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t 92 MUTH ET AL. TABLE l.-Relative intensity of fluorescent marks in otoliths from Colorado squawfish larvae that were im- mersed in tetracycline hydrochloride (TC} solutions. Mark intensities were determined for five larvae from each treatment and sampling time (days after treatment) and were ranked 0-3 (absent-bright). Values were summed within each sample lot to produce a batch mark-intensity value. All fish immersed in TC solutions of 350 or 500 mg/L for 36 h died during treatment. Mark intensity by TC concentration and exposure time Days after 200 mg/L 350 mg/L 500 mg~L treatment 4 b 12 h 36 h 4 b 12 h 4 h 12 h 0 5 9 10 10 14 !0 14 7 5 10 12 12 15 13 14 28 5 8 12 15 14 14 15 56 5 10 13 13 IS 14 77 5 9 13 14 t4 14 Figure 2). Marks appeared as yellow fluorescent bands against adark-green background and were retained throughout the 11-week experiment with no apparent decrease in intensity over time. Flu- orescent marks in sagittae became slightly more diffuse as otoliths changed shape with growth; however, mark intensity was not affected (Figure 2). Overall, mark intensity was higher for the 350- and 50(1-mg/L treatments than for the 200-mg/L treatments, but mark intensity increased with exposure time at all treatment concentrations. In the second experiment, otoliths of all fathead minnow larvae examined by UV-light microscopy had fluorescent mazks similar to those observed in otoliths of Colorado squawfish larvae. Mark in- tensity was high for all three light-intensity expo- sures throughout the 7-d experiment. Discussion Our results demonstrated that otoliths of proto- larval Colorado squawfish can be successfully marked by whole-body immersion in TC solu- tions. Tetracycline deposited in otoliths of larval fish has been successfully used as a mark for several species (e.g., Hettler 1984; Tsukamoto 1985), but, to our knowledge, this demonstration is the first documented use for marking the early larvae of cyprinids. The technique is relatively easy to use; reasonable in cost, and suitable for the mass marking offish in the field; it offers 100% marking success and at lower concentrations and exposure times has no adverse effect on fish mortality, growth, or development. For the best survival and mark intensity, larvae should be immersed in a solution of 350 mg TC/L for 4-12 h. Hettler (1984) and Tsukamoto (1985) reported similar results. Tsukamoto (1985) observed that larval ayu survival decreased as exposure time in 500 mg TC/L increased. This relationship was not noted at lower TC concentrations. The lowest ayu survival occurred in TC concentrations of 500 mg/L or greater for all exposure times tested. Tsukamoto also reported that TC treatment had no affect on ayu growth, and he recommended whole- body immersion in 200-300 mg TC/L for 3-24 h for otolith marking. Hettler (1984) found that the per- centage of larval spots with otoliths that were suc- cessfully marked by oxytetracycline hydrochloride (OTC, a compound closely related to TC) increased as exposure time was increased, and it reached 100% in OTC concentrations of 250 and 500 mg/L at exposures of 120 and 60 min, respectively. Mark Retention The knowledge of mark-retention time is nec- essary for mark-recapture studies. In our labora- toryexperiments, TC deposits in otoliths of larval Colorado squawfish were retained in detectable amounts at least 77 d after treatment in all of the fish that were examined. Larval ayu that were treated with 100 mg TC/L for 7 h and then reared in a greenhouse retained fluorescent marks in their otoliths for at least 164 d (Tsukamoto 1985). Several authors (Choate 1964; Trojnaz 1973; Laird and Scott 1978; Hettler 1984; Brothers 1985) re- ported that tetracycline-produced marks are labile in light because sunlight deactivates the fluores- cent properties of tetracycline compounds. How- ever, they provided no confirmatory evidence that marks in internal bony structures would be lost or degraded under natural conditions. Deactivation of tetracycline is presumably caused by molecular excitation and subsequent decay through absorp- tion of solar energy in the UV spectrum (particu- larly at 360 nm, Weber and Ridgway 1967). Ex- cited molecules of fluorescent substances are usu- ally unstable and have a mean lifetime of only about 10-s-10_9 s (Lehninger 1975). If this were true, the reliability of tetracycline marking of larval fish for use in field mark-recapture studies would be suspect. However, this presumption is questionable, particularly for TC deposited in the otoliths of fish released into natural waters. In our experiment using fathead minnow larvae treated with TC, exposure of larvae to artificial white light (in the 340-650 nm range) at surface light intensi- ties as high as 1,000 lx during rearing in pure tap water had no effect on the presence or intensity of fluorescent marks in otoliths. Water absorbs UV