American Fisheries Society Symposium 7:541-546, 1990

Inorganic Chemical Marks Induced in Fish

U.S. Fish and Wildlife Service
Mississippi Cooperative Fish and Wildlife Research Unit
Post Office Drawer BX, Mississippi State, Mississippi 39762, USA
U.S. Fish and Wildlife Service
Southeastern Fish Culture Laboratory
Route 3, Box 86, Marion, Alabama 36756, USA
U.S. Fish and Wildlife Service
Tunison Laboratory of Fish Nutrition, Cortland, New York 13/)45, USA

1Present address: Route 3, Box 299, Moneta, Virginia 24121, USA.
2Present address: Texas Cooperative Fish and Wildlife Research Unit, Department of Range and Wildlife Sciences, Texas Tech University, Lubbock, Texas 79409-2125, USA.

Abstract. -Researchers have attempted to mark batches of fish internally through feeding, immersion, and injections with stains, dyes, radioactive isotopes, rare earth compounds, metallic elements, and fluorescent compounds. In general, immersion in dyes and stains produced short-term marks detectable for days rather than years. Injections of dyes and stains provided marks detectable for longer periods, but they subjected fish to greater stresses during handling and marking. Rare earth salts administered in fish feeds were deposited in the body structure at a level of a I ,ug/g or less, and detection by neutron activation analysis was difficult after 6 months. When oxytetracycline and calcein were injected or fed to fish, they produced marks that remained detectable after several years in both captive and wild individuals.

Internal marking with chemicals has been considered for rapidly marking large numbers of fish of various sizes without handling them individually (Emery and Wydoski 1987). Research on fish nutrition, physiology, and toxicology has increased knowledge of the uptake and internal metabolism of chemical compounds tested as internal markers. In general, when fish are fed or immersed in solutions of chemicals, metabolically active compounds are taken up more rapidly, reach higher concentrations in the body, and are dispersed and excreted faster than are metabolically inactive compounds. Fish metabolism and growth can dissipate or dilute chemicals not strongly bonded within more stable systems such as bone. Direct injection of less reactive metallic compounds (such as rare earth salts), liquid latex,by immersion, injection, and feeding (Babb et al. 1967; Shibuya 1979; Michibata 1981; Michibata and Hori 1981; Muncy and D'Silva 1981; Zak 1984; Kato 1985). Incorporation of visible and fluorescent chemicals into new bone has sparked continued interest in marking fish by immersion, injection, and feeding of such substances as lead versenate (Fry et al. 1960; Jensen and Cummings 1967), tetracycline compounds (Weber and Ridgway 1962; Bilton 1986; Koenings et al. 1986; Babaluk and Campbell 1987), and calcein (Wilson et al. 1987).

Guillou and de La Noue (1987) suggested that hatchery-reared fish released in the wild be identified by their levels of specific elements and by statistical techniques that detect combinations of elements absorbed from hatchery diets. Microanalytical and statistical techniques were applied to identify different elements or combinations of compounds known as chemoprints (Calaprice et al. 1971) in wild fish stocks (Lapi and Mulligan 1981; Mulligan et al. 1983; Schroeder 1983; Wiener et al. 1984; Guillou and de La Noue 1987). Behrens Yamada et al. (1987) cautioned that stock and treatment effects, as well as influences of most elements, have not been well studied.

Application and Detection Techniques

Researchers have tested dyes and stains, rare earth elements, heavy metals, calcein, and tetracycline compounds for incorporation into fish eggs (Muncy and D'Silva 1981), larval fish (Jessop 1973; Hettler 1984; Behrens Yamada and Mulligan 1987), and juvenile fish (Ennis and Ziebell 1965; Zak 1984; Koenings et al. 1986; Babaluk and Campbell 1987; Wilson et al. 1987). Dyes and stains such as Bismark brown Y. natonal fast blue 8GXM, and hydrated chromium oxide have been used mainly for short-term marking because they fade over time (Kelly 1967; Jessop 1973; Everhart et al. 1975; Wydoski and Emery 1983). Fluorescent compounds of tetracycline and calcein bind to alkaline earth metals in fish tissues (Wilson et al. 1987) and to bones during periods of growth. Alkaline earth metals, when bonded to recently formed bone, reduced the dispersal of fluorescent compounds (Koenings et al. 1986).

Externally visible dyes and stains can be detected without instruments, but they tend to increase predation on marked fish. Injections of metallic cadmium sulfide, mercuric sulfide (Hansen and Stauffer 1964), and chromium oxide (Kelly 1967) lasted more than I year, whereas most dyes and stains dispersed more rapidly. Fluorescent marks can be examined under ultraviolet light (Hettler 1984; Bilton 1986; Wilson et al. 1987) and detected at levels of less than 0.2 Ug/g by fluorometric techniques (Koenings et al. 1986).

Detecting low concentrations of nonvisible chemical compounds requires sophisticated support facilities and trained operators. Rare earth elements have been analyzed in fish samples by means of neutron activation analysis, X-ray-excited optical luminescence, dye laser techniques, resonance ionization spectroscopy, and atomic absorption spectroscopy. X-ray fluorescecse spectroscopy has proved effective in detecting small (ug/g) concentrations of strontium (Behrens Yamada and Mulligan 1987). Lapi and Mulligan (1981) were able to identify the freshwater lakes from which sockeye salmon Oncorhynchus nerka originated by the chemical composition of the scales as revealed by X-ray scanning electron microscopy.

Retention of Internal Chemical marks

Injection of dyes and stains (Kelly 1967) usually has resulted in short-term marks (Laird and Stott 1978; Wydoski and Emery 1983). In comparative tests summarized by Table 1, rare earth compounds lasted longer at higher concentrations when injected than when administered by immersion or feeding techniques (Miller 1963; Michibata and Hori 1981; Zak 1984). Injection of metallic compounds (Fry et al. 1960; Hansen and Stauffer 1964) produced intensive color patterns recognizable for up to 4 years. Injection of tetracycline (Smith 1984; Babaluk and Campbell 1987) provided internal marks recognizable longer than 2 years.

Researchers have successfully marked small fish by immersing them in strontium (Behrens Yamada and Mulligan 1987), tetracycline (Hettler 1984), and calcein solutions (Wilson et al. 1987), but postimmersion exposure of small fish to sunlight may reduce detectable levels of oxytetracycline (Lorson and Mudrak 1987). Eggs of walleyes Stizastedion vitreum immersed in europium and terbium salts retained up to 5 ug/g, and sac fry hatched from marked eggs contained from 5 to 50 ng/g (Muncy and D'Silva 1981).

When salts of the rare earth elements terbium and europium were fed to striped bass Morone saxatilis for 84 d, they accumulated in scales at levels (detectable by neutron activation analysis) below I ug/g. The levels corresponded to dietary concentrations and duration of feeding. Relative concentrations of rare earth elements decreased during a 400-d period after termination of the labeled diets probably because of dilution within the scale mass by growth (Muncy et al. 1988). Dilution of rare earth elements in fish scales was also reported by Michibata (1981), Zak (1984), and Kato (1985). Feeding of oxytetracycline (OTC) to fry of rainbow trout Oncorhynchus mykiss (Trojnar 1973) and chum salmon O. keta (Bilton 1986) produced concentrated fluorescent bands still visible under ultraviolet light I and 2 years later.

Koenings et al. (1986) demonstrated that concentrations of OTC in juvenile sockeye salmon O. nerka, detected by fluorometric procedures, declined in muscle and other soft tissues soon after treatment stopped but remained stable in bones; also, uptake of OTC by bones increased over the period of feeding. Moser et al. (1986) found little uptake of stains or tetracycline from one feeding. Improvements in technique allowed Koenings et al. (1986) to measure the uptake, loss, and retention of fluorescent compounds at concentrations as small as 0.17 Agog. The percentages of fish marked by different tetracycline formulations and concentrations, treatment techniques, and lengths of treatment have been determined by several investigators, including Weber and Ridgway (1962), Scidmore and Olson (1969), Troinar (1973), Odense and Logan (1974), Hettler (1984), Bilton (1986), Babaluk and Campbell (1987), Lorson and Mudrak (1987). The use of tetracycline to validate age marks for fish would require increased levels if the OTC marks were to be located visually (Smith 1984; Babaluk and Campbell 1987).

TABLE l.-Chemical methods and detection techniques used for marking fish internally. Application techniques: In, injection; F. feeding; Im, immersion. Detection techniques: V, visual; Uv, ultraviolet; NAA, neutron activation analysis; AAS, atomic absorption spectroscopy; XEOL, X-ray-excited optical luminescence; XFS, X-ray fluorescence spectroscopy; DL, dye laser; Fl, fluorometric; EM, electron microscopy; RIS, resonance ionization spectroscopy. The time required to apply a mark and the period of detectability for a specific concentration are given when applicable.

Chemical Application Detection Amount detected(ug/g) Reference
Method Time Method Time
Dyes and stains Im 3h V 6d Jessop (1973)
Moser et al. (1986)
Kelly (1967)
F ? V 77d
In V 1 year
Rare earth elements Im 0.5 h XEOL 21d 5 Muncy and D'Silva (1981)
Zak (1984)
Muncy et al. (1988)
Coutant (1990)
Kato (1985)
Michibata and Hori
Im 0.5 h DL 10 d 0.0002
Im,F 30 d AAS 30 d 2,000
F 84 d NAA 1.5 year 0.6
F 84 d RIS 1.5 year 0.1
F 40 d NAA 2 years 0.1
In NAA 2 years 1
Tetracycline Im 2 h V, Uv 8 d 0.6 Hettler (1984) 
Bilton (1986)
Koenings et al. (1986)
Babaluk and Campbell (1987)
F 14 d Uv 2 years
F 40 d Fl 1 year
In Uv 2 years
Calcein Im 2 h Uv 27 d Wilson et al. (1987)
Pollutants F EM ? Lapi and Mulligan (1981)
F AAS 0.05 Wiener et al. (1984)
Lead In V 2 years Fry et al. (1960)
Cadmium In V 4 years Hansen and Stauffer (1964)
Mercury In V 4 years Hansen and Stauffer (1964)
Cobalt Im 1 d NAA 36 d ? Hoss (1967)
Strontium F 42 d AAS 42 D 200 Guillou and de la Noue (1987) Behrens Yamada et al. (1987) Behrens Yamada and Mulligan (1987)
F 80 d XFS 75 d 1
Im 49 d XFS 169 d 1
Manganese F 60 d XFS 75 d 1 Behrens Yamada et al. (1987)
Natural mixtures N EM 1 Calaprice et al. (1971)
Calaprice et al. (1971)
Mulligan et al. (1983)

a Unpublished. McWilliams, R. H. 1986. Natural lakes investigations. Iowa Conservation Commission, Federal Aid Fish Restoration, Project F-95-R, Annual Report, Des Moines.




The experience of Muncy and D'Silva (1981) and Muncy et al. (1988), plus this review of the published literature on marking fishes with chemicals, indicates that application of chemical markers continues to be limited by inadequate fielddetection techniques. Compounds such as rare earth salts are not normally abundant in natural environments of fish and may not be readily absorbed at levels above 5 p~g/g (Zak 1984; Kato 1985; Muncy et al. 1988). Nutritional studies on mammals (Hutcheson et al. 1979; Kennelly et al. 1980) have demonstrated low absorption of lanthanide markers from the digestive tract. Compounds that are both readily available and absorbed, such as strontium (Behrens Yamada et al. 1987; Guillou and de La Noue 1987), can be masked by background levels taken up by fish from some environments.

Lapi and Mulligan (1981) used microanalytical techniques to distinguish sockeye salmon by differences in the chemical composition of their scales and to classify mixed stocks of these fish according to their natal lakes. Fluorescent compounds (OTC and calcein) bonded to particular sites in bone during active growth appear to have better potential now for field studies of fish because detection techniques are more readily available and less expensive than those used for rare earth elements. Identification of fish marked with fluorescent compounds may be complicated by the presence of hatchery-reared fish fed OTC for disease control and the presence of natural fluorescent compounds in study areas.

Growth of the aquaculture industry has increased the need to distinguish farm-raised from wild stocks, both to ensure that marketed fish are legal and to provide quality control; these additional incentives may stimulate the development

of improved detection techniques for fluorescent compounds (Parker 1988). Analyses of soft tissues and bones of fish from highly variable natural and aquaculture environments (Behrens Yamada et al. 1987; Coutant 1990, this volume) present many challenges and opportunities for improving instrumentation (Skoog 1985) to detect chemical fingerprints. To obtain the maximum value of tools and techniques available, researchers must understand the basis of analytical techniques and be aware of improvements that lower detection limits, mask background levels, remove interferences, and determine the metabolic pathways of uptake and incorporation of chemicals used to mark fish internally.


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Hansen, M. J., and T. M. Stauffer. 1964. Cadmium sulfide and mercuric sulfide for marking sea lamprey larvae. Transactions of the American Fisheries Society 93:21-26.

Harrell, R. M. 1985. Experimental marking techniques for young-of-year, hatchery-reared striped bass. Proceedings of the Annual Conference Southeastern Association of Fish and Wildlife Agencies 37:295-303.

Hettler, W. F. 1984. Marking otoliths by immersion of marine fish larvae in tetracycline. Transactions of the American Fisheries Society 113:370-373.

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Hoss, D. E. 1967. Marking post-larval paralichthid flounders with radioactive elements. Transactions of the American Fisheries Society 96:151-156.

Hutcheson, D. P., B. Venugopal, D. H. Gray, and T. Luckey. 1979. Lanthanide markers in a single sample for nutrient studies in humans. Journal of Nutrition 109:702-707.

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Jessop, B. M. 1973. Marking alewife fry with biological stains. Progressive Fish-Culturist 35:90-93.

Kato, M. 1985. Recent information on europium marking techniques for chum salmon. NOAA (National Oceanic and Atmospheric Administration) Technical Report NMFS (National Marine Fisheries Service) 27:67-73.

Kelly, W. H. 1967. Marking freshwater and a marine fish by injected dyes. Transactions of the American Fisheries Society 96: 163-175.

Kennelly, J. J., F. X. Aherne, and M. J. Apps. 1980. Dysprosium as an inert marker for swine digestibility studies. Canadian Journal of Animal Science 60:441-446.

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Miller, W. P. 1963. Neutron activation analysis of stable dysprosium biologically deposited in the bone of chinook salmon fingerlings. Master's thesis. University of Washington, Seattle.

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Scott, D. P. 1961. Radioactive iron as a fish mark. Journal of the Fisheries Research Board of Canada 18:383-391.

Scott, D. P. 1962. Radioactive caesium as a fish and lamprey mark. Journal of the Fisheries Research Board of Canada 19:149-157.

Shibuya, M. 1979. Non-destructive activation analysis for Eu in fish materials. Radioisotopes 28:64-68. (In Japanese.)

Skoog, D. A. 1985. Principles of instrumental analysis, 3rd edition. Saunders, Philadelphia.

Smith, S. E. 1984. Timing of vertebral-band deposition in tetracycline-injected leopard sharks. Transactions of the American Fisheries Society 113:308-313.

Trojnar, J. R. 1973. Marking rainbow trout fry with tetracycline. Progressive Fish-Culturist 35:52-54.

Weber, D., and G. Ridgway.1962. The deposition of tetracycline drugs in bones and scales of fish and its possible use for marking. Progressive Fish-Culturist 24:150-155.

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Zak, M. A. 1984. Mass-marking American shad and Atlantic salmon with the rare earth element, samarium. Master's thesis. Pennsylvania State University, State College.






and pigments bypasses metabolic barriers but exposes individual fish to increased handling stress (Hoss 1967; Harrell 1985). Batch-marking precludes the recognition of individual fish (Wydoski and Emery 1983).

Recently improved radioisotope techniques (Heydorn 1984) and detection instruments (Willard et al. 1981; Skoog 1985) have expanded research capabilities to include more of the 12 characteristics listed by Everhart et al. (1975) for the "ideal mark." Radioactive iron and cesium (Scott 1961, 1962) and cerium and cobalt (Hoss 1967) have been used to mark fish; however, human health and safety concerns and adverse public reaction toward radioactivity (Everhart et al. 1975) have constrained the use of radioactive isotopes in field studies. Nonradioactive rare earth compounds of dysprosium were tested first by Miller (1963) as markers for fingerling chinook salmon Oncorhynchus tshawytscha. Subsequent studies addressed the uptake, retention, and detection potentials and problems of rare earth compounds administered to various fish species