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Environmental Biology of Fishes

, Volume 89, Issue 3–4, pp 427–432 | Cite as

Bromine patterns in Norwegian coastal Cod otoliths—a possible marker for distinguishing stocks?

  • Karin E. Limburg
  • Hans Høie
  • Darren S. Dale
Article

Abstract

Bromine was found to accumulate in otoliths of Norwegian coastal Cod Gadus morhua that were reared under known conditions. Despite the fact that the Cod were moved from one rearing environment to another, causing marked changes in some otolith elemental concentrations, bromine appeared to accumulate continuously along certain growth axes as revealed by 2-D elemental mapping. In contrast, North Sea and Baltic Sea Cod showed little to no patterning in Br. We suggest that Br uptake in otoliths may be under physiological and genetic control, and as such, may prove useful as a stock identification tool.

Keywords

Otolith chemistry Bromine Synchrotron-based x-ray fluorescence Cod 

Introduction

Although over 30 elements have been documented in fish otoliths (Campana 1999), most of the otolith chemistry literature deals with a handful (Sr, Ba, Mn, Mg) and a few isotopic ratios (O, C, Sr). Bromine, which can occur in low ppm concentrations (Campana 1999) is rarely reported. We posit that this is because it is difficult to analyze with the most widely used analytical methods in this field (i.e., electron microprobe analysis and inductively coupled plasma mass spectrometry [ICPMS]).

We have mapped concentrations of a number of different elements, sometimes down to sub-ppm levels, in several fish species using scanning x-ray fluorescence microscopy (SXFM, Limburg et al. 2007). Our mapping creates 2-dimensional (2-D) displays of spatial configurations of detectable elements (Fig. 1), and in so doing, literally provides another dimension unobtainable by 1-D transect analysis. We report here on finding strong patterning in bromine within otoliths of Atlantic Cod (Gadus morhua). More specifically, we have seen these patterns in individuals from the Norwegian coastal Cod stock, but the patterns do not appear to hold across other stocks.
Fig. 1

A transverse section of a Norwegian coastal Cod otolith, showing bromine patterns (left) and masking polygons created in ArcGIS to extract data beneath the masks

SXFM is a spectral analysis. Unlike ICPMS or wavelength-dispersive electron microprobe analysis, for which specific isotopes and elements must be targeted for analysis, SXFM scans for fluorescence along a spectrum of energies. We did not target bromine, but rather we noticed strong fluorescence of this element in certain Cod otoliths we were analyzing for a study of manganese and strontium. Furthermore, the patterns were suggestive of a systematic incorporation of bromine over the lifetime of the fish, because they begin at the otolith core and extend through the otolith along particular growth axes (Fig. 1). The patterns appeared specific to a particular group—Norwegian coastal Cod—and were not as strongly observed in specimens from other stocks that we were surveying (Fig. 2). Hence, we provide one of the few reports on otolith bromine, and propose that at least in Atlantic Cod, its uptake may have a genetic component.
Fig. 2

Optical images (left and right columns) and corresponding elemental maps of bromine in Cod otoliths. White horizontal bar = 1 mm. Left panels are the four Norwegian coastal Cod. Top right is a North Sea Cod; the remaining otoliths come from Baltic Sea Cod captured in ICES sub-division 25. Note that the bottom left otolith was over-ground, exposing material behind the core. Laser ablation trenches are visible in the second-uppermost otolith on the right

Materials and methods

We obtained otoliths of four Norwegian coastal Cod that were reared for experimental purposes at the Norwegian Institute of Marine Research facility at Austevoll in Hordaland County, approximately 35 km southwest of Bergen. The fish were initially hatched and reared in a land-locked fjord (or “poll”) at the Parisvatnet Field Station ca. 25 km northwest of Bergen; they were subsequently moved at 6 months of age to rearing pens at Austevoll. Two six-year-old males from the 1990 year class, and two fish from the 1992 year class (one male, one female), were used in our study.

For comparison, we examined otoliths of four wild Cod from the Baltic Sea. All of these Cod were captured in ICES fishery sub-division 25 in the southwestern Baltic. Additionally, we analyzed an otolith from a Cod captured in November 1974 in the North Sea.

All otoliths had been previously cleaned and stored in envelopes. Otoliths were mounted on very clean glass (fused quartz) and sectioned in the transverse plane, and polished to or close to the core (one was excessively ground). The sections were cleaned with ethanol followed by a de-ionized water rinse.

Elemental analysis was carried out using SXFM on the F3 bending magnet beamline at the Cornell High Energy Synchrotron Source (CHESS). A double-bounce multilayer monochromator with 0.6% bandpass was used to produce a 16.1 keV X-ray beam, which was focused with a single-bounce glass capillary optic (Bilderback et al. 2003; Cornaby 2008) to produce a photon flux of ca. 1011 counts per second in a spot varying between 15 µm and 30 µm in diameter at the sample, depending on the level of resolution desired. Scans were made initially at 10 keV and 17.5 keV to augment lower and higher energy fluorescences respectively, but later we found that 16.1 keV produced a good compromise. The beam was focused to a spot on the specimen and the fluorescence spectrum was integrated for a fixed amount of time (typically 1 s to 10 s), then the beam was moved to an adjacent spot, and the process repeated until the entire surface of the sample had been mapped. The fluorescent X-rays were collected using an energy-dispersive Vortex silicon drift detector. An aluminum foil attenuator was placed over the detector to reduce the overwhelming intensity due to calcium fluorescence, to increase sensitivity to elements present in trace amounts. Calibrations were carried out using a variety of standards, including NIST Standard Reference Materials 8704 (Buffalo River Sediment) and 1572 (Citrus Leaves), and the MACS-3 carbonate standard developed at the USGS Reference Materials Project (Wolf and Wilson 2007). Data were processed with PyMCA (Solé et al. 2007) running under XPaXS (Dale 2009) to produce elemental maps that could be exported as numerical data for further analysis. Detection limits were ca. 0.1–0.2 ppm (± 0.1 ppm).

Because of the spatial nature of the data, we imported the data files into a geographical information system (ArcGIS, ESRI 2009) and clipped out data from five areas on each map using polygon masks that could be modified to fit each individual otolith (Fig. 1). The Spatial Analyst ™ ArcGIS extension was used to compute mean Br ±s.d. within each polygon. Statistica (Statsoft 2003) was used for further statistical analysis. Repeated measures ANOVA was conducted on mean values within the five zones; groups were the Norwegian coastal Cod, Baltic Cod, and North Sea Cod.

Results

All four of the Norwegian coastal Cod otoliths showed strong Br patterning in the transverse plane (Fig. 2). Bromine appears to concentrate along particular axes of otolith growth, starting from the core and radiating out along the ventral and dorsal sides of the sulcus acusticus. Additionally, bromine is visible in growth bands that correspond more-or-less to optical growth zones. In contrast, Br patterning is low to non-existent in the Baltic and North Sea otolith sections (Fig. 2), and one otolith (BaltCod-1222) registered no detectable bromine at all. High concentrations on the edges may be artifacts due to the glancing angle of the X-ray beam catching on the specimen edge, which is a function of sample placement.

Spatial analysis of otolith bromine concentrations showed that, for the Norwegian coastal Cod, highest concentrations occurred in the dorsal area on the sulcal side (Zone 3, Table 1). Repeated measures ANOVA found no difference between Baltic Cod and the single Cod from the North Sea, so they were combined and tested against the Norwegian coastal Cod and found to be statistically different (F4,24 = 12.22, p < 0.0001; Fig. 3). Dropping the North Sea Cod from the analysis led to weaker inferences when all five spatial zones were included in the analysis (p < 0.07), but any combination of four zones was always significant at the 0.05 level.
Table 1

Mean (ppm) ±standard errors of Br in five zones of Cod otolith transverse sections. Zonal statistics were calculated on pixels (N is given for each zone in each otolith); pixels ranged from 30–50 microns on a side. BaltCod-1222 had no Br detectable, so zonal statistics were not calculated, as all pixels were measured as 0 ppm (i.e., below the limit of detection)

 

Zone 1

s.e.

N

Zone 2

s.e.

N

Zone 3

s.e.

N

Zone 4

s.e.

N

Zone 5

s.e.

N

Norwegian coastal Cod

NCC-1

1.47

0.027

182

5.37

0.043

2064

6.79

0.112

1903

2.72

0.020

2998

2.21

0.010

4477

NCC-2

4.04

0.144

180

8.12

0.166

1477

8.78

0.176

885

2.92

0.022

1762

3.46

0.029

909

NCC-3

3.34

0.060

577

3.47

0.015

2525

4.53

0.022

1695

2.28

0.013

2230

2.53

0.018

1933

NCC-4

2.15

0.055

246

4.97

0.029

1093

6.59

0.058

1931

2.77

0.023

2004

2.84

0.019

2690

Baltic Sea Cod

BaltCod-1131

0.74

0.042

194

2.25

0.133

584

0.74

0.013

1043

0.58

0.007

1731

1.23

0.055

1440

BaltCod-1222

0.00

  

0.00

  

0.00

  

0.00

  

0.00

  

BaltCod-0780

0.70

0.020

92

0.81

0.013

433

0.68

0.011

379

0.68

0.005

1821

0.68

0.007

1169

BaltCod-1720

0.45

0.009

228

0.52

0.006

840

0.74

0.008

687

0.51

0.005

1676

0.43

0.003

1839

North Sea Cod

0.29

0.008

131

0.26

0.007

245

0.39

0.008

431

0.35

0.006

720

0.30

0.003

1060

Fig. 3

Results of repeated measures analysis of variance of five zones defined on the transverse sections of Cod otoliths in the study (see Fig. 1). “Other” combines the four Baltic Cod otoliths and the single North Sea Cod otolith

Discussion

Bromine is rarely reported in the otolith chemistry literature—so rarely, in fact, that we have found almost no mention of it. Campana (1999), reviewing the literature on reported values of 28 elements found in otoliths, includes a median concentration of slightly more than 1 ppm for Br but does not provide a specific citation.

Aragonitic bromine is difficult to quantify with inductively coupled plasma mass spectrometry due to polyatomic interferences, and it is well below the detection limits of wavelength-dispersive electron probe microprobe analysis. On the other hand, more sensitive fluorescence methods, such as particle induced X-ray emission analysis (PIXE) and SXFM, have been successfully employed to quantify Br (Pingitore et al. 2002; Svedäng et al. 2010; this study). From our experience, the bromine K-alpha line of 11.92 keV has few if any interferences in typical otolith aragonite, making it possible to quantify to sub-ppm concentrations via SXFM (Table 1).

One possible complication is that bromine in aragonitic structures may be only loosely bound to the protein matrix. Pingitore et al. (2002), using K-edge XANES (X-ray absorption near-edge structure) to examine the form of bromine in corals, suggested that corals had incorporated NaBr which could readily be washed away. In a similar vein, Proctor and Thresher (1998) suggested that Br (which they did not quantify) would behave like Cl in otoliths, and therefore should be easily removed by certain preservation or cleaning methods.

Although we did not test this systematically in our study, we handled all otoliths similarly. Also, we performed a trial on one of the Norwegian coastal Cod otoliths, attempting to knock out Br by vigorous ultrasonication in ethanol for 6 min. We then re-scanned the otolith, and found that the concentrations were <10% different from the original scan, which was within our calibration error. Importantly, the spatial patterns of Br were preserved (i.e., the Br did not “migrate” around in the aragonitic matrix). We thus concluded that Br was not lost from our otoliths through handling effects, but in fact remained bound in place.

The Norwegian coastal Cod used in this study were transferred from rearing facilities in a land-locked fjord to sea-pens at a marine station approximately 70 km to the south when they were 6 months old (Høie et al. 2004). This change in environment could be seen in the concentrations of strontium and manganese (Limburg et al. in prep.), but bromine concentrations appear to accumulate in continuous fashion from the otolith core to the outer edge, along growth axes (Figs. 1 and 2). This suggests that bromine was not taken up in these otoliths passively, but rather was under physiological control through some mechanism we do not as yet understand.

In contrast, the otoliths of Baltic and North Sea Cod we examined had far lower levels of Br. The differences may at face value suggest genetic differences. In another study reported in this issue, Svedäng et al. found that Br was one of seven trace elements (along with Co, Cr, Fe, Mn, Sr, and Ti) that served to discriminate among spawning stocks of Cod in the Skagerrak-Kattegatt-Baltic region. Although those authors were not able to confirm a genetic basis of Br incorporation, the fact that Br was detected and varied significantly among stocks suggests that it may prove useful as a tool for stock discrimination among Atlantic Cod.

As mentioned above, there is little information as to what factors, environmental or endogenous, affect bromine incorporation into otoliths. We presume that Br, like Na, Sr and Cl, varies with salinity. Sr variation in Cod otoliths is a function of temperature (Townsend et al. 1995) and likely salinity as well, although no explicit tests have been reported for the latter factor. We have observed changes in Sr concentrations in Cod otoliths that are consistent with movements into cooler, more saline waters, but have not observed concomitant changes in bromine concentrations (Limburg et al. in prep.). Experimental manipulation of such factors as temperature, salinity, and genetic origin would be a next logical step.

Two-dimensional elemental mapping is not commonly done in fish otolith chemistry applications, but has great potential to help answer questions about mechanisms of chemical incorporation. For example, Limburg and Elfman (2010) recently showed that spatial patterning in another trace element, zinc, can be used to classify Salmoniform fishes and agrees with a recent revision of their phylogeny. Mapping can also reveal changes that may occur in otoliths after death, as in the case of burial of archaeological materials. In a study of Stone Age Cod otoliths which had been buried ca 5,000 yr, post mortem alterations in Fe, S, P, and Sr were evident due to diagenetic effects (Olson et al. 2002).

SXFM 2-D mapping and other similar methods (e.g., micro-PIXE) are still relatively little used, due to the rareness of instrumentation and length of time for analysis (our scans took 6–24 h, depending on the size of the otolith section). However, this may change in the future, as improved detector technology is developed and implemented. Another advantage of the method is that it is non-destructive of the otolith material; hence, samples may be re-analyzed multiple times (as in the case of our test of removing Br by ultrasonication, and subsequent re-analysis).

Conclusions

We have shown here that synchrotron-based scanning X-ray fluorescence microscopy is an excellent method to quantify trace levels of bromine with high sensitivity and high spatial resolution. Further, we have demonstrated that the spatial patterns of elemental uptake can be quite complex and different from patterns observed in growth rings. In general, we suggest that 2-D mapping of otolith chemistries—currently only elements, but perhaps stable isotopes in the future—will enhance our understanding of the mechanisms of incorporation and help to parse out physiological from environmental influences.

Notes

Acknowledgments

We thank Y. Walther and H. Svedäng, Swedish Fisheries Board, for providing otoliths of Cod from the Baltic and North seas, respectively, and Ø. Karlsen, Institute of Marine Research Norway, for the Norwegian coastal Cod otoliths. We also thank S. Cornaby for producing the glass capillaries used in the analysis. Finally, we thank two anonymous reviewers for constructive criticism of an earlier draft. This work is based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS) which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under NSF award DMR-0225180. Partial support (to KL) was from NSF grant DEB-0238121.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Karin E. Limburg
    • 1
  • Hans Høie
    • 2
    • 3
  • Darren S. Dale
    • 4
  1. 1.State University of New YorkCollege of Environmental Science and ForestrySyracuseUSA
  2. 2.Department of BiologyUniversity of BergenBergenNorway
  3. 3.EWOS ASBergenNorway
  4. 4.Cornell High Energy Synchrotron Source, Wilson LaboratoryCornell UniversityIthacaUSA

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