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EXPERIMENTAL AND METHODS Samples Herring Oil Heron Eggs Arochlor 1254 Supplied by van den Berghs and Jurgens Led., Purfleet, Essex. Collected from Heronry at Tumby, Woodstide, Lincolnshire, March 1967, by Toxic Chemicals Section, Nature Conservancy, Monks Wood. Supplied by Monsanto Ltd. Reagents Dimethy1formamide (DMF) Hexane Florisil Acetic acid, glacial Nitric acid Sulphuric acid Acetone Sodium sulphate Redistilled under vacuum at 50C. Petroleum fraction SBP 60-70. Redistilled and fraction boiling between collected. Synthetic silicic acid - magnesia adsorbent manufactured by Floridin Co. . Activated. Heat to 120C overnight. Deactivated. Activate as above, cool and add 3% w/v distilled water. (See Appendix) Analytical Reagent Grade. Analytical Reagent Grade, SG - 1.42 Analytical Reagent Grade, SG - 1.84 Redistilled General Purpose Reagent, anhydrous, granular washed with hexane prior to use. Extraction and "clean-up" of samples for gas-liquid chromatography - mass spectral analysis Fish Oil The crude herring oil (220 g) was dissolved in 500 ml of hexane and dried over anhydrous sodium sulphate. Eggs The total egg weight was 67 g and the final hexane extract volume prior to the DMF partition was 100 ml. MGNS 048998 o -6- o Chromatographic "clean-upM Fish Oil The solution in hexane was concentrated to 50 ml and percolated through a column 1 cm wide containing 20 g of activated Florisil. The column was eluted with hexane until there was no further elution of peaks corresponding to those occurring in the first fraction of routinely analysed samples (see Appendix A). The resulting eluate was concentrated to 10 ml and passed down an activated Florisil column (6 g) eluted with hexane and fractions collected until no further peaks were eluted, as shown by GLC electron capture. This process was repeated three times. The hexane eluate obtained as above contains pp'-ODT and its congeners polychlorinated biphenyls, non-oxygenated "drin" insecti cides. The more polar compounds such as HEOD, endrin and heptachlor epoxide are not eluted under these conditions. The solvent was evap orated from the eluate and the residue was nitrated with a mixture of nitric and sulphuric acid A : 1 v/v. The acid mixture was poured into water and extracted with hexane. The resulting hexane solution was again chromatographed through activated Florisil and the eluate was evaporated to 1 ml. Heron Egg Purification of the heron egg extract, following DMF partition, was achieved by chromatography down a deactivated Florisil column (25 g) using hexane (150 ml) as eluent. The concentrated eluate (10 ml) was further purified by chromatography down activated Florisil (6 g) using hexane as eluent. Portions of the above samples were examined by gas-liquid chroma tography with electron capture detection, micro-coulometric detection and mass spectrometric and flame ionisation detection. Standards of Arochlor 125A were also examined by GLC with the same column conditions and detection procedures. HONS 048999 o - 7- ) Gas chromatographic operating conditions for electron capture detection and micro-coulometric detection were as follows:** * Instrument: Column: Inlet pressure: Temperature; Carrier Gas: Electron capture detector: Microcoulometric detector: Electrolyte Oxygen flow rate: Pye 104 152 cm x 0.3 cm glass tube packed with 3.8% SE30 on Diatoport S. 18 psi. 185C. Oxygen free Nitrogen. 63 Ni source with pulsed DC supply and 150 microsecond pulse interval. Inlet temperature 200C Furnace temperature 900C 702 glacial acetic acid : 32 water. 60 ml/minute The GLC/mass spectrometer operating conditions were:- Inetrument: Chromatographic column: Carrier gas: Flow rate Temperature Ionisation voltage Pye 104 chromatograph with flame ionisation detector and 1 : 1 split between detector and mass spectrometert an AEI MS 12. 152 cm x 0.3 cm glass column packed with 22 Silicone SE30 and 0.22 Epikote 1001 on 85-100 mesh Diatoport S. Helium 40 ml/minute 184C 70 volts. HONS 049000 RESULTS AND DISCUSSION Chromatograms of extracts of heron egg and herring oil and Arochlor 1254 obtained by gas-liquid chromatography and electron capture detec tion are given in Figures 2-4 respectively. The chromatograms obtained by microcoulometric detection are given in Figures 5-7. These show that many of the components of the live samples respond both to electron capture and microcoulometric detection and are probably halogenated compounds Mass spectral - GLC examination was carried out using a gas chroma tograph with a SE30 column identical to chat used for electron capture, the gas chromatograph was coupled to a MS12 mas spectrometer with a Wateon-fiiemann separator. The chromatogram was recorded with a flame ionisation detector such that there was a 1:1 gas flow split between the detector and the separator. The chromatograms obtained are given in Figures 8-10 and portions of the mass spectra in Figures 11-13. The extracts were also examined by thin layer chromatography and the R^ values obtained together with those of various chlorinated insecticides are given in Table 2. The isotopes of chlorine, Cl35 and Cl37 , occur in a ratio of 3 to 1 (precisely 75.4 : 24.6) and a compound containing one Cl atom per molecule will exhibit a doublet parent peak one doublet corresponding to an ion with a Cl chlorine atom of mass 35 and the other to an ion with a chlorine atom mass 37. Further, the intensity of these peaks will be in the ratio of 3:1. Generally the expression for the probability of ions of the type R Cln3_5ft Cl3fl7 is:- ram co-75>n'a (0-25)* ' (where n * number of chlorine atoms per ion). Using this expression it is possible to calculate the number and relative intensity of the peaks in the group due to a chlorinated ion existing in these several isotopic forms. This is illustrated in Figure 13 and illustrates * 1tet4*Tt.y sAtcr/tettere*. HONS 049001 \ o o- 9 - how the number of chlorine atoms per ion may be determined from a characteristic peak pattern. The assignment of the chlorine atoms per molecule and the molecular weight for compounds in each peak is given in Table 3 and the number of Cl atoms associated with each peak is illustrated in Figures 7-9. From the results obtained there can be no doubt that the interference encountered in the determination of organochlorine insecticides was due to polychlorinated biphenyls. A comparison of the relative retention values of Arochlor 1254, heron egg and herring oil extract and some organochlorine insecticides is given in Table 4 and this illustrates that with SE 30 it is virt ually impossible to separate the insecticides of the DDT group from some of the components of Arochlor 1254. The separation of Arochlor 1254 from the less polar insecticides such as aldrin and pp'-DDE is also not possible by the technique of thin layer adsorption chromatography, and column chromatography em ploying adsorption is not likely to prove any more helpful. However, the determination of organochlorine residues in environmental samples requires that some separation procedure is employed prior to gas- liquid chromatography. The routine analysis of samples in Tunstall Laboratory makes use of the difference in polarity between compounds containing the oxirane group, heptachlor epoxide, HEOD and endrin and the non-oxygenated chlorine compounds to separate the two groups of compounds (See Appendix). Since the non-oxygenated compounds are eluted in the first fraction by hexane there is no interference in the determination of oxygenated chlorine insecticides by the poly chlorinated biphenyls. This is not the case in all analytical tech niques for the determination of organochlorine insecticides, for example, that of the Government Chemists (13) where all the insecti cides are eluted in one fraction. Although different stationery phases are then used to overcome this difficulty they are not completely success*fu,l(14) The occurrence of chlorinated biphenyls in the environment raises questions as to the source of such pollution, how long this pollution HONS 049002 (..) ) - 10 - has existed, its relationship to changes in particular ecosystems and the possibility of pollution by other chlorinated compounds. The causes of pollution are less obvious but it is possible that direct discharge into the environment occurs directly into the atmos phere as in jet turbine lubrication, or directly into rivers by in dustrial waste disposal. Once in the environment the polychlorinated biphenyls are biologically very hard and will persist for some years. Polychlorinated biphenyl pollution has not been detected or inferred prior to 1963 but it would appear likely that it has occurred previously. If this pollution had been recognised earlier it is likely that there would not have been the pressure against the DDT group of insecticides. It seems very likely that with the analytical pro cedures employed for organochlorine insecticide determination at that time any interference in the region of DDT would be reported as DDT and hence in some of the predatory birds particularly fish eating birds they appeared to have high organochlorine insecticide levels. The toxicity of polychlorinated biphenyls has been studied with respect to rat and guinea-pigs^*^. The compounds studied would appear to be at least as toxic as DDT. Also Risebrough, et al^^ state that these compounds are more powerful inducers of hepatic microsomal ensymes than is DDT. Since polychlorinated biphenyls occur in the environment it is to be expected that compounds with similar industrial usage will . also occur. These include the chlorinated paraffins and the chlorin ated naphthalenes. The chlorinated naphthalenes also interfere with the determination of organochlorine insecticides. MQNS 049003 APPENDIX 1 The determination of organochlorine insecticides in animal products The method described is suitable for the determination of mixed insecticide residues in animal products. It is suitable for the determination of yBHC, HHDN, heptachlor, pp*-DDE, pp'-TDE, pp'-DDT, heptachlor epoxide, endrin and HEOD at concentrations of one part per hundred million in animal tissues, fats, oils and eggs. The pro cedure consists of extraction with hexane or hexane/acetone followed by clean-up with dimethylformamide., hexane partition, liquid solid chromatography on Florisil and determination of the chlorinated insecti cides by gaa liquid chromatography with electron capture detection. Method Apparatus Separating funnels Volumetric flasks: Graduated stoppered cylinders Soxhlet extraction apparatus Filter funnel Chromatographic column: High speed homogeniser Centrifuge Beakers 100 ml and 250 ml and 1 litre capacity. 100 ml, 50 ml, 25 ml, 20 ml and 10 ml capacity. 25 ml capacity. 4" short stem (for buffer). The column consists of thick wall glass tubing 55 cm long x 0,5 cm bore. A female S29 ball joint is fused at the bottom. The outlet of the column is made from a femal S13 joint. The top of the column is connected to a S29 male joint which fits to an air line. MONS 049004 - 12 - o. Reagents Sodium eulphatet anhydrous granular. General purpose reagent Wash with hexane prior to use. - Hexane petroleum fraction SBF 60-70 Redistill and collect fraction boiling between 64 and 66C. Acetone. General purpose reagent, redistilled. Sand. Horticultural Sharp sand. Hexane washed. Diethyl ether. Analytical reagent. Florisil 60-100 mesh. Activate by heating overnight at 120C. Deactivate by addition of 3 v/w distilled water into conical flash stopper and shake for 30 minutes. Distilled water. Potassium oxalate. General purpose reagent. Methanol. Analytical reagent grade. All reagents and apparatus should be examined by gas liquid chromatography of a hexane extract to ensure freedom from contamination. Extraction Animal Fats, muscle tissue, kidney, liver and brain. Freese sample by storing in polystyrene box with Cardice. With sharp knife chop up frozen sample, mix well and weigh 4 g into a 250 ml beaker. Add 10 g of sand and grind with a heavy glass rod with flattened end, add sufficient sodium sulphate and grind again to give a dry uniform granular mass. Warm the ground material with successively 50, 20, and then 20 ml portions of hexane on a steam bath until it is gently boiling, stir carefully, decant the solution through a Whatman Ho. 1 filter paper into a 100 ml volumetric flask and carefully transfer the solid to the filter paper. Wash the beaker, filter paper and its contents with a further 10 ml warm hexane, cool the flask and its contents to 20C and make up to the mark with hexane. Treat a 25 ml solution of this by DMF partition. Butter. Dissolve 1 g of clarified butter in 10 ml hexane. Trans fer to 100 ml separating funnel using 15 ml hexane for transfer and washing. Carry out DMF partition on this solution. MONS 049005 Errs. If the eggs are fluid incorporate sufficient sodium sulphate to give a granular mass. If the contents are dehydrated and solid grind them with sand and sodium sulphate. Transfer the granular mass to a suitable extraction thimble and extract for 26 hours in a Soxhlet extractor with acetone - hexane (1 + 2). - Cool the extract, filter through a filter paper containing sodium sulphate into a concentrator filter with a tube. Concentrate to a small volume and transfer to 100 ml flask. Make up to volume with hexane. Take the volume equivalent of 1 g weight for DMF partition. Fish Oil. Dissolve 4 g of fish oil in hexane in graduated 100 ml flask and make up to volume with hexane. Take 25 ml for DMF partition. Milk. Weigh 100 g milk into a 250 ml beaker. Transfer the sample to a 1 litre separating funnel. Rinse the beaker several times . with water and add the water washings to the milk. Add 10 ml of 5% aqueous potassium oxalate solution and mix. Add 200 ml diethyl ether and shake for 2 minutes. Add 100 ml redistilled hexane and shake for one minute. Allow the mixture to stand for ten minutes discard the lower layer, stand for a further minute and discard the remaining aqueous layer. Filter the solvent layer through a 4" funnel plugged with .glass wool which is covered with granular sodium sulphate into a 600 ml beaker. Wash the separating funnel with three volumes of hexane 15 ml, and filter into the 600 ml beaker. Evaporate to dryness, dissolve the residue in 10 ml hexane and transfer to 100 ml volumetric flask. Make up to volume with hexane. Add 10 ml of this solution and 15 ml hexane to e 100 ml separating funnel for the DMF partition procedure. Liquid-liquid partition process Place 10 ml of DMF in a 100 ml separating funnel. Allow a little of this to run through into the lower 100 ml separating funnel such that both taps are lubricated by clean DMF. This ensures that any leaking taps can be discarded without loss of sample. Place 25 ml MQNS 049006 o ) - 14 - of the hexane extract into the upper 100 ml separating funnel, shake vigorously for 1 minute, allow the layers to separate and run off the DMF phase into the lower 100 ml separating funnel. Shake the hexane extract with two further 10 ml of DMF and add these to the first DMF extract. Discard the hexane phase. Vash the combined extracts with 10 ml of hexane saturated with DMF and run off the DMF layer into a 250 ml separating funnel. Shake the aqueous sodium sulphate/DMF/hexane mixture and allow to settle for 40 minutes. Run off the aqueous phase and wash the hexane with two further 20 ml of distilled water. Dry the hexane extract by using a small quantity of sodium sulphate before the next clean up procedure. Liquid-solid chromatography Using a plug of hexane washed cotton wool as a support add to the column 3 g of Florisil and top this with 1 cm of sodium sulphate. Run on to the column the hexane solution obtained from the DMF partition and wash in with a little hexane. Using a 25 ml graduated flask as a receiver elute with hexane to a volume of 20 ml. Replace the receiver with another 25 ml flask and elute with 25 ml of 102 diethyl ether in hexane. These two eluates are examined by gas-liquid chromatography. The first fraction will contain any yBHC, heptaohlor, HHDN, pp'-DDE, pp'-DDD and ppf-DDT which may be present in the original extract. The second fraction contains any heptachlor epoxide, HEOD and endrin which may be present. Gas-liquid chromatography Instrument conditions for 1st fraction: Column: 1 m x 3 ton i.d. all glass Column packing: 3.81 SE30 on Diatoport S 80-100 mesh Carrier gas: Inlet pressure: N2, oxygen free 1.75 kg/em2 Detector: Electron capture Temperature: MONS 049007 - 15 - o Instrument conditions for 2nd fraction: Column: 1*5 1x3m i.d. all glass Column packing IX Oronite polybutene + 0.2X Epikote 1001 on Celite 85-100 mesh Carrier gas: Inlet pressure N^, oxygen free 1.75 kg/cm2 Detector: Electron capture Temperature: 1B4C Standard solutions: Standard 1 (a) A hexane solution containing 0.004 y/ml yBHC, 0.004 y/ral HHDN and 0.04 y/ml pp'-DDT. Standard 1 (b) A hexane solution containing 0.004 y/ml heptachlor 0.02 y/ml pp'-DDE and 0.02 y/ml pp'-DDD. Standard 2 A hexane solution containing 0.01 y/ml heptachlor epoxide, 0.02 y/ml HEOD and 0.02 y/ml endrin. Method Inject 40 pi of sample into the appropriate column and record the chromatogram. The sample then may be diluted or concentrated to give a peak of the same height as in appropriate standard. Due to the decrease in sensitivity of the detector during pro longed running it is impracticable to construct a calibration curve for the estimation of insecticide present. To overcome this difficulty it is essential to inject a standard after every two samples and to mean the two standard peak heights for calculating the concentration of the insecticide in the egg samples between the standard samples. MONS 049000 { O - 16 - I 0 ' Calculation: Let mass of egg Initial volume of hexane containing W g - wg - 100 ml Volume taken for clean up - 5 ml Final volume after clean up - Z ml (20 or 25 ml) Volume of standard * volume of sample injected - 40 ill Concentration of standard " C Tf/ml Dilution or concentration of final volume -D 1 Mean peak height of standard at " PS cm Peak height of sample at Hy ' PE c" Then ppm of each component - PgXlOOxZxDxC ppm Pg x W x 4 x Z HUNS 049009 \0 - 17 - REFERENCES 1. Lovelock, J., (1961), Nature, 189, 729. 2V Roburn, J., (1965), Analyst, 90, 467. 3. Eidelman, M., (1963), J. Ass. Off. Agric. Chem., 46, 182. 4. Harrison, R. B.v (1966), J, Sci, Fd. Agric., ^7, 10. 5. Jensen, S., (1966), u New Scientist, ^2, 612. . 6. Holmes, D. C., Simoons, J. H. and Tatton, J. O'G., (1967), * Nature, 216, 227. 7.y Holden, A. V. and Marsden, K., (1967), Nature, 216, 1274. 8. Robinson, J. 9. Koeman, J., (in press) 10. / Risebrough, R. W., et al, (1968), Nature, 220, 1098. , 11. Quayle, A., (1956), Thornton Research Report R.722. 12. Weingarten, H,, et al. (1962), Analytics Chim. Acta., _26, 391. ` MGNS 049010 - 18 - -J 13. / de Faubert Maunder, M, J., et al, (1964), Analyst, JJ9, 168. 14. Simmons, J. H., and Tatton, J. O'G., (1967), J. Chromatography, 27, 253. 15. Treon, J. F., et al, (1956), American Industrial Hygiene Assoc. Quarterly, 17:2 , 204. MONS 049011 19 - V\ i) Table t - Assignment of molecular weight and number of chlorine atoms per molecule to components separated bv gas-1iould chromatography Peak No. R.R.V. DOE 1.0 Aroclor 1254 1 0.42 2 0.50 3 0.53 4 0.70 5 0.82 6 1.00 7 1.07 8 1.30 9 1.58 10 1.85 M 2.25 Fish Oil 1 2 3 4 5 6 7 8 9 10 11 0.42 0.50 0.53 0.70 0.82 1.00 1.07 1.30 1.58 K85 2.22 Heron Eoa 1 2 3 4 5. 6 7 8 9 10 II 12 13 0.35 0.45 0.69 0.81 1.00 I.30 1-52 1.84 2.05 2.42 2.76 3.07 3-75 No. of Cl2 atoms/mol 3 and 4 4 5 5 5 3 and 6 5 and 6 5 and 6 6 6 6 and 7 4 N.D. 5 5 4 5 6 6 6 7 7 ; 3 4 4 and 5 4 5 6 and 5 and 4 6 and 5 6 6 7 and 6 and 5 8 and 7 and 6 7 and 6 and 5 7 and 6 and 5 N.D. Not determined. Molecular weight 256 - 290 290 324 324 324 N.D. N.D. N.D. N.D. N.D. N.D. 290 N.D. 324 324 316 - DDE 324 N.D. 358 N.D. 392 392 256 290 324 316 - DDE 324 358 N.D. N.D. N.D. N.D. N.D. N.D. N.D. Sflo MUNS 04901,; Table 2 - Rf values of Aroclor I25k oroanochlorine insecticides and heron ego extract Solvent: Hexane ' Adsorbent Alumina Type E (Merck) Aroclor 1254 Heron Egg pp'-DDE pp'-DDT HHDN Heptachlor 7BHC Endrln HE00 Rf 0.63 0.55 - 0.76 (Streak) 0.62 0.50 0.67 0.59 0.26 0.20 0.15 Table 3 - Relative retention volumes DDE 1.0 of Aroclor 1254* Heron eoa. Herrlno 0M and some oroanochlorine insecticide Microcoulometrlc Detection Column: Mt x 3.9% SE30 on DIatoport S. . T - 190C 7BHC HHDN Heptachlor HE0D pp'-DDE Endrln pp'-TDE pp'-DDT 0.27 0.45 0.55 0.96 1.00 1.09 1.28 1.77 Aroclor 125^ 0.50 0.57 0.75 0.95 1.07 1.22 1.58 1.70 . Fish Oil 0.40 0.54 0.72 0.79 1.00 1.25 1.45 1.71 2.02 2.28 Heron Egg 0.36 0.51 0.61 0.85 1.00 1.2.3 1.40 1.76 2.02 HONS 049013 \ O i i Fig 1 Structural Formulae of Nine Chlorinated Hydrocarbon Pesticides i HONS 049014 ODE HONS 049015 Fig 4 G.l.C.-E.C. Chromatogram Aroclor 1254 MONS 04901b 0 f VJ DDE Fig 6 G.L.C.-M.C. Chromatogram Heron egg extract Fig 6 G.L.C.-M.C. Chromatogram Herring oil extract HONS 049017 ; \ i I I. MOWS 049018 MONS 0 4 9 0 1 9 Fig 8 Haron egg F.I.D.-M.S. split Fig 9 Herring oil F.I.D.-M.S. split Fig 10 Aroclor 1254 F.I.D.-M.S. split Heron egg meee spectrum of peek 2 (SCI) Fig 11 Mass Spectrum of Heron egg HONS 049020 \0 ) Herring oil man spectrum ol peak 4 I Fig 12 Mass Spectrum of Herring oil MONS 049021 Arochlor 1254 mass spectrum of psak 2 288 (40) 324 (50) Arochlor 1254 mass spectrum of peak 9 Fig 13 Mass Spectrum of Aroclor 1254 MQNS 049022