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Mercury isotope ratio measurements of methylmercury in fish


Species-specific Hg isotope ratio data has increasingly become an important tool in understanding biogeochemistry of mercury. Such species-specific isotope data provides information on natural sources of CH3Hg production, enables the tracing of species pathways in the environment, and supports the assessment of the potential impact on the aquatic ecosystems. In the past, large variations in the bulk isotopic composition of Hg were observed in fish. Due to the ability of CH3Hg to bioaccumulate and biomagnify in aquatic biota, species-specific Hg isotope ratio data in fish can hopefully facilitate discriminating source relating isotopic signatures from those formed during biotransformation processes including degradation.

Analysis of mercury species is most often performed by speciation analysis using a separation technique such as chromatography, electrophoresis and others combined with element selective detection techniques such as ICP-MS. For precise and accurate Hg isotope ratio determinations only multi-collector instruments can be used. While gas chromatography coupled to ICP-MS (GC-ICP-MS) is the most sensitive technique for Hg speciation analysis, it requires derivatization to convert the different Hg species into volatile ones. Also since the Hg isotope ratio should be used to trace reactions it is not a good idea to rely on reactions for derivatization for detection.

The new study:

Scientists from LGC (UK) now developed a method based on HPLC. Especially problematic is to obtain accurate isotope ratio data from transient signals created by on-line separation techniques. Many steps of optimization were necessary to obtain the required sensitivity and  accuracy. It involved developing improved, quantitative Hg species extraction incurring minimal dilution, matrix matching between the extraction and separation procedures and high capacity HPLC separation of Hg species as well as instrumental protocols to account for the aforementioned features of Hg isotopic analysis involving the use of cold vapor generation (CVG) for effective sample introduction. It turned out that offline separation produced more reliable data than on-line separation.

The analytical steps of the whole procedure are shown in figure 1.

Figure 1: Workflow of the analytical procedure for the mercury isotope ratio determination of methylmercury in fish

The chromatographic separation of Hg species was carried out under isocratic conditions using a reversed phase C18 column avoiding changing conditions that could have an influence on sensitivity. Low detection limits were obtained by using a high sample injection volume of 500 µL. Also the collected analyte fraction were preconcentrated by freeze drying and digested with a small volume (0.5 mL) of nitric acid and 0.1 ml of hydrogen peroxide. The digestion of the fractions avoided any matrix effects produced by the binding partners of mercury.

The digested fractions were measured by multi-collector ICP-MS using CVG. The authors evaluated the uncertainties of the measurements by propagating individual uncertainty components according to the ISO/GUM guide. The isotope data obtained in this study show that fractionation can occur during dissociation of CH3Hg as well as Hg° volatilisation from solution containing reduced sulphur. Lighter Hg isotopes were preferentially partitioned into reaction products in both cases.

The authors concluded that the developed method could be helpful for certifying reference materials for mercury isotopes in biological materials.

The original study:

John Entwisle, Dmitriy Malinovsky, Philip J. H. Dunn, Heidi Goenaga-Infante, Hg isotope ratio measurements of methylmercury in fish tissues using HPLC with offline cold vapour generation MC-ICPMS, J. Anal. At. Spectrom., 33/10 (2018) 1645-1654. DOI: 10.1039/c8ja00099a

Used techniques and instrumentation:

Agilent Series 1100 HPLC

Related studies (newest first)

S. Bérail, J. Cavalheiro, E. Tessier, J. P. G. Barre, Z. Pedrero, O. F. X. Donard and D. Amouroux, Determination of total Hg isotopic composition at ultra-trace levels by on line cold vapor generation and dual gold-amalgamation coupled to MC-ICP-MS, J. Anal. At. Spectrom., 32 (2017) 373–384. DOI:  10.1039/C6JA00375C

  Q. Huang, J. Chen, W. Huang, P. Fu, B. Guinot, X. Feng, L. Shang, Z. Wang, Z. Wang, S. Yuan, H. Cai, L. Wei and B. Yu, Isotopic composition for source identification of mercury in atmospheric fine particles, Atmos. Chem. Phys., 16 (2016) 11773–11786. DOI: 10.5194/acp-16-11773-2016

S. Y. Kwon, J. D. Blum, D. J. Madigan, B. A. Block and B. N. Popp, Quantifying mercury isotope dynamics in captive Pacific bluefin tuna (Thunnus orientalis), Elem. Sci. Anth., 2016, 4, 88. DOI: 10.12952/journal.elementa.000088

  M. Amde, Y. Yin, D. Zhang and J. Liu, Methods and recent advances in speciation analysis of mercury chemical species in environmental samples: a review, Chem. Speciat. Bioavailab., 28 (2016) 51–65. DOI: 10.1080/09542299.2016.1164019

V. Perrot, J. Masbou, M. V. Pastukhov, V. N. Epov, D. Point, S. Bérail, P. R. Becker, J. E. Sonke and D. Amouroux, Natural Hg isotopic composition of different Hg compounds in mammal tissues as a proxy for in vivo breakdown of toxic methylmercury, Metallomics, 2016, 8, 170–178. DOI: 10.1039/C5MT00286A

  C.C. Brombach, Z. Gajdosechova, B. Chen, A. Brownlow, W T. Corns, J. Feldmann and E. M. Krupp, Direct online HPLC-CV-AFS method for traces of methylmercury without derivatisation: a matrix-independent method for urine, sediment and biological tissue samples, Anal. Bioanal. Chem., 407 (2015) 973–981. DOI: 10.1007%2Fs00216-014-8254-1

  C. C. Brombach, B. Chen, W. T. Corns, J. Feldmann and E.M. Krupp, Methylmercury in water samples at the pg/L level by online preconcentration liquid chromatography cold vapor-atomic fluorescence spectrometry, Spectrochim. Acta, Part B, 105 (2015) 103–108. DOI: 10.1016/j.sab.2014.09.014

R. Jagtap and W. Maher, Measurement of mercury species in sediments and soils by HPLC–ICPMS, Microchem. J., 121 (2015) 65–98. DOI: 10.1016/j.microc.2015.01.010

S.Y. Kwon, J.D. Blum, C.Y. Chen, D.E. Meattey, R.P. Mason, Mercury Isotope Study of Sources and Exposure Pathways of Methylmercury in Estuarine Food Webs in the Northeastern U.S, Environ. Sci. Technol., 48/17 (2014) 10089–10097. DOI: 10.1021/es5020554

  B. A. Bessinger, Use of Stable Isotopes to Identify Sources of Mercury in Sediments: A Review and Uncertainty Analysis, Environ. Forensics, 15/3 (2014) 265–280. DOI: 10.1080/15275922.2014.930939

R. Wang, X.-B. Feng, W.-X. Wang, In Vivo Mercury Methylation and Demethylation in Freshwater Tilapia Quantified by Mercury Stable Isotopes, Environ. Sci. Technol., 47/14 (2013) 7949– 7957. DOI: 10.1021/es3043774

S.Y. Kwon, J.D. Blum, M.A. Chirby, E.J. Chesney, Application of mercury isotopes for tracing trophic transfer and internal distribution of mercury in marine fish feeding experiments, Environ. Toxicol. Chem., 32/10 (2013) 2322–2330. DOI: 10.1002/etc.2313

J. Masbou, D. Point and J.E. Sonke, Application of a selective extraction method for methylmercury compound specific stable isotope analysis (MeHg-CSIA) in biological materials, J. Anal. At. Spectrom., 28 (2013) 1620–1628. DOI:  10.1039/C3JA50185J

M. Jiménez-Moreno, V. Perrot, V. Epov, M. Monperrus and D. Amouroux, Chemical kinetic isotope fractionation of mercury during abiotic methylation of Hg(II) by methylcobalamin in aqueous chloride media, Chem. Geol., 336 (2013) 26–36. DOI: 10.1016/j.chemgeo.2012.08.029

Y. Gao, R. Liu and L. Yang, Application of chemical vapor generation in ICP-MS: A review, Chin. Sci. Bull., 2013, 58, 1980–1991. DOI: 10.1007/s11434-013-5751-0

P. Rodríguez-González, V. N. Epov, C. Pecheyran, D. Amouroux and O. F. X. Donard, Species-specific stable isotope analysis by the hyphenation of chromatographic techniques with MC-ICPMS, Mass Spectrom. Rev., 31/4 (2012) 504–512. DOI: 10.1002/mas.20352

S.Y. Kwon, J.D. Blum, M.J. Carvan, N. Basu, J.A. Head, C.P. Madenjian, S.R. David, Absence of Fractionation of Mercury Isotopes during Trophic Transfer of Methylmercury to Freshwater Fish in Captivity, Environ. Sci. Technol., 46/14 (2012) 7527–7534. DOI: 10.1021/es300794q

G.E. Gehrke, J.D. Blum, D.G. Slotton, B.K. Greenfield, Mercury Isotopes Link Mercury in San Francisco Bay Forage Fish to Surface Sediments, Environ. Sci. Technol., 45/4 (2011) 1264–1270. DOI: 10.1021/es103053y

J. E. Sonke, A global model of mass independent mercury stable isotope fractionation, Geochim. Cosmochim. Acta, 75 (2011) 4577–4590. DOI: 10.1016/j.gca.2011.05.027

D. Malinovsky and F. Vanhaecke, Mercury isotope fractionation during abiotic transmethylation reactions, Int. J. Mass Spectrom., 307 (2011) 214–224. DOI: 10.1016/j.ijms.2011.01.020

N. Estrade, J. Carignan, J. E. Sonke and O. F. X. Donard, Measuring Hg Isotopes in Bio‐Geo‐Environmental Reference Materials, Geostand. Geoanal. Res., 34 (2010) 79–93. DOI: 10.1111/j.1751-908X.2009.00040.x

L. S. Sherman, J. D. Blum, K. P. Johnson, G. J. Keeler, J. A. Barres and T. A. Douglas, Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight, Nat. Geosci., 2010, 3, 173–177. DOI: 10.1038/ngeo758

R. Yin, X. Feng and W. Shi, Application of the stable-isotope system to the study of sources and fate of Hg in the environment: A review, Appl. Geochem., 25 (2010) 1467–1477. DOI: 10.1016/j.apgeochem.2010.07.007

  D. Malinovsky, K. Latruwe, L. Moens and F. Vanhaecke, Experimental study of mass-independence of Hg isotope fractionation during photodecomposition of dissolved methylmercury, J. Anal. At. Spectrom., 25 (2010) 950–956. DOI:  10.1039/B926650J

D.B. Senn, E.J. Chesney, J.D. Blum, M.S. Bank, A. Maage, J.P. Shine, Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the Northern Gulf of Mexico, Environ. Sci. Technol., 44/5 (2010) 1630–1637. DOI: 10.1021/es902361j

V. Perrot, V.N. Epov, M.V. Pastukhov, V.I. Grebenshchikova, C. Zouiten, J.E. Sonke, S. Husted, O.F.X. Donard, D. Amouroux, Tracing Sources and Bioaccumulation of Mercury in Fish of Lake Baikal– Angara River Using Hg Isotopic Composition, Environ. Sci. Technol., 44/21 (2010) 8030–8037. DOI: 10.1021/es101898e

V. Epov, S. Berail, M. Jimenez-Moreno, V. Perrot, C. Pecheyran, D. Amouroux and O. F. X. Donard, Approach to Measure Isotopic Ratios in Species Using Multicollector-ICPMS Coupled with Chromatography, Anal. Chem., 82 (2010) 5652–5662. DOI: 10.1021/ac100648f

  B.A. Bergquist and J.D. Blum, The Odds and Evens of Mercury Isotopes: Applications of Mass-Dependent and Mass-Independent Isotope Fractionation, Elements, 5 (2009) 353–357. DOI: 10.2113/gselements.5.6.353

  A.L. Buchachenko, Mercury isotope effects in the environmental chemistry and biochemistry of mercury-containing compounds, Russ. Chem. Rev., 2009, 78, 319–328. DOI: 10.1070/RC2009v078n04ABEH003904

P. Rodriguez-Gonzalez, V.N. Epov, R. Bridou, E. Tessier, R. Guyoneaud, M. Monperrus, D. Amouroux, Species-specific stable isotope fractionation of mercury during Hg(II) methylation by an anaerobic bacteria (Desulfobulbus propionicus) under dark conditions, Environ. Sci. Technol., 43/24 (2009) 9183–9188. DOI: 10.1021/es902206j

K. Kritee, T. Barkay, J.D. Blum, Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury, Geochim. Cosmochim. Acta, 73/5 (2009) 1285–1296. DOI: 10.1016/j.gca.2008.11.038

N. Gantner, H. Hintelmann, W. Zheng, D.C. Muir,  Variations in stable isotope fractionation of Hg in food webs of arctic lakes, Environ. Sci. Technol., 43/24 (2009) 9148–9154. DOI: 10.1021/es901771r

R. Das, V.J.M. Salters, A.L. Odem, A case for in vivo mass-independent fractionation of mercury isotopes in fish, Geochem. Geophys. Geosystems, 10 (2009) Q11012. DOI: 10.1029/2009GC002617

  L. Laffont, J.E. Sonke, L. Maurice, H. Hintelmann, M. Pouilly, Y.S. Bacarreza, T. Perez and P. Behra, Anomalous Mercury Isotopic Compositions of Fish and Human Hair in the Bolivian Amazon, Environ. Sci. Technol., 43/23 (2009) 8985–8990. DOI: 10.1021/es9019518

M. Dzurko, D. Foucher and H. Hintelmann, Determination of compound-specific Hg isotope ratios from transient signals using gas chromatography coupled to multicollector inductively coupled plasma mass spectrometry (MC-ICP/MS), Anal. Bioanal. Chem., 2009, 393, 345–355. DOI: 10.1007/s00216-008-2165-y

L. Yang and R.E. Sturgeon, Isotopic fractionation of mercury induced by reduction and ethylation, Anal. Bioanal. Chem., 2009, 393, 377–385. DOI: 10.1007/s00216-008-2348-6

T.A. Jackson, D. M. Whittle, M.S. Evans and D.C.G. Muir, Evidence for mass-independent and mass-dependent fractionation of the stable isotopes of mercury by natural processes in aquatic ecosystems, Appl. Geochem., 23 (2008) 547–571. DOI: 10.1016/j.apgeochem.2007.12.013

  V. Epov, P. Rodriguez-Gonzalez, J. E. Sonke, E. Tessier, D. Amouroux, L. M. Bourgoin and O. F. X. Donard, Simultaneous Determination of Species-Specific Isotopic Composition of Hg by Gas Chromatography Coupled to Multicollector ICPMS, Anal. Chem., 80 (2008) 3530–3538. DOI: 10.1021/ac800384b

L.H. Reyes, G.M. Mizanur Rahman, T. Fahrenholz and H.M. Skip Kingston, Comparison of methods with respect to efficiencies, recoveries, and quantitation of mercury species interconversions in food demonstrated using tuna fish, Anal. Bioanal. Chem., 390 (2008) 2123–2132. DOI: 10.1007/s00216-008-1966-3

B.A. Bergquist, J.D. Blum, Mass-dependent and-independent fractionation of Hg isotopes by photoreduction in aquatic systems, Science (Washington, DC, U. S.), 318/5849 (2007) 417– 420. DOI: 10.1126/science.1148050

J.D. Blum, B.A. Bergquist, Reporting of variations in the natural isotopic composition of mercury, Anal. Bioanal. Chem., 388/2 (2007) 353–359. DOI: 10.1007/s00216-007-1236-9

  C.W. Shade and R.J.M. Hudson, Determination of MeHg in Environmental Sample Matrices Using Hg−Thiourea Complex Ion Chromatography with On-line Cold Vapor Generation and Atomic Fluorescence Spectrometric Detection, Environ. Sci. Technol., 39/13 (2005) 4974–4982. DOI: 10.1021/es0483645

R. Clough, S. T. Belt, E.H. Evans, B. Fairman and T. Catterick, Uncertainty contributions to species specific isotope dilution analysis. Part 2. Determination of methylmercury by HPLC coupled with quadrupole and multicollector ICP-MS, J. Anal. At. Spectrom., 18 (2003) 1039–1046. DOI:  10.1039/B305454N
C.F. Harrington and T. Catterick, Problems Encountered During the Development of a Method for the Speciation of Mercury and Methylmercury by High-performance LiquidChromatography Coupled to Inductively Coupled Plasma MassSpectrometry,  J. Anal. At. Spectrom., 12 (1997) 1053–1056. DOI: 10.1039/A701453H

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