Determination of mercury in fish otoliths by cold vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS)

Abstract

A method based on cold vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS) has been developed for determination of inorganic mercury, Hg(II), and total mercury in fish otoliths. Sodium borohydride (NaBH₄) was used as the only reducing agent and its concentration was optimized across an acidity gradient to selectively reduce Hg(II) without affecting methylmercury, CH₃Hg(I). Inorganic Hg was quantitatively reduced to elemental mercury (Hg⁰) with 1 × 10⁻⁴% (m/v) NaBH₄. CH₃Hg(I) required a minimum of 0.5% (m/v) NaBH₄ for complete reduction. Increasing the HCl concentration of solution to 5% (v/v) improved the selectivity toward Hg(II) as it decreased the signals from CH₃Hg(I) to baseline levels. Potassium ferricyanide solution was the most effective in eliminating the memory effects of Hg compared with a number of chelating and oxidizing agents, including EDTA, gold chloride, thiourea, cerium ammonium nitrate and 2-mercaptoethylamine chloride. The relative standard deviation (RSD) was less than 5% for 1.0 μg L⁻¹ Hg(II) solution. The detection limits were 4.2 and 6.4 ng L⁻¹ (ppt) for Hg(II) and total Hg, respectively. Sample dissolution conditions and recoveries were examined with ultra-pure CaCO₃ (99.99%) spiked with Hg(II) and CH₃HgCl. Methylmercury was stable when dissolution was performed with up to 20% (v/v) HCl at 100 °C. Recoveries from spiked solutions were higher than 95% for both Hg(II) and CH₃Hg(I). The method was applied to the determination of Hg(II) and total Hg concentrations in the otoliths of red emperor (CRM 22) and Pacific halibut. Total Hg concentration in the otoliths was 0.038 ± 0.004 μg g⁻¹ for the red emperor and 0.021 ± 0.003 μg g⁻¹ for the Pacific halibut. Inorganic Hg accounted for about 25% of total Hg indicating that Hg in the otoliths was predominantly organic mercury (e.g., methylmercury). However, as opposed to the bioaccumulation in tissues, methylmercury levels in otoliths was very low suggesting a different route of uptake, most likely through the deposition of methylmercury available in the water.

Introduction

Fish otoliths are calcium carbonate minerals (as aragonite) in the head of fish that aid in balance and hearing to the fish [1], [2]. These aragonite minerals grow throughout the life of fish by deposition of calcium carbonate in concentric layers on a proteinaceous matrix. In the meantime, trace elements from the surrounding water successively incorporate into the newly forming aragonite layer. The aragonite polymorph is not susceptible to resorption. Therefore, the temporal concentrations of the trace elements (so-called fingerprints) remain unchanged throughout the fish's lifetime, and consequently integrate over the fish's life history when a whole is dissolved [1], [2], [3], [4], [5], [6], [7].

Trace elements and heavy metals make up less 1% (by mass) of an otolith. With the exception of Sr, their concentrations range from low ng g⁻¹ to a few μg g⁻¹ [3], [8], [9], [10], [11], [12], [13], [14]. The utilization of trace elements as robust biological tags in otolith micro-chemical analysis has been largely due to the use of inductively coupled plasma mass spectrometry (ICP-MS) as a highly sensitive tool. Nevertheless, accurate determination of most trace elements and heavy metals, with the exception of relatively abundant Mg, Cu, Mn and Zn, from the otoliths is still a challenging task by direct analysis [3], [4], [6], [8], [9], [10]. Various analytical methods, including isotope dilution [4], [15], solvent extraction [16], solid phase extraction [6], [17], [18], [19], [20], co-precipitation [21] and hydride generation [22] have been developed to overcome the difficulties associated with low elemental concentrations in a highly saline calcium matrix.

Mercury (Hg) in the aquatic ecosystems mainly originates from the deposition of atmospheric Hg released from the anthropogenic activities [23], [24], [25]. Inorganic Hg in water is converted by bacteria to highly toxic methylmercury that accumulates in the sediments [23], [25], [26], [27]. While microorganisms, such as phytoplankton and zooplankton ingest methylmercury (CH₃Hg) from water, dietary uptake is the major route of exposure of fish to methylmercury [25], [28], [29], [30]. It is now well-documented that most Hg in fish tissue is in the form of methylmercury, although total body burden could vary with geological, biological and physiological differences among species [31], [32], [33].

The concentrations for a number of minor and major elements (Al, Na, Cl, Sr, Ca, Si) in bluefin tuna otoliths were reported to vary with total Hg body burden [34]. Further, laboratory exposures conducted with different fish indicate that the uptake of Hg into otoliths is related with its concentration in the water [35]. To date, however, Hg has not been considered as a biological tracer in otolith microchemistry, which is due in part to measurement difficulties and relatively low sensitivity of solution-based ICP-MS to this element. Thus, there is no information about the chemical forms of Hg in fish otoliths and whether otolith Hg could aid in population studies or not.

In this study, we have developed a cold vapor generation method for determination of Hg(II) and total Hg in otoliths by ICP-MS in an attempt to elucidate the chemical forms of Hg in fish otoliths, and to provide an insight about its source and utility in otolith microchemistry. Sodium borohydride (NaBH₄) was used as reducing agent to discriminate between the Hg(II) and total Hg levels. Studies were performed with a number of chelating and oxidizing reagents to eliminate the memory effects. Effects of acid dissolution on the stability and recoveries of Hg species were examined by spiking Hg(II) and CH₃HgCl to ultra-pure calcium carbonate. The method was applied to the determination of Hg(II) and total Hg in otolith samples from two different oceanic fish species, red emperor and Pacific halibut.