38 



Fishery Bulletin 98(1 



liths; therefore transverse sections are used to deter- 

 mine ages of older fish (Gunn et al.^). In the future, 

 as strontium-marked otoliths are returned from older 

 fish that have been at liberty for longer periods, we 

 will locate the strontium mark in transverse sections 

 with the SEM and increase the number of increments 

 that have been validated. 



The recapture rates of orange-tagged and injected 

 SBT were not significantly different ft-om the recapture 

 rates of yellow-tagged SBT; therefore the Sr injections 

 apparently did not affect survival rate. Although the 

 dosages of Sr varied between 65 and 250 mg/kg of 

 fish, there is no evidence to suggest that higher doses 

 increased mortality because Sr-injected fish with the 

 highest doses were among those recaptured. A direct 

 relation between the dosage and the intensity of the 

 mark had been found in trials with three other species 

 in 1990-9 1 ( CSIRO, unpublished datai^ ). The increased 

 dose for large SBT resulted in much more distinct 

 marks on their otoliths, which have a larger surface 

 area than that in smaller fish. The less distinct marks 

 could also be attributable in some large fish to a loss 

 of strontium solution fi-om the muscle after injection. 

 Although more solution was injected if a loss was 

 noticed, there may have been further loss of solution 

 afler the fish was returned to the water, resulting in 

 a less effective dose. Thus, as a general guideline, we 

 recommend a dose of 100 mg Sr/kg for marking otoliths 

 in SBT. We note, however, that tissue area around the 

 injection should be observed to ensure that there is no 

 loss of injected solution fi-om the muscle tissue. 



The problem of detecting indistinct marks that re- 

 sult from low dosage levels are eliminated by using 

 SrCl2 as a marker. Unlike fluorescent marking, where 

 it is very difficult to evaluate faint or ambiguous marks 

 objectively (particularly if they are close to the outside 

 edge of the otolith), it is possible to evaluate Sr marks 

 objectively by x-ray analysis. Because the concentra- 

 tions of the Ca and Sr on the Sr mark are high, very 

 simple energy dispersive spectroscopy systems, which 

 are available in many SEM facilities, can be used. Al- 

 though not a trivial procedure, x-ray analysis requires 

 preparation methods similar to those used for examin- 

 ing fluorescent markers and can usually be contracted 

 to facilities at a low cost. Given the ofi;en substantial 

 investment in tagging progi'ams, and the common 

 combination of low recapture rates and even lower oto- 

 lith sampling rates, every sample is extremely valu- 

 able in a marking experiment. The safety net of chem- 

 ical analysis is thus very advantageous. 



Comparison of strontium and fluorescent markers 



At the beginning of this project we chose strontium 

 chloride over the more commonly used fluorescent 



markers because previous work on SBT with oxytet- 

 racycline had been unsuccessful. Although immersion 

 in high concentrations of strontium or feeding with 

 strontium-laced food (or both) had been used success- 

 fully for marking hard parts of larvae and juveniles of 

 hatcheiy-reared salmon (Behrens Yamada and Mul- 

 ligan, 1982; 1990), salmonids and a variety of tropi- 

 cal fish species (Brothers, 1990) and squid (Hurley et 

 al., 1985), strontium had not previously been used to 

 mark otoliths of large fish. On the basis of his experi- 

 ments. Brothers ( 1990 ) concluded that, for mass mark- 

 ing of fish in captivity, detection of strontium marks 

 was expensive and involved more difficult preparation 

 than did fluorescent markers and other marking tech- 

 niques such as thermal inducement (Volk et al., 1990). 



Brothers' (1990) comment on expense is certainly 

 pertinent but the expense of analyzing marked oto- 

 liths is often a small fraction of the cost of a marking 

 experiment, particularly one where large numbers of 

 fish have been tagged, injected, and released. Perhaps 

 most important in the cost equation should be the rate 

 of success of detecting marks in the otoliths of marked 

 fish rather than the comparative cost of analysis. In 

 otoliths from large tuna whose time at liberty has 

 been long, the strontium marks are covered by a large 

 amount of otolith material deposited after the time of 

 injection. Sectioning is necessary for either marker; 

 thus preparation times in these cases are much the 

 same. For smaller tuna and those at liberty for short 

 periods, fluorescent markers can be detected in the 

 whole otolith, whereas detection of strontium with- 

 out an EDS system would require sectioning, which 

 would increase preparation time. The equipment for 

 fluorescent markers is cheaper and comprises a light 

 microscope equipped with an ultraviolet illumination 

 source and filters to match the wave length of the fluo- 

 rescence emitted from the marker when excited by the 

 light source (see Wild and Foreman, 1980). For stron- 

 tium, an SEM equipped with a Robinson detector is 

 the minimum requirement; an EDS system is a use- 

 ful extra. Although an SEM is a common apparatus in 

 large research laboratories, hourly charges to the user 

 can be high, although we have found that, with well 

 prepared specimens, as many as four otoliths can be 

 examined and analyzed per hour with an SEM. 



Apart from preparation time and costs, strontium 

 marking for age validation has clear advantages over 

 fluorescent marking. One benefit of a technique that 

 requires both a light microscope and an SEM is that 

 measurements of increments and strontium marks 

 are independent: the strontium cannot be detected 

 in whole otoliths under the light microscope and the 

 annual increments cannot be observed in the SEM. 

 Allergic reactions by humans to compounds such as 

 oxytetracycline have led the U.S. FDA to ban their use 



