13C and 180 Isotopic Disequilibria in Fish Otoliths: Metabolic and Kinetic Effects

MARINE ECOLOGY PROGRESS SERIES Vol. 75: 191-203, 1991 Published September 11 Mar. Ecol. Prog. Ser.

13c and 180 isotopic disequilibria in fish otoliths: metabolic and kinetic effects

John M. Kalish*

Department of Zoology, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia

ABSTRACT Distnbution of oxygen and carbon stable isotopes in fish otoliths from a wide range of species was invesugated to determine if the isotopes were deposited in equilibnum with ambient seawater and the potential utihty of isotope data to studies of fish biology Companson of 6180 and 6I3c data from otoliths wth estimates of equilibnurn aragonite indicate that ''0, but not I3C was deposited near equdibnum and this confirms the utllity of otolith oxygen isotopes as an indicator of ambient temperature The departure of 613C from equilibrium was considered In light of several current theones on 13c isotope disequilibrium There was a strong correlalon between bI3C and temperature and, concomitantly a strong relationship between 6180 and 6I3C Evldence is presented to support the Importance of metabolic effects in medialng I3C disequdibna and a strong relationship between metabohc rate (VO?) and 6I3c is hypothesized F~sheswith low metabolic rates or those living at low temperatures had 13C deposited near to equilibnum whereas fishes living at higher temperatures showed extreme depletion in otolith I3C Trends were also evident in 613C values in luveniles and adults of a species with the juveniles presumably with a higher metabolic rate being more depleted in I3C than the adults The study shows that both oxygen and carbon stable isotopes have great potential in studies of fish biology and are worthy of further investigation

INTRODUCTION an ecosystem. The use of stable isotope data to gain insight into these problems would be particularly valu- The oxygen and carbon stable isotopic composition able to studies of both deep-sea and large pelagic of marine biogenic carbonates can provide information fishes, where the ability to sample populations and on the biology of marine organisms and insight into carry out investigations with live individuals is limited. mechanisms of calcification. This principle has been Despite the possible benefits of oxygen and carbon widely applied to studies of marine invertebrates and isotope data derived from fish otoliths, the suitability of has only received minor attention in relation to fishes. these data to studies of most marine fish species is still Mulcahy et al. (1979), Radtke (1984a,b), Radtke et al. uncertain, particularly when the range of potential (1987) and Kalish (1991) have shown the potential isotopic fractionation effects is considered. applications of fish otolith oxygen isotope data to During precipitation of calcium carbonate from solu- studes of fish ecology and physiology. Environmental tion thermodynamic relationships exert the primary temperature information derived from oxygen isotope influence on the partitioning, or fractionation, of oxy- data may be useful in studies of migration and distribu- gen and carbon isotopes (O'Neil et al. 1969). In this tion. They may also help researchers locate life history study, fractionation refers to the partitioning of carbon stages that have eluded capture, but are important in and oxygen isotopes between dissolved inorganic car- studies of basic biology and stock assessment of fish bon (DIC) and aragonite, and between seawater oxy- species. Carbon isotope data may provide information gen and aragonite, respectively. When thermodynamic on metabolic rates and diet, both important in under- relationships are the only factors affecting the fraction- standing the productivity of a species and its position in ation of carbon and oxygen isotopes during precipitation of CaC03 from solution, the isotopes are precipitated in equilibrium. Precipitation of biological car- ' Present address: MAF Fisheries Greta Point, PO Box 297, bonates, however, frequently results in the deposition Wellington, New Zealand of both carbon and oxygen isotopes out of predicted

O Inter-Research/Printed in Germany Mar. Ecol. Prog. Ser. 75: 191-203, 1991

equilibrium. Non-equilibrium deposition of oxygen corals, were considered by McConnaughey (1989a). and carbon isotopes in biogenic carbonates can be Epstein et al. (1953) estimated oxygen isotope fraction- attributed to 'metabolic' or 'kinetic' isotope effects. In ation in biogenic calcite by determining isotopic ratios biological systems these effects are generally grouped in molluscs collected from environments of known as the potential mechanisms of biological fractionation. temperature and from shell material grown in Metabolic effects result from changes in 613c and b180 laboratories under controlled temperature conditions. of CO2 and HC03- in the region of the calcium car- They obtained results which are in close agreement bonate precipitate, due to biological processes such as with determinations of oxygen isotope fractionation in respiration or photosynthesis. Kinetic effects result inorganically precipitated calcite (O'Neil et al. 1969). from the discrimination against heavy carbon and oxy- The close agreement between these studies of inor- gen isotopes during diffusion of the carbonate isotopic ganic and biogenic calcite indicated that the biogenic species to the crystal surface (Turner 1982, Grossman carbonate of the mollusc shells was formed in isotopic 1984b) or, during hydration and hydroxylation of CO,, equilibrium with the seawater environment and that which results in HC03- (McConnaughey 1989a, b). there was no biological fractionation of oxygen Many researchers have not differentiated between isotopes. metabolic and kinetic effects and have simply referred The fractionation of ''0 in inorganic aragonites has to biological fractionation or vital effects (Weber & only been estimated at a single temperature (25 "C) Woodhead 1970, Land et al. 1977, Kahn 1979, Kahn & (Tarutani et al. 1969). Tarutani et al. found that the Williams 1981). Deviations from equilibrium deposition aragonite polymorph of calcium carbonate was of oxygen and carbon isotopes have been attributed to enriched in 180 by 0.6%0relative to calcite at 25 "C. the relative contributions of seawater versus metaboli- Several relationships describing the temperature cally derived carbon and oxygen, with metabolically dependent fractionation of oxygen isotopes in biogenic derived carbon and oxygen being depleted in the aragonite have been determined since the study of heavier isotopes. Using this assumption it becomes Tarutani et al. (1969): possible to estimate the relative contributions of metabolic carbon and seawater derived carbon to the makeup of biogenic carbonates (Williams et al. 1977, from Horibe & Oba (1972), based on shells of the Tanaka et al. 1986, Kalish 1991). mollusc Anadara sp.; This study considers the theoretical and empirical basis for the estimation of isotopic equilibrium in cal- from Sommer & Rye (1978) and Rye & Sommer (1980), cium carbonate and applies this information to isotopic based on the fractionation between aragonite/calcite data collected on fish otoliths from a wide range of foraminifera shell pairs, their data actually show the species. These data are critical in determining the fractionation between aragonite and calcite and these utility of isotopic data to studies of a fish's environment results must be combined with data on calcite fraction- and physiology. Data collected from different fish ation (e.g. Epstein et al. 1953) as has been done in Eq. species living at a wide range of temperatures are used (2); to investigate the utility of oxygen isotope thermometry to studies of fish biology and carbon isotope data is a,, - 6, = 4.42 - 0.219 (TC) (3) considered in light of its potential as a source of infor- from Grossman (1982), based on the Foraminifera mation on metabolic processes and somatic and otolith Hoeglundina elegans; growth. Finally, the relationship between oxygen and bar 6, = 4.70 0.228 (TC) carbon isotopes collected from a wide range of species - - is investigated with respect to current theories on from Grossman & Ku (1986), based on the Foraminifera isotopic fractionation and in relation to physical and H. elegans; biological processes. a,,-6, = 4.65-0.213 (TC) from Grossman & Ku (1986), based on a series of coeval molluscs; DETERMINATION OF EQUILIBRIUM FRACTIONATION IN ARAGONITE bar - 6, = 5.10 - 0.268 (TC) (6) from Dunbar & Wefer (1984), based on the Foraminif- Before discussing, in detail, the basis for isotopic era H, elegans; dsequilibria in fish otoliths it is important to consider the problems encountered in estimating carbon and bar - 6, = 4.82 - 0.226 (TC) (7) oxygen isotopic fractionation in biogenic carbonates, from Aharon & Chappell (1983), based on tridacnid particularly aragonite. Similar points, in relation to clams; Kalish Otolith isotopic disequilibria 193 -

bar - b,, = 6.69 - 0.326 (TT) (8) bon (DIC) in the water where precipitation occurred. Eqs. (10) and (11) are in close agreement, whereas Eq. from Kalish (1991), based on the otoliths of the percoid (9) results in fractionations enriched in the heavier teleost Arripis trutta. 13c isotope relative to Eqs. (10) and (11). Eq. (9) indicates The variable S,, is the Si80 of the carbonate sample that aragonite enrichment in 13C increases with and S, is the Si80of CO2 gas equilibrated with a water increasing temperature, while Eqs. (10) and (11) pre- sample from which the carbonate was precipitated. dict the opposite effect. The effect of temperature on Lines representing the isotopic temperature scales carbon isotope fractionation in carbonates, based on for each of the above relationships and the data from the above relationships, is both small and uncertain. Tarutani et al. (1969) are plotted in Fig. 1. Although these relationships are based on different organisms from far removed higher taxa, all but one are in agree- MATERIALS AND METHODS ment, on average, to within about 1.5%O and, with the 180 fractionation determined by Tarutani et al. (1969) at Otolith material used in this study was obtained 25 "C. The oxygen isotope fractionation equation deter- during 1986 and 1987 from fish caught in Storm Bay, mined by Horibe & Oba (1972) shows a significant Tasmania and along the continental slope of Tasmania depletion in "0 relative to the other calibrations and, by line fishing or trawling. Antarctic fish were collected furthermore, indicates that aragonite is depleted in 180 by trawl in the Southern Ocean off Heard Island in July relative to calcite. This result disagrees with theoretical 1987. Otoliths were extracted, the adhering otolith cap- determinations of relative isotopic fractionation in cal- sule removed and the otoliths were cleaned ultrasoni- cite and aragonite and inorganic precipitate studies cally in deionised water, oven dried at 50 "C and stored (Tarutani et al. 1969). Horibe & Oba's (1972) data, in glass vials. Where isotope data is reported from both obtained from the mollusc Anadara sp., are probably the juveniles and adults of a species, inner and outer the result of disequilibrium fractionation due to biologi- layers of the otoliths were isolated with a dental drill. cal effects (Grossman & Ku 1986). Otoliths were roasted in a vacuum at 370 "C for h Equations for equ~librium I3C fractionation in l and then reacted with l00 '10 phosphoric acid at 24 "C biogenic aragonite include: for 24 h under vacuum. The CO2 resultinq from the reaction with phosphoric acid was purified and any non-condensables removed by a series of 3 freezing/ from Grossman (1984a), based on H. elegans; transfer steps. The purified CO2 samples were analysed on a VG Micromass 602C mass spectrometer. All val- 6I3ca,- bi3CDIC= 2,40 - 0,108 (T°C) (10) ues are reported in standard 6 notation relative to the from Grossman & Ku (1986), based on H. elegans; and PDB-1 standard (Epstein et al. 1953):

from Grossman & Ku (1986), based on a series of coeval molluscs. The variable 613Ca,is the 6I3C of the aragonite sam- where R = mass ratio (46/44 for oxygen and 45/44 for ple and 613CD1C is the 613C of dissolved inorganic car- carbon) of the sample or standard. Isotopic ratios were

--c Horbeand ODa l972 Sommerand Rye 1978 - Grossman 1982

I Fig 1. Relationships describing the temperature dependent fractionation of oxygen isotopes in biogenic aragonite. One line, Epstein et al. (1953) is based on biogenic calcite. The star shows the oxygen isotope fractionation datum measured in an inor- -4.0r....'~~~-1..-.~~..8...l..~8 ganic system by Tarutani et al. (1969). De- 0 5 10 15 20 25 30 tails of these relationships appear in the text Temperature("C) 194 Mar. Ecol. Prog. Ser. 75- 191-203, 1991

related to the PDB standard through analysis of the equilibrium for aragonite (6180eq and 613ceq)in each of Biggenden calcite standard (BCS) which had been cali- the environments was calculated using Eq. (4) for oxy- brated to the PDB standard via the international stand- gen and Eqs. (9) and (10) for carbon. The oxygen ards NBS-19 (National Bureau of Standards) (Craig isotope versus temperature relationship from Gross- 1957) and TKL (Te Kulti Limestone) (Blattner & Hulston man & Ku (1986) was used, rather than the relationship 1978). Analytical precision of the reported measure- between Arripis trutta otoliths and temperature (Kalish ments is & 0.03 % (1 SD) or better. 1991) to avoid any potential bias in the estimation of Values for seawater 6180 (6,) and 613C (613cDIC)for isotopic equilibrium. The resulting values are shown in each of the species investigated was approximated on Table 1, which also presents the mean isotope data the basis of data available in the literature (Epstein & measured in otoliths (6''0, and 8l3cm) from each Mayeda 1953, Craig & Gordon 1965, Kroopnick et al. species considered in this study. For example, for shal- 1972, Kroopnick 1974a, b, 1980, Kroopnick & Craig low-dwehng fishes from southeast Australian waters 1976, Williams et al. 1977, Grossrnan 1984a, b). Isotopic (e.g. Arripis trutta) the isotopic composition of arago-

Table 1. Summary of isotopic composition of fish otoliths, water, and dissolved inorganic carbon for species used in Figs. 2 to 7 and data used to estimate deviations from equilibrium isotopic fractionation. Values are means based on the number of replicates at each species. Full species names appear in Figs. 2 & 3. Isotopic values are reported in %o notation relative to the PDB standard. Headings are defined in the 'Materials and Methods' section

Species" Depth Temp. S1'O, S, b'%O,,h A'~O,,, b13C, b'3CDICb13ceq1 813~0q~a13ceq1 (m) ("Cl

C. acrolepis (23jb AnornrnaC NomeusC Cubjceps' PampusC Pepnlus' Stromateus' PsenopsiS SeriolellaC Schedophilus' CentrolophuS HyperglypheC Slenotorn usC RoccuS CentropristeS PijonotuS MerlucciusC Melanograrnrn usc GadusC Ceratoscopelus' OsmerusC IM cephalus (21d T thynnus (6)' P hlarnentosus (3)' I. ~llecebrosus(3)g A. trutta (25) H. atlanticus (10) T atun (4) N. rnacropterus (4) T declivjs (2) B. brarna (2) M novaezeland~ae(4) P barbatus (4) T macro@ (2) A. esper (2) N. squarnifrons (2)

" Number of replicates in parentheses h Mulcahy et a1 (1979); C Degens et al. (1969); Radtke (1984a1; " Radtke et al. (1987); 'Radtke (1987); Q Radtke (1983) h Using Grossman & Ku (1986) ' Uslng Grossman (1984a) l Using Grossman & Ku (1986) Kalish: Otolith isotopic disequilibria

nite precipitated in equilibrium (6180,, and b13C,,) with laboratories, there is still a strong correlation between Tasman Sea surface water varying from 10 to 20 'C was 6I3C and 6180. The least squares regression line for all estimated as follows. The value for b'3CDrc was esti- data is 6180 = 2.73 + (0.45)b~~C(r2 = 0.50, p < 0.001) mated to be 2.0 %o,based on depth and apparent oxy- and, for data collected in this study only, it is 6180 = gen utilization (AOU) (Williams et al. 1977). Seawater 2.64 + (0.41)b13C (r2 = 0.58, p < 0.001). The majority of

6180 (6,) was estimated to be 0.086 %O for Tasman Sea the 6180 and 613C data from the various fish species fall surface water (using data from the South Pacific Ocean close to the regression line, with the greatest scatter from Craig & Gordon 1965). Using Eqs. (g), (10) and attributable to the data of Degens et a1.(1969) who only (ll), 6I3Ceq of otolith aragonite is estimated to range made a single isotopic measurement for each species. from 2.04 to 4.23%0 and, 6180e, ranges from 0.13 to Data on mullet Mugil cephalus from Radtke (1984a) 2.32%0based on Eq. (4). The departures from isotopic also fall relatively far from the regression llne, perhaps equilibrium (~'~0,~= 6180,-618~eq and A~~C~~= due to the fact that these data are from fish reared at a 613C,,, - 613Ceq) also appear in Table 1. salinity of 32%0. In general, it must be assumed that When estimating the isotopic composition of arago- intercalibration through the international standard nite precipitated in equilibrium for Atlantic bluefin PDB is satisfactory and that these deviations are real. tuna Thunnus thynnus and southern bluefin tuna The significance of the relative distribution of the vari- Thunnus maccoyii otoliths, an approximation of these ous species along the regression line will be considered fishes' body temperature was used rather than the further in the discussion. ambient water temperature. This was done because Plots of carbon isotopes against temperature show tuna maintain muscle, eye and brain temperatures that otolith carbonate becomes more depleted in 13C significantly above ambient temperatures (Stevens & with increasing temperature (Fig. 4). Although the data Fry 1971, Linthicum & Carey 1972, Carey & Lawson show a negative correlation with temperature (r2 = 1973). 0.50, p < 0.001) as described by Grossman & Ku (1986) (Eqs. 10 & ll), the slope of the otolith carbon data versus temperature is approximately 2 times what they RESULTS observed for a single species of foraminifera and several related mollusc species. If deviations from equi- Plots of 6180 as a function of 613C for the sagittal librium precipitation (A'~C,~)are plotted against tem- otoliths of 35 marine fish species and the statoliths of perature (Fig. 5)an even stronger negative correlation one squid species appear in Figs. 2 & 3; the mean (r2 = 0.62, p < 0.001) results. This appears to indicate values are tabulated by species in Table 1. Data are that changes in b13C with temperature are probably broken down into 2 graphs (Figs. 2 & 3) so that it is due to biological fractionation of isotopes and are not possible to distinguish which data are attributable to a the result of the influence of 613CDIC. TO draw this particular species. The bulk of the data are from conclusion, it must be assumed that errors in the esti- isotopic measurements made in this study with the mation of both 613CDICand 613Ceq are insignificant remainder of the data being obtained from the litera- relative to biologically mediated fractionation effects. ture. Despite the fact that the data are from numerous The 613CDICvalues were obtained from the literature species encompassing a wide range of higher taxa, and, although every effort was made to obtain values collected in different oceans, at different depths and, representative of the sample locations in question, that the samples were analysed by several different these values are only estimates. Given the range of

..OOO.&.S. Fig. 2. Plot of 6180versus 613C # .B0 for the aragonitic otoliths of 35 - ..C# g 0.00 a fish species and the aragonitic a . Coryphaemidesacmlepis (jw) statoliths of one squid species. g ., . 0 A -2.00 q. 0 m Degens et al. (1969) Data identified by species in 0 Mugil cephalus m this graph are from literature. 00 I Thnwlhynnus @v) Data sources indicated in 1 T Ihynnus (adull) Table 1. For comparison data o Pnnomopoidesfilarnemosus lllex illecebrosus(sq~quid) collected in this study are also plotted on the graph and these data are identified to species in Fig. 3 Mar Ecol. Prog. Ser. 75: 191-203, 1991

Arripis fruna Quv) A.truna (adult) 0 Ampis esper 0 Hopbstethusallaniis Ouv) H. atlanticus(adult) 0 Thyristesatun 6 Trachurusdeclivis Q Brama brarna + Macruronusnovaezelandiae Quv) 0 M. novaezelardiae(adult) Pseudophycisbarbatus Thunnusrnacmyii m Nototheniasquarnilmns A Nernadactylusrnacropterus (juv) * N. rnacropterus(adult1

Fig. 3. Plot of 6180 versus 613c for the aragonitic otoliths of 11 fish species. For some specles data have been collected from both juveniles and adults; this is indicated in the inset; all data were collected In this study

613CDICvalues measured in the ocean, however, poten- line is in reasonable agreement with inorganic precipi- tial errors in the estimation of 6'3CDICwould be small tate data from Tarutani et al. (1969) and relationships compared with the devlations from b13C,, measured in derived from other studies of biogenic aragonites fish otoliths. Some studies have shown a small temper- shown in Fig. 1. It must be remembered, however, that ature effect on the precipitation of carbon isotopes in 6180, values for individual fish otolith data points rep- foraminifera (Williams et al. 1977, Grossman 1984b, resent a mean value accumulated over the life of a fish, Grossman & Ku 1986) and molluscs (Mook & Vogel which, in this study, might range from some fraction of 1968, Fritz & Poplawslu 1974), however, as for the 1 yr to, perhaps, over 20 yr. Furthermore, the estimates estimation of 613CDIC,the magnitude of this effect of temperature are based on current knowledge on the would not be significant in view of the large deviations distribution of each species over the fish's lifetime, and, from equilibrium in the fish otolith data. of course, have not been measured. Under these cir- Otolith oxygen isotopes become more depleted in the cumstances, the strong correlation appears to provide heavy isotope ("0) at higher temperatures (Fig. G), good evidence for a relationship between the b180 similar to the trend for carbon isotopes. Least squares measured in fish otoliths and temperature. These linear regression shows that the relationship between points also apply to the carbon isotope data above, but oxygen isotopes and temperature for all otolith data is it is clear that differences between the carbon data 6180, = 3.58 - 0.196 (TC),(r2 = 0.69, p < 0.001). This presented here and data appearing in the literature

3 H allaflDCuS 3 H. atlanticus 0.00 Mulcahyet al. 1979 m^ Radlke (various) n 4 -5.00 3" 0 L0 , -10.00 oE $! CO -15.00 0 5 10 15 20 25 30

-1ooo-I~~~~l----T.~~~,~~~7,,,l'. 1 Temperature ("C) 0 5 10 15 20 25 30 Temperature ("C) Fig. 5. Carbon isotope enrichment between otol~tharagonite and estimated equilibrium aragonite as a function of tempera- Fig. 4. Relationshp between 813C and ambient water temper- ture. Data used to estimate equilibnum appear in Table 1 ature. Solid line based on linear regression for all data points; Solid line based on linear regression for all data points; dashed line, only on data collected in this study. Eight data dashed line, only on data collected in this study. Eight data points from Hoplostethusatlanhcus are obscured and their points from Hoplostethusatlanticus are obscured and the11 location 1s Indicated in the figure location 1s mdcated in the figure Kalish: Otolith isotopic disequ~libria 197

that the otoliths of these fish are maintained at temperatures above ambient and, presumably, at tem- 2.00 peratures similar to the brain and body. Therefore, they cannot be used as recorders of environmental tem- 0.00 0 - peratures as suggested by Radtke & Morales-Nin i I - - .2.00 Th~sstudy : • 8 (1989). At present, the utility of bI80 data in the deter- o Mulcahvet al. l979 n Degenset al. 1969 mination of environmental temperatures is restricted 400 41 8 Radlke(var~ous) primarily by the sample size requirements of stable

-6.00 isotope mass spectrometers. Developments in laser 0 5 10 15 20 25 30 ablation mass spectrometry and other, microanalytical Temperature ("C) related methods may make it possible to investigate variations in 6"O over small portions of an otolith. Fig. 6. Relationship between b180 and ambient water temperature. Solid line based on linear regression for all data points; dashed line, only on data collected in this study Relationship between 6% and 6180

(Grossman 1984a, Grossman & Ku 1986) are more The interpretation of the relationship between 613C extreme than differences between oxygen isotopes and 6180 (Fig. 8) has been open to debate since Keith & measured in otoliths and Literature values for other Weber (1965) first suggested that the correlation might biogenic carbonates. be due to calcification processes incorporating carbon A plot of the differences between 6180, and bl'O,, and oxygen compounds originating from 2 isotopically for all data (Fig.?) shows that deviations from oxygen different sources or, the incorporation of specific por- isotopic equilibrium (~'~0,~)are not related to temper- tions of oxygen and carbon compounds that displayed a ature (r2 = 0.03, p > 0.08) and the mean deviation from correlation between 613C and 8l80. In those corals that equilibrium fractionation approaches zero. Considera- contain symbiotic algae (hermatypic corals) the corre- tion of data from this study alone (Fig. 7) indicates that lation between 613C and 61a0 is not always evident there may be evidence for a positive correlation because only carbon is fractionated during photosyn- between ~'~0,~and temperature (r2 = 0.27, p < 0.01) thesis (Swart 1983). Furthermore, the combined effect with relatively large depletions in the heavier oxygen of respiration, which depletes both oxygen and carbon isotope occurring at the lower temperatures. Closer heavy isotopes and, photosynthesis results in there examination of the data, however, indicates that this often being no apparent relationship between 6I3C and relationship is largely the result of a few data points at 6180 in hermatypic corals (Weber & Woodhead 1970, the extreme high and low temperatures. Furthermore, Goreau 1977, Swart & Coleman 1980, Swart 1983). there was no evidence of a linear relationship among Most researchers have concluded that the correlation these data (F = 1.23, df = 1,59, p > 0.25). between carbon and oxygen isotopes in ahermatypic corals and other organisms is the result of the incorporation of metabolically derived CO2 in the biogenic DISCUSSION carbonate (Keith & Weber 1965, Weber & Woodhead 1970, Vinot-Bertouille & Duplessy 1973, Land et al. Use of 6180 data as an environmental thermometer 1977, Kahn 1979, Kahn & Williams 1981, Williams et al. 1981a, b, Grossman 1984b). Data collected from a wide range of fish species Kinetic effects, due to varying weights and, thus, provide convincing evidence for a relationship between diffusion rates of different isotopes, are one of the main 6180 and temperature. Furthermore, the precipitation of factors affecting isotopic fractionation. finetic effects oxygen isotopes in the aragonite of fish otoliths appears could result in the depletion of the heavier carbon and to be in equilibrium with seawater 6"O. Similar conclu- oxygen isotopes, rather than incorporation of metabolic sions were made in Kalish (1991), however, more pre- CO2 alone. For example, Turner (1982) found that cal- cise determination~from further laboratory experiments cium carbonate, specifically calcite, precipitated in an carried out under controlled temperature conditions are inorganic system, could be depleted in I3C by 0.35 to needed to conclusively determine the equilibrium 3.37 %O depending on the rate of carbonate precipita- deposition of otolith oxygen isotopes. tion with the magnitude of the 13C depletion increasing Values of bl'O from fish otoliths can be used to with increased rate of precipitation. Unfortunately, he investigate the temperature of environments experi- was only able to measure 6I3c and, thus, it is not enced during the fish's life. The 6180 data obtained possible to directly interpret these data with respect to from the otoliths of Thunnus sp., however, confirms the relationship between b180 and 6I3C. Mar. Ecol. Prog. Ser. 75: 191-203, 1991

Degens et al. 1969 n Radtke(various) ---

M. Fig. 7 Oxygen isotope enrichment between - 8 M a . otolith aragonite and estimated equilibrium coo B' aragonite as a function of temperature. Data r D used to estimate equilibrium appear in Table 1. -4.00 I ~"'I~'"l"r'l r'''lr'''l Solid line based on linear regression for all data 0 5 10 15 20 25 30 points; dashed llne, only on data collected in this Temperature("C) study

The magnitude of the kinetic fractionations, in car- versus '80:'60, where the heavy isotopes are approxi- bonates, due to different rates of diffusion can be mately 1.1 % (Nier 1950) and 0.2 % (Garlick 1969) of approximated by estimating the ratio of diffusivities of the total carbon and oxygen, repectively. The net result the ~0~-~isotopic species involved in carbonate pre- is that a molecule with 13C is 5 times more likely cipitation. The ratio of diffusivities, D/D' is approxi- to occur at the crystal surface than a molecule with "0, mated by: indicating net depletions of 8 and 3.2 YW for carbon and oxygen, and a slope of 0.4. The mean slope of 8180 versus 813C data obtained from the literature for several higher taxa (Fig. 8) is approximately 0.37 and, for where D and D', and mL and mH = diffusivities and the fish otolith data the slope is 0.45, very close to masses of light and heavy carbonate isotopic species, the approximations based on the kinetiddiffusion respectively. The 13C and 180 fractionation factors due hypothesis. to diffusion are 0.992 (12C1603 -2/13~1603-2) and 0.984 The significance of a different kinetic effect, during (12c160 3-2/12~1801602-2). The ratios of the molecular hydration and hydroxylation of COz, in altering 613C velocities during diffusion indicate that depletions of and 6180 and causing isotopic disequilibria in biogenic 8.0 and 16.0%0occur in b13C and 8180, which corre- carbonates, has recently been investigated by sponds to a slope of 2.0 for the regression of blaO McConnaughey (1989a, b). He concluded that the against 613C. Grossman (1984b) rejected the kinetic linear relationship between 6180 and 6l3C indicates the hypothesis, as outlined above, on the basis that his data influence of a kinetic mechanism that would result in did not have the required slope. However, this calcula- simultaneous depletions and that such a mechanism is tion fails to consider the relative abundance of '3C:12C responsible for mediating isotopic disequilibria in

4.00

2.00 S 40 0.00 g m 2.00 L0

-4W

-6.00 .Q W -10W .a W -600 4m .zm ooo 2.00 400 i3c(%.PDB)

Fig. 8. Plot showing general trend in 6180 versus 6I3Cfrom several taxa, reported in the literature and from fish otoliths analysed in ths study. Only data from the photosynthetic planktonic foraminifera (Williams et al. 1981a) deviate from the general pattern. Of the remaining groups, only the algae are photosynthetic Kalish: Otolith isotopicdisequilibria 199

biogenic carbonates, particularly during rapid precipi- seawater dissolved inorganic carbon (Fig. 5) and oxy- tation. McConnaughey's (1989a) data from photo- gen isotope enrichment between the otoliths and sea- synthetic and non-photosynthetic corals show extreme water 6180 (Fig. ?), both as a function of temperature. departures from isotopic equilibrium and he states that As mentioned previously, otolith 6180 values do not isotopic disequilibria increase with increased growth differ significantly from predicted equilibrium values. rate in these organisms. He does not consider the The highly significant regression between 613c and additional effects of a kinetic/diffusion effect acting at 6180 may indicate that some mechanism, perhaps the crystal face. related to temperature (e.g. metabolic rate) is resulting Other sources present evidence that contradicts the in this relationship in fish otoliths. kinetic hypothesis and provide support for the role of On the basis of the relationships described to this metabolism in influencing the carbon isotopic composi- point it seems reasonable to draw the same conclusion tion of calcium carbonate. Fritz & Poplawski (1974) as Grossman (1984b), Tanaka et al. (1986) and many cultured freshwater molluscs in water with 613CDrCof others, that carbon isotopic disequilibria, in fish otoliths

-35.5, -13.1, and +5.4 %D. Those animals cultured in in this case, are due to the incorporation of metaboli- aquaria with 613cDICof -35.5 %O had shells that were cally derived COz. This further supports calculations enriched in 13C relative to the water, while animals made to estimate the relative contributions of DIC and cultured in water with 6l3CDICof -13.1 and +5.4 %D metabolic carbon to fish otoliths as in Kalish (1991). were depleted in 13C relative to the culture media. This result implies the inclusion of carbon with an isotopic composition of between -35.5 and -13.1 %D, presum- Variations in carbon and oxygen stable isotopes ably metabolically derived carbon. The isotopic com- among species position of metabolically derived carbon in molluscs should fall within this range based on measurements of There are several points relating to the carbon and 613 in molluscan tissue (Gearing et al. 1984). Tissue oxygen isotope data that support the hypothesis that 613c is an accepted estimator of the 613c of metaboli- 613C values measured in the otoliths are related to the cally derived COz present in the body fluids. metabolic rate of the fish. The relationship between Studies by Pearse (1970), Sikes et al. (1981), Tanaka 613C and temperature is not likely to be directly due to et al. (1986) and Spero & Williams (1988, 1989) on sea temperature as discussed in the previous section. In the urchins, corals, molluscs, barnacles and foraminifera majority of studies on biogenic carbonates a significant have all shown that both metabolic carbon and DIC are relationship between temperature and 613C has not incorporated into shell calcium carbonate. Pearse been found. Those studies that found 613C to be a (1970) and Sikes et al. (1981) showed the importance of function of temperature are not consistent, even with metabolic carbon by providing organisms with 14C- regard to the slope (positive or negative) of the relation- labelled food which was subsequently detected in the ship. Also, the magnitude of the temperature effect on shell. Tanaka et al. (1986) looked at naturally occurring 613C that has been found is much smaller than the levels of 14C in DIC and, various food sources in a apparent relationship in fish otoliths. natural system and concluded that up to 85 % of the Several trends in the isotope data add support to the carbon in shell carbonate was derived from metabolic hypothesis that there is a relationship between 613c and sources. Spero & Williams (1988, 1989) found that 613C metabolic rate. Those species that display the smallest values measured in foraminifera were not influenced departures from 6I3c equilibrium appear to have low by temperature, but that the values were a function of metabolic rates, live in deep, cold waters and possess metabolism in the form of symbiont photosynthetic relatively large otoliths, most notably the macrourid activity and irradiance levels. Coryphaenoides acrolepis and orange roughy Hoplo- The fish otolith isotope data presented here, when stethus atlanticus. At the opposite end of the spectrum plotted with isotope data from other organisms follows are the fast growing, highly active, homeothermic tunas, a trend similar to that described by McConnaughey which have, relative to body size, small otoliths. It is (1989a) (Fig. 8) and the overall distribution of the otolith notable that the older macrourids are at the point most isotope data is very similar to that of the ahermatypic opposite from the tunas, whereas the younger fish, corals (Swart 1983). However, my estimates of 613C and which presumably have a higher metabolic rate, display 6180 for aragonite precipitated in isotopic equilibrium a greater tendency to be more depleted in 13C, thus, with the various fish environments represented in this showing that the potential relationship between 613C data set (Table l), indicate that only carbon isotopes and temperature/metabolic rate may also be manifest are fractionated out of isotopic equilibrium. This point within a species. A similar relationship is also evident is illustrated by graphs of the carbon isotope enrich- among samples from juvenile and adult Atlantic bluefin ment between biogenic aragonite of fish otoliths and tuna Thunnus thynnus (Radtke et al. 1987). Australian Mar Ecol. Prog. Ser. 75: 191-203, 1991

salmon Arripis trutta, jackass morwong Nemadactylus basis of habitat temperature and the postulated rela- macropterus and blue grenadier Macruronus tionship between metabolic rate and b13c (Fig. 5). The novaezelandiae. In each of these species, 13C was more mean 613c value for N. squamifrons otoliths, collected depleted in the otoliths of juveniles than in the adults. in the Southern Ocean off Heard Island (mean temper- The differences in both 613c and 6180 between juvenile ature ca. 1 "C), is -3.75 2 0.13, a value that would be and adult orange roughy are very small and may be expected for fishes living at a temperature of about directly related to the biology of these deepwater fishes, 11.5 "C and with oxygen consumption rates between which are very slow growing both as juveniles and those determined for the deep-sea species (2.8 + 0.5 p1 adults (Mace et al. 1990). h-') and the estimated mean for a range of species The isotopic data from the otoliths of other fish (62 f 24 p1 g-1 h-'). This deviation can be explained species readily falls within the spectrum of metabolic by the fact that cold-adapted fishes have metabolic rates and 613C values outlined by Coryphaenoides rates above what would be expected on the basis of acrolepis and Thunnus spp. (Figs. 2 & 3). Less active temperature and size alone (Macdonald et al. 1987). species with large otoliths, such as red cod Pseudophy- This property is frequently attributed to metabolic cold cis barbatus and blue grenadier have 613C values about adaptation (MCA), although the exact nature of this

4.0 %O depleted in 13C relative to equilibrium. The 613c phenomenon is still open to debate (Macdonald et al. values measured in a wide range of pelagic species, 1987). There are no published data on the V02 of N. which might be classified as moderately active and squamifrons, but an estimate can be derived by cal- have medium sized otoliths, are clustered towards the culating the mean V02 from oxygen consumption data tuna end of the range and show a depletion of ca 7.0 to (measured below 3 "C) on 10 species in the genus

12 %O relative to 613C,,. Notothenia appearing in Macdonald et al. (1987). The Differences between 6I3C measured in juveniles and mean value for V02 for these 10 species is 43.4 f 17.0 adults of a single species are very small when com- p1 g-' h-', placing these species within the range of pared with differences among species such as Thunnus metabolic rates and 613C values expected for fishes maccoyii, Coryphaenoides acrolepis and Hoplosteth us occurring in warmer waters. This information provides atlanticus. This may be due to the relatively small further support for the idea that variations in 613c are differences in metabolic rates between juveniles and due to some metabolic effect and that they are not adults, when compared with the metabolic rates of directly related to temperature. other species. Information on the metabolic rates of The data presented above provide the initial each of these species is not available but, in many framework for development of a relationship between cases, these rates can be inferred from data available 613C and oxygen consumption. Although few data are on other species. The mean oxygen consumption rate available, I have plotted bt3C versus oxygen consump- (VOz) for Coryphaenoides acrolepis, Coryphaenoides tion (p1 g-' h-') in Fig. 9. The data for Coryphaenoides armatus and Sebastolobus altivelis measured at depths acrolepis, Notothenia spp. and Thunnus spp. are based of more than 200 m was 2.8 + 0.5 p1 g-' h-' (Smith & Hessler 1974, Smith 1978, Smith & Brown 1983). Oxy- gen consumption rates for skipjack Katsuwonus pelamis and albacore Thunnus alalunga tuna have Coryphaenoidesacrolepis

been measured as 505 (at 23 to 25 "C) and 212 p1 g-' Nololhen~aspp. h-' (at 15 to 19 "C), respectively (Gooding et al. 1981, Mean VOpand 613c for 34 species Graham & Laurs 1982), more than 75 times the rate 4.0 measured for the 3 deepwater species. Brett & Groves (1979) calculated a mean V02 of 62 k 24 wl g-' h-' with a range of 18 to 160 ,p1 g-' h-' for 34 fish species (not including tunas or deep-sea species). Clearly, the fish represented in the isotope data cover the widest -10.0 4 possible extremes of metabolic rates. Taking this range 0 50 100 150 200 250 300 into consideration, it is not surprising that differences Oxygenconsumption (p1 %/@h ) in metabolic rates within a species are not always re- Fig. 9 Relationship between bI3c and oxygen consumption solved by the 613C data. (VOz).Data points are explained in the text. There is a s~gnifi- A notable exception to the relationship between cant correlation between 613C and oxygen consumption (r2 = temperature/b'80 and metabolism/613c in this study is 0.92, n = 4, p<0.05]. Logarithmic curve is described by equation b13C = 1.1979 + (-3.5161)log(VO2). The logarithmic form the data collected from the Antarctic fish Notothenia of the relationship is similar to that representing the relation- squamifrons, where the deviation from equilibrium ship between temperature and metabolic rate (Brett & Groves 613C is much greater than would be expected on the 1979) Kalish: Otolith isotopic disequilibria

on multiple observations of both metabolic rates and depleted in I3C of those measured. Of course, in draw- otolith isotopic composition. An additional point, rep- ing this conclusion it is necessary to assume that a unit resentative of a mean value for 'all fish', is also plotted. of otolith material, such as a daily increment (Campana This point is based on the mean oxygen consumption for & Neilson 1985) is produced over a similar time frame 34 species (62 + 24 p1 g-1 h-') from Brett & Groves in all species, e.g. both tuna and orange roughy otolith (1979) and the mean value for bI3C obtained from daily increments are formed over a period of 12 h. isotope data collected in this study (-4.51 L 2.15). If the The kineticeffect of isotope fractionation, and its link to data from the isotope studies of Mulcahy et al. (1979), isotopicdisequilibria, is basedon the idea thatsimultane- Radtke (1984a, 1987) and Radtke et al. (1987) are ous depletions in "0 and I3C occur during CO2hydration included the mean value for 613C is 4.25 f 2.32. I have and hydroxylation, presumably in the presence of some chosen not to include the data from Degens et al. (1969) catalyst such as carbonic anhydrase. In fish, it seems because they did not do replicate analyses, thus, reduc- unnecessary to carry out the process of CO2 hydration ing the reliability of their data. The resultant relation- and hydroxylation given the very large pool of available ship (Fig. 9) is a reasonable approximation of models HC03-. Mugiya (1977, 1986) and Mugiya et al. (1979) that relate temperature to metabolic rate (Brett & Groves investigated the rollof the enzyme carbonic anhydrasein 1979). The relationship between oxygen consumption fish otolith formation by a combination of in vivo and and Sl3C is also very similar to a relationship between in vitro experiments. In a study of otolith formation minimum depth of occurrence (which can be related to in goldfish it was found that moderate doses of temperature) and oxygen consumption found by Torres acetazolamide (Diamox),a carbonic anhydrase inhibitor, et al. (1979) for migratory and non-migratory midwater did not effect otolith growth, whereas large dose multiple fishes. On the basis of this initial result, it would be injections reduced otolith growth by less than 17 %. He worthwhile to investigate further the significance of concluded that the catalyzed hydration and hydroxyala- both intra- and interspecific variations in 613C. This tion of CO:, was not necessary for otolith formation in could be accomplished by carrying out isotopic analyses goldfish (Mugiya 1977). A subsequent study on in vitro on the otoliths of those species for which oxygen con- preparations of intact rainbow trout sacculi found rela- sumption data are available. This exercise should be tively low levels of carbonic anhydrase activity, and it carried out over a wide range of species from different was concluded that, perhaps, carbonic anhydrase was habitats and should also consider intraspecific varia- not required at low rates of otolith deposition (Mugiya et tions, related to age and sex, in a species for which there al. 1979).These studies indicate that carbonic anhydrase is detailed information on oxygen consumption rates. and CO2 may play a non-essential role in fish otolith Such research is necessary before it is possible to establ- formation. If this is so than kinetidhydration-hydroxyla- ish a firm relationship between metabolic rate and 613C. tioneffects wouldnot be significant in carbon andoxygen The relationship between 613C and temperature, the isotope disequilibria, thus, providing further evidence potential association with metabolic rate and the fact that the correlation between 613C and 6180 for fish otolith that, in many cases, otolith size relative to body size data is not due to a kineticisotope fractionation mechan- decreases as the otolith aragonite becomes more ism as proposed by McConnaughey (1989a, b) for corals. depleted in 613C, are points not consistent with recent Furthermore, in light of our present knowledge on data on isotopic fractionation in corals (McConnaughey processes of otolith growth and otolith growth rates it 1989a, b). There are several points that support this seems unlikely that isotopic disequilibria are due to the conclusion with regard to fish otoliths. Firstly, although kinetic/diffusion mechanism. there is a strong correlation between 6180 and 613C, the departures of 6180 and 613C from equilibrium do not Acknowledgements. I thank Mike Power and Christine Cook seem to be attributable to the same mechanism. The for assistance with the stable isotope analyses, and Dick 8180 values are satisfactorily explained on the basis of Williams, Australian Antarctic Division, for the nototheniid otoliths. This study was supported by the University of Tas- the temperature fractionation effects first discussed by mania Research Grant Scheme. Urey (1947). The kinetic/hydration and hydroxylation mechanism of isotope fractionation would result in simultaneous depletions in oxygen and carbon isotopes LITERATURE CITED independent of temperature. Furthermore, the kinetic effect is postulated to result in greater isotopic dis- Aharon, P., Chappell, J. (1983). 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This article was presented by R. L. Haedrich, St. John's, Nfld, Manuscript first received. August 21, 1990 Canada Revised version accepted. June 4, 1991