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In this chapter the different techniques are described briefly. The terminology used by the different researchers is adopted when referring to grading or grinding equipment. A common terminology would be preferable also in this matter. We also discuss briefly what efforts have been made to validate the different methods.

3.1 Grinding and sliceing

Grinding convex side: French “Groupe National Anguille”. First the otolith is measured using an ocular micrometer and transmitted light. Then each otolith is embedded in methyl metacrylate and the convex surface is ground with carborundum powder. The grinding is checked microscopically during the process using reflected light. The otolith is then examined immersed in camomil essence (“essence de Camomille”) against a dark background using reflected light. The ground otolith may also be stained with dilute aqueous toluidine blue after etching with 1% HCl for 30 to 60 seconds (Lecomte-Finiger, 1985 and references therein). Using this staining procedure translucent zones appear blue.

Lecomte-Finiger (1985) confirmed that the formation of opaque and translucent zones in the otoliths of eels from the Gulf of Lion, in the south of France, was annual. By following the deposition of opaque and translucent zones on the otoliths of glass eels, elvers and yellow eels during the seasons she verified her interpretation of annual and supernumerary zones.

Grinding convex side: Institute of Freshwater Research, Drottningholm, Sweden. The otolith is embedded in thermoplastic (thermoplastic quartz cement; No. 70C Lakeside Brand) on a microscope slide with the convex side up. This side is ground with a series of 280, 400, 800 and 1200 grit wet-grinding paper with a constant supply of water. The grinding continues until the surface is approximately parallel to the otolith's concave side and until the surface reaches the focus of the otolith. The ground otolith is then etched in 1% HCl for 5 to 45 seconds depending on the size of the otolith. The treated otolith can then be viewed using either reflected or transmitted light (Mosegaard and Wickstrøm, 1984).

By stocking elvers in eel-free lakes, Wickstrøm (1987) found that zones of fast growth in the otoliths roughly corresponded to the known age, but problems in interpretation could occur due to false checks in summer and late resumption of otolith growth in spring.

Grinding convex side: Finnish Game and Fisheries Research Institute. The otolith is placed convex side up on a microscope slide and glued with clear nail varnish after having been gently ground on the concave side to get an even plane. The otolith is then ground with grinding paper (240, 600 and 1200 grit) and finally etched for 20–25 seconds with a 1% solution of HCl. Reading can be done using either reflected or transmitted light. Normally an otolith have to be ground and etched several times, between each grinding a photograph is taken (Tulonen, 1987). Tulonen (1987) used otoliths from eels of known age to check his interpretation of otolith zonation. He found that his ageing was reliable up to age 7 or 8, but for older eels the interpretation becomes more difficult and more unreliable (J. Tulonen, pers. comm.).

Grinding convex side: Sinha and Jones (1967). The otolith is placed in distilled water for one minute. The convex side is then ground by hand on a fine carborundum stone on which there is a trace of dilute hydrochloric acid. During this grinding the otolith is periodically examined by microscope to check the appearance of the rings. When the rings show up clearly the otolith is placed in water for 1 to 2 minutes to remove the acid, then in 70% alcohol, from this in absolute alcohol for 1 to 2 minutes and finally in medicinal creosote for 5 to 10 minutes. Age determination is made whilst the otolith is in the creosote and illuminated by reflected light against a dark background (Sinha and Jones, 1967). This method, or modifications of it, has been in common use in North America (Gray and Andrews, 1971; Bieder, 1971; Hurley, 1972; Facey and LaBar, 1981; Harrel and Loyacano, 1982; Levesque and Whitworth, 1987). Penaz and Tesch (1970) used this method, but they also ground the concave side of the otolith. This modified method is used by the Italian workers (Rossi, 1979; Rossi and Colombo, 1976; Colombo and Rossi, 1978; Rossi and Villani, 1980; Rossi and Cannas, 1984).

The validity of age assessment using this method has not been ascertained. However, Penaz and Tesch (1970) and Tesch (1977) have compared growth rates obtained using this method with growth rates obtained through tagging experiments. The annual length increment from otolith readings was 2.8 cm per year, whereas the annual length increment was calculated at 2.0 cm using internal magnetic tags. These results are nearly similar, especially if we take into account the possibility of a tagging effect on the growth rate of tagged eel (see Nielsen, 1988). Further studies on the validity of this method have not been made.

Grinding both sides: Danish Institute for Fisheries and Marine Research. The otolith is placed in a mould, embedded in epoxy, and ground from both sides until the focus is reached. The grinding is done using a rotating machine with grinding paper of fineness 220 and 1000 grits. The ground otoliths, or more properly the otolith slices, are then viewed and photographed using transmitted light (Steffensen, 1980). The method has not been verified or validated, but Dahl (1967) found two annual zones in eels with known age two years.

Grinding both sides: Balaton Limnological Research Institute, Hungary. The otolith is degreased in 70% alcohol for 2 hours and then stuck to a microscope slide with a heat-softening synthetic resin (thermoplastic quartz cement; No. 70C Lakeside Brand), 10–15 mm from the margin of the slide. Grinding and polishing is done using a grinding machine consisting of a rotating mechanism, an otolith-holding assembly unit and grinding and polishing discs. For rough grinding the disc is fitted with abrasive material of 30 um grain size. For fine grinding an abrasive film of 3 um is used. For fine polishing a disc fitted with an abrasive film of 0.3 um grain size is used. During grinding, the surface of the disc is repeatedly sprayed with distilled water and the plane of grinding adjusted. The grinding has to be checked regularly under a microscope. After grinding the otolith on one side it can be removed from the resin using a solvent mixture of chloroform:acetic acid:alcohol in a ratio of 3:1:6. This solvent also partly decalcifies and brightens the otoliths. The otolith is then again embedded in resin for grinding on the reversed side. Finally, the thin otolith slice is removed from the slide with the solvent and embedded inCanada balsam to obtain a permanent preparation (Vero, Paulovits and Biro, 1986; Paulovits and Biro, 1986). The method has not been verified or validated.

Grinding: North American laboratories. Each pair of otoliths is mounted on standard microscope slides with Buehler epoxide (resin and hardener) and heated to speed up the hardening process. The otoliths are then ground close to the focus with a Hillquist grinder with a diamond-impregnated brass cup wheel. In most cases the surfaces are polished with fine carborundum powder, but rings occasionally show up before the otoliths are polished. The otoliths are immersed in methyl-benzoate to improve resolution and examined in a microscope using transmitted light (Bouillon and Haedrich, 1985).

Hansen and Eversole (1984) ground dried otoliths on a fine sharpening stone. Otoliths were examined in xylene with reflected light against a dark background.

Neither of these methods have been verified or validated.

Sliceing. Otolith slices (0.2 mm thick) are cut from otoliths embedded in polyester resin with a low speed circular saw covered with diamond dust as used in metallurgy. The otolith is cut through its longitudinal axis passing through the focus so as to make clearly visible the circumference of the leptocephalus otolith (Deelder, 1976, 1981; Berg, 1985). The slice is mounted between two pieces of glass. Deelder (1976) describes how the optical density of the transect from the focus to the margin is obtained using a microscope fitted with an adjustable motor and a light-dependent resistance connected to a recorder. Using this method the interpretation of the otolith is shifted from the direct viewing phase to a later period. This method was tested on eels of known age by Moriarty and Steinmetz (1979). The method underestimated the age of the slow growing eels in these tests. Moriarty and Steinmetz (1979) concluded that the graphs of this scanning method were difficult to interpret, and that the problem of interpreting doubtful rings when reading otoliths is transferred to the moment when the graphs have to be read. Thus this method does not overcome the subjective element in the interpretation. This method has been used in studies of Norwegian eels (Haraldstad, 1984; Bergersem, Klemetsen and Sommerseth, 1987), both studies concluded that this method underestimated the age of the eels.

Berg (1985, 1987) and Jarmatz, Winkler & Biester (1986) also cut slices of thickness 0.2 mm of the otolith after embedding the otoliths in plastic (EPON or Kalloplast), but they read the otoliths using standard microscopes.

Berg (1985, 1987) used otoliths of eels recaptured up to 5 years after tagging to verify the growth measurements made on untagged eels. It was possible to locate false annuli caused by the tagging procedure and subsequently to define annual rings in otoliths within the known period between first tagging and final catch. This is very close to a validation of the ageing technique, and it obviously gave a good indication on how to interpret the otolith zonation.

Casselman (1982) analysed otolith sections along a line from the focus to the edge with an electron microprobe. The procedure tests for calcium concentration (percent dry weight). Calcium content is inversely related to growth rate and directly related to translucency in eel otoliths, as in the otoliths of other species (Casselman, 1978, 1982). Electron microprobe X-ray analysis of the surface of sections that appear to be uniformly translucent indicate that calcium zonation exists even though optical zonation is not apparent. Hence, microprobe analysis could be used to detect zonation to mechanize and automate the age assessment of eels (Casselman, 1982). However, the technique is laborious and very expensive. The method has not been verified or validated.

Scanning electron microscopic (SEM) examination have been used by Liew (1974), Berg (1985, 1987) and Lecomte-Finiger (in press). Lecomte-Finiger (in press) concluds that SEM is a useful technique for examination of the external texture of the otoliths and for investigation of its microstructure. She adds that the method is inappropriate for routine ageing, owing to the high costs and the long time needed for otolith preparation.

Acetate peel (Liew, 1974). Otoliths from elvers and small eels less than 20 cm require no grinding or polishing prior to examination. The otoliths of the larger eels are placed in distilled water and cut into two through the focus. The posterior part of the otolith is ground by holding down the convex surface of the otolith with one finger on a fine carborundum powder (3 micron equiv.). The ground otolith is etched with 1% HCL solution for times ranging from one to three minutes. The etched otoliths are then immersed in acetone and placed on a piece of 0.05 mm thick acetate sheet. After the acetone has evaporated the peel is removed from the otolith and mounted between a glass slide and a cover glass. Slides are viewed by transmitted light (Liew, 1974; Helfman, Bozeman and Brothers, 1984a).

The validity of this method for ageing American eels was demonstrated by the annual formation of the winter translucent zones for fish kept in ponds and by comparing back-calculated lengths with the empirical lengths for samples taken from the pond. In addition to the translucent winter and opaque summer zones, supplementary translucent zones were quite often observed in the otoliths. The check usually appeared as an isolated narrow zone and could be distinguished from the true winter zone. The formation of supplementary translucent zones was found to be correlated with periods of starvation, induced either by low temperatures, sudden temperature changes, handling or starvation under laboratory conditions (Liew, 1974). The study of Liew (1974) is central when discussing ageing of eels. He documented beyond doubt the annual formation of translucent and opaque zones, as well as describing the microstructure of different types of zonation.

3.2 Burning and cracking

This method was first described by Christensen (1964) and has since then received considerable popularity, both for ageing of eels (Moriarty, 1973, 1983; Rasmussen and Therkildsen, 1979; Todd, 1980; Hu and Todd, 1981; Mills and Hussein, 1985; Aprahamian, 1986, 1987, 1988; Jarmatz et al., 1986) and for ageing slow-growing arctic species such as whitefish (Skurdal, Vøllestad and Qvenild, 1985). The otolith is burnt either before or after cracking. In the first case the otolith is placed on the blade of a heavy scalpel in a bunsen flame for 30 to 60 seconds (Moriarty, 1973; Hu and Todd, 1981) or in a flame from an alcohol burner (O.T. Sandlund, pers. comm.). Burning on a low temperature flame such as on an alcohol burner is often to be preferred (O.T. Sandlund and T.F. Næsje, pers. comm.), as exposure to too high temperatures may cause complete incineration of the otoliths (Hu and Todd, 1981).

During the burning treatment the otolith cracks and may be divided easily along the crack into two pieces across the short axis (Moriarty, 1973). After burning the otolith is very delicate and liable to be reduced to powder if roughly handled (Moriarty, 1973). Alternatively the otolith may be broken in half transversly by placing it, convex side up, between the folds of a piece of thin, clear plastic (or alternatively wet blotting paper), and pressing across the centre with a scalpel blade. Usually a clean break is obtained. The plastic or the wet paper prevents the pieces from scattering. The otolith halves are then burnt by holding the broken edge in a bunsen flame until the edge turns brown, or alternatively by heating both halves on a thick scalpel blade (Todd, 1980). Also here the use of an alcohol flame is preferred by some workers (Chilton and Beamish, 1982).

After cracking, the broken surface of the otolith can be placed in a mould and viewed in a microscope using reflected light and either 96% ethanol or 1.-2. propandiol as refracting medium (Vøllestad, 1985), or painted with cedar wood oil, cooking oil, or a creosote-toluene mixture (Chilton and Beamish, 1982; Jessop, 1987). To prepare the burnt and broken otoliths for viewing and storing, the otoliths can be embedded in a clear silicone rubber sealant (Hu and Todd, 1981) or a resin called Isopon (Moriarty, 1973). After burning, the otolith fragments are picked up with a mounting needle, the top of which has been coated with a small quantity of the clear rubber sealant. The fragments are then carefully embedded in a small portion of sealant on a microscope slide with the reading surface pressed against the slide surface. The sealant remains sufficiently flexible for about 3 minutes at room temperature for the otolith portions to be mounted. The slide is sufficiently optically dense to permit the otoliths to be read through it. For reading, the slide is inverted and the otoliths are read using reflected light.

Several attempts have been made to test the validity of this method. The ageing of eels of known age (two years) from ponds confirmed that the method gave good results (G. Rasmussen, pers. comm.). The multi-method test by Moriarty and Steinmetz (1979) showed that this method can overestimate the age of the young eels. For the older eels this method was the best of the five tested. But there were apparent problems in interpretation of double or multiple rings (Moriarty and Steinmetz, 1979). Eels of known age (15 years old) from a pond in Kolderveen, the Netherlands, were studied by Vøllestad and Nasje (1988) (eels from this pond was also studied by Moriarty and Steinmetz (1979), but at a far younger age). About 26% of the specimen were aged correctly. This figure should probably be much higher since some introduction of eels of unknown age to the pond must have occurred. The growth of the eels was very slow, and the result indicate that burning and cracking is a good method for slow growing eels. Mean annual growth of Irish eels calculated using this method agrees closely with estimates of length increment determined from tagged eels from the same location (Moriarty, 1983). When reading the same otoliths twice it is found that in over 80% of the cases the difference between readings are one year or less (Moriarty, 1983; Vøllestad, 1985). Using tetracycline labelled otoliths Dekker (pers. comm.) found that the last annulus was correctly identified in over 90 % of the burnt and cracked otoliths. Aprahamian (1987) was able to recognize year classes of stocked elvers in the upper reaches of the River Severn using this method.

All these results taken together seem to indicate that this method usually is acceptable in order to take correct management decisions.

3.3 Whole otoliths

The otoliths are stored dry. Storing in ethanol is not advisable. Before ageing the otolith is cleared in 96% etanol. Vøllestad (1985) recommends clearing the otoliths for 18 – 24 hours, whereas Barak (pers. comm.) recommend clearing times up to 48 hours especially in the larger otoliths. The otoliths are read using reflected light against a dark background with 96% ethanol or propandiol as refracting medium (Aass, 1972; Vøllestad, 1985, 1986; Vøllestad and Jonsson, 1986, 1988). Lecomte-Finiger, Mallawa and Ximenes (in prep.) read the otoliths in camomil essence, without any treatment prior to reading. After reading the otoliths must be stored dry, and no clearing is usually necessary prior to subsequent analysis.

When reading the same otoliths twice it is found that in over 80% of the cases the differences between readings are less than or equal to one year (Vøllestad, 1985). It is also confirmed that one translucent and one opaque zone is deposited in the otoliths annually (Vøllestad, 1986). This study involved brackish water eels with a comparatively fast growth rate. The study by Vøllestad and Næsje (1988) on eels of partly known age also include results with cleared otoliths. They concluded that this method was unsuitable for ageing these slow growing eels. Vøllestad and Jonsson (1988) on the other hand was able to give indirect evidence for the methods validity in the Imsa River, Norway. The mean age at descent of silver eels was estimated using regression models on data on number of ascending elvers and number of silver eels some years later. This analysis indicate a mean age at descent of 8 years, whereas age determinations of silver eels give mean ages of 7.9 years. Also mortality estimates for single year classes estimated from age data was very close to the total mortality rate estimated from the total ascent and descent data. They could also follow a weak year-class through six successive seasons.

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