Methylmercury In Fish Tissue Research Articles

Hg isotope ratio measurements of methylmercury in fish tissues using HPLC with off line cold vapour generation MC-ICPMS

Species-specific Hg isotope ratio data has increasingly become an important tool in understanding biogeochemistry of mercury.

Bioaccumulation of methylmercury in fish tissue from the Roosevelt River, Southwestern Amazon basin

Mercury is a major pollutant in the Amazon River system, and its levels in fish and human hair are usually above the limit recommended by health agencies. The objective of this study was to analyze the methylmercury (MeHg) concentration in fish tissue from the Roosevelt River. The river’s water velocity, depth, pH, temperature, electrical conductivity, dissolved oxygen and substrate type were measured, and fifty specimens distributed in 14 fish species were collected. A total of 64.3% of the sampled species were of the order Characiform and 71.4% of the species were carnivores. Fifty percent of the species had MeHg concentrations above threshold limit (Hg-T 0.5 mg kg-1) established for food by the World Health Organization. Cichla monoculus had the highest value of MeHg (2.45 mg kg-1). The MeHg concentration in fish varied according to dietary habits. The study also found bioaccumulation of MeHg in fish tissue in the following descending order: carnivorous > detritivorous > frugivore. Low significant correlations were found between fish weight or length and MeHg. Further studies on MeHg contamination are recommended in tissues of fish consumed in human riverine communities in the Roosevelt River Basin.

Anatomical mercury speciation in bay scallops by thio-bearing chelating resin concentration and GC-electron capture detector determination

The highly toxic methyl-, ethyl- and phenylmercury species that may exist in the three main anatomical parts – the adductor muscle, the mantle and the visceral mass – of bay scallops (Argopecten irradias) were quantitatively released by cupric chloride, zinc acetate, sodium chloride and hydrochloric acid (HCl) under ultrasonic extraction. After centrifugation, the mercury species in the supernatant were concentrated by thio (SH)-bearing chelating resins, eluted with HClO4 and HCl and extracted with toluene. Separation was achieved by capillary GC equipped with programmed temperatures, a constant nitrogen flow and detected by a micro-electron capture detector (μECD). Under optimised conditions, the LODs for methyl-, ethyl- and phenylmercury in bay scallop samples were 1.1, 0.65 and 0.80 ng g−1, respectively. The maximum RSD for three replicate determinations of methyl-, ethyl- and phenylmercury in bay scallop samples were 13.7%, 14.0% and 11.2%, respectively. In the concentration range of 4–200 ng g−1 in bay scallop samples, the calibration graphs were linear with correlation coefficients not less than 0.997. Recoveries for spiked samples were in the range of 92.7–103.5% (methylmercury), 87.5–108.3% (ethylmercury) and 91.6–106.0% (phenylmercury), respectively. The method was verified by the determination of methylmercury in a CRM GBW10029 (Total Mercury and Methyl Mercury in Fish Tissue), with results in good agreement with the certified values. Methylmercury – the only existing species in bay scallops – was successfully determined by the method.

Development of an Empirical Nonlinear Model for Mercury Bioaccumulation in the South and South Fork Shenandoah Rivers of Virginia

Mercury is a globally distributed pollutant that biomagnifies in aquatic food webs. In the United States, 3781 water bodies fail to meet criteria for safe fish consumption due to mercury bioaccumulation. In the risk assessment and management of these impairments (through the total maximum daily load program), an important step is evaluating the relationship between aqueous mercury and mercury in fish tissue. Often, this relationship is simplified to a bioaccumulation factor (BAF): the ratio of fish tissue mercury to aqueous mercury. This article evaluates the relationship between aqueous mercury and fish tissue mercury across a contamination gradient in the South and South Fork Shenandoah rivers of Virginia. The relationship was found to be nonlinear, with BAFs decreasing as the level of contamination increased. This means that protective water column mercury concentration targets established from site-specific BAFs will be overestimated in contaminated areas and will not be sufficiently protective. To avoid this over-prediction in the South and South Fork Shenandoah rivers, an empirical nonlinear Michaelis-Menten model was used to establish a protective water-quality target. Among other models and variables, the Michaelis-Menten model, relating total mercury in the water column to methylmercury in fish tissue, achieved the best empirical fit (r(2) = 0.9562). The resulting water-quality targets using this model were 3.8 and 3.2 ng/l for the South and South Fork Shenandoah rivers, respectively. These values are 2.1-2.5 times lower than the water-quality target developed using a site-specific BAF. These findings demonstrate the need to consider nonlinear BAF relationships in mercury-contaminated areas.

Multiple Spiking Species-Specific Isotope Dilution Analysis by Molecular Mass Spectrometry: Simultaneous Determination of Inorganic Mercury and Methylmercury in Fish Tissues

This work demonstrates, for the first time, the applicability of multiple spiking isotope dilution analysis to molecular mass spectrometry exemplified by the speciation analysis of mercury using GC(EI)MS instrumentation. A double spike isotope dilution approach using isotopically enriched mercury isotopes has been applied for the determination of inorganic mercury Hg(II) and methylmercury (MeHg) in fish reference materials. The method is based on the application of isotope pattern deconvolution for the simultaneous determination of degradation-corrected concentrations of methylmercury and inorganic mercury. Mass isotopomer distributions are employed instead of isotope ratios to calculate the corrected concentrations of the Hg species as well as the extent of species degradation reactions. The isotope pattern deconvolution equations developed here allow the calculation of the different molar fractions directly from the GC(EI)MS mass isotopomer distribution pattern and take into account possible impurities present in the spike solutions employed. The procedure has been successfully validated with the analysis of two different certified reference materials (BCR-464 and DOLT-4) and with the comparison of the results obtained by GC(ICP)MS. For the tuna fish matrix (BCR-464), no interconversion reactions were observed at the optimized conditions of open focused microwave extraction at 70 degrees C during 8 min. However, significant demethylation was found under the same conditions in the case of the certified dogfish liver DOLT-4. Methylation and demethylation factors were confirmed by GC(ICP)MS. Transformation reactions have been found to depend on the sample matrix and on the derivatization reagent employed. Thus, it is not possible to recommend optimum extraction conditions suitable for all types of matrices demonstrating the need to apply multiple spiking methodologies for the determination of MeHg and Hg(II) in biological samples. Double spike isotope dilution analysis methodologies using widespread GC(EI)MS instrumentation are proposed here for the routine analysis of inorganic mercury and methylmercury in fish samples. The estimated method detection limits were below 10 ng g(-1) for both mercury species. Precision was evaluated for the concentrations present in the certified reference materials (CRMs) which vary from 0.1 to 5 microg g(-1), achieving values of coefficients of variation ranging from 7% to 2%. The concentrations obtained in both CRMs analyzed were in excellent agreement with the certified values, demonstrating the accuracy of the method at these concentration levels.

Robust microwave-assisted extraction protocol for determination of total mercury and methylmercury in fish tissues

A rapid and efficient closed vessel microwave-assisted extraction (MAE) method based on acidic leaching was developed and optimized for the extraction of total mercury (Hg), inorganic mercury (Hg 2+) and methylmercury (CH 3Hg +) from fish tissues. The quantitative extraction of total Hg and mercury species from biological samples was achieved by using 5 mol L −1 HCl and 0.25 mol L −1 NaCl during 10 min at 60 °C. Total Hg content was determined using inductively coupled plasma mass spectrometry (ICP–MS). Mercury species were measured by liquid chromatography hyphenated with inductively coupled plasma mass spectrometry (LC–ICP–MS). The method was validated using biological certified reference materials ERM-CE464, DOLT-3, and NIST SRM-1946. The analytical results were in good agreement with the certified reference values of total Hg and CH 3Hg + at a 95% confidence level. Further, accuracy validation using speciated isotope-dilution mass spectrometry (SIDMS, as described in the EPA Method 6800) was carried out. SIDMS was also applied to study and correct for unwanted species transformation reactions during and/or after sample preparation steps. For the studied reference materials, no statistically significant transformation between mercury species was observed during the extraction and determination procedures. The proposed method was successfully applied to fish tissues with good agreement between SIDMS results and external calibration (EC) results. Interspecies transformations in fish tissues were slightly higher than certified reference materials due to differences in matrix composition. Depending on the type of fish tissue, up to 10.24% of Hg 2+ was methylated and up to 1.75% of CH 3Hg + was demethylated to Hg 2+.

Rapid determination of methylmercury in fish tissues

The aim of the present study was to develop a rapid and inexpensive method for the determination of methylmercury in fish tissues based on GC/ECD instrumentation. The new method is based on acidic digestion in hydrochloric acid and subsequent extraction with toluene. Methylmercury is determined by the GC/ECD technique using a DB-608 capillary column. The following parameters of the method were established: detection limit 13 μg/kg, limit of quantification 22 μg/kg, linearity 0.2–200 ng/ml, reproducibility 9.4%, and recovery 90%. The method was developed and verified using CRM 464 reference material and was successfully tested in inter-laboratory comparisons IMEP – 20 “Trace elements in tuna fish” organised by the Joint Research Centre – Institute for Reference Materials and Measurements (Belgium), with the success rate of E<sub>n</sub>=0.43.    

Uncertainty contributions to single and double isotope dilution mass spectrometry with HPLC-CV-MC-ICP-MS for the determination of methylmercury in fish tissue

A full uncertainty budget has been used to compare the performance of species specific single and ‘approximate matching’ double isotope dilution mass spectrometry (IDMS). Two fish tissue reference materials were employed in this study, DORM-2 (NRC, Canada) and BCR464. Methylmercury was separated from inorganic mercury by HPLC, which was coupled to multicollector ICP-MS via a cold vapour generation system for the measurement of isotope amount ratios. For each CRM the mass fraction of methylmercury determined by the two IDMS methods was not statistically different, within the limits of uncertainty, from the certified values. Double IDMS was found to be more precise than single IDMS when a full uncertainty budget was estimated, the converse applied when the method precision was estimated only by the standard deviation of replicate analysis. For single IDMS the major uncertainty contribution was derived from the standard uncertainty of each analytical replicate, Uwithin. The between blend standard uncertainty, Ubetween, was the major contributor to the expanded uncertainty for approximate matching double IDMS.

Blank correction considerations for isotope dilution and reverse isotope dilution calibration: Determination of methylmercury in fish tissue

A mathematical approach to the accurate correction of the blank when applying isotope dilution (ID) and reverse ID is presented. The manner in which blank correction is undertaken is critical to the quality of the final results. Direct subtraction of a procedural blank from the gross analyte concentration is only valid when the blank contributes to the primary ID process and not to the reverse ID process. When the blank contributes to both processes, typically only a fraction of this blank concentration should be subtracted. The approach developed here was illustrated and validated by the determination of MeHg in tuna fish using ID and reverse ID SPME GC-ICP-MS and an enriched Me198Hg spike. Despite a 150-fold higher blank (equivalent to 7% of the analyte concentration in the sample) arising from use of a 1 M NaOAc/HOAc buffer solution compared to that obtained with use of a 0.5 M NH4OAc/HOAc buffer, final concentrations of 19.90 ± 0.34 and 19.88 ± 0.10 μmol kg−1 (one standard deviation, n = 3) respectively, were derived. These data are in good agreement with the assigned value of 19.91 ± 0.82 μmol kg−1 (as 95% confidence interval, derived from an international intercomparison exercise). The methodology was also applied to the determination of a 50-fold lower concentration of MeHg in a salmon fish. A method detection limit (3SD) of 0.9 nmol kg−1 based on processing of a 0.40 g subsample was obtained.

Application of isotope dilution to the determination of methylmercury in fish tissue by solid-phase microextraction gas chromatography–mass spectrometry

Species-specific isotope dilution (ID) calibration using solid-phase microextraction (SPME) in combination with gas chromatography–mass spectrometry (GC–MS) for separation and detection of methylmercury (MeHg) in fish tissue is described. Samples were digested with methanolic potassium hydroxide. Analytes were propylated and headspace sampled with a polydimethylsiloxane-coated SPME fused-silica fiber. ID analysis was performed using a laboratory-synthesized 198Hg-enriched methylmercury (Me 198Hg) spike. Using selective ion monitoring (SIM) mode, the intensities of Me 202HgPr + at m/ z 260 and Me 198HgPr + at m/ z 256 were used to calculate the m/ z ratio at 260/256, which was used to quantify MeHg in NRCC CRM DORM-2 fish tissue. A MeHg concentration of 4.336±0.091 μg g −1 (one standard deviation, n=4) as Hg was obtained in DORM-2, in good agreement with the certified value of 4.47±0.32 μg g −1 (95% confidence interval). A concentration of 4.58±0.31 μg g −1 was determined by standard additions calibration using ethylmercury (EtHg) as an internal standard. The three-fold improvement in the precision of measured MeHg concentrations using ID highlights its superiority in providing more precise results compared to the method of standard additions. A method detection limit (3 S.D.) of 0.037 μg g −1 was estimated based on a 0.25 g subsample of DORM-2.

Determination of methylmercury in fish tissues by isotope dilution SPME-GC-ICP-MS

A method is described for the accurate and precise determination of monomethylmercury (MMHg) by species specific isotope dilution (ID) calibration using solid phase microextraction (SPME) in combination with gas chromatographic (GC) separation and inductively coupled plasma mass spectrometric (ICP-MS) detection. Samples were digested with methanolic potassium hydroxide, derivatized in aqueous solution with sodium tetrapropylborate and headspace sampled with a polydimethylsiloxane coated SPME fused silica fiber. The analyte was then directly transferred from the fiber to the head of the GC column for desorption by insertion of the fiber through the heated injection port. Reverse spike ID analysis was performed to determine the accurate concentration of an in-house synthesized 198Hg-enriched monomethylmercury (MM198Hg) spike [candidate Certified Reference Material (CRM) EOM-1] using two natural abundance MMHg standards. Concentrations of 0.719 ± 0.015 and 4.484 ± 0.029 µg g−1 (one standard deviation, n = 4) as Hg were obtained for MMHg in NRCC CRMs DOLT-2 and DORM-2, respectively, using the present method. These are in good agreement with the certified values of 0.693 ± 0.053 and 4.47 ± 0.32 µg g−1 (as 95% confidence interval). A MMHg concentration of 4.69 ± 0.12 µg g−1 (one standard deviation, n = 4) as Hg in DORM-2 was subsequently determined by standard additions calibration using ethylmercury (EtHg) as an internal standard. A nearly 4-fold improvement in the precision of determination using ID was obtained, clearly demonstrating its superiority in providing more precise results compared to the method of standard additions. The method was applied to the determination of MMHg in a new biological CRM DOLT-3 and a concentration of 1.540 ± 0.025 µg g−1 (one standard deviation, n = 6) as Hg was obtained. A method detection limit (3σ) of 2.1 ng g−1 was estimated for a 0.25 g of subsample, sufficiently low for the routine determination of MMHg in most biological samples.

Measurement of inorganic and methylmercury in fish tissues by enzymatic hydrolysis and HPLC-ICP-MS

The measurement of mercury(II) and methylmercury(I) in fish muscle tissues after protease type XIV extraction by HPLC-ICP-MS is described. A SphereClone 5 μm ODS2 80A PEEK column (100 mm × 4.6 mm, Phenomenex, USA) and a 5% v/v CH3OH–water mobile phase containing 0.06 M CH3COONH4 and 0.1% w/v cysteine, pH 6.8 (flow rate, 1.0 ml min−1; temperature, 25 °C) was used for the separation of mercury species. The addition of cysteine during incubation with protease and HPLC-ICP-MS separation was essential to eliminate adsorption, peak tailing and memory problems. The recovery of mercury from two reference materials (NRCC DORM-2 Dogfish and NIST RM 50 Albacore tuna) and sample fish tissues was 92–107%. The calibration curves for mercury(II) and methylmercury standards were linear in the range of 0.5–20 µg L−1 (r2 = 0.9992, r2 = 0.9999, respectively). The lowest measurable mercury was 0.5 µg l−1 and this corresponds to 0.025 µg g−1 in fish tissues. Methylmercury measured in NIST RM 50 (0.87 ± 0.03 µg g−1) was similar to that reported in the literature (0.89–0.91 µg g−1). The methylmercury concentration measured in DORM 2, when results are corrected for the mercury extracted during incubation with the enzyme, was similar (4.4 ± 0.1 µg g−1) to the certified methylmercury concentration (4.47 ± 0.32 µg g−1). Most of the mercury in fish tissues is present as methylmercury (88–100%).

Determination of methylmercury in fish tissue by gas chromatography with microwave-induced plasma atomic emission spectrometry after derivatization with sodium tetraphenylborate.

The detection of methylmercury species (MeHg) in fish tissue was investigated. Samples were digested with KOH-methanol and acidified prior to extraction with methylene chloride. MeHg was back-extracted from the organic phase into water. An aliquot of this aqueous solution (buffered to pH 5) was subjected to derivatization with sodium tetraphenylborate (NaBPh4) and then extracted with toluene. The organic phase containing MePhHg was injected into a gas chromatograph (GC) which is on-line with a microwave-induced plasma atomic emission spectrometer (MIP-AED). The quantification limit was about 0.6 microg/g and 0.1 microg/g of MeHg (as Hg) for 0.08 g of freeze-dried fish powder and 0.5 g of fresh samples, respectively. Two certified reference materials, CRM 464 (tuna fish) from Community Bureau of Reference-BCR and DORM-2 (dogfish muscle) from National Research Council Canada-NRC were selected for checking the accuracy of the method. This methodology was applied to the determination of MeHg in some kinds of fish from the Carmo river with alluvial gold recovery activities ("garimpos") in Mariana, Minas Gerais, Brazil.

Glow discharge atomic emission spectrometry as a detector in gas chromatography for mercury speciation

The analytical potential of radiofrequency (rf) and direct current (dc) glow discharges (GD), with atomic emission spectrometry (AES), as detectors in capillary gas chromatography (GC) is investigated for the speciation of low levels of mercury (methylmercury, ethylmercury and inorganic mercury). The design of the interface between the GC and the GDs is discussed and the effect of the operating parameters on the analyte signals is studied for both types of GDs. The figures of merit obtained with both GC–GD–AES systems (rf and dc) are presented. The detection limits, for 1 µl sample injections, were found to be 1.3, 1.3 and 3.0 ng ml–1 of methylmercury, ethylmercury and inorganic mercury, respectively. The reproducibility was below ±7% for the three species. The coupling of GC to dc-GD-AES was applied successfully to the determination of methylmercury in fish tissue certified reference material after extraction with sodium diethyldithiocarbamate and Grignard derivatization.

Methylmercury in hair of fishermen from Turkish coasts.

Environmental methylmercury mainly arises from the methylation of inorganic mercury. The change in speciation of mercury from inorganic to methylated forms is the first step in the aquatic bioaccumulation processes. The bioconcentration factor of methylmercury in fish tissue to that in water is usually between 10{sup 4} and 10{sup 5}. Among seafood, fish products are the main source of methylmercury absorbed by men from the environment. Since Minimata epidemic health injuries and deaths in relation to mercury pollution, environmental and biological monitoring of inorganic and organic mercury species has gained importance through out the world and many reports have been published on the health effects and biological monitoring of mercury compounds including some Mediterranean countries. This study focuses on methylmercury in hair of fisherman living in different geographical Turkish coasts and relationship to eating fish habit. 17 refs., 1 fig., 2 tabs.

Methylmercury in hair of fishermen from turkish coasts

Environmental methylmercury mainly arises from the methylation of inorganic mercury. The change in speciation of mercury from inorganic to methylated forms is the first step in the aquatic bioaccumulation processes. The bioconcentration factor of methylmercury in fish tissue to that in water is usually between 10{sup 4} and 10{sup 5}. Among seafood, fish products are the main source of methylmercury absorbed by men from the environment. Since Minimata epidemic health injuries and deaths in relation to mercury pollution, environmental and biological monitoring of inorganic and organic mercury species has gained importance through out the world and many reports have been published on the health effects and biological monitoring of mercury compounds including some Mediterranean countries. This study focuses on methylmercury in hair of fisherman living in different geographical Turkish coasts and relationship to eating fish habit. 17 refs., 1 fig., 2 tabs.

Determination of methylmercury in fish samples using GC/AA and sodium tetraethylborate derivatization.

A simple technique is described for the rapid determination of methylmercury in fish tissue. Following simple dissolution in methanolic KOH solution, aqueous phase ethylation by derivatization with NaB(C2H5)4, cryogenic trapping on a packed chromatographic column, and GC separation, volatile mercury species are detected by atomic absorption spectrometry. Absolute detection limits are 4 pg of Hg for CH3Hg+ and 75 pg of Hg for labile Hg2+. Concentration detection limits for this optimized procedure are 4 ng of Hg for CH3Hg+ and 75 ng of Hg for labile Hg2+ per gram of pulverized dried fish tissue. Analysis of standard reference materials demonstrates the accuracy, precision, and reproducibility of the analytical method.

Impacts of acid rainon aquatic birds

Studies of toxicological and ecological effects of acidification on aquatic birds in Europe and North America are reviewed. Heavy metals are deposited by acid emissions, which also increase solubility and mobility of heavy metals in soil and water. Aluminium is leached from soil and mobilized from lake sediments under acid conditions; it removes susceptible fish and invertebrate species and contaminates remaining invertebrates. It is not highly toxic to birds, but may interfere with their regulation of calcium and phosphorus. Mercury is concentrated as methylmercury in fish tissues, and tends to be biomagnified in aquatic food chains. Experimental studies have demonstrated negative effects on reproduction of birds, and wild Common Loons Gavia immer breed less successfully in territories contaminated by mercury.The clearest demonstrable effect of acidification on aquatic birds is the disruption of their food chains. The loss of invertebrates and fish affects both the food-webs and the predators and competitors of aquatic birds. Cyprinid fish are important food resources for fish-eating birds, in Europe as well as North America, and are particularly sensitive to acidification. Fish-eating waterfowl in Ontario are scarcer, and breed less successfully, in areas of high acidic deposition. Experimental studies of imprinted young Black Duck Anas rubripes showed that they grew more slowly on acidic lakes, apparently due to competition from acid-tolerant fish for a reduced invertebrate resource. Negative effects of acidified habitats on growth and reproduction, again through depletion of the food-web, have also been demonstrated in field studies of Tree Swallows Tachycineta bicolor and European Dippers Cinclus cinclus.

Spectrophotometric determination of methylmercury in fish tissue with dithizone using a dual-wavelength procedure.

A dithizone spectrophotometric procedure is described for the measurement of trace concentrations of methylmercury salts. The application of a simple equation using absorbance measurements taken at two wavelengths cancels out small differences in excess of dithizone arising between blank and sample, thus ensuring good precision in the range 0.1–4.0 µg ml–1.The developed method is used in combination with the Westoo extraction procedure to determine methylmercury concentrations in fish tissue. A crab meat sample contained less than 0.04 µg g–1, and values for eight tuna fish ranged from 0.08 to 0.41 µg g–1.

Molecular activation analysis for methylmercury compounds

The use of neutron activation analysis for the selective determination of mercury in various mercury compounds is described as well as the application of this technique to the determination of methylmercury in fish tissue.