Estimating the timing of growth rings in Atlantic cod otoliths using stable oxygen isotopes
H. HØ I E* A N D A. FO L K V O R D
University of Bergen, Department of Biology, P. O. Box 7800, N-5020 Bergen, Norway
(Received 14 January 2005, Accepted 15 September 2005)
A technique involving micro-scale sampling of otolith carbonate and analyses of stable oxygen isotope composition was used to relate the zone appearance of the otolith to the seasonal temperature cycle. Otolith opacity could then be related to the timing of zone formation.
Otoliths from two groups of Atlantic cod Gadus morhua held under known temperature conditions over a period of 4 and 6 years were examined. The otolith translucency followed the same pattern as the estimated temperature (from otolithd18O values) in the yearly incre- ments three and four, meaning that the translucent zones were deposited at the seasonal highest temperature in late summer and early autumn. The relative light intensities of otolith yearly increments five and six of older fish (deposited in the same years), however, were not signifi- cantly correlated to the estimated temperatures since increased otolith translucency also occurred at low temperatures. This might have been caused by stress in connection with gonad development or starvation during the spawning period. The results showed that this method of coupling otolith opacity and stable oxygen isotope composition can be used to estimate the timing of zone formations in otoliths. #2006 The Fisheries Society of the British Isles
Key words: age estimate; corroboration;Gadus morhua; otolith chemistry; otolith macrostructure.
INTRODUCTION
Since Reibisch (1899) first described the periodic pattern of growth structures in otoliths and related it to fish age, ageing of fishes using otolith growth structures has become routine work worldwide andc. 800 000 otoliths were aged in 1999 (Campana & Thorrold, 2001). An accurate estimate of fish age is essential for age-based management of fish stocks. When using macroscopic opaque and translucent zones in otoliths to age fishes, it should be based on knowledge of the periodicity and timing of the formation of the zones (Campana, 2001). The interpretation of annual growth zones in otoliths (and hence also the age estimate), however, is to a high degree based upon subjective human interpreta- tion. In addition, knowledge of the biological basis for otolith zone formation is incomplete. The temporal significance of the opaque and translucent zones is
*Author to whom correspondence should be addressed. Tel.:þ47 55 58 46 04; fax:þ47 55 58 44 50;
email: [email protected]
doi:10.1111/j.1095-8649.2006.00957.x, available online at http://www.blackwell-synergy.com
unclear and there is substantial variability in the timing of zone formation at the individual, population and species level (Beckman & Wilson, 1995). Analysis of otolith chemical composition has in recent years provided new information of the life history and ambient environmental history of fishes, but there have been few attempts to use such information to facilitate interpretation of otolith macrostructure. Weidman & Millner (2000) related stable oxygen isotope com- position (d18O) in Atlantic cod Gadus morhua L. otoliths to translucent and opaque bands and found that the translucent zones were generally deposited at seasonal d18O minima, corresponding to the warmest times of the years. They did not, however, have detailed independent measurements of temperature to relate ambient temperature to seasonality, and they did not measure the otolith luminosities. In the present study a method to obtain information on the tem- poral significance of otolith macrostructure formation by relating visual appear- ance of otolith macrostructure to otolith stable oxygen isotope composition based on fish reared at known temperatures was investigated. A computer controlled micromill that allowed eight to 10 discrete samples to be taken within each yearly otolith increment was used, and the samples for oxygen isotope composition analysed. Otolith opacity at the sampling sites could then be related to the ambient temperature experienced by the fish when the otolith carbonate was deposited, and the time of otolith carbonate deposition relative to the seasonal temperature cycle could therefore be estimated.
MATERIALS AND METHODS
Otoliths of sexually mature pen-reared Atlantic cod were used in this experiment. The fish were released into 5 m deep pens in September 1990 and 1992, and both groups were sampled in autumn 1996 when the Atlantic cod were 4 and 6 year sold. The daily monitored temperature at 2 m depth in the pen varied seasonally from 3 to 5C at seasonal minimum in the spring to 17–20 C at seasonal maximum in late summer and early autumn. Four otoliths from each of two groups of fish that were 6 (6L) and 4 years old (4L) were analysed. The fish were used as broodstock, and all fish were placed in spawning bags from January to April in 1995 (Huse & Jensen, 1983). Fish number 1, 3 and 4 in the 6L group were also placed in spawning bags from January to April in 1994.
No food was offered to the fish during the period they were kept in the spawning bag.
The experimental fish consisted of two males and six females. The fish standard length (LS) in the 4L and 6L groups ranged from 630 to 820 mm and from 780 to 845 mm respectively (Table I). A detailed description of the fish and method involved when analysing the stable oxygen isotope composition of the otoliths from group 4L and 6L is given in Høie et al. (2004a). Briefly described, a 500mm section of the otoliths was cut through the transverse plane at the core and both sides were ground and polished with P600–P4000 grit paper followed by 1mm diamond suspension to a final thickness of 140–160mm. Ten and eight samples of otolith material deposited in 1994 and 1995 were sampled from the otoliths respectively (Table I). This corresponds to increments formed at age 2 and 3 years for the 4L fish and increments formed at age 4 and 5 years for the 6L fish. The yearly increments were defined as an adjacent opaque and translucent zone. An opaque zone restricts the passage of light in comparison to a translucent zone. In this study transmitted light was used, so areas with relative high measured light intensities were the translucent zones and areas with relative low measured light intensities were the opaque zones (Fig. 1).
The otolith sampling was performed using a computer-controlled micromill (New Wave Research, Fremont, CA, U.S.A.) system where samples were collected by milling sequential layers at equal distances along the growth increments on the otoliths, while the
TABLEI.Mass,standardlengthandsexofthefishinthetwogroups[4(4L)and6(6L)yearsold],andinformationofotolithmassand micron-samplingintheotoliths.ModifiedfromHøieetal.,2004a FishOtolith GroupNumberSexMass (g)LS (mm)Mass (mg)Yearly incrementYear depositedNumber ofsamplesDistance betweenscans(mm)Sample volume(mm3 103 ) 4L1Male37927305917319941050135 41995840140 2Male30546305561319941047133 41995842147 3Female37646305551319941034110 41995834122 4Female67158205551319941045125 41995834111 6L1Female57388457908519941023805 61995825918 2Female50068006978519941022844 61995823947 3Female65307806981519941021811 61995829817 4Female61558156650519941019814 61995828854
co-ordinates of each sample were noted. The carbonate was sampled in the distal side at the ventral part of the otolith where the yearly increments were largest, starting at the distal side and moving towards the nucleus. A Finnigan Mat 252 mass spectrometer at the Laboratory of Geological Mass Spectrometry at the University of Bergen, Norway, was used to analyse the otolith powder for oxygen isotope composition (d18O), expressed relative to a VPDB standard. Precision of the otolith carbonated18O measurements was 007% (S.D. of repeated measurements of the standard) which corresponds to 03 C
when estimating temperature using the relationship for inorganic calcite (Kim & O’Neil, 1997). The seasonal maximum and minimum otolithd18O were related to time (month).
This was done by assigning the seasonal otolithd18O sample with the lowest value to the month with highest recorded mean temperature, and the otolith sample with the seasonal maximumd18O was assumed to be deposited in the month with lowest recorded mean temperature. This was based on the inverse relationship that exists between the oxygen isotopic composition in fish otoliths and temperature (Høieet al., 2004b). The otolithd18O values were related to temperature by the equation:d18OCd18OW¼390020 C whered18OCis thed18O of the otolith samples andd18OWis thed18O composition of the sea water (Høie et al., 2004b). The d18OW values reported by Gao et al. (2001) from the same area were used. The estimated temperature based on otolith d18O values reproduced the ambient temperature with high precision (Høie et al., 2004a), and the accuracy of the temperature estimate corresponded to the mean monthly temperature experienced by the fish. (Fig. 2).
The relative light intensity was measured in the yearly increments where the samples for isotope measurements were taken. Prior to milling, a digital photograph was taken of each otolith section when viewed in a dissecting microscope at25 magnification with transmitted light (Olympus 3040 digital camera with 20481536 pixels resolution). The images were converted to 8 bit grey scale and the light intensity in the area where the samples were collected was measured by using an image analysis programme, ImagePro Plus. Line thickness was equal to one pixel, and no smoothing was applied to the light intensity curves. The light intensities of individual otolith sections were converted to relative light intensities (minima set as 0%, maxima as 100% for each otolith).
RESULTS
The four otoliths of the 4L Atlantic cod showed the same pattern with one translucent zone deposited at the seasonal high temperature in summer and one opaque zone deposited at the seasonal low temperature in early spring (Fig. 3).
The Pearson product-moment correlation coefficient of the estimated tempera- ture (byd18O analyses) and the mean relative light intensity in the sample area varied fromþ0
57 toþ077 (p<005, Table II). Highest relative light intensity was found at the start of the sampling area, which corresponded to August 1995 when the highest temperature was recorded that year. The light intensity(a) (b)
FIG. 1. Photographs of otoliths of (a) 4 and (b) 6 year old Atlantic cod viewed with transmitted light. The scale bars¼1 mm.
thereafter decreased to a level ofc. 40% of maximum level, and this area corre- sponded well with the lowest temperature in 1995 that was found in March 1995.
An increase and new maximum in the relative light intensity of c. 80–90%
appeared in the otolith area that was deposited close to the seasonal maximum temperature in 1994 that occurred in July. The lowest light intensity for otolith material deposited in 1994 was found in the area that was deposited at the seasonal lowest temperature in February. These results confirm that the opaque zones in these Atlantic cod otoliths were deposited at the time of the year with the lowest water temperature, and the translucent zones were deposited at the time of the year with the highest water temperature. For all four otoliths, the relative light intensity was lower in the area that was deposited at seasonal lowest temperature in 1994 as 2 year olds compared to in 1995 deposition as 3 year olds.
For otoliths of the 6 year old fish, the relative light intensities produced a less distinct signal of one translucent and one opaque zone per otolith yearly incre- ment (Fig. 3). The Pearson product-moment correlation coefficient between estimated temperature and the mean relative light intensity in the sample area varied fromþ0
12 toþ037, and was not significantly correlated at thea¼005level (Table II). Generally there was a continuous increase in relative light intensity as the fish grew older and the variations did not follow the estimated temperature in the same manner as for the otoliths of the 4L fish. The relative light intensity peaked when the seasonal temperature was highest and the lowest
February 1994 July 1994 March 1995 Date
0 2 4 6 8 10 12 14 16 18 20 22
Temperature (°C)
FIG. 2. Recorded mean monthly temperature ( ) and temperature estimated from otolithd18O values for two typical otoliths represented with fish number 1 from the 4L ( ) and 6L ( ) groups respectively.
Distance in the otolith sampling area, from the core towards the edge (µm)
Relative light intensity (%) Estimated temperature (
° C)
0600500400300200100
Febuary 1994July 1994March 1995August 1995 020406080100 4812164L-1 040035030025020015010050 040035030025020015010050 035030025020015010050 035030025020015010050
Febuary 1994July 1994March 1995August 1995 020406080100 4812166L-1 0700600500400300200100 0700600500400300200100
Febuary 1994July 1994March 1995August 1995 020406080100 4812164L-2
Febuary 1994July 1994March 1995 020406080100 4812166L-2 0600500400300200100
Febuary 1994March 1995August 1995 020406080100 4812164L-3
Febuary 1994July 1994March 1995 020406080100 4812166L-3 Febuary 1994July 1994March 1995August 1995 020406080100 4812164L-4 Febuary 1994July 1994March 1995 020406080100 4812166L-4
(a)(b) FIG.3.Relativelightintensity()andestimatedtemperature(,fromotolithd18 Ovalues)at(a)otolithdistancefromstartofthethirdyearlyincrementtotheend ofthefourthyearlyincrementinotolithsofthe4yearoldAtlanticcod(4L),and(b)fromstartofthefifthyearlyincrementtotheendofthesixthyearlyincrement inotolithsofthe6yearoldfish(6L)infishnumbers1,2,3and4.Oneotolithcarbonatesamplefrom4Lfishnumber3and6Lfishnumber2waslost.
relative light intensity occurred at seasonal minimum temperature in 1994. The relative light intensities in the otoliths corresponding to the low seasonal tem- perature in 1995, however, was higher than expected from the pattern seen in the otoliths of the 4L fish. This was particularly evident for fish number 1 where a peak of the relative light intensity occurred in the area corresponding to March 1995 (Fig. 3).
The relative light intensities in yearly increments 3 and 4, deposited in 1992 and 1993, in otoliths of the 6L fish were also measured. They showed the same pattern as the relative light intensities found in yearly increments 3 and 4 of the 4L fish (Fig. 4). The range of relative light intensity between minimum and maximum values in yearly increment 3 and 4 also showed the same pattern for the two fish age groups (Table III). Both age groups had the largest difference in yearly increment 3 and less difference in yearly increment 4.
DISCUSSION
The analysis of otolith stable oxygen isotope composition can be used to gain information of the timing of otolith zone formation if the fishes experience seasonal temperature cycles. The method used in this study has two main advantages. First, information of the whole otolith can be extracted by milling from the margin to the nucleus of the otolith. The otolith structures of long-lived species are especially difficult to interpret since the optical properties of otoliths often change as the fishes grow older (Campana, 2001). By relating the otolith optical properties to a seasonal temperature signal across the whole fish life span, the timing of opaque and translucent zone formation and its change with age can be studied. The change in the relative optical properties of the zones as the fishes grow older (Mina, 1968) can also be studied by this method. Secondly, unlike many other elements in the otoliths that are influenced by fish growth (Campana, 1999), the relative deposition of the two stable oxygen isotopes18O and 16O on a growing otolith is not affected by fish growth and otolith pre- cipitation rate (Thorroldet al., 1997; Høieet al., 2003). The isotope signals in the TABLEII. Pearson product-moment correlation coefficient (r) between mean relative light intensity of the otolith and estimated temperature. Significant correlations at the
a¼005 level are in bold
Age group (years) Yearly increment Fish number N r
4 3 and 4 1 18 077
4 3 and 4 2 18 061
4 3 and 4 3 17 058
4 3 and 4 4 18 057
6 5 and 6 1 18 012
6 5 and 6 2 17 035
6 5 and 6 3 18 037
6 5 and 6 4 18 023
N, number of estimated temperatures (using otolithd18O values) and mean relative light intensities and that are compared for each fish.
otoliths are therefore well suited to infer the periodicity of otolith growth increments relative to the ambient temperature cycle, independently of somatic growth that may also influence the otolith zone formation (Beckman & Wilson, 1995).
In this study, the otolith optical properties were related to estimated tempera- ture since data of both otolith and water d18O were available. When studying otoliths of wild fishes, thed18O of sea water is often unknown and an absolute temperature can therefore not be estimated. The coupling between otolithd18O
TABLEIII. Difference (maximumminimum) in relative light intensity measured in otolith yearly increment 3 and 4 of fish in age groups 4 and 6 years
Age group Fish
Range (maximumminimum value of relative light intensity) (years) number Yearly increment 3 Yearly increment 4
4 1 936 762
4 2 879 624
4 3 902 673
4 4 760 632
6 1 980 657
6 2 100 753
6 3 886 603
6 4 100 550
100 80 60 40 20
0 0 20 40 60 80 100
100 80 60 40 20
0 0 20 40 60 80 100
0 20 40 60 80 100
0 20 40 60 80 100
Relative light intensity (%)
0 20 40 60 80 100
0 20 40 60 80 100
Relative distance (%) YI6
YI5 YI4 YI3
YI6 YI5 YI4
YI3 YI3 YI4 YI5 YI6
YI6 YI5 YI4
(a) (b) YI3
(c) (d)
FIG. 4. Relative light intensity ( ) at relative distance in otoliths of 6 year old Atlantic cod (6L) in fish number (a) 1, (b) 2, (c) 3 and (d) 4. YI 3 to YI 6 refers to yearly increments from 3 to 6 respectively.
The relative light intensity in yearly increments 5 and 6 (to the right of the vertical dotted lines) are also shown in Fig. 3.
and otolith opacity, however, can still be a powerful combination since the relative temperature difference and hence seasonality can be deduced from the otolithd18O values.
Otolith structures that do not conform to the translucent and opaque zones of a yearly increment are called secondary growth structures (Panfiliet al., 2002).
The secondary growth structures are a substantial source of error when ageing fish based on otolith macrostructure. Little is known about the causes of these secondary structures, although feeding, spawning, temperature and developmen- tal changes have been suggested to play a role (Wrightet al., 2002). A coupling of the optical properties and the stable isotope composition of the otoliths can also provide more information of the timing and environmental basis of the secondary growth structures.
Generally, the Atlantic cod in this study deposited the translucent zones at seasonal highest temperature although the 6L fish also showed increased otolith translucency at cold temperatures. Thorough examination of field-caught Atlantic cod otoliths in southern Norway by Dannevig (1933) also showed that fish deposited the translucent otolith zones at the seasonal highest temperature in the period August to October. This is in contrast to other studies of young Atlantic cod in southern Norway where the translucent zones were assumed to be deposited during the winter when backcalculating fish size (Smestad & Holm, 1996; Olsenet al., 2004). This illustrates the importance of proper understanding of the temporal significance of otolith zone formation.
There is no obvious explanation for the difference in otolith opacity between the two fish age groups found in this study. Two factors that have major influence on fish physiology and growth, however, can possible explain the difference in otolith opacity between the two age groups: (1) gonad development and (2) starvation.
In terms of gonad development, all fish became sexually mature at age 2 years so the presence or absence of spawningper secan be ruled out. Older fishes have lower size-specific metabolism than younger fishes, however, and older fishes generally also allocate relative more energy into their gonads than younger fishes (Wootton, 1990). Differences in the pattern of otolith opacity between the two fish age groups were found in otolith carbonate deposited in winter and early spring, which is the time of final gonad development and spawning in Atlantic cod. This might suggest that relative energy allocation in the gonads influence otolith opacity, and explain the high relative light intensity in otoliths of the 6L fish in the low temperature period in early spring. On the other hand, energy demanding processes like oocyte development and vitellogenesis occur in an extended period prior to spawning and not only in late winter and early spring when the eggs ovulates (Tyler & Sumpter, 1996). Physiological stress associated with gonad development seems therefore unlikely to be the primary reason for the increased translucency in otolith carbonate of the 6L fish deposited in early spring.
Starvation is a second explanation for the unexpected high relative light intensity values found in otolith carbonate deposited at low temperatures in the 6L group. The observed translucent zones at seasonal high temperature can be caused by reduced fish appetite at such high temperatures. Optimum temperature for growth for the Atlantic cod size range of this study is 5–7 C,
and fish feed and grow poorly above 15–16 C due to high maintenance cost and lack of appetite (Brett, 1979; Jobling, 1988; Bjørnsson & Steinarsson, 2002). The formation of translucent zones at high temperature can therefore be caused by an indirect temperature effect, i.e. lack of feeding. Increased translucency of otolith carbonate deposited at low temperatures of fish from the 6L group can also be explained by starvation (Huse & Jensen, 1983). The 6L fish (except number 2) were kept in spawning bag from February to April in both 1994 and 1995 and thereby starved. The increased translucency in otolith carbonate deposited in spring could therefore be caused by starvation-induced stress. For the 4L fish there seem to be less correlation between relative light intensity in the otoliths deposited in spring in 1995 when the fish were placed in the spawning bags and starved compared to spring 1994 when the fish were fed. Controlled laboratory experiments have shown that reduced feeding causes translucent zone formation in otoliths (Neilson & Geen, 1985), and field observations also sup- port this explanation (Admassu & Casselman, 2000; Colloca et al., 2003;
Johnson & Belk, 2004). The effect of starvation is a factor that seems to be important for otolith opacity, and should therefore be further examined.
Change in otolith opacity by fish age support the results by Mina (1968) who concluded that the terms opaque and hyaline (translucent) are only relative in their nature and do not give an absolute value of otolith opacity. This phenom- enon is particular evident when seen over a longer time span, as in Fig. 4, where the relative light intensity typical for a translucent zone in yearly increment 3 is generally not higher than the relative light intensity that is typical for an opaque zone in yearly increment 6. Age estimates based on visual examination of otolith structures by the human eye will therefore always have an element of subjective interpretation included, so the need of validation or corroboration of otolith structures must not be underrated.
This work has been financed by the Norwegian Research Council grant no. 130192/140 and the EU-project Q5RS-2002–01610 (IBACS). We wish to thank Ø. Karlsen for providing the otoliths, C. Andersson Dahl for good support and help when using the micromill, and A. Geffen, K. Limburg and K. Nedreaas for constructive comments that significantly improved an earlier version of the manuscript. We also wish to thank U. Ninnemann and R. Søra˚s at the GMS laboratory for valuable help.
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