MARINE ECOLOGY PROGRESS SERIES PubIished November 3 Mar Ecol Prog Ser l

REVIEW Chemistry and composition of otoliths: pathways, mechanisms and applications

Steven E. Campana*

Marine Fish Division, Bedford Institute of Oceanography. PO Box 1006. DartmouU~,Nova Scotia B2Y 4A2, Canada

ABSTRACT:The fish otolith (earstone) has long been known as a timekeeper, but interest in its use as a metabolically inert environmental recorder has accelerated in recent years. In part due to technolog- ical advances, applications such as stock identification, determination of rnigralon pathways, recon- struction of temperature and salinity history, age validation, detection of anadromy, use as a natural tag and chemical mass marking have been developed, some of which are difficult or impossible to imple- ment using alternative techniques. Microsamphg and the latest advances in beam-based probes allow many elemental assays to be coupled with daily or annual growth increments, thus providing a detailed chronologcal record of the environment. However, few workers have critically assessed the assump- tions upon which the environmental reconstructions are based, or considered the possibility that ele- mental incorporation into the otolith may proceed differently than that into other calcified structures. This paper reviews current applications and their assumptions and suggests future directions. Particu- lar attention is given to the premises that the elemental and isotopic composition of the otolith reflects that of the environment, and the effect of physiological filters on the resulting composition. The roles of temperature, elemental uptake into the fish and the process of otolith crystahzation are also assessed. Drawing upon recent advances in geochemistry and paleoclirnate research as points of contrast, it appears that not all applications of otolith chemistry are firmly based, although others are destined to become powerful and perhaps routine tools for the mainstream fish biologst.

KEY WORDS: Otolith . Element. Isotope . Composition . Stock identification. Temperature history

INTRODUCTION addition to the continued and widespread use and research on the timekeeping properties of the otolith, Otoliths (earstones) are paired calcified structures a new and largely independent sub-discipline of used for balance and/or in all . otolith chemistry has emerged which promises to While they are clearly more important to the fish than address a suite of questions that have proven difficult to the fish biologist, that point is easily forgotten. The or impossible to resolve through other means. Dis- use of otoliths as indicators of fish age has now reached cussed in only 6 papers prior to 1980, the otolith the century landmark, starting with Reibisch's obser- chemistry field has since entered a period of exponen- vations of otolith annuli in 1899. In 1971, Pannella's tial growth, resulting in 157 papers on the topic hav- discovery of daily growth increments helped propel ing been published by the end of 1998. It is the objec- the interpretation of otolith microstructure into the tive of this review to highlight the basis for this mainstream of fish biology. Since that time, interest in rapidly growing interest, and to critically assess the the otolith as a metabolically inert timekeeper and emerging suite of applications involving otolith com- environmental recorder has accelerated, culminating position and chemistry. in 2 recent international symposia devoted solely to The same 2 properties of the otolith which allow it to fish otoliths (Secor et al. 1995a, Fossum et al. 1999). In form and retain daily growth increments are also responsible for its ability to record aspects of the envi- ronment to which the fish is exposed. The fact that the otolith is acellular and metabolically inert means that

O Inter-Research 1999 Resale of fuLl article not permitted [Mar Ecol Prog Ser 188: 263-297, 1999

any elements or compounds accreted onto its growing COMPOSITION OF THE OTOLITH surface are permanently retained, whereas the contin- ued growth of the otolith from before the time of hatch Otolith composition is relatively pure compared to to the time of death implies that the entire lifetime of most biological and mineralogical structures, being the fish has been recorded (Campana & Neilson 1985). dominated by carbonate in a non-collagenous In principle then, the otolith may contain a complete organic matrix. A total of 31 elements have been record of exposure to both the temperature and com- detected in otoliths to date, not including radioactive position of the ambient water. When coupled with age- elements such as Th and Ra. The elemental composi- or date-structured examination of otolith growth incre- tion is dominated by the major elements calcium, oxy- inents (either annual or daily), the potential for a gen and carbon, which make up the calcium carbon- detailed chronological record of the environment to ate (CaC03) matrix. However, the majority of the which the fish was exposed is obvious. In practice, the elements are present at the minor (> 100 ppm) and assay and interpretation of this chemical record is less trace (

OTOLITH MINERALOGY AND CRYSTALLIZATION to be clarified, periodic secretion of the soluble pro- teins to the mineralization front could result in an asso- A mechanism for otolith growth is beyond the scope ciation which would then either initiate or inhibit crys- of this review, but some details are important for under- tal growth (Wheeler & Sikes 1984). standing the incorporation of trace elements. In brief, Calcium carbonate can crystallize as any 1 of 3 crys- of otoliths differs somewhat from that tal polymorphs: calcite, or vaterite. Aragon- of bone, molluscan shell and coral skeleton ite and vaterite are normally considered to be crystal- in that the otolith epithelium is not in direct contact with lographically unstable, reverting to the calcite poly- the region of . As a result, the calcification morph, although the transformation of aragonite to process is heavily dependent upon the composition of calcite is slow (Carlstrom 1963). Yet unlike the calcitic the endolymphatic fluid surrounding the otolith, and to otoconia of mammals, X-ray diffraction has confirmed a certain extent, can be described on the basis of purely that the vast majority of sagittal otoliths are composed physical principles. The key physical, regulating factor of polycrystalline aragonite (Irie 1955, Carlstrom 1963, appears to be the pH of the , which is deter- Degens et al. 1969, Mann et al. 1983, Morales-Nin mined by the concentration of bicarbonate ions in the 1986, Maisey 1987, Lecomte-Finiger 1992, Oliveira et endolymph (bicarbonate is one of the ion products of al. 1996). The orientation of the individual crystal faces carbon dioxide in solution) (Romanek & Gauldie 1996, is sometimes referred to as being twinned (Degens et Payan et al. 1997, 1998). Reduced alkalinity in the al. 1969, Gauldie & Nelson 1988). Until recently, an endolymph is regulated by proton secretion through explanation for the overwhelming predominance of the saccular epithelium, which then reduces the rate of aragonite over calcite in otoliths was unclear; despite calcification (Payan et al. 1997). Temperature influ- the fact that calcite is rhombohedral and aragonite is ences calcification rate as well. By itself however, a orthorhombic, the 2 polymorphs have very similar solely inorganic process cannot account for many of the crystal structures, differing primarily in the organiza- features of biomineralization in general, or otolith tion of the carbonate molecules sandwiched between growth in particular (Wheeler & Sikes 1984), suggest- layers of calcium (Falini et al. 1996). Small quantities of ing that the small percentage of the otolith composed of divalent ions such as Sr and Mg favour formation of proteins plays a pivotal role in otolith calcification. aragonite over calcite (Carlstrom 1963), but it was not Recent findings suggest that the composition and clear that such conditions were by themselves suffi- orientation of otolith proteins conform to those in other cient to explain the aragonitic otolith. However, a biomineralized structures, including squid statoliths recent study indicates that selection of the polyrnorph (Morris 1991). Approximately half of the otolith pro- of calcium carbonate to be precipitated is mediated, teins are water-insoluble (Asano & Mugiya 1993), and and perhaps controlled, by organic molecules (Falini et presumably make up the structural framework for sub- al. 1996). Under identical experimental conditions, sequent calcification. These proteins are characterized macromolecules extracted from aragonitic shell layers by a high abundance of acidic amino acids, and were induced aragonite formation, while macromolecules termed 'otolin' by Degens et al. (1969). However, it is extracted from calcitic shell layers induced calcite for- the more recently characterized water-soluble proteins mation (Falini et al. 1996). These results provide the which appear to be the most influential in regulating best evidence to date for organic control of crystal for- the rate of calcification. These calcium-binding glyco- mation in organisms, and suggest that both the rate proteins are also characterized by acidic amino acids, and type of calcium carbonate crystals formed in and appear to serve as regulators of calcification rate otoliths is regulated by proteins. (Asano & Mugiya 1993), perhaps through inhibition of Curiously, different polymorphs of calcium carbon- crystal nucleation (Wright 1991). Support for this sug- ate appear to be linked to the different otolith organs. gestion comes from observations of molluscan shell While aragonite is the norm for sagittae and lapilli, formation, in which a soluble matrix rich in acidic most asteriscii are made of vaterite, thus accounting for amino acids binds calcium and forms an anti-parallel their glassy appearance (Lowenstam & Weiner 1989, P-pleated sheet in the polymerized state (Morris 1991). Oliveira et al. 1996). Vaterite is also the principal poly- In such a conformation, all of the amino acid side morph in many aberrant, or 'crystalline', otoliths chains lie on 1 side of the corrugated layer and project (Mugiya 1972, Gauldie et al. 1997, but see Strong et al. in the same direction; the spacing between these side 1986). There is no accepted explanation for the forma- chains closely matches that of the calcium atoms in tion of vateritic regions within a largely aragonitic aragonite crystals, suggesting that the soluble matrix otolith (Strong et al. 1986), but vaterite is known to pre- could act to either encourage or inhibit crystal nucle- cipitate in supersaturated solutions which are far from ation (Morris 1991, Falini et al. 1996). While the link- equilibrium (Carlstrom 1963). Alternatively, vaterite age between the soluble and insoluble proteins needs formation may be mediated by matrix proteins in the Mar Ecol Prog Ser 188: 263-297, 1999

same way described earlier for calcite and aragonite ments might be included. However, the rate of otolith (Falini et al. 1996). Calcitic regions in otoliths are much precipitation might have a different influence on these rarer (Davies et al. 1988),and are probably a product of elements than those more directly associated with cal- anomalous inorganic precipitations or slip planes cium carbonate crystals. Perhaps more importantly, to (Oliveira et al. 1996). the extent that a change in temperature changes the The exact location of elemental impurities in the balance between organic matrix and calcium carbon- otolith matrix has not been studied, but would be ex- ate formation, it might also be expected to change the pected to be similar to that of other arayonitic carbon- ratio of elements which is incorporated. ates. Three sites are possible: within the crystal lattice as a substitute for calcium, as an inclusion in the inter- stitial spaces, or in association with the proteinaceous PATHWAYS AND BARRIERS OF ELEMENTS INTO matrix. The first of the three (the within-lattice inclu- THE OTOLITH sion) can occur through a simple ion substitution for Ca or through CO-precipitation of another carbonate The pathway of any given element or ion fl-om the en- (e.g. MgC03). High correlations (positive or negative) vironment into the otolith is a multi-stage process, and among elements such as Sr, Mg, Li and Ba suggest a is characterized by a sequence of more or less indepen- common mode of inclusion, while uncorrelated ele- dent barriers. Elemental and ionic barriers are an obvi- ments such as Mn and Zn probably differ in their in- ous prerequisite of a highly osmoregulated organism clusion site. Of the 3 possible sites, the calcium substi- such as a fish, but the pathway into the otolith is even tution/~~-precipitationis most studied, and appears more regulated than is the case for other tissues. For ex- directly applicable to divalent metal ions of con~parable ample, calcified tissues such as bone (Ishikawa et al. size. carbonate is virtually isostructural with 1991, Schmitz et al. 1991, Miller et al. 1992) and scale aragonite, thus explaining strontium's affinity for arag- (Johnson 1989, Pender & Griffin 1996) contain higher onite, but in light of the lower Sr concentration in concentrations of most elements than do otoliths (Sr is a otoliths compared to corals, substitution of Sr ions for conspicuous exception), while uncalcified structures Ca may be more likely than CO-precipitation of %CO3 such as the eye lens are often characterized by a differ- (Greegor et al. 1997). Ions slightly larger than calcium ent suite of elements altogether (Dove & Kingsford (such as Ba and Pb) can also be expected to be substi- 1998).This high degree of regulation is most evident in tuted or CO-precipitated(Morse & Mackenzie 1990). For a phylogenetic comparison, in which the aragonitic such elements, the extent of incorporation can often be skeleton of the most primitive animals (e.g. corals) predicted with temperature-sensit~ve partition coeffi- tends to reflect the composition of the ambient water, cients, although the rate of precipitation can also be in- while animals of increasing complexity show increas- fluential. Anions such as chloride and sulfate can also ing discrimination against the most abundant elements be CO-precipitated, but less predictably. The spacing (Table 1). Interestingly, there appears to be relatively between calcium ions in the crystal lattice is important little discrimination against the incorporation of at least to both substitution and CO-precipitation,explaining the some of the trace elements into the otolith. very different affinities of vateritic otoliths for some ele- The basic pathway of the bulk of inorganic elements ments compared to aragonitic otoliths (Gauldie 1996b). into the otolith is from the water into the blood plasma In contrast, ions such as those of sodium do not behave via the gills or intestine, then into the endolymph, and as normal CO-precipitantsand are probably found at finally into the crystallizing otolith. Water passing over crystal defects (Morse & Mackenzie 1990). In general, the gills (branchial uptake) is the primary so'urce of ion impurities trapped in interstitial spaces are difficult to predict or model in terms of partition coefficients Table 1. Phylogenetic comparison of representative trace ele- Otoliths are well known for their micro-channel archi- ment concentrations in marine biogenic aragonite of corals, tecture (Gauldie et al. 1998), and the presence of poorly bivalves, squid statoliths and fish otoliths. Elemental discrim- bound elemental inclusions in these interstitial regions ination tends to be least for the most primitive animals (e.g. corals) and for physiologically unimportant trace elements could well explain why elements such as Na, Cl, Zn and (e.g.Ba). AU concentrations (in pm01 mol-') are approximate, K are so easily leached out (Proctor & Thresher 1998, and are for dustration only Campana et al. 1999a). Elements incorporated into a calcium carbonate Element Coral Bivalve Squid Otolith matrix as a result of covalent bonding or other associa- skeleton shell statolith tions with organic molecules are virtually unstudied, but are expected to be present. Aside from the major SrlCa (C, H, N) and minor elements (S) that make up the 0, Ba/Ca constituent amino acids, it is not clear what other ele- Campana: Chemistry and composition of fish otoliths 267

most elements in , while the continual values of D = 1 would be expected when there is no drinlung of marine fish is the main source of water- elemental dscrirnination, and values of D = 0 when borne elements for assimilation via the intestine none of the element is incorporated into the otolith. (Olsson et al. 1998). A small but unknown proportion Observed values for many of the major ions (e.g. Na, K, of elements is undoubtedly assimilated from food Cl) are less than 0.05, while that for Sr is about 0.14. sources; for example, at least some of the Sr in the diet Distribution coefficients for many of the trace elements can be incorporated into the otolith (Limburg 1995, exceed 0.25, and may even approach 1.0. Farrell & Campana 1996, Gallahar & lngsford 1996). Undoubtedly, one of the biggest barriers to elemen- However, other experiments suggested that otolith up- tal uptake occurs at the gill-water (in freshwater fishes) take of a suite of minor elements from food was mini- or intestine-water (in saltwater fishes) interface, where mal (Hoff & Fuiman 1995). The majority of the inor- osmoregulation regulates movement of water-borne ganic otolith elements are probably derived from the ions into the fish. Plasma concentrations of Ca and water: 80 to 90 % in the case of Ca and Sr, respectively, other major ions are approximately one-third of that of in freshwater fishes (Simkiss 1974, Farrell & Campana marine waters, but flux rates between water and blood 1996). are even lower, since only excreted ions are replaced. As shown schematically in Fig. 2, elemental discrim- In saltwater fishes, trace elements are probably assim- ination can occur at any or all of the 3 interfaces: water- ilated from the intestine in direct proportion to their gill, blood-endolymph and endolymph-crystal. For relative concentration in the water, albeit with low effi- most elements, the e1ement:Ca ratio in the otolith is far ciency (Olsson et al. 1998). Salinity, pH, dissolved oxy- lower than that in the blood plasma or ambient water, gen concentration and other factors can also influence but there are large differences in the degree of discrim- elemental uptake into the fish (Mayer et al. 1994). In ination among elements, and where that discrimina- freshwater fishes, however, it is calcium concentration, tion occurs. Using the distribution coefficient between or water hardness, which has one of the largest effects water and otolith as a measure of elemental discrimi- on elemental uptake through the gills. While the nation (where D = (element:Ca),,,/(element:Ca)WaLer),mechanism remains unclear, it appears that quite a few elements (particularly the divalent elements) are taken up through chloride cell calcium channels in the Branchial Cellular Crystallization Uptake Transport gill (Mcfim 1994). The net result is that branchial uptake of metals generally decreases as the relative concentration of calcium in the water increases (Mayer et al. 1994). Where the ambient calcium concentration is low, a greater proportion of both the calcium and the Seawater* Plasma$dolymph J.otollth other elements will be taken up. For this reason, the absolute concentration of a dissolved element is often ..S? @J an unreliable indicator of environmental availability to ca 36% * 50%- Sol~d the freshwater fish; the e1ement:Ca ratio is more rele- Maior ions :\ I--,,A, +&r vant for such elements. In the case of salt water, Ca concentration is highly correlated with salinity, imply- ing that either the e1ement:Ca or e1ement:salinity ratio can be used as an indicator of environmental availabil- ity. This effect is shown in Fig. 2, in which the concen- C,? - tration of major ions (such as Na and K) in the water differs substantially between estuarine and marine environments, yet uptake into the blood is constant in the 2 environments due to the fact that the ion:Ca ratio in the water remains unchanged. Such is not the case for trace elements such as Pb, whose ratio with Ca Fig. 2. Ove~ewof elemental pathways and barriers between varies widely among locations or with salinity. seawater and the otolith, with coarse estimates of transfer rates (distribution coefficients) for selected elements at each Elemental discrimination also occurs during the physiological barrier. Elemental concentrations have been movement from plasma to endolymph (Fig. 2), result- referenced to calcium concentration, since the initial uptake ing in an endolymph composition which is depleted in of many elements is often inversely proportional to the rela- all major ions other than K (Fange et al. 1972, Mugiya tive calcium concentration. Elemental discrimina.tion is great- est for major and physiologically regulated ions, and least for & Takahashi 1985, Kalish 1991a, Gauldie & Romanek trace elements, but the site of maximum discrimination is 1998). In general, the composition of the endolymph often unpredictable appears to be closer to the composition of the otolith Mar Ecol Prog Ser 188. 263-297, 1999

than is the water or blood plasma. While the factors ically between 1.06 and 1.15 (de Villiers et al. 1994, influencing endolymph composition remain poorly Stecher et al. 1996). However, the D,, between endo- understood, the transfer of calcium and other ions Into lymph and otolith is close to 0.25 (Kalish 1989). In light the endolymph may occur via a transcellular route, of the similarity between the coefficients for water- thus ensuring significant regulation of both the selec- otolith and endolymph-otolith (Fig. 2), it appears that tion of elements and their concentrations in the most of the discrimination against Sr occurs during endolymph (Mugiya & Yoshida 1995). Recent experi- otolith crystallization, rather than during Sr uptake into ments by Payan et al. (1998) indicate that the elemen- the blood plasma from the water. A similar argument tal composition of the endolymph is less affected by may explain the observed discrimination against Sr in starvation than is the plasma. bivalves (Stecher et al. 1996). The final stage of the elemental pathway from envi- The extent to which otolith trace element concentra- ronment to otolith occurs during the otolith crystalliza- tion reflects (or fails to reflect) that of the ambient water tion process, as discussed above in the section 'Otolith can be inferred from a comparison of water, blood and mineralogy and crystallization'. Even without regula- otolith composition from 2 very different chemical envi- tion by otolith proteins, significant discrimination ronments: fresh water and salt water. For example, the against some elements could be expected at this stage. concentrations of many of the most common elements For example, the distribution coefficient of Sr:Ca (e.g. Ca, Na, K, Mg, Cl) differ substantially between between water and coral or inorganic aragonite is typ- fresh and salt water, even when normalized to Ca con-

Water

Blood Fig 3. Examples of oto- lith trace elements un- likely to be influenced by concentrations in the -I water, as ind.icated by 3.2 2- IU inconsistencies between ll 9 the published composi- 6000' ?oouo* 392' tion of the water (top - panels), blood plasma (middle panels), or oto- lith (bottom panels). All - m 200. 4000 I~UOO* elemental concentra- 38.6' tions other than that of C % Ca are in units of pmoles Otolith element per mole Ca. (@IFreshwater spectes; 00 1 I000- - ! (0) marine species. Error - bar = 1 SE. Numbers -1 below x-axis indicate number of papers upon 0. . - I 1000. 37.8. - - I - 14 I 1.1 I 1' whlch the elemental CU P Na Ca concentration was based Campana: Chemistry and composition of fish otoliths 269

centration, yet those differences do not appear to be re- quired of the fish's survival. Since the elements de- flected in the otolith (Fig. 3). Other physiologically irn- posited in the otolith are derived penultimately from portant elements, such as P, Cu and S, also appear to re- the blood plasma, it is clearly unrealistic to expect the main uninfluenced by the relative concentration in the otolith content of physiologically regulated elements to environment (Fig. 3). The lack of correspondence be- reflect environmental abundance. tween environmental and otolith differences in fresh Based on differences in concentration between fresh and saltwater fishes becomes more understandable water and salt water, there are several elements whose when the corresponding blood concentrations are ex- environmental abundance may well be reflected in the amined (Fig. 3). For most of the elements noted above, otolith. Although the data are limited, the relative con- there is little or no difference in plasma concentrations centration of trace elements such as Sr, Zn, Pb, Mn, Ba between freshwater and marine fishes. This stability in and Fe in freshwater and marine otoliths is consistent blood plasma composition is well documented in the with an environmental effect (Fig. 4). These are also physiological literature (Evans 1993), and is completely elements whose uptake is more likely to be unregu- consistent with the strict osmoregulatory control re- lated compared to those of the common salts. Other

Water

Blood

Fig. 4. Examples of otolith trace elements which are probably influenced by concentrations h the 300C water, as indicated by consistency between the published composition of the water (top panels), 2000 blood plasma (middle panels), andlor otolith Otolith (bottom panels). All ele- - mental concentrations are 1000 in units of pmoles element per mole Ca. (.) Preshwa- ter species; (0) marlne species. Error bar = 1 SE. Numbers below x-axis 7 I4 indicate number of papers upon whlch the elemental Ba concentration was based 270 Mar Ecol Prog Ser 188: 263-297, 1999

Table 2. Summary of published otolith trace element compositions (pg g-') from 3 major habitat types

Element Marine species Freshwater species Estuarine species Mean SE N Mean SE N Mean SE I\;

trace elements such as Li, Cd, Ni and the less abundant BASIS FOR TRACE ELEMENT VARIATIONS IN elements may well fall into this category as well, THE OTOLITH although data for these elements are even more scarce (Table 2). Note, however, that the e1ement:calcium Environmental availability ratios in the plasma (excluding Sr) tend to be much higher than in the otolith (Figs. 3 & 4), indicating that For those elements where the composition of the otolith composition is not merely a passive reflection of otolith reflects the elemental composition of the water, plasma composition, even if correlated. a broad range of otolith concentrations can be ex-

t Otolith

Fig. 5.Mean elemental composition (m01 I-') of seawater (SW) and fresh water (W) as drawn from the literature. Only recent (1987 to present) publications (n = 80) were used so as to take advantage of improvements in analytical accuracy. Elements which have been reported as being detected in otoliths are indicated Element by an arrow Carnpana: Chemistry and composition of fish otoliths 27 1

pected (Johnson et al. 1992).Elemental concentrations The answer is an unequivocal 'sometimes'. While sev- in seawater range over at least 16 orders of magnitude eral experiments have been carried out to the for the 78+ elements that have been quantified (Fig. 5). response of otolith composition to the composition of With the exception of the common marine salts, fresh- the water, the majority have been limited to simple water concentrations are reasonably similar (Fig. 5); dilutions of seawater so as to modify absolute elemen- the apparent abundance of the rarer elements in fresh tal concentrations. These experimental salinity treat- water compared to seawater may reflect recent ad- ments have resulted in modest changes in the otolith vances in marine analytical procedures more than true composition (somewhat greater for Sr), but not of the differences (Benoit et al. 1997). Many of the more scale which might have been expected based on the abundant elements have also been detected in otoliths change in absolute concentration (Fowler et al. 1995a, (Fig. 5), suggesting that the less common elements Hoff & Fuiman 1995, Secor et al. 199513, 1998, Tzeng simply await detection in the otolith. For those otolith 1996). In retrospect, this is not surprising given that the elements which have already been detected, the vari- e1ement:Ca ratio in the experimental water did not dif- ance around the mean values is reasonably large fer among treatments. Experiments which have mani- (Table 2), suggesting the presence of inter-specific dif- pulated ambient elemental concentrations indepen- ferences, methodological problems and heterogeneity dent of Ca have largely been restricted to Sr, but have in environmental concentrations. Of course, it is the shown much more pronounced effects on otolith com- geographic variation in water-borne concentrations position (Ennevor & Beames 1993, Brown & Harris which is of greatest interest to those using otolith com- 1995, Schroder et al. 1995, Farrell & Campana 1996, position as an indicator of past location. Such geo- Gallahar & Kmgsford 1996, Dove 1997, Geffen et al. graphic variations in water concentration are the norm 1998). Increased concentrations of trace elements such for many elements (Balls et al. 1993, Benoit et al. 1997), as La, Sm, Ce, Ba, Hg and Mn have all been reported although variability in coastal and inland waters is in the otolith after exposure to elevated concentrations often much larger than that of the open ocean. With in fresh water (Ennevor & Beames 1993, ~ove1997, elements such as Ca, Sr and Mg showing conservative Geffen et al. 1998). Single-element additions of Sr, Cd profiles (reflecting salinity) and others such as Ba, Zn and Ba to salt water also resulted in significant incor- and Cd showing nutrient-type profiles (surface deple- poration into the otolith (S. R. Thorrold, G. Bath, C. tion with enrichment at greater water depths), broad Jones & S. E. Campana unpubl.). Similarly, Sr isotope patterns of relative concentration are often predictable ratios in the otolith increased with the ratios present in (Bruland 1983).Elements such as Hg and Pb are more the surrounding water (Kennedy et al. 1997). In each of generally associated with anthropogenic activity. How- 5 independent experiments which manipulated Sr:Ca ever, regional variability associated with river dis- ratios in the water, ambient Sr:Ca was well correlated charge, upwelling, volcanic activity, pollution, biologi- with otolith Sr:Ca (Brown & Harris 1995, Schroder et al. cal activity and inter-annual differences can all 1995, Farrell & Campana 1996, Gallahar & Kingsford interact to confound any expected relationship. Vari- 1996, Dove 1997).When the data from all of these stud- ability of a factor of 10 or more is not unusual when ies are pooled, the overall relationship also shows a comparing aqueous elemental concentrations among significant positive relationship between ambient and locations (Balls et al. 1993, Benoit et al. 1997). otolith Sr:Ca (Fig. 6);this experimental relationship is It is important to note that the ambient concentration consistent with observed differences between fresh- of an element, even when referenced to calcium, is not water and saltwater fish otoliths (Fig. 6).However, the necessarily a good indication of its availability to either linear increase in the otolith Sr:Ca ratio relative to the the otolith or the fish. In general, dissolved ions which ratio in ambient water is not proportional; the distribu- are free of ligands are the only chemical species avail- tion coefficient, D,,, of 0.14 from Fig. 6' is significantly able for uptake by the gills (Knezovich 1994). Elemental less than 1, in keeping with the earlier discussion of concentrations referenced to calcium concentrations physiological discrimination. Distribution coefficients are most relevant for such elements. As an example, the between water and otolith have not yet been calcu- absolute concentration of Sr ranges from about 9 X lated for elements other than Sr. Otolith calcium, how- 10-7 M in fresh water to about 8.7 X 10-5 M in salt water, ever, does not respond to variability in water concen- an almost 100-fold difference. However, the molar ratio of Sr:Ca in fresh water (1.8 X 10-~)is only 4.8 times less than that of salt water (8.6 X 10-~).It is this latter ratio that is more relevant to the question of relative rates of 'The D,, reported here, as well as that of Kalish (1989), is based on the slope of the relationship between ambient Sr uptake in fish between fresh water and salt water. and otolith Sr:Ca, since the regression intercept was signif- Do the elemental concentrations in the otolith reflect icant. However, D is usually calculated as (element:Ca),,,/ the elemental concentrations of the ambient water? (element:Ca),,l,t,o, 272 Mar Ecol Prog Ser 188: 263-297, 1999

trations, at least under normal conditions (Ichii & Mugiya 1983, Farrell & Campana 1996). The presence of geographic variations in water chemistry, combined with partial cor- respondence between environmental avail- ability and the incorporation rate into the otolith, bodes well for those applications of otolith elemental analysis that reconstruct environmental history. However, there are caveats to this statement. The first such caveat is that 5 of the 6 minor elements 0.ml." I which are most easily measured with the 0.m 0.002 0.034 0.m 0.038 0.010 0 012 widely available electron microprobe (Gunn Water Sr:Ca et al. 1992) are likely to be under strict phys- iological regulation, and thus unsuitable for Fig. 6. Summary of published molar Sr:Ca values as a function of the Sr:Ca use as environmental indicators. The only ratio in the water (e).The fitted regression is statisticaily significant. Also shown are the mean (295% Cl) ratios for freshwater (FMr)and saltwater exception is Sr The second caveat concerns lSWl otolilhs/water as drawn from the literature the analysis of the less abundant trace ele- ments, which appear to be more suitable as environmental indicators. While their lower concentra- This equation does not apply to aragonitic corals, tions makes them less likely to be osmoregulated by where some form of biological fractionation is pre- the f~sh,it also makes them more difficult to assay with sumed to influence the underlying kinetic relationship. accuracy and without contamination during handling. Nevertheless, some form of temperature dependence Finally, few if any trace elements (even when normal- in coral Sr:Ca seems to be well accepted, despite con- ized to Ca) are likely to be incorporated into the otolith cerns over the nature of the Sr incorporation (Greegor in direct proportion to availability in the environment. et al. 1997) and the likelihood of confounding physio- Both temperature and growth rate are known to be at logical effects (de Villiers et al. 1994, Hart & Cohen least as influential as ambient concentration in modify- 1996). Shen et al. (1996) presented the following equa- ing otolith elemental composition, as is discussed in tion for generalized use with the Porites genus of arag- the following section. onitic corals: Sr:CaN (in mm01 mol-') = 10 286 - 0.0514 SST Temperature where SST = sea surface temperature and N = Sr:Ca normalized to that of Hawaiian seawater. Note that Perhaps more than any other variable, the influence here, as in all published temperature calibrations for of temperature on the rate of trace element incorpora- corals, the Sr:Ca of the coral is directly proportional tion into calcified tissues has been studied and to that of the seawater, independent of any tempera- debated. The driving force behind much of this interest ture effect. Even in the open ocean, variability in the is the potential application of trace element concentra- Sr:Ca of the water among locations or throughout the tions as paleoindicators of oceanographic conditions year can introduce variability in the Sr:Ca of the and for reconstructing the temperature exposure his- coral equivalent to temperature variations of 0.2 to tory of individual organisms. Several elements have 0.7"C (de Villiers et al. 1994, Shen et al. 1996). Vari- been reported to vary systematically with temperature ability in coastal waters would, of course, be much in aragonitic corals, including Mg (Mitsuguchi et al. larger. 1996) and U (Shen & Dunbar 1995). But aside from In light of the strong correlation between ternpera- '80:'60ratios (which are discussed below under 'Sta- ture and e1ement:Ca concentrations in corals, it is ble isotopes'), it is Sr:Ca ratios that have been been understandable that similar relationships were ex- considered to be the most promising. The underlying pected to be uncovered in otoliths. Indeed, significant basis of the Sr:Ca temperature dependence is pre- and often substantial temperature effects have been sumed to be a kinetic effect, since the rate of Sr:Ca reported in many otolith studies, although the relation- incorporation into inorganic aragonite varies inversely ships have not necessarily been consistent among with temperature according to the following equation studies. Examples of otolith elements influenced by (Kinsman & Holland 1969): temperature include Mg (Fowler et al. 1995a, Hoff & (Sr:Ca) (in mm01 mol-'1 = 10.66 - 0.039 X Temperature Fuiman1995), K (Hoff & Fuiman 19951, Na (Kalish Campana: Chemistry and compos~tlonof fish otoliths 273

itive (Kalish 1989, Arai et al. 1995, 1996, Fowler et al. 1995a, Hoff & Fuiman 1995. Limburg 1996), negative (Townsend et al. 1989, 1992, 1995, Radtke et al. 1990, Sadovy & Severin 1992, Secor et al. 1995b), and non-existent (Gallahar & Kings- ford 1996, Tzeng 1996) correlations with temper- ature have been reported. A meta-analysis of the existing literature does not support an overall relationship between otolith Sr:Ca and tempera- ture for either saltwater or freshwater fish (Fig. ?a). Of course, interspecies differences could I 0.m l mask such a relationship. Therefore, the analysis 0 10 20 30 49 was repeated using only the within-study differ- Temperature ence in Sr:Ca ratio versus the within-study differ- ence in temperature, so as to maximize statistical 0001 power (Fig. 7b). Once again, no relationship was m m b m evident. While this result does not prove that tem- 0m' a @mm m perature-dependent Sr:Ca fractionation in oto- m m m m liths is absent, it does indicate that it is not a gen- m m 0 eralized phenomenon. &j -c.ml1 • m m C .- m Townsend et al. (1992) postulated that temper- a, ature-dependent Sr:Ca fractionation may only F0 002' m occur at low temperatures, perhaps because of 5 some reduced ability to discriminate against Sr 0.W' • during entry into the endolymph. To test this m hypothesis, the slopes of all published Sr:Ca ver- -0.004. 0 2 4 6 8 10 12 14 sus temperature relationships were regressed against the median temperature of the study Change in temperature (Fig. 8), with the expectation that fractionation Fig. 7. Relationship between marine otolith Sr:Ca (pm01 mol-l) and (negative be apparent at low temperature. Each point represents the results of an experimental temperatures. The slope in Fig. 8 is significant treatment, as drawn from the literature. Neither relationship is sta- (p = 0.03), although only those experiments car- tistically significant. (a) Observed values. (b) Within-study change ried out below 100~produced temperature coeffi- in Sr:Ca across within-study temperature treatments cients significantly different from zero. If real, such a relationship suggests that Sr:Ca ratios 1989, Hoff & Fuiman 1995), Mn and Zn (Arai et al. decrease with increasing temperature at low tempera- 1995, Fowler et al. 1995a), and Fe (Gauldie et al. 1980, tures, but the ratios increase with temperature at high Arai et al. 1995, Fowler et al. 1995a). However, partic- temperatures. As will be seen later, however, there is ular attention has been focused on Sr:Ca, in which pos- an alternative explanation for these results.

Pagrus l

Alosa m

Fig. 8. Temperature sensitivity of the Sr:Ca ratio In otoliths as a function of mean experimental temperature. Temperature sensitivity was de- Anguilla Ul -- Clu~ea I fined as the slope of the within-study regression of Sr:Ca on temperature, as determined from published values. The regression is significant (p = 0.03), suggesting that temperature sensitiv- ity declines with increasing temperature. Also shown are the temperature coefficients for inor- ganic aragonite (Kinsman & Holland 1969) and an aragonitic coral (Shen et al. 1996) Mean temperature 274 Mar.Eco1Prog Set 188: 263-297, 1999

Given the controversy in the Literature concerning but not necessarily correlated with, temperature cycles the utility of otolith Sr:Ca ratios as indicators of either (Sadovy & Severin 1992, 1994, Fuiman & Hoff 1995). salinity (Kalish 1990, Halden et aI. 1995, Secor et al. The common factor among all of these observations is 199%) or temperature (Radtke 1989, Townsend et al. variability associated with the rate of protein synthesis 1992, 1995), there has been disagreement over which in relation to the crystallization rate of the otolith. of the 2 environmental factors would be expected to be The rate of calcium carbonate crystallization would most influential in modifying otolith composition. Sim- normally be considered a major source of Sr:Ca vari- ple calculations, however, suggest that changes in ability in fish otoliths, just as it is in calcite (Lorens salinity would generally be more detectable than 1981), bivalves (Stecher et al. 1996), and corals (de Vil- would changes in temperature. On the basis of the liers et al. 1994). However, various studies have failed observed difference in ololith Sr:Ca ratios (shown in to find a relationship between Sr:Ca and otolith incre- Fig. 6) between fresh water and salt water, each l %O ment width (Kalish 1989, Gallahar & Kingsford 19921, increase in salinity would be expected to produce a indicating that otolith Sr:Ca is not a simple function of 0.05 X 10-~increase in the otolith Sr:Ca molar ratio. calcification rate. To some extent, this may be ex- Given the 30 to 35% difference in salinities between plained by the fact that otolith and endolymph compo- marine and riverine waters, corresponding to a 1.5 X sition appear to be more tightly regulated than are the 10-3 change (or 3-fold) in the otolith Sr.Ca ratio, it is same processes in other taxa. in addition, the role of obvious why otolith Sr:Ca is such an effective indicator water soluble, calcium binding prote~ns in regulating of anadromy in fishes. Mean observed Sr:Ca tempera- otolith growth appears to be substantial (Wheeler & ture slopes of about -0.1 X le3per degree for Clupea Sikes 1984, Wright 1991, Asarlo & Mugiya 1993). sp. (Fig. 8) are twice those observed in corals (Shen et Therefore, while the rate of crystallization may well be al. 1996) and nearly 3 times that observed in inorganic one of the factors influencing Sr:Ca incorporation into aragonite (Kinsman & Holland 1969), and are thus the otolith, it seems likely that the crystallization rate is somewhat larger than would otherwise be expected. controlled by the formation rate of proteinaceous Even if accepted as given, a slope of -0.1 X 10-3implies matrix on the growing otolith surface. The rate of that each degree of rising temperatnre would only matrix formation in turn is highly correlated with the result in a 0.1 X IO-~decline in the otolith Sr:Ca ratio. rate of somatic growth, since the latter reflects the rate Thus a 15°C temperature shift would be required to of net protein synthesis. produce a change in otolith composition equivalent to Our current understanding of the otolith mineraliza- that produced by anadromy. Using the temperature tion process is too scanty to allow the development of a effect observed in corals, a 30°C shift would be detailed hypothesis to explain otolith Sr:Ca ratios. required. Note, however, that a temperature effect has However, a crystallization process which is controlled only been observed in otoliths at median temperatures by the rate of matrix protein formation, and inversely of less than 10°C (Fig. 8). proportional to otolith Sr:Ca, is consistent with most of the observations. Since the rate of protein synthesis is often highly correlated with metabolic rate, tempera- Integrated effects on otolith Sr:Ca ture and somatic growth rate, explanations for pre- sumed temperature (Radtke 1989, Townsend et al. Notwithstanding the controversial effects of temper- 1989, 1992, 1995) and growth rate effects (Sadovy & ature on otolith Sr:Ca, it is clear that there is a wide Severin 1992, 1994) on otolith Sr:Ca become readily range of observed ScCa ratios among species, habitats apparent. However, the link between temperature and and studies, even within a given salinity regime. Thus protein synthesis often fails at higher temperatures, any generalized hypothesis for Sr:Ca incorporation due to higher metabolic losses. Such losses would into the otolith must reconcile the following apparently explain the absence of Sr:Ca temperature sensitivity in unrelated observations: (1) a broad tendency towards fish maintained at higher temperatures (Fig. 8). By lower Sr:Ca ratios in the faster growing individuals of a corollary, both Sr:Ca ratios and their sensitivity to tem- species (Sadovy & Severin 1994); (2) the apparent tem- perature should be maximal in otoliths with low pro- perature effect in low-temperature larvae, but not in tein synthesis (growth) rates. This prediction of the mid- and high-temperature larvae (Fig. 8); (3) the very hypothesis is consistent with the elevated Sr:Ca ratios high Sr:Ca ratio in larvae of eels, AnguiUa species, in eel leptocephalus otoliths, which exhibit extremely even at hgh temperatures (Otake et a1 1994, Tzeng low growth rates even at high temperatures (Otake et 1996); (4) the commonly observed increase in otolith al. 1994, Tzeng 1996). It also explains, for the first time, Sr:Ca with fish age (Radtke & Targett 1984, Radtke the peak in the Sr:Ca ratio at the time of metarnorpho- 1987, Sadovy & Severin 1992, Proctor et al. 1995); and sis from the larval stage, since metamorphosis is a life (5) annular variations in Sr:Ca which are in phase with, history stage with markedly curtailed growth (Toole et Campana: Chemistry an .dcomposit~on of fish otoliths 275

al. 1993, Otake et al. 1994, Tzeng 1996). Similarly, pro- al. 1993, Thorrold et al. 1997b), differentiate among tein synthesis and otolith growth tend to decline with groups of fish (Edmonds & Fletcher 1997, Kennedy et age in adult fish (Mulcahy et al. 1979, Schwarcz et al. al. 1997, Dufour et al. 1998, Thorrold et al. 1998b), infer 1998), which is consistent with the observed long-term metabolic history (Radtke et al. 1987, Kalish 1991c, increase in Sr:Ca (Radtke & Targett 1984, Radtke 1987, Gauldie 1996a, Schwarcz et al. 1998), and reconstruct Sadovy & Severin 1992, Proctor et al. 1995). Even migration history (Northcote et al. 1992). otolith checks, which are regions of reduced otolith The source of stable isotopes incorporated into the growth (Campana & Neilson 1985), are characterized otolith varies with the element. Elements such as oxy- by elevated Sr:Ca levels (Kalish 1992).Thus it appears gen and strontium appear to be incorporated into the that variations in the rate of otolith matrix formation otolith with isotopic ratios which are nearly identical to are at least partially responsible for the observed range that expected of crystallization from the ambient water of Sr:Ca ratios across species within a given salinity (Kalish 1991c, Kennedy et al. 1997, Thorrold et al. regime, with temperature and/or growth being occa- 1997b), suggesting that the water is their primary sional, and often frequent, correlates. Of course, Sr source. In contrast, approximately 10 to 30% of otolith incorporation into the accreting otolith is also a func- carbon may be derived from metabolic sources (Kalish tion of the Sr:Ca ratio in the endolymph (Kalish 1989). 1991c, Schwarcz et al. 1998), suggesting a dietary ori- Since the latter varies with the salinity of the ambient gin. The remainder of the otollth carbon would pre- water, variation in otolith Sr:Ca due to protein-regu- sumably come from dissolved inorganic carbon (DIC) lated crysta.llization would overlay, not replace, varia- in the water. tion due to salinity effects. The chemical processes that lead to isotopic enrich- The key to testing the hypothesis linking otolith ment or depletion in biogenic carbonates have been Sr:Ca with the rate of proteinaceous matrix formation carefully described elsewhere (Leder et al. 1996, Swart would appear to lie with experiments in which otolith et al. 1996), and do not bear repeating here. In brief, Sr:Ca, matrix formation and calcification rate are thermodynamic principles (e.g. solution concentration simultaneously monitored. In light of the 'uncoupling' and temperature) regulate the degree of isotopic frac- of fish and otolith growth noted in many studies, it is tionation in inorganic aragonite; where these principles quite possible that the rate of matrix formation pro- are the only ones Influencing fractionation in a biologi- vides threshold limits for calcification rate, but that the cal system, the system is said to be in equilibrium. The effect of temperature on calcification rate is additive effect of these purely physical factors is easily predicted within those limits (Mosegaard et al. 1988). If such a when the temperature and the isotopic composition of mechanism exists, it is not at all obvious how otolith the precipitating solution (e.g. ambient water) is Sr:Ca might respond. Further research in this area is known. Thus carbonates precipitated under equilib- clearly required. rium conditions are often used to estimate past temper- atures and/or water composition (Kim & O'Neil 1997). However, many biogenic carbonates undergo addi- STABLE ISOTOPES tional fractionation due to 'vital effects', which includes both kinetic and metabolic effects (Kalish 1991c, Thor- Stable isotopes are neutral, non-radioactive variants rold et al. 199713). These effects have been carefully of an element whose relative uptake can be modified studied in corals and bivalves, but recent studies have by the environment or biological activity due to their indicated that fractionation in corals and otoliths differ slightly different atomic mass. Processes that enhance in some key respects. For example, corals are often iso- the uptake of 1 isotope over another thus result in an topically depleted (typically -3 to -5 %o) in 6180 relative isotopic ratio which differs from that of the source. to marine fish otoliths (-2 to +4%0) from comparable Accordingly, stable isotope ratios have a long history of habitats. For this reason, corals are probabIy a poor use as geological and biological tracers, recorders of model for isotope fractionation in otoliths. temperature, salinity and pH, and indicators of feeding Oxygen isotopes in otolith aragonite are deposited history, trophic level and metabolic rate (Peterson et al. in, or very near to, equilibrium with the ambient 1985, Hesslein et al. 1991). Carbon and oxygen are the water (Kalish 1991b,c, Patterson et al. 1993, Radtke et 2 elements with the most extensive history of interpre- al. 1996, Thorrold et al. 1997b), suggesting similar tation, although stable isotope ratios of nitrogen, sul- behaviour to that of inorganic aragonite. Inorganic fur, lead, strontium and boron are increasingly being aragonite fractionation has not been studied at more used (Vogel et al. 1990, Spivak et al. 1993, Gannes et than 1 temperature, but the recent study of Kim & al. 1997). In otoliths, stable isotope ratios have been O'Neil (1997) rigorously quantified the temperature used to reconstruct temperature history (Devereux dependence of the oxygen isotope fractionation factor 1967. Mulcahy et al. 1979, Kalish 1991b. Patterson et in calcite. Since a 0.6%0 enrichment in aragonite rela- 376 Mar Ecol Prog Ser 188: 263-297, 1999

(calcite equation from km& O'Neil 119971 apply- ing their revised acid fractionation factor, en- riched by 0.6 % for calcite-aragonite): lOOOlna = 18.03 (1000 TK-') - 31.82 1a3 where a = the fractionation factor = (S,, + 1000)/!6,,, + 1000)

I- 001 or approximately: 6010- 6, = 3.71 - 0.206 T°C Normally, the temperature term would refer to the temperature of the ambient water, but in . thermo-regulating fishes such as , it must be -4 26 referred to the internal temperature of the fish 0 10 20 30 (Radtke et al. 1987). As is clearly evident from Temperature (OC) both of the above equations, it is not possible to estimate temperature from otolith oxygen isotopic Fig. 9. Relationship between the estimated oxygen isotope frac- ratios unless the isotopic cornposit~onof the water tionation factor (10001n a) and temperature fcr inorganic aragonite (heavy solid line = calcite of Klm & O'Neil 1997, enriched by 0.6% at the lime of fOrnlatiOnis (Or can be for aragonite). Also shown are the published otolith relationships of estimated). Broad correlations between the iso- ~alish(l99lb)(- - - - -) and T'horrold et al. (1997b) (- - - -)Ithe topic composition of water and salinity exist, but otolith-based relationships of Patterson et al. (1983). Kalish (1991~) these often vary substantially with water depth, and Radtke et al. (1996) are also shown, but are indistinguishable latitude and other factors (Tan et al. 1983). As a from that of inorganic aragonite. The overall relationship of arago- nitic forams and molluscs studied by Grossrnan & Ku (1986) (. . . . .) result, interpretation of otolith oxygen isotopic has been commonly used for otoliths in the past, but no longer ratios can be dangerous in the absence of inde- seems appropriate pendent information on the isotopic composition of the water (Thorrold et al. 199813). tive to calcite has been well documented (Tarutani et There is broad agreement that carbon isotopes are al. 1969, Grossman & Ku 1986, Kim & O'Neil 1997), deposited in otoliths under con-equilibrium condi- the fractionation equation for aragonite can be calcu- tions, probably due to metabolic generation of the car- lated. A comparison between the resulting inorganic bonate ion incorporated into the endolymph from the aragonite fractionation relationship and that esti- blood plasma (Kaiish 1991c, Gauldie 1996a, Thorrold mated in several otolith studies shows a striking et al. 1997b, Schwarcz et al. 1998). Reported values for resemblence (Fig. 9). Furthermore, there is no evi- otolith 6I3C range from -9 to +l, and are typically 5% dence of a relationship between precipitation rate and more depleted than inorganic aragonite (Romanek et fractionation (Thorrold et ai. 199713). Deviations al. 1992). In contrast, blood and other fish tissues are among studies in the intercept of the fractionation highly depleted in 613C due to their metabolic origin, relationship shown in Fig. 9 are unlikely to be biolog- with values of about -17%0 (Fry 1983). The large dis- ically significant, since some studies inferred, rather crepancy in the 6I3C composition between otolith and than measured, the isotopic composition of the water blood is attributable to the fact that only 10 to 30 % of (Radtke 1984, Kalish 1991b). An additional report of the otolith carbon is derived from metabolic sources non-equilibrium deposition was probably the result of (Kalish 1991c, Schwarcz et al. 1998); the remainder a calculation error (Radtke et al. 1996). Most of the probably comes from DIC which is typically about lob remaining studies show good agreement with the in marine surface waters (Schwarcz et al. 1998).Over- eqnation for inorganic aragonite (Kalish 1991c [across lying all of these effects is a weak positive relationship all species]; Patterson et al. 1993, Radtke et al. 1996), between otolith precipitation rate and 813C, which is but differ slightly from that based on aragonitic further evidence of a metabolic influence on fractiona- foraminifera and molluscs, which was previously tion (Thorrold et al. 1997b). applied to otoliths (Grossman & Ku 1986). In light of Interpretation of the otolith 6% signal is compli- the close similarity of the recent experimental results cated by more than just metabolic fractionation. As on otoiiths with the theoretical expectation, there is noted by Schwarcz et al. (19981, the metabolic compo- no reason to continue to use the Grossman & Ku nent of otolith 6I3C is in turn influenced by the 613C of (1986) equation with otoliths. Therefore, the relation- the diet and the metabolic rate of the fish. In general, ship between temperature and the oxygen isotopic the 613C of the diet can be expected to increase with ratio is best described by that of inorganic aragonite trophic level, whereas the metabolic rate (and meta- Campana: Chemistry and composition of fish otoliths 277

bolic contribution) would be expected to decrease with 228Ra, 'lOPb, 210Po, 228Th, 238U). Other radioisotopes age. These 2 processes are probably responsible for such as "Sr and I3'Cs have been detected in fish tis- the oft-observed increase in otolith F13C until the age of sues (Beddington et al. 1989, Forseth et al. 1991), sug- maturity (Mulcahy et al. 1979, Kalish 1991c, Gauldie gesting that they may also be incorporated into the 1996a, Schwarcz et al. 1998), since both would result in otolith. Nevertheless, the only attention which has an increase in otolith 6I3C with age. Temperature been given to otolith radioisotopes has been in the con- effects on otolith 8I3C appear to be small, resulting in a text of age determination and validation, and virtually decline of 0.2%0 for each degree of warming (Kalish all of that attention has been focused on only 2 isotope 1991c, Thorrold et al. 199713). A previous report of no pairs: 210Pb:22%a(Bennett et al. 1982, Campana et al. temperature effect appears to be based on an error in 1990, Fenton et al. 1990, 1991, Kastelle et al. 1994, Mil- calculation (Radtke et al. 1996). Variations in the 613C ton et al. 1995) and 228Th:228Ra(Smith et al. 1991, Cam- of DIC due to water depth are slightly larger, declining pana et al. 1993). A third isotope pair, 2'0Po:2'0Pb,has by about 2%oover the upper 300 m of the water column been used to age molluscs (Cochran & Landman 1984, (Schwarcz et al. 1998). Fabry & Delaney 1989), but has not yet seen use in otoliths. All radioisotopes have characteristic half-lives which RADIOISOTOPES reflect their exponential decay rates and the rates at which they approach secular equilibrium (when the Radiochemical dating of calcified structures has a rate of loss [through decay] of the daughter comes to long history in corals and molluscs (Moore & Krish- equal the rate of loss of the parent). The radiometric naswami 1972, Druffel & Linick 1978, Turekian et al. equations describing their decay and interpretation in 1982, Cochran & Landman 1984). The same underlying otoliths have been described elsewhere (Campana et concepts apply to fish otoliths, and are based on well al. 1993, Kimura & Kastelle 1995). It is the rate at which established physical principles governing radioactive secular equilibrium is approached which determines decay. Radioisotopes are incorporated into fish otoliths the preferred age range for any given isotope pair. For in exactly the same way as are stable isotopes of any example, 210Pb:226Raactivity ratios, where the half- given element. Once incorporated into the otolith, the lives of 'I0Pb and 226Ra are 22.3 and 1600 yr respec- radioisotopes decay into radioactive daughter prod- tively, approach a secular equilibrium of 1 over a ucts, which are themselves retained within the acellu- period of more than 100 yr (Fig. 10). Yet the activity lar crystalline structure. Since the half-lives of the par- ratio changes more and more slowly as the equilibrium ent and daughter isotopes are known (and fixed), the is approached, limiting the useful range of 210Pb:226Ra ratio between them is an index of elapsed time since to about 0 to 50 yr. For example, a 0.1 uncertainty in incorporation of the parent isotope into the otolith. In the case of man-made radioiso- 1.6. topes (e.g. those from nuclear explosions), ---- _H------an additional application is possible; the 1.4' / , / , presence of the parent isotope can be used 12' / :.:n0.0 / as a dated marker in the calcified structure, / m ,; ,; ' . . - - .. . . . - . . . - . . - . - . . - . - - . - - . - . ------. . . ------. . - - . taking advantage of independent knowl- .$ ,,0 " ,l edge of the year of introduction of the 3 ' I radionuclide into the fish's environment. : , .S m' I Since natural radioisotopes are generally 2 0.61; I interpreted in terms of the extent of . I radioactive decay, while radioisotope prod- ucts of nuclear explosions are used as dated marks, they will be treated separately in the following discussion. 0 10 40 Core age (yr)

Natural radioisotopes Fig. 10. Relationship between the activity ratio of 228~h:228~a,210Po:210Pb and 2'0~b:226Raas a function of fish age when the radiochemical assay is ~i~~~ their extremely low concentrations restricted to an otolith core (age 0) with an initial activity ratio of 0. Since age categories become increasingly difficult to separate as the activity ratio in both the environment and the Only a approaches secular equilibrium. Po:Pb, Th:Ra and Pb:Ra ratios are best handful of radioisoto~eshave been suited for age determinations of 0 to 1, 0 to 8 and 0 to 50 yr, respectively. detected in otoliths (e.g. I4C, 226Ra and Insert shows full age range for the 210Pb:226Raactivity ratio 278 MarEcol ProgSer 188: 263-297, 1999

the activity ratio may correspond to an uncertainty of or measured. An otolith core can be dated even if allo- just over 3 yr in a young otolith core, but would corre- genic uptake of the radioisotopic daughter is large dur- spond to 20 yr in a core from a S0 yr old fish. Similar ing the period of core formation, as long as the initial principles control the useful age range of both the daughterparent ratio is known. Moderate uncertainty 2'0Po:2'0Pb (0 to 1 yr) and the "8Th:*%a (0 to 8 yr) iso- (e.g. 10%) around some mean value of the ratio will tope pain (Fig. 10). have little influence on the predicted decay weif the Unlike many other aspects of otolith science, the allogenic daughtcr:parent ratio is small (e.g. <0.2). assumptions central to radiochemical dating in otoliths However, the same relative uncertainty can introduce have been given careful attention by a number of serious error in age estimation if the mean ratio is high, authors (Fenton et al. 1990, Fenton & Short 1992, Cam- since the ability to discriminate among adjacent age pana et al. 1993, Kastelle et al. 1994, West & Gauldie groups decreases as secular equilibrium is approached 1994, Francis 1995, Kimura & Kastelle 1995). Some (Fig. 10). The initial value of the daughter:parent ratio problematic assumptions must be made to interpret can be inferred using otoliths of young fish (of the radioisotope ratios in whole otoliths. However, since same age as the core) from several year-classes from the otolith core was formed when the fish was very an appropriate location. Where these measurements young, the age of the extracted otolith core is very sim- have been made, 210Pb:226Ra ratios have generally ilar to the age of the fish (Campana et al. 1990, Smith et been less than 0.2 (Bennett et al. 1982, Carnpana et al. al. 1991, Kim.ura & Kastelle 1995). Thus interpretation 1990, Fenton et al. 1990, 1991, Kastelle et al. 1994, Mil- of the otolith core avoids problematic assumptions, and ton et al. 1995). However, large variations in the first is widely acknowledged to provide more reliable few years of life have been noted (Milton et al. 1995), results than would the whole otolith (Kimura & Kastelle suggesting that significant uptake of allogenic Pb or 1995). The assumptions for dating otolith cores are as 210Po (the intermediate daughter) is quite possible follows: (Fabry & Delaney 1989, Fenton et al. 1990). (1) The otolith constitutes a closed chemical system, Radiochemical dating of whole otoliths makes the such that radionuclides are neither galned nor lost same 2 assumptions as listed above, although the after incorporation, except through decay. Further- assumption dealing with negligible internal migration more, there is no appreciable migration of radionu- of radioisotopes within the otolith does not apply. How- clides within the otolith (e.g. into or out of the core ever, assays of whole otoliths make the following 2 region). Clear violations of this assumption have been additional assumptions, both of which can be trouble- observed in vertebrae (Weldon et al. 1987), in some: which remobilization of calcified material distorted the (3) The rate of uptake of both the parent and daugh- expected daughter gradient between the older centre ter isotopes remains in constant proportion to the mass and the younger edge. However, such remobilizations accumulation rate of the otolith. As was the case with do not occur In otoliths, since they are both acellular the otolith cores, the initial daughter:parent ratio must and metabokcally inert (Campana & Neilson 1985). either be low or known. The additional constraint with The possibility of loss of 222Rn,the gaseous intermedi- the interpretation of whole otoliths is that this ratio ate of the 2"~a + "OPb decay chain, through the must remain constant and in fixed proportion with cal- charinel architecture of the otolith has been vigorously cium deposition as the otolith grows. Otherwise, it is debated in the literature (Fenton et al. 1990, 1991, impossible to know the proportion of decay which Gauldie et al. 1992, Campana et al. 1993, West & occurred in each year of growth. Only Fenton et al. Gauldie 19941, although neither side has presented (1990, 1991) have tested this assumption, but they doc- experimental evidence in support of their position. umented age-dependent trends in the 226~a:Caratio, Nevertheless, there is compelling empirical evidence thus invalidating the assumption. In addition, Fowler in support of a closed system: if '"Rn is lost during et al. (1995a) demonstrated that the rate of Pb incor- decay, age estimates based on daughter:parent isotope poration into the otolith increased with decreasing ratios should be skewed towards lower ratios, and growth rate, suggesting that Pb incorporation probably hence younger ages. Yet all published 210Pb:226Ra increases with fish aye. ratios to date have resulted in extremely old age esti- The assumption associated with a fixed rate of mates, some of which have exceeded 100 yr (Carnpana radioisotope uptake can be relaxed if stable, more eas- et al. 1990, Fenton et al. 1991, Francis 1995). Therefore, ily measured proxies can be found for the parent and if Rn emanation is occurring in these otoliths, it must be daughter radioisotope (Fenton & Short 1992). The use occurring at very low levels. of stable Pb as a proxy for ''qb seems reasonable, (2) Uptake of daughter isotopes from external since there is little reason to expect age-related frac- sources (allogenic) is smdcompared to that of f3e par- tionation between 2'%b and the far more abundant ent. Where this uptake is non-zero, it must be known stable isotopes of Pb. However, the application of Ba as Campana: Chemistry and c:omposit~on of fish otoliths 239

an analog of Ra assumes that the chemical behaviour has been well described at large spatial scales of Ba in the water column is similar to that of Ra. There (Broecker et al. 1985, Duffy et al. 1995). Through is no evidence to suggest that this is the case (Bruland analysis of annular growth rings in coral, various work- 1983). Re-analysis of the data of Milton et al. (1995) ers demonstrated that bomb radiocarbon was incorpo- showed no significant correlation between Ba and Ra rated into the accreting coralline skeleton in concen- concentrations, whether withn otolith cores or the trations proportional to those present in the water whole otolith. Therefore it is premature to consider Ba column at the time (Druffel & Linick 1978, Nozaki et al. as a stable equivalent of 226Ra. 1978). Thus the time series of bomb radiocarbon in the (4) The mass growth rate of the otolith is either con- coral was shown to reflect that present in the marine stant or its rate of change is known. Since the otolith environment, which increased by about 20 % between core decays at a fixed and known rate over a length of 1950 and 1970. During the same time period, radionu-

time almost equivalent to the age of the fish, it is inde- clides such as 'OS~suddenly appeared in bones, antlers pendent of any subsequent otolith growth. However, and other calcified structures (Beddington et al. 1989, when the entire otolith is analyzed, the outer layers of Schonhofer et al. 1994). In the case of both radiocarbon the otolith will have been more recently deposited and and the other bomb-produced radionuclides, the hence will have experienced less radioactive decay period corresponding to their sudden increase in the than the inner growth layers. Modification of the decay environment serves as a dated marker for those calci- equations to accommodate variable otolith growth fied tissues in which they were incorporated. rates are straightforward and uncontested as long as Kalish (1993) was the first to demonstrate that fish the relative otolith growth rate is known (Bennett et al. otoliths also incorporated bomb I4C, and that the time 1982, Campana et al. 1993, Francis 1995, Kimura & series of radiocarbon reconstructed from presumed Kastelle 1995).However, it is circular reasoning to use otolith annuli was similar to that present in nearby annulus counts to calculate the growth rate required of corals. Thus he was able to infer that the otolith annuli the decay equation, which is then used to validate the had been interpreted and aged correctly, because sys- annulus counts (West & Gauldie 1994, Francis 1995, tematic under- or over-ageing would have resulted in a Kimura & Kastelle 1995). In a simulation study of the phase shift between the otolith A'~Cand the coral A14C effect of ageing error on the calculated radiochemical time series. Subsequent work (Kalish 1995a, b, Kalish age, Kimura & Kastelle (1995) demonstrated that incor- et al. 1996, 1997, Campana 1997, Campana & Jones rect annulus-based ages readily translated into incor- 1998) has confirmed the value of the bomb radiocarbon rect radiochemical ages, with each supporting the technique for solving problems of age validation in a 'accuracy' of the other. Francis (1995) also recognized variety of fish species. A similar approach has been this problem, and proposed the use of 2 alternative used by workers in other disciplines to infer age and approaches which provide most-probable andlor mini- the frequency of growth ring formation in both bi- mum age estimates, thus avoiding the need for annu- valves (Turekian et al. 1982, Peck & Brey 1996) and lus-based ages. While a notable improvement over mammals (Bada et al. 1990). Note that this approach is previous whole-otolith equations, these alternative unlike traditional radiocarbon dating, since there is no approaches still assume 2-stage linear otolith growth, appreciable radiocarbon decay over periods of less an assumption whose validity remains to be tested. than 100 yr. The synchrony in the appearance of the bomb AI4C signal in surface waters throughout the global ocean is Radioisotopes from nuclear explosions striking, particularly since it has been observed in a diverse array of organisms (Fig. 11). Corals, bivalves The widespread atmospheric testing of atomic and fish otoliths from the North Atlantic (Druffel 1989, bombs in the 1950s and 1960s released more than a Weidman & Jones 1993, Campana 1997) through to the dozen radioactive isotopes into the environment. Iso- South Pacific (Kalish 1993, Guilderson & Schrag 1998, topes such as 'OSr and 239P~tended to be associated Peng et al. 1998) all recorded a sharp increase in AI4C with particulates (nuclear 'fallout'), and thus were most between the late 1950s and early 1970s. Such large- concentrated in the vicinity of the bomb site. On the scale synchronicity implies that the 6I4C time series other hand, radiocarbon (14C or carbon-14) was intro- reconstructed from the otolith cores of old fish can duced as carbon dioxide gas, spread rapidly through- safely be compared (and validated) against a baseline out the atmosphere, and soon resulted in a doubling of chronology from any other species in the region from a background atmospheric radiocarbon levels (Nydal comparable habitat. As will be discussed later, subtle & Lovseth 1983). Rain and atmosphere-ocean gas differences in the phase of the various AI4C chronolo- exchange quickly introduced the radiocarbon into the gies may exist, attributable primarily to depth (Fig. 11) surface layer of the world's oceans in a manner which and water source (Kalish 1995b, Campana & Jones Mat Ecol Prog Ser 188: 263-297.1999

there may have been limited contamination of the cores with more recently deposited otolith (and &I4C) material. The second assumption is implicit in all age- ing studies, sincc it implies that a given growth in- crement is interpreted in the same way, whether observed in a young or an old fish. However, the third assumption, that of synchronicity among the AI4C chronologies, is more interesting. This assumption has been tested and confirmed in all reported bomb radio- carbon otolith ageing studies on marine fishes living in the surface mixed layer, whether in the south Pacific (Kalish 1993, 1995a) or the north Atlantic (Campana 1997). However, lack of synchrony has been reported in both estuarine (Carnpana & Jones 1998) and deep-sea (Kalish et al. 1997) fishes. In the Year case of the estuarine fish, the At4C chronology recon- structed from otolith cores was phase shifted 2 to 4 yr Fig. 11. Tkme series of bomb 6I4C uptake into Bermuda coral towards earlier years, consistent with an atmospheric (-), South Pacific coral (--- ) and Georges Bank rather than a marine input. Because estuaries are bivalves ( - - -), as well as known-age Melanogrammus shallow, well-mixed areas with strong riverine input, aeglefinus(m) otoliths from the NW Atlantic and Pagrus aura- tus (0) otoliths from the South Pacific. Note the synchrony in there is a rapid and relatively complete exchange of the timing of the bomb 6I4C signal increase among the radiocarbon between the atmosphere and the water. diverse taxa and locations, but the differences in pre- and As a result, the AI4C chronology of an estuary is a post-bomb levels due to differences in water residence times. much closer reflection of the atmospheric chronology Insert sholvs typical relationshp between depth and A'~C (data drawn from Peng et al. 1998). The Bermuda and South (which started to increase in the mid-1950s) than of Pacific coral chronologies are drawn from Drullel (1989) and the marine chronology (Erlenkeuser 1976, Spiker Gullderson et al. (1998), respectively, while the bivalve time 1980, Tanaka et al. 1986). The opposite is true of series 1s from Weidman & Jones (1993).The Melanogrammus deep-sea waters, which were much slower to mix with and Pagrus data are from Campana (1997) and Kalish (1993), 1114C respectively surface waters containing the homb signal, and thus were characteiized by a delayed 3I4C chronol- ogy. As a result, interpretation of the A"C signal in 1998). However, the most substantial differences are the otolith core of a deep-sea fish can he problen~atic, associated with the post-bomb AI4C history, which is and must be carefully interpreted according to depth strongly i~fluenced by water mixing times (Weidman unless the fish's juvenile phase (corresponding to the & Jones 1993, Guilderson & Schrag 1998). For this rea- otolith core) was spent in near-surface waters (Kalish son, the post-bomb AI4C level requires more careful 1995b, Kalish et al. 1997). interpretation than does the period of increase, Interpretation of an otolith-based chronology will not although it can be valuable when used as a tracer of generally be influenced by the magnitude of the pre- upwelling and circulation (Kalish 1995b). In contrast, and post-bomb AI'C levels, since it is the period of the phase (rather than the magnitude) of the chronol- increase that is of interest for age determination. How- ogy is of greater importance for age validation. ever, calibration of individual A14C values against the Three assumptions underlie the use of the AI4C year of formation is also possible as long as additional chronology for the validation of otolith annuli: (1) the information is available. Post-bomb, and to a lesser extracted otolith care must not be contaminated with extent pre-bomb, A'~Cvalues vary significantly and material of more recent origin; (2) annulus interpreta- substantially with latitude, upwelling, circulation, tions and any associated errors must be made consis- depth and any other factor influencing the mixing of tently across all ages examined; and (3) the period of sub-surface, 14C-poor waters with surface waters increase in the dI4C 'reference' chronologies must be (Druffel 1989, Toggweiler et al. 1991, Peng et al.1998). synchronous with that of the fish species under study. The variation typically associated with water depth is The first assumption has been both modelled (Kalish shown in the insert of Fig. 11. In the absence of infor- et al. 1996) and tested (Campana 1997). In the latter mation on the fish's habitat at the time of otolith annu- study, intact age 1 otoliths and the extracted otoIith lus formation, it would be difficult to assign a year of core of older fish from the same year-class were simi- formation to an individual A'~Cvalue. Additional vari- lar but not identical. Since the cores from the older ability due to diet might be expected, in light of the fact fish contained somewhat more recent A"C values, that about one-third of otolith carbon is metabolically Campana: Chemistry and composition of fish otoliths 281

derived (Kalish 1991c, Schwarcz et al. 1998). However, must be considered given the exceedingly low concen- diet is unlikely to be a significant modifier as long as trations of many otolith trace elements. the prey live in a water mass comparable to that of the Assays of whole dissolved otoliths have become pop- fish. Examples of habitats where diet might be ex- ular for differentiating among stocks and tracking mi- pected to be a confounding issue would include deep- grations, since the elemental composition can be used sea habitats, where fish could feed on dead prey sink- as a natural tag even without knowledge of the original ing from surface waters above, and estuarine habitats, source of the component elements (Edmonds et al. where otolith composition could reflect a combination 1989, 1995, Campana et al. 1995, 1999b, Gillanders & of terrigenous food sources (which tend to be enriched Kingsford 1996). Advantages of whole-otolith assays in 14C, similar to the atmosphere) and 14C-depleted include ease of preparation, absence of error associ- marine sources. In such environments, the A'~Csignal ated with sampling or identifying growth increments, would be expected to be more variable than in a more and the availability of accurate and precise assay pro- homogeneous environment like the open ocean (Cam- tocol~.The major disadvantage is associated with the pana & Jones 1998). inability to take advantage of the chronological growth sequence recorded in the otolith. Atomic absorption spectrometry (AAS) (Grady et al. 1989, Hoff & Fuiman Sampling and analyses 1995), inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Edmonds et al. 1995), neu- A distinctive and powerful feature of the field of tron activation analysis (Papadopoulou et al. 1980), otolith chemistry is that the assays can either be Raman spectroscopy (Gauldie et al. 1994) and induc- restricted to some portion of the fish's life history or tively coupled plasma mass spectrometry (ICPMS) integrated across the entire lifetime of the fish. In other (Edmonds et al. 1991, Dove et al. 1996) are among the words, the scale of sampling can be modified to techniques which have been used to analyze otoliths. address the hypothesis being tested, through analysis However, it is ICPMS whlch has emerged as the instru- of either the entire otolith or through a targeted assay ment of choice for such assays, due largely to its capa- of a specific region. In general, analyses of whole bility for rapid and accurate isotopic and elemental otoliths are best suited to questions of stock discnmi- assays over a wide range of elements and concentra- nation, since the primary question is one of overall dif- tions (Table 3). Isotope dilution ICPMS (ID-ICPMS),a ferences between groups of fish, integrated across variant of ICPMS often used to certify reference rnate- their lifetimes. In contrast, microsampled or beam- rials (Fassett & Paulsen 1989), is the most accurate of based assays can target a particular range of ages or the otolith analytical techniques currently available dates, and thus take advantage of the chronological (Campana et al. 1995). Sample sizes required for most growth sequence recorded in the otolith. Currently, of the above assays are on the order of 5 to 10 mg of bulk and/or solution-based elemental assays are capa- otolith material, although ICPMS units outfitted with ble of better accuracy, precision and/or sensitivity than high efficiency nebulizers are capable of handling are most beam-based assay techniques, a factor that otolith weights as low as 0.3 mg (Thorrold et al. 1998a).

Table 3. Summary of the most frequently measured otolith elements which can be detected and quantified by each of the most popular analytical instruments. Mean llrnits of detection (LOD in pg g-') are hsted where available, as drawn from unpublished data and the literature. Values in brackets represent elements with a record for both reliable and unreliable quantification, while blank entries are either unreliable or unmeasured. A = reliable quantification, but with an unknown LOD. Elements are listed in declining order of relative abundance. Since there is no established equation for ED-EM LODs, the listed LODs for ED-EM are approximate only

Type of lnstru- Beam dia- Element assay ment meter(pm) Ca Na Sr K S C1 P Mg Zn B Fe Mn Ba Ni Cu Li Pb

Whole AAS 0.5 0.2 2 2 0.1 otol~th ICP-AES - A 50 1 150 90 100 20 (7) (5) (5) ICPMS - 4.3 (1) 0.3 0.06 0.05 A (4) 0.002 0.005 0.006 0.03 0.0070.009

Beam- ED-EM 2-20 A (12000) (6000) (20001 based WD-EM 2-20 525 240 300 220 200 200 600(300) PIXE 3-20 0.8 0.5 5 (81 1.5 1.3 (51 LA-ICPMS" 5-30 A 1.3 0.1 0.2 (A) 0.04 (0.3) (4

"LODs based on Ar gas blank, with no attendant laser pulse Mar Ecol Prog Ser 188: 263-297.1999

With analytical sensitivity comes the potential for age or date range in the otolith is required, but beam- contamination. Factors such as the mode of fish or based assay techniques are either inappropriate or otolith preservation, composition of the instruments insufficiently sensitive. For these applications, the best used to remove the otolith from the fish, cleaning alternative often involves microsampling or coring methods, handling and even dust are all potentially techniques whlch physically remove a portion of the major modifiers of the perceived trace element compo- otolith for subsequent analysis. Computerized micro- sition. Preservation fluids such as ethanol and formalin milling machines have proven effective in stable iso- appear to have the greatest potential for contamina- tope studies, whereby seasonal or annual growth tion, given the microchannel architecture of the otolith zones visible in otolith cross-sections are milled to a and the relative impurity of most preservatives (Milton discrete depth and the powder collected for assay & Chenery 1998, Proctor & Thresher 1998). Small but (Prezbindowski 1980, Patterson et al. 1993, Schwarcz significant effects due to freezing have been observed et al. 1998). Earlier studies used hand-held dental drills in one study (Milton & Chenery 1998), but not in for the same purpose (Meyer-Rochow et al. 1992). another (Proctor & Thresher 1998). Therefore, trace Where coarser sampling is adequate, otolith cores element analysis of otoliths removed from fish shortly (e.g. first year of growth) can be extracted using either after capture and stored dry appears to be safest. Cur- a stepwise grinding (Campana et al. 1990), drilling rent protocols for handling and preparing otoliths are (Campana 1997) or sculpturing (Kalish 1995a) drawn from the water analysis literature (Benoit et al. approach. Controlled acid dissolution of overlying 1997), and always involve isolation from skin, metallic material has also been reported, although the acid instruments, and solutions which are of other than apparently leached some material from the core (Dove trace metal grade. In general, decontamination based et al. 1996). The advantages of microsampling or cor- on brushing and sonification in a series of distilled, ing include access to bulk analytical techniques of high deionized, reverse osmosis water baths (Super-Q or sensitivity and accuracy: accelerator mass spectrome- Milli-Q water) in a positive flow, laminar flow fume try (AMS) in the case of I4C (Kalish 1993), mass spec- hood (Class loo), followed by storage in dry, acid- trometry for '80:'60 and 13C:12Cratios (Patterson et al. washed polyethylene vials, results in minimal contam- 1993), and ICPMS for trace element assays (Campana ination. Minor elements such as Na, K, Cl, and S are & Gagne 1995). The disadvantage is one of limited likely to be affected by the water sonification stage sampling resolution, since the ten~poral resolution of (Proctor & Thresher 1998), perhaps because these ele- the extracted sample is seasonal at best (Campana et ments are incorporated by occlusion and are not lattice al. 1990, Patterson et al. 1993:. This limited resolution bound. However, it is equally probable that such is in part a constraint of the physical sampling, since it poorly bound elements would be severely affected by is extremely difficult to trace the 3-dimensional, non- exposure to any fluid, including the endolymph if it linear form of otolith growth zones. However, a more shifts its composition daring the death of the fish. As a telling limitation is that of the sample size required result, such elements would probably not be well for the assays: current limitations for stable oxygen suited for use as stable biological tracers. Acid washing isotope and radiocarbon assays are about 30 pg of otoliths does not appear to be necessary for elements (Schwarcz et al. 1998) and 5 mg (Kalish 1995b), respec- such as Ba, Mg, Sr, and Li (Campana et al. 1999a), tively. It appears unlikely that microsampling or coring despite the fact that it is an important step in the would introduce contaminants which would confound decontamination of sechment-laden forams (Boyle stable isotope or radiocarbon assays, as long as the 1981). extracted samples were treated carefully. On the other Of particular relevance to ICPMS, but applicable to hand, there is potential for contamination from the all analytical techniques, is the likelihood of instru- sampling process associated with trace element assays, ment drift (change in sensitivity) during the analysis of despite the fact that Dove et al. (1996) reported no arti- large numbers of samples or between analysis ses- facts due to sectioning with an Isomet saw. sions. Since the estimated elemental concentration can Beam-based assays target a particular age or date be significantly affected by this drift despite the analy- range in the sectioned otoiith, and thus take advantage sis of analytical standards, it is important that the of the chronological growth sequence recorded in the analysis sequence be blocked and randornized so that otolith. These types of targeted assays have been used the order of analysis for any one sample group is to reconstruct migration histories (Secor et al. 1995b. spread over the entire analysis sequence. Use of ID- Thorrold et al. 1997a), identify nursery areas (Thresher ICPMS minimizes (although it may not eliminate) the et al. 1994, Milton et al. 1997), and determine Sr:Ca effects of instrumental drift. ratios in order to infer temperature history (Radtke et Assays for bomb radiocarbon and stable isotope al. 1990, Townsend et al. 1995). The advantages of an ratios are examples of applications where a particular age-structured approach are obvious, particularly Campana: Chemistry an d composition of fish otoliths 283

since the beam sizes of the current generation of in- I Laser I struments approach the width of a typical daily incre- I 3.0 ' I Solution ment (Table 3). As a result, the assay can be limited to the time scale of interest, whether it is weekly, annual 25' or at an intermediate scale. Disadvantages of the approach include the requirement for sectioning to 20 ' expose the growth sequence, the potential for contam- > 1.5' ination from the sectioning and polishing procedure, and some degree of beam penetration into underlying 1.0' growth layers. The method also makes 2 significant assumptions: firstly, that there is no ageing error, and 0.5' that the probe assays have been correctly aligned with 00. ? the appropriate growth increment; and secondly, that Ba Cu Mg Sr Zn Li Mn the estimated elemental concentration in any given Element growth increment is independent of the growth axis that is sampled. This latter assumption is an interesting Fig. 12 Superior sensitiv~tyand precision (CV) of solution- based over laser-based ICPMS assays in a matched compari- one, since it implies that the rate of elemental incorpo- son of otolith pairs reared under a constdnt environment. LA- ration is independent of the rate of calcification, con- ICPIMS assays for LI and Mn were not significantly above the trary to our current understanding of trace element 1.imit of detect~on.All precision values ~nrludevariability incorporation. The few studies that have examined among fish, and thus are not measures of analytical precision. these assumptions in otoliths reported varying levels of Data are drawn from Fowler et al. (1995a. b) consistency in minor element concentrations along a variety of transects within an otolith, or between Table 3. Also evident from Table 3 is the reduced paired otoliths (Gallahar & Kingsford 1992, Gunn et al. range or sensitivity of most beam-based assays com- 1992, Secor 1992, Thresher et al. 1994). It was not clear pared to their solution-based counterparts, in part due from these studies whether the variation was real, or if to the much lower sample weights being analyzed. it merely reflected difficulties in aligning the micro- This difference becomes even more apparent in a probe beam to the appropriate increment. Trace ele- matched comparison of otolith pairs analyzed with 2 ment concentrations, in which an effect of calcification complementary techniques: solution-based ICPMS rate might be expected to be more pronounced, have and LA-ICPMS (Fig. 12). Not only did the sensitivity of not yet been examined in this manner. However, there the solution-based assays extend over a broader range are reports of extension rate along a given growth of elements, but the precision of those assays was sig- layer influencing trace element concentrations in coral nificantly better than those of the laser-based assays. (de Villiers et al. 1994). Until this issue is examined in Of course, such a comparison of precision is not strictly further detail, the selection of standardized otolith valid, since the probed assays incorporate within- growth axes for probe assays appears wise. otolith heterogeneity which is not present in the solu- There are a wide variety of sophisticated instruments tion-based assays. However, such a caveat does not available for probed assays of the otolith (Coutant apply to the comparison of sensitivity. 1990, Jackson et al. 1993, Gauldie et al. 1994, Jambers The absence of otolith reference material has un- et al. 1995), but the most frequently used include the doubtedly hindered progress in otolith trace element energy-dispersive (ED-EM) and wavelength-disper- analyses, both solution-based and probe-based. While sive (WD-EM)electron microprobes (Gunn et al. 1992), analytical standards are used routinely with all instru- proton-induced X-ray emission (PIXE) (Sie & Thresher ment types (Campana et al. 1997), they lack the arago- 1992), and laser ablation ICPMS (LA-ICPMS) (Cam- nite-dominated matrix of an otolith, and thus do not pana et al. 1994). In a detailed experimental compari- provide the matrix matching so important to quantita- son among the above instruments, Campana et al. tive assays. The result is often an assay which is accu- (1997) noted that no one instrument type was sensitive rate relative to other samples being analyzed at the to each element, nor was any one instrument preferred same time, but quantitatively in error (Campana et al. for use in all assays. In general, however, the minor 1997). Pressed carbonate pellets (Perkins et al. 1991, elements such as Na and K could only be measured van Heuzen & Morsink 1991, Pearce et al. 1992) and accurately with an electron microprobe, while the otolith powder fused into glass beads (Campana et al. trace elements required PIXE or LA-ICPMS. Sr was 1997) have both been used for beam-based assays, but measured accurately and precisely with either WD- neither completely addresses the requirements for EM, PIXE or LA-ICPMS. An overview of the capabili- matrix matching or surface smoothness. Homogeneous ties of the most popular instrumentation is presented in otolith powders have been developed to ensure com- 284 Mar Ecol Prog Ser 188: 263-297. 1999

parability among solution-based assays (Campana et Edmonds et al. 1989, Grady et al. 1989, Hoff & Fuiman al. 1999a), but are not available in quantities suitable 1993, Campana et al. 1995, 1999a, Begg et al. 1998). for broad distribution. Until a certified otolith reference Since the elemental fingerprint reflects the exposure of material, analogous to those used by analyhcal the individual fish to both the environment and its own chemists in other fields peauchemin et al. 1988), is physiology, it would be expected to differ among any developed, comparability among published otolith groups of fish which have experienced different histo- trace element studies is likely to be suspect. ries, whether or not the groups come from the same population. Logically, the presence of different finger- prints could not be used to infer the length of time that Applications the groups of fish remained separate, since even occa- sional residency in a different environment would Stock identification have the potential to introduce a detectable difference in the elemental composition. By corollary, the absence To the extent that groups of fish inhabit different en- of differences would not necessarily imply that the vironments, the otolith elemental composition should groups of fish are of common origin. As a result, it is senre as a natural marker or tag of those groups. Two fair to categorize otolith elemental fingerprints as pow- assumptions cnderlie the use of the otolith elemental erful discriminators of groups when differences exist, composition as a marker (1) material deposited on the but of negligible value when differences cannot be otolith is metabolically inert after deposition, and is not detected. Where differences are detected, additional susceptible to resorption; and (2) the physical and information would be required to determine if the chemical environment influences the rate of trace ele- groups actually corresponded to stocks or populations. ment incorporation into the growing otolith surface. Two types of elemental fingerprinting are in general Elements under strong physiological regulation (e.g. use: one based on whole dissolved otoliths, and the Na, K, S, P, Cl) probably do not meet the second other based on analysis of the otolith core. Since the assumption, and are thus of limited value for tracking otolith grows continually throughout the life of the fish, or stock identification studies (Thresher et al. 1994, the whole-otolith fingerprint i.ntegrates across the Proctor et al. 1995).However, both assumptions appear entire lifetime, and thus serves as a marker for groups to be met with respect to elements such as Sr, Ba, Mn, of fish which have experienced different overall envi- Fe and Pb (and perhaps Li, Mg, Cu and Ni), in which ronmental exposures (Edmonds et al. 1989, 1991, 1992, both ambient e1ement:Ca concentrations and tempera- 1995, Northcote et al. 1992, Campana & Gagne 1995, ture produce significant effects on otolith composition Campana et al. 1995, 1999a,b, Bronte et al. 1996, (Fowler et al. 1995a, Farrell & Campana 1996, Dove Gillanders & Kingsford 1996, Edmonds & Fletcher 1997). Similar effects of environmental availability and 1997, Kennedy et al. 1997, Begg et al. 1998, Dove & temperature apply to isotopic ratios of elements such Kingsford 1998, Secor & Zdanowicz 1998, Thorrold et as strontium (Kennedy et al. 1997) and oxygen (Thor- al. 1998a). While the elemental fingerprint of a group rold et al. 199713). Environmental responses such as of fish may or may not be characteristic of a stock or these, recorded permanently in the otolith, imply that population, u.se of the fingerprint a.s a long-tern~ stock the otolith concentrations of selected elements and iso- discriminator may be justified in instances where envi- topes (the 'elemental fingerprint') can be used as a bio- ronmental differences among stock areas are larger logical tag to discriminate among groups of fish which than those within areas or across year-classes, acd have spent at least part of their lives in different envi- where differences in the fingerprint due to fish size ronments. While the application of otolith composition have been statistically removed. The assumption of as a natural tag appears to be relatively well founded, long-term stability in the fingerprint is probably met in use as a stock or population marker requires consider- some, but not all, stocks (Campana et al.1999a). ably more assumptions [Campana et al. 1999a). Analysis of the otolith core is generally intended as a It is probably inappropriate to refer to the use of ele- more direct measure of stock origin than is the analysis mental fingerprints as stock discriminators, since of the whole otolith. As is the case with whole-otolith genetic differences are not implied and spatial hetero- elemental fingerprints, the presence of fingerprint dif- geneity in the stock environment can result in different ferences implies differences in the history of environ- fingerprints for different stock components (Thorrold mental exposure which may or may not correspond to et al. 1998b, Campana et al. 1999a). Perhaps more genetic differences. In this application, however, the importantly, ontogenetic effects and age-related dif- environmental exposure is limited to the period of ferences in exposure history can result in very differ- growth represented by the otolith material which is ent fingerprints for fish of different size-classes from assayed, whether that is the period around hatch, the the same population (Papadopoulou et al. 1980, first few months of life, or some other period. The sub- Campana: Chemistry and compos~tionof fish otoliths 285

sequent life history is not sampled, and is therefore (3) The marker remains stable over the interval be- irrelevant. To the extent that spawning grounds are tween characterization and mixing. Long-term stability characterized by different temperature or chemical of an environmentally induced marker would not be environments, this approach has proven effective in expected, nor has it been observed (Edmonds et al. distinguishing among groups of fish with different ori- 1995, Campana et al. 1999a). However, short-term sta- gins (Kalish 1.990, Sie & Thresher 1992, Campana et al. bility over the interval between characterization (e.g. 1994, Thresher et al. 1994, Proctor et al. 1995, Severin spawning group) and mixing is both expected and ob- et al. 1995, Dove et al. 1996. Gillanders & Kingsford served, particularly with respect to differences among 1996, Milton et al. 1997, Thorrold et al. 1997a). groups (Campana et al. 1995, 1999a, Kennedy et al. The most robust application of whole-otolith finger- 1997, Thorrold et al. 1998a, but see Edmonds et al. prints is one which is targeted at questions of stock 1995). In the case of the Atlantic Gadus morhua, mixing or for tracking stock migrations, in which the this interval was less than 6 mo, and thus much less fingerprints are used as natural tags of pre-defined than the period required for a noticeable change in the groups of fish (Campana et al. 1995, 1999a, b, G~llan- elemental fingerprint (Campana et al. 199913). Longer ders & Kingsford 1996, Kennedy et al. 1997). Use of an intervals may be possible in some instances, but the po- elemental fingerprint as a natural tag takes advantage tential for drift in elemental composition becomes of the fact that otolith size and composition cannot greater as the interval length IS extended (Edmonds et change appreciably over a brief time period. Once the al. 1995). By corollary. shorter interval lengths would elemental fingerprint of all potential source groups has presumably be required in analyses of young fish, in been determined, fish should remain identifiable as to which the proportional annual change in otolith weight their source group, despite any mixing with other (and.,potentially, composition) would be more marked. groups, until the elemental composition of later otolith growth has significantly altered overall elemental composition. An appealing feature of this application is Migration history that the elemental fingerprint need not be linked to potential sources or locations in the environment. Reconstruction of migration histories using the ele- Use of otolith elemen.ta1 fingerprints as natural tags men.taI or isotopic composition along an otolith growth makes 3 central assumptions: (1) There are characteris- axis is similar in many respects to stock discrimination tic and reproducible markers for each group. If the ele- based on analysis of the otolith core, and shares many mental fingerprints of the groups of interest do not dif- of the same assumptions. However, migration analysis fer significantly, little more can be accomplished. expands the scope of the interpretation, by linking a However, group-specific variation in elemental compo- series of elemental assays along an otolith transect to sition appears to be common (Edmonds et al. 1989, the growth chronology recorded in the otolith, thus 1991, 1992, 1995, Northcote et al. 1992, Campana & allowing the reconstruction of migration pathways Gagne 1995, Campana et al. 1995, 1999a, Bronte et al. structured by age or date. An additional assumption of 1996, Thorrold et al. 1998a). Since smaller fish seem far this application is that there is negligible ageing error more likely to contain elevated (or depressed) concen- and that growth increments have been accurately trations of any given element than larger fish (Papa- linked to assay locations. In principle, migration analy- dopoulou et al. 1980, Edmonds et al. 1989, Grady et al. sis is one of the most powerful applications of otolith 1989, Hoff 81 Fuiman 1993), it is important that differ- microchemistry, although its successes to date have ences in elemental concentration among groups are not been largely limited to the detection of anadromy confounded by size differences among groups. Statisti- (Kalish 1990, Meyer-Rochow et al. 1992, Secor 1992, cal removal of the effect of otolith weight on elemental Halden et al. 1995, 1996, Limburg 1995, Secor et al. concentration is a ready solution to this problem (Cam- 1995b. Tzeng et al. 1997, Otake & Uchida 1998, Tsuka- pana et al. 1999a); (2) All possible groups contributing moto et al. 1998). Subtler migration patterns are now to the group mixture have been characterized. This as- being detected with instrumentation such as PIXE and sumption applies as much to genetic studies as it does LA-ICPMS (Proctor et al. 1995, Blaber et al. 1996, to otolith elemental fingerprints, with the implication Halden et al. 1996, Thorrold et al. 1997a), which are being that uncharacterized groups of fish present in the more sensitive to non-physiologically regulated trace mixture could be mistakenly interpreted as one or more elements (Campana et al. 1997). Past difficulties in of the reference groups (Wood et al. 1987, 1989, Wirgin interpreting migration histories may have been due to et al. 1997). Careful selection of reference groups can the use of the electron microprobe, which is more help minimize this problem, particularly if they are suited to assays of physiologically regulated elements. sampled at a time when the groups are known to be Several forms of migration analysis have been discreet (e.g. on the spawning or nursery grounds); reported. One of the most successful has been the com- Mar Ecol hog Ser 188: 263-297,1999

parison of elemental trajectories among fish in search all have been used to validate more traditional, less of a common history, or, alternatively, the deterrnina- expensive methods of age determination. tion of the age or date at divergence (Thresher et al. The incorporation of at least some isotopes and ele- 1994, Limburg 1995, Proctor et al. 1995, Thorrold et al. monts into the otolith is likely to vary with a yearly 1997a). This is a particularly robust application, since it periodicity, but studies to date have provided mixed requires no knowledge of the fish's past environment, results. Annular Sr:Ca ratios in particular might be and is relatively insensitive to any ontogenetic shifts expected based on yearly cycles in temperature, in otolith elemental composition which may have migration patterns, distribution, sexual maturation, occurred. In many respects, this comparative approach growth rate or other physiological variables. Indeed, is analogous to the use of elemental fingerprints as bio- clear periodicity in Sr:Ca ratios, inferred to be annual, logical tracers, as described earlier. A potentially more have been reported in several studies (Radtke & Tar- powerful approach is one in which the otolith elemen- gett 1984, Gauldie et al. 1991, 1992, Sadovy & Severin tal trajectory is linked to shifts in temperature and 1992, Secor 1992, Toole et al. 1993, Halden et al. 1995). water chemistry along possible migratory routes. Such However, other studies reported no consistent correla- an approach is the basis for the detection of anadromy tion between the Sr:Ca value and the annuli visible in using Sr:Ca ratios, in which the shift between freshwa- the otolith (Fuiman & Hoff 1995, Gauldie et al. 1995). ter and saltwater environmerits is clearly evident in the Oxygen isotope cycles are evident in both freshwater otolith (Kalish 1990, Secor 1992, Halden et al. 1995, and saltwater otoliths, due to annual patterns in tem- 1996, Limburg 1995, Secor et al. 1995b, Tzeng et al. perature and/or distribution (Iacumin et al. 1992, Pat- 1997, Otake & Uchida 1998, Tsukamoto et al. 1998). terson et al. 1993). Annual cycles in elements such as Migration through a more homogeneous environment Na, K and S, out of phase with temperature cycles and should also be detectable. However, interpretation of perhaps due to seasonal reproductive activity, have such subtle shifts in composition is complicated by the also been reported (Kalish 1939, Fuiman & Hoff 1995) assumption that a given elemental concentration While annual isotopic and elemental cycles undoubt- reflects the same environmental condition at different edly occur in some species, the environmental (and points along an otolith transect. In other words, onto- therefore transient) basis for their formation suggests genetic changes in otolith elemental composition are that their use for age vahdation is no more firmly assumed not to exist. Yet such changes have been grounded than are the annuli which are visible in clearly documented, even in fish held under constant otoliths. environmental condit~ons(Gallahar & Kingsford 1992, Radiochemical dating of otoliths is based on the Fowler et al. 1995b. Fuiman & Hoff 1995, Hoff & radioactive decay of naturally occurring radioisotopes Fuiman 1995). As a result, observed trends in concen- which are incorporated into the otolith during its growth. tration across an otolith could reflect either a shift in As discussed above under 'Natural radioisotopes', assays the fish's environment, an age-related change in incor- of the extracted otolith core appear to provide objective, poration rate independent of the environment, or both. accurate age estimates (Bennett et al. 1982, Campana et With further experimentation, it may be possible to al. 1990, 1993, Fenton et al. 1990, 1991, Smith et al. 1991, factor out ontogenetic shifts in elemental composition, Kastelle et al. 1994, Milton et al. 1995).Nevertheless, the thus simplifying the interpretation of possible migra- isotopic concentrations requiring measurement are ex- tion pathways. To date, however, it is not always clear ceedingly low, resulting in assay precisions which are from published reports whether reconstructed distrib- often less than optimal. Current discriminatory power utions have been adjusted for ontogenetic effects is on the order of 5 yr for 2'0Pb:226~aand 1 to 2 yr for (Townsend et al. 1992, 1995, Blaber et al. 1996). 228Th:'28Ra, over age ranges of 0 to 50 and 0 to 3 yr, respectively. Therefore, this approach is best suited to species where the candidate age interpretations are Age determination widely divergent, such as in Sebastes or Hoplostethus (Campana et al. 1990, Fenton et al. 1991). Age determination studies based on elemental or Age validation on the basis of radiocarbon derived isotopic concentrations in the otolith are motivated by from nuclear testing is currently one of the best age the ongoing requirement for accurate age-structured validation approaches available (Kalish 1993, 1995a, b, information in support of many fish studies. The objec- Kalish et al. 1996, 1997, Campana 1997, Carnpana & tivity of elemental assays is appealing in light of the Jones 1998). As described above under 'Radioisotopes subjectivity that can confound or invalidate the inter- from nuclear explosions', marine waters with A'~Cval- pretation of annuli in otoliths or other structures. As a ues greater than O%e did not generally exist prior to the result, all chemically based age determination studies onset of nuclear testing in the late 1950s. Even otolith to date have focused on the yearly scale, and virtually contamination with material of more recent origin Carnpana: Chemistry an~d composition of fish otoliths 287

could only increase the A'~Cvalue, not decrease it. believed to have been constant, the difference be- Thus the otolith A'~Cvalue sets a minimum age to the tween 2 isotopic assays can be used to estimate the sample, and the years 1958 to 1965 become the most temperature differential, but not the absolute tempera- sensitive years for A'~C-based ageing. For fish born ture (Thorrold et al. 199713). Temperature reconstruc- during this time period, bomb radiocarbon can be used tions made without any knowledge of water composi- to confirm the accuracy of more traditional ageing tion necessarily assume some given value (Edmonds & approaches with an accuracy of at least * 1 to 3 yr; the Fletcher 1997), although variations in salinity are discriminatory power of samples collected before or sometimes used as a proxy for variations in 6"0 (Tan after this period is more than an order of magnitude et al. 1983). lower. Since the 14C signal recorded in deep-sea and Otolith-based reconstructions of recent salinity his- freshwater environments is different from that of sur- tory have generally been restricted to anadromous face marine waters (deep-sea = delayed; freshwater = migrations on the basis of Sr:Ca ratios (Kalish 1990, advanced), reference chronologies appropriate to the Secor 1992, Halden et al. 1995, 1996, Limburg 1995, environment experienced during the period of otolith Secor et al. 1995b, Tzeng et al. 1997, Otake & Uchida core formation must be used (Kalish 1995b, Campana 1998) or stable oxygen isotopes (Meyer-Rochow et al. & Jones 1998). 1992, Northcote et al. 1992), although Dufour et al. (1998) were able to distinguish between hypersaline lagoonal water and that of the open ocean using stable Reconstruction of environmental history oxygen and carbon isotopes. Detection of the fine- scale salinity differences evident in coralline Sr:Ca Otolith-based reconstruction of individual tempera- ratios (de Villiers et al. 1994) are likely to be more dif- ture histories is now possible, albeit with some caveats. ficult in otoliths given the other factors that are known Despite the initial promise, otolith Sr:Ca ratios do not to influence Sr:Ca deposition in otoliths. Strontium iso- appear to reflect temperature in a consistent manner. tope ratios have been used to reconstruct salinity his- As noted in the section 'Temperature', temperature tory in fish bones and other taxa (Schmitz et al. 1991, reconstructions based on Sr:Ca have performed well in Holmden et al. 1997), but have not yet been tested for only a handful of coldwater examples (Townsend et al. this purpose in otoliths. 1989, 1992, 1995, Radtke et al. 1990), and are readily Paleoclimate reconstructions take advantage of the confounded by changes in salinity. However, oxygen same suite of temperature and salinity indicators noted isotope ratios do not appear to suffer the same con- above, but require additional assumptions concerning straints, and their relationship to temperature has been the 6180 and trace element composition of the water at experimentally validated in several fish species (Kalish the time. Nevertheless, there is a long history in recon- 1991c, Thorrold et al. 199713). Temperature reconstruc- structing past temperature regimes on the basis of cal- tions based on oxygen isotope ratios have been cified tests (Rye & Sommer 1980, Weid- reported for both freshwater (Patterson et al. 1993) and man & Jones 1994), and initial results on otoliths saltwater (Mulcahy et al. 1979, Kalish 1991b, Iacumin promise similar conclusions (Devereux 1967, Elder et et al. 1992) fishes, and have been based both on whole al. 1996). Reconstruction of paleosalinity on the basis of otoliths (to derive a mean temperature) and rnicrosam- strontium isotopes has not yet been attempted with ples taken along an otolith transect (to derive a tem- otoliths, despite reports of their use in other structures perature history). These successful applications are in (Schmitz et al. 1991, Holmden et al. 1997). However, keeping with the extensive literature on the use of oxy- Sr:Ca ratios in fossil otoliths have been used to recon- gen isotopes to reconstruct the temperature history of struct flooding events in inland lakes (J. Kalish, Aus- corals, bivalves and other calcified poikilothermic tralia National University, Canberra ACT 0200, Aus- aquatic organisms (Weidman & Jones 1994, Leder et trala, pers. comrn.). Of course, attention must always al. 1996). However, oxygen isotopes in the otoliths of be given to possible diagenetic shifts in fossil composi- temperature-regulating fish such as tuna appear to tion, although Gauldie et al. (1991) noted that such reflect body temperature more than that of the ambient changes were often visually obvious. water (Radtke et al. 1987). The accuracy of oxygen isotope-based temperature reconstructions can be limited by insufficient informa- Pollution tion concerning the 6180 of the water. Since otolith 6180 is deposited in equilibrium with that of the water, Trace elements are often considered to be excellent variations in the latter must be accounted for before pollution indicators, given their relatively high concen- the temperature at deposition can be calculated. trations in industrial effluent (Phillips & Rainbow Where the composition of the water is unknown but is 1993). Accordingly, the trace element composition of Mar Eml Prog Ser 188 263-297, 1999

soft tissues of aquatic organisms is used routinely to other factors which can result 1.n increased Sr:Ca ratios, monitor pollution (Cutshall et al. 1978, Settle & Patter- it would probably be difficult to attribute a particular son 1980). Calcified tissues in organisms such as Sr:Ca peak to a stressful event without pcor knowl- bivalves and corals preserve a longer, less refractory edge of the event. in principle, reproductive events record of exposure, and are thus preferred for use should be easier to reconstruct, since the increase in where available (Scott 1990, Brown & Luoma 1995). In calcium-binding proteins (and concomitant decrease fish, there is a similar precedent for use of calcified in free calcium) associated with spawning would be structures such as scales and bones (Moreau et al. expected to occur over the entire spawning season 1983, Johnson 1989). Yet initial reports suggest that (Kalish 1989). In practice, however, the generalized the otolithis not an obvious indicator of pollution (Gef- increase in the Sr:Ca ratio might he difficult to disen- fen et al. 1998. Hanson & Zdanowicz 1999. but see tangle from background variability. For example, Dove & Kingsford 1998). Since the partition coeffi- Friedland et al. (1998) were not able to discriminate cients for most pollution-related metals into the otolith between mature and immature sea-run Atlantic are much lower than those into other calcified tissues, Salmo salar based on otolith Sr:Ca ratios. the otolith is probably less than ideal for use as a proxy for pollution. Chemical marking Physiological indicators Chemicals incorporated icto otoliths have been used Although there are several physiologically important as dated markers or as batch markers of groups of fish life history events that can influence elemental or iso- for at least 30 yr (Kobayashi et al. 1964, Meunier & topic uptake into the otolith, not all can be interpreted Boivin 1978). When chemically tagged individuals are as such in the absence of supporting information. The recaptured, time at liberty can be compared with the most robust of the physiological indicators is the gen- number of growth increments deposited distal to the eralized increase in otolith 613C with declining meta- chemical check (Casselrnan 1983). This approach to bolic rate (Mulcahy et al. 1979, Kalish 1991c, Gauldie age validation has proved effective at both yearly 1996a, Schwarcz et al. 1998). This pattern has been (Fowler 1990, McFarlane & Beamish 1995) and daily observed in a variety of taxa across a range of habitats (Wild & Foreman 1980, Campana & Neilson 1982) lev- and appears to be indicative of a reduced contribution els. The objective of batch or mass marking is com- of metabolic carbon associated with increased age and pletely different, in that it is intended as a rapid, cost- declining growth rate (Kalish 1991c, Schwarcz et al. effective means of tagging large cumbers of fish for 1998). Peak or asymptotic 613C levels appear to be subsequent identification (Secor & Houde 1995, Tsuka- indicative of the onset of sexual maturity (Gauldie moto 1985). Batch markers are generally applied 1996a, Schwarcz et al. 1998). While diet and ambient through immersion, although incorporation into the 613C may also influence otolith 613C, the relationship diet has also proven successful. In many ways, this with metabolic rate appears to be sufficiently strong to method of marking is analogous to thermal marking of provide a relatively clear signal over the lifetime of the otoBths, although thermal marking leaves a visual, individual. rather than a chemical, mark (Mosegaard et al. 1987, Metamorphosis of an eel leptocephalus to a juvenile Hagen et al. 1995). An advantage of chemical marks is glass eel also leaves a clear signal in the otolith, in this that they can be applied at any life history stage, case in the form of an elevated Sr:Ca ratio (Otake et al. including that of the embryo (Geffen 1992). 1994, 1997, Tzeng et al. 1997). However, metamorpho- Fluorescent calcium-binding chemicals are among sis in other taxa will not necessarily leave an elemental the most popular of the chemical markers, since they mark: Toole et al. (1993) could find no elemental evi- are easily introduced into the fish and produce clear dence of metamorphosis in 5 otoliths, and flat- fluorescent marks in the otolith and other calcified fish settlement was often associated with depressed, structures when viewed with ultraviolet Light. Oxy- rather than elevated, Sr:Ca ratios. Further studies on tetracycline hydrochloride has the longest history of other species are clearly required, but the unarnbigu- use with otoliths, and has been administered through ous Sr:Ca peak deposited in the eel otolith may in part injection (Wild & Foreman 1980, Campana & Neilson reflect the unusual and extreme nature of metamor- 1982, Geffen 1992, Lang & Buxton 1993). feeding (Ped- phosis in this group of fishes. ersen & Carlsen 1991, Thomas et al. 1995) and imrner- Other physiological processes which may leave a sion (Hettler 1984, Schmitt 1984, Tsukamoto 1985, chemical mark on the otolith include stressful events Muth et al. 1988, Secor et al. 1991, Nagiec et al. 1995). [Kalish 1992) and reproductive activity (Kalish 1989), Alizarin red and alizarin complexone have a shorter both in the form of elevated Sr:Ca ratios. In light of the history of use, but are generally considered to produce Carnpana: Chemistry an~d composition of fish otoliths 289

a mark superior to that of oxytetracyline when applied tion purposes (Thorrold et al. 1998b) and further devel- through immersion (Tsukamoto 1988, Blom et al. 1994, opments are expected. Nevertheless, there remain Nagiec et al. 1995, Secor & Houde 1995, Szedlmayer & applications of otolith chemistry which have received Howe 1995, Thomas et al. 1995, Beckman & Schulz little or no attention from otolith researchers to date, 1996). ALizarin incorporated into feed tended to pro- and some of these areas are described below. duce a less visible mark (Lang & Buxton 1993, Taka- Methodological advances will continue to play a hashi 1994, Thomas et al. 1995), while injected alizarin pivotal role in this field in light of the continued resulted in poor marking success (Thomas et al. 1995, requirement for improvements in assay accuracy, sen- but see Geffen et al. 1998). In contrast, the mark qual- sitivity and spatial resolution. High-resolution mag- ity of injected calcein is reported to be very good netic sector ICPMS systems already offer more pre- (Monaghan 1993, Thomas et al. 1995), although cal- cise isotopic ratios and significant improvements in cein application through immersion has also been suc- assays of elements subject to isobaric interference cessful (Wilson et al. 1987, Alcobendas et al. 1992). (e.g. Fe, Sn, As) (Moens & Jakubowski 1998). When Irrespective of the mode of application, there have coupled with W lasers, or, alternatively, using the been no reports of mark loss from the otolith through new generation of ion microprobes, beam-based the lifetime of the fish, nor would any be expected assays of a broader suite of elements and more pre- based on the chemical stability of the otolith. cise isotope ratios in otolith growth increments are Elemental supplements incorporated into the otolith likely (Christensen et al. 1995). Initial demonstrations have also been used as chemical tags or as dated mark- of laser-based '3C:'2C assays (Murnick & Peer 1994), ers, although detection is necessarily limited to analyt- laser-based Sr isotope assays (Christensen et al. 1995) ical methods. Immersion of fish in strontium-enhanced and ion microprobe-based '80:'60 assays (Saxton et solutions has been used sucessfully in several studies, al. 1995) in minerals and bivalves have already been either to leave a dated mark on the otolith detectable reported, suggesting that age-structured metabolism, with an electron microprobe (Proctor et al. 1995. salinity and temperature reconstructions at the weekly Schroder et al. 1995) or as a batch marker of groups of or even daily level may soon be possible. In the fish (Brown & Harris 1995). Strontium-enhanced interim, computer-controlled microsamplers have been checks may even form in wild fish in response to stress developed for fine-scale collection of otolith material (Kalish 1992), presumably in response to abruptly for stable isotope and radioisotope assays (Dettman & lower calcification rates. Indeed, Mugiya & Murarnatsu Lohmann 1995). (1982) used the calcium antagonist acetazolamide to The interpretation of isotopic ratios other than C and produce a visual check on the otolith, although there 0 provides many useful insights in geochernical stud- was no report of a matching elemental mark having ies, but is largely unknown to fisheries science. Exam- been formed. However, a dated mark detectable with ples of potentially valuable otolith applications include autoradiography was produced after fish were ex- assays of Sr isotopic ratios as indicators of proximity to posed to a radioactive isotope of calcium (Yoklavich & land, use of U isotopes as salinity indicators, and inter- Boehlert 1987). Mass marking with other elements is pretation of Pb isotopes as proximity indicators for pol- also possible (Ennevor & Beames 1993), although lution. The isotopes of many elements are fractionated given the selectivity of the otolith to specific elements, on the basis of temperature: can the isotopes of the certain elements might be expected to be incorporated predominant element in otoliths, Ca, be used as a tem- more easily than others. perature indicator which is insensitive to salinity varia- tions? What about temperature reconstructions based on otolith oxygen isotopes using an independent salin- Future directions ity indicator as a proxy for the oxygen isotopic compo- sition of the water? These and other applications Many of the applications described in the previous remain to be developed. sections have not yet reached their full potential. For Our current understanding of the factors influencing example, age-structured assays which take advantage the composition of the otolith is constraining our of the otolith growth sequence have not developed as progress in several areas. For example, the influence of quickly as anticipated, in part due to methodological otolith proteins on trace element incorporation has not limitations. This in turn has slowed research on prob- yet been studied, but may explain the affinity (or lack lems such as reconstruction of migration pathways, thereof) for certain elements. Similarly, a better under- which requires trace elements and isotopes other than standing of the uptake dynamics of many elements is those accessible to the commonly used electron micro- required to determine which might serve best as envi- probe. Progress has been made in integrating trace ronmental indicators, and which serve only to confuse element and stable isotope assays for stock identifica- our interpretations. 290 Mar Ecol Prog Ser 188: 263-297,1999

As with any ecological methodology, applications longevity in Sebastes diploproa (Pisces: Scorpaenidae) based on otolith chemistry are often used to the grcat- from Pb-210/Ra-226 measurements in otoliths. Mar Biol est advantage when combined with other, comple- 71:209-215 Benoit G, Hunter KS, Rozan TF (1997) Sources of trace metal mentary tools. Examples of such a multidisciplinary contamination artifacts during collection, handling, and approach might include the use of both otolith elemen- analysis of freshwaters. Anal Chem 69:1006-1011 tal fingerprinting and microsatellite DNA for stock Blaber SJM, Milton DA, Pang J, Wong P, Boon-Teck 0,Nyigo identification, or use of b0t.h externally attached tags L, Lubim D (1996) The life history of the tropical shad Ten- ualosa toli from Sarawak: first evidence of protandry in the and laser-based otolith assays for reconstruction of ? Environ Biol Fishes 46:225-242 migration routes. Ilere as in other fields, multiple inde- Blom G, Nordeide JT,Svasand T, Barge A (1994) Application pendent approaches to a problem will often yield the of two fluorescent chemicals, alizarin complexone and best results, and are to be encouraged. aliarin red S, to mark otoliths of Atlantic cod, Gadus morhua. Aqua Fish Manage 25 (Suppl 1):229-243 Boyle EA (1981) Cadmium, zinc, copper, and barium in foram- Acknowledgements. Discussions with Cynthia Jones, John inifera tests. Earth Planet Sci Lett 53:11-35 Kalish, Bill Patterson, Simon Thorrold and Ron Thresher Broecker WS, Peng TII, Ostlund G, Stuvier M (1985) The dis- helped me crystallize my thoughts for this paper. I also thank tribution of bomb radiocarbon in the ocean. J Geophys Res Joanne Harncl and Linda Marks for their expert technical 90:6953-6970 assistance. The constructive comments of Karin Limbwg and Bronte CR, Hesselberg KJ, Shoesmith JA, Hoff MH (1996) 2 anonymous referees were very helpful in revising the man- Discrimination among spawning concentrations of Lake uscript. Superior lake based on trace element profiles in sagittae. Trans Am Fish Soc 125:852-859 Brown CL, Luoma SN (1995) Use of the bivalve LITERATURE ClTED Potamocorbula amurensis as a biosentinel species to assess trace metal contamination in San Francisco Bay. Alcobendas M, Lecomte F, Francillon-Vieillot H, Castanet J, Mar Ecol Prog Ser 124:129-142 Meunier FJ, Maire P (1992) Marquage vital en masse chez Brown P, Harris JH (1995) Strontium batch-marking of golden l'anguille (Angda anguilla) a I'aide d'une technique de perch (Macquana ambigua Richardson) and trout cod balneation rapide. In: Bagliniere JL, Castanet J, Conand F, (Maccullochella macquariensis) (Cuvier). 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Editorial responsibility: Otto Kinne (Editor). Submitted: February 2, 1999; Accepted: June 10, 1999 Oldendorf/Luhe, Germany Proofs received from author(s): October 8, 1999