (~e2onus

( T i l a o i a mossambica) from Manoa Stream, Hawaii, and b l o a t e r s

hovi) from Lake Michigan.

These f i s h were c o l l e c t e d , s t o r e d , p r e p a r e d , and ground

a c c o r d i n g to the procedures o u t l i n e d by Schmitt e t a l . 4.

Extraction.

For analysis, I0 g of fish tissue was mixed with 40 g of anhydrous Na2SO ¢.

The

dried mixture was ground to a fine powder and packed into a chromatographic column (Ace Glass), 30 cm x 2 cm i.d, fitted with a 200-mL reservoir and removable Teflon stopcock. extracted with 200-mL of dichloromethane at an adjusted flow of 3 mL/min.

The samples were

The lipid extracts were

collected in a 250-mL round-bottomed flasks fitted with a 10-mL reservoir at the bottom (double reservoir flask) II, and the solvent was reduced to about 2-3 mL by rotary evaporation. concentrated extracts were then diluted to 10 mL with

a

The

I:1 mixture of cyclohexane in

dichloromethane; 2 mL of this diluted extract was transferred to a pre-weighed 2-dram vial and evaporated to dryness overnight before the percent lipid was determined gravimetrically.

a-/References to companies or brand names do n o t imply GoverT-.ent endorsement of commercial products.

1231

Cleanup and ~ractionation.

Automated gel permeation chromatography (GPC) was used to

separate the organochlorine insecticides and PCBs from the bulk of the lipid.

A 60-g bed of SX-3

Bio Beads gel resin (Bio Rad) was used with a 1:1 mixture of cyclohexane in dichloromethane.

The

resin was packed in a glass column, 2.5 cm i.d. x 48 cm, fitted with two adjustable plungers (G1enco Scientific).

The column was placed on an automated low-pressure GPC Autoprep I001

chromatograph (ABC Labs), and solvent was pumped through the column at 5 mL/min.

Five milliliters

(but not more than I g of lipid) of the sample extract was placed on the GPC column.

The first

150 mL of eluate was discarded and the next 150 mL was collected in a 250-mL double reservoir flask.

The GPC eluates were rotoevaporated to approximately I mL and subsequently diluted to 5 mL

with hexane before Florisil fractionation. Florisil adsorption chromatography further cleans up fatty extracts and fractionates the nonpolar compounds before residues are determined by GLC.

Florisil columns were prepared by

placing I cm of anhydrous Na2SO 4 on a pledget of glass wool in a chromatography column (Ace Glass), I cm i.d. x 30 cm, fitted with a 75-mL reservoir at the top.

Five grams of 60/80 mesh

Florisil (Fisher Scientific), activated at 130°C for 16 h, was added and topped with another 1-cm layer of Na2SO 4.

Each column was then washed with 20 mL of hexane.

When the hexane layer reached

the top of the upper Na2SO 4 layer, the GPC concentrate was quantitatively transferred to the column and allowed to drain onto the bed of Florisil.

The column walls were washed with 5 mld of

the first eluant (40 mL of 6% diethyl ether in petroleum ether).

When the solvent reached the top

of the Florisil, the remaining eluant (35 mL) was added, and the eluate was collected for further separation.

Polar compounds (i.e., dieldrin and endrin) were collected with 40 mL of a 40%

solution of the same solvents. The majority of the chlordane components were separated from interfering PCB's by using 70/230 mesh Silica Gel 60 (E. Merck) activated at 130 C for 16 h. prepared in the same way as the Florisil columns.

The columns and extracts were

The first eluate, 0.5% benzene in hexane (PCB

fraction), contained the PCB's, heptachlor, and the monochloro adduct of pentachlorcyclopentadiene with cyclopentadiene.

The second eluate, 25% diethyl ether in hexane (pesticide fraction),

contained the rest of the major chlordane components and metabolites, along with toxaphene, ~,R-DDT, ~,R'-DDD, the benzene hexachloride (BHC) isomers, and other organochlorines.

Total

volume of the eluant depended on the activity of the silica gel and was calculated on a per-batch basis.

In general, 35-45 mL of the PCB eluant and 25-35 mL of the pesticide eluant were needed.

The eluate volumes were reduced by rotoevaporation, and the resulting concentrates were diluted to 5 mL with isooctane prior to GC/ECD and GC/MS.

Gas Chromatography.

All GC/ECD analyses were performed with a Varian 3700 gas chromatograph

with a direct capillary injection system and a standard 63-Ni electron capture cell and constant current amplifier. columns.

For the chromatography we used both wall coated open tubular (WCOT) and packed

The WCOT columns were 30 m x 0.25 mm i.d. SE-30 (methysilicone) fused silica (J & W

Scientific) and 24 m x 0.25 mm i.d. OV-17 (50Z phenyl- + 50Z methyl silicone) fused silica (Quadrex Corp).

Hydrogen carrier gas with a linear velocity of 60 c ~ s

(pressure fixed) at

maximum program temperature was used to chromatograph samples; an initial temperature of 120 C was held for 2 min, programmed to 250 C at 3 C/min, and held at the final temperature for I0 min.

The

1232

injector temperature was 220 C and the detector temperature 350 C, and a flow of 4 mL/min was used to sweep the septum.

The detector makeup gas was nitrogen at a flow rate (when added to the

carrier gas) of 40 mL/min.

To reduce the detector dead volume, we inserted the column up to the

detector source and introduced the makeup gas before the end of the column.

For packed-column GC

analysis we used glass columns, 1.83 M x 2 mm i.d., packed with (a) 1.5% SP-2250/1.95% SP-2401, (b) 3% or-101, and (c) 3% OV-17 on 100/120 mesh Supelcoport (Supelco). argon/methane (9:1) at a flow of 20 mL/min; no makeup gas was used.

The carrier gas was

The samples were

chromatographed at 190 C (isothermally). We collected capillary data with a Model 3600 Data Station/Chromatography Intelligent Terminal (Perkin-Elmer), using programs supplied by the vendor for peak detection and integration and ultimately for storage of chromatograms and replotting and reintegration.

Mass Spectrometry.

All mass spectromery was done with a Finnigan 4023 GC/MS and INCOS 2300

Data System in either electron-impact (El) or chemical-ionization (CI) mode with a methane plasma for ionization.

Samples were analyzed by GC/MS with a WCOT glass column (about 20 m x 0.25 ---

i.d.) coated with OV-17 (Chrompak U.S.A.) with helium carrier gas chromatography; the initial temperature of i00 C was held for 3 min, programmed to 240 C at 3 C/min, and then held for 8 min. Injection temperature was 250 C; the splitless injections were made through a standard Finnigan injection port.

Septom purge was 4 mL/min.

When E1 spectra were acquired, the manifold pressure

was about 4 x 10-7 torr; during negative ionization (NI), the manifold pressure was about 0.3 torr (all values read from standard Finnigan gauges).

Other variables, which remained constant for

both E1 and NI, were temperatures (source, 250 C; manifold, 120 C; transfer oven, 240 C), emission current (0.25 mA), and electron energy (70 eV).

Spectra were acquired with the lucos data system

scanning from 50 to 550 AMU every 2 s for E1 and from 60 to 500 A ~

every 2 s for NI.

A methane

plasma (99.99% methane, supplied by Matheson), maximized with perfluorotributylamine (FC43), was used for NI/MS.

RESULTS AND DISCUSSION

Technical chlordane is produced by a Diels-Alder condensation of cyclopentadiene and hexachlorocyclopentadiene to yield cblordene (Fig. I), followed further by chlorination addition or substitution reactions to form a number of endo-4,7-methanoindene compounds.

Addition of

chlorine across the 2-3 olefinic bond of chlordene forms cis- and trans-chlordane.

Substitution

of chlorine into position 1 of chlordene forms heptachlor, and further addition of chlorine across the 2-3 olefinic bond forms cis- and trans-nonachlor.

A number of other compounds are formed in

these reactions, as is evidenced by a typical packed column chromatogram of technical chlordane on the mixed phase substrate (Fig. 2).

We determined that this phase gave the best separation of the

individual components of chlordane and it is commonly used for pesticide residue analysis. Sovocool et al. 9 used an equivalent column in determining the composition of chlordane.

Peaks C,

D, E, and F (see Table I) are the major chlordane components detected in NPMP fish samples.

Peak

A represents a product from an impurity in the hexachlorcyclopentadiene used in the production of technical chlordane.

Peak B represents heptachlor, which readily forms the epoxide by adding

1233

Cl

Ct

Cl C!

3

CI 21

FiB. I

Diels-Alder condensation product of cyclopentadiene and hexachlorocyclopentadlene,

= ....

;

I

:

1

~:I ~-~-~

" -I

t.0

Fig. 2

"

2.0

3.0

4.0

S.O

Gas chromatogram of technical chlordane taken from a mixed phase column in series with an electron capture detector.

Fig.

3

I~0

2.0

3.0

4:0

50

, a

Gas ehromatograms of chlordane and metabolites in (A) a standard pesticide mixture and (B) an extract of Cuban limia.

I

Ill

O

Fig. 4

,

I

tTJl~

1115

4OOO

Tech Chlordane

Blootef

L Michigan

ll4D

¢lloItlle

4]J

Total ion gas chromatograms of Cuban limia from Hawaii, bloater from L. Michigan, and technical chlordane analyzed by negative ion mass spectrometry.

~

117

Llmll

Cuban LimJa

Hawaii

t'O

Fig. 5

o' |

I

":"

4

I

E

'~+ I~+ii~l,

,I

.-,..Lit + ~ +

A

Gas chromatograms of (A) technical chlordane, (B)Cuban limia pesticide fraction, and (C) Cuban limia PCB fraction taken from an OV-17 capillary column.

_L_;+- ..... .,_L],,t_,.,,J.'! ,+:'.!mLl.,-..~....

II

lot

,I

+~]l!i l,!li ° _,__._ ..++_.+_jjjjtj'tt~ U,'tL~L+,,+,._+,_

+,,

_+++I.++.JJ++++L~_,.++.,J._+,,,., .....

i

C

Fig. 6

,

J

4Jll

II

~

i t~:t+IL

't Io

1

,',

IP !'

I+I,

bi

,3, +flilP "t

Io

, f

LT il

o1

311

+~....... .+.~+_++~,.L~+#i +~,+'LL..,+.........'+.+.....

,

Z5

__

B

Gas chromatograms of (A) technical chlordane, (B) Cuban limia pesticide fraction, and (C) Cuban limia PCB fraction taken from a SE-30 capillary column.

. . . .

21

I+ ! ,

..-,.~,...v.,+f+ + _..+'+L,...,.,J.'J++'t~',,,,r~-_-~,,.~,J~+,+..

]

I

I

C

LO

1236

oxygen across the olefinic 2-3 bond 12.

A and B are the only major components found in the PCB

fraction of the silica gel chromatographic fractionation. Figure 3B shows a chromatogram of the pesticide fraction of an extract from Cuban limia, and Figure 3A is a chromatogram of a pesticide standard. detectable in this sample.

Chlordane components C, D, E, and F are

The amounts of compounds C through F in the standard approximate the

relative residue composition in NPMP fish samples and are considerably altered from the original technical chlordane mixture (Fig. 2).

On closer examination of the chromatogram of the Hawaiian

sample, we noticed that the retention time of cis-chlordane (E) was consistently longer than in the standard (Fig. 3B).

This indicated that the peak represented either an entirely different

residue or that it was cis-chlordane plus one or more unresolved unknown.

Chromatographic

analysis on OV-101 and OV-17 packed columns failed to adequately resolve components

C, D, E and

the unknown. A preliminary and cursory screen of the sample, in which GC/NI/MS was used on an OV-I7 capillary column, resolved peak E into two components. of samples analyzed by NIMS.

Figure 4 shows the total ion chromatograms

The solid peak after cis-chlordane (E) is the interference, and was

tentatively identified as an isomer of chlordane.

This component represents a relatively minor

one in the technical standard (also shown in Figure 4).

This chlordane analog was also detected

in the Lake Michigan bloater (Fig. 4); however, the chromatogram for the bloater reflects the usual residue composition of chlordane observed in NPMP fish samples, whereas that of the Cuban limia differs drastically.

The other darkened peaks in the chromatogram for the Cuban limia are

unidentified chlordane contaminants that were not observed in the packed column GC/ECD analysis. The Lake Michigan sample also has a number of toxaphene component peaks in the later portions of the chromatogram. Figure 5 shows the GC/ECD analyses of technical chlordane and Cuban limia chromatographed on a high resolution fused silica capillary column coated with OV-17. The PCB fraction (Fig. 5C) contained one major residue, peak I, that matched with the chlordane "peak A" component.

The

pesticide fraction (Fig. 5B), however, contained a number of residues that matched with chlordane components.

Analysis of tilapia from the same location gave similar chromatograms.

Figure 6 is a

GC/ECD chromatogram of Cuban limia extract obtained on an SE-30 fused silica capillary column. The relative retention order of some of the peaks -- notably peak Ol with 02 and D with E (cf. Fig. 5) -- has changed. peaks 6 and 7.

Also~ the SE-30 phase had a large effect on the relative retention of

Identities were assigned to some of these peaks by comparing the retention order

and relative retention times with published data 13 and comparing them with standards.

The

chlordane components in the Cuban limia for which individual standards were available are identified as follows: ~rans-chlordane, peak C; trans-nonachlor, peak D; cis-chlordane peak E; cis-nonachlor, peak F; compound "K", peak G; oxychlordane, peak O1; and heptachlor epoxide, peak O2.

Oxychlordane is an oxidation product of trans-chlordane and heptachlor epoxide results from

the oxidation of heptachlorl2. compounds.

Individual standards were not available for the remaining

Therefore, a more detailed analysis was conducted with the aid of GC/MS in both the El

and NZ modes. Components found in the Hawaiian fish extracts are listed in Table I with major m/e from El/MS and NI/MS.

The GC/EI/MS analysis of peaks E and C yielded fragmentation patterns identical

1237

Table I.

Analysis of components in Hawaiian fish.

GC Peak

Mol Wt

Formula

A,I

336

CIOH6C16

EI-336(6CI), 301(5CI), 236(5CI), 230(3CI), 196(3CI), 100(ICI)

Monochloro adduct of pentachlorochlordene

2

336

CIOH6C16

EI-336(6CI), 301(5CI), 265(4CI), 196(2CI), 169(3CI)

beta-Chlordene

3

372

CIOH7C17

EI-372(7CI), 337(6Ci), 302(5Ci), 230(3CI), 202(4CI), 167(3CI) NI-372(7CI), 264(4CI), 230(3CI), 201(4CI), 167(3CI)

Dihydroheptachlor isomer of the tetrachlorocyclopentadiene series

4

406

CIOH6C18

EI-406(8CI), 371(7CI), 337(6CI), 301(5Ci), 236(5CI) NI-406(8CI), 298(5C1), 264(4CI), 201(4CI), 167(3CI)

Chlordane isomer of the pentachlorocyclopentadiene series

5

406

CIOH6C18

EI-406(8CI), 371(7CI), 270(6CI) NI-406(8CI), 298(5CI), 264(4CI), 235(5CI)

Chlordane isomer

6

406

CIOH6C18

EI-371(7CI), 236(5CI), NI-406(8CI), 235(5CI),

Chlordane isomer of the pentachlorocyclopentadlene series

7

372

CIOH7C17

EI-337(6CI), 300(5CI), 265(4CI), 230(3C1),

Significant m/e and Chlorine Content

230(3CI),

335(6CI), 298(5CI), 264(4CI), 201(4CI) 371(7ci), 298(5CI), 264(4CI), 201(4Ci)

202(4CI), 167(3CI) NI-372(7CI), 337(6Ci), 264(4CI), 230(3C1),

Compound Identity

Dihydroheptachlor isomer of the tetrachlorocyclopentadiene series

167(3CI) Ol

420

CIOH4C180

EI-385(7CI), 149(2CI), NI-420(8CI), 279(4CI),

02

386

CIOH5C170

EI-386(7CI), 385(7CI), 351(6CI), 315(5CI), 270(6CI), 235(5CI) NI-316(5C1), 280(4CI), 246(3CI), 235(5CI)

Heptachlor epoxlde

C

406

CIOH6C18

EI-406(8CI), 371(7CI), 335(6CI), 270(6CI), 235(5CI) NI-406(8CI), 372(7CI), 298(6CI), 264(4CI), 235(5CI)

t rans-Ch io rdane

D

440

CIOH5C19

EI-440(9CI), 405(8Ci), 270(6Ci), 235(5CI) NI-440(9CI), 372(7Ci), 332(6CI), 298(5CI), 264(4CI), 235(5CI)

trans-Nonachlor

E

406

CIOH6C18

EI-406(8CI), 371(7Ci), 335(6CI), 270(6CI), 235(5CI) NI-406(8CI), 372(7CI), 298(5CI), 264(4CI), 235(5CI)

cis-Chlordane

F

440

CIOH5C19

EI-440(9CI), 405(8Ci), 369(7CI), 270(6CI), 235(5CI) NI-440(9CI), 372(7CI), 332(6CI), 298(5CI), 264(4CI), 235(5CI)

cis-Nonachlor

G

406

CIOH6C18

EI-406(8CI), 335(6CI), 298(6CI), 262(5CI), 214(4CI) NI-406(8CI), 298(5CI), 264(4CI)

Compound '%["

349(6CI), 235(5CI), 185(3Ci), I15(ICi) 348(6CI), 313(5CI), 314(5CI), 246(3CI), 235(5CI)

Oxychlordane

1238

with those of cis- and trans-chlordane.

The spectra of ¢is-chlordane in fish (Fig. 7A) had the

characteristic (M-C1) +, m/e 271 (7 CI) followed by chlorine clusters m/e 335 (6 CI), 299 (5 CI), 270 (6 CI) and a weak molecular ion cluster at ~ e

406.

Peaks D and F were confirmed as trans-

and cis____-nonacblor, with the (M-C1) + ion cluster containing the base peak, m/e 405 (8 CI). addition the spectra contained a weak M + cluster at m/e 440 (9 CI). had fragments corresponding to hexachlorocyclopentadiene

In

All four of these components

(m/e 270, C5CI 6) .

The formation of this

fragment results from the retro-Diels-Alder (RDA) decompositon, which is common to these types of compounds9,14.

Figure 8A shows the NI/MS spectra of cis-chlordane.

Both the chlordanes and

nonachlors had stronger molecular anion clusters in NI/MS and much weaker (M-CI) anions than the corresponding cations in El/MS.

Negative chemical ionization is a less energetic ionization

process than El, involving resonance capture of low energy thermal electrons by electronegative species to form stable anions.

Most of the fragmentation processes are due to elimination

reactions involving dissociation of H, CI, and HCI neutral species.

Figure 8A shows the presence

of a cluster at m/e 235 resulting from a pentachlorocyclopentadienyl

radical (C5C15).

This

fragment was also observed in the NI nonachlor spectrum and results from RDA decomposition accompanied by a loss of chlorine.

In NI/MS, the chlordanes and nonachlors had a 10-fold increase

in response over El/MS, thus demonstrating that NI/MS is a useful tool for detecting these compounds at trace levels in environmental samples.

Also, chlordane components are easier to

distinguish from toxaphene in NI/MS, which has fragmentation similar to chlordane in El/MS 15. El/MS analysis of peak 6, the largest of the unknowns detected, gave a base cluster at m/e 371 (7 CI); indicative of an octachloro component (Fig. 7B).

But no hexachlorocyclopentadiene

fragment ion was observed at m/e 270, as seen in the cis-chlordane spectra. fragment ion at mass 236 corresponds to a pentachlorocyclopentadiene

However, an RDA

(C5HC15) species.

RDA

fragments accompanied by prominent (M-CI) + ions, in El/MS, permits the assignment of molecular formulas, even when the M + ion intensity is very weak or nonexistent 9.

Thus a molecular formula

of CIoH6CI 8 is assigned to this compound, the same as that of cis-chlordane (Fig. 7A). assignment of this formula was verified by NIMS analysis. cluster was observed at mass 406.

The

In Figure 8B, an intense molecular ion

The masses at 201, corresponding to a tetrachlorocyclo-

pentadienyl ion (C5HC14), gave more intense ion currents than those at 235, which follows the observation that the major RDA fragments in NI/MS are accomp@nied by loss of chlorine. compound is an analog of chlordane, and is derived from pentachlorocyclopentadiene in the hexachlorocyclopentadiene used to manufacture technical chlordane.

This

contamination

It undergoes the same

Diels-Alder condensation with cyclopentadiene, with subsequent chlorine substitution and addition. Peak 4 was also observed to be an octachloro analog of pentachlorocyclopentadiene.

A series of

these compounds were recognized by Saha and Lee 16 and Sovocool et al. 9 in technical chlordane. However, they have never been reported in NPMP fish samples. Peak I, which corresponds to peak A in technical chlordane, is also a product from pentachlorcyclopentadiene.

In agreement with previous workl6,17, it was identified as a

monochloro adduct of pentachlorochlordene with a molecular formula CIoH6CI 6.

This was the only

chlordane component found in the non-polar PCB fraction, especially since heptachlor -- also a non-polar component -- is easily oxidized to the more polar epoxide.

The El/MS spectrum (Fig. 9)

shows that, in addition to the major RDA fragment (C5HC15, m/e 236), a monochlorocyclopentadiene

Fig. 7

"'

wll

~t-

_.oit~

'

'

350

100

i/

37'1 ///t

100

335

. . . . .

,.

'

. . . . . .

, ,i[,..

400

150

150 '

4,50

. . . . . .

235

. . . . .

200

:~0

500

27O

. . . . .

250

21o

550

300

a,~o

t Bo____ i

Mass spectra of (A) cls--chlordane, peak E, and (B) the octachloro isomer, peak 6, taken ~n the electron impact (El) mode.

[

:1

Fig.

•

8

2

:i

I

I

.I~L

250

,,,,~o'''

. '

300

298

372

~

350

e~

~

"dil

400

406 \~, 450

~oI . . . . . . . . . ,t,o ' .... gO'

.,to

o _ _

. . . . 3;, ....

isomer

Mass spectra of (A) ci___~s-chlordane,peak E, and (B) the octachloro isomer, peak 6, taken in the negative ionization (Nil mode.

M/E '00

201

210 M/E ....... o

2~5

B-octachlor

pa ~J

1240

fragment (C5H5CI) gives an intense ion current at mass I00.

This intense ion results from the

olefinic 2-3 bond imparting stability to the monochlorocyclopentadiene

fragment.

In El/MS the RDA

decomposition of chlordane and nonachlors did not give abundant dichlorocyclopentene (C5H6C12, m/e 136) and trichlorocyclopentene (C5H5C13, m/e 170) ions, respectively, which would correspond to the other major fragment ions.

These compounds lack the 2-3 olefinic bond.

From NMR spectral

data, Cochrane and Greenhalgh 17 postulated that the monochloro adduct of pentachlorochlordene must have one H at position 4, as shown in Figure 9.

This compound has eluded detection in other NPMP

fish samples in which high chlordane residues have been found.

NI/MS analysis was not performed

on the PCB fraction of the Hawaiian fish extracts. In ELMS, two components (3 and 7) were characterized by their formation of intense m/e 202 (4 CI) fragments.

These compounds were identified as dihydroheptachlor isomers of a tetrachloro-

cyclopentadiene series, which undergo prominent RDA fragmentation to C5H2CI 4. intense (M-CI)* ion clusters at ~ e molecular weight of 372.

337.

In NIMS the tetrachlorocyclopentadiene

However, a three-chlorine cluster at ~ e

These compounds had

The assigned molecular formula is CIOH7C17, with a fragment is not seen (Fig. I0).

167 is derived from a trichlorocyclopentadienyl

fragment.

This observation is consistent with the loss of one chlorine from the major RDA fragments under NI/MS conditions.

Peaks 3 and 7 are found in technical chlordane and originate from

tetrachlorocyclopentadiene

impurity in the starting material 9.

Component 7 is more polar than

component 3 because of its greater retention on OV-17 (Fig. 5), a polar phase; however, the isomeric configuration of chlorine and hydrogen in these compounds, which is the probable cause for the difference in polarity, is difficult to determine from these mass spectral data. Peak 2 was determined to be isomeric with chlordene, with the formula C6H6CI 6.

However, the

El/MS fragmentation pattern was dominated by neutral losses of CI, and HCI and had no major RDA fragment for any of the hexa-, penta-, or tetra-chlorocyclopentadiene was very similar to that for beta-chlordene9.

series.

The mass spectrum

This compound does not have the 4,7-methanoidene

structure, common to chlordane (Fig. l), but has a different ring fusion, which accounts for the absence of RDA fragmentation.

NI/MS spectra were not obtained for this component.

Our data for

peak G indicate that this component (compound "K") must have an unusual ring fusion that was not observed for chlordane.

Upon rearrangement it gave an intense ~ e

214, consistent with a C6H2CI 4

(6 carbon fragment) in El/MS, and no RDA decomposition to form the five carbon ring fragment.

It

has been theorized 9 that this species results from direct decomposition of the molecular ion (M +) in ELMS.

This theory is supported by the fact that the NI/MS spectra for compound "K" were

dominated by neutral losses of CI and HCI, with no rearrangement or RDA fragments. The last unknown determined in this study was component "5", which was isomeric with chlordane (CIoH6CI8).

Both the E1 and NI spectra gave similar m/e to chlordane (Table I), which

is a product of the hexachlorocyclopentadiene

series of the Diels-Alder condensation reaction.

In Hawaii, chlordane and heptachlor are used in pineapple culture. insecticides are some of the highest observed in NPMP samples.

Residues from these

However, occurrence of abnormal

residue compositions and previously unrecognized components have not been reported.

These

occurrences may be a result of different metabolic behavior between species, a different source of commercial chlordane, or environmental factors, all of which are undetermined.

That we were able

to identify the majority of these compounds in the environmental samples without available

1241

lO0.1

100 k

50.0,

196 '

23011

265

I.,[,.!: !I:

M/E

O0

IlO.O -

150

200

250

, ......... 450

500

300

58.0,

01

336 I

i,,..,,

M/E

Fig. 9

,11,., . . . . . . . . . . . . . . . . . . 350 400

.........

550

Mass spetrum of peak 1 in the Cuban llmia PCB fraction extract taken in the electron impact (EI) mode.

IN •

~41,il

230 167

....

/

ii ,,,,

lee .41 -

51.1

264

Fig. I0 Mass spectrum of peak 7 in the Cuban llmia pesticide fraction extract taken in the negative ionization (NI) mode.

1242

standards, but by using a combination of GC column substrates and modes of mass spectrometry, speaks well for the promise of screening environmental samples and determining the residue composition and identity of unknown contaminants by instrumental methods.

ACKNOWLEDGMENT

We thank James Johnson for mass spectrometry analysis and George Tegerdine for preparing the samples and standards used in this work.

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(8)

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(Received in T h e N e t h e r l a n d s

26 M a y

1983)