Electron probe microanalysis of fish otoliths — evaluation of techniques for studying age and stock discrimination

doi:10.1016/0022-0981(92)90306-U

Journal of Experimental Marine Biology and Ecology

Volume 158, Issue 1, 12 June 1992, Pages 1-36

https://doi.org/10.1016/0022-0981(92)90306-U Get rights and content

Abstract

Electron probe microanalyzers fitted with energy-dispersive (ED) and wavelength-dispersive (WD) X-ray spectrometers are the standard instruments for measuring microscale differences in the chemical composition of the calcified tissues of fishes. We review the principles of using these spectrometers, detail procedures for preparing otoliths for microanalysis, and compare the accuracy and sensitivity of the two instruments. ED systems on electron probes are prone to produce spectral artifacts, peak overlaps and difficulties in modelling nonlinear backgrounds. Current generation ED systems do not, therefore, appear suitable for quantitative analysis of trace elements (<5000 ppm) in otoliths. For the species we examined, WD systems could measure six elements (Ca, Na, Sr, K, S and Cl) accurately and precisely to levels of several hundred ppm. However, electron probes operating at the beam powers required for WD analysis damage otoliths, causing pitting and chemical change, which increases measurement errors. This damage and the consequent loss in data quality vary directly with the power density of the electron beam, which, in turn, determines in part the rate of data acquisition. Use of a WD spectrometer to analyze otoliths, therefore, requires making a trade-off between acquisition time and data quality, which has to be evaluated against the magnitude of natural variability in the composition of the otoliths of each species studied. In the species we examined, we concluded that beam power densities >3μW · μm⁻² resulted in unacceptable levels of specimen damage.

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The ambient concentrations of trace elements are influenced by a range of external factors that vary at both the spatial and temporal scales (Campana and Thorrold, 2001; Rogers et al., 2019). Most studies of the elemental composition of fish otoliths have reported levels of elements, such as Ca, Sr, Na, K, Mg, Ba, P, S, and Cl (Edmonds et al., 1989; Kalish, 1991; Gunn et al., 1992; Edmonds et al., 1995; Fuiman and Hoff, 1995; Limburg, 1995; Secor et al., 1995), or the concentrations of trace elements like Cu, Zn, Fe and Sn (Campana et al., 1994; Campana and Gagne, 1995; Kalish et al., 1996; Thorrold et al., 1997). In the marine environment, Ba, Mg, U, B, and Sr in various calcitic tissues show strong correlations with ocean water temperatures (Sadekov et al., 2005, 2009; Montagna et al., 2007; Long et al., 2014), while other elements like Sr and Ba are used successfully to reconstruct environmental and coastal-estuary migration histories for individual fish (Fowler et al., 2016), as their concentrations reflect local availability in seawater.
Coastal ecosystems are increasingly threatened by a wide range of human activities. Fish otolith chemistry, by creating a unique specific signature, can be used as a natural tag for determining life stage dispersal, spatial connectivity and population structure. In this study, we tested whether differences in otolith composition among juveniles of gilthead sea bream, Sparus aurata, could enable their proper allocation to polluted areas based on higher concentrations of elements related to contaminants. Otoliths were embedded, sectioned and analysed by LA-ICP-MS in line scan mode. Multivariate analysis confirmed clear separation between sites and elements. Samples from the site under the strongest anthropogenic impact from industrial and agricultural river input were characterized by higher values of Pb/Ca and Zn/Ca. However, these relatively low values likely do not have a negative effect on S. aurata recruitment, though they could serve for identifying the contribution of polluted nurseries to stock dynamics.
Connectivity between the marine coast and estuary for white mullet (Mugil curema) in northeastern Brazil revealed by otolith Sr:Ca ratio
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The right otolith was embedded in epoxy resin and a longitudinal section including the nucleus was cut using an Accutom-50 micro-cutting instrument (Struers). Each section was sanded up to the nucleus, using a TegraPol 35 (Struers) with abrasive discs of 15 μm, 10 μm and 5 μm calibers, then polished with a diamond solution (0.25 μm) (Gunn et al., 1992). The preparations were cleaned by ultrasound using milliQ water and stocked in a desiccator cabinet.
Microchemical analyses were carried out in order to estimate the Strontium:Calcium (Sr:Ca) ratio in the otolith of the white mullet, Mugil curema, in the Pernambuco (at the Santa Cruz channel, Brazil) in order to determine its connectivity between the estuary and ocean. Variation in the otolith Sr:Ca ratio was directly related to salinity, with greater salinity denoting a higher otolith Sr:Ca value. Data on the otolith Sr:Ca ratio demonstrates that the individuals analyzed are born in areas of salinity that are characteristic of the estuary, where they develop until approximately one year of age, at which point they migrate to areas of greater salinity until reaching sexual maturity (3 years of age) in the sea. Spawning occurs in the ocean, after which M. curema individuals may either remain or return to the estuary until the next spawning. Differences in estuarine salinity were found for young-of-year individuals and may be related to the season when spawning took place, since M. curema females are found spawning throughout the year. The hypothesis is that higher salinity in the dry season leads to a greater otolith Sr:Ca signature among individuals spawned in this season from birth until one year of life. On the other hand, the lower salinity in the rainy season leads to a lower otolith Sr:Ca signature among individuals spawned in this season. These information are important for the adequate management of the white mullet stock in northeastern Brazil.
Biogenic and diagenetic indicators in archaeological and modern otoliths: Potential and limits of high definition synchrotron micro-XRF elemental mapping
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Current common techniques for elemental analyses of otoliths include Laser Ablation Inductively-Coupled Plasma Mass Spectrometry (LA-ICP-MS), Electron Probe Microanalysis (EPMA), and Proton-Induced X-ray Emission (PIXE). LA-ICP-MS and EPMA are both destructive and/or damaging to the sample, the former as a fact of its operation, and the latter as a result of the operating conditions to measure concentrations of minor elements (Gunn et al., 1992). PIXE has also been observed to produce discolouration (Huszank et al., 2009).
Fish otoliths are biogeochemical archives of environmental conditions and are a valuable tool for examining life traits. These sclerochronological life history records, accessed through geochemical analyses, are widely used in fisheries sciences, and are seeing growing use as palaeoenvironmental archives or for reconstruction of past human activities. The fidelity of such reconstructions relies on the preservation of the biogenic geochemistry, which may be altered through diagenetic processes. In this work, a methodology is provided that enables measurement of elemental concentrations with a high precision with simultaneous quality control of the data to ensure high reliability and a multi-technique sample marking strategy to ensure precise alignment. Thin sections of four archaeological and two modern otoliths from the Pacific coast of South America were examined by synchrotron μXRF. High-definition elemental maps of sample sections up to 3.2 × 1.0 mm² (V × H) were produced with an on-sample spot size of 6.0 × 16 μm². Thirteen elements were detected, some of which may be useful indicators of diagenetic alteration. Strontium was accurately quantified and one sample presented local concentrations above the previously described range in literature. The quantity of collected data allows the use of statistical approaches to examine the intra- and inter-sample Sr distribution. A highly reliable profile, produced by integrating multiple points, additionally permits identification of defects and potential alteration along its length through changes in median absolute deviation. Contamination at the edge of some of the specimens was evidenced from preparation and other post-mortem alterations, even in modern samples, however diagenetic alteration in the elemental signals was not evidenced significantly further than a few hundred micrometres deep in the samples. Medium-energy SR-μXRF therefore provides a fast and sensitive elemental probe without a visible effect on the sample, and the integration volume which limits the attainable lateral resolution in this energy range is discussed. This paper discusses the methodological limitations and opens new perspectives in the analysis of biomineral carbonate palaeoproxies.
Chemical Composition of Fish Hard Parts as a Natural Marker of Fish Stocks
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The chemical composition of calcified structures in fish (otoliths, spines, rays, scales, and vertebrae) has been identified as a useful natural marker of stock structure. This application depends on geographic variation in water chemistry or other factors (e.g., temperature) that influences the chemistry of the calcified structure such that fish that inhabit different environments exhibit differences in their chemical signature. The use of this technique in stock discrimination requires that a number of assumptions must be met, including that groups of interest possess characteristic and reproducible markers and all possible groups contributing to the group mixture have been characterized. In some instances, structural chemistry can permit observation of subtle population structure that may not be detected by genetic applications alone. Chemical analysis of calcified structures is most effective when used as part of a holistic approach to stock identification.
Rapid and cost-effective assessment of connectivity among assemblages of Choerodon rubescens (Labridae), using laser ablation ICP-MS of sagittal otoliths
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Citation Excerpt :
Solid-state analysis of otolith slices can provide additional time-resolved elemental data. Initially, microprobe techniques, such as energy dispersive and wavelength dispersive x-ray analyses (Mulligan et al., 1987; Gunn et al., 1992) were used, but, since first employed (Campana et al., 1994), laser ablation ICP-MS (LA ICP-MS) has been the method most used. In this case, a laser is used to volatilise material from the otolith surface that is swept by an inert gas into the ICP-MS for analysis.
A rapid and cost-effective assessment was required to provide advice to management on the connectivity between juvenile and adult life cycle stages of Baldchin Groper Choerodon rubescens, a labrid endemic to the west coast of Australia, which has high social value, but relatively low commercial fishery importance. To minimise costs we used laser ablation ICP-MS to analyse levels of a small suite of elements (Ca, Mg, Mn, Cu, Zn, Sr, Rb, Ba and Pb) at the margin (adult phase) and core (juvenile phase) of the same otoliths of adult C. rubescens, collected at ten locations in five management zones. The elemental composition of both otolith margins and cores differed significantly among management zones and in some cases among locations within zones. Similarity of the pattern of among-zone elemental composition in otolith margins and cores indicated that, when cores are laid down, individuals have already recruited to the zones they will occupy as adults and there is no evidence of discrete juvenile nurseries. Thus, movement of juvenile or adult C. rubescens is likely to occur at relatively small spatial scales. Monitoring and management of adult stocks at the management zone level may be appropriate to sustain stocks broadly, but may not detect more localised depletion. Methods of elemental analyses are discussed and costs and benefits of this study vs an equivalent tagging study were compared.
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We test for and calibrate a proxy for ocean temperature based on the skeletal composition of the widely distributed, deep-sea gorgonians in the family Isididae (bamboo corals), through use of three complementary methods: a short-term comparison of element/Ca ratios to a four-year temperature record, a long-term comparison with oceanographic records spanning forty years, and a geographic comparison of Isidids collected at sites ranging from the tropics to Antarctica. The assays consistently support a temperature-dependency for Mg/Ca ratios and suggest S/Ca is indirectly affected by temperature, but indicate little or no effect of temperature on P/Ca and Sr/Ca. The consensus relationship between Mg/Ca and temperature for Isidid calcite from the comparisons with the temperature time-series is T = −0.505 + 0.048 Mg/Ca, where T is in °C, Mg/Ca is in mmol/mol, and the applicable range is 3–6 °C. The results of the geographic assay, though imprecise, suggest the applicable range extends to temperatures below freezing. The scatter of data points around the regression of temperature and Mg/Ca is wide in all assays. This could reflect the effect of factors other than temperature on Mg/Ca ratios, but is also likely to reflect limitations of the field data, the effects of assumed constant growth rates in the corals and instrumental analytical error. The combined effects of micro-scale variability in growth rates and wide confidence intervals for each data point suggests that environmental reconstruction from Isidid internode calcite from sparse data or at time scales less than decades be done with caution. Comparisons within and among colonies do not indicate strong vital effects on ontogenetic variability in the corals, other than possibly close to the central pore of the coral. However, similar Mg/Ca ratios for Isidids from Antarctic and more temperate regions suggest adaptation to local conditions and hence a role for physiology at higher taxonomic levels, at least. Taxonomically higher level vital effects are also suggested by large differences between gorgonian families in their regressions between Mg/Ca and temperature, by Mg/Ca ratios that overlap over a wide temperature and habitat range, and for a non-linear relationship between temperature and the slope of the Mg/Ca-temperature relationship across the order.

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Electron probe microanalysis of fish otoliths — evaluation of techniques for studying age and stock discrimination

Abstract

Comp. Biochem. Physiol. A

N.Z.J. Mar. Freshwater Res.

Mar. Ecol. Prog. Ser.

Mar. Poll. Bull.

Thalassia Jugosl.

Mar. Ecol. Prog. Ser.

J. Phys. Sci. Inst.

Mineralogy and rare earth geochemistry of apatile and xenotime from the Gloserheia granite pegmatite, Froland, Southern, Norway

Am. Mineral.

Applications of statistical methods in microanalysis

The distinction between brown trout and sea trout by the strontium content of their scales

J. Fish Biol.

Role of environment and stock on the elemental composition of sockeye salmon (Oncorhynchus nerka) vertebrae

Can. J. Fish. Aquat. Sci.

Confirmation of the longevity in Sebastes diploproa (Pisces: Scorpaenidae) from 210Pb/226Ra measurements in otoliths

Mar. Biol.

Strontium uptake in shell aragonite from the freshwater gastropod Limnaea stagnalis

Science

X-ray spectrometric and multivariate analyses of sockeye salmon (Oncorhynchus nerka) from different regions

J. Fish. Res. Bd Can.

X-ray fluorescence study of stock variation in bluefin tuna

Status report submitted to NMFS

Chemical variability and stock variation in northern Atlantic bluefin tuna

Collect. Vol. Sci. Pap. ICCAT/Recl. Doc. Sci. CICTA/Collecc. Doc. Cient. CICAA

Stock identification using X-ray spectrometry and multivariate techniques

Bull. INPFC

Radioisotope X-ray fluorescence spectrometry in aquatic biology: a review

J. Fish. Res. Bd Can.

210Pb/226Ra determination of longevity in redfish

Can. J. Fish. Aquat. Sci.

A crystallographic study of vertebrate otoliths

Biol. Bull.

Molecular structure and composition of fish otoliths

Mar. Biol.

Temperature measurements from oxygen isotope ratios of fish otoliths

Science

Trace element analysis of fish sagittae as an aid to stock identification: pink snapper (Chrysophrys auratus) in Western Australian waters

Can. J. Fish. Aquat. Sci.

Study on identification of the Pacific salmon by neutron activation analysis

Salmon stock identification based on elemental composition of vertebrae

Can. J. Fish. Aquat. Sci.

Atomic emission and proton microprobe studies of the ion content of otoliths of chinook salmon aimed at recovering the temperature life history of individuals

Comp. Biochem. Physiol. A

Scanning electron microscopy and X-ray microanalysis

Geochemistry and mineralogy of the rare earths

Electron beam X-ray microanalysis

The response of coral growth and skeletal strontium to light intensity and water temperature

Metals in fish scales collected in Lake Opeongo, Canada, from 1939 to 1979

Trans. Am. Fish. Soc.

Otolith microchemistry: validation of the effects of physiology, age and environment on otolith composition

J. Exp. Mar. Biol. Ecol.

Confirmation of the longevity in Sebastes diploproa (Pisces: Scorpaenidae) from ²¹⁰Pb/²²⁶Ra measurements in otoliths

²¹⁰Pb/²²⁶Ra determination of longevity in redfish