Environ. Sci. Jechnol. 1995, 29, 267-271
Cyclodiene Compounds in Ringed Seal Blubber, Polar Bear Fat, and Human Plasma from Northem Quebec, Canada: Identification and Concentrations of Photoheptachlor J I P I N G Z H U , + ' t ROSS J . N O R S T R O M , * ~ t ~ ' DEREK C. G. MUIR," LILIANE A. F E R R O N , l JEAN-PHILIPPE WEBER,' A N D E R I C DEWAILLYl Centre for Analytical and Environmental Chemistry, Carleton University, Ottawa, Ontario KlS 5B6, Canada, Canadian Wildlife Service, Environment Canada, Hull, Quebec KlA OH3, Canada, Department of Fisheries and Oceans, Freshwater Institute, Winnipeg, Manitoba R3T 2N6, Canada, and Centre de Toxicologie d u Qulbec, CHUL, Sainte-Foy, Qulbec G1 V 4G2, Canada
Introduction Heptachlor, a chlorinated cyclodiene, was used as an agricultural and domestic insecticide in North America during the 1950s- 1970s. The actual amount of heptachlor consumed is hard to determine, but the usage of heptachlor in the United States alone was estimated at 16 kt during the period 1971-1976 (1). Heptachlor is also a major component of technical chlordane, another heavily used pesticide in human history (2,3). Because it is difficult to distinguish the relative importance of technical heptachlor and chlordane as sources of heptachlor and its environmentally important degradation products and metabolites, we have considered them together. Although it was demonstrated in the laboratory that heptachlor can be transformed into a caged photoisomer, photoheptachlor, under ultraviolet (UV) irradiation at wavelengths longer than 290 nm (Figure 11, the early 1970s work failed to link photoheptachlor to the presence in the environment (4-6). The first possible occurrence in biological samples was proposed in the 1980sby Norstrom et al. (7) and Muir et al. (8). However, due to lack of standards, the peak could be only tentatively identified as photoheptachlor from its mass spectrum and retention time relative to chlordane compounds and heptachlor. It was therefore designated as U-1 (unknown 1). More recently, Buser and Miiller reported the presence of photoheptachlor and other chlordane components in Baltic salmon, Baltic herring, and Antarctic penguin (9). * Author to whom correspondence should be addressed. Carleton University. address: JP Ztech Company, 6058 Pineglade Cresc., Gloucester, Ontario KIW 1H1, Canada. § Environment Canada. I1Freshwater Institute. CHUL. +
* Present
0013-936~95/0929-0267$09.00/0
D 1994 American Chemical Society
The main heptachlor-related compound found in the environment is heptachlor epoxide, which is the major heptachlor metabolite formed through epoxidation of the nonchlorinated double bond in the molecule (Figure 1). The toxicity, including carcinogenicity, of both heptachlor and heptachlor epoxide have been studied (10,11). Photoheptachlor was found to be the most toxic compound among the chlordane components, including their metabolites (12). Compared to heptachlor, photoheptachlor is 47 times more toxic to bluegill, 19 times to rat, 4 times to housefly, and 264 times to goldfish. It is also much more toxic to bluegill (4 times) and rat (16times) than heptachlor epoxide. The same study also revealed a longer half-life of photoheptachlor in fat in the investigated rats and rabbits than that of heptachlor and heptachlor epoxide (12). The fate of photoheptachlor in rabbits, including tissue distribution, elimination, and metabolism of the compound, has also been studied (13). The prevalence of U-1 in Arctic biological samples (79) and the toxicity of photoheptachlor prompted this investigation. Since high contaminant levels in native food have caused concerns for the health of aboriginal people in northern Quebec (141, the study focused on identifying and quantitating the U-1 peak in biological samples from that area using a photoheptachlor standard prepared by photocyclization of heptachlor and comparing its relative importance to other heptachlor- and chlordane-related residues. Ringed seals are important in the diet of both polar bears and humans (7, 14).
Experimental Section Synthesis and Characterizationof Photoheptachlor. Heptachlor (7 mg) was irradiated under long Wwavelength at 365 nm for 24 h in 50 mL of acetone in a Pyrex glass bottle. An aliquot of the acetone solution was analyzed by gas chromatography /electron impact mass spectrometry (GCI EIMS). The GUMS chromatogram showed that over 80% of heptachlor has converted to photoheptachlor, based on the ratio of the peak areas of the two compounds. The acetone solutionwas evaporated to dryness,and the residue was dissolved in 1 mL of hexane. The raw product was then purified on a 25-g Florisil (deactivatedwith 1.2% water) column. The heptachlor was eluted first with 70 mL of hexane, and the photoheptachlor was eluted with a further 30 mL of hexane. A total of 4 mg of photoheptachlor was obtained by crystallizationfrom an acetone-water mixture. Full-scan GClEIMS analysis of the purified product showed a single peak to be present. Based on the GC/EIMS detection limit of heptachlor, the purity was 299%. The proton signals in the nuclear magnetic resonance (NMR) spectrum were consistent with the photoheptachlor structure. No extraneous proton signals from impurities were observed. The NMR and MS data were collected from Bruck AMX-400 and HP 5988 instruments, respectively. Spectroscopic data: lH NMR (CDClS): 4.72 ppm (H-1, dd, 1.8 Hz, 1.7 Hz); 3.62 ppm (H-5, dd, 7.3 Hz, 4.8Hz); 3.57 ppm (H-2,dd,7.3Hz,5.6Hz);3.46ppm(H-4,m);3.38ppm(H-3, m). 13CNMR (CDCL3): 62.48 ppm (C-l),58.53 (C-5),56.60 (C-2), 48.08 (C-4), 43.44 (C-3). Signals of nonprotonattached carbons (C-6 to C-10) were not observed due to low sample concentration. EIMS and electron capture
VOL. 29.
NO. 1, 1995 I ENVIRONMENTAL SCIENCE & TECHNOLOGY 267
-
CI
CI
101* ; c
CI
+ hv & ;c
CI
CI
CI
CI
CI
CI
Heptachlor epoxide
Heptachlor
Photoheptachlor
FIGURE 1. Environmentally important oxidation and photocyclization reactions of heptachlor.
A
FIGUREZ. Map of 0uehec.Samplingsites are indicated hy[r)seal. [A)bear, and IO) human.
negative ionization (ECNII-MS spectra are illustrated in Figures 3 and 4, respectively. The GC retention time of photoheptachlor (16.36 minl on a 30-m DB-5 column was close to that of octachlorostyrene (16.61 minl and greater than that of heptachlor (14.55 min). Under the same GC conditions, heptachlor epoxide and oxychlordane peaks appeared at 16.95 min. The oven temperature was programmed as follows: 3 min hold at 100 “C, 20 “Clmin increase to 180 “C and then 5 “Clmin to 300 “C. Sample Collection. Ringed seal blubber was collected fromKangiqsujuaq, Inukjuak, and Kangiqsualujjuaq in 1990 and 1991; polar bear fat was collected from Sanikduaq, Sleeper Islands, Iqaluit, Lake Harbour, and Pangnirtung in 1989-1990: and human plasma was collected from the villages of Kuujjuaq, Kuujuuaraapik, Povungnituk, Tasiujaq, and Umiujaq in 1992. All are located in northern Quebec and the surrounding areas of Canada (Figure 2). Analysis of Biological Samples. Three laboratories participated in the study. Each of the laboratories used their own quantitation standards except for photoheptachlor. A standard of photoheptachlor (16.6 nglpL in hexane) was prepared from crystals and distributed to the threelaboratories. Good comparabiliryofdataamongthese three laboratories was demonstrated through an interlaboratory comparison on polychlorobiphenyls (PCBs)and organochlorine pesticides (OCsl in 1993 (15). Seal blubber samples were analyzed using gas chromatographylelectron capture detector (GC/ECD) at the Freshwater Institute according to the procedure of Muir et a/.18). Polar bear fat sampleswere analyzed at the National 268 m ENVIRONMENTAL SCIENCE & TECHNOLOGY IVOL. 29. NO. 1.1995
Wildlife Research Centre using gas chromatography/mass selectivedetector (GCIMSDIunder the conditions described by Zhu et a/. (16). Human plasma samples were analyzed at le Centre de Toxicologie du Quebec under the following conditions: 2 mL of plasma samples fortified with two internal quantitation standards (PCB 198 and endrin) was mixed with 2 mL of ethanol, 2 mL of aqueous saturated ammonium sulfate, and 6 mL of hexane. The mixture was vigorously shaken for 4 min and centrifuged to separate the phases. Extraction with 6 mL of hexane was repeated twice. The combined organic phases were evaporated to about 1 mL and then passed through two Florisil (0.5% waterdeactivated) columns arranged in tandem. PCBs and OCs except dieldrin and heptachlor epoxide were eluted with 14 mL of hexane/dichloromethane (7525). Dieldrin and heptachlor epoxide were eluted with 20 mL of hexane/ acetone (928). The two fractions were taken to 100 and 500 pL, respectively. Samples were then analyzed on a HP-5890 Series I1 GC with a 63NiECD using dual columns (Ultra-l and Ultra-2, 50 m x 0.20 mm, 0.33 pm film thickness), which were connected by a two-hole graphic ferrule. The collection and processing of data were performed with a HP-3365 chemstation.
Results and Discussion Mass Spectra of Heptachlor and Photoheptachlor. The two double bonds in heptachlor can react via I2 + 21 intramolecular photocyclization under W light to form a caged photoisomer, photoheptachlor (Figure 1). The E1 mass spectra of these two compounds are rather similar (Figure31with afewimportant differences. Theheptachlor spectrum is dominated by retro-Diels-Alder cleavage (RDAI to form ions of mi2270 IC5Cl61+and mlz 235 IC5Clsl+. The loss of a chlorine from the molecular ion occurs, but IM Cl]+has relatively low intensity. In the case of photoheptachlor, RDA fragments were less dominant, while loss of CI and HCI was the main fragmentation, producing ions of mlz 335 [M CIl+, mlz 299 [M - 2CI - HI+, mlz 264 IM - 3CI - HI+, and mlz 230 [M - 4Cll+. In the low mass range, ions ofm/z65,100,133/135.160,167/169,and 194 were present in both molecules. The E1 spectrum of photoheptachlor is very similar to that reported by Buser and Muller (9)and that which we obtained for U-1 (7). However, the [M - C1]+peak is more abundant compared to an earlier work (51,which could be due to different mass spectrometric conditions. Greater dissimilarity was observed in the ECNI mass spectra of heptachlor and photoheptachlor than in the El spectra (Figure 4). As was the case for the E1 spectrum, RDA ion mlz 235 was the main peak in the ECNI spectrum of heptachlor, alongwith m / z 298 [M - 2C1-2Hl-, m/z 264 ~
I
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272
237
\
135
337
\
i60
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100
140
120
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:eo
200
220
240
280
280
300
320
340
360
337
1
io0
\ 266
i
133 /
FIGURE 3. Electron impact mass spectrum of heptachlor (top) and photoheptachlor(bottom). Electron energy, 70 eV; ion source temperature,
200 "C. 237
266
/
eo
io0
120
140
:eo
ieo
200
220
240
260
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300
320
340
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300 196
i
[M - 3C1 - HI-, and mlz 230 [M - 4C1]-. In the photoheptachlor spectrum, there were no RDAfragments. The only fragmentationwas the loss of chlorines to form mlz336 [M + H - Cll-, mlz298 [M - 2C1-2H]-, mlz264
\
I
[M - 3C1 - HI-, mlz 230 [M - 4C1]-, and mlz 196 [M 5C1+ HI-. The abundance of mlz 336 clearly indicates a process associatedwithproton attachmentto the molecule intheionsource. Such[M-Cl+H]ionsweredsoobserved VOL. 29, NO. 1, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 1269
TABLE 1
Concentration of Photoheptachlor, Related Contaminants, and Total PCBs in Biological Samples from Northern Quebec (Units in ng/g lipid Weight) photoheptachlor heptachlor epoxide oxychlordane nonachlor-Ill trans-nonach lo r cis-nonachlor trans-chlordane cis-chlordane total CHL total PCB
seal blubber ( n = 41)
bear fat ( n = 52)
human plasma ( n = 57)
15 f 15 (2.2Ia 91 i 87 (12.8) 207 f 226 (29.3) 59 f 90 (8.3) 268 f 378 (38.0) 27 f 34 (3.8) 5 i 8 (0.7) 34 f 40 (4.9)
145 f 95 (3.4) 475 f 326 (11.1) 2652 f 2002 (61.9) 545 f 531 (12.7) 470 f 224 (11.O) NDC ND ND
11 f 11 (1.3) 51 f 51 (6.1)b 242 f 230 (28.8) 141 f 134 (16.8) 333 f 292 (39.6) 62 f 45 (7.4) ND ND
706 762
4287 10293
840 6819
* M e a n value f 1 SD; number in parentheses is the concentration as percentage of total chlordane (total CHL). detection limit was 2 ng/g.
in the ECNI spectra of other types of compounds such as polychlorobornanes (1 7). Formation of [M H - Cll- in the spectra of chlorinated cyclopentadiene derivatives was explained as an addition-elimination mechanism (18).We also noticed a higher abundance of mlz 336 in this study compared to the spectrum recently published by Buser and Muller (9) that used argon gas as a buffer in the ion source. Many factors, such as ion source design, source temperature, tuning parameters, concentration of reagent gas, nature of makeup gas, and sample concentration, can affect ECNI spectra. Levels of Photoheptachlor and Other Chlorinated Cyclodiene Compounds in Biological Samples. The concentration of photoheptachlor in ringed seal blubber, polar bear fat, and human plasma is summarized together with concentrations of other chlordane compounds and total PCBs in Table 1. The values in Table 1were adjusted to a lipid weight basis for all three matrices. The total chlordane concentration in bear fat was five to six times higher than that in human plasma and seal blubber (Table 1). Although the total chlordane concentration in human plasma was at same level as that of seal blubber, the total PCB concentration in human plasma was more than 10 times higher than in seal blubber. The total PCB concentration in human plasma was about half of that in bear fat. The overall contaminant pattern in seal blubber and human plasma was similar. trans-Nonachlor and oxychlordane comprised 70% of the total chlordane concentration measured (Table 1, values in parentheses). Among other chlordane components, nonachlor-I11 (7), which is also called MC-6 (3), and cis-nonachlor increased the most from seal to human. The proportion of heptachlor epoxide decreased, and photoheptachlor remained at the same level. Both cis- and trans-chlordane were not detectable in human plasma. In contrast, the pattern in polar bear fat was dominated by oxychlordane (62%)followed by almost equal levels of nonachlor-I11 (13%),trans-nonachlor (11961, and heptachlor epoxide (11%). Photoheptachlor is more significant in polar bear (3.4% of total chlordane measured) than in the other two species. The ratio of photoheptachlor to heptachlor epoxide was 0.16 in seal blubber, 0.30 in bear fat, and 0.22 in human plasma. The ratio of photoheptachlor to oxychlordane in the same species was 0.07,0.05, and 0.04, respectively. The bioaccumulation factor (BAF)of photoheptachlor is greater than that of heptachlor epoxide and similar to that of oxychlordanefrom seal to bear and seal to human, assuming
+
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n = 29.
ND = not detected,
that ringed seal was the main source of contamination in humans as well as polar bears (19). The pattern of chlordane- and heptachlor-related compounds in biota varies considerably. Species at the low end of food chains, such as fish, contain complex mixtures of compounds mostly derived from the commercial pesticides at relatively low concentrations. Species at the top, such as seals, bears, and humans, bioaccumulate only a few original chlordane components and their metabolites (8, 20). Nonachlordanes (C19 compounds) are the most persistent chlordane components, followed by octachlordanes (GIB compounds). Some less chlorinated compounds are not biomagnified and are therefore quickly eliminated during passage through the food chain. Among the chlordane metabolites, heptachlor epoxide and oxychlordane are the two major persistent compounds. Nonachlor-111 (or MC-6) was first identified in technical chlordane by Miyazaki et al. (6 in ref 21). It is a minor constituent of technical chlordane, but makes a more important contribution to total chlordane residues in high trophic level animals, presumably because it is not easily metabolized (7). Photoheptachlor was only recently confirmed to be an environmentally persistent chlordane contaminant even though it was synthesized three decades ago (9). Photoheptachlor is usually not detectable in Arctic char and cod (22),but its propensity to biomagnify in high trophic level Arctic species such as seal, bear, and humans and its high toxicity make it a potentially significant contaminant. Age-CorrelatedContaminant Levels in Humans. The samples collected from people living in northern Quebec show clearly the increase of total chlordane levels in plasma with age (Figure 5). The same trend of total PCB levels was also observed but not included in the figure. The contaminant level is relatively low in cohorts under age 35 and then increases to a maximum loading in age group 56-60. The older people (age 51 and above) have almost four times the concentration of younger people (age 35 and below). Studies on Inuit food and diet show that old people take 1.5-2 times more traditional food than young people (23). This may partly account for the higher concentration of contaminants in old people. While the contaminant level is correlated to age, the pattern of the contaminants remained similar for all age groups. For example, the concentrations of photoheptachlor, cis-nonachlor, nonachlor-111, and trans-nonachlor normalized to that of oxychlordane show no apparent difference in pattern
.-"g 3
0
18-20
26-30 36-40 51-55 61-66 " 31-35 46.50 56-60 Age Group
21-25
1 +Ph.HP
+c-CHL
Non-111
+bNON
*Total-CHL]
FIGURE 5, Relative concentrations of four chlordane components to oxychlordane(=lW) in each age group in human plasma samples. Ph.HP, photoheptachlor; c-CHL cis-chlordane; Non-Ill, nonachlor111; t-NON, trans-nonachlor; total CHL, total chlordane. Population in each age group: n = 4 (age group 16-20), n = 10 (a-251, n = 7 (26-30), n = 10 (31-35), n = 4 (36-40),n = 6 (46-50), n = 5 (51-55). ti = 7 (56-60), n = 4 (61-66).
among age groups (Figure5). There were some fluctuations in relative concentrations, but no clear relationship with age. The relative concentration of these four components to oxychlordane was about 5,30,60, and 150, respectively. Significant correlation between age and total chlordane or total PCB concentration in seals and bears was not observed in this study.
Acknowledgments This project was partly funded by the Canadian Arctic Environmental Strategy Greenplan. We thank Michael M. Mulvihill, Canadian Wildlife Service, for his laboratory assistance;Keith Bourgue, Chemisuy Department, Carleton University, for the NMR analysis; and Craig E. Hebert, Canadian Wildlife Service, for handling of the polar bear data. We thank Makivik Corporation for providing the ringed seal and polar bear samples.
Literature Cited (1) Fendick, E.A.; Mather-Mihaich, E.; Houck, K.A.; St. Clair, M. B.; Faust, J. B.; Rockwell, C. H.; Owens, M. Rev. Enuiron. Contamin. TOX~CO~. 1990, 111, 61-142.
(2) Buchert, H.; Glass,T.; Ballschmiter, K. FreseniusZ. Anal. Chem. 1983, 333, 211-217. (3) Dearth, M. A.; Hites, R. A. Enuiron. Sci. Technol. 1991,25,245254. (4) Parlar, H.; Korte, F. Chemosphere 1977, 10, 665-705. (51 Onuska, F. I.; Comba, M. E. Biomed. Mass Spectrom. 1975,2, 176-182. (6) Benson, W. R.; Lombardo, P.; E m , I. J.;Ross, R. D., Jr.; Barron, R. P.;Mastbrook, D. W.; Hansen, E.A. 1.Agric. Food Chem. 1971, 19, 857-862. (7) Norstrom, R. J.; Simon, M.; Muir, D. C. G.; Schweinsburg, R. E. Enuiron. Sci. Technol. 1988, 22, 1063-1071. (8) Muir, D. C. G.; Norstrom, R. J.; Simon, M. Enuiron. Sci. Technol. 1988,22, 1071-1079. (9) Buser, H.-R.; Muller,M. D. Enuiron.Sci. Technol.1993,27,12111220. (10) HealthAdvisory.Rev. Enuiron. Contam. Toxicol. 1988,104,131145. (11) Cavender, F. L.; Cook, B. T.; Norbert, P. P. Office of Health and Environmental Assessment, US. Environmental Protection Agency: Washington, DC, 1987; EPA/600/S6-87/004. (12) Khan, M. A. Q.; Feroz, M.; Podowski, A. A.; Martin, L. T. In Dynamics, Exposure and Hazard Assessment of Toxic Chemicals; Haque, R., Ed.; Ann Arbor Science Publishers: Ann Arbor, MI, 1980; pp 392-415. (13) Feroz, M.; Khan, M. A. Q. 1.Agnc. Food Chem. 1979, 27, 108113. (14) Dewailly, E.;Ayotte, P.; Bruneau, S.; LalibertB, C.; Muir, D. C. G.; Norstrom, R. Enuiron. Health Perspect. 1993, 101, 618-620. (15) Zhu,J.P.FinalReportof 1992/93and 1993194QualityAssurance Program to Indian and Northern Affairs Canada. 1994. (16) Zhu, J. P.; Simon, M.; Mulvihill, M. J.; Norstrom, R. J. Manuscript in preparation. (17) Zhu, J. P.; Norstrom, R. J. Chemosphere 1993, 27, 1923-1936. (18) Stemmler, E. A.; Hites, R. A. Anal. Chem. 1985, 57, 684-692. (19) Cameron, M.; Weis, M. Arctic 1993, 46, 42-48. (20) Norstrom, R. J.; Muir, D. C. G. Sci. Total Enuiron. 1994, 154, 107-128. (21) Miyazaki, T.; Yamagishi, T.;Matsumoto, M. Arch. Enuiron. Contam. Toxicol. 1985, 14, 475-483. (221 Muir, D. C. G. Unpublished data. (23) Kinloch, D.; Kuhnlein, H.; Muir, D. C. G. Sci. Total Enuiron. 1992, 122, 247-278.
Received for review June 7,1994.Revised manuscript received October 13, 1994. Accepted October 20, 1994.
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