Analysis of polychlorinated terphenyls in marine samples

~

' Pergamon

Chemosphere,Vol.36, No. 14, pp. 2941-2948, 1998 © 1998ElsevierScienceLtd All rightsreserved.Printedin GreatBritain 0045-6535/98$19.00+0.00 PII: S0045-6535(97) 10250-8

ANALYSIS O F POLYCHLORINATED TERPHENYLS IN MARINE SAMPLES

M.A. Fernindez 1, L.M. Hernindez t, M'.J. Gonzilez l, E. Eljarrat~, J. Caixach 2 and J Rivera 2. 1 Department of Instrumental Analysis and Environmental Chemistry, Institute of Organic Chemistry, C.S.I.C. C/Juan de la Cierva, 3. 28006-Madrid, Spain. 2 Laboratory of Mass Spectrometry, C.I.D.-C.S.I.C. C/Jordi Girona, 18-26.08034-Barcelona, Spain. (Receivedin Germany27 Ocu3ber1997;accepted1 December1997)

ABSTRACT Polychlorinated terphenyls (PCTs) are compounds related to Polychlorinated biphenyls (PCBs), because of their physical and chemical properties. They have been used for similar applications, although to a lower degree. As a consequence, PCTs have been sprayed in the environment. In this paper, we have analyzed some samples of marine origin that are part of the spanish diet, purchased in municipal markets of Madrid. Levels found range between 146.6 ng/g to 32,966.8 ng/g (fat weight). The analytical technique employed is HRGC using ECD for quantification and HRMS (SIM) for correct identification due to the large number of peaks. ©1998 ElsevierScienceLtd. All rightsreserved

INTRODUCTION Polychlorinated terphenyls (PCTs) are a family of compunds with similar characteristics to polychlorinated biphenyls (PCBs). PCTs have a basic structure of three benzene rings linked among them, adopting three possible positions (ortho, meta and para) and a number of chlorosubstitutions ranging between 1 and 14 chlorine atoms. So, the possible number of different expected congeners is higher than the theoretical 209 PCB congeners. They have an anthropogenic origin and have been produced under different commercial names: Aroclor (Monsanto Co., USA), Kanechlor C (Japan), Leromoli and Clophen (Bayer, Germany), CloresiU (Caffaro, Italy), Electrophenyl T-60 and Phenoclor (Prodelec, France) and Terphenyl Chlore T-60 (unknown). The main manufacturer of polychlorinated terphenyls was the Monsanto Co., that between 1959 and 1972 produced 114.54 x 103 Ibs in a plant in Anniston (Ala, USA)(1). In 1972 the production of PCTs ceased in USA and, from this year, PCTs were imported into the country from other producers, mainly France. The last documented importation of PCTs into the United States occurred in 1979(2). In addition, these chemicals have been marketed commercially under different degrees of chlorination. In the case of Aroclor, they were designated by three different numbers (5432, 5442 and 5460) which contained 32, 42 and 60 % of chlorine by weight, Aroclor~->5460 being one of the most extensively used. 2941

2942 Due to their high stability, the polychlorinated terphenyls have been applied in hydraulic fluids, electrical equipment, plasticizers, paints, sealants, adhesives and investment casting waxes because of their desirable electrical and flame-retardant properties (3). Due to their chemical structure, similar to PCBs, but with higher level of chlorination, they have high resistance to degradation and, therefore, they are capable of accumulating both in living organisms and in food chains, The PCTs have been detected in various environmental samples, such as water (4,5), soils and sediments (5,6,7,8), oysters (4,6,8,9), mussels (10), clams (10), crabs (9), eels (4), estuarine fishes (9), cod (11), herring gulls (12), hen's eggs (13), and humans (14) and due to the similarity to PCBs, fish consumption may be the major path of human exposure. The aims of this paper are the analysis of biological marine samples for polychlorinated terphenyls, evaluating the levels and comparing their toxicological significance, measuring the percentage composition of homologues and comparing them with the results obtained by other authors working with similar samples.

MATERIAL AND METHODS

(Tapes decusata), mussels (Mytilus edulis), bonito (white tuna) (Thunnus thynnus) and salmon (Salmo salar)~ purchased in For covering the objectives, we obtained of several samples of commercial clams

municipal markets in Madrid (Spain). Clams and mussels, were homogenized and lyophilized, whilst the bonito and salmon were mixed and homogenized with granulated anhydrous sodium sulphate 12-60 mesh (J.T. Baker, Deventer, Holland). All the completely dry samples were introduced to a column (30 c m x 2,5 cm i.d.) filled with several layers of anhydrous sodium sulphate 12-60 mesh, activated silica 70-230 mesh (Merck, Darmstadt, Germany), silica 70-230 mesh modified with H2SO4 40% (Merck, Darmstadt. Germany)(w/v), silica 70-230 mesh modified with KOH (33%) (Quimicen, Madrid, Spain) and a top layer of anhydrous sodium sulphate 12-60 mesh. The samples were extracted with 330 ml of a mixture of cyclohexane (Promochem, Wessel, Germany) and dichloromethane (Merck, Darmstadt, Germany)(80:20; v/v). The purification of the extract was carried out on a column filled with Florisil~"~(Floridin Company, Florida, USA) activated at 450°C during 24 h and kept at 135°C for at least 18 h, using 20 ml of 1% of dichloromethane (Merck, Darmstadt, Germany) in n-hexane (Fisher Scientific, New Jersey, USA) as solvent. The identification and percentage composition of the homologes of the polychlorinated terphenyls were made by mass spectrometry HRGC/HRMS (SIM). The quantification was carried out by HRGC/ECD comparing the sum of the areas of some selected peaks in the standard Aroclor r~ 5460 (Alltech Associated, Inc., Deerfield, IL, USA), composed of isomers containing between 7 and 11 atoms of chlorine, with the sum of the same peaks in the sample. The chromatographic analysis was made in a chromatograph Hewlett-Packard (Palo Alto, CA, USA) 5890 Series I1 equipped with an 63Ni-ECD and a capillary column of 30 m length, 0.32 mm of inner diameter and 0.25~tm of DB-5 (J& W Scientific, Folsom, CA, USA) film thickness. The conditions of working were the following: initial temperature of 60°C maintained for 0.6 min and a first ramp rate of 20°C/min up to a

2943 second temperature of 200°C, kept 1 min and a second ramp rate of 4°C/min to 240°C as third temperature, held during 3Q'min and afterwards raised at 8*C/min as third ramp rate to 280°C. This forth temperature was maintained during 50 min more. The injector and detector temperatures were 280°C and 300°C, respectively. Time of splitless was 0.6 min and N2 was the cartier gas. All the chromatographic data were acquired and analysed using the System Gold software from Beckman TM (Fullerton, CA, USA). The mass spectrometry was carried out in a gas chromatograph Fisons series 8000 coupled to a mass spectrometer AUTOSPEC-ULTIMA (VG Instruments, Manchester, UK). The chromatograph mounted a capillary column ofDB-5 (J&W Scientific, Folsom, CA, USA) of 60 m length and 0.25 mm of inner diameter and a film of 0.25 Jam. The carrier gas was He at a linear velocity of 25 crn/s. The program of temperatures and time conditions of chromatograph were: temperature 1, 90°C (during 3 min); ramp rate 1, 20°C/min; temperature 2, 200°C (during 1 min); ramp rate 2, 4°C/min; temperature 3, 310°C (during 60 min). The temperature of the injector was 280°C. For the High Resolution Mass Spectrometer (HRMS) operating in the selected ion monitoring mode (SIM) these were the conditions: temperature of ion source: 250°C; temperature of interface: 280°C; ionisation energy: 39 eV (electron impact mode); power of resolution: 20,000-25,000 (10% valley definition); voltage acceleration: 8,000 V; dwell time: 50 ms; monitorized ions: 435.8728/437.8698

(HexaCTs),

469.8338/471.8308

(HeptaCTs),

503.7948/505.7919

(OctaCTs),

537.7558/539.7529 (NonaCTs), 573.7139/575.7110 (DecaCTs) and 607.6750/609.6720 (UndecaCTs); lock mass: m/z 454.9728 (Pentafluorokerosene, PFK). All solvents used in this work were of analysis of residues quality and all glassware was thoroughly rinsed with a battery of solvents.

RESULTS AND DISCUSSION

In table I appear the data belonging to the levels of polychlorinated terphenyls found in all samples analysed, expressed in ng/g wet weight and in fat weight. As can be deduced from these data, the maximum level corresponds to the bonito, with 32,966.8 ng/g fat weight (the percentage of fat on fresh weight was 2% for bonito and 15-16% for salmon), but a further two samples exhibited much higher concentrations, than ten thousands, exactly 13,428.8 and 16,228.9 ng/g, respectively. One clam showed 1,309.0 and another 1,702.2 ng/g f.w. It is necessary to take in account the low levels of fat in mussels and clams (1,3% and 0,8%, respectively) in order to interpretate the data. Referred to fresh weight, all of the levels found were lower than 260 ng/g. Thanks to the coupled HRGC/HRMS technique we have been able to study more carefully the composition of each sample concerning the percentage of each isomer. In this sense, we have examined the proportion of each isomer in the samples and in the standard of reference, Aroclor 5460.

2944

Table I. Levels of PCTs in all the samples analysed, expressed in ng/g. ilil)iiil) iiliZ i i iii i?)i~i~ii!i!iiii!i!i!i!iiii!iiiiiiii ::i::i::i::i::i::!:;i :;:i:.ii;:iiii::i::i:.i::i::iii~ ~i:::i:i:i:~;:i~!~/ii i~/i~ii ii]i)ii 534.3-1,039.0 9.84-17.64

~i~

))i

124_2419

146.6-3,581.5

633%2594

499.0-32,966.8

21.05-64.24

182.9-487.8

The figure 1 shows the chromatogram corresponding to a standard mix of Aroclor 5460 and a sample of bonito in which the profile of PCTs is clear, appearing at 35 rain approximately, after that of the polychlorinated biphenyls. There is a very close relation between the two chromatograms explaining the choice of this standard for comparison. Though the properties of PCTs are so close to PCBs, the gas-chromatographic separation is complicated by two main reasons: on one hand the number of homologues and congeners is higher than those of PCBs and, therefore, this is translated in a complex mixture of peaks; on the other hand, due to the higher weight and boiling points of the higher chlorinated congeners, the elution on gas-chromatography is delayed, with long retention times. The number of papers that have appeared in the literature on PCTs is very low: however, improved techniques have been applied over the years. Initial investigations on the analysis of PCTs used classic packed columns (4, 5, 11, 12, 15). The introduction of the capillary columns for gaschromatography in the analysis of PCTs brought as consequence the appearance of analyte peaks, although a great part of these were unresolved. For overcomming this notorious complexity, the best solution has been the recent application of high thermal stability stationary phases in capillary columns and the coupling of GC with MS (6, 10, 16), using the Selected Ion Monitoring (SIM) mode. In addition, the low chlorinated homologes of PCTs can interfere with high chlorinated PCBs. To solve this drawback, HRGC/HRMS technique is the best weapon, that permits working at a resolving power of 20,000-25,000. In figure 2 we can observe the ffagmentograms obtained from a bonito sample for isomers with 6 to 11 chlorine atoms by HRGC/HRMS working at a resolution power of 25,000. This high resolution overcomes the problems during the ionisation process in which the PCTs lose 2 CI atoms for each ion cluster which give a signal at low resolution with the subsequent disturbing effect. The main families of congeners detected have been those that appear in table I1. As can be seen, the percentage distribution of isomers for mussels, salmons and bonitos have followed the order: Octa-CTs :> Nona-CTs > Hepta-CTs > Deca-CTs > Undeca-CTs > Hexa-CTs. However, for the clams, this order were slightly different: Nona-CTs > Octa-CTs > Deca-CTs > Hepta-CTs > Undeca-CTs > Hexa-CTs. Therefore, the percentage isomerical composition is distributed exactly the same for clams as found in Aroclor 5460,

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(bottom) obtained by H R G C / E C D

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5460 (top) and a sample of bonito

.;.,, ,;.,,, ,.,,,,,, ,,,,,, .,,,

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Aroclor

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Fig. l.- Chromatograms of

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sample of bonito by HRGC/HRMS at a resolution power of 25,000.

Fig 2.- Fragmentograms o f Hexa-CTs to Undeca-CTs obtained from a

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2946 but it is similar in the other three types of samples. All this confirms the close relation between the standard Aroclor 5460 and the profiles found out in the environmental samples. These organisms studied are part of the common diet in Spain. It is therefore important to evaluate the possible influence of the consumption of this marine food.

Table II. Comparison of the percentages of most representative isomers of Polychlorinated Terphenyls between the standard Aroclor 5460 and the different samples, obtained by HRGC/HRMS (S.I.M.)

~ T s

:

"

-

........

1.2

-

0.2

0.3

7.8

10.4

4.1

15.7

15.1

28.8

44.2

32.9

48.4

42.3

i

-N~n~CTs : -

42.3

"

339

40.1

28.8

296

"i:: ~ c a - C T s

177

'

8.3

19.3

6.2

9.5

2.0

3.5

0.5

2.4

r

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{UNde¢~e~:

3.2

There is not a specific rule or guideline for PCTs in Spain (only, one that limits the maximum admissible concentration for PCBs and PCTs in waters destined for human consumption, established at a level of 0,5 g/L by C.E.E. in 1980 (17)) and few or very. few in the rest of the world. However, some countries have given maximum levels for PCBs in fish~ crustaceans, shellfish or their derivatives fir for human comsumption In the USA (18), this maximum level is situated in 2 ~tg/g of PCBs in fish, crustaceans or shellfish ; in Canada, the objective of quality in Ontario (19) for PCBs in fish is 2 p.g/g ; in Belgium (20), this limit is situated at 3,0 p.g/g fat weight; and 10 !ug/g is the set in Finland (21) for PCBs in cod liver oil. As can be deduced, assuming the lack of rules for PCTs, we conclude, that the concentrations quantified in this paper do not imply risks for the population, because has been estimated (22) that the induction and toxicological effects appear at very. high doses (5,000 Mg/g), that is, ten fold less than for PCBs mixtures. There is little known about the biotransformation of PCTs. Addison et al. (1 I) noted that PCTs had a short retention time in the excreta of a cod dosed orally with Aroclor 5460. Sosa-Lucero et al. (23) observed that a similar loss in rat faeces after the administration of a diet containing Arodor 5460. Gallagher et al. (24) found that the PCTs injected to fish (Aroclor 5432 and 5460) provoked an induction of the P4501A and EROD enzymes at 32 mg/kg level of Aroclor 5432, but did not induce these enzymes significantly with Aroclor 5460. There is not much information about the toxicological effects caused by the polychlorinated terphenyls. However, a significant liver enlargement has been noted at level of 1,000 mg/kg in rats (23) Theoretically, the ortho or meta isomers of PCTs might still fit the TCDD receptor if they contained a

2947 biphenyl moiety chlorinated in the appropriated meta and para positions. However, the biological effects of PCT isomers is very complex, because of the large number of possible isomers, the relatively low toxicity of PCT mixtures compared to PCBs, and the decrease in the usage of PCTs. The pathways of human exposure to PCTs appear to resemble those of PCBs. As PCTs have been detected and quantified in food of marine origin, and by analogy with PCBs, fish consumption may be a main route of PCT exposure. Up to this moment, no episodes of high-dose exposures of populations to PCTs have been reported.

Table IlL Comparison of the PCT levels found in this study and those obtained by other authors in similar species, expressed in fat weight (f.w.), wet weight (w.w.) and dry weight (d.w.). ~z::::i:::::?.:.::::::::::::::::::::::::::: 1~i} i~

....

.............. :

::i 0.2-0.5 mg/kg ([w.)

Holland

4

6-210 lag/kg (w.w.)

Holland

25

10-790 ng/g (d.w.)

Spain

10

3-58 ng/g (h.w.)

Spain

26

3-118 ng/g (d.w.)

Spain

10

o.o8-o.15 gg/g (fw.)

U.S.A.

6

U.S.A.

6

3 gg/kg (w.w.)

Holland

25

47-85 ng/g (d.w.)

Spain

t s.

9-97 ng/g (d.w.)

Spain

t s.

183-488 ng/g (f.w.)

Spain

ts.

499-32,967 ng/g (f.w.)

Spain

t.s.

......... ............................................................ a . vn nv_ ~~. a~vv n n ~tg/kg ~ ,tw.w3 :|

:5:555::::5:::5:::55,:2

5 :.

:::5:5

:

25:

t.s.: this study.

The table III shows the limits for the values found out in the samples analysed in this study and those contributed by several authors in similar marine species. In general, we can say that our results are of a similar order. The data from salmon and, especially, the highest value for the bonito, are clearly higher than the rest of levels, although these were measured in fat weight. In this sense, the quantities of PCTs in oysters from USA found by Hale et al. (6) were nearly 1,000 fold higher than we have observed in our samples

Bibliography

I R. D. Kimbrough (Editor), Halogenated bipheny&, terphenyls, naphtalenes, dibenzodioxins and related

products', Elsevier/North-Holland Biomedical Press, Amsterdam, 1980.

2948 2. Fed. Regist. 4_.99:11181-11184(1984).

3. Jensen, A.A. and Jorgensen, K.F. Sci. Total Environ. 22:231-250 (1983). 4. Freudenthal, J. and P.A. Greeve. Bull. Environ. Contain. Toxicol. 1__00:108-111(1973). 5. Stratton, C.L. and Soseebe, B. Jr. Environ. Sci. and Technol. 1Q0:1229-1233 (1976). 6. Hale, R.C, Greaves, J., Gallagher, K. and Vadas, G.G. Environ. Sci. and Technol. 24:1727-1731 (1990) 7. Risebrough, R.W., de Lappe, B.W. and Youghans-Hang, C. Mar. Pollut, Bull. 21:523-529 (1990). 8. Hale, R.C., Greaves, J., Vadas, GG., Harvey, E., Gallagher, K., Mayer, M.A., Barron, M.G. (eds.). Aquatic Toxicology and Risk Assessment: l%urteenth Volume., American Society for Testing and

Materials, Philadelphia, PA (USA), pp: 305-312, (1991 ). 9. Gallagher, K., Hale, R C , Greaves, J., Bush, ED. and Stilwell, DA. Ecotoxicol. Environ. Saf 2_66:302312 (1993). 10 Galcer~in, MT., Santos, F.J., Caixach, J., Ventura, F. and Rivera, J. J. Chromatog. 643:399-408 (1993). 11. Addison, R.F., Fletcher, GL., Ray, S. and Doane, J. Bull. Environ. Contain. Toxicol. 8:52-60 (1972). 12. Zitko, V., Hutzinger, O., Jamieson, WD. and Choi, P.MK. Bull. Environ. (?ontam. Toxicol. _7:200-201 (1972). 13. Jan, J and Josipovic, D. ('hemo,~phere, _7:863-866 (1978). 14. Doguchi, M. Ecotoxicol. Envrion. Saf i: 239-248 (1977). 15. Putnam, T.B., Gulan, MP., Bills, D.D. and Libbey, L M Bull. Environ. (7ontam. Toxicol. 1_!: 309-311 (1974). 16. Caixach, J., Rivera, J, Galceran, M.T. and Santos, F.J..1. ('hromatog. 675:205-211 (1994). 17 C . E E Daily of the Communities, N° -L-299/1 I. (1980). 18. F.D.A. Food and Drug Administration. Federal Register, vol. 49, no 100, 21514-21520, 22.4.1984 (Rectification: ibid. N °. 118, 24892, 18.6. 1984), USA, (1984). 19. M.O.E Ministry of the Environment of Canada, Guide to eating Ontario fish. Canada (1992). 20. Belgium. Reglament du 22 .lanvier 1985 relat~faux residues biologiques. Texte n° 9 de 1985 (1985). 21. Finland. Finlands Forfattningssamling, 15 of november 1984, n°. 759-763, 1528-1538 (1984). 22 Jensen, A.A. and Jorgensen, KF. Sci. "IotalEnviron. 2 7 231-250 (1983). 23. Sosa-Lucero, J., de la Iglesia, F.A., and Thomas, G H Bull. Environ. Contain. "l'oxicol. 1 0 248-255 (1973). 24. Gallagher, K., Van Veld, P.A., Hale, R.C. and Stegeman, J.J. Environ. 7bxicol. (7hem. L4:405-409 (1995). 25. Westir, P.G and J. de Boer. Dioxin '93. vol. 14, Viena, 1993, pp. 121. 26. ~dvarez Pifieiro, M.E, Lage Yusty, M.A, Simal Lozano, J. and Carril Gonz~ilez-Barros, S.T. Toxicol. l~'nviron. (7hem. 5 ~ 3 1 - 3 6 (1996).