Abstract

A variety of tissues, including liver and kidney, were obtained from four species of pinnipeds which had died in captivity. Three of the five animals had been rehabilitated strandings from the California coast. The animals included a Steller's sea lion, a Northern elephant seal, two Northern fur seals and a California sea lion. The tissues were analyzed for levels of cadmium, chromium, copper, lead, selenium and zinc. This paper discusses the possibility that the deaths of these animals may have been due to the influence of metal loadings. The results of these data indicate that the adult Northern fur seal may have died as a result of cadmium poisoning, in addition to general septicemia. The Northern elephant seal, whose death was caused by broncho-pneumonia, was further stressed by a cadmium/zinc imbalance. The authors compared necropsy results with the symptoms of metal toxicity.

Introduction

Over the past six years, we have been studying the levels of heavy metals in a variety of marine mammals. The desired end result of gathering this data has been to document "normal" metal burdens in tissues from these types of animals. This information could then be utilized to determine a possible cause of death in stranded animals, or to aid in rehabilitation of animals suspected of having metal poisoning. We have documented one stranding of Northern fur seals in the Puget Sound area as having been caused by a chemical spill of a toxic metal compound. (1).

With the cooperation of Mystic Marinelife Aquarium (MMA) we obtained tissues, along with the necropsy reports and medical histories, of five captive pinnipeds which had died. We analyzed the tissues for cadmium, chromium, lead, selenium, copper and zinc. These metals were chosen since they highly influence immune responses in animals and are considered to be both synergistic and anti-synergistic toxic agents. We were primarily interested in comparing ratios between the metals and comparing metal toxic symptoms with those symptoms in the necropsy reports.

When an animal is under physiological stress, such as caused by injury, disease or even surgery, the metabolic rate increases enormously. This increase will cause the migration of many substances, including the heavy metals, from tissue storage sites. These substances will be transported throughout the body and, in the case of toxins, will normally concentrate in either the liver for detoxification or in the kidneys for excretion. However, the transportation itself will also expose sensitive tissues to the toxic effect of metals, including both the T cell and B cell portions of the immune system.(2) This would further stress the animal and probably interfere with normal medical procedures, because if the animal was being treated for infectious agents the immune responses could be interfered with.

In this paper we find that in at least two of the cases, not only could the reported symptoms be due to metal poisoning, but also the cause of death could be attributed to metal poisoning. Diagnosing heavy metal poisoning is not a simple task since analytic and sampling techniques are not readily available to the veterinarian during the treatment for a suspected cause of illness. We do, however, suggest that awareness of metal toxic problems might assist the veterinarian in diagnosis and treatment of a visible problem.

Materials and Methods

Frozen tissue samples, along with the necropsy reports, were obtained from 5 pinnipeds which had died during captivity. These samples were as follows: 1) adrenal, diaphragm, heart, kidney, liver, lung, lymph, pancreas, spleen and testes samples from a 13 yr. old male Steller's sea lion; 2) heart, kidney, liver and lung from a 17 yr. old female Northern fur seal; 3) from a 4 day old male Northern fur seal, the samples included heart, kidney, liver and skin (fat and fur included); 4) heart, kidney, lung, spleen and liver from a 4-6 month old Northern elephant seal; and 5) tissues supplied from an 18 yr. old female California sea lion were brain, liver, heart, Muscle and fat. The frozen samples were cut in duplicate, weighed (approx. 5 gm) and dried by microwave, ground and the dry weight taken. This dried material was then digested for 24 hrs. in a water bath at 60 C using a 1:1:1:1 mixture of deionized-distilled water, sulfuric, nitric and perchloric acids. The analysis for the metals was accomplished by flame atomic absorption spectrophotometry using both air/acetylene and nitrous-oxide/acetylene techniques. The digestion technique was developed by this laboratory and has been used by our lab for the past few years.

Results

The results of the analyses are presented in Table 1, Figures 1 through 6 are graphical presentations for comparisons of tissue loads in heart, kidney, liver, lung, and spleen. We did not receive the same tissues from all animals. All results are expressed in parts per million per gram dry weight (ppm).

Figure 1. Cadmium

Cadmium levels in the Steller's sea lion were highest in the kidney (13.3 ppm), while in the other tissues the cadmium concentrations were relatively low (0.49 to 1.115 ppm). The female Northern fur seal had unusually high cadmium levels in the kidney (478.11 ppm), the liver (5.89 ppm) and in the lung (2.04 ppm) compared to the other animals. The California sea lion also showed a high concentration in the liver (3.03 ppm) while the other tissues ranged from 0.18 to 0.68 ppm. Both the Northern elephant seal and the Northern fur seal pup had low values in their tissues ranging from 0.25 to 0.56 ppm.

Figure 2. Chromium

The chromium levels in the tissues of the Steller's sea lion were variable but considerably higher than in any of the other animals, ranging from 1.88 ppm in lung to 18.76 ppm in heart tissue. The variability of this metal in the Northern fur seal and the California sea lion was less noticeable and values ranged from 1.00 to 1.60 ppm in the comparative tissues. The Muscle from the California sea lion, however, contained 2.39 ppm. The Northern elephant seal had no detectable amounts, in the kidney, spleen and liver, while the heart contained 1.58 ppm and the lung 0.71 ppm. The Northern fur seal pup had no detectable amounts in the kidney, while the other tissues ranged from 0.98 to 2.06 ppm.

Figure 3. Copper

There were variable amounts of copper within each animal for the tissue samples analyzed as well as variable amounts of copper in particular tissue for all five pinnipeds. The one outstanding feature was that in all the animals studied, except for the Northern fur seal, the liver always contained higher concentrations of copper than in the other tissues.

Figure 4. Lead

Ranges varied within each animal for the tissues analyzed, but when considering the similar tissues as a group, the lead levels appeared to be relatively consistent. For heart tissues the range was from 94 0.51 to 103 .16 ppm, kidney levels fell between 101 . 85 to 123 ppm, for liver the levels were 72.48 to 91.58 ppm, lung was 96.53 125.55 ppm and lastly levels of 90.8 to 104.16 ppm were found in spleen.

Figure 5. Selenium

There was only one value for selenium which was too high to calculate and that was in the liver of the California sea lion. Remaining concentrations were variable but quite consistent, with the one exception noted above, in each of the animals for the tissue analyzed. Heart selenium values ranged from 253.17 to 267 .64 ppm, kidney from 290.18 to 324.44 ppm, the liver (excluding the above) 217.7 to 273.99 ppm, lung tissue ranged from 260.53 to 348.45 ppm, and the spleen 256.39 to 217.12 ppm.

Figure 6. Zinc

Zinc values showed considerable variation. Zinc was higher in the female Northern fur seal in heart (273.8 ppm), kidney (336.84) and lung (180.98 ppm) than in tissues from the other four animals. In the Northern fur seal pup, however, the levels were higher in the liver (402.78 ppm) and spleen (194.57 ppm) than in the other animals.

Discussion

For purposes of discussion we will concentrate on the liver and kidney metal levels. Although the values obtained in this study for selenium and lead are much higher than in previous studies, the authors do not believe that the animals all had lead or selenium poisoning. The data available for comparison in the literature on previous studies may reflect the differences in both sampling and analytical (including instrumentation) techniques used by different researchers. It is for these reasons that we believe that to a certain extent the numbers calculated should not be taken literally, but used as a tool to observe trends, as we have attempted in this paper. The values for the other metals we analyzed are comparable to to previous reported results with some exceptions.

We include in this discussion the cause of death as stated in the, necropsy reports. In no instance are we disputing the findings in these reports, rather we are presenting our findings from the standpoint of metal toxicology, using the reported symptoms as a guideline in an attempt to determine whether or not a particular metal might have contributed or perhaps caused the animal's death.

Steller's Sea Lion

This animal was a 13 yr. old male who was scheduled for castration, but due to the location of the testes the surgery was not attempted and the animal was not fully sedated. Cause of death was reported as "probable anaesthetic idosyncrasy".

It has been shown that cadmium levels tend to accumulate in both liver and kidney with increasing age in the Harbour seal.(3) Despite the fact that the body burden of cadmium increases with age and that young animals absorb the metal to a greater degree than older animals, there does not appear to be a corresponding increase in cadmium excretion.(4) As to be expected, cadmium was found to be highest in the kidney of this animal. The data here reported does not, however, show a significantly high level in the liver. From the data one cannot determine whether or not there was a cadmium excess. The necropsy report, nonetheless, stated the presence fluids in the esophagus, pharynx and trachea and a hard yellow mass in the spleen, both of which do occur in cadmium poisoning. Proteinuria and heavy renal output which can result in dehydration and cardio-vascular problem are also symptoms of cadmium poisoning. ( 4 ) It is possible then that an animal being subjected to a surgical procedure, as this animal was to be, and if there was an unusual cadmium body burden, there could have been a contributing cause to its death. of further interest are the zinc levels in the liver and kidney. The zinc level in the liver was somewhat high but of greater interest is the high level in the kidney. Since zinc and cadmium are known anti-synergists,(5) the authors do not believe that this animal's death was caused by a metal poisoning, but the low value of cadmium in the liver in relation to zinc may indicate that the animal may have been suffering from a cadmium/zinc imbalance prior to the initiation of the surgical procedure.

The chromium levels in this animal were significantly higher than in the other animals in this study. However, when compared to levels reported by Richard and Skoch(5) in the livers of Northern fur seals, the values do not seem "abnormal". In general the remaining metals also seem to be within normal levels for pinnipeds.

Northern Fur Seal

This animal was a female (approx. 17 yrs.) who died within two clays after the ingestion of 3 lbs. of fish. She had apparently been healthy prior to this, since she had interacted with a male the previous day. Cause of death was tentatively listed as "septecemia".

This animal did not have unusual levels of chromium, lead or selenium. The other metals, however, seem to be "out of synch". The cadmium levels were unusually high in kidney, liver, and lung; while the copper levels were unexpectedly low in the liver, a normal storage site for the element.(4) Zinc also appeared higher than expected heart, kidney and lung while normal in the liver.(6,5).

The liver of this animal was the most affected by these three metals. Cadmium and zinc are anti-synergistic, and the data from kidney and lung gave a normal ratio for these two metals. In the liver, however, cadmium was higher than expected for the amount zinc present. This indicates potential cadmium poisoning. Another factor which indicates the potential for cadmium poisoning is the apparent reduction of copper in the liver.(7) We strongly believe the animal's burden of cadmium contributed to her death.

The fact that the animal was reported as lethargic and refused to eat may indicate gastric distress which can be attributed to cadmium-zinc toxic reaction.(4) Also, the liver was reported to be discolored. Since copper deficiency leads to a form of anemia that characterized by a reduction of hemoglobin,(7) the low copper level may account for the discoloration.

The necropsy report further mentions that the teeth were easily a removed, that the lung was bloody and that there was a prominent swelling in the shoulder area which proved to be filled with clotted blood and serum (with no evidence of external injury). These symptoms can all be accounted for by vasodilation caused by cadmium poisoning. A test for proteinuria might have confirmed this possibility.(4) The question that arises is whether the animal was subjected to an acute poisoning from the feeding or whether the animal had simply succumb age related stress and the metabolism of stored metal burdens, producing a death due to chronic poisoning.

Northern Elephant Seal

This 4 to 6 month old animal had been found stranded and subsequently been transferred to MMA from Marineland of the Pacific 10 days before its death. It had not been feeding well and blood samples showed leukocytosis with absolute neutrophilia. Antibiotics and feedings were started about 4 days prior to death. Cause of death stated as "broncho pneumonia".

The levels of cadmium, copper, selenium and zinc in this animal appeared to be relatively normal for pinnipeds. Chromium levels were not detectable in kidney, liver and spleen, however, which can increase susceptibility to lead poisoning. As a result, the potential of lead as being a catalyst or causative agent in the death of this animal was examined, along with the chromium deficiency.

Aortic plaques and diabetes melitis, both indicators of chromium deficiency, were not reported. There was however damage to the kidneys which suggest to us a problem with lead poisoning. For instance, interstitial fibrosis and glomular atrophy were reported in the histological findings, both of which are indicative of prolonged lead exposure.(4) The pathologist did state that these renal abnormalities were not the cause of death. In our viewpoint, however, these changes may well have contributed to the animals' difficulties. We believe that the animal was sufficiently deficient in chromium to allow lead to act as a toxic agent on the kidney. In our opinion the changes in the kidney were caused by a chronic exposure to lead well before the stranding. Siegmund and Fraser(8) advise that determinations of lead levels in kidney, liver or in unclotted blood can provide sufficient confirmation of lead poisoning. It should be noted that the lead levels in this animal were not that different from the other animals in this study, however, they were significantly different from the levels in the Northern fur seals (15.8 to 30.03ppm) reported by Richard and Skoch(5) and for kidney samples from the California sea lion containing lead concentrations of 2.0 to 2.4 ppm.(2).

Northern Fur Seal Pup

This male pup had been born four days prior to its death. It had not been nursing and was being tube fed. The cause of death was reported as "neonatal isoerythrolysis".

The only metal appearing to be abnormal in its concentration was 80.27 ppm of copper in the liver. This level seems unusual for what appears to be a possible neonate. The reported jaundice is an example of either copper and/or lead poisoning.(4) The pathology report also lists unusual erythrocyte formation which can be attributed to either neonatal isoerythrolysis as diagnosed, or to copper poisoning.(8) Our interpretation is that the animal was suffering from a copper poisoning which caused the reported intravascular hemolysis. This copper loading would have developed in utero. The lack of feeding then prevented the animal from getting sufficient iron and calcium, compounds which act as a protective agent against copper poisoning, thereby elevating the toxic effects of the copper. We suggest the possibility that a copper imbalance in utero might be the cause for neonatal isoerythrolysis.

California Sea Lion

This 18 yr. old female was a rehabilitated, stranded animal obtained from Marineland of the Pacific and had been at MMA for six years. It had a long history of epileptiform seizures which were being controlled by low medicinal doses. Anorexia occured two days prior to death. Cause of death was stated as "drowning subsequent to seizure".

This animal did not have unusual levels of the metals except in the case of liver levels of selenium and cadmium. Selenium is noted for its neurological toxicity in high doses (blind staggers in cattle) which could possibly account for the epileptiform seizures. The large amounts of clear yellow fluid in the thoracic cavity (ascites?) as well as a report of anorexia two days before its death also suggest a chronic exposure to selenium.(8) Normally cadmium or zinc can be used to detoxify non-critical levels of selenium poisoning, but the animal probably stranded initially due to a sub-lethal accumulation of selenium which permanently damaged its nervous system.

Summary

Due to new information and the increasing possibility of metal contamination in the marine environment, the authors stress the need in the future for more metal analyses on tissues from marine mammals. The potential for contamination in food sources used by marine parks and zoos should be a concern for husbandry programs. In suspected metal poisonings in the live animal, urine, fecal material and blood should be collected for metals analysis. Animals not responding to treatment for known infections should also be surveyed for metal loadings. Finally rehabilitated animals, which die in captivity, should also have metal loadings determined in the tissues. For these animals, the authors suggest the following tissues: blood, feces, both lobes of the liver, both kidneys, lymph nodes, bone and skeletal Muscle. The tissues should be frozen if possible or preserved in 95% alcohol. Again, the authors have not attempted to change the recorded causes of death, nor suggest any such thing in this paper.

Acknowledgement

We wish to acknowledge the cooperation of Dr. Larry Dunn and the people at Mystic Marinelife Aquarium, without whose help this paper would not have been presented.

References

1.  Skoch, E.J., Hoste, R. and Bral, C. 1985. Titanium, Aluminum and Fluoride Levels in Tissues of the Northern Fur Seal, Callorhinus ursinus. Proc. IAAAM 16th Conf. & Wkshp.

2.  Casarett Doull's T oxicology. The Basic Science of Poisons 3rd ed.1986. ed. C.D. Klaassen, M.O. Amdure and J. Doull. Macmillan Publishing Co. New York.

3.  Eisler, R. 1981. Trace Metal Concentrations in Marine Organisms. Pergamon Press. New York. pp. 651-678.

4.  Hammond, P.B. and Beliles, R.P 1980. Metals. In Casarett and Doull's Toxicology, The Basic Science of Poisons and 2nd ed. pp.409-462.

5.  Richard, C.A. and Skoch, E.J. 1986. Comparison of Heavy Metal Concentrations Between Specific Tissue Sites in the Northern Fur Seal. Proc. IAAAM 17th Conf. and Wkshp. pp. 94-103.

6.  Hoste, R., Skoch, E.J. and Grills, J. 1984. Heavy Metal Analysis in the Northern Fur Seal, Callorhinus ursinus. Proc. IAAAM 15th Ann. Conf.& Wkshp. 1:89-96.

7.  West, G.P. 1985. Black's Veterinary Dictionary, 15th ed. Barnes and Noble Books. New Jersey. pp. 7,188-190,450.

8.  Siegmund, O.H. and Fraser, C.M. 1979. The Merck Veterinary manual 5th ed. Merck and Co., New Jersey.