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Lipid-free tuna muscle samples are suitable for total mercury analysis

https://doi.org/10.1016/j.marenvres.2021.105385Get rights and content

Highlights

  • Scarcity of tuna samples makes essential to get the most out of a single sample.

  • Dichloromethane is more efficient than cyclohexane to extract lipids.

  • Dichloromethane extraction has no effect on Hg levels.

  • Bulk and lipid-free tropical tuna samples can be used jointly to infer Hg levels.

Abstract

Tropical tunas are largely consumed worldwide, providing major nutritional benefits to humans, but also representing the main exposure to methylmercury, a potent neurotoxin that biomagnifies along food webs. The combination of ecological tracers (nitrogen and carbon stable isotopes, δ15N and δ13C) to mercury concentrations in tunas is scarce yet crucial to better characterize the influence of tuna foraging ecology on mercury exposure and bioaccumulation. Given the difficulties to get modern and historical tuna samples, analyses have to be done on available and unique samples. However, δ13C values are often analysed on lipid-free samples to avoid bias related to lipid content. While lipid extraction with non-polar solvents is known to have no effect on δ15N values, its impact on mercury concentrations is still unclear. We used white muscle tissues of three tropical tuna species to evaluate the efficiency and repeatability of different lipid extraction protocols commonly used in δ13C and δ15N analysis. Dichloromethane was more efficient than cyclohexane in extracting lipids in tuna muscle, while the automated method appeared more efficient but as repeatable as the manual method. Lipid extraction with dichloromethane had no effect on mercury concentrations. This may result from i) the affinity of methylmercury to proteins in tuna flesh, ii) the low lipid content in tropical tuna muscle samples, and iii) the non-polar nature of dichloromethane. Our study suggests that lipid-free samples, usually prepared for tropical tuna foraging ecology research, can be used equivalently to bulk samples to document in parallel mercury concentrations at a global scale.

Introduction

Mercury (Hg) is a widespread heavy metal of particular concern to wildlife and human health. In oceans, it is naturally converted into methylmercury (MeHg), its organic and highest neurotoxic form, well known for its persistence and unique bioaccumulation properties in food webs (Hintelmann, 2010). Consumption of contaminated seafood is considered as the main route of human exposure to MeHg. Top predators like tunas are known to display relatively high MeHg concentrations, sometimes exceeding food safety guidelines (1 μg g−1 fresh tissue) (WHO and UNEP Chemicals, 2008) depending on the oceanic basin and the tuna species. Yet, tunas are also among the most popular marine species consumed worldwide, particularly tropical species that account for more than 90% of the global tuna fishery (FAO, 2018). In terms of food and nutrition security, they provide a major source of proteins, essential fatty acids, vitamins and minerals (Di Bella et al., 2015; Sirot et al., 2012).

Knowing their economic importance, nutritional benefits and potential impact on human health, tropical tunas have been studied broadly but at relatively small spatial scale to document their Hg exposure and characterize the processes driving Hg biomagnification along food webs (Bodin et al., 2017; Chouvelon et al., 2017; Houssard et al., 2019; Kojadinovic et al., 2006; Nicklisch et al., 2017). Complex regional interplay between physical (e.g. light intensity), geochemical (e.g. redox status), physiological (e.g. organism's length and age), and ecological factors (e.g. tuna's foraging depth) have been identified to govern Hg concentrations in these top predators (Choy et al., 2009; Houssard et al., 2019; Kojadinovic et al., 2006; Médieu et al., 2021; Wang et al., 2019). Nevertheless, key global aspects remain poorly understood, in particular biogeochemical methylation/demethylation mechanisms controlling MeHg bioavailability at the base of the food web, as well as factors driving both fate and accumulation through the food web. Global studies combining Hg concentrations and ecological tracers are therefore needed to clarify these points, especially in the context of the UNEP Minamata Convention since monitoring studies in marine biota have become essential the better characterize Hg cycle and fate in oceans.

Pelagic food web structuration and functioning have been broadly investigated, mainly through the use of carbon and nitrogen stable isotopes data (δ13C and δ15N values) (Fry, 2006). Recently, collaborative and global studies relying upon δ13C and δ15N values enabled identifying broad-scale patterns of trophic structure, movements and trophodynamics of tunas in relation to environmental conditions (Logan et al., 2020; Lorrain et al., 2020; Pethybridge et al., 2018). Based on individual records with associated metadata (i.e. fish length, fishing date and position) (Bodin et al., 2020), these collaborative and global studies also represent a gold mine of already collected and preserved samples to characterize spatial and/or temporal Hg trends in tunas.

An issue with global modern and historical datasets is that samples from different laboratories are not always processed the same way. To account for the influence of lipids on δ13C values (DeNiro and Epstein, 1977) while making a single analysis for both δ13C and δ15N values, δ13C values are either produced from i) bulk tissue and a mathematical correction (Sardenne et al., 2015), or from ii) lipid-free tissue, with lipids removed through various methods and solvents selected not to alter δ15N values. In the latter case, manual or automated (high temperature and pressure) methods are generally applied with solvents of low polarity such as dichloromethane and cyclohexane (Bodin et al., 2009; Ménard et al., 2007). While these methods do not affect δ15N values and provide valuable data on lipid content, nothing is known regarding their effect on Hg content, restricting the development of global studies on tuna Hg concentration with preserved samples prepared for tuna foraging ecology research.

Here, we investigated i) the efficiency and repeatability of two common lipid extraction methods (manual and automated) and two neutral solvents (dichloromethane and cyclohexane) on lipid content determination (experiment A), and ii) the influence of the most efficient solvent for lipid extraction on total Hg concentrations (experiment B). Experiments were carried out in three tropical tuna species, i.e. bigeye (Thunnus obesus), yellowfin (T. albacares) and skipjack (Katsuwonus pelamis), and on white muscle tissues, the commonly used tissue in studies investigating both trophic ecology and Hg bioaccumulation and the final storage for MeHg in fish. .

Section snippets

Sample collection

All bigeye, yellowfin, and skipjack tuna samples (ntotal = 33) were collected in the western Indian Ocean during the unloading of commercial vessels (purse seine) at Victoria port (Seychelles). To test for efficiency and repeatability of different lipid extraction protocols (experiment A), we used three individuals, one per tuna species (nexp A = 3). To test for the effect of lipid extraction on Hg concentrations (experiment B), we used 10 other individuals per tuna species (nexp B = 30), all

Efficiency and repeatability of lipid extraction methods and solvents

TLC values measured in white muscle varied between 0.4 and 1.7% dw (Supplementary Information Appendix S1). These values are similar to levels reported in same tropical tuna species from the Indian (~3%, Sardenne et al., 2016), Atlantic (~6%, Sardenne et al., 2019) and Pacific (~2%, Peng et al., 2013) Oceans, which confirms tropical tunas are lean species.

For the three tropical tuna species, TLC values were significantly higher when lipids were extracted with dichloromethane than with

Conclusion

This study reveals the higher efficiency of dichloromethane compared to cyclohexane to extract lipids in tropical tuna white muscle tissue, which may be attributed to its slightly higher polarity index. Automated method appeared more efficient than manual method to extract lipids, especially when used with dichloromethane, which may be attributed to the combined conditions of elevated temperature and pressure. Repeatability of all experiments were in acceptable ranges considering that we were

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the fishermen and crews of the SAPMER fishing company who gave the tuna samples. We are grateful to Jean-Marie Munaron from LEMAR (Plouzané, France) for his help with lipid extractions and the access to his lab. We thank Laure Laffont from GET (Toulouse, France) for mercury analyses.

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