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loggerhead marine turtles, we used sodium diatrizoate, a commonly
used radiographic contrast agent, injected subcutaneously either
in the neck or hind limb followed by serial filming. The kidneys
were not visualized after 24 hr. The contrast agent was absorbed
more quickly from the neck injection site than from the hind limb
site. These absorption rates could be useful for determining
injection site selection for other purposes, such as antibiotic
therapy in suspected or proven infections.

In this paper we report the use of sodium diatrizoate to
demonstrate differential absorption from two subcutaneous sites,
the neck and the hind limb in the Atlantic ridley turtle. The
kidneys were also observed to determine if opacification of the

urinary tract occurred.

MATERIALS AND METHODS

After the earlier studies involving two loggerhead turtles of
approximately 300 gq each, six Atlantic ridley turtles from four
to six months of age, and ranging in weight from 100 to 320 g,
and in apparent good health were chosen at random from the
rearing tanks at the Galveston Laboratory. The drug used for
injection was sodium diatrizoate (50% W/V) (Hypaque® Sodium 50%
brand of diatrizoate sodium injection, USP sterile aqueous injec-
tion, Winthrop Laboratories, 90 Park Avenue, New York, N.Y.
Reference to trade names or commercial companies does not imply
endorsement by the National Marine Fisheries Service, NOAA).
Sodium diatrizoate is freely miscible with blood, absorbs x-rays
in part of the diagnostic x-ray spectrum (due to its high atomic
number), and is therefore opaque (Blaufox and Freeman, 1978). A

dosage of 150 mg (0.5 cc of sodium diatrizoate 50%) for turtles



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Acknowledgments

We appreciate the help, encouragement, and support of Dr.

M. H. Schreiber, Department of Radiology, University of Texas
Medical Branch, as well as his comments on preparation of this
paper. We also wish to thank Dr. Robert Perry, Department of
Radiology, UTMB, for his assistance in taking and calculating
the radiation doses, and Dr. Raymond Sis, School of Veterinary
Medicine, Texas A&M Universitv, College Station, Texas, for his
review and constructive criticisms of the manuscript.

The Atlantic ridley turtles (Lepidochelys kempi) were
hatched and supplied to the Galveston Laboratory, National Marine
Fisheries Service under the joint efforts of the Mexican
Government, the U.S. Fish and Wildlife Service, and the U.S.
National Park Service.

The investigations involving the ridley turtle were conducted
under Permit No. PRT. 2-1770 (U.S. Federal Fish and Wildlife
Service), Scientific Permit No. 359 (Texas Parks and Wildlife
Service), Permits 1978-ABC-IV0751, No. 27611-8786, and
1979-ABC-IV-1258, Exp. 4287 (Mexican Government).



change in size of the opacified area and decrease in the opacity
of the contrast material, using the 5-min and 60-min films. The
grading was done on a scale of 1 to 6 in the following manner:
l-unchanged, 2-mild, 3-moderate, 4-marked, 5-nearly complete
absorption, and 6-no contrast detected. The data were then com-
piled into a frequency distribution curve and the mode, range,

and mean determined.

RESULTS

The range, mode, and mean for the change in the size of the
opacified area for the neck injections are given in Table 2 and
for the hind limb injections in Table 3. The mode for the change
in size of the contrast area was 2 (mild) and 4 (marked) for the
hind limb and neck, respectively, with the neck injection mean
being 3.9 and the hind limb being 2.5. The change in opacity of
the area showed similar results with a mode of 3 (moderate) and 5
(nearly complete absorption) for hind limb and neck, respec—
tively, with the mean being 4.3 for the neck and 2.9 for the hind
limb. The kidneys were never observed on any of the radiographs.

We observed no untoward side effects in these animals, and
there was no evidence of tissue necrosis or cutaneous sloughing
at the injection sites. Radiation exposure was of concern.
However the total dose was approximately 6.8 milliroentgen per
exposure at 65 kVp and 2 MAS at a distance of 90 cm. Measure-
ments were made using a 15 cc pancake ionization chamber
(Keithley 15 cc pancake ionization chamber, Keithley Co.,
Cleveland, Oh.) on a digital dosimeter (Keithley 35055 digital
dosimeter, Keithley Co., Cleveland, Oh.). This is well below
the LDs5q in some chelonians shown by Cosgrove (Cosgrove, 1971).
It is considerably less than that used in turtle reproductive
studies by Gibbons and Green, (1979).



ranging in weight from 266 to 320 g and a dosage of 75 mg (0.25
ec of sodium diatrizoate 50%) in the two turtles weighing
approximately 100 q were given (less than 85% of maximum animal
dose) (Gillette, 1977). The contrast material was administered
with disposable plastic syringes, using 26-qauge needles. All
radiographic exposures were made on standard radiologic equipment
available in the Department of Radiology, University of Texas
Medical Branch, Galveston, Texas, using standard radiographic
techniques, regular radiographic cassettes (Kodak X-omaticTM
regular cassette, Eastman Kodak Co., Rochester, N.Y.) equipped
with high-speed intensifying screens (Kodak X-omaticTM regular
intensifying screens, Eastman Kodak Co., Rochester, N.Y.) and
standard medical radiographic film (Dupont CronexR Four safety
film, E. I. Dupont de Nemours and Co, Photo Products Dept.,
Wilmington, De.). (Table 1).

On each day of the study, two healthy ridley turtles were
selected and were injected subcutaneously (with sodium
diatrizoate), one in the ventral surface of a hind limb and the
other in the dorsal aspect of the neck after sterile preparation
of the overlying skin with an iodophor solution. Satisfactory
injection was determined by two criteria: little or no leak of
contrast through the needle track after the needle was withdrawn,
and after the 5-min film, the retention by the opacified area of
a nearly round or ovoid shape with little dissection along
fascial planes, thus allowing for a similar volume and surface
area for absorption. If the criteria were met, filming was con-
tinued for 15, 30, and 60 min after injection. The films on each
animal were made in the dorsol-ventral projection with the
radiographic technique detailed in Table 1. Upon completion, all

studies were reviewed by five radiologists and were graded on the



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and 3 we show that, in general, there were marked changes from
the 5-min to l-hr films in the two parameters seen in the animals
injected in the hind limb (Figs. 1 and 2). Considering these
findings we believe that the faster absorption of sodium diatri-
zoate from the neck would result in higher blood levels of this
compound and therefore of other drugs with related phar-
macokinetics.

Although the absorption of any drug from a subcutaneous or
intramuscular site depends on its peculiar pharmacology and the
environment at the site, any of the commonly used drugs that are
water soluble and do not cause unacceptable tissue damage when
injected subcutaneously would be absorbed more rapidly from neck
injection sites due to that site's greater vascularity as has
been indirectly shown by this study. Therefore, the choice of
the neck as opposed to the limb as an injection site where high
levels of therapeutic agents are needed is recommended.

Unfortunately, the urinary tract was not opacified in these
animals. This may be due to many factors including inadequate
dose, peculiar distribution, and metabolism of contrast material
in these animals, and inadequate filming time (though filming was
carried out to 24 hr in the preliminary studies with the

loggerhead turtles).



NOAA Technical Memorandum
NMFS-SEFC-93

Radiologic Evaluation of the

Differential Absorption of

Diatrizoate in Marine Turtles

By Garey L. McLellan and Jorge K. Leong

MAY 1982

U. S. DEPARTMENT OF COMMERCE
Malcolm Baldrige, Secretary

National Oceanic and Atmospheric Administration
John V. Byrne, Administrator

National Marine Fisheries Service
William G. Gordon, Assistant Administrator for Fisheries

Technical Memorandums are used for documentation and timely communication of
preliminary results, interim reports, special-purpose information, and have not
received complete formal review, editorial control, or detailed editing.



Gillette, E. L. 1977. Special procedures, p. 210-212. In:
Carlson's Veterinary Radiology, ed. 3., E. L. Gillette et al.
(ed.). Lea and Febiger, Philadelphia.

U.S. Department of the Interior. 1978a. Fact sheet: The
Endangered Species Act - The green, loggerhead and olive
(Pacific) ridley sea turtles, p. 1-3. Fish and Wildlife

Service, U.S. Dept. Interior.

- 1978b. Fact sheet: Reptiles,

amphibians, fishes, snails, clams, insects listed in the
appendices to the Convention on International trade in
Endangered Species of Wild Fauna and Flora. Fish and
Wildlife Service, U.S. Dept. Interior.

Witten, D. M., G. H. Meyers, and D. C. Utz. 1980. Clinical
Urology, Vol. 1. W. B. Saunders Co., Philadelphia.

10



Literature Cited

Altman, R. C. 1978. Perching birds, parrots, cockatoos, and
Macaws (psittacines and passerines), p. 347-394, In: Z00
and Wild Animal Medicine, M. E. Flower et al. (ed.). W. BL
Saunders Co., Philadelphia.

Blaufox, M. D. and L. M. Freeman (ed.). 1978. Diatrizoate
sodium 50%, p. 176-179. in: Physicians' Desk Reference for
Radiology and Nuclear Medicine, ed. 8. Medical Economics
Co., Ordell, N.Y.

Cerny, J. C., A. R. Kendall, and R. M. Nesbit. 1967.
Subcutaneous pyelography in infants: A reappraisal. J.
Urol. 98:405-409,.

Cosgrove, G. E. 1971. Reptilian radiobiology. J. Am. Vet. Med.
Assoc. 159:1678-1684.

Fingle, E., and D. M. Woodbury. 1975. General principles, p.
5-8. In: The Pharmacological Basis of Therapeutics, L. S.
Goodman, and A. Gilman (ed.). MacMillan Publishing Co.,
Philadelphia.

Frye, F. L. 1978. Hematology of captive reptiles, p. 146-148.
in: Zoo and Wild Animal Medicine, M. E. Fowler et al.
(ed.). W. B. Saunders Co., Philadelphia.

Gibbons, J. W. and J. L. Green. 1979. X-ray photography: A
technique to determine reproductive patterns of fresh water

turtles. Herpetologica 35:86-89.



DISCUSSION

Although the initial studies on the loggerhead turtles failed
to opacify the urinary tract, they did show nearly complete
absorption of contrast material from the neck and moderate
absorption from the hind limb at 1 hr. We believed this to be an
important finding when considering the need to attain the highest
possible blood levels of therapeutic agents in turtles that are
known to have or are suspected to have serious or life threaten-
ing systemic or localized disease amenable to drug therapy.
Traditional parenteral drug administration in sea turtles is
often done by injection into the hind limb area. Results in the
present study indicated that subcutaneous injection in the dorsal
neck is perhaps more desirable in order to attain maximal thera-
peutic effects.

Vascular access in these animals is quite limited, and
repeated subcutaneous or intramuscular injections become
necessary. The absorption of drugs from either of these sites
depends on several factors. Vascularity, blood flow, solubility,
and concentration all have important roles in druq absorption
(Fingle and Woodbury, 1975). Sodium diatrizoate is hypertonic
and highly soluble in water. Both of these factors lend them-
selves to its rapid absorption. These factors, of course, are
constant when the same drug and drug concentration are used at
different injection sites. The vascular supply to the area and
the blood flow then become predominant factors. In turtles that
died for various reasons, at the Galveston Laboratory, we
observed at necropsy a denser vascular network in the neck than
in the limbs. We believe this directly accounts for the observed

difference in absorption demonstrated in this study. In Tables 2



graphy is based upon the use of water soluble iodinated contrast
agents. These agents are administered parenterally, excreted
almost exclusively by goloerular filtration, not resorbed by the
tubules, and concentrated by the normal solute and water
resorbing processes of the kidney, thereby providing radiographic
contrast in the kidney due to the high density of iodine within
it, i.e., the kidneys can be seen on radiographs, a process known
radiographically as opacification (Witten et al., 1980).

The opacification technique is a well-known and widely-used
radiologic procedure in humans (Witten et al., 1980) as well as
in some animal species such as the dog and cat (Gillette, 1977).
It has been attempted in birds but has not been successful
(Altman, 1978). The preferred route of administration of contrast
is intravenous. However intravenous access in small turtles,
although possible, is not always successful or satisfactory,
(Fry, 1978). Therefore, a repeatable, satisfactory and safe
means of administration with reproducible results is needed.

Cerny and associates, in a two-phase study using sodium
diatrizoate in rabbits, demonstrated that the subcutaneous admi-
nistration of contrast material produced satisfactory results as
well as an acceptable level of side effects and minimal tissue
injury (Cerny et al., 1967). Afterward they obtained satisfac-
tory excretory urograms in 98 of 100 human patients with sub-
cutaneous injections, while minimal side effects and no tissue
slough were observed. Further, sodium diatrizoate and similar
compounds have little observed toxicity in animals (Gillette,
1977). Since the subcutaneous method of administration is a
recognized safe alternative to intravenous injection, it was cho-
sen for this study.

In a preliminary study to opacify the kidneys in two



Figure 2.

Enlargement of the neck of an Atlantic ridley turtle
at 5-min and 1-hr post injection. The arrowheads
point to the opacified areas. Comparison of the 5-min
and 1-hr films show that at 1 hr the contrast material
is only faintly visible and has been almost completely
absorbed (turtle weight 100 g and carapace length 8.4

cm).

12



Table 1. Radiographic Technique. Kilovoltage peak mass 65.

Milliampere seconds was 2, with a tube film distance

of 90 cm.
Turtle Turtle Turtle
Turtle Injection Weight Carapace Dorso-ventral
Number Site (g) Length (cm) Thickness (cm)
446 Hind Limb 266 11.3 4.0
465 Neck 320 12.2 5.2
469 Neck 275 11.6 45
479 Hind Limb 100 8.0 3.6
470 Neck 100 8.4 3.5
473 Hind Limb 310 12.0 4.2

13



RADIOLOGIC EVALUATION OF THE DIFFERENTIAL ABSORPTION
OF DIATRIZOATE IN MARINE TURTLES

INTRODUCTION

Since September 1977, the National Marine Fisheries Service
(U.S. Department of Commerce) has been raising hatchlings of the
Atlantic ridley (Lepidochelys kempi) and loggerhead (Caretta
caretta) marine turtles at the Southeast Fisheries Center's
Galveston Laboratory. The hatchlings were raised to provide a
better understanding of the early life ‘of those animals, and to
engage in a headstart program as part of an overall project
attempting to help repopulate the Atlantic ridley in nature. At
the time the rearing was started, the Atlantic ridley had been
classified as an endangered species under the U.S. Endangered
Species Act of 1973. The loggerhead was later designated
"threatened," effective 6 Sept. 1978 (U.S. Dept. of the Interior,
1978a). Both species of turtles have been listed in Appendix 1
to the Convention on International Trade in Endangered Species of
Wild Fauna and Flora (U.S. Dept. of the Interior, 1978b).

Of immediate concern was evaluation and treatment of various
diseases found in the above marine turtles, ranging from der-
matologic disorders, lung infections, bowel perforation, perito-
nitis, bacteremia, to emaciation. Radiologic examination was
employed as a research and diagnostic modality to investigate
these problems.

At necropsy the kidnevs of some turtles were found to be
enlarged and edematous. Therefore, we reasoned that excretory
urography might be of benefit in demonstrating abnormalities in

living turtles with suspected kidney disease. Excretory uro—



ABSTRACT

The differential absorption, in the hind limb and neck was
evaluated in six Atlantic ridley turtles. A commonly used excre-
tory urographic iodinated contrast material was injected subcuta-
neously and serial radiographs were obtained. The 5-min and
70-min films were evaluated independently by five radiologists.
The data obtained were compiled and the mode, mean, and range for
the hind limb and neck absorption rates were analyzed. The neck
site showed more rapid absorption than did the hind limb.

Further, the area of the urinary tract was serially radio-
graphed in these animals to evaluate whether the urographic
contrast agent would opacify the tract. No opacification of the
kidneys was seen on serial films made up to 2 hr after injec-

tion.



Figure 1.

Enlargement of the hind limb area of an Atlantic
ridley turtle at 5-min and 1-hr post injection. The
arrowheads point to the opacified area. Comparison of
the 5-min and 1-hr films show that a substantial quan-
tity remains at 1 hr (turtle weight 310 g and carapace
length 12 cm).

11



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