Camp. Biochem. Physiol., 1972, Vol. 42A, pp. 205 to 211. Pe~gamon Press. Printed in Great Britain

THE FATE OF p,p’-DDT [2,2-BIS(p-CHLOROPHENYL)- l,l,l-TRICHLOROETHANE] IN THE DOGFISH,

SQUALUS ACANTHIAS”t

BARRY H. DVORCHIK and THOMAS H. MAREN

Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida; and Mount Desert Island Biological Laboratory,

Salisbury Cove, Maine

Abstract-l. Ring labeled 14C-p,p’-DDT was administered via the caudal vein or artery or by stomach tube to Squalus acunthius; tissue distribution and excre- tion of total radioactivity was followed with time.

2. The liver, which contains about 60 per cent lipid, was found to sequester the drug quantitatively.

3. No radioactivity was found in the gill effluate or urine over a 48-hr period. 4. The drug appears to be well absorbed from the gastrointestinal tract of

the dogfish. 5. Preliminary studies indicate that the drug may be bound to and trans-

ported by the plasma lipoproteins. 6. Preliminary studies showed that there is a marked toxicity, of a delayed

type, following injection of 5 mg/kg of p,p’-DDT.

INTRODUCTION

THE EXTENSIVE use of DDT over the past 30 years, along with its high stability toward various chemical, physical and biological agents has led to its ubiquitous distribution in the environment. Little work, however, is available on the dynamic movement of DDT in marine life. In order to understand the fate of p,p’-DDT in marine life, we undertook to examine the pharmacology of this drug in a repre- sentative elasmobranch, the dogfish, Squahs acanthias. This work is part of an overall marine pharmacology program instituted at the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine.

MATERIALS 4ND METHODS

Male dogfish, weighing l-3 kg, were caught by trawl in Frenchman’s Bay, off Bar Harbor, Maine, placed in circulating sea water in the laboratory launch and brought to the dock, where they were placed in live cars. The fish were used either on the day of capture or the following day.

* Supported by N.I.H. Training Grant GM 00760-10. t Reported in part in Fedn Proc. Fedn Am. Sots Exp. Biol. 30, 448 (1971) and Bull. Mt

Desert Isl. biol. Lab. 10, 12-15 (1970).

205

206 BARRY H. DVORCHIK AND THOMAS H. MAREN

The general protocol was as follows: Fish were placed in a box that contained 50 1. of sea water which was constantly renewed. The urinary papilla was catherized and a balloon attached for collection of urine. After about 1 hr, 60 pg/kg of ring labeled i4C-p,p’-DDT (Amersham/Searle or New England Nuclear) was administered, in ethanol, via the caudal vein or artery or by stomach tube. The flow of water was stopped for 2 hr after administra- tion of drug intravascularly and 1 hr following oral administration, to allow for the deter- mination of excretion of drug via the gills or regurgitation.

Determination of drug excretion via the gills, after intravascular administration, or regurgitation, after oral administration, was accomplished by sampling of the water in the closed system. A 25-ml aliquot of the ambient sea water was withdrawn at intervals and extracted once with an equal volume of scintillation counting solution containing 4 g of PPO (2,5-diphenyloxazole, New England Nuclear) and 50 mg POPOP (p-bis[2-(S-phenyl- oxazolyl)]-benzene, New England Nuclear) per 1. of toluene. The extraction efficiency was greater than 99 per cent. Blood was withdrawn at intervals, 0.1 ml of a 20% potassium oxalate solution added, and plasma obtained. The balloon containing urine was removed at various intervals and a new one attached. Urine volume was measured, the balloon rinsed once with a small volume of water, which was added to the urine, and the balloon was discarded. Fish were killed, by severing the spine, at 10 min, 1, 4 and 48 hr and various tissues were removed. Tissue distribution of total radioactivity utilized entire organs with the exception of muscle, where a representative sample was removed.

After weighing, the tissues were homogenized with water in a Waring Blender (50 per cent homogenate for all tissues except muscle where a 33 per cent homogenate was utilized), an aliquot removed and placed in a glass scintillation vial, I.0 ml of Nuclear Chicago Solubilizer (NCS) added and the mixture shaken at room temperature or at 40°C overnight until a clear solution was obtained. Twenty ml of the scintillation counting solution was then added to the vial and all samples were refrigerated for at least 1 hr at 5°C before being counted in a refrigerated liquid scintillation counter (Nuclear Chicago Mark I). Total muscle weight and plasma volume were estimated using the data of Burger (1967). All data are in terms of the parent compound, p,p’-DDT.

Preliminary binding studies utilized density gradient ultracentrifugation. To 40 ml of shark plasma, 0.17 PC of *V-p,p’-DDT was added. The density of the plasma was raised to 1.21 by the addition of 0.3 g of solid KBr/ml of plasma. An aliquot was then centrifuged at 105,OOOg for 24 hr at 12-15°C (Have1 et al., 1955), and the upper layer removed. Ali- quots of the upper and lower layers were taken, appropriate dilutions made and counted for total radioactivity. Radioactivity in the upper layer was taken as an indication of drug bind- ing to plasma lipoproteins.

Toxicity studies utilized nonradioactive p,p’-DDT (Aldrich) and ‘*C-DDT (New England Nuclear). Dogfish were administered various doses, in l-2 ml of ethanol, via the caudal vein or artery. Brain concentrations of DDT at death were analyzed by counting total radioactivity as described above. Control studies were run with ethanol only; no toxicity was noted.

RESULTS AND DISCUSSION

The concentration of 14C, as p,p’-DDT in the plasma, following an intra- arterial dose of 60 pg/kg, decayed rapidly (Figs. 1 and 2); less than 1 per cent of the drug remained in the plasma 6 hr after administration. A polyexponential fit of these data yielded at least three early linear components, with half lives of about 3, 30 and 95 min. The plasma concentration 5 min after intravascular admini- stration of 14C was about 1 pg/ml (Fig. l), approximately 10s fold greater than the water solubility of DDT (O’Brien, 1967). Preliminary binding studies, utilizing density gradient ultracentrifugation, indicate that greater than 90 per cent of the

FATE OF p,p’-DDT IN THE DOGFISH 207

2000

1000

z 500

‘0 c

200

i 1

f

f

f

i f

5 IO 15 20 25 x) 35 40 45 50 55 60

2oOc

1000

500

200

FIG. 1. Plasma concentration f S.E. of r4C, as p,p’-DDT in S. acunthius, as a function of time, following 60 pg/kg of 14C-p,p’-DDT intravascularly. Drug was injected at zero time, in ethanol (1-2 ml) over a lo-set interval. The syringe was then-,rinsed two to three times with the circulating blood for a period of about

30 sec.

1000 v_ - IOOC

I

500 - - 500

-I

- f

IOO- - 100

f

50 - i

- 50

is . ?

1

IO: ! z IO r-

FIG. 2. Same as Fig. 1 except over a longer time interval.

I?3456 7 6 9 IO

HR

208 BARRY H. DVORCHIK AND THOMAS H. MAREN

label is in the fraction of density less than or equal to 1.21 and thus the drug is probably bound to plasma lipoproteins. Further quantification of the binding of p,p’-DDT to shark plasma is currently under investigation.

The percentage of administered dose found in various tissues as a function of time is shown in Fig. 3. Tissues other than those shown in Fig. 3 which were followed were gonads, intestine, heart, brain and bile. These tissues accounted for 3-5 per cent of drug at any one time, with brain never exceeding O-5 per cent.

loo I

/

I

I

-100

-1 ,, -60

1-W

-40

-20

Y. FIG. 3. Percentage of dose I!I SE. of l&C, as p,p’-DDT, in various tissues of S. acunthias, as a function of time, following 60 pg/kg of ‘*C-p,p’-DDT intravascularly. Key: 0, kidney; 0, muscle; A, liver; A, plasma; ( ), total percentage recovery.

The drug was taken up quite rapidly by all tissues, with a final redistribution to the liver where it remained for an indefinite period of time. After 48 hr, 85 _C 7 per cent of the dose was found in the liver (Fig. 3). Experiments over a 2-week period indicate that from 72 hr to 2 weeks virtually all of the administered radio- activity can be accounted for by that present in the liver (not shown).

An analysis of the data of Fig. 3 shows that in the time interval 10 min to 1 hr, the increase in drug in liver and muscle agrees quite well with that lost by the plasma and kidney (51 vs. 45 per cent). The high kidney drug concentration at 10 min is probably due to the mode of administration. For the time interval 1 hr to 4 hr, the loss in plasma (12 per cent) was balanced by a gain in liver (10 per cent).

FATE OF p,p’-DDT IN THE DOGFISH 209

In this period there was also a small increase in muscle and kidney. In the final period, 4 hr-48 hr, the increase of drug in liver agrees well with that lost from plasma, muscle and kidney (25 vs. 26 per cent). Thus, the initial half-life of 3 min may be ascribed to distribution of drug from plasma to all tissues. The second component, half-life of 30 min, is probably due to a redistribution from tissues of low affinity to tissues of higher affinity. The third component, half-life of 2 hr, probably reflects final redistribution of drug to the liver.

That the liver quantitatively sequesters the drug is not surprising. In the dog- fish this organ is quite large, comprising lo-12 per cent of body weight (Burger, 1967); 40-60 per cent of this being lipid (Guarino, 1971). DDT is an extremely iipid soluble agent; the oil : water partition coefficient being 923 (O’Brien, 1967). Thus the liver acts as a large sink for the lipid-soluble drug and its metabolites, a function served by adipose tissue in mammals. The muscle of S. munthks com- prises about 43 per cent of body weight and even if rather low in lipid content (currently being studied) might provide some sink for DDT. It could not be more than about 20 per cent of the dose.

Urine, collected over a 48-hr period, showed no significant radioactivity, nor did samples of gill effluate collected over a 2-hr period, when plasma levels were the highest, indicative of no significant excretion of drug. This is supported by the tissue distribution studies which showed that over the time interval 4 hr-2 weeks, all the administered drug could be accounted for by that present in muscle and liver.

The fact that there was no excretion of drug via the gills is explicable on the basis of the very low water solubility of DDT and its high plasma binding. These factors permit only a very small amount of free drug, if any, into the plasma to diffuse across the gill. This, in conjunction with the rapid uptake of drug by tissues from plasma, severely limits diffusion across the gills.

Urinary excretion of DDT residues in mammals occurs only after the liver has metabolized the drug to compounds which can be handled by the kidney. Morello (1965) h as shown that DDT is metabolized, at least in part, by the liver microsomes. A survey of the literature on the in vitro hepatic metabolism of various drugs by fish and rats, at the respective temperature optima, indicate that fish hepatic metabolism is at least one-tenth that of rats. Since there are at least three modifications that must be made on the DDT molecule before it can be handled by the kidney (Datta & Nelson, 1970), it is quite probable that the absence of any detectable urinary excretion of radioactivity, over the time interval investigated, may be explained by a slow rate of hepatic metabolism.

The initial rapid decay of radioactivity in the plasma following intravascular administration of 14C-p,p’-DDT does not allow for any estimation of the final elimination half-life of DDT in S. acunthius. Gakstatter (1966) has shown that in goldfish and bluegills, the half-life for elimination of DDT is greater than 5 weeks. Pritchard & Kinter (1970) reported that in the flounder, Pseudopleuronectes americanus, the urinary excretion rate of radioactivity following 100 pg/kg of W- DDT was about 2 per cent per week. Assuming a similar rate of excretion of DDT

210 BARRY H. DVORCHIK AND THOMAS H. MASEN

for the dogfish, one arrives at a half-life of 25 weeks. Long-term studies to assess the rate of elimination of DDT in S. acunthias are currently in progress.

Preliminary studies on the rate of absorption of p,p’-DDT by the gastro- intestinal tract of S. acanthius were conducted. The data (Table 1) show that 6 per cent was absorbed 4 hr after oral administration and 22 per cent 24 hr after

TABLE I--PERCENTAGE DOSE OF ‘“C AS p,p’-DDT, IN VARIOUS TISSUES OF s. ac~nthius

FOLLOWING ORAL ADMIN;STRATION OF 60 pg/kg OF i4C-p,p’-DDT

Dose Tissue (%)

4 hr 24 hr

Liver 2 12 Muscle 3.3 9 Others * 0.7 1

- -

Recovered (%) 6 22

Stomach (and contents) 34 17 Intestine (and contents) t 12 7

-

Recovered (%) 46 24

In the two experiments given, analysis of the ambient water at 1 hr showed 7 and 17 per cent of the dose.

* Heart, brain, plasma, bile, gonads and kidney. t Duodenum, valvular intestine and colon.

administration. In the dogfish, about 72 hr is required for the digestion and absorption of a meat meal (Van Slyke & White, 1911). If DDT in the diet is absorbed at the same rate as protein, one obtains an average figure of 33 per cent per 24 hr, the data, 22 per cent per 24 hr, is in rough agreement with this figure. Van Slyke & White (1911) 1 a so showed that the stomach emptying time of the dogfish is about 6 hr, with food still remaining in the stomach after 48 hr. The recovery of drug following oral administration (Table 1) was 60 per cent. The flow of water to the box was stopped only for a period of 1 hr ; thus drug remaining in the stomach after the first hr might still be lost by regurgitation. Further experiments are planned where flow to the box will be stopped for a period of at least 6 hr, with constant monitoring of 14C levels in the ambient water.

Preliminary acute toxicity studies, utilizing a mixture of radioactive and non- radioactive DDT, were done on groups of at least four dogfish at 5, 10 and 20 mg/kg. These studies indicate that there is a marked toxicity, of a delayed type, following injection of 5 mg/kg of DDT.

In the fish given 5 mg/kg, no symptoms were noted until 48 hr after admini- stration. At this time, four out of eight fish had become rigid. Three days following administration of this dose, four of the eight were dead. Of the other four, one

FATE OF p,p’-DDT IN THE DOGFISH 211

died 2 days after administration, another died 4 days after administration, and two were still alive and showing no toxicity signs 5 days after administration. In the fish given 10 and 20 mg/kg, the pattern of toxicity was similar, occurring earlier than at the 5 mg/kg dose, but all the fish ultimately died. Studies are currently underway to quantify the acute toxicity of DDT in S. acanthias; thus far it appears that the concentration found at death in the brains of the fish given 5 mg/kg

(0.4 LLg/g) f 1 is ar ower than that found in rats, even following a non-toxic dose. Thus Henderson & Woolley (1969) found that 100 mg/kg per OS in rat yielded about 20 pg/g in brain. It appears that the shark brain is far more sensitive to DDT than the mammal.

SUMMARY

These studies have shown that in the dogfish, S. acanthias, a tracer dose of p,p’-DDT is accumulated, stored in the liver and probably not metabolized nor excreted at any appreciable rate. Furthermore, DDT appears more toxic to fish than to mammals. Thus, in a contaminated environment, the fish becomes a repository of DDT (Adamson et al., 1970) and upon death, the unchanged drug and metabolites (themselves insecticides) are returned to the sea to perpetuate another cycle.

REFERENCES

ADAMSON R. H., GUARINO A. M. & RALL D. P. (1970) Further studies on the disposition of DDT in Squalus acanthias. Bull. Mt Desert Isl. Biol. Lab. 10, 1-2.

BURGER J. W. (1967) Some parameters for the dogfish, Squalus acanthias. Bull. Mt Desert Isl. Biol. Lab. 7, 5-9.

DATTA P. R. & NELSON M. J. (1970) p,p’-DDT detoxication by isolated perfused rat liver and kidney. Ind. Med. 39, 54-57.

GAKSTATTER J. (1966) The uptake from water by several species of fresh-water fish of p,p’-DDT, dieldrin and lindane: their tissue distribution and elimination rate. Ph.D. dissertation, University of North Carolina.

GUARINO A. M. & CALL J. B. (1971) Preliminary studies on the distribution of i4C-DDT in the lobster, Homarus americanus. Fedn Proc. Fedn Am. Sots exp. Biol. 30, 448 (Abstr.).

HAVEL R. J., EDER H. A. & BRAGDON J. H. (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Inwest. 34, 1345- 1353.

HENDERSON G. L. & WOOLLEY D. E. (1969) Studies on the relative insensitivity of the immature rat to the neurotoxic effects of l,l,l-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT). J. Pharmac. exp. Ther. 170, 173-180.

MORELLO A. (1965) Induction of DDT-metabolizing enzymes in microsomes of rat liver after administration of DDT. Can.J. Biochem. 43, 1289-1293.

O’BRIEN R. D. (1967) Insecticides, Action and Metabolism (Edited by O’BRIEN R. D.), p. 109. Academic Press, New York.

PRITCHARD J. B. & KINTER W. B. (1970) Fate and distribution of i4C-DDT in the winter flounder, Pseudopleuronectes americanus. Bull. Mt Desert Isl. Biol. Lab. 10,64-67.

VAN SLYKE D. D. & WHITE G. F. (1911) Digestion of protein in the stomach and intestine of the dogfish. J. biol. Chem. 9,209-217.

Key Word Index-DDT; Squalus acanthias; detoxification; excretion of DDT.