Development of a Common Procedure for the Determination of Methylmercury, Ethylmercury, and Inorganic Mercury in Human Whole Blood, Hair, and Urine by Triple Spike Species-Specific Isotope Dilution Mass Spectrometry.

We report the first common methodology for the simultaneous determination of methylmercury (MeHg), ethylmercury (EtHg), and inorganic mercury (Hg(II)) in human blood hair and urine. With the exception of the initial sample mass (0.15 g for blood, 0.5 g for urine, and 0.1 g for hair), the same sample preparation and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS) measurement conditions are employed for the three matrixes providing experimental values in agreement with the certified values in the analysis of NIST SRM 955c (Caprine Blood) Level 3 and the certified human hairs IAEA 085 and IAEA 086. Also, the method provides quantitative recoveries for the three Hg species in the analysis of fortified human urine samples at 1, 2, and 5 ng Hg g-1. Mercury species concentrations for levels 2 and 4 of SRM 955c are reported here for the first time. A systematic interconversion of EtHg into Hg(II) was obtained for all matrixes reaching values up to 95% in blood, 29% in hair, and 11% in urine. MeHg dealkylation was also observed in a lesser extent in blood and hair analyses, but it was not observed when analyzing urine samples. Hg methylation was not observed in any matrix. The amount of NaBPr4 added for derivatization has been found to be the main factor responsible for Hg species interconversion. This work demonstrates for the first time that experimental conditions optimized for SRM 955c (caprine blood) are not valid for human blood samples as the optimum initial sample amount for a real sample is more than 3 times lower than that for SRM 955c.

Human biomonitoring reference values for metals and trace elements in blood and urine derived from the Canadian Health Measures Survey 2007-2013.

Human biomonitoring reference values are statistical estimates that indicate the upper margin of background exposure to a given chemical at a given time. Nationally representative human biomonitoring data on 176 chemicals, including several metals and trace elements, are available in Canada from 2007 to 2013 through the Canadian Health Measures Survey (CHMS). In this work, we used a systematic approach based on the reference interval concept proposed by the International Federation of Clinical Chemistry and Laboratory Medicine and the International Union of Pure and Applied Chemistry to derive reference values (RV95s) for metals and trace elements. These RV95s were derived for blood and urine matrices in the general Canadian population based on the latest biomonitoring data from the CHMS. Biomarkers were chosen based on specific selection criteria, including widespread detection in Canadians (≥66% detection rate). Reference populations were created for each biomarker by applying appropriate exclusion criteria. Age and sex were evaluated as possible partitioning criteria and separate RV95s were derived for the sub-populations in cases where partitioning was deemed necessary. The RV95s for metals and trace elements in blood ranged from 0.18µg/L for cadmium in young children aged 3-5 years to 7900µg/L for zinc in males aged 20-79 years. In the case of urinary biomarkers, the RV95s ranged from 0.17µg/L for antimony in the total population aged 3-79 years to 1400mg/L for fluoride in adults aged 20-79 years. These RV95s represent the first set of reference values for metals and trace elements in the general Canadian population. We compare the RV95s from other countries where available and discuss factors that could influence such comparisons.

Critical perspectives on mercury toxicity reference values for protection of fish.

Environmental management decisions at mercury-contaminated sediment sites are predicated on the understanding of risks to various receptors, including fish. Toxicity reference values (TRVs) for interpreting risks to fish have been developed to assess mercury concentrations in fish or fish prey. These TRVs were systematically evaluated based on several lines of evidence. First, their conceptual basis and specific derivation were evaluated, including a close review of underlying toxicity studies. Second, case studies were reviewed to investigate whether TRVs are predictive of effects on fish populations in the field. Third, TRVs were compared with available information regarding preindustrial and present-day background concentrations of mercury in fish. The findings show that existing TRVs are highly uncertain, because they were developed using limited data from studies not designed for TRV derivation. Although field studies also entail uncertainty, several case studies indicate no evidence of adverse effects despite mercury exposures that exceed the available TRVs. Some TRVs also fall within the range of background mercury concentrations in predatory or prey fish. Lack of information on the selenium status of mercury-exposed fish is a critical confounding factor, and the form of methylmercury used in toxicity testing may also contribute to differences between TRV-based predictions and field observations of mercury effects on fish. On balance, the available information indicates that several of the TRVs reviewed are lower than necessary to protect fish populations. The 20% effect concentration from a previously published dose-response analysis appears closer to an effect threshold, based on available laboratory data. Additional research is needed to provide a stronger basis to establish dose-response relationships for mercury effects on fish.

Determination of methylmercury in marine sediment samples: method validation and occurrence data.

The determination of methylmercury (MeHg) in sediment samples is a difficult task due to the extremely low MeHg/THg (total mercury) ratio and species interconversion. Here, we present the method validation of a cost-effective fit-for-purpose analytical procedure for the measurement of MeHg in sediments, which is based on aqueous phase ethylation, followed by purge and trap and hyphenated gas chromatography-pyrolysis-atomic fluorescence spectrometry (GC-Py-AFS) separation and detection. Four different extraction techniques, namely acid and alkaline leaching followed by solvent extraction and evaporation, microwave-assisted extraction with 2-mercaptoethanol, and acid leaching, solvent extraction and back extraction into sodium thiosulfate, were examined regarding their potential to selectively extract MeHg from estuarine sediment IAEA-405 certified reference material (CRM). The procedure based on acid leaching with HNO3/CuSO4, solvent extraction and back extraction into Na2S2O3 yielded the highest extraction recovery, i.e., 94±3% and offered the possibility to perform the extraction of a large number of samples in a short time, by eliminating the evaporation step. The artifact formation of MeHg was evaluated by high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS), using isotopically enriched Me(201)Hg and (202)Hg and it was found to be nonexistent. A full validation approach in line with ISO 17025 and Eurachem guidelines was followed. With this in mind, blanks, selectivity, working range (1-800 pg), linearity (0.9995), recovery (94-96%), repeatability (3%), intermediate precision (4%), limit of detection (0.45 pg) and limit of quantification (0.85 pg) were systematically assessed with CRM IAEA-405. The uncertainty budget was calculated and the major contribution to the combined uncertainty (16.24%, k=2) was found to arise from the uncertainty associated with recovery (74.1%). Demonstration of traceability of measurement results is also presented. The validated measurement procedure was applied to the determination of MeHg incurred in sediments from a highly polluted and scarcely studied area in the Caribbean region.

[Fish and seafood as a source of human exposure to methylmercury]. / Ryby i owoce morza jako zródlo narazenia czlowieka na metylortec.

Fish and seafood are recommended diet constituents providing high quality protein, vitamins, minerals and omega-3 fatty acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, these foodstuffs can also be the major source ofmethylmercury intake in humans. In general, more than 90% of the mercury in fish is found as methylmercury, but contents of methylmercury can vary considerably between species. Predatory species that are at the top of the food chain and live a long time, may accumulate higher levels of methylmercury. This paper contains information about sources of human exposure to organic compounds of mercury, toxicity, metabolism and transformation of mercury in the environment. Assessment of methylmercury by international risk assessment bodies such as the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and U.S. National Research Council (NRC) were presented. Climate changes and their influence on the mercury cycle in the environment especially mercury methylation and concentrations of methylmercury in marine species were also presented. Consumer advice prepared by European Commission and Member States as regards consumption of predatory fishes such as swordfish, tuna, shark, marlin and pike, taking into account the most vulnerable groups of population e.g. women planning pregnancy, pregnant or breastfeeding women and children were presented. Mercury and methylmercury contamination of fishes and seafood on the basis of the literature references as well as intake of mercury with fish and fish products in Poland and other European country were discussed. The role of selenium as a factor which counteracts methylmercury toxicity and protects against some neurological effects of methylmercury exposure in humans, as well as information on potential etiological factors connected with autism disorder were also described. Attention has also been drawn to increasing number of notifications to Rapid Alert System for Food and Feed (RASFF) concerning the contamination of fish and fish products with total mercury. European and national regulations concerning maximum permissible levels of mercury in food were also presented. Possibility of selection of different fish and seafood species, taking into account low methylmercury contamination and high contents of omega-3 fatty acids e.g. sardine, mackerel, anchovy, salmon, periwinkle, have been discussed.

A physiologically based toxicokinetic model for methylmercury in female American kestrels.

A physiologically based toxicokinetic (PBTK) model was developed to describe the uptake, distribution, and elimination of methylmercury (CH(3)Hg) in female American kestrels. The model consists of six tissue compartments corresponding to the brain, liver, kidney, gut, red blood cells, and remaining carcass. Additional compartments describe the elimination of CH(3)Hg to eggs and growing feathers. Dietary uptake of CH(3)Hg was modeled as a diffusion-limited process, and the distribution of CH(3)Hg among compartments was assumed to be mediated by the flow of blood plasma. To the extent possible, model parameters were developed using information from American kestrels. Additional parameters were based on measured values for closely related species and allometric relationships for birds. The model was calibrated using data from dietary dosing studies with American kestrels. Good agreement between model simulations and measured CH(3)Hg concentrations in blood and tissues during the loading phase of these studies was obtained by fitting model parameters that control dietary uptake of CH(3)Hg and possible hepatic demethylation. Modeled results tended to underestimate the observed effect of egg production on circulating levels of CH(3)Hg. In general, however, simulations were consistent with observed patterns of CH(3)Hg uptake and elimination in birds, including the dominant role of feather molt. This model could be used to extrapolate CH(3)Hg kinetics from American kestrels to other bird species by appropriate reassignment of parameter values. Alternatively, when combined with a bioenergetics-based description, the model could be used to simulate CH(3)Hg kinetics in a long-term environmental exposure.

Certification of methylmercury in cod fish tissue certified reference material by species-specific isotope dilution mass spectrometric analysis.

A new cod fish tissue certified reference material, NMIJ CRM 7402-a, for methylmercury analysis was certified by the National Metrological Institute of Japan in the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). Cod fish was collected from the sea close to Japan. The cod muscle was powdered by freeze-pulverization and was placed into 600 glass bottles (10 g each), which were sterilized with gamma-ray irradiation. The certification was carried out using species-specific isotope dilution gas chromatography inductively coupled plasma mass spectrometry (SSID-GC-ICPMS), where (202)Hg-enriched methylmercury (MeHg) was used as the spike compound. In order to avoid any possible analytical biases caused by nonquantitative extraction, degradation and/or formation of MeHg in sample preparations, two different extraction methods (KOH/methanol and HCl/methanol extractions) were performed, and one of these extraction methods utilized two different derivatization methods (ethylation and phenylation). A double ID method was adopted to minimize the uncertainty arising from the analyses. In order to ensure not only the reliability of the analytical results but also traceability to SI units, the standard solution of MeHg used for the reverse-ID was prepared from high-purity MeHg chloride and was carefully assayed as follows: the total mercury was determined by ID-ICPMS following aqua regia digestion, and the ratio of Hg as MeHg to the total Hg content was estimated by GC-ICPMS. The certified value given for MeHg is 0.58 +/- 0.02 mg kg(-1) as Hg.

Environmental quality standards for water framework directive priority substances: challenges and opportunities.

The recently published Daughter Directive on priority substances is the culmination of several years of effort by the European Commission to develop consistent, Europe-wide environmental quality standards (EQS) that are scientifically based and protective of Europe's surface waters. This commentary describes progress to date in deriving EQS under the Water Framework Directive and identifies some remaining challenges and opportunities. In the Daughter Directive, annual average EQS are set for surface waters for all 33 priority substances. No sediment standards are included, and biota EQS are proposed for methyl-mercury, hexachlorobenzene and hexachlorobutadiene. Important remaining questions include whether the standard chemical risk assessment methodology is appropriate for deriving water column EQS and whether it would be appropriate to derive EQS for sediment or biota or to adopt alternative strategies. Moreover, some cross-cutting issues about the cessation of discharges of naturally occurring or ubiquitous substances and the advantages and disadvantages of member states setting their own EQS are briefly addressed.

Simultaneous multi-species determination of trimethyllead, monomethylmercury and three butyltin compounds by species-specific isotope dilution GC-ICP-MS in biological samples.

An accurate and sensitive multi-species species-specific isotope dilution GC-ICP-MS method was developed for the simultaneous determination of trimethyllead (Me3Pb+), monomethylmercury (MeHg+) and the three butyltin species Bu3Sn+, Bu2Sn2+, and BuSn3+ in biological samples. The method was validated by three biological reference materials (CRM 477, mussel tissue certified for butyltins; CRM 463, tuna fish certified for MeHg+; DORM 2, dogfish muscle certified for MeHg+). Under certain conditions, and with minor modifications of the sample pretreatment procedure, this method could also be transferred to environmental samples such as sediments, as demonstrated by analyzing sediment reference material BCR 646 (freshwater sediment, certified for butyltins). The detection limits of the multi-species GC-ICP-IDMS method for biological samples were 1.4 ng g(-1) for MeHg+, 0.06 ng g(-1) for Me3Pb+, 0.3 ng g(-1) for BuSn3+ and Bu3Sn+, and 1.2 ng g(-1) for Bu2Sn2+. Because of the high relevance of these heavy metal alkyl species to the quality assurance of seafood, the method was also applied to corresponding samples purchased from a supermarket. The methylated lead fraction in these samples, correlated to total lead, varied over a broad range (from 0.01% to 7.6%). On the other hand, the MeHg+ fraction was much higher, normally in the range of 80-100%. Considering that we may expect tighter legislative limitations on MeHg+ levels in seafood in the future, we found the highest methylmercury contents (up to 10.6 microg g(-1)) in two shark samples, an animal which is at the end of the marine food chain, whereas MeHg+ contents of less than 0.2 microg g(-1) were found in most other seafood samples; these results correlate with the idea that MeHg+ is usually of biological origin in the marine environment. The concentration of butyltins and the fraction of the total tin content that is from butyltins strongly depend on possible contamination, due to the exclusively anthropogenic character of these compounds. A broad variation in the butylated tin fraction (in the range of <0.3-49%) was therefore observed in different seafood samples. Corresponding isotope-labeled spike compounds (except for trimethyllead) are commercially available for all of these compounds, and since these can be used in the multi-species species-specific GC-ICP-IDMS method developed here, this technique shows great potential for routine analysis in the future.

Simultaneous sample preparation and species-specific isotope dilution mass spectrometry analysis of monomethylmercury and tributyltin in a certified oyster tissue.

A rapid, accurate, sensitive, and simple method for simultaneous speciation analysis of mercury and tin in biological samples has been developed. Integrated simultaneous sample preparation for tin and mercury species includes open focused microwave extraction and derivatization via ethylation. Capillary gas chromatography-inductively plasma mass spectrometry (CGC-ICPMS) conditions and parameters affecting the analytical performance were carefully optimized both for species-specific isotope dilution analysis of MMHg and TBT and for conventional analysis of MBT and DBT201Hg-enriched monomethylmercury and 117Sn-enriched tributyltin were used for species-specific isotope dilution mass spectrometry (SIDMS) analysis. As important, accurate isotope dilution analysis requires equilibration between the spike and the analyte to achieve successful analytical procedures. Since the spike stabilization and solubilization are the most critical and time-consuming steps in isotope dilution analysis, different spiking procedures were tested. Simultaneous microwave-assisted spike stabilization and solubilization can be achieved within less than 5 min. This study originally introduces a method for the simultaneous speciation and isotope dilution of mercury and tin in biological tissues. The sample throughput of the procedure was drastically reduced by fastening sample preparation and GC separation steps. The accuracy of the method was tested by both external calibration analysis and species-specific isotope dilution analysis using the first biological reference material certified for multielemental speciation (oyster tissue, CRM 710, IRMM). The results obtained demonstrate that isotope dilution analysis is a powerful method allowing the simultaneous speciation of TBT and MMHg with high precision and excellent accuracy. Analytical problems related to low recovery during sample preparation are thus minimized by SIDMS. In addition, a rapid procedure allows us to establish a performant routine method using CGC-ICPMS technique.

Methods and rationale for derivation of a reference dose for methylmercury by the U.S. EPA.

In 2001, the U.S. Environmental Protection Agency derived a reference dose (RfD) for methylmercury, which is a daily intake that is likely to be without appreciable risk of deleterious effects during a lifetime. This derivation used a series of benchmark dose (BMD) analyses provided by a National Research Council (NRC) panel convened to assess the health effects of methylmercury. Analyses were performed for a number of endpoints from three large longitudinal cohort studies of the neuropsychological consequences of in utero exposure to methylmercury: the Faroe Islands, Seychelles Islands, and New Zealand studies. Adverse effects were identified in the Faroe Islands and New Zealand studies, but not in the Seychelles Islands. The NRC also performed an integrative analysis of all three studies. The EPA applied a total uncertainty factor (UF) of 10 for intrahuman toxicokinetic and toxicodynamic variability and uncertainty. Dose conversion from cord blood mercury concentrations to maternal methylmercury intake was performed using a one-compartment model. Derivation of potential RfDs from a number of endpoints from the Faroe Islands study converged on 0.1 microg/kg/day, as did the integrative analysis of all three studies. EPA identified several areas for which further information or analyses is needed. Perhaps the most immediately relevant is the ratio of cord:maternal blood mercury concentration, as well as the variability around this ratio. EPA assumed in its dose conversion that the ratio was 1.0; however, available data suggest it is perhaps 1.5-2.0. Verification of a deviation from unity presumably would be translated directly into comparable reduction in the RfD. Other areas that EPA identified as significant areas requiring further attention are cardiovascular consequences of methylmercury exposure and delayed neurotoxicity during aging as a result of previous developmental or adult exposure.

Challenges from speciation analysis for the development of biological reference materials.

Starting with explanations for the increasing demand for speciation analysis the need of appropriate certified reference materials is justified. Specific aspects of biological reference materials for speciation QA/QC are discussed from the point of view of the total analytical process. Examples of unwanted species transformations during analysis are given and critical analytical steps are highlighted. Unchanged biological materials as CRMs and appropriate pure standard compounds are indispensable for the further development of this analytical field.

Uncertainties in the reference dose for methylmercury.

This paper critically examines the National Academy of Sciences and the National Research Council report on the toxicological effects of methyl mercury and the recently published US Environmental Protection Agency Reference Dose (RfD) for Methylmercury. Particular scrutiny is placed on the choice of the critical study and the underlining assumptions utilized in the selection of specific uncertainty factors (UFs) and the rationale for using a less-than-default factor of 10. The UFs that were utilized or considered by other agencies and organizations are also critically examined, explained and compared to one another. Based on these analyses, the authors suggest research that could be performed that would ameliorate the uncertainty of choosing a more precise partial UFor that may even provide completeness of database to allow for selecting of a UF for unity, thus improving the precision of the current published RfD.

Fluorimetric determination of methylmercury as an ion-association complex with rhodamine B in the presence of iodide.

A fluorimetric method for the determination of methylmercury was established. The method was based on the formation of an ionic pair between iodide-methylmercury-rhodamine B in hydrochloric acid, which can be extracted with benzene. The fluorescence emission was measured at lambda(ex)/lambda(em) 575/590 nm, and the experimental variables and possible interference were studied. The linear calibration range was 4 x 10(-8) mol/L to approximately 5 x 10(-7) mol/L with a correlation coefficient of 0.9992. The detection limit was 1 x 10(-8) mol/L. The method was used to determine methylmercury in human hair. The recovery was in the range of 91% to 105% and the relative standard deviation was 2.8%. The results agreed with those obtained by gas chromatography with electron capture detection.