Biogeochemistry of Environmentally Important Trace Elements

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Chapter 21

Determination of Stable Mercury Isotopes by ICP/MS and Their Application in Environmental Studies 1

Holger Hintelmann and Nives

1,2

Ogrinc

1

Department of Chemistry, Trent University, Peterborough, Ontario K9J 7B8, Canada Jozef Stefan Institute, 1000 Ljubljana, Slovenia 2

Inductively coupled plasma mass spectrometry (ICP/MS) is a powerful tool for mercury determinations. Introducing mercury in form of gaseous species into a dry plasma greatly reduces memory effects, achieving absolute detection limits of less than 100 pg of Hg. The capability of ICP/MS to take advantage of speciated isotope dilution methods makes this technique suitable for very precise and accurate measurements. Equally important, multiple stable tracer experiments to study the fate of Hg species in the environment become possible. This novel concept allows the investigation of multiple transformation processes simultaneously. However, since isotope enrichments are often well below 100 %, a system of linear equations must be solved to exactly calculate individual isotope concentrations. This paper describes the necessary calculations and demonstrates how stable isotopes improve methylmercury measurements.

© 2003 American Chemical Society In Biogeochemistry of Environmentally Important Trace Elements; Cai, Yong, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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322 Mercury is an ubiquitous pollutant in the environment and of great concern as a large number of aquatic organisms in many ecosystems from all over the world show elevated levels of Hg. Severalfreshwaterfish are known to be contaminated with high levels of toxic and bioavailable methylmercury (MeHg) (/). Consequently, many countries have issued advisories to manage the consumption offish,representing the main entry of MeHg into the human diet and thus, being the main concernfroma human health point of view. Numerous studies have been conducted to elucidate the fate of Hg species in the environment and to understand the factors controlling MeHg formation and bioaccumulation. Similarly, many different methods have been developed to accurately determine the concentrations of Hg species in various environmental matrices (2). Only recently has inductively coupled plasma mass spectrometry (ICP/MS) emerged as a competitive technique for Hg species analysis (3-6). A couple of unfounded myths regarding the applicability of ICP/MS for Hg (species) analysis had to be overcome, namely its perceived lack of sensitivity (compared to the de-facto standard of highly sensitive atomic fluorescence measurements) and suspected potential for exhibiting severe memory effects. However, the appeal of employing highly precise isotope dilution techniques and the intriguing possibility of conducting stable isotope tracer experiments to study the fate of Hg have prompted us early on to develop ICP/MS methods (7). This paper gives a comprehensive description of the various methods in use in our laboratory for Hg species determinations in a variety of matrices, focusing especially on water column measurements. We will furthermore describe the concept of speciated isotope dilution and stable tracer techniques introducing a novel approach to accurately calculate individual Hg isotope concentrations in multiple isotope addition experiments.

Materials and Methods Accurate and precise Hg species measurements at ultra trace levels, typically encountered in pristine water samples, presents the analytical chemist with a number of challenges. Most notably is the need for vigorously cleaned laboratory-ware and the challenge to preserve the integrity of the individual Hg species during all sample preparation and measurement steps (no degradation or de-novo formation of Hg species). Cleaning of Teflon and glassware for ultra trace level measurements Cleaning methods for equipment used in Total Hg and MeHg measurements are identical, except that Teflon bottles used for MeHg procedures are not subjected to the BrCl soaking step. Teflon bottles designated for MeHg analysis must never been in contact with such solutions, since BrCl might

In Biogeochemistry of Environmentally Important Trace Elements; Cai, Yong, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

323 diffuse into Teflon, leach back out into subsequently prepared samples and effectively decomposes MeHg in the new sample. Teflon bottles, vials and tubing are rinsed first with tap water, then with Milli-Q water and are immersed in (or filled with) a 50% (ν/ν) HNO3 solution and heated to 65°C for 2 days. This is followed by at least 4rinseswith Milli-Q water and soaking in 10% (v/v) cone. HC1 at room temperature for another 3 days. The Teflon is rinsed 4x with Milii-Q water and again soaked in a solution of 250 ml of Milli-Q water, 1 ml of concentrated HC1 and 2 ml 0.2 Ν BrCl. Prior to the nextrinsingstep, 100 μΐ of NH OH*HCl per 250 mL of solution is added to neutralize BrCl. After repeated rinsing with Milli-Q water (at least 4x), all Teflon bottles are filled to 25% of their volume with 1% low Hg-grade HC1, capped and double bagged in new polyethylene bags for storage. For new bottles and equipment, the initial nitric acid soaking may be omitted. Glass-ware isfirstrinsed4x with Milli-Q water and then transferred into a container filled with 10% (v/v) HC1. After overnight soaking at room temperature, the equipment is rinsed 4x with Milli-Q water and soaked for another night at room temperature in a BrCl solution (16 1 water, 160 ml HC1 cone, and 80 ml 0.2 Ν BrCl). Finally, the equipment is rinsed 4x with Milli-Q water. Distillation bridges are stored in plastic containers, while vessels are filled to 25% with 1% low Hg-grade HC1, capped and stored in the plastic container. Quartz fiber filters (QF/F, 47 mm diameter) used to collect particles from water samples are cleaned prior to use by heating overnight in a muffler furnace at 500 °C. Individualfiltersare stored in precleaned (procedure as for new teflon equipment) polyethylene petri-dishes.


2

Sample digestion for total mercury determinations Water samples and particles collected on QF/F quartzfiberfiltersare treated with a strong oxidant (0.2 Ν BrCl) to oxidize all Hg species to Hg . A l l sample manipulation are performed in a clean room under a class 100 laminar flow hood. Water samples are digested by adding 0.5 mL of BrCl per 100 mL of sample. At the same time, 20 μΐ, of an internal Hg(II) standard solution (2.3 ng/mL) are added. Bottles are sealed again in plastic bags for a minimum digestion period of 12 h (room temperature). Oxidation is considered to be complete if excess of BrCl is present after 12 h as determined by a yellow tint in the sample. If BrCl has been consumed, additional BrCl must be added and allow to react for another 12 h. A slightly modified (8) method for digesting particles collected on QF/F filters has been employed. Thefilterpaper isfittedinto 30 mL Teflon vials and 20 μΐ, of the internal Hg(II) standard solution (2.3 ng/mL) are added directly onto the filter, which is then covered with 10 mL of Milli-Q water. The particles are digested by adding 0.1 mL HC1 (cone.) and 0.1 mL BrCl (0.2N). Vials are tightly closed and heated overnight to 65°C on a hot plate. 2+

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324 Vegetation, sediment and biota samples are digested by weighing the sample (typically 100 mg of freeze dried plants, 1 g of wet sediment or 0.5 g of wetfish)into 25 mL glass or Teflon vials. After spiking with an appropriate amount of Hg(II) standard solution (approximately equaling the amount of Hg expected to be in the sample), samples are digested by adding 5 to 10 mL of a HNO3/H2SO4 (7:3) mixture. Samples are heated to 80°C until the formation of brown NO gases has ceased. Samples are diluted to volume and analyzed. 201


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Distillation of methylmercury from water and particles Methylmercury can be isolated from complex sample matrices by atmospheric pressure water vapor distillation. Our procedure has been slightly modifiedfromprocedures described in detail elsewhere (9, 10). This distillation step is required for water and particle samples to remove matrix components that may interfere with the subsequent analytical procedures. Water samples are distilled in an all glass distillation apparatus consisting of two 50 mL glass tubes connected via an air cooled glass distillation bridge (i.d. 8 mm). 50 g of water are weighed into the distillation tube and 20μ1 of 1 ng/ml CH Hg solution (internal standard), 500 μΐ of 9M H S0 and 200μ1 of 20% KC1 solution are added to each sample. 5 mL of Milli-Q water are placed into the receiver tube. Both tubes are connected to the distillation bridge and the set-up is placed into an aluminum heating block set to 140 °C. Hg-free N gas (60 - 80 mL/min, controlled by individualflowmeters) is connected to each unit to hasten the distillation process. Approximately 85 % (42.5 mL) of the sample are distilled, which should take between 5 and 7 h. Samples can be stored in the dark at 4 °C in the receiver tubes without additional preserving agents for up to one week. Sediments and particles collected onfiltersare distilled in a similar fashion, except that 30 mL Teflon bottles are used instead of the glass distillation system. Filters (or sediments) are placed in the vial, 10 mL of Milli-Q water, 20 μΐ of the CH Hg standard solution (1 ng/ml), 0.5 ml of H2SO4 (9 M) and 0.2 ml of KC1 (20%, w/v) are added. 9 mL of sample are distilled within 60 to 90 min. 201

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Measurement of total mercury in water and particles Total Hg in water and particles is determined using an off-line cold vapor method involving preconcentration of Hg on gold traps. Cleaned gas-wash bottles (glass, 125 mL) arefilledwith 100 mL of Milli-Q water and 1 mL of SnCl reducing solution (20% w/v in 20% HC1, v/v) and are purged with Hgfree N for 1 hour to remove Hgfromthe system. Another 0.5 mL SnCl is added, pre-cleaned gold traps are attached to the bubbler outlet and the solution is purged at 100 mL/min for 20 minutes to give the system (or bubbler) blank. 2

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325 An additional 0.5 ml of 0.2N BrCl, together with 0.1 ml of NH OHHCl, 0.5 mL of SnCl are measured to obtain the reagent blank. Typically, 100 mL of the BrCl treated sample is weighed into the bubbler and excess BrCl is pre-reduced by addition of 0.1 mL of NH&H-HCl. 0.5 mL of SnCl are added and the sample is purged for 20 minutes collecting liberated Hg° on the gold traps. Particle digests can be measured by transferring the 10 mL digest to the bubbler without removing previous samples. Sample analysis will only proceed if the following two criteria are met: a) no bubbler blank must contain more than 20 pg of Hg and b) the standard deviation of the mean response for three replicate standards must be S0 ) and the formed CH HgBr was extracted 3x with 400 μΐ, of toluene. Tne combined toluene extracts were dried over sodium sulfate and 100 μΐ, of this primary stock solution were diluted with 10 mL of isopropanol. Working solutions were prepared fresh daily by diluting the secondary isopropanol stock with deionized water as needed. 3

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Tuning and optimisation of the ICP/MS for mercury measurements The ratios of Hg isotopes in different environmental samples were determined using a Platform-ICP/MS (Micromass Ltd., Manchester, UK). The instrument consists of a standard ICP source and quadrupole mass analyser with a unique ion transfer system based on an RF-only hexapole collision cell. The instrument provides computer control of the xyz-torch positions. In addition to an RF-voltage, a DC-voltage may be applied to the hexapole as a bias to reject low energy ions originatingfromnon-plasma sources. Typically, He and H are introduced simultaneously into the collision cell at flow rates of 0-10 mL min" . 2

1



328 The platform is uniquely equipped with a scintillator-photomultiplier type of detector, which has a large dynamic linear range extending over 8 orders of magnitude. In contrast to commonly used electron multiplier detectors, it does not switch between pulse and analogue counting modes and hence, no crosscalibration or dead time corrections are needed. Both parameters must be diligently controlled on instruments sporting electron multipliers to obtain the most precise isotope ratios over a wide range of concentrations. Although modem ICP-MS instruments provide relatively good short- and medium-term signal stability, there is a strong dependence of the signal response on plasma operating conditions. For optimum performance the instrument requires daily optimisation and monthly tuning. For this purpose a continuous stream of elemental mercury is introduced into a dry plasma to determine optimal operation conditions. The use of a dry plasma avoids many problems, in particular extended wash-out times associated with introduction of ionic mercury species into a wet plasma. A steady mercury signal was generated by gently heating a quartz tube containing a small HgS crystal. Parameters most critical for obtaining maximum sensitivity are nebuliser gas flow rate, torch position, hexapole gas flow rates, cone voltage and hexapole bias. The dependence of signal intensity as a function of the nebuliser flow rate is illustrated in Figure 2. Optimal flow rates ranged between 0.8 and 1.05 L min" with the analyte intensities between 5xl0 and 1.3xl0 counts per second (cps). Beyond these limits a reduction in sensitivity is observed and measured Hg isotope ratios deviate randomly.

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He, H gas (mL/min) 2

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600

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900 1200

Nebuliser gas (mL/min)

Figure 2: Influence of nebuliser and collision cell gas flows The measurement of sensitivity as a function of the collision cell gas flow rates was performed changing both gas flow rates independently. Figure 2 shows that an increase in He leads generally to an improvement of ion sensitivity in this instrument. This observation was expected as intensities of heavy ions are generally improved under such conditions. This effect is commonly referred to as the thermalization effect in collision cell instruments (13). On the other hand the presence of H in the collision cell had an unexpected effect. Even the 2


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slightest amount of H (the minimum allowed flow rate is 0.1 ml min" ) significantly quenched the Hg signals and changed measured Hg isotope ratios. This peculiar effect could not be explained satisfactorily and is still under investigation. Hence, the ICP/MS instrument is operated with zero H in the collision cell, when used for Hg measurements. The influence of the cone lens voltage and the retarding hexapole DC-bias potential (HBP) on the intensity of Hg ions are illustrated in Figure 3. The HBP is employed as a barrier, which low energy ions are not able to surmount. Changing the HBP to only -0.1 V will already reduce the Hg signal by 50%. Since none of the mercury ions is interfered by any residual molecular gas ions, the HBP is set to 0 V to achieve the highest sensitivity possible (13). Table I summarizes ICP/MS operating conditions providing the highest signal response for Hg measurements. 2

HBP (V) 0.0 -0.5 -1.0 -1.5 -2.0 _ I 1 4 -. —«—Cone — " B P J ο 12

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* 4 2 200 400 600 800 1000 Cone lens (V)

Figured .Influence of cone lens and hexapole bias potential

Table I. Optimized parameters for Hg isotopes measurements ICP/MS ICP Incident power Plasma gas Nebulizer gas Carrier gas

1350 W 13.98 L min' 1.03 L min 0.03 L min 1

1

1

Analyser/Collision cell Cone lens 550V Hexapole exit lens 400 V H gas flow 0 mL min" He gas flow 10 mL min" 1

2

1

All methods will generate transient signals, where quantification is based on integrating peak areas of Hg isotopes. Generally, a minimum of 10 data points is required to adequately describe a peak for integration purposes. However, we feel that this minimum requirement is insufficient to determine isotope ratios with high precision. We typically adjust scan times to obtain one data point per second per isotope. Average peak width are in the order of 30 sec,


330 resulting in a minimum of 30 points to fully describe the peak. Faster scan rates introduce a higher baseline noise level, which makes reproducible peak-start and peak-end detection and isotope ratio measurements less precise.


Figures of merit The described methods and techniques proved to be extremely sensitive, overcoming the perceived shortcomings of ICP/MS for Hg species analysis and allowed ultra trace determinations of Hg species in pristine water samples. Using a dry plasma and introducing only gaseous Hg species (Hg or peralkylated organomercury species) resulted in relatively short washout times with negligible memory. Even after introducing high concentrations of Hg for continuously for more than one hour, the baseline went back to acceptable low values within minutes after removing the Hg source. Absolute instrumental detection limits for Hg were determined to be 100 fg (30 pg of Hg) for both methylmercury and total Hg using the gold trap preconcentration technique. These values compare favorable with instrumental LOD's reported for AFS (300 fg, 14), the previous state-of-the-science technique for Hg measurements. Typical procedural blanks routinely achieved in water analysis are 1.9 ± 0.6 pg of MeHg and 6.9 ±3.5 pg for total Hg. These blanks translate into detection limits of 35 pg/L for MeHg measurements (50 mL sample volume) and 100 pg/L for total Hg determinations (100 mL sample volume). Table Π compares results obtained with methods described in this paper with certified values of reference materials. Generally, there is no statistical difference among measured and certified concentrations. 202

Table Π: Comparison of measured with certified total Hg values in CRM Reference material NIST 1515 (apple leaves) NIST 1515 (apple leaves) MESS-3 (marine sediment) MESS-3 (marine sediment) DORM-2 (dogfish muscle)

Method Certified (ng/g) Measured (ng/%) 48 ± 5 FIA 44 ± 5 43 ± 2 Au-trap FIA 91 ± 9 87 ± 9 Au-trap 96 ± 8 FIA 4640 ± 260 4350 ± 350

Most of our measurements are based on isotope ratio determinations. Hence the techniques and ICP/MS methods are all optimised with the premise to give most precise ratio determinations. This approach also introduces a new concept for limits of detection. Particularly in tracer experiments, we are faced with the challenge to detect a variation in the isotope ratio from the natural ratio, which would then be an indication for the presence of the tracer isotope. Since we will always measure a background of ambient Hg species, the detection limit is


331 affected with our precision to measure those isotope ratios. Hence, our predisposition with isotope ratio measurements. Generally LOD'$ for isotope concentrations are limited by the precision of the isotope ratio determination and the background concentration of Hg (the more Hg already in the sample, the more tracer isotope is needed to change the natural ratio by 1%). LOD's for isotopes can be expressed as:


LOD (isotope) = ambient Hg Χ A X 3RSD (of ratio measurement) with A = natural abundance of tracer isotope. Using optimum RSD's (0.5 %) and H ^ as the tracer isotope, at least 0.35 % of the total Hg must be in form of the Hg tracer isotope to change the isotope ratio enough allowing to detect its presence in the sample (77). 2 0 0

2

Stable tracer studies In contrast to a traditional isotope dilution experiment, where the amount of tracer isotope is exactly known, a stable tracer experiment designed to study the fate of Hg species follows a different concept and requires a different approach for calculating individual isotope concentrations. In an stable isotope experiment, known amounts of one or more stable Hg isotopes are added to the system under investigation. This will change the isotopic composition of the added species in the sample or in the source compartment. At the beginning of the experiment (at time zero) the isotopic composition of the species to be generated or of Hg in the target compartment is identical to the natural Hg pattern. Only if the tracer isotope reacts to a new species (by means of methyiation, demethylation or redox processes) or is transferred from the source to the target compartment (e.g. uptake into organisms or adsorption from water onto particles) it will alter the isotopic Hg pattern of the new species or in the target compartment. The magnitude of the change in isotope ratios is then related to the amount of new Hg species generated or transferred. Figure 4 illustrates this concept, showing the isotope pattern of ambient Hg according to IUPAC (7 7) (4a) and the composition of a typical isotope enriched solution of Hg (4b). Here, the solution is enriched with Hg(II) (97.36 %). As an example, adding this solution to a sediment sample will change the isotope pattern of the inorganic Hg in the sediment. The concentration of CH Hg is now enriched in the sample relative to the fraction of CH Hg in the original sample and the magnitude of the relative change relates to the concentration of Hg(D) methylated. At the same time, any other isotope can be used to determine the concentration of ambient MeHg in the same sample (e.g. ambient MeHg = A X CH Hg , A = natural abundance of Hg). However, at the beginning of the experiment (time zero), the isotope pattern of MeHg in the sediment remains unchanged, since no isotope enriched MeHg was added. Only if afractionof the added Hg(II) is converted to CH Hg will the isotope pattern of MeHg change. Figure 5 represents a GC-ICP/MS chromatogram of a sediment extract from a methyiation assay. 199

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Biogeochemistry of Environmentally Important Trace Elements

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