990 SHORTPAPERS Analyst, Vol. 105 Rapid Extraction of Some Persistent Chlorinated Hydrocarbons from Biological Material with Low Fat Content Gunnar Norheim and Elisabet Mo Okland National Veterinary Institute, P.O. Box 8166 De#., Oslo 1, Norway Keywords: Chlorinated hydrocarbon extraction; hexachlorobenzene; octachloro- styrene; biological material; low f a t content Persistent chlorinated hydrocarbons of agricultural and non-agricultural interest, such as l,l,l-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT), polychlorinated biphenyls (PCBs) and hexachlorobenzene, have obtained a global distribution, and can be detected in wildlife samples in variable amounts. PCBs together with l,l-dichlc~ro-2,2-bis-(~-chlorophenyl)- ethylene (DDE) are the main types of chlorinated hydrocarbons found in Norwegian avian fauna and in fish along the Norwegian In Frierfjorden, a fiord in South-Eastern Norway, heavy local contamination with chlo- rinated hydrocarbons of industrial origin has been detected.The contaminants most often found in the fish in this area are hexachlorobenzene, octachlorostyrene and decachlorobiphenyl. In addition, complex mixtures of PCBs and also of chlorinated naphthalenes have been detectede415 Decachlorobiphenyl has previously been detected in. arctic fox ( A l o p e x lagopus) from Svalbard,6 and octachlorostyrene was first detected in birds from The Netherlands. '18 In a monitoring programme over the last 6 years, the above chlorinated hydrocarbons have been determined in samples from cod (Gadus morhua) obtained from the area.Flesh samples from cod have a low fat content (about 0.3%) and extraction of such samples is not especially reproducible. Some procedures are also very time consuming. A rapid and simple procedure has therefore been developed for this type of sample for the gas-chromatographic determina- tion of some chlorinated hydrocarbons resistant to concentrated sulphuric acid. Experimental Apparatus and a 2 m x 3 mm i.d. glass column was used. 15.9% SP-2401 on 100-120-mesh Supelcon AW DMCS. temperatures were 200, 250 and 275 "C, respectively. the carrier gas, the flow-rate being 55 ml min-l. Reagents A Carlo Erba 2100 gas chromatograph equipped with a nickel-83 electron-capture detector The column material was 1.5% SP-2250 - The column, injector and detector Argon - methane (95 + 5 ) was used as The electrometer attenuation was x 128.S u l p h u r i c acid, 95-97%. Heptane. Pro analysi grade (Merck). Hexachlorobenzene. Pract. grade (Fluka) . octachlorostyrene. Standard Solutions Amounts of 100 mg each of hexachlorobenzene and octachlorostyrene were dissolved in 100 ml of heptane and the mixture was diluted 1 + 50000 with heptane. Pro analysi grade (Merck). Obtained as a gift from Norsk Hydro.Gctober, 1980 SHORT PAPERS 99 1 Procedure A 0.5-g amount of sample was accurately weighed into a 10-ml Soveril glass tube fitted with a screw-cap, and 6 ml of concentrated sulphuric acid were measured into the tube. The tube was placed in a thermostatically controlled oven a t 60 "C for 4 h, during which time it was shaken lightly a few times to ensure complete solubilisation of the sample.After cooling, 1.0 ml of heptane was pipetted into the tube, the screw-cap put on and the tube shaken for about 3 min. Finally, the tube was centrifuged with the screw-cap on, after which the sample was ready for gas chromatography. An injection volume of 5 p1 was used. TABLE I RECOVERY OF HEXACHLOROBEKZEKE (HCB) AND OCTACHLOROSTYRENE (OCS) FROM HOMOGENISED COD FLESH AFTER ISOLATION BY THE PROPOSED METHOD AND EXTRACTION WITH DRY DIETHYL ETHER OR HEXANE - PROPAN-8-OL Recovery, yo r--L- Method HCB ocs\ Present method . . . . . . 95 95 Dry diethyl ether . . . . . . 32 58 Hexane - propan-2-01 . . . . 87 86 Results and Discussion The proposed method is rapid and requires only small amounts of reagents and little equipment.The recoveries of hexachlorobenzene (HCB) and octachlorostyrene (OCS) were determined after a standard addition by a method described previou~ly.~ The recovery of each component was 95%. A large number of samples can be extracted simultaneously. i C B l ocs ocs - 0 4 8 0 4 8 0 4 8 Time/min Fig. 1. Chromatograms of 5-p1 injec- tions of hexachlorobenzene (HCB) and octachlorostyrene (OCS). (a) Standard solution of HCB and OCS (0.1 ng of each) ; ( b ) extract of cod flesh from a contami- nated area (in addition to HCB and OCS this chromatogram has peaks for penta- chlorobenzene and heptachlorostyrene) ; ( c ) extract of a blank sample of cod flesh. The gas-chromatographic conditions are stated in the text.992 SHORT PAPERS Analyst, Viol. 105 TABLE I1 RESULTS OF A COLLABORATIVE STUDY IN WHICH PENTACHLOROBENZENE (5CB), HEXACHLOROBENZENE (HCB) AND OCTACHLOROSTYRENE (ocs) WERE DETERMINED IN HOMOGENISED COD FLESH Concentration/yg 8-l (wet mass) Laboratory 5CB HCB OCS This work .. . . 0.002 0.13 3.7 Laboratory 2 . . . . 0.003 0.17 4.2 Laboratory 3 . . . . 0.002 0.10 1.3 Laboratory 4 . . . . - 0.08 1.3 Laboratory 1 . . . . 0.001 0.10 3.7 The recovery of HCB and OCS from homogenised cod flesh using the proposed method was compared with two other methods, namely column extraction with dry diethyl etherlo and direct extraction for 4 h with hexane - propan-2-01 and subsequent partition with water.4 The results are presented in Table I. The reproducibility of the method was calculated after the determmation of HCB and OCS in eight parallel samples of cod flesh.The following results were obtained after manual injection of the extracts into the gas chromatograph: HCB, 0.25 & 0.014 pg g-l; and OCS, 0.18 f 0.017 pg g-1. The coefficients of variation for HCB and OCS were 5.5% and 9.5%, respectively. The retention time under the conditions used was 1.6 min for HCB and 4.5 min for OCS. Typical gas chromatograms are presented in Fig. 1. A blank sample from a non-contaminated area gave no interfering peaks. The accuracy of the proposed method was tested in a collaborative study involving five laboratories.11 The other four laboratories used other extraction procedures. The results of this study are presented in Table 11. Agreement was fairly good for HCB, but less good for OCS, two of the laboratories finding lower levels. The method was also tested on homo- genised herring containing about 10% of fat, and agreement with the results of ordinary extraction techniques was better than that for the cod flesh, especially for OCS.With increasing fat content in the sample, emulsion formation after the extraction with heptane causes problems. This may be partly overcome by increasing the volume of heptane, although sensitivity is thereby decreased. Heptane was preferred to hexane as the solvent because of its lower volatility. However, if a high reproducibility is to be achieved, it is essential that centrifugation be carried out with the screw-cap on. The sensitivity of the method is dependent on the gas-chromatographic equipment used.In our laboratory we can routinely determine HCB and OCS in cod flesh at levels down to 0.001 pg g-l wet mass. An important limitation of the proposed method is that DDE is partly broken down during the digestion and cannot be determined accurately. PCB:j, on the other hand, can be recovered as efficiently as HCB and OCS. In our studies on HCB, OCS and decachlorobiphenyl in fish from Frierfjorden and the surrounding fiords, the proposed method has proved useful in the gas-chromatographic determination of these chlorinated hydrocarbons. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. References Holt, G., Fralie, A,, and Norheim, G., Acta Vet. Scand., 1979, Suppl. 70, 28 pp, Brevik, E. M., Bjerk, J. E., and Kveseth, N. J., Bull. Environ. Contam. Xoxicol., 1978, 20, 715. Kveseth, N. J., Bjerk, J. E., Fimreite, N., and Stenersen, J., Arch. Environ. Contam. Toxicol., 1979, Lunde, G., and Baumann Ofstad, E., Fresenius 2. Anal. Chem., 19768, 282, 395. Baumann Ofstad, E., Lunde, G., Martinsen, K., and Rygg, B., Sci. Total Environ., 1978, 10, 219. Norheim, G., Acta Pharmacol. Toxicol., 1978, 42, 7. Koeman, J . H., ten Noever de Brauw, M. C., and de Vos, R. H., Natuve (London), 1969, 221, 1126. ten Noever de Brauw, M. C., and Koeman, J . H., Sci. Total Environ., 19'72/73, 1, 427. Norheim, G., and 0kland, E., paper presented at Workshop in Gas Chromatography, Sandefjord, Bjerk, J. E., and Holt, G., Acta Vet. Scand., 1971, 12, 429. Martinsen, K., Baumann Ofstad, E., Lunde, G., Brevik, E. M., Kveseth. N. J., Bse, B., Egaas, E., 8, 201. Norway, 1978. Crowo, J . A., Murer, K., Frmlie, A., and Norheim, G., Report, Oslo, Norway, 1978. Received May 6th, 1980 Accepted May 29th, 1980