Abstract

Replicate samples of kidney and Muscle tissue from one California sea lion were analyzed for heavy metal concentrations using three different methodologies at the Marine Mammal Lab at John Carroll University. The metals were analyzed via A.A. spectroscopy and included lead, copper, iron, cadmium, chromium, and nickel. This analysis was the basis for a methodological comparison study between an established procedure and two other relatively recent modifications. The recoveries for all metals in tissues improved from 0.00 % to 567.3% between the techniques, but varied with each metal and tissue studied. Results, therefore, point to the need to establish standard procedures for this type of analysis while not ideal in every case, provides the most improved recoveries.

Introduction

Heavy metal accumulations in members of the cetacean and pinniped orders are sparse throughout literature. Few metals have been analyzed, primarily lead and mercury, and few tissue types, mostly liver, kidney and Muscle have been studied. Furthermore, most investigations involved only small numbers of individuals (usually four or five). The concern being addressed here, however, is the difficulty in correlating such information due to the variation of methods used in determining heavy metal concentrations in tissues.

Some existing literature shows that California sea lions, (Zalophus Californianus) appearing healthy from the Oregon coastal waters had a cadmium content of 10.2 ppm (Buhler Et Al., 1975). Harbor seals (Phoca Vitulina), and Northern fur seals have shown kidney levels of 0.17 ppm (Buhler, Et Al., 1975), and 15.6 ppm (Anas, 1974) respectively. Lead was found in Harbor seal bone at 3.5 ppm (Roberts Et Al., 1976), and in liver at 0.74 ppm (Harms Et Al., 1978).

This study has been done in order to arrive at a consistent and accurate technique for heavy metal analysis by comparing an established digestion procedure and two other relatively recent modifications.

Materials and Methods

Replicate samples of kidney and Muscle from one California sea lion were analyzed for heavy metal concentrations using three different methodologies at the Marine Mammal Research Lab at John Carroll University. The metals were analyzed via A.A. spectroscopy and included lead, copper, iron, cadmium, chromium, and nickel.

The techniques for digestion of the tissues were as follows:

Method A: was adopted and modified from the acid digestion/ashing techniques found in Detection and Determination of Trace Elements (Pinta, 1962) and Analytical Methods for Atomic Absorption Spectrometry (Anonymous, 1973).

In this method, frozen tissue samples were cut, weighed (approximately 5 grams wet weight) and placed in acid washed 25x100mm Pyrex culture tubes. To these samples, a mixture of concentrated nitric acid, sulfuric acid and distilled/deionized water (1:1:1) was added. The samples were then loosely capped (to prevent pressure build-up), and placed in a 60 c water bath for 24 hours, enough time to digest the organic material. They were then capped with inverted 20ml beakers and placed into the ash furnace. The samples were slowly ashed from an initial temperature of 80 c until 2-3 cm of fluid remained. The temperature was then raised to 100 c until no fluid remained. At that point the temperature was raised 25 c every hour to a final temperature of 545 c, at which it was maintained for 24 hours. The samples were cooled, and 50ml of a 3% nitric acid solution was added to each tube in order to dissolve the remaining ash. If samples were incompletely ashed (black residue), the tubes and contents were re-digested and re-ashed in the furnace as stated previously. Analysis was done using Flame Atomic Absorption Spectroscopy with an Instrumentation Laboratory (Jarell-Ash) Video 11 Flame Atomic Absorption Spectrophotometer with nitrous oxide-acetylene, and air-acetylene gas mixtures. The recommended element specifications for the Jarell-Ash Video 11 were used for each metal in each of the three methods.

Method B: The samples were cut frozen and weighed (approximately 5 grams wet weight). These tissues were placed in small Pyrex petrie dishes and dried in a microwave at 20% power for 20 minutes, then at 60% power for 10 minutes. The dried tissue was then ground to a fine powder with the use of a microsampler cup adapter placed on a commercial Waring blender. The powdered tissue samples were transferred to a tared screwcap, 50ml culture tube, and a dry weight was obtained. A twelve milliliter solution containing distilled/deionized water, concentrated nitric, sulfuric, and perchloric acids, in a 3:3:2:2 ratio respectively, was added to each test tube. The tubes were then thoroughly vortexed and placed in an 80 c water bath (loosely capped) for 24 hours, until all of the organic material dissolved. The resulting clear, dark yellow solution was then diluted with distilled/deionized water to a final volume of 50 mi. The analysis for this digestion technique was also performed using the Jarell-Ash Video 11.

Method C: The samples were treated in the same manner as in Method B, previously stated. The acid solution, however, was changed to one of concentrated sulfuric, nitric, perchloric and distilled/deionized water in a 1:1:1:1 ratio.

Accuracy of technique was determined by standard additions and additions of blanks. Percent recovery for all metals ranged between 87% and 99%. The data was analyzed for significance with a one-tailed and two-tailed 'IT" test, using the Minitab Handbook (Ryan Et Al., 1985). These tests allow one to compare the means of a certain trait between populations, most accurately when a sample size is small. The one-tailed test demonstrates the magnitude and direction of a value as it varies from the overall mean. The two-tailed test demonstrates the significance of a value in a population in comparison to the overall mean. The confidence interval used for all the data was 95%. A third test, the "Z" test, was also performed to check the results of the two previous analyses. The "Z" test, also from the Minitab Handbook (Ryan Et Al., 1985), provides the same comparisons and is used for "normal" data. The confidence interval for the "Z" test was also 95%. There were no differences found between the "Z" and 'IT" tests used for statistical accuracy.

Results

Lead is a metal with no known essential value within a mammal. It is a strong neurotoxin, and if concentrated in high amounts within a tissue, can be lethal. The mean values for lead were found to be 1.82 ppm for Method A, 9.82 ppm for Method B and 16.33 ppm for Method C in Muscle tissue. In kidney tissue, the mean values for lead in Methods A, B and C were 1.68 ppm, 13-08 ppm and 24.52 ppm, respectively (Figure 1). The percent differences between the methods ranged from 49.8% to 159.8% for Muscle tissue, and 60.9% to 567.3% in kidney tissue (Table 1).

Table 1. Percent Differences Between Methods of Acid Ingestion

Braham (1973) reported a mean value of 8.1 ppm in brain tissue from California sea lions, yet a higher mean value of 8.8 ppm was found in kidney tissue. In addition, lead was detected in gonad tissue (5.6 ppm) and liver tissue (3.0 ppm). Richards (1985), on Northern fur seals from the Pribilofs, reports a mean lead value of 30.03 ppm in kidney, and 21.50 ppm in Muscle.

Copper and iron are usually found in highest concentrations in the liver. Concentrations may vary depending on which lobe of the liver is sampled and analyzed. The obvious reason is that the liver does not concentrate copper and iron equally. This may also be true for other metals and tissues, including brain and kidney. Iron is also highly concentrated in Muscle due to the myoglobin present. Thus, copper and iron values, without adequate medical background on the animal, may be difficult to interpret.

The mean values for iron in Muscle tissue for Methods A, and C were found to be 31.57 ppm, 957.15 ppm and 932.73 ppm, respectively. The mean values for iron in the kidney tissue found to be 0.00 ppm, 35.08 ppm and 38.51 ppm for Methods A, E and C respectively (Figure 2). The percent difference for the three methods for iron ranged from 2.6% to 187.2% for Muscle tissue, and 9.3% to 200% for the kidney tissue (Table 1).

The mean background values for cadmium in tissues analyzed ranged from 0.00 ppm to 26.91 ppm. No cadmium was found in Muscle tissue for any of the methods. The mean values for cadmium in kidney tissue was found to be 8.46 ppm, 26.91 ppm, 26.71 ppm for methods A, B and C respectively (Figure 4).

The major target organs for cadmium are the liver and the kidney. This is due to the presence of metallothionein in organs, to which cadmium binds. The percent differences for cadmium between the methods ranged from 0.00% to 0.00% for Muscle, and 0.75% to 104.3% for kidney tissue, (Table 1).

The mean levels for chromium in the Muscle tissue analyzed were 7.93 ppm,

26.04 ppm, and 33.92 ppm for Methods A, B and C respectively. The mean levels for kidney tissue in Methods A , and C were found to be 9.77 ppm, 56.25 ppm, and 38.45 ppm respectively (Figure 5). The percent differences for chromium between methods for Muscle tissue ranged from 26.3% to 1.214.2%, and from 9.5% to 119.0% for kidney tissue (Table 1).

Very little chromium analysis in marine mammal tissue has been noted in published reports. Because chromium in man readily passed through the gastrointestinal tract, little chromium should be expected in the internal organs. These results, however, point to a need for a conclusive study for marine mammals. Richards (11985), does report mean chromium values on Pribilof fur seals as 111.56 ppm in kidney, and 30-43 ppm in Muscle.

The mean values obtained for nickel in Muscle tissues were 1.9-3 ppm, 19.4 ppm, and 11.84 ppm for Methods A, B and C respectively- The mean values for kidney tissue in Methods A, B and C were found to be 3.16 ppm, 29.59 ppm, and 17.67 ppm respect respectively (Figure 6). The percent difference in Muscle ranged from 16 . 5 % to 146.5%, and 50 . 5 % to 161.4% for kidney tissue (Table 1).

Discussion

It has been important to look at trends and parallel phenomena that surface in each report of metal levels in tissues. One major reason for this approach is the variety of ways concentrations are reported. For example, some use wet weight instead of dry weight. Values reported as ppm/g wet weight vs. dry weight may vary by 10 to 100 times with respect to each other. But at issue here is that various extraction techniques are suggested for metal studies, but they are specific for the metal to be determined. Thus, metal concentrations can vary greatly from one paper to the next. The data in this is report is important in the establishment of a more accurate, reliable and less time consuming method for the analysis of metals. Of the methods used by the Marine Mammal Research Lab at John Carroll University, the third method, using microwave drying and the acid mixture of concentrated nitric, sulfuric, perchloric and distilled/deionized a ratio, proves to be the most reliable and less time consuming in use. This reliability is reflected in both the raw data results, mean yield, and in statistical analysis for all the metals tested in this paper. These results are believed to carry over to other metals, which has persuaded the authors to recommend the use of this technique consistently for more accurate results. This method, although it may not prove to be in every case, provides the most consistent improved.

Figure 1

Figure 2

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Figure 5

Figure 6

References

1.  Anonymous. 1973. Analytical Methods for Atomic Absorption Spectrometry. Perkin Elmer Corporation, Norwalk, Conn.

2.  Anas, R.E. 1974. Heavy Metals in the Northern Fur Seal Callorhinus Ursinus and the Harbour Seal Phoca Vitulina Richardi. 2(1) 133-137.

3.  Braham, H.W. 1973. Lead in the California Sea Lion (Zalophus Californianus). Environ. Poll. 5: 253-258.

4.  Buhler, D.R., R.R. Claeys and B.R. Mate. 1975. Heavy Metals and Chlorinated Hydrocarbons Residues in California Sea Lions (Zalophus Californianus). J. Fish. Res. Bd- Canada. 32: 2391-2397.

5.  Harms, U.H. Drescher and E. Huschenbeth. 1978. Further Data on Heavy Metals and Organochlorines in Marine Mammals from the German Coastal Waters. Meers Fonsch. 26: 153-161.

6.  Pinta, M. 1962. Detection and Determination of Trace Elements. Dunod, Paris.

7.  Richards, C.A., Skoch, E.J. 1985. Comparison of Heavy Metal Concentrations Between Specific Tissue Sites in the Northern Fur Seals. IAAAM Proceedings; 17th Annual Conference.

8.  Roberts, T.M., P.B. Heppleston and R.D. Roberts. 1976. Heavy Metals and Tissues in the Common Seal. Marine Pollution Bulletin. 7: 194-196.

9.  Ryan, B.F., B.L. Joiner and T.A. Ryan, Jr. 1985. Minitab Handbook. PWS Publishers, Boston.