Document 8bMq8mgZ5GzQDe7qzmjV3wwd

PCB PHARMACODYNAMICS IN THE RING DOVE AND EARLY GAS CHROMATOGRAPHIC PEAK DIMINUTION Jeffkey L. Ljnctr* Si David 8. Peakall Section of Ecology A Systematic!, Langmuir Laboratory, Cornell University, Ithaca, New York 14850, USA ABSTRACT Ring Doves (Streptopelia risoria) were maintained on a daily dietary dose of 10 ppm of the polychlorinated biphenyl {PCB), Aroclor 1254. Sequential sampling and residue analysis of eggs 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 was observed in experimentals 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 was not the primary factor 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 33581, USA. 59 E*e!ron. Polltel. (4) (>973) pp. 59-61-- Applied Science PublHhen Ltd. Emlind, 1973--Primed m Gmt Britain individual and the ecosystem. Within the individual this means that the bulk of the organochlorine is to be found in the adipose tissue. While stored the PCB is com paratively inert, but if the fat is mobilised then the organochlorine is released into circulation. Studies with organochlorine pesticides have shown that release of DDT (Bernard, 1966), dieldrin (Stickel ef a/., 1969) and heptachlor (Stickel et at., 1965) caused by reduced food intake can be fatal to birds, PCB, like the organo chlorine pesticides, is able to accumulate up food chains (see Pcakall & Lincer. 1970; Peakall, in press, for reviews). Holdgate (1971) considered that 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-offs such as that in the Irish Sea (Holdgate, 1971), of particular comparative interest are the PCB tissue residues resulting from known dosage and the possible lethal effects 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 (Koeman et at., 1969a; Grant er a!., 1971; Nimmo et al., 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 at a temperature of 20 1C. Aroclor 1254 was given in pellets of turkey grower mash (Agway) at a dosage 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 days which led to a weight loss of approximately 10% per week. After three weeks of this regimen the birds were completely starved until death occurred. The objective W8S 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 earlierone(US Dept. ofHealth, Education & Welfare, 1970). Specifically, samples were dried for 48 h at 40-45C, ground with a coarse anhydrous Na2S04, placed in Soxhlet extractors and extracted for 8 h with a 1:3 mixture of ethyl ether and petroleum ether. The extracts were concentrated to 50 ml and a 5 ml aliquot was evaporated to dryness at 40-45C for 2 h to arrive at an extractable fat estimate. Sample clean-up was accomplished with a Fiorisil column (USDHEW, 1970). As the second (15% ethyl ether in petroleum ether) fraction did not contain any PCB peaks, only the first (6% ethyi ether) fraction was analysed. Determination was STLCOPCB4009972 carried out on a Varian Aerograph model 2100 gas chromatograph equipped with a 63Ni 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 7O/80; 2% SE-30 on Chrom. G A/W DMCS 7O/80. Nitrogen gas flow rates of 35 and 150 ml/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 Sc Murphy, 1971) are 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) 1 % QF-1 on Chrom. G. A/W DMCS 7/80, 4 mm (id) x 2 m, injector--238'C, column--203"C. detector--28IC. nitrogen flow--30 ml/min. (B) 1 % silicone GE XE-OOon Varaport #30 100'120,2 mm x 1 66 m. injector--238C. column--203;C. detector-- 28PC,nltTogen--25ml/min. (C)4%SE-30 + 6%QF-1,Chrom. G /80,2mm x 2 m,injector-- 250'C, column--225C. detector--281C, nitrogen--90 ml/min. (D) 2% SE-30 on Chrom. G A/W DMCS 70/80, 4 mm x|2 m, injector--2J0"C, column--225C, detector--281C. 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-I (Fig. 1A) was compared between sample types. A relative diminution value was calculated for each sample as follows: 100 - (100 x (A/B)]; 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 et 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 iI I STLCOPCB4009973 62 JEFFREY L. LINGER, DAVID B. PEAKALL approximately 15 %. Recoveries made in this laboratory ofcommon organochlorines (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 :} .V* *t Fig. 2. Aroclor 1254 residues (ppm oven dry weight basis (doited square) and extractable (at weight basis (dotted circle!) in dove eggs versus time (each point represents one residue value (or two pooled eggs of a clutch; two points shown as (CTossed square) or (crossed circle) were not considered in curve-fitting respective curves because they came from a single female which showed predisposition to laying eggs containing inordinately high residues). In the equationy ~ residues: x -- time. DSU 026012 STLCOPCB4009974 ihe onset of the experiment. The equations for that increase, based on the oven dry and extractable fat egg sample weights (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 31-7 118% 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 are shown in Fig. 3. PCB equilibrium residues (ppm, OD) were as follows: fat--797; eggs--73-7; liver--51-8; muscle--28-1; f i .. STLCOPCB4009975 TABLE 1 arocvo* 1254 residues in tissues and taecej at fquiubkium (a) Arm ttabyatton () and the ratio or b/a (c) Sampfc Mutch Brain Liver Fat Faeces ppm based on trtean A s(d. dev. A Jtd. error EF i.d.t mean B std. dev. a std. error EF i.<Lt c BMrr WW OD EF 80S 28 1 318 2-72 904 121 136 4 52 60-3 91* 2-91 172 703 31300 43 177 19900 19 79 3 8900 2-91 1 34 21-4 250 98 4 WW OD EF 5-33 23 4 102 185 888 36-3 0-92 4-44 182 23-3 3 14 293 1430 3910 315 125 900 14-1 558 402 24-5 i 2 20 53 0 56 3 579 WW OD EF 153 51 8 380 991 35-0 274 4-96 17 5 137 13 4 0-17 1120 4020 37200 422 1370 8450 188 613 3780 10-7 2-48 73 2 77 6 97 9 WW OD EF 736 797 833 253 273 296 126 136 148 96-1 1-39 * WW OD EF _ 1-27 224 ___ QOt 7-78 -- 0-00 5 50 1-02 003 719 124 1830 __ ___ -- -- 152 - 3-49 8 20 N * 4 for tmua at equilibrium; 5 for (issues after starvation period; 2 Tor faeces at equilibrium: I Tor faeces after tumlion. WW wet weight; OD oven dry weight: EF extractable fat 'Aright. " No fat available for analytic, t Extractable fat (mean) i standard deviation, ft Ratio of sturvalion/equilibrium residue levels. STLCOPCB4009976 PCB PHARMACODYNAMICS IN THE 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. Extractable 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 1). With the exception of one control, all 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). TABLE 2 W EIGHT CHANGE IN DOVES* IN STARVATION EXPERIMENT FIGURES ARE MEAN STANDARD DEVIATION (STANDARD ERROR) Controls PCB-fed Significance Inilial weight (g) Weight at death (g) Weight at death (% of initial wt) 171 9-78(4 37) 162 7-65(3-42) 86 8 5-77(2-58) 831 8-13(3-64) 52 4 2 18(0-97) 47-3 4-45(1-99) ns at OTO ns at 010 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 (Hutzinger el al., 197!) but also the apparent differential metabolism of early-arising gas chromatographic peaks (Koeman et a!., 1969a; Heath el al., in press; Grant et al., 1971; Nimmo el 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. This same pattern emerged with each of the four different columns used (Fig. 1). CONCLUSIONS AND DISCUSSION PCB residues increased to, and levelled off at, 80 ppm (OD) or 17 ppm wet weight (\VW), in eggs from doves fed 10 ppm Aroclor 1254 (Fig. 2). Scott et 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 DSM 026015 STLCOPCB4009977 66 JEFFREY L. LINCER, DAVID B. PEAKALL leads to the following ratios: 1:3 8 (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 Prestt et 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 at., 1968; Koeman et a!., 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 (/>. 15-3 to 1120 ppm, WW). Since there was no apparent differential toxicity between controls and experimentals and the liver levels of experimentals were roughly twenty times higher than those in livers from birds found dead after the Irish Sea wreck (i.e. average - 56 ppm; Holdgate, 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 (1) toxic biological effects between different PCB (Street et al., 1969; Viileneuve et al., 1971; 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 at., 1970); and (4) analytical and quantitation techniques used for PCB (Peakall Sc Lincer, 1970; Lincer, 1971), the last of which alone can lead to a difference of over 100% (Rote & Murphy, 1971). In addition, the possibility of: synergistic or additive action of PCB and other environmental pollutants (Lichenstein et al., 1969) or infectious diseases (Friend & Trainer, 1970); interactions affecting the retention of body residue burdens (Street et a/., 1966); gross reproductive effects (Dahlgren & Linder, 1971; Scott et al.. 1971) and defects in proper embryonic development (Hays & Risebrough, 1972) stemming possibly from aberrant chromosome formation (Peakall et at., 1972) must be considered very carefully before classifying PCB as innocuous. The diminution of early-arising gas chromatographic peaks occurred in all tissues sampled, as well as eggs and faeces. The presence oflate-arising `metabolic' peaks from the various samples (Fig. 1) and the similarity of peak-diminution (Table 3) in the faeces (compared with other sample types) suggests that an in vit-o transformation, possibly microsomal (Lincer & 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. Early 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 QSW 026016 EARLY PEAK DIMINUTION* Or PCB'S (aROCLOR 1254) IN DOVE TISSUU, EOOJ AND FAECES. (EXPRESSED AS % CHANGE RELATIVE TO EQUIVALENT STANDARD; SAMPLE SIZES IN PARENTHESES.) Parameter Muscit{4) Brain{4) Uverii) Fat (A) Fggl4) Fatcex[2) X i.d. t.e. -68-8 6 85 3-42 -58-3 3-86 1-93 -57-4 3 13 1 40 -70-8 263 1-31 -54-0 9-49 4-74 See `Methodology' for calculation of values in table and operating parameters. -54 5 0-71 0-50 notable differences (/.e. the lack of their prominent peak 2 in ours, and vice versa the lack of our peak 13 in theirs). The fact that this kind of early peak diminution has also been described in fish and invertebrate (shrimp) tissues (Nimmo et a!., 1971) suggests that this is a widespread mechanism among the animal kingdom. Con sidering the possibility of differential toxicity of individual components (Vos et al, 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. AC KNOWLEDGEMENTS Some of the work presented here was carried out under NTH Grant ES00306, Dr T. J. Cade, Principal Investigator. This research was 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. appl. Ecol. 3 (Suppl.), 193-8. Dahlgren, R. B. A Linder, R. L. (1971). Effects of polychlorinated biphenyls on pheasant repro duction, behavior, and survival. J. Midi. Mgml, 33, 315-9. Friend, M. A Trainer, D. O. (1970). Polychlorinated biphenyl: interaction with duck hepatitis virus. Science, N.Y., 170, 1314-4. Grant. K. L., Fhiuips. W. E. i. A Viixeneuv*. D. C. (1971). 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