Marking Striped Bass with Rare Earth Elements

Robert J. Muncy, Mississippi Cooperative Fish and Wildlife Research Unit,' P.O. Drawer BX, Mississippi State, MS 39762.

Nick C. Parker, U.S. Fish and Wildlife Service, Southeastern Fish Culture Laboratory, Marion, AL 36756

Hugh A. Poston, U.S. fish and Wildlife Service, Tunison Laboratory Of Fish Nutrition, Contend, NY 13045

Abstract Non-radioactive rare earth elements (REE) were evaluated as potential markers in scales of hatchery-reared juvenile striped bass Morone saxatilis over a 12-week feeding study. Uptake and retention levels of europium (Eu) and terbium (Tb) detected by neutron activation analyses al below I m g/g could be related directly to dietary concentrations of the 2 elements and duration of feeding. Decreased relative concentrations following post-feeding could be related to scale mass increases and the inherent problem with detection techniques which analyze for amounts per unit mass. We compared our 1981 study with more recent similar studies as well as studies using different applications and detection techniques for REE. Low levels of REE Uptake limited availability and high cost of detection technology, masking effects resulting during poll-release growth, and potential wild-sources of REE place tagging techniques by feeding REE beyond practicability for most field fisheries studies at the present lime.

Proc. Annul Cord. Southeast. Assoc. Fish and Wildl. Agencies 41:244-250

Increased recreational and commercial fishing, as well as suspected adverse localized and global impacts from man-caused environmental changes, have refocused interests of biologists on improved tagging and marking techniques to evaluate potential roles for hatchery fish. The non-radioactive rare earth elements (REE) samarium (Sm) and europium (Eu) have been suggested by Michibula (1981) as potential and by Kato (1985) as successful markers in enriched diets when fed to fish for I to 2 months. However, Sm levels in American shad (Alosa sapidissima) and Atlantic salmon (Salmo salar) were only slightly higher than in control fish 30 to 60 days after feeding with Sm-enriched diets ( 1984).

Neutron activation analyses (NAA) were conducted on 3 occasions by personnel of the Phoenix Memorial Laboratory Ford Nuclear Reactor at the University of Michigan, Ann Arbor. Known U.S. Geological Survey rock standards (USGS- I, USGS-G-2) and blanks were used as checks for peak energy and area for 22 elements. Spectrum detection time varied from 30 to 100 minutes. One-sigma (SD) levels, calculated as the square root for that element against the background level of all other radiation at the detection peak, varied over all scale samples. Skoog (1985) reported possible sensitivities of 5 ph for Eu and 50 mg for Tb. The actual sample composition, especially without chemical separation, as well as the blank signal affect the prior detection limits (Heydorn 1984).

Scales of striped bass, fed high levels of Eu and Tb for 84 days, were examined by personnel of Mississippi State University Electron Microscope Center using an electron scanning microscope with x-ray spectrometer (ESM) operating at accelerating voltages of 10, I S. 20, and 30 kv and computer analyzed at 30 to the detector for 2 hours when examining whole scales as well as recent scale edge growth zones. Although Skoog (1985) suggests that electron spectroscopy information is restricted largely to surface layers, Postek (1980) indicated that x-ray production initially increased with specimen thickness. The voltage and analysis times used in our study would insure x-ray penetration and analyses to variable depths. Skoog (1985) suggested that x-ray photoelectric spectroscopy for qualitative analysis can resolve peaks leading to unambiguous identification if the element is present in concentrations greater than 0.1 % (100 m g).

Secondary ion mass spectroscopy (SIMS) analysis of striped bass scales by Dr. George Morrison, Department of Chemistry at Cornell University, Ithaca, New York, was prevented by charged ion buildup on scale surfaces. Scales ashed under vacuum could not be maintained as to position nor shape when the vacuum was released; therefore, SIMS proved impractical for whole scale analyses.

Results and Discussion

Relative concentrations of Eu and Tb in striped bass scales detected by NAA increased over feeding time (day 28 and 84) in relation to the levels of REE in experimental diets (Table 1). Accuracy of values not detected above l-sigma error cannot be verified and actual values may be much less than listed value. Differences between relative concentrations detected in scales from high REE and low REE diets was 4 to 5 times, whereas differences in diet levels were 8 to 10 fold. Detected levels remained below I ug/g as other researchers (Miller 1963, Anonymous 1974, Shibuya 1979, Zak 1984, Kato 1985) have generally found when examining bony substances during and following feeding trials. It does not appear possible to increase REE levels much above I m g/g in bones when feeding REE salts or oxides.

Retention of REE within the bony matrix as fish grow has been a matter of concern if absorped REE are to be useful long-term markers. Michibuta (1981) stated that the mechanism for the retention of Sm remains to be resolved even Mulligan (1981) used ESM differences in chemical composition of freshwater growth regions of scales from adult sockeye salmon (O. nerka) to classify their lakes of origin based upon elemental scale composition.

Since a simple presence or absence criteria would be highly desirable, we attempted in 1986 to learn whether concentrated Eu or Tb zones were deposited during feeding and post-feeding growth of scales as has been found in fish fed tetracylines (Bilton 1986). Earlier NAA analyses had detected less than I m g/g (Table 1) Eu and Tb in the whole scales but we hoped to see whether higher concentrations could be detected by SEM near recent growth zones of scales collected on day 84. Our unsuccessful efforts, with SEM x-ray electron probe and with SIMS, indicated Eu and Tb concentrations were below milligram (0.1%) levels in recent growth zones.

Our experimental feeding trials as well as those by Kato (1985) and Zak (1984), demonstrated that feeding diets enriched with 500 to 1,000 m g/g REE salts resulted in defection at less than 1 m g/g REE depositions in scales of fish. Subsequent growth of test fish caused dilutions of those relative concentrations by increased scale mass. Kato (1985) apparently has been able to overcome this dilution and masking effect for Eu in chum salmon scales by allowing scales to cool for a 6-month period following thermal neutron flux of 8 x 1013 neutrons/cm2s for 20 minutes. The longer cooling time reduced background radiation, especially from 45Ca. Although we were not aware of Kato's (1985) techniques and results when securing NAA services in 1983, we cannot be assured that the small differences noted between treatments in 530-day samples (Table 1) could be considered valid following additional growth when compared with wild stocks at later time intervals (Table 2). Our failure to locate Eu or Tb concentration by SEM x-ray or SIMS techniques eliminated the possibility of a distinctive concentrated REE zone in scales from treated fish as demonstrated on vertebrae of chum salmon by Bilton (1986) following feeding of oxytetracycline in the diet for 14 to 21 days.

Although an ideal method for marking any fish is probably illusory (Laird and Scott 1978), evaluation of older as well as new techniques continues to evolve with improved technology. Wydoski and Emery (1983), mentioning metallic compounds and radioisotopes as methods of marking by immersion, injection, tattooing, and feeding presented some favorable but mostly unfavorable evaluations. The array of analytical methods available for laboratory analyses of the structural, physical, chemical, and molecular properties of biological materials have increased to a bewildering degree (Skoog 1985). Detection limits depend upon the sensitivity of the instrument, operator skills, as well as the materials being analyzed. According to Skoog (1985:5), "Data from physical measurements are always plagued by uncertainties or errors.... The work required in evaluating the quality of data is frequently comparable IO the effort that goes into obtaining them. Ultimately, the scientist can only make a judgement as to the probable accuracy of a measurement; with experience, judgements of this kind tend to become rather harsher and less optimistic."

Skoog, D. A. 1985. Principals of instrument analysis. 3rd ed. Saunders Coll. Publ., Philadelphia, Pa. 509pp.

Wydoski, R. and L. Emery. 1983. Tagging and marking. Pages 215-237 in L. A. Nielsen and D. L. Johnson. eds. Fisheries techniques. Am. Fish. Soc., Beheads, Md.

Zak, M. A. 1984. Mass marking American shad and Atlantic salmon with the rare earth element, samarium. MS Thesis, Pa. State Univ., State College. 88pp. 1987 Proc. Annul Conf. SEAFWA