Environmental Biology of Fishes 61: 445–453, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Seasonal stable isotope records of otoliths from ocean-pen reared and wild cod, Gadus morhua Yongwen Gaoa,e , Henry P. Schwarczb , Uwe Brandc & Erlend Moksnessd Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, BC V9R 5K6, Canada b School of Geography and Geology, McMaster University, Hamilton, ON L8S 4M1, Canada c Department of Earth Sciences, Brock University, St. Catharines, ON L2S 3A1, Canada d Institute of Marine Research, Flødevigen Marine Research Station, N-4817 His, Norway e Current address: Makah Fisheries Management, P.O. Box 115, Neah Bay, WA 98357, U.S.A. (e-mail:
[email protected])
a
Received 9 February 2000
Accepted 1 February 2001
Key words: stable isotope ratios, δ 18 O and δ 13 C, temperature, seasonality, environmental constraints, diet Synopsis Otoliths of ocean-pen reared cod, Gadus morhua, provide a unique opportunity to examine the lifetime history of the fish. Here we report 18 analyses of such otoliths on seasonal (winter and summer) stable oxygen and carbon isotope ratios. The calculated isotopic temperatures from otoliths of reared cod were essentially in agreement with the temperature record during rearing, suggesting that temperature is a dominant factor in the precipitation of otolith aragonite. Carbon isotope ratios increased with age and leveled off at 4 years old, presumed to correlate with sexual maturity of cod. As compared with otoliths of wild-caught cod, significant differences were found in isotope variation and the correlation between δ 13 C and δ 18 O. These differences were probably attributable to the different environmental constraints and food supply.
Introduction The study of otoliths from fishes reared in the laboratory can yield useful information about optimal growth and early life history. Under known temperature and salinity conditions, the survival of larvae and juveniles is a good indicator of their living environment (Laurence 1978, Brown et al. 1989, Lambert et al. 1994). The otolith extracted preserves a record of early development of teleost fishes (Lecomte-Finiger 1992, Hare & Cowen 1994). Most laboratory experiments, however, are conducted only a couple of months after hatching. As compared with a fish’s lifetime history, the information derived from the larva period may be very useful for aquaculture, but is too limited to extend to the adults. In this sense, otoliths of ocean-pen raised cod, Gadus morhua, surviving as long as 6 years near Bergen, Norway, are exceptionally valuable in studying
the lifetime history and influence of environmental factors. The environmental temperature that a marine organism experienced can be determined from the oxygen isotopic fractionation between biogenic carbonate (both calcite and aragonite) and the ambient seawater. This is based on the theoretical proposition of Urey (1947) and many empirical calibrations of the temperature dependent fractionation of 18 O/16 O ratios (e.g., Epstein et al. 1953, Horibe & Oba 1972, Grossman & Ku 1986). Similar efforts have been made for fish otoliths (Kalish 1991, Patterson et al. 1993, Thorrold et al. 1997). Stable carbon isotopes in otoliths are generally related to isotopic disequilibrium fractionation, and the variation of 13 C/12 C ratios reflects changes in metabolism and dietary shift of the fish (Mulcahy et al. 1979, Schwarcz et al. 1998). Stable isotope studies on cod otoliths have been previously
446 conducted by Radtke (1984), Radtke et al. (1996) and Weidman & Millner (2000). As a cold-water groundfish species, cod are sensitive to temperature changes and seem to prefer certain temperature ranges (Scott 1982). The known-history otoliths of Norwegian cod, therefore, provide an exceptional opportunity to gain insights into the aspect of temperature dependence during otolith aragonite precipitation. Comparison of otoliths between reared cod and a previously studied wild cod from the northeast Scotian Shelf, Atlantic Canada (or 4Vs cod for convenience; Gao 1997), will further broaden our knowledge of stable isotope studies on cod. In this paper, we report the result of stable isotope analyses on 18 otoliths of ocean-pen reared Norwegian cod and 10 otoliths of wild 4Vs cod from the same age group and year, using a new microsampling technique that can obtain seasonal (winter and summer) isotope records. The first objective was to examine the isotopic composition of individual otoliths from the initial stage to age six that involved lifetime isotope variation and the temperature dependence of otolithseawater fractionation. The second goal was to evaluate the environmental imprints on isotope indicators, such as the isotope range and relationship between 13 C/12 C and 18 O/16 O of otoliths from the reared and wild cod.
Materials and methods The ocean-pen reared cod otoliths were collected from the IMR Austevoll Aquaculture Research Station, Norway. These cod were reared from eggs in the laboratory, raised in mesocosms and transferred to meshed ocean-pens when the young were about 10 cm in fork length. The pens were about 10×10 m2 and 5 m deep with recording thermometers and salimeters. Temperature in the pens was naturally homogeneous and salinity varied with fluctuations of seawater. Cod were reared for 6 years (November 1988–1994) to an average length of about 70 cm. The wild 4Vs cod otoliths were selected from the 1994 archival of the Bedford Institute of Oceanography (BIO) in Dartmouth, Department of Fisheries and Oceans Canada (cf. Gao 1997). Otoliths of both origins were initially sectioned at BIO and clearly showed growth increments: the natural time series as daily, seasonal and annual otolith rings (Pannella 1971). Yearly increments in cod otoliths are composed of two seasonal zones: a translucent and an opaque zone that correspond to the winter and summer precipitation,
Figure 1. Photograph of a typical otolith section of the reared Norwegian cod, showing the clearness of translucent (winter) and opaque (summer) growth zones. Scale bar ≈ 1 mm.
respectively (Figure 1). Each sectioned otolith was cut with a jewelry saw and mounted on a frosted glass slide. After polishing, the otolith section was measured and sampled from seasonal zones using a computerdriven micromilling technique (DM 2800) that has been described by Gao (1999). Since the milling tool is about 25 µm at its tip and the milling width and depth can be predetermined, this method has a high resolution to evaluate both seasonal and annual otolith zones. In general, about 30 µg of aragonite powder was collected from each seasonal zone (cf. Figure 1). Powder samples were reacted with 100% phosphoric acid to liberate CO2 gas in an Autocarb carbonate analyzer attached to a VG Optima mass spectrometer in the Stable Isotope Laboratory, McMaster University. All analyses were reported in the conventional δ notation (‰): δ 18 O = {[(18 O/16 O)X /(18 O/16 O)S ] − 1} × 1000, for example, where X is sample and S is standard (VPDB). Precision of the analysis was better than ±0.06‰ for both δ 18 Oand δ 13 C. Seawater samples from one pen were collected in November 1996 to measure oxygen isotope ratios (δ 18 OW ) for temperature calculations. These samples (n = 16) were prepared using a modified procedure from Epstein & Mayeda (1953) for small samples (0.2 ml). After preparation, the samples were equilibrated with CO2 in a 25◦ C constant shaking water bath for at least one week and analyzed by the VG Optima mass spectrometer. The results were expressed with respect to standard mean ocean water (VSMOW) in per mil (‰) and the overall analytical precision was better than 0.15 ‰ (1 SD). The isotopic temperature was computed using Grossman & Ku’s (1986) equation for otolith aragonite
447 and a constant δ 18 OW value of −1.06‰ (the mean δ 18 OW value of 16 seawater samples). The relationship between the VPDB and VSMOW scales was corrected by −0.26‰ (Coplen et al. 1983). T -test and analysis of variance (ANOVA) were used to interpret the isotope difference, and p < 0.05 was considered statistically significant. All tests were conducted using the Minitab (1996) statistical software.
Results The δ 18 O values of otoliths of reared Norwegian cod ranged from −0.21 to +1.98‰, whereas the δ 13 C values ranged from −6.08 to −1.01‰ (Table 1). The mean δ 18 O values of 18 otoliths generally showed
small variation (0.59 to 1.14‰), but the range of mean δ 13 C values was much larger (−2.33 to −3.87‰). Isotope variations of the wild-caught 4Vs cod and reared Norwegian cod were significantly different (t-test, p < 0.0001). In particular, otoliths of Norwegian cod were depleted in both 18 O and 13 C (Table 2). During the analytical process sample 2B3 was too brittle to provide enough aragonite powder and was discarded from the data series, even though its isotopic composition was within the normal range (cf. Table 1). Seasonal temperature variation in ocean-pens was uniform (Figure 2), from about 4 to 16◦ C throughout the growth history of the Norwegian cod. The midsummer temperatures varied by up to ±4◦ C around their mean value of 16◦ C. Salinity was also uniformly distributed around a mean value of about 30‰, with
Table 1. Summary of the isotopic composition from 18 otoliths of ocean-pen reared cod. Sample no.
Fork length (cm)
Weight (g)
δ 13 C range (‰)
Mean (‰)
δ 18 O range (‰)
Mean (‰)
2B2 2B3 2B5 2B6 2B7 2B8 3B9 3B13 3B14 3B16 3B17 3B18 4C20 4C21 4C22 5B25 5B26 5B28
69 74 72 80 81 84 68 76 83.5 73 72.5 88 73 80.5 72 80 89 84.5
2640 3435 3875 4140 4905 4790 3455 4895 5490 5395 3210 5515 3430 4420 4720 4110 6710 6630
−5.13 to −2.39 −3.72 to −1.36 −4.85 to −2.23 −4.40 to −2.07 −4.83 to −2.28 −4.15 to −1.29 −3.79 to −1.33 −4.52 to −1.41 −3.86 to −1.66 −4.74 to −2.01 −6.08 to −2.41 −4.12 to −1.67 −4.34 to −1.42 −3.91 to −1.01 −4.42 to −2.12 −4.51 to −1.82 −5.32 to −3.01 −4.92 to −1.66
−3.70 −2.33 −3.30 −2.86 −3.30 −2.58 −2.50 −2.77 −2.76 −3.22 −3.87 −3.04 −2.69 −2.51 −3.07 −2.80 −3.84 −2.89
0.03 to 1.75 0.80 to 1.13 0.16 to 1.40 0.10 to 1.98 0.31 to 1.88 0.24 to 1.87 0.16 to 1.37 0.49 to 1.55 0.31 to 1.56 0.47 to 1.53 −0.07 to 1.73 0.17 to 1.43 0.20 to 1.61 −0.04 to 1.59 −0.21 to 1.76 0.29 to 1.47 0.28 to 1.81 0.26 to 1.89
0.80 0.96 0.78 0.59 1.12 0.99 0.90 1.14 0.92 1.01 0.81 0.91 1.03 0.76 0.93 1.06 1.04 1.01
Table 2. Summary of the isotopic composition from 10 otoliths of wild 4Vs cod. Sample no.
Age (y)
Fork length (cm)
δ 13 C range (‰)
Mean (‰)
δ 18 O range (‰)
Mean (‰)
9432 9433 9434 9437 9439 9441 9442 9444 9445 9452
5 5 5 5 6 6 5 5 6 6
57 51 56 62 59 63 54 63 65 69
−2.48 to −1.00 −2.65 to −0.73 −3.78 to −0.75 −2.14 to −0.82 −4.56 to −0.43 −2.86 to −0.98 −2.56 to −0.68 −2.29 to −0.48 −2.59 to −0.58 −3.33 to −0.47
−1.49 −1.40 −1.43 −1.24 −1.38 −1.41 −1.15 −1.20 −1.26 −1.04
1.20 to 2.50 1.27 to 2.43 0.89 to 2.60 0.96 to 2.62 0.24 to 2.70 1.30 to 2.66 0.34 to 2.50 0.71 to 2.68 0.35 to 2.82 0.94 to 2.63
2.22 2.01 2.14 2.26 1.77 2.29 2.02 1.98 2.29 2.19
448
Figure 2. Variation in temperature and salinity recorded in ocean-pens from November 1988 to November 1994 during rearing.
more variation in summer than winter. Seasonal δ 18 O values (winter and summer) of Norwegian cod and the temperature record during rearing showed an inverse relationship over the first 4 years; thereafter the δ 18 O variation was much less (Figure 3). This pattern of δ 18 O variation was similar among single otoliths and essentially independent of the number of samples examined (cf. Figure 3b). The largest seasonal δ 18 O shift was from ages 1–1.5, even though there had been no particular temperature shift over those years. In general, the seasonal δ 18 O variation during early growth years
was consistent with the variation in temperature, but decreased in amplitude with increasing fish age. As a result, the calculated isotopic temperature profile essentially matched the record for the first 3–4 years, but was biased towards summer values for later years (Figure 4). Apparently the isotopic effect of salinity variation observed in Figure 2 averaged out to give correct isotope temperatures when we used the mean δ 18 OW of seawater from one open. The average δ 13 C values of these reared cod varied from low starting values (from −4.15 to −3.60‰) to
449
Figure 4. Comparison of calculated and recorded temperatures for otoliths of reared cod. The calculation was based on isotopic temperature equations of Grossman & Ku (1986) and a mean measured δ 18 OW value of −1.06‰ (see text for details).
Figure 3. Temperature records and δ 18 O variations of otoliths of reared cod. The temperature profile (a) was based on seasonal average readings from 1988–1994, whereas the δ 18 O variations (b) were based on average isotope values from 10 and 17 otoliths, respectively (error bar = 1 SD).
a plateau (−2.50 to 2.20‰), with a transition from ages 3.5–4 years (Figure 5). This pattern of δ 13 C variation was also independent of the number of otoliths examined, and similar to that observed in the average δ 13 C values of wild 4Vs cod that displayed a more 13 C-enriched isotope range (−2.88 to −0.76‰). However, the Norwegian cod showed a large degree of variation over ages 3–4, while the 4Vs cod increased their δ 13 C values steadily. Furthermore, seasonal δ 13 C
variations from the opaque (summer) and translucent (winter) zones were significantly different (ANOVA, F3,198 = 75.3, p < 0.001) between the two cod samples. Cod from 4Vs revealed consistent 13 C-enrichment in summer with respect to winter, but there was no such relationship in reared Norwegian cod (Figure 6). Significant isotopic differences were also found in the relationship of δ 13 C and δ 18 O between the two cod samples (ANOVA, F3,324 = 1092.2, p < 0.001). Both δ 13 C and δ 18 O values of individual 4Vs cod were higher than those of the Norwegian cod (cf. Table 2), and showed two distinct data groups (Figure 7). These differences suggest the causes of the positive relationship between δ 13 C and δ 18 O are more complicated than previously believed.
Discussion Stable isotope studies in biogenic carbonate have shown that δ 18 O composition can be used as a proxy for reconstruction of seawater temperature, particularly for regions not affected by large changes in salinity. As a result, δ 18 O values of otoliths can be used as a measure of growth temperature: with decreasing temperature, δ 18 O increases (Kalish 1991). The ocean-pen
450
Figure 5. Lifetime δ 13 C variation of reared and wild 4Vs cod of the same age group and timing (error bar = 1 SD, respectively), showing the age of sexual maturity (ages 3–4) as they reach the maximum δ 13 C values.
Figure 6. The different δ 13 C variations between seasonal otolith zones. The 4Vs cod show consistent 13 C-enrichment in summer with respect to winter, but there is no such relationship in the Norwegian cod.
reared Norwegian cod show that seasonal δ 18 O variations in otoliths can be divided into two intervals: prior to the 4 years old, δ 18 O varies with ambient temperature; after age 4, δ 18 O remains approximately constant (cf. Figure 3). Our results clearly indicate that
temperature is an important factor in otolith aragonite precipitation, with age 4 as a transitional period in this feature. Biological studies show there is an important relationship between water mass character and age-4 year cod in the 4Vs area (Smith et al. 1991). The 4Vs
451
Figure 7. Relationship between δ 13 C and δ 18 O from the reared and wild 4Vs cod, showing two distinct groups of data in the isotope range and variation.
cod have the ability and preference to choose certain water layers to live after maturity by moving to the bottom layers where the temperature shift is minimal (Scott 1982). For the ocean-pen reared cod, it is difficult to link the decrease in isotope variation to external oceanic changes because of the limited dimension of the pens (about 5 m deep). However, the lifetime δ 18 O variations of the Norwegian cod may suggest that these cod change their growth physiology around age 4, probably the time of sexual maturity. The environmental temperature of cod can be quantitatively predicted if their otoliths are formed in oxygen isotopic equilibrium conditions (Radtke et al. 1996). From the isotopic temperature calculation, we see that the calculated temperatures essentially parallel the recorded ones over the first 3–4 years (cf. Figure 4). The bias is probably due either to sampling effect or the constant δ 18 OW values we used in the temperature computation. The value of −1.06‰ in δ 18 OW was analyzed on the 1996 water collection from one pen and is only an estimate for the rearing period (1988–1994). Fortunately, there are no large variations in salinity as recorded, so the bias may not hinder our purposes to examine how well the isotope variations capture the variation in temperature for the reared cod. The sampling effect is similar to that discussed by Leder et al. (1996) for corals. The fact that all the calculated
temperatures after age 4 agree well with the summer records, indicates that otolith aragonite is mainly deposited in the summer after the age of maturity. The largest δ 18 O shift from ages 1–1.5 years may have corresponded to an exceptionally intense seasonal change in temperature, but is more likely due to the very fast growth in the reared cod that allowed for better resolution of summer and winter deposits. Overall, the close approximation between the calculated isotopic temperature profile and the record suggests that temperature is the dominant factor in δ 18 O variations between seasonal otolith zones. The lifetime δ 13 C variation in the reared cod also showed a marked transition at about age 4, as indicated by the leveling off of δ 13 C values at about −2.20‰ (cf. Figure 5). In contrast, the wild 4Vs cod at the same age group showed a maximum δ 13 C value of about −0.80‰ while in recent years, they reached a similar maximum δ 13 C value at age 3 (Schwarcz et al. 1998). These authors attributed the attainment of maximum δ 13 C values to sexual maturation of the cod, which is known to occur at approximately that age. Although the ranges of δ 13 C variations in both settings are identical (about 2‰), their values from the reared Norwegian cod (−6.08 to −1.01‰) and the wild 4Vs cod (−4.56 to −0.43‰) are significantly different (t-test, p < 0.0001). Because the environmental conditions did not change the fundamental patterns of the lifetime δ 13 C variation, these differences were more likely attributed to the diet of the fish (DeNiro & Epstein 1978). The food used for the Norwegian cod is a commercial dry feed that consists of dry matter, protein, carbohydrates and lipids (Karlsen 1997, personal communication). Thus, the lower δ 13 C values obtained in this study may be simply due to the fact that the average δ 13 C of the dietary level of the reared cod was much lower than that of 4Vs cod. The relationship between δ 13 C and δ 18 O has been extensively discussed in other organisms such as corals (McConnaughey 1989), Australian salmon (Kalish 1991) and Atlantic cod (Radtke et al. 1996), as being either a kinetic or a metabolic effect. Among the 17 Norwegian cod otoliths we examined, the linear relationship of δ 13 C and δ 18 O was generally weak. Other independent research has reported that there is no such relationship for the laboratory-reared fish (Kalish 1991). The lack of a correlation between δ 13 C and δ 18 O suggests that the pronounced increase in δ 13 C before maturity is not due to a kinetic isotope effect, as this would in general lead to a strong correlation between the two isotope ratios (Hoefs 1997). Kinetic isotope
452 effects have been attributed to metabolic production of CO2 ; some authors suggested that the positive correlation between δ 13 C and δ 18 O in otoliths of marine fishes might be related to growth temperature (e.g., Kalish 1991). Schwarcz et al. (1998) argued that at maturity the δ 13 C in cod otoliths approach equilibrium with seawater dissolved inorganic carbon (DIC), while still including some metabolic HCO−3 . Since the seasonal δ 13 C differences in otoliths of both reared and wild cod are small, it in turn indicates that there is no effect of seasonal variation in photosynthetic activity on δ 13 C of seawater DIC. Some other mechanisms must account for the different correlation between δ 13 C and δ 18 O in the reared and wild cod, such as the more complex life-paths of the wild cod (Brown et al. 1989). The lower correlation between δ 13 C and δ 18 O for the Norwegian cod was probably evidence that the degree of activity was limited by the dimensional and environmental constraints in ocean-pens. These isotopic signals, as demonstrated by Gao & Beamish (1999), would be potentially useful for identifying different stocks and populations in cod fisheries and management. In summary, we have demonstrated that seasonality effects occur in δ 18 O but not in δ 13 C of cod otoliths. The lack of seasonality in the older year growth may be partly an artifact of the difficulty in isolating pure winter or summer layers, but the general bias towards summer δ 18 O values suggests that this is the time when most otolith aragonite is deposited. We also observed that there are pronounced differences between the pen-reared Norwegian cod and the wild 4Vs cod in respect to isotopic composition, lifetime variation, and relationship between δ 13 C and δ 18 O. These differences have probably arisen from the different environmental constraints and food supply, suggesting that stable isotopic signatures derived from otoliths could serve as sensitive and useful indicators of these factors. Acknowledgements We are indebted to the IMR Austevoll Aquaculture Research Station, Norway, and Steven Campana at BIO for providing samples in this study. We thank Martin Knyf for training in isotopic analysis and Ron Lodewyks for assistance in otolith microsampling. Two anonymous reviewers made comments and editorial suggestions that greatly improved the quality of the manuscript. Financial support provided by HPS and UB through their grants from the Natural Sciences and
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