Document Z40YqYROXM8yQKoZ159No6wX0

Sent by: MOTE MARINE LABORATORY 9801 BLIND PASS ROAD Sarasota, tlqrioa ss&bi, u.s.a PCB PHARMACODYNAMICS IN THE RING DOVE AND EARLY GAS CHROMATOGRAPHIC PEAK. DIMINUTION Jeffrey L. Linger* <fc David B. Peakall Section of Ecology & Systematic, Langmuir Laboratory, Cornell University, Ithaca, New York 14850, USA ABSTRACT Ring Doves (Streptopclia risoria) were maintained on a daily dietary dose of 10 ppm of the polychlorinated biphenyl (PCB), Aroclor 1254. Sequential sampling and residue analysis ofeggs indicated a linear increase in PCB levels until approximately 105 days, after which an asymptote was reached at 80 ppm, oven dried weight (OD). At equilibrium PCB residues {ppm, OD) were, as follows: fat 797, liver 51-8, muscle 28-1, brain 25-4, faeces 2-27. At death, following starvation stress, residue levels increased by the following factors: liver 78, muscle 25, brain 56, faeces 5-5. No differential toxicity attributable to PCB mobilisation h'qj observed in experimental.1: compared with controls. Experimental birds tended to lose relatively more weight than the controls. The high levels in the livers of our starvation-stressed birds com pared with those found in the livers of seabirds involved in the Irish Sea kill suggests that PCB .,ui ><o< .he prUnuryfactor in their death. Early gas-liquid chromatography peaks"showed considerable diminution in all samples compared with Aroclor 1254 standard and this was particularly marked in muscle andfat samples. INTRODUCTION The widespread distribution of polychlorinated biphenyls (PCB) is well known. Their properties, uses, distribution, toxicity and the means of detection and quanti fication have been reviewed previously (Peakall & Lincer. 1970; Reynolds, 1971; Risebrough, 1971; Lincer, 1971; Peakall, in press). One of the most important properties of organochlorines is their high lipid-low water solubility. This property, combined with their considerable stability, governs their movements both within the Present address-. Mote Marine Laboratory. 9501, Blind Pass Road. Sarasota, Florida 335R1, USA. . 59 Environ. Collin (4) (1973) pp. 39-68-- C Applied Science Publisher* Lid. England. 1973--Primed m Great Britain HOMS 064112 60 JEFFREY L. UNCER, DAVID B. PLAKALL individual and (he ecosystem. Within the individual this means that the hulk of (he organochlorinc is to be found in the adipose tissue. While stored the PCB is com* paratively inert, but if the fat is mobilised (lien the organochlorinc is released into circulation. Studies with organochlorinc pesticides have shown that release of DDT (Bernard,1966), dieldrin (Stickel et ai, 1969) and hcptachlor (Shekel et ai, 1965) caused by reduced food intake can be fatal to birds. PCB, like the organo chlorinc pesticides, is able to accumulate up food chains (sec Pcukall &. Lincur, 1970; Peakall, in press, for reviews). Holdgatc (1971) considered (hat one of the most striking findings in the sea birds found dead in the Irish Sea area in the autumn of 1969 was the unusually high levels of PCB in their livers. This paper is concerned with the distribution, metabolic transformation, storage and excretion of Aroclor 1254 (a PCB supplied by Monsanto) in the Ring Dove In view of the coincidence of high PCB levels and bird die-olFs such as that in the Irish Sea (Iloldgate, 1971), of particular comparative interest are the PCB tissue residues resulting from known dosage and the possible lethal cfTccts contributed by mobilisation of these stored poisons upon starvation stress. Because many metabo lites of pesticides are known to be biologically active (e.g. parathion-paraoxon; DDT-DDE) and PCB undergo in vivo metabolic transformation (Kocman et ai, 1969a; Grant et ai., 1971; Nimmo et ai, 1971) this facet of the problem is also considered. METHODOLOGY For the first experiment, single pairs of doves were maintained in cages 115 x 134 x 229 cm, on 14 h light, 10 h dark photoperiod and al a temperature of 20 1C. Aroclor 1254 was given in pellets of turkey grower mash (Aeway) at a dosace of 10 ppm. For the starvation experiment which followed, five doves were maintained per cage. The birds were fed between 15 and 50 g of food per cage on alternate clays which led to a weight loss of approximately 100^ per week. After three weeks of this regimen the birds were completely starved until death occurred. The objective was to mimic likely field conditions wherein birds obtained an inadequate amount of food and were finally too weak to search for food. The analytical procedure used was a modification by WARF (Wisconsin Alumni Research Foundation) of an earlier one (US Dept, of Health, Education & Welfare. 1970). Specifically, samples were dried for 48 h at 40-45%', ground with a coarse anhydrous Na2S04, placed in Soxhlet extractors and extracted for 8 h with a 1:3 mixture of cth> I ether and petroleum ether. The extracts were concentrated to 50 ml and a 5 ml aliquot was evaporated to dryness at 40-45rC for 2 h to arrive at an extractable fat estimate. Sample clean-up was accomplished with a Florisil column (USDHEW, 1970). As the second (15% ethyl ether in petroleum ether) fraction did not contain any PCB peaks, only the first (6% ethyl ether) fraction was analysed. Determination was HONS 084113 PCB PHARMACODYNAMICS IN THE RING DOVE 61 carried out on a Varian Aerograph model 2100 gas chromatograph equipped with a *JNi electron-capture detector. Two 4 mm (id) x 2 m glass columns were used, the liquid phases and solid supports were: 1 % QF-1 on Chrom. G A/W DMCS 70/80; 2% SE-30 on Chrom. G A/W DMCS 7O/80. Nitrogen gas flow rates of 35 and 150 ntl/min were used, respectively. Injector, column and detector tempera tures were 225', 200 and 285C, respectively. Although the dangers of varied detector response depending on degree of chlorination (Rote & Murphy, 1971) arc recognised, it was felt that, for within-group comparison, quantification based on the combined height of only a percentage of the total number of peaks would be quite adequate. Therefore, peaks 7 and 10 on the QF-1 column and peaks 7 and 8 on the SE-30 column (Fig. 1) were used. MINUTES Fig. 1. Comparison of chromatograms of Aroclor 1254 (above) and extract of muscle from Aroclor 1254-dosed dove (below). (A) J % QF-1 on Chrom. G. A/W DMCS 70/8O. 4 min (id) ' 2 m. injector--238C. column--203:C. detector--281 C. nitrogen (low--30ml/min.(B) I % silicone GE XE-60 on Varaport J30 100/120,2 mm x I -66 m. injector--238'C. column--203 C. detector-- 2Bi*C, nitrogen--25 ml/min. <C) 4% SE-30 + 6% QF-1, Chrom. G ;80.2 mm x 2 m. injector-- 250*C, column--225C. detector--28I'C, nitrogen--90 ml/min. tL>) 2% SE-30 on Chrom. O A/W DMCS 7/80. 4 mm x 2 m. injector--250C. column--225r`C, detector--281#C. nitrogen- 90 ml/min. In an attempt to elucidate PCB metabolism as reflected by chromatographic profiles of various tissues, egg and faeces samples, the relative diminution of the first two major peaks of QF-1 (Fig. 1A) was compared between sample types. A relative diminution value was calculated for each sample as follows: 100 -- [100 x (AIB)]; where A is the ratio of the combined heights of peaks (4 + 6)/(7 + 10) in the sample and B is the same ratio in a similarly-concentrated standard of Aroclor 1254. Since there is evidence for a change in peak ratios over time (Grant el al., 1971), peak diminution data are based on tissues presumed to be at equilibrium. Except in the peak diminution experiment, samples were routinely measured on both columns and the lower of the two results chosen. Results agreed within HONS 084114 62 JEFFREY L. LINCOR, DAVID B. PEAKALL approximately 15 %. Recoveries made in this laboratory ofcommon organochlorincs (e.g. p.p'-DDE, -TDE and -DDT) exceeded 88%. WARF's recovery values for Aroclor 1254 using this technique ranged between 85% and 90,'. (personal communication: D. L. Hughes). No corrections were made for recovery. RESULTS Doves fed 10 ppm Aroclor 1254 usually produced a pair of eggs every ten days if eggs were removed as laid. The sequential sampling and analysis of these pooled eggs indicated a linear increase in residue levels until approximately 105 days after Fie. 2. Aroclor 1254 residues (ppm oven dry weight basis (doited square) and eurucublc fat weight basis (dotted circle)) in dove eggs versus time (each point represents one residue value tor two pooled eggs of a dutch: two points shown as (crossed square) or (crossed arete) were not considered in curve-filling respective curves because they came from a single female which showed predisposition to laying eggs containing inordinately high residues). In the equation t - residues. x - lime. MQNS 084115 PCB PHARMACODYNAMICS IN THE RING DOVE 63 the onset of the experiment. The equations for that increase, based on the oven dry and extractable fat egg sample weiglits (OD and EF, respectively), refer only to the initial linear portions of the curves. After that time an asymptote was reached at approximately 80 ppm (OD) or 240 ppm (EF) (Fig. 2). Extractable fat in eggs was much more consistent within egg samples from the same female than between females. There was no apparent trend in extractable fat from the beginning of the experiment to 200 days, the average standard deviation being 31-7 + 1-18 "/,, for 13 samples (two pooled eggs/sample). Residue levels in tissues, eggs and faeces sampled from doves at this time of equilibrium and after starvation stress arc shown in Fig. 3. PCB equilibrium residues (ppm, OD) were as follows: fat--797; eggs--73-7; liver--51-8; muscle--281; HONS 084116 JEFFREY L, UNCER, D AV ID B. PCAKALL MONS 0 8 4 1 1 1 % TABLE I AAOCIOA 1234 RESIDUES IN TUSVCS ANO FAECES AT IQUIUMUUM (A) AFTER STARVATION (*1 ANO Till RATIO OP R/A (C) Stunpk Muscle Rr*i Liter Fi ppr* bated on WW OD EF WW OD EF WW OD EF WW OD EF WW OD EF mean A sld. dev. *05 28 1 318 272 404 121 1 36 4 32 60-3 9 It 291 3 53 23 4 102 113 18* 36 3 0*2 4 44 1*2 23 3 i 3 14 13-3 991 496 SIS 3 35-0 2 17 5 I 13 4 017 736 797 *33 253 273 296 126 136 14* 96 I 1 39 2 27 224 0-01 7-7* O00 3 30 I 02 = 003 8 Ud! error EF a ,.d t 172 703 31300 43 177 19900 19 79 3 *900 291 _t 1 34 243 31 3 14 1 14 30 3910 123 900 S3* 402 24 5 i 2 20 1120 422 188 4020 37200 1370 *430 613 3780 10 7 i 2 4* 7 19 124 1830 I 52 C 0/4t7 21 4 25 0 9* 4 53 0 5 J 57 9 73 2 77 6 97 9 N 4 lot tissues at equilibrium; 5 fur tissues after starvation period; 2 for faeces at equilibrium: 1 for faeces after starvation WW wet weight; OD - oen dr* weight; EF eatractabte fat weight. * n0 fat available for analysis, t Esuaoab* fat inseam standard deviation. Ratio ol starvation equilibrium residue levels. -- 5 44 *20 1 PCD PIIARMACODYNAMICS IN TUI: RING DOVE 65 brain--25-4; faeces--2-27. Although the relationships of residue levels in various samples varied, depending partially on the amount of extractable fat, the above descending order remained, regardless of basis of ppm calculation, bxtractable fat decreased in stressed birds to varying degrees, depending on tissue. There was no fatty tissue remaining in stressed birds. In terms of per cent of original extractable fat, there was 32% in muscle tissues of stressed birds, 80%, in livers and 97% in brain tissues. These decreases tended to exaggerate the increase in residue levels in muscle tissue and, to a lesser extent, livers based on extractable fat. At death, following starvation stress, residue levels increased by the following factors (ratios based on ppm,OD): liver--78;muscle--25; brain--56; faeces--5-5 (Table I). With the exception of one control, nil birds died within the same six-day period and gave no suggestion of differential mortality between groups. These was, how ever, an indication that the experimental group lost, on the average, more weight relative to their own original weight (Table 2). TABLEJ2 WEIGHT CHANGE IN OOVfS* IN STARVATION EXVER1MFNT FIGURES AKt MEAN STANDARD DEVIATION (S1ANDARD ERROR) Controls PCB-frtf Significance Initial weight (g) ' 171 9-78<4-37) 162 Weight at death (g) 86 8 5-77(2 58) 83 1 Weight at death (% of initial wi) 52 4 2-18(0-97) 47-3 + 7 650 42) ns at 010 ) 8-13(3 64) ns at 0*10 4 45(1-99) signif. at 0 10 N 5 for each group. Until recently, the analytical picture concerning PCB has been clouded not only by the past lack of synthesising and characterising the individual peaks (Huizingcr et al, 1971) but also the apparent differentia! metabolism of early-arising gas chromatographic peaks (Koeman et al, 1969a; Heath et a/., in press; Grant ct at., 1971; Nimmo et al., 1971). Although chromatogram profiles from dove sample extracts resembled each other superficially, the relative early peak diminution varied considerably, depending on type of sample (Table 3). Concurrent with the reduction or removal of the first three peaks came the appearance of three new peaks following what would normally be the last Aroclor 1254 standard peak. Tins same pattern emerged with each of the four different columns used (Fig. I). CONCLUSIONS AND DISCUSSION PCB residues increased to, and levelled off at, 80 ppm (OD) or 17 ppm wet weight (WW), in eggs from doves fed 10 ppm Aroclor 1254 (Fig. 2). Scott ct al. (1971) noted a similar levelling off of PCB residues at approximately 3 ppm (WW) in the eggs of chickens fed 10 ppm Aroclor 1248. A comparison of egg to fat residue levels MONS 004110 66 JEFFREY L. LINCER, DAVID B. PEAKALL leads to the following ratios: S :38 (EF); 1:11 (OD); 1:47 (WW). For comparison, Scott's group found the same ratio to be 1:12 (WW). Further comparison is difficult due to differences in both species and Aroclor used. With regard to wild bird populations, these egg residue levels appear comparable to those reported by Jensen et al. (1969) for some marine birds and Prcstt ct al. (1970) for a variety of wild birds from many families. Dove liver residue levels (15 ppm, WW) were close to liver residue levels reported in birds of prey (Risebrough et al., 1968: Kocman ct al., 19696) but somewhat lower than those found in great crested grebe livers (Prestt & Jefferies, 1969). The starvation stress experiment conducted on the doves and the subsequent mobilisation of stored fat resulted in an increase in liver PCB residues of almost two orders of magnitude (.e. 15-3 to 1120 ppm, WW). Since there was no apparent differential toxicity between controls and experimental and the liver levels of experimental were roughly twenty times higher than those in livers from birds found dead after the Irish Sea wreck (i.c. average * 56 ppm: Hoidgatc, 1971) it appears that PCB was probably not the major cause of death in those wild birds. Due caution should be applied, however, as wide differences have been observed in the (I) toxic biological effects between different PCB (Street ct al, 1969; Villeneuve et al1971; Heath et al., in press; Nagai et al., 1971); (2) species sensitivities to toxicants (Heath et al., in press); (3) ability of various organs to reflect the cause of death (Stickel et al., 1966; Prestt et al., 1970); and (4) analytical and quantitation techniques used for PCB (Peakall A Lincer, 1970; Lincer. 1971), the last of which alone can lead to a difference of over 100% (Rote A Murphy, 1971). In addition, the possibility of: synergistic or additive action of PCB and other environmental pollutants (Lichenstcin et al., 1969) or infectious diseases (Friend A Trainer, 1970); interactions affecting the retention of body residue burdens (Street ct a!., 1966); gross reproductive effects (Dahlgren & Linder, 1971; Scmt et ni 1971) and defects in proper embryonic development (Hays A Risebrough, 1972) stemming possibly from aberrant chromosome formation (Peakall ei al., 1972) must be considered very carefully before classifying PCB as innocuous. The diminution of eariy>arising gas chromatographic peaks occurred in all tissues sampled, as well as eggs and faeces. The presence of late-arising 'metabolic' peaks from the various samples (Fig. I) and the similarity of peak-diminution (Table 3) in the faeces (compared with other sample types) suggests that an in nt o transformation, possibly microsomal (Lincer A Peakall, 1970) dechlorination or hydroxylation, is taking place and not a differential excretion of those PCB com ponents associated with early peaks. It is, of course, difficult to say whether later peaks are increasing, early peaks decreasing or a combination of the two. F-arly peak diminution was particularly acute in muscle (68-8%) and fat (70*8".,) samples. Although one should be especially cautious in comparing chromatograms origi nating from different laboratories, there appears to be a basic similarity between those from the rat (Grant et al., 1971) and ours from the dove (Fig. 1C) with some HONS oa'tH9 PCB PHARMACODYNAMICS IN TIIL RING DOVC 67 TABLE 3 EARLY PEAK DIMINUTION* Of PCB'S (AROCLOR J254) IN DOVE TISSUES, fOCiS AND FAECES. (EXI'RCSSEO At */9 CHANCE RELATIVE TO EQUIVALENT STANOAKO; SAMPLE SIZES IN PARENTHESES.) Parameter Muscie{4) Brain{4) l.ivcr(5) Fat (4) Fgg(4) FaecesiD * -68 8 - 58-3 - 57 4 - 70 8 - 54 0 sd. 6-85 3 86 3 13 2 63 9-49 s.e. 3-42 1 93 1-40 131 4-74 * Sec 'Methodology' for calculation of values in (able and operating parameters. - 54 5 0 71 0-50 notable differences (/.<*. the lack of their prominent peak 2 in ours, and vice versa the lack of our peak 13 in (heirs). The fact that this kind of early peak diminution has also been described in fish and invertebrate (shrimp) tissues (Nirnmo ei ai, 1971) suggests that this is a widespread mechanism among the animal kingdom. Con sidering the possibility of differential toxicity of individual components (Vos et ai, 1970; Yoshimura & Oshima, 1971) the need and desirability of investigations on the toxicity of individual components in PCB parent compounds, and especially metabolically-derived compounds, is evident. It would indeed be unfortunate if, after evolving the necessary apparatus to break down foreign compounds, an organism produced more toxic compounds, and thereby was killed. ACKNOWLEDGEMENTS Some of the work presented here was carried out under NIH Grant ES00306, Dr T. J. Cade, Principal Investigator, This research wus carried out while one of us (DBP) was an Established Investigator, American Heart Association. Thanks go to Miss D. Zalkind for technical assistance and Mrs C. V. Lincer for maintaining the dove colony. REFERENCES Bernard. R. F. (1966). DDT residues in avian tissues. J. appi. coI. 3 (Suppl ). 193-8. Dahlgren. R. B. & Linden. R. L. (1971). Effects of polychlorinated biphenyls onpheasam repro duction. behavior, and survival. J. IVIMI. Mgmr, 35, 315-9. ~ Friend, M. & Trainer. D. O. (1970). Polychlorinated biphenyl: interaction with duck hepatitis virus. Science, N.Y., 170, 1314-6. Grant. K. L.. Phillips. W. F. J. St Vili eneuve. D. C. (1971). Metabolism of a polychlorinated biphenyl (Aroclor 1254) mixture in the rat. Bull, environ. Contam. & Toxicol., 6, 102-12. Hays, H A Risebrough. R. W. (1972). Pollutant concentrations in abnormal young terns from Lone Island Sound. Auk, 89, 19-35. Heath, R. G., Spann, J. W., Kreitzcr. J. F. A Vance. C (in press). Effects of polychlorinated biphenyls on birds. Proc. irtt. orn, Congr., 15th, The Hague. 1970. HoldGatE. M. W. (1971). The sea bird wreck in the Irish Sea, autuvn 1969. 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