such as cold shock (Mugiya and Muramatsu 

 1982), or by simply bringing field-captured fish 

 into the laboratory (Boehlert and Yoklavich 

 1985). Comparing increment counts with number 

 of days following the time-mark accurately esti- 

 mates frequency of occurrence of the growth in- 

 crements. Our study evaluates the commonly 

 used OTC and an alternate chemical, the ra- 

 dioisotope calcium-45, in terms of their success as 

 time-markers to validate daily growth increment 

 formation in the otoliths of juvenile black rock- 

 fish, Sebastes melanops. 



Materials and Methods 



Young-of-the-year black rockfish, Sebastes 

 melanops, ranging from about 2 to 5 g wet body 

 weight and 47 to 64 mm standard length (SL), 

 were collected from a rocky, intertidal area 8 km 

 south of Newport, OR, in July 1982 and from 

 Yaquina Bay, OR, in July 1983. Fish were held in 

 200 L tanks under ambient water temperature 

 conditions which fluctuated between 13° and 

 17°C; a ration of ground squid and shrimp was 

 offered ad libitum and photoperiod was main- 

 tained at 13 h light and 11 h darkness. After at 

 least 10 days of acclimation to laboratory condi- 

 tions, fish were anesthetized with MS-222 and 

 injected intramuscularly (midbody below dorsal 

 fin) with a solution of either OTC or calcium-45. 

 Fish continued to feed immediately following in- 

 jection and handling. 



Calcium-45 



Fish were injected with a solution of low- 

 calcium physiological saline and calcium-45 

 (^^CaCls dissolved in HCl; New England Nu- 

 clear).^ Through preliminary experiments, a dose 

 of 0.1 ixCi '*^Ca/g wet body weight proved to be 

 optimum for isotope uptake and retention. Four 

 fish each were sacrificed at 1, 4, 12, 24, 48, 72, 96, 

 120, 144, 168 hours and at subsequent 4-d inter- 

 vals for 63 days following injection. Three fish 

 were sacrificed after having maintained good 

 health and growth for 1 year following injection. 

 Four nonradioactive fish were sacrificed on the 

 first day of the experiment and used as blanks or 

 controls in determining activity levels of the in- 

 jected fish. 



At the time intervals specified above, fish were 



anesthetized, blotted dry, measured (nearest mm, 

 SL), and weighed (nearest 0.01 g). Both sagittal 

 otoliths were removed from each fish, rinsed thor- 

 oughly in water to remove surface contamination 

 of calcium-45, and stored dry for liquid scintilla- 

 tion counting (LSC) or autoradiography. One 

 otolith from each fish, with the exception of the 

 three 1-yr-olds, was weighed, dissolved in 0.1 mL 

 concentrated HCl, diluted with 10 mL of Beck- 

 man Ready-solv EP liquid scintillation cocktail, 

 and assayed for calcium-45 activity in a Beckman 

 LS 8000 liquid scintillation counter. Activity was 

 corrected for decay and quench and expressed as 

 disintegrations per minute (DPM) per mg of sam- 

 ple. The perceived decrease in radioactivity due to 

 the increase in weight of otolith over the experi- 

 mental period was corrected using the following 

 equation: 



Corrected activity = 



DPM 



1 References tx) trade names do not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



mg tissue^. 



weight tissue^, 

 mean weight tissue^ 



where tf is time at sacrifice and ti is time of exper- 

 iment initiation. Mean weight of otolith at i, was 

 obtained from the 4 fish sacrificed prior to injec- 

 tion; since all fish were of similar length at the 

 onset of experimentation, these 4 fish adequately 

 represented size of injected fish. 



Four otoliths from time interval 1 hour and two 

 otoliths from each of intervals 4 and 12 hours and 

 1, 4, 11, 19, 39, 55, and 63 days were prepared for 

 autoradiography. The right otolith of each pair 

 was affixed to a microscope slide with histological 

 mounting medium. The proximal surface of the 

 otolith was ground with 600 grit carborundum 

 paper on a rotating wheel until the focus was just 

 visible and most of the curvature of the otolith 

 was removed. The mounting medium was gently 

 heated and the otolith was turned to expose the 

 distal surface. Grinding was continued until most 

 of the mounting medium was removed from the 

 margins of the otolith. The external surface was 

 polished using jeweler's rouge (3 ^JLm) and the 

 whole slide was immersed in an ultrasonic 

 cleaner to remove all loose particles from the 

 otolith surface. The resulting sagittal section was 

 coated with Kodak NTB3 nuclear emulsion and 

 exposed in a light-tight box for 8 days at 4°C. The 

 autoradiographs were developed in Kodak D-19 

 developer for 2 minutes, fixed for 5 minutes in 



827 



