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Fishery Bulletin 93(2). 1995 



ined the efficacy of sectioning and polishing juvenile 

 menhaden otoliths on two orientations of transverse 

 planes. Greater attention to otolith preparation can 

 significantly improve ageing accuracy (Campana and 

 Moksness, 1991), and section orientation can affect 

 the ability to read otoliths (Secor et al., 1992). 



Previous menhaden validation studies have used 

 only a minor amount of otolith processing, and the 

 material has been examined on the sagittal plane. 

 Maillet and Checkley ( 1990 ) and Warlen ( 1992 ) used 

 known-age, lab-spawned and reared larvae, whereas 

 Simoneaux and Warlen (1987) examined the outer- 

 most growth increments of juvenile Atlantic menha- 

 den injected with oxytetracycline (OTC). Maillet and 

 Checkley ( 1990) examined growth/increment forma- 

 tion from hatching through 36 days of age, Warlen 

 (1992) through 41 days of age, and Simoneaux and 

 Warlen (1987) used juveniles (63-98 mm in fork 

 length) with an experimental duration of 7-14 days 

 after marking with OTC. With the exception of one 

 test group (Maillet and Checkley, 1990), results of 

 all studies indicated that, on average, one growth 

 increment was formed daily. 



However, these approaches have not resulted in a 

 method that will permit precise and accurate ageing 

 of older juvenile Atlantic menhaden otoliths. While 

 Simoneaux and Warlen ( 1987) were able to validate 

 the daily periodicity of increment formation for a 

 short period of time, their otolith processing method 

 could not be used to determine the actual age of the 

 juveniles examined. The periodicity of increment for- 

 mation in otoliths should be validated over the ranges 

 in age and size that can potentially be encountered 

 with unknown-age, field-collected material. 



We use two of the preferred validation methods, 

 known-age and otolith-marking, to bridge the gap in 

 age and size among the studies of Maillet and 

 Checkley ( 1990), Warlen ( 1992), and Simoneaux and 

 Warlen (1987) and to provide an estimate of the un- 

 certainty in deriving age from older juveniles. Our 

 known-age study provides a continuous validation 

 from first feeding through metamorphosis, to juve- 

 niles up to 9 months old. 



52 to 136 days after hatching, then preserved in 70% 

 ethyl alcohol. 



Oxytetracycline (OTC) marked fish 



In 1988, larval Atlantic menhaden collected at Pivers 

 Island, Beaufort, North Carolina, were acclimated 

 to 100-L indoor laboratory tanks and immersed on 7 

 April in OTC by a procedure modified from Hettler 

 ( 1984). Salinity was slowly reduced to 0%c by adding 

 tap (well) water over several hours. A premixed, buff- 

 ered (sodium bicarbonate) stock solution of OTC was 

 added to the tank. The resulting treatment condi- 

 tions were 300 mg-L" 1 OTC at pH 6.3. After four hours 

 of immersion, ambient seawater flow was restored; 

 the test solution was thus diluted within an hour 

 and salinity slowly increased. The fish remained in 

 this tank for the duration of the study. Samples of 

 postlarvae and juveniles were taken periodically, 13 

 to 147 days following treatment, and preserved in 

 95% ethyl alcohol. 



Alizarin complexone (ALC) marked fish 



In 1992 we conducted validation trials with marked 

 fish under high and low feeding rations to examine 

 further the effect of growth rate on increment depo- 

 sition. Larval menhaden were captured with a neus- 

 ton net at Pivers Island, NC, on 1 April 1992 and 

 held at ambient temperatures and salinities until 21 

 April, then immersed for 14 hours in 100 mg-L -1 ALC 

 buffered with sodium bicarbonate to pH 6.5. After 

 immersion, 1,026 larval menhaden were divided be- 

 tween two 2, 100-L outdoor holding tanks and 

 sampled monthly, May through December. The lar- 

 vae were fed cultured, live Artemia franciscana and 

 increasing additions of dry food until 29 May, when 

 only dry food (Ziegler salmon starter) was added in 

 a ratio of 3 (high food tank) to 1 (low food tank). The 

 amount of dry food initially added for the low food 

 treatment was 25 mLday -1 in April; this amount was 

 increased to approximately 90 mLday -1 by July and 

 held constant after this period. 



Materials and methods 



Known-age fish 



Atlantic menhaden brood stock were held in the labo- 

 ratory and induced to spawn in February 1987 by 

 Hettler 's (1981) methods. Eggs were hatched and 

 larvae were reared under laboratory conditions as 

 described by Warlen (1992). Samples of postlarvae 

 and later juveniles were sampled periodically from 



Otolith preparation and increment counting 



Some otoliths from late larvae were mounted whole 

 in a mounting medium (Flo-tex) on glass microscope 

 slides. After increments were counted on the sagit- 

 tal plane, many were removed from the mounting 

 medium and sectioned on one of two planes as de- 

 scribed below. 



We generally followed the sectioning techniques 

 described in Epperly et al. (1991) and Secor et al. 

 (1992). Our processing techniques for the ALC- 



