SW-846 Update V 3200 - 1 Revision 1 July 2014 METHOD 3200

MERCURY SPECIES FRACTIONATION AND QUANTIFICATION BY MICROWAVE ASSISTED EXTRACTION, SELECTIVE SOLVENT EXTRACTION

AND/OR SOLID PHASE EXTRACTION

SW-846 is not intended to be an analytical trainin g manual. Therefore, method procedures are written based on the assumption that they will be performed by analysts who are formally trained in at least the basic principles o f chemical analysis and in the use of the subject technology.

In addition, SW-846 methods, with the exception of required method use for the analysis of method-defined parameters, are intended to be gu idance methods which contain general information on how to perform an analytical procedu re or technique which a laboratory can use as a basic starting point for generating its own de tailed standard operating procedure (SOP), either for its own general use or for a specific pr oject application. The performance data included in this method are for guidance purposes o nly, and are not intended to be and must not be used as absolute QC acceptance criteria for purp oses of laboratory accreditation.

1.0 SCOPE AND APPLICATION

For a summary of changes in this version from the p revious version, please see Appendix

A at the end of this document.

1.1 This method contains a sequential extraction a nd separation procedure that may be used in conjunction with a determinative method to differentiate mercury species that are present in soils and sediments. This method provid es information on both total mercury and various mercury species.

1.2 The speciation of a metal, in this case mercur y, involves determining the actual form of the molecules or ions that are present in t he sample. When combined with an appropriate determinative method, this procedure is designed to provide varying degrees of mercury species information. All metal speciation methods are operationally defined by the level of post-extraction processing and the chosen method of analysis.

The environmental mobility and toxicity of mercury in a soil profile depend on its speciation. Alkyl mercury species such as methylme rcury are at least an order of magnitude more mobile than inorganic mercury species, and thu s are more toxic and more readily bioaccumulated. Soluble inorganic mercury species such as mercury chloride are more easily transported by natural process than the other inorg anic mercury species and serve as the substrate for mercury methylation process (Ref. 1). These extractable organomercury species and extractable inorganic species contribute the ma jor portion of mercury potential toxicity in the soils. The mercury species that fall into the "sem i-mobile" category such as elemental mercury are less toxic than extractable mercury species. T he "non-mobile" mercury species such as mercury sulfide are chemically stable in the soil f or geologic time periods and thus are least

toxic.SW-846 Update V 3200 - 2 Revision 1 July 2014 Examples of the operationally-defined mercury frac tions and individual species that may be determined using this procedure are presented in the table below.

Operationally-Defined Mercury Fractions Individual Mercury Species CAS No. a

Total Mercury

Extractable Mercury Extractable Organic

Mercury CH 3 HgCl

CH 3 CH 2 HgCl 115-09-3

107-27-7 Extractable Inorganic Mercury HgCl 2 Hg(OH) 2 Hg(NO 3 ) 2 HgSO 4 HgO Hg 2+ complexes c 7487-94-7

b

 10045-94-0

13766-44-4

21908-53-2

 Non-extractable Mercury Semi-mobile

Mercury Hg 0
Hg 0 -M d
Hg 2+ complexes c Hg 2 Cl 2 (minor) 7439-97-6



 10112-91-1

Non-mobile Mercury Hg 2 Cl 2 (major) HgS HgSe 10112-91-1

1344-48-5

20601-83-6

a Chemical Abstracts Service Registry Number

b Not registered by the Chemical Abstracts Service

c Certain inorganic mercury complexes may be present in both the organic and inorganic

extractable fractions

d This represents a mercury-metal amalgam

1.3 Quantification of mercury in the different fr actions may be performed using any suitable technique with appropriate precision and a ccuracy, for example Method 6800, 7473, 1631, or Methods 7470 and 7471. Other analytical t echniques, such as gas chromatography/mass spectrometry (GC/MS), ion chrom atography or high performance liquid chromatography (HPLC) with either GC/MS or inductiv ely coupled plasma-mass spectrometry (ICP-MS) detection (Method 6020), or other hyphenat ed and/or mass spectrometric techniques, may be employed if performance appropriate for the intended application can be demonstrated. This method may also be applicable to other matrice s, such as industrial and municipal waste materials, but its performance on such matrices has not yet been evaluated. Method 6800 (Elemental and Molecular Speciated Isotope Dilution Mass Spectrometry) may also be applicable as a diagnostic and validation tool for quantification of selectively extracted mercury species, especially when species transformations oc cur in the sample preparation or analysis procedures.

1.4 Prior to employing this method, analysts are a dvised to consult each method that may be employed in the overall analysis (e.g., Meth od 6800, 7473, 7470, or 7471) and the

manufacturer’s instructions for additional informat ion on quality control procedures, development of QC acceptance criteria, calculations , and general guidance. Analysts also

should consult the disclaimer statement at the fron t of the manual and the information in Chapter Two for guidance on the intended flexibilit y in the choice of methods, apparatus, materials, reagents, and supplies, and on the respo nsibilities of the analyst for demonstrating that the techniques employed are appropriate for th e analytes of interest, in the matrix of interest, and at the levels of concern.

In addition, analysts and data users are advised t hat, except where explicitly specified in aSW-846 Update V 3200 - 3 Revision 1 July 2014 regulation, the use of SW-846 methods is not mandatory in response to Federal testing requirements. The information contained in this me thod is provided by EPA as guidance to be used by the analyst and the regulated community in making judgments necessary to generate results that meet the data quality objectives for t he intended application.

1.5 This method is restricted to use by, or under supervision of, personnel appropriately experienced and trained in the use of metal speciation and analysis techniques. Each analyst must demonstrate the ability to genera te acceptable results with this method.

2.0 SUMMARY OF METHOD

2.1 For the determination of extractable mercury s pecies, a representative sample aliquot is extracted with an appropriate volume of solvent at elevated temperatures. Extraction is accomplished with the aid of either microwave ir radiation or ultrasound.

2.2 Following initial extraction the resultant ext racts are separated from the remaining sample matrix for analysis of extractable mercury b y an appropriate technique. The residual sample matrix may be analyzed for non-extractable m ercury using an appropriate technique.

2.3 The method also has provisions for the separat ion of the extractable mercury fraction into inorganic and organic mercury fractio ns or individual species. The inorganic and organic mercury fractions may be separated by using a solid-phase extraction procedure. Individual species may be separated and determined by using an HPLC or other appropriate separation device coupled to an appropriate detecto r.

2.4 The method also has provisions for the separat ion of the non-extractable mercury fraction into semi-mobile and non-mobile mercury fr actions using sequential acid extraction and digestion.

3.0 DEFINITIONS

3.1 Species - The actual form of a molecule or ion that is present in a sample. Ref. 2 contains additional definitions for “species.”

3.2 Sub-speciation - The process by which mercury species in the extractable mercury

or non-extractable fraction are further subdivided. Ref. 2 contains additional definitions for “subspeciation.”

3.3 Total mercury - Total mercury content within t he sample, including all inorganic, organic, and complex forms of mercury.

3.4 Extractable mercury - An operationally-defined fraction of mercury which can be extracted from the sample using the protocols descr ibed within this method. The extractable mercury is meant to represent both organic and inor ganic forms of mercury which are more

labile.

3.5 Extractable inorganic mercury -- An operationa lly-defined subset of the extractable mercury species, i.e., the fraction of inorganic me rcury species which can be extracted from the sample using the protocols described within this me thod.

3.6 Extractable organic mercury - An operationally -defined subset of the extractable mercury species, i.e., the fraction of organic merc ury species which can be extracted from theSW-846 Update V 3200 - 4 Revision 1 July 2014 sample using the protocols described within this me thod.

3.7 Non-extractable mercury - The operationally-de fined fraction of mercury remaining in the sample after using the extraction protocols described within this method. The nonextractable mercury is meant to represent the least labile forms of mercury.

3.8 Semi-mobile mercury - An operationally-defined subset of the non-extractable mercury species, i.e., the fraction of mercury spec ies which can be extracted from the sample using the mild-acid extraction protocol(s) describe d within.

3.9 Non-mobile mercury - An operationally-defined subset of the non-extractable mercury species, i.e., the fraction of mercury spec ies which can be extracted from the sample using the harsh-acid extraction protocol(s) describ ed within.

3.10 Refer to Chapter One, Chapter Three, and the manufacturer’s instructions for other definitions that may be relevant to this procedure.

4.0 INTERFERENCES

4.1 Solvents, reagents, glassware, and other sampl e processing hardware may yield artifacts and/or interferences to sample analysis. All of these materials must be demonstrated

to be free from interferences under the conditions of the analysis by analyzing method blanks. Specific selection of reagents and purification of solvents by distillation in all-glass systems may be necessary. Refer to each method to be used for specific guidance on quality control procedures and to Chapter Three for general guidanc e on the cleaning of glassware.

4.2 Transformations among mercury species have bee n reported and experimentally verified. For example, methylmercury formed during sample processing from inorganic mercury, may cause positive biases in the methylmer cury results (Ref. 3 through 5). Also, a conversion of methylmercury and ethylmercury to ino rganic mercury has been observed under certain sample processing conditions (Figures 1 and 2) (Ref. 6).

4.3 The possibility of species interconversions ca nnot be eliminated due to the necessary reagents, sample matrix, the combination of reagents and matrix, and/or the

extraction method used. Method 6800 has successful ly been used to monitor and correct for such species transformations during speciation of c hromium and mercury (Ref. 7-9).

4.4 When non-specific detection techniques are emp loyed, the analyst must be aware of possible interferences. For example, certain or ganic compounds may absorb at the wavelength of interest when ultraviolet (UV) detect ion is used.

NOTE : The adjustment of the pH during certain sample p rocessing steps may result in the formation of a precipitate. Filtration (10-μm pore size or less) may be applicable to remove such a precipitate. Low recoveries may resu lt from co-precipitation of mercury and other sample components. Rinsing the precipita te on the filter with a 0.1% HCl solution has been demonstrated to minimize this pro blem.

5.0 SAFETY

5.1 This method does not address all safety issues associated with its use. The laboratory is responsible for maintaining a safe wo rk environment and a current awareness file of OSHA regulations regarding the safe handling of the chemicals listed in this method. ASW-846 Update V 3200 - 5 Revision 1 July 2014 reference file of material safety data sheets (MSDS s) should be available to all personnel involved in these analyses.

5.2 The proper handling of inorganic and organomer cury compounds cannot be overemphasized. Exposure to organo (alkyl) mercury compounds may cause damage to the central nervous system, emotional disturbances, or irritation of the eyes and skin, and may even lead to death (Ref. 10, 11).

5.3 The use of commercially-available protective g ear, such as gloves made of nitrile, polyethylene (PE) and ethylvinyl alcohol (EVA) lami nate or other appropriate material is required. Latex gloves are not suitable for the ha ndling of organomercury compounds (Ref. 11)

and must

not be used. In addition, appropriate eye protect ion should be used.

5.4 If any organomercury compound makes direct con tact with the gloves, remove the gloves immediately, dispose of them properly, and p ut on new gloves immediately. These materials only provide temporary protection. Consu lt the glove manufacturer for permeation

rates and times. If contact should occur with the skin or eyes, flush with large amounts of water and seek medical attention immediately. Further in formation on the safety guidelines on the handling of inorganic and organo (alkyl) mercury ca n be obtained from the material safety data

sheets for the substances.

5.5 The preparation and use of concentrated soluti ons or samples should be carried

out in a fume hood (some organomercury compounds are odorless) . It is advisable to work inside a plastic container with absorbent pads so t hat accidental spills will be contained.

5.6 In the event of a spill, the area should be we ll ventilated and any ignition sources removed. If the spill is a solid, collect and disp ose of the material in a sealed container. If the spill is a liquid, absorb on paper towels and disca rd in a designated waste bin. The contaminated area can also be cleaned with 0.05% (v /v) 2-mercaptoethanol solution to complex and remove the spilled mercury.

5.7 The extraction process may generate a moderate amount of pressure. Vessels used for extraction should be capable of withstandi ng these pressures or have pressure relief mechanisms or should be vented during the extractio n process.

5.8 For safety precautions associated with the det erminative methods, consult those methods directly.

6.0 EQUIPMENT AND SUPPLIES

The mention of trade names or commercial products in this manual is for illustrative purposes only, and does not constitute an EPA endor sement or exclusive recommendation for use. The products and instrument settings cited in SW-846 methods represent those products and settings used during method development or subs equently evaluated by the Agency. Glassware, reagents, supplies, equipment, and setti ngs other than those listed in this manual may be employed provided that method performance ap propriate for the intended application

has been demonstrated and documented.

This section does not list all common laboratory g lassware (e.g., beakers and flasks) that might be used.

6.1 Analytical balance - Accurate to 0.01 gSW-846 Update V 3200 - 6 Revision 1 July 2014 6.2 Vials/bottles, amber glass - Sizes as appropri ate, e.g., 20-mL, with PTFE-lined screw-caps or crimp-tops for storage of extracts.

6.3 Heating sources - Non-sonicating heating sourc es equipped with adjustable heating control able to maintain a temperature of 9 5 ± 2 °C (e.g., microwave heat unit, hot block, hot water bath or other equivalent)

6.4 Sonication heating source - Bath or horn type

6.5 Graduated cylinder or equivalent volume measur ing device

6.6 Volumetric flasks - Sizes as appropriate

6.7 pH measuring device - Universal pH paper or ca librated pH meter

6.8 Solid-phase extraction system - Visiprep solid -phase extraction manifold (Supelco or equivalent system). Consult the manufacturer’s recommendations for the glassware and hardware necessary to perform sample extractions.

6.9 Solid-phase extraction column - 6-mL glass rea ction tubes (Supelco) or equivalent, complete with PTFE frits (2 per tube)

6.10 Filtration device - Vacuum or manual

6.11 Filters - The filter media should have an eff ective pore size of 1.0 μm or less (glass fiber filters are known to work effectively).

6.12 Temperature measurement device - Device shoul d be capable of measuring up to

100 °C accurate to ± 0.1 °C.

6.13 Vortex mixer or equivalent

6.14 Centrifuge - Maximum speed of at least 3200 r pm and capable of handling 10-mL or greater centrifuge tubes

6.15 Centrifuge tubes - Disposable glass centrifug e tubes with capacity of at least 10 mL with snap-on cap

6.16 Microwave solvent extraction apparatus

6.16.1 The temperature performance requirements ne cessitate that the laboratory microwave extraction system be capable o f sensing the temperature to within ± 2.5 oC and automatically adjusting the microwave fi eld output power within 2 sec of sensing. Temperature sensors should be accurate to ± 2 oC. Temperature feedback control provides the primary performance mechanism for the method. Measurement in at least one vessel is required and a rotating turntab le to homogenize the microwave field to all samples is required for most systems.

6.16.2 Microwave extraction vessels are needed. V essels are available that can accommodate 1-g to 10-g samples. In addition the v essel apparatus must accommodate the necessary amount of solvent and if appropriate an inner vessel and stir bar and secondary microwave energy absorber. Vessels shoul d be essentially transparent to microwave energy (with the exception of purposeful microwave absorbing apparatus components), relatively inert to reagents and sampl e components, and capable ofSW-846 Update V 3200 - 7 Revision 1 July 2014 withstanding the temperature and pressure requireme nts (minimum conditions of 200 ° C and 442 psi) necessary to perform this procedure. Follow the manufacturer’s instructions regarding cleaning, handling, and sealing the vesse ls (Ref. 12).

7.0 REAGENTS AND STANDARDS

Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, it is intended that all reagents conform to the specifica tions of the Committee on Analytical Reagents of the American Chemical Society, where su ch specifications are available. Other grades may be used, provided it is first ascertaine d that the reagent is of sufficiently high purity to permit its use without lessening the accuracy of the determination. Reagents should be stored in glass to prevent the leaching of contamin ants from plastic containers.

7.1 Hydrochloric acid (concentrated, 12 M), HCl - C ertified ACS Plus grade or equivalent

7.2 Sodium hydroxide, NaOH - Certified ACS grade or equivalent

7.3 Sodium chloride, NaCl - Certified ACS grade or equivalent

7.4 Copper (II) chloride dihydrate, CuCl 2 2H 2 O - Analytical reagent grade or equivalent

7.5 Nitric acid (concentrated, 16 M), HNO 3 - Certified ACS Plus grade or equivalent

7.6 Hydrochloric acid (6 M), HCl - Prepared by dilu tion of 12 M HCl in reagent water

7.7 Sodium hydroxide (10 M), NaOH - Prepare by diss olution of solid NaOH pellets of appropriate purity in reagent water

7.8 Ethanol, CH 3 CH 2 OH - HPLC grade or equivalent

7.9 Silver nitrate, AgNO 3 - Certified ACS grade or equivalent

7.10 HPLC subspecies option mobile phase component s

7.10.1 Methanol, CH 3 OH - HPLC grade or equivalent

7.10.2 2-Mercaptoethanol, HSCH 2 CH 2 OH - reagent grade

7.10.3 Ammonium acetate, NH 4 CO 2 CH 3 - reagent grade

7.11 Acetic acid, CH 3 CO 2 H - HPLC grade or equivalent

7.12 Sulphydryl cotton fiber (SCF) separation and concentration option

7.12.1 Materials for preparing SCF

7.12.1.1 Cotton - Mercury-free cotton fiber

7.12.1.2 Mercaptoacetic acid (97+%), HSCH 2 CO 2 H - analytical reagent

grade

7.12.1.3 Acetic anhydride, (CH 3 CO) 2 O - HPLC grade or equivalentSW-846 Update V 3200 - 8 Revision 1 July 2014 7.12.1.4 Sulfuric acid, (concentrated, 18 M), H 2 SO 4 - analytical reagent

grade or spectrograde quality

7.12.2 SCF eluent solutions

7.12.2.1 SCF eluent 1, an aqueous solution contain ing 1.0 M HCl and 1.0 M NaCl -- Prepare by diluting 20.7 mL of concen trated HCl to 250 mL in reagent water, then dissolving 14.6 g of NaCl in th e prepared 1.0 M HCl.

7.12.2.2 SCF eluent 2, an aqueous solution contain ing 6 M HCl,
saturated NaCl, and 0.1% CuCl 2 ⋅ 2H 2 O -- Prepare by diluting 124 mL of concentrated HCl to 250 mL in water, then adding 0. 25 g of CuCl 2 ⋅ 2H 2 O and 11.0 g

of NaCl.

NOTE : A portion of the solid NaCl may remain un-dissol ved and settle to the

bottom of the vessel. The top portion of this solu tion should be used.

7.13 Reagent water - Reagent water must be interfe rence free. All references to water in the method refer to reagent water unless otherwi se specified.

7.14 2.0% (v/v) HCl + 10% (v/v) ethanol extraction solution - Prepared by dilution of the proper amount of concentrated HCl and ethanol in re agent water.

7.15 1:2 (v/v) HNO 3 extraction solution - Prepare by combining 1 part concentrated

HNO 3 with 2 parts reagent water.

7.16 1:6:7 (v/v/v) HCl : HNO 3 : reagent water - Prepare by combining 1 part concentrated HCl, 6 parts concentrated HNO 3 with 7 parts reagent water

7.17 Chloride ion test solution - 0.1 M silver nit rate in 0.1 M HNO 3 . Commercially prepared 0.1 M silver nitrate solution is available

.

7.18 4 M HNO 3 extraction solution

- Prepared by dilution of the proper amount of

concentrated HNO 3 in reagent water.

7.19 Inorganic mercury standard solutions - Commer cially-prepared standards containing natural isotopically-abundant inorganic mercury species and isotopically-enriched inorganic mercury species, such as Hg 2+ , are available (Ref. 13, 14). Standard solutions can also be prepared by dissolution of the selected pur e solid mercury compound in an appropriate solvent. Mercury compounds must be of known concen tration. All solvents used for standards preparation must be analytical reagent grade or equ ivalent.

7.20 Organic mercury standard solutions - Commerci ally prepared standards containing natural isotopically abundant CH 3 HgCl or other organic mercury species and/or isotop icallyenriched CH 3 HgCl or other organic mercury species are available (Ref. 13, 14). Standard solutions can also be prepared using the pure organ ic mercury compound and appropriate solvents (Ref. 4). Pure compounds must be of known concentration. Solvents used for standards preparation must be analytical reagent gr ade or equivalent.

7.21 Sodium acetate, anhydrous, NaCH 3 CO 2 - Certified ACS grade or equivalent

7.22 Sodium acetate trihydrate, NaCH 3 CO 2 3H 2 O - Certified ACS grade or equivalentSW-846 Update V 3200 - 9 Revision 1 July 2014 7.23 0.2 M acetate buffer solution (pH 3.0) - Mix 11.4 mL of acetic acid and 0.2789 g of

anhydrous sodium acetate or 0.4627 g of sodium acet ate trihydrate in reagent water. Dilute to 1 L with reagent water. Measure the pH of the buffer solution and if needed adjust the pH with a strong acid or base.

8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE

Sample collection, preservation and storage requir ements may vary by EPA program and may be specified in a regulation or project plannin g document that requires compliance monitoring for a given contaminant. Where such req uirements are specified in the regulation, follow those requirements. In the absence of speci fic regulatory requirements, use the following information as guidance in determining the sample c ollection, preservation and storage requirements.

8.1 See the introductory material of Chapter Three , “Inorganic Analytes.”

8.2 Samples should be collected and placed in cont ainers that are made of glass or other appropriate material.

8.3 Sample extracts should be analyzed within 5 da ys (Ref. 15). This unique holding time recommendation will be added to the Update V v ersion of Chapter Three.

9.0 QUALITY CONTROL

9.1 Refer to Chapter One for guidance on quality a ssurance (QA) and quality control (QC) protocols. When inconsistencies exist between QC guidelines, method-specific QC criteria take precedence over both technique-specif ic criteria and those criteria given in Chapter One, and technique-specific QC criteria take preced ence over the criteria in Chapter One. Any effort involving the collection of analytical data should include development of a structured and systematic planning document, such as a Quality Ass urance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which translates project o bjectives and specifications into directions for those that will implement the project and asses s the results. Each laboratory should maintain a formal quality assurance program. The l aboratory should also maintain records to document the quality of the data generated. All da ta sheets and quality control data should be maintained for reference or inspection.

9.2 Initial demonstration of proficiency

Each laboratory must demonstrate initial proficien cy with each sample preparation and determinative method combination it utilizes by gen erating data of acceptable accuracy and precision for target analytes in a clean reference matrix. This will include a combination of the sample extraction method and the determinative meth od (e.g., a 7000 series method). The laboratory must also repeat the demonstration of pr oficiency whenever new staff members are trained or significant changes in instrumentation a re made. See Method 8000 for information on how to accomplish a demonstration of proficiency.

9.3 Initially, before processing any samples, the analyst should demonstrate that all parts of the equipment in contact with the sample a nd reagents are interference-free. This is accomplished through the analysis of a method blank . As a continuing check, each time samples are extracted, cleaned up, and analyzed, an d when there is a change in reagents, a method blank should be prepared and analyzed for th e analytical species of interest as a safeguard against chronic laboratory contamination. The blanks should be carried through allSW-846 Update V 3200 - 10 Revision 1 July 2014 stages of sample preparation and analysis. When ne w reagents or chemicals are received, the laboratory should monitor the preparation and/or an alysis blanks associated with samples for any signs of contamination. It is not necessary to test every new batch of reagents or chemicals prior to sample preparation if the source shows no prior problems. However, if reagents are changed during a preparation batch, separate blanks need to be prepared for each set of reagents.

The laboratory should not subtract the results of the method blank from those of any associated samples. Such “blank subtraction” may l ead to negative sample results. If the method blank results do not meet the project-specif ic acceptance criteria and reanalysis is not practical, then the data user should be provided wi th the sample results, the method blank results, and a discussion of the corrective actions undertaken by the laboratory.

9.4 Sample quality control for preparation and ana lysis

The laboratory must also have procedures for docum enting the effect of the matrix on method performance (precision, accuracy, method sen sitivity). At a minimum, this should include the analysis of QC samples including a meth od blank, a duplicate, a matrix spike and a laboratory control sample (LCS) in each analytical batch. Any method blanks, matrix spike samples, and replicate samples should be subjected to the same analytical procedures (Secs. 4.0 and 11.0) as those used on actual samples. An e xample of Quality Control is described in section 13.8 (ref 26), demonstrates a mass balance method for mercury species where the interchanges between mercury species are quantified by measuring each species and then correcting for the inter-species transformations wi th method 6800 and obtaining mass balance by thermally grouping remaining non-extractable spe cies as a single mercury species. These species are added together and compared with total mercury values independently measured by methods 3200 and 6800. The species added togeth er equal the total mercury to, within the confidence interval, total combined measurement unc ertainty to achieve mass balance. Method 3200 should not be used with other analytical metho ds except 6800 if the mercury species are active and transform from one species to another as demonstrated in Reference 26 and Method

6800. 

### Species spike recovery is also a method of demonstr ation of Species stability.

9.4.1 Method blank - For each analytical batch of samples processed, at least one method blank should be carried throughout the e ntire sample preparation and analytical process. A method blank is prepared usi ng the same reagents and quantities used with samples and processed through the appropr iate steps of the procedure with the samples. These steps may include, but are not limi ted to extraction, chromatographic separation, concentration, dilution, filtering, and analysis. If the method blank does not contain target analytes at a level that exceeds the project-specific criteria requirements, then the method blank would be considered acceptabl e. In the absence of project-specific criteria, if the blank is less than the lower limit of quantitation, less than 10% of the regulatory limit, or less than 10% of the lowest sa mple concentration, whichever is greater, then the method blank is considered acceptable. If the method blank cannot be considered acceptable, the method blank should be r e-analyzed at once, and if still unacceptable, then all samples after the last accep table method blank must be reprepared and re-analyzed along with the other appro priate batch of QC samples. These blanks will be useful in determining if samples are being contaminated. If the method blank exceeds the criteria, but the samples are all either below the reporting level or below the applicable action level or other criteria, then the data should not be rejected based on this analysis.

9.4.2 Matrix spike (MS)/ Duplicate - For each batc h of solid phase samples extracted using this method, at least one matrix sp ike (MS) and one duplicate unspiked sample or one matrix spike/matrix spike duplicate ( MS/MSD) pair should be carriedSW-846 Update V 3200 - 11 Revision 1 July 2014 through the entire sample extraction, preparation a nd analytical process. An MS is an intra-laboratory split sample spiked with a known c oncentration of each analytical species of interest (see Secs. 7.19 and 7.20). Spiking is performed directly on the solid sample matrix prior to extraction in order to assess the e fficiency of the extraction procedure. The decision on whether to prepare and analyze duplicat e samples or a MS/MSD must be based on knowledge of the samples in the sample bat ch. If samples are expected to contain the target analyte, laboratories may use a matrix spike (MS) and a duplicate analysis of an unspiked field sample. If samples a re not expected to contain the target analyte, the laboratories should use a MS/MSD pair. A separate spiked sample and a separate duplicate unspiked sample may be analyzed in lieu of MS/MSD analyses. MS samples should be spiked at the project-specific ac tion level or, when lacking projectspecific action levels, between the low- and mid-le vel calibration standards. Acceptance criteria should be set at a laboratory-derived limi t developed through the use of historical analyses per matrix type analyzed. In the absence of historical data, the accuracy limit should be set at ±25% of the spiked concentration f or the MS and 25% RPD for the MS/MSD or duplicate precision. Matrix spike/duplic ate or MS/MSD samples should be included whenever a new matrix type is being analyz ed.

9.4.3 LCS - A laboratory control sample (LCS) shou ld be included with each analytical batch of samples processed. The LCS con sists of an aliquot of a clean (control) matrix similar to the sample matrix and of the same weight or volume. The LCS is spiked with the same analytes, at the same concentrations, as the matrix spike, when appropriate. When the results of the matrix spike analysis indicate a potential problem due to the sample matrix itself, the LCS results ma y be used to verify that the laboratory can perform the analysis in a clean matrix.

NOTE : A standard reference material (SRM), certified r eference material (CRM) or reference material (RM) may be used in place of an LCS. The SRM, CRM or RM generally consists of a commercially-prepared, well characterized matrix similar to the sample matrix and containing the analytical spe cies of interest at established

reference concentration levels.

9.5 It is recommended that the laboratory adopt ad ditional quality assurance practices for use with this method. The specific p ractices that are most productive depend upon the needs of the laboratory and the nature of the s amples. Whenever possible, the laboratory should analyze standard reference materials and par ticipate in relevant performance evaluation

studies.

10.0 CALIBRATION AND STANDARDIZATION

The laboratory microwave system should be calibrat ed to measure within ± 2 ° C. Consult the manufacturer’s instructions for microwave syste m calibration.

11.0 PROCEDURE

11.1 The specific procedures that are applied to t he sample depend on the extent of the mercury speciation information that is required.

11.1.1 If only total mercury data are required, th en analyze the sample using Method 7473 or other suitable procedures, or sum th e results for the extractable and nonextractable mercury fractions from Sec. 11.8.SW-846 Update V 3200 - 12 Revision 1 July 2014 11.1.2 If extractable mercury data are required, t hen proceed to Sec. 11.2 for the mercury extraction procedures and use either the mi crowave-assisted extraction procedure (Sec. 11.2.1) or the ultrasound-assisted extraction procedure (Sec. 11.2.2).

11.1.3 If non-extractable mercury data are require d, then proceed to Sec. 11.2 for the mercury extraction procedures, but analyze the residual solids from the extraction procedure as stated in Sec. 11.2.1.5 or 11.2.2.8.

11.1.4 If data are required for extractable organi c mercury, extractable inorganic mercury, or both fractions, proceed to Sec. 11.2 fo r the extraction procedures and then to Sec. 11.4 for the separation and concentration of t he extractable mercury by solid-phase extraction, followed by Sec. 11.5 for the sub-speci ation of the extractable organic and extractable inorganic mercury fractions.

11.1.5 If data for individual extractable mercury species are required, proceed to Sec. 11.2 for the extraction procedures and then pr oceed to Sec. 11.6 for the subspeciation of the individual extractable mercury sp ecies by HPLC or appropriate hyphenated technique (see Sec. 11.8).

11.1.6 If data are required for semi-mobile mercur y, non-mobile mercury, or both fractions, proceed to Sec. 11.2 for the mercury ext raction procedures. Then, process the residual solids generated in Sec. 11.2 through the separation and extraction procedures in Sec. 11.7 for the sub-speciation of the semi-mobile and non-mobile mercury.

11.1.7 For some projects, it may be appropriate or required to report the results of the mercury speciation analyses relative to the dry weight of the original sample. The calculation of the dry weight of the sample is desc ribed in Sec. 12.1.

11.2 Extractable mercury

11.2.1 Microwave-assisted extraction

This extraction involves the use of a solution of 4.0 M HNO 3 to extract mercury species from soil or sediment samples (Ref. 16-18).

11.2.1.1 Weigh approximately 1 g ( ± 0.2 grams, to at least 3 significant figures) of homogenized soil or sediment sample int o microwave extraction

vessels.

11.2.1.2 Add 10.0 mL of 4.0 M HNO 3 to each sample. Add a magnetic stirring bar to each vessel for thorough mixing of solvent with the sample.

11.2.1.3 Seal microwave vessels and irradiate at 100 ° C for 10 min with magnetic stirring on. A 2-min ramping time should be used to reach the desired temperature of 100 ° C.

NOTE : Longer ramping time may be used depending on the microwave power

loading of the system. The extraction time of 10 m in should be maintained independently of the ramping time.

11.2.1.4 Let the vessels cool for safe handling, and then filter the extracts through a 0.22-μm glass fiber filter. Sto re extracts in the cold at ≤ 6 ° C until analysis (within 5 days). This extract conta ins the extractable mercury species and can be analyzed directly, concentrated and then analyzed or dividedSW-846 Update V 3200 - 13 Revision 1 July 2014 for further processing. See Sec. 11.8 for analysis , Sec. 11.4 for separation and concentration and Sec. 11.5 for further speciation.

11.2.1.5 The residual solids may be saved for the direct analysis of the non-extractable mercury by Method 7473 or may be fu rther extracted as semimobile and non-mobile mercury species. See Sec. 11 .8 for analysis and Sec. 11.3 and 11.7 for further speciation.

11.2.2 Ultrasound-assisted extraction

This extraction involves the use of a solution of 2.0% HCl + 10% ethanol to extract mercury species from solid or sediment samples.

11.2.2.1 Weigh approximately 1 g ( ± 0.5 g, to at least 3 significant figures) of sample into a centrifuge tube.

11.2.2.2 Add 2.5 mL of 2.0% HCl + 10% ethanol extraction solvent to each sample. Make sure each tube is properly seale d.

11.2.2.3 Vortex the samples for 1 min then centri fuge the samples for 1 min. Test the pH value of the supernatant. If the pH is greater than 3, add concentrated HCl drop-wise; then vortex, centrifuge , and again test the pH. Repeat this step until the pH value of the supernat ant is between 1.5 and 3. It is recommended to add a few drops (1 drop = 0.05 mL; n ot more than 5 drops) of concentrated acetate buffer (0.2 M, pH 3.0) to the supernatant, before adding HCl, to achieve this pH with minimal reagent addition.

11.2.2.4 Vortex the samples for 1 min. Sonicate samples for 7 min at

60 ± 2 °C.

11.2.2.5 Centrifuge the samples for 5 min at 3100 ± 100 rpm. Decant the supernatant into an appropriate container.

11.2.2.6 Repeat the extraction (Secs. 11.2.2.2, 1 1.2.2.4 and 11.2.2.5) three more times. Additional pH adjustment (Sec. 1 1.2.2.3) is not necessary. Combine all collected extraction supernatants into the same vessel.

11.2.2.7 Add 2.5 mL of reagent water to the sampl e residue. Vortex the samples for 1 min, and then centrifuge the samp les for 1 min. Combine the water rinse with extraction supernatants from 11.2. 2.6. This combined solution contains the extractable mercury species and can be analyzed directly, concentrated and then analyzed, or divided for furt her processing. See Sec. 11.8 for analysis, Sec. 11.4 for separation and concentr ation and Sec. 11.5 for further speciation.

11.2.2.8 The residual solids may be saved for the direct analysis of the non-extractable mercury by Method 7473 or further e xtracted as semi-mobile mercury species and non-mobile mercury species. Se e Sec. 11.8 for analysis and Secs. 11.3 and 11.7 for further speciation.

11.3 Further speciation of extractable mercury and non-extractable mercury

Determine if the extractable mercury fraction is t o be divided into organic and inorganic fractions or into individual mercury species. To c oncentrate the extractable mercury fractionSW-846 Update V 3200 - 14 Revision 1 July 2014 and/or separate it into inorganic and organic mercu ry fractions, proceed to Sec. 11.4. To separate the extractable mercury fraction into indi vidual mercury species, proceed to Sec. 11.6. To separate the non-extractable mercury fraction in to semi-mobile and non-mobile mercury fractions, proceed to Sec. 11.7.

Sub-speciation by alternative techniques, such as GC-MS or chelation resins, may also be appropriate, provided that adequate performance can be demonstrated.

11.4 Isolation of extractable mercury by solid-ph ase extraction

This procedure is appropriate for the separation a nd concentration of the extractable mercury fraction using solid-phase extraction. Ade quate separation has been achieved using SCF as the solid-phase extraction medium (Ref. 6, 1 5, 16, 19-21). The medium is commercially available (Ref. 13) or can be prepared in house fol lowing the procedure described below.

11.4.1 Prepare a mixture containing 50 mL of merca ptoacetic acid, 35 mL of acetic anhydride, 16 mL of acetic acid, 0.15 mL of sulfuric acid and 5 mL of reagent water in a clean vessel. Immerse a 15-g portion of cotto n fiber in this mixture. Cover the vessel and place it in a constant temperature apparatus ca pable of maintaining a temperature of 40 ± 2 °C for four days. Remove the cotton fiber f rom the reagent mixture and place it in a filter funnel (or alternative vacuum filtration dev ice) and rinse with reagent water until the pH of the washings is neutral. Dry the cotton fibe r (now "SCF") at 40 ± 2 °C for two days.

Store the SCF in a cold location at ≤ 6 ° C or in a refrigerator in a dark bottle wrapped wit h aluminum foil. The SCF product is stable for at le ast 3-4 months (Ref. 20). Alternatively, purchase SCF solid phase columns commercially.

11.4.2 Place a PTFE frit at the bottom of an appr opriate size column. Add a 0.2-g portion of the SCF, along with 3 mL of water. Place a second PTFE frit on the top of the SCF. Apply pressure to the top frit to compact the SCF into a homogeneous disk

between the two frits.

NOTE : The bed volume of the SCF disk is approximately 0.7 mL per 0.1 gram of SCF

solid-phase material. For example, in a column wit h an internal diameter of 1.1 cm, the height of the disk is about 1.4 cm. Hi gher capacity can be achieved by using more SCF; however the reagents for separat ion and fractionation should be proportionally increased.

NOTE : The column should be packed on the same day that the separation procedure will

be performed and the SCF disks should be prepared w ith care to avoid channeling. If using commercially available SCF di sks, then same day packing is not required.

11.4.3 Just prior to use condition the SCF disk by passing 10 mL of reagent water, then 10 mL of 6 M HCl and finally 15 mL of r eagent water sequentially through the medium using a flow rate of 1 mL/min.

NOTE : The conditioning procedure should be performed j ust before the separation. After

conditioning, the SCF media must be used within 30 min.

11.4.4 Adjust the pH of the extract from Sec. 11.2 .1.4 or Sec. 11.2.2.7 of the extraction procedure to pH = 6 ± 1 with 10 M NaOH. Filter the solution to retain the particles whose sizes are larger than 1.0 μm. Rins e the retained particles with 3 mL 0.1%

HCl. Combine the filtered solution with the rinse solution.SW-846 Update V 3200 - 15 Revision 1 July 2014 NOTE : The presence of large amount of Fe 3+ in the extracted solution will degrade the

retention and separation ability of the SCF media. It may also induce the transformation from alkyl mercury to inorganic merc ury under acidic condition. After adjusting the pH to 6 ± 1, the precipitation of Fe(OH) 3 is achieved. 1.0-μm filtration will minimize the presence of Fe 3+ in the solution before it is passed through the SCF column. The 0.1% (v/v) HCl rinse w ill minimize the loss of Hg species of interest during the filtration procedure (Ref. 6).

NOTE : 10 M NaOH is preferred over concentrated NH 4 OH for pH adjustment because

NH 4 OH may precipitate out Hg 2+

.

11.4.5 Adjust the pH of the filtered solution to p H = 3 ± 1 with 6 M HCl and pass through the SCF column using a flow rate of ≤ 1 mL/min.

11.4.6 If only the extractable mercury fraction is of interest, remove the SCF disk from the column and analyze it directly using an ap propriate determinative method such as Method 7473 (Ref. 6, 22) to determine the amount of extractable mercury.

11.4.7 Alternatively, the concentrated extractable mercury fraction can be further separated into extractable inorganic mercury and ex tractable organic mercury fractions. If such separation is needed, proceed to 11.5.

11.5 Sub-speciation of inorganic and organic extr actable mercury

11.5.1 Elute the organic mercury species from the SCF solid phase extraction medium by passing 8 mL of eluent 1 (see Sec. 7.12.2 .1) through the SCF column at a

flow rate of ≤ 1 mL/min, followed by 2 mL of water. This eluent i s analyzed using an appropriate determinative method to determine the a mount of organomercury that has been extracted from the sample. See Sec. 11.8 for details.

11.5.2 The extractable inorganic mercury fraction will remain on the SCF and may be analyzed by an appropriate mercury detection method, either by direct analysis of the solid SCF medium or by appropriate dissoluti on of the medium and subsequent analysis. See Sec. 11.8 for details.

11.5.3 As another option, the remaining inorganic mercury fraction can also be eluted from the SCF medium by passing 8 mL of eluen t 2 (see Sec. 7.12.2.2) through

the SCF column at a flow rate of ≤ 1 mL/min, followed by 2 mL of water. This eluent i s analyzed using an appropriate determinative method to determine the amount of inorganic mercury that has been extracted from the sample. See Sec. 11.8 for details.

11.6 Sub-speciation of extractable mercury by HPL C

This procedure is appropriate for the separation o f the extractable mercury fraction into individual mercury species: HgCl 2 , Hg(OH) 2 , Hg(NO 3 ) 2 , HgSO 4 , HgO, Hg 2+ complexes as Hg 2+

ion, and CH 3 Hg + and CH 3 CH 2 Hg + ions.

11.6.1 Adequate separation has been achieved by HP LC using a 30 cm x 4.0 mm, 5-μm pore size C-18 column and a mobile phase c ontaining 30:70 methanol:H 2 O, 0.005% 2-mercaptoethanol, 0.6 M ammonium acetate (R ef. 8, 9).

11.6.2 To ensure compatibility with the HPLC colum n, adjust the pH of the

extract from either Sec. 11.2.1.4 or Sec. 11.2.2.7 or Sec. 11.5.1 or Sec. 11.5.3 to a value in the range of 3 to 7. Dilute samples to an appro priate volume using reagent water.SW-846 Update V 3200 - 16 Revision 1 July 2014 NOTE : Certain metals which may be extracted from the s oil along with the mercury

species may precipitate when the pH is raised.

11.6.3 A precipitate formation due to the pH adjus tment step may interfere with the separation or the detection method, in which ca se it may be necessary to filter the sample. Refer to Sec. 11.4.4

11.6.4 If the presence of a large amount of inorga nic mercury interferes or masks the organic mercury peak then the SCF separat ion described in Sec. 11.4 can be used to eliminate this problem.

11.6.5 Refer to Method 8000 for additional informa tion on HPLC analysis, Method 6800 for speciated isotope dilution, and oth er appropriate analytical detection

methods.

11.7 Sub-speciation of non-extractable mercury

If needed, the remaining matrix material from Sec. 11.2.1.5 or Sec. 11.2.2.8 may

be further divided as semi-mobile and non-mobile me rcury fractions.

Sub-speciation of non-extractable mercury by alter native techniques, such as Method 3052 or other acid leaching and digestion pr ocedure (Ref. 12, 23-25), may also be appropriate.

11.7.1 Semi-mobile mercury species

11.7.1.1 Add 5 mL reagent water to the remaining s ample portion from Sec. 11.2.1.5 or Sec. 11.2.2.8. Vortex the sample continuously for 1 min.

11.7.1.2 Sonicate the sample for 1 min at 60 ± 2 ° C.

11.7.1.3 Centrifuge the mixture for 5 min at 3200 rpm. Transfer the supernatant to a container.

11.7.1.4 Test the supernatant for chloride ions by adding 2-3 drops of chloride ion test solution (Sec. 7.17). Formation of a white precipitate or white turbidity indicates the presence of chloride ions.

11.7.1.5 Repeat the steps in Secs. 11.7.1.1-11.7.1 .4 until the decanted supernatant is free of chloride ions. After testin g for the presence of the chloride ion the supernatant should be discarded.

NOTE : The presence of chloride ions in the soil sample portion may result in the

extraction of both semi-mobile and non-mobile mercu ry species in the same extraction step.

11.7.1.6 Add 5.0 mL of 1:2 (v/v) HNO 3 extraction solution to the remaining soil sample portion from 11.7.1.3. Make sure each vessel is properly sealed. Vortex the sample for 1 min.

11.7.1.7 Heat the mixture for 20 min at 95 ± 2 °C in a heating device

described in Sec. 6.3.SW-846 Update V 3200 - 17 Revision 1 July 2014 NOTE : Gas may evolve, therefore, the vessel should be capped loosely during heating, unless the vessel is pressurizable.

11.7.1.8 Centrifuge the extracted mixture for 5 mi n. Transfer the supernatant to an appropriate container.

11.7.1.9 Repeat the steps in Secs. 11.7.1.6-11.7.1 .8 once more. Combine all collected extraction supernatants in th e same vessel.

11.7.1.10 Add 5.0 mL of reagent water to the remai ning soil sample portion. Vortex the sample for 1 min and then cent rifuge for 5 min. Decant and combine the rinse water with the extract supernatan ts. This solution contains the semi-mobile mercury species and can be analyzed by an appropriate mercury

detection method. See Sec. 11.8 for details.

11.7.1.11 The residual solids may be saved for the analysis of the nonmobile mercury species.

11.7.2 Non-mobile mercury species

11.7.2.1 Analyze the solids from 11.7.1.11 directly for non-mobile

mercury species (Sec. 11.8).

11.7.2.2 Alternatively, perform the following extra ction (Secs. 11.7.2.3-

11.7.2.7) and analyze the extract by appropriate me ans (Sec. 11.8).

11.7.2.3 Add 5.0 mL of 1:6:7 (v/v) HCl:HNO 3 :reagent water extraction solution (Sec. 7.11) to the residual soil sample po rtion from 11.7.1.11. Make sure each vessel is properly sealed. Vortex the sample for 1 min.

11.7.2.4 Heat the mixture for 20 min at 95 ± 2 ° C in a heating device

described in Sec. 6.3.

NOTE : Gas may evolve, therefore, the vessel should be capped loosely during

the heating, unless the vessel is pressurizable.

11.7.2.5 Centrifuge the extracted mixture for 5 mi n. Transfer the supernatant to an appropriate container.

11.7.2.6 Repeat the steps in Secs. 11.7.2.3-11.7.2 .5 once more. Combine all collected extraction supernatants into the same vessel.

11.7.2.7 Add 5.0 mL of reagent water to the remain ing soil sample portion. Vortex the sample for 1 min and then cent rifuge for 5 min. Decant and combine the rinse water with the extract supernatan ts from Sec. 11.7.2.6. This solution contains the non-mobile mercury species an d can be analyzed by an appropriate mercury detection method. See Sec. 11. 8 for details.SW-846 Update V 3200 - 18 Revision 1 July 2014 11.8 Analysis of specific mercury fractions

Many mercury detection techniques are appropriate detection techniques, such as Methods 7473, 1631, 7470, 7471, 6020 and 6800. Oth er mercury detection techniques may also be appropriate, provided that adequate per formance can be demonstrated.

11.8.1 Extractable mercury -- Analyze the extrac table mercury fraction from Sec. 11.2.1.4, 11.2.2.7 or 11.5.3, using an appropriate detection technique.

NOTE : Pre-concentration may be necessary in some cases . The SCF solid-phase

extraction procedure in Sec. 11.4 can be used for t his purpose.

11.8.2 Extractable organic mercury -- Analyze the eluent from Sec. 11.5.1 by an appropriate detection technique. Alternatively, su m the individual organic species

determined in Sec. 11.6.

11.8.3 Extractable inorganic mercury -- Analyze th e SCF disk from Sec. 11.5.2 directly by Method 7473. Alternatively, analyze th e eluent from Sec. 11.5.3 by an appropriate analytical detection technique. As ano ther alternative, analyze either of the sample aliquots from Sec. 11.2.1.4, 11.2.2.7 or 11. 5.3 by quantitating the inorganic peak

as described in Sec. 11.6.

11.8.4 Non-extractable mercury -- Analyze the rema ining solids from Sec. 11.2.1.5 or 11.2.2.8 directly by Method 7473 or by suitable decomposition such as Method 3052 and appropriate analysis procedures. Alternat ively, determine the non-extractable mercury as the difference between the total mercury concentration (Sec. 11.8.5) and the extractable mercury concentration (Sec. 11.8.1).

11.8.5 Total mercury -- Analyze the unaltered sam ple by Method 7473 or by suitable decomposition such as Method 3052 and appr opriate analysis procedures. Alternatively, sum the results from Secs. 11.8.1 an d 11.8.4.

11.8.6 Semi-mobile mercury -- Analyze the extract ed mercury fraction or a portion thereof from Sec. 11.7.1.10 using an approp riate detection technique.

11.8.7 Non-mobile mercury -- Analyze the remainin g solids from Sec. 11.7.1.11 directly by Method 7473 or by other suitable decomp osition such as Method 3052 and appropriate analysis procedures. Alternatively, an alyze the extracted mercury fraction or a portion thereof from Sec. 11.7.2.7 using an appropr iate detection technique (Ref. 12, 22).

12.0 DATA ANALYSIS AND CALCULATIONS

12.1 If appropriate for the specific project, calc ulate the sample dry weight fraction as

follows:

3 1 3 2 

### W W W W Fraction Wt. Dry

−

−

=

where:

W 1 = Weight of sample + vessel before drying, in g

W 2 = Weight of sample + vessel after drying, in gSW-846 Update V 3200 - 19 Revision 1 July 2014 W 3 = Weight of empty, dry vessel, in g

12.2 The results of the analyses of the various me rcury fractions can be converted to the concentration of the fraction in the solid samp le as follows:



### S W D V C ion Concentrat Sample

× × ×

=

where:

C = Concentration in the extract in mg/L

V = Volume of the extract in mL D = Dilution factor from the analysis of the extrac t, if any W = Wet weight of original sample aliquot that was extracted, in g S = Dry-weight fraction of the sample, g/g (if dry- weight reporting is required)

The factors of 1000 in the numerator and the denom inator convert mL to L and g to kg, effectively canceling each other out.

13.0 METHOD PERFORMANCE

13.1 Performance data and related information are provided in SW-846 methods only as examples and guidance. The data do not represent r equired performance criteria for users of the methods. Instead, performance criteria should be developed on a project-specific basis, and the laboratory should establish in-house QC per formance criteria for the application of this method. These performance data are not intended to be and must not be used as absolute QC acceptance criteria for purposes of laboratory accr editation.

13.2 The extractability of pure mercury species ar e summarized in Table 1 (Ref. 6). These data are provided for guidance purposes only.

13.3 The 2.0% HCl + 10% ethanol extraction procedu re was evaluated by spiking natural matrices (mercury species spiked at 25 μg m ercury per 1 g of sample). The natural matrices selected for spiking were silica (SiO 2 ), NIST SRM 2709 (San Joaquin Soil), and two soil matrices. The samples were analyzed by Method 7473 and HPLC-ICP-MS. The results are

summarized in Tables 2 to 5. The "extractable" mer cury fraction in 2.0% HCl + 10% ethanol extraction is compared with Method 1311 (TCLP) by u sing BCR 580 and SRM 2709. The

results are summarized in Table 6. These data are provided for guidance purposes only.

13.4 The SCF solid-phase extraction was optimized. The optimal amount of SCF packed in column was determined to be 0.2 g with a bed volume of 0.7 mL/0.1 g SCF. The best pH range was 3 ± 1. The optimal flow rate was 1.0 mL/min. The optimal eluent 1 was a 1.0 M HCl + 1.0 M NaCl solution which minimizes the inorg anic mercury interference on the organic mercury measurement. The optimal eluent 2 was a so lution of 6 M HCl, saturated NaCl and

0.1% CuCl 2 ⋅ 2H 2 O. The overall performance of SCF solid-phase extr action procedure is

summarized in Table 7. See Ref. 6 for details. T hese data are provided for guidance purposes only.

13.5 This method was validated with Institute for Reference Materials and Measurements (IRMM) reference material BCR 580. Th e results are summarized in Table 8. The samples were analyzed by EPA Method 7473 and HP LC-ICP-MS (Ref. 6). These data are provided for guidance purposes only.SW-846 Update V 3200 - 20 Revision 1 July 2014 13.6 The microwave-assisted extraction method was validated with two specifically prepared reference soil materials, three National I nstitute of Standards and Technology (NIST) standard reference materials (SRM 1941a, SRM 2704 a nd SRM 2709) and one IRMM reference material (BCR 580). The samples were analyzed by E PA Method 7473 and HPLC-ICP-MS. The results are summarized in Table 9 (Ref. 16, 17) . These data are provided for guidance purposes only.

13.7 The microwave-assisted extraction method was also validated by using EPA Method 6800. These results indicate that, based on the matrices tested, the Method 3200 protocol does not induce any transformation from me thylmercury to inorganic mercury or vice

versa. The results are summarized in Table 10. Se e Ref. 16, 17. These data are provided for guidance purposes only.

13.8 The microwave-assisted extraction method was validated by mass balance, that is to say, all species were added up to equal the tota l mercury content in the samples by analysis of two crude oil samples using EPA Method 6800. Th e extractable and non-extractable fractions of mercury species were summed to calcula te the total mercury content in those samples. Method 3200 was used to extract the extra ctable mercury species which consisted of inorganic, methyl-mercury and ethyl-mercury. The t otal mercury contents were determined by Method 6800 SIDMS after digestion with Method 3052. These results indicate that Method 3200 is suitable for mass balance study of mercury species. Non-extractable mercury was spiked in the extracted sample by Method 6800 IDMS and thermally converted to metallic mercury, then subsequently analyzed (Ref. 26). In a ddition, this research paper demonstrates that Method 3200, in combination with method 6800, facilitates measurement of mercury species’ concentrations and including corrections o f the interconversions among these mercury species and achieves mass balance of the mercury sp ecies. Mass balance is the independent measurement of total mercury and its comparison, by adding up all mercury species, a value that is compared and confirmed by the independent t otal mercury measurement. Table 11 and Reference 26 demonstrate that the mercury species i n the sample equal the total, within the stated confidence interval, to the summed measureme nts; that is the 95% confidence interval in the case of the referenced research paper. The res ults are summarized in Table 11. These data are provided for guidance purposes only.

13.9 Both extraction protocols outlined in Method 3200 were demonstrated and validated by analyzing human hair reference materia l (IAEA-085). The results are summarized

in Table 12 and Ref. 27. These results demonstrate that both extraction protocols are capable of extracting approximately 100% of several extract able mercury species from human hair, such as inorganic and methylmercury and that when Method 3200 is coupled with Method 6800 to correct for these species conversions, mass balance is achievable. These data are provided for guidance purposes only.

13.10 The microwave-assisted extraction method was demonstrated and validated by analyzing tuna fish reference material (ERM-CE-464) . The tuna fish SRM was extracted and analyzed by using HPLC-ICP-MS. The percentage recov ery of both mercury species was approximately 80% of the certified value. However, when the SRM was spiked with isotopically enriched analogues of mercury species and Method 68 00 SIDMS is used for quantification, both mercury species were corrected to certified values. These data and those in related publications demonstrate that after equilibration ( as described in Method 6800), the level of analyte recovery is no longer a factor affecting ac curacy when Method 3200 is used in

combination with Method 6800. The results are summa rized in Table 13 (See Ref. 28). These data are provided for guidance purposes only.SW-846 Update V 3200 - 21 Revision 1 July 2014 14.0 POLLUTION PREVENTION

14.1 Pollution prevention encompasses any techniq ue that reduces or eliminates the quantity and/or toxicity of waste at the point of g eneration. Numerous opportunities for pollution prevention exist in laboratory operations. The EPA has established a preferred hierarchy of environmental management techniques that places pol lution prevention as the management option of first choice. Whenever feasible, laborat ory personnel should use pollution prevention techniques to address their waste generation. When wastes cannot be feasibly reduced at the source, the Agency recommends recycling as the next best option.

14.2 For information about pollution prevention th at may be applicable to laboratories

and research institutions consult Less is Better: Laboratory Chemical Management for Waste

Reduction , a free publication available from the American Ch emical Society (ACS), Committee on Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_01229 0/pdf/WPCP_012290.pdf.

15.0 WASTE MANAGEMENT

15.1 The Environmental Protection Agency requires that laboratory waste management practices be conducted consistent with all applicab le rules and regulations. The Agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the lett er and spirit of any sewer discharge permits and regulations, and by complying with all solid an d hazardous waste regulations, particularly

the hazardous waste identification rules and land d isposal restrictions. For further information on waste management, consult the ACS publication li sted in Sec. 14.2.

15.2 All inorganic mercury and organomercury waste should be stored separately in dedicated glass bottles. The waste bottles should be kept in a hood or ventilated area in containers equipped with absorbent pads. Gloves, p ipette tips, etc., which might have come in contact with mercury containing compounds should be disposed in a dedicated waste receptacle fitted with a tight lid.

REFERENCES

1. Bloom, N. S.; Gill, G. A.; Cappellino, S.; Dobbs , C.; McShea, L.; Driscoll, C.; Mason, R.; Rudd, J., "Speciation and Cycling of Mercury in Lav aca Bay, Texas, Sediments." Environ.

Sci. Technol.

, 1999, 33, (1), 7-13.

2. Templeton, D.; Ariese, F.; Cornelis, R.; Daniels son, L. G.; Muntau, H.; van Leeuvan, H. P.; Lobinski, R., "Guidelines for terms related to chem ical speciation and fractionation of elements: Definitions, structural aspects, and met hodological approaches." Pure Appl.

Chem.

, 2000, 72, (8), 1453-1470.

3. Hintelmann, H.; Falter, R.; Ilgen, G.; Evans R. D., "Determination of Artifactual Formation of Monomethylmercury (CH 3 Hg + ) in Environmental Samples Using Stable Hg 2+ Isotopes with ICP-MS Detection: Calculation of Contents App lying Species Specific Isotope

Additions." Fresenius J. Anal. Chem.

, 1997, 358, (3), 363-370.

4. Bloom, N. S.; Coleman, J. A.; Barber, L., "Artif act formation of methyl mercury during aqueous distillation and alternative techniques for the extraction of methyl mercury from environmental samples." Fresenius J. Anal. Chem.

, 1997, 358, (3), 371-377.

5. Rahman, G. M. M.; Kingston, H. M. S.; Bhandari, S., "Synthesis and Characterization ofSW-846 Update V 3200 - 22 Revision 1 July 2014 Isotopically Enriched Methylmercury (CH 3 201 Hg + )." Appl. Organomet. Chem.

, 2003, 17, (12), 913-920.

6. Han, Y.; Kingston, H. M.; Boylan, H. M.; Rahman, G. M. M.; Shah, S.; Richter, R. C.; Link, D. D.; Bhandari, S., "Speciation of mercury in soil and sediment by selective solvent and

acid extraction." Anal. Bioanal. Chem.

, 2003, 375, (3), 428-436.

7. Huo, D.; Kingston, H. M. S.; Larget, B., "Applic ation of Isotope Dilution in Elemental Speciation: Speciated Isotope Dilution Mass Spectr ometry (SIDMS)." In: Comprehensive Analytical Chemistry, Volume 33 (Elemental Speciati on: New Approaches for Trace Element Analysis) , Elsevier, New York, 2000; 277-313.

8. Rahman, G. M. M.; Kingston, H. M. S., "Applicati on of Speciated Isotope Dilution Mass Spectrometry to Evaluate Extraction Methods for Det ermining Mercury Speciation in Soils

and Sediments." Anal. Chem.

, 2004, 76, (13), 3548-3555.

9. Rahman, G. M. M.; Kingston, H. M. S.; Kern, J. C .; Hartwell, S. W.; Anderson, R. F.; Yang, S. -Y., "Inter-laboratory Validation of EPA Method 3200 for Mercury Speciation Analysis using Prepared Soil Reference Materials." Appl. Organomet. Chem.

, 2005, 19, 301-307.

10. Labor, U.S. Department of, Occupational Health Guideline for Organo (Alkyl) Mercury

, Occupational Safety and Health Adminstration, Washi ngton, D.C., 1978.

11. Nierenberg, D. W.; Nordgren, R. E.; Chang, M. B .; Siegler, R. W.; Blayney, M. B.; Hochberg, F.; Toribara, T. Y.; Cernichiari, E.; Cla rkson, T., "Delayed Cerebellar Disease and Death after Accidental Exposure to Dimethylmerc ury." New Engl. J. Med.

, 1993, 338,

1672-1675.

12. Kingston, H. M.; Haswell, S. J., Microwave Enha nced Chemistry: Fundamentals, Sample Preparation, and Applications , 10th ed.; American Chemical Society, Washington, DC,

1997.

13. Applied Isotope Technologies, Inc., www.sidms.c om , e-mail: info@sidms.com.

14. Kingston, H. M. Speciated Isotope Dilution Mass Spectrometry of Reactive Species and

Related Methods. US Patent 6 790 673 B1 , September 14, 2004.

15. Jain, W.; McLeod, C. W., "Rapid Sequential Dete rmination of Inorganic Mercury and Methylmercury in Natural Waters by Flow-injection - Cold Vapor-Atomic-Fluorescence Spectrometry." Talanta

, 1992, 39, (11), 1537-1542.

16. Rahman, G. M. M.; Kingston, H. M. S., "Developm ent of Microwave-Assisted Extraction Method and Isotopic Validation of Mercury Species i n Soils and Sediments." J. Anal. At. Spectrom.

, 2005, 20, 183-191.

17. Rahman, G. M. M. "Development, Research and Val idation of Environmental Speciation Methods: Evaluation by Speciated Isotope Dilution Mass Spectrometry in Mercury and Chromium Speciation Analysis." Ph. D. Dissertation , Duquesne University, Pittsburgh, PA,

2004.

18. Fischer, E.; Tu, Q.; Nagourney, S.; England, R. ; Buckley, B. "Microwave-assisted Solvent
Extraction for Quantitative Simultaneous Extraction of Inorganic Mercury and Methylmercury from Soils." 20 th Annual National Environmental Monitoring Conferenc e

, Washington, DC, July 19-23, 2004; Washington, DC, J uly 19-23, 2004.SW-846 Update V 3200 - 23 Revision 1 July 2014 19. Jain, W.; McLeod, C. W., "Field Sampling Techni que for Mercury Speciation." Anal. Proc.

, 1991 , 293-294.

20. Lee, Y. H.; Mowrer, J., "Determination of Methy lmercury in Natural Waters at the Subnanograms-per-litre Level by Capillary Gas Chromato graphy after Adsorbent Preconcentration." Anal. Chim. Acta

, 1989, 221, (2), 259-268.

21. Mena, M. L.; McLeod, C. W., "Mercury Species Im mobilized on Sulphydryl Cotton: A New Candidate Reference Material for Mercury Speciation ." Mikrochim. Acta.

, 1996, 123, (1-4),

103-108.

22. Boylan, H. M.; Richter, R. C.; Kingston, H. M.; Ricotta, A. C., "Rapid Analysis for the Field: Method Development and Application to Natural Gas U tility Sites." Water Air Soil Poll.

, 2001, 127, (1-4), 255-270.

23. Davis, A. Bloom, N. S.; Hee, Q.; Shane, S., "Th e Environmental Geochemistry and Bioaccessibility of Mercury in Soils and Sediments- A Review." Risk Anal.

, 1997, 17, (5),

105-113.

24. Revis, N. W.; Osborne, T. R.; Holdworth, G.; Ha dden, C., "Distribution of Mercury Species in Soil from a Mercury-Contaminated Site." Water Air Soil Poll.

, 1989, 17, (5), 557-569.

25. Revis, N. W.; Osborne, T. R.; Sedgely, D.; King , A., "Quantitative Method for Determining the Concentration of Mercury (II) Sulphide in Soils and Sediments." Analyst

, 1989, 114,

823-826.

26. Rahman, G. M.; Fahrenholz, T. M.; Kingston, H. M. Skip; Pamuku, M.; Hwang, J. D.; Young, L.A., "Speciation of Mercury in Crude Oil ui sng Speciated Isotope Dilution Mass Spectrometry." Spectroscopy , 2010, 25(1), 36-45.

27. Rahman, G.M.; Fahrenholz, T.M.; Kingston, H. M. Skip, "Application of Speciated Isotope Dilution Mass Spectrometry to Evaluate Methods for Efficiencies, Recoveries, and Quantification of Mercury Species Transformation in Human Hair." J. Anal. At. Spectrom., 2009, 24, 83-92.

28. Reyes, L. H.; Rahman, G. M.; Fahrenholz, T.; Ki ngston, H. M. Skip, "Comparison of Methods with Respect to Efficiencies, Recoveries, a nd Quantitation of Mercury Species Interconversions in Food Demonstrated using Tuna Fi sh." Anal. Bioanal. Chem. , 20008, 390, 2123-2132.

17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION D ATA

The following pages contain the tables and figures referenced by this method.SW-846 Update V 3200 - 24 Revision 1 July 2014 TABLE 1

EXTRACTABILITY OF MERCURY SPECIES

Extractability (%) Species* Operationallydefined Mercury

Fraction 2.0% HCl +

10% Ethanol;

60 °C and

sonication

for 30 min 1:2 HNO 3 :

reagent water;

95 °C for 40

min 1:6:7 HCl:HNO 3 : reagent water;

95 °C for 40 min

CH 3 HgCl Extractable

Organic 98 99 104

C 2 H 5 HgCl 96 94 93 HgCl 2 Extractable

Inorganic 96 99 99 HgO 97 97 102 Hg Semi-mobile 4 95 102 HgS Non-mobile 0.15 0.04 97 Hg 2 Cl 2 1.6 11 96

*10 mg of each pure mercury species in each 10-mL e xtract solutions

Data obtained from Ref. 6. These data are provided for guidance purposes only.

TABLE 2

RECOVERIES OF EXTRACTABLE MERCURY SPECIES SPIKED IN TO A SILICA MATRIX

Mercury spiked species Recovery range* (%) HPLC-ICP-MS HgCl 2 96 ± 2

CH 3 HgCl 98 ± 6

C 2 H 5 HgCl 95 ± 7 HgO 98 ± 4

*95% confidence level, n = 3.

Data obtained from Ref. 6.

These data are provided for guidance purposes only.SW-846 Update V 3200 - 25 Revision 1 July 2014 TABLE 3

RECOVERIES OF EXTRACTABLE MERCURY SPECIES

SPIKED INTO SRM 2709 SOIL MATRIX

Mercury spiked species Recovery* (%) HPLC-ICP-MS Recovery* (%) Method 7473 HgCl 2 98 ± 7 Total extractable, 92 ± 9

CH 3 HgCl 92 ± 17

C 2 H 5 HgCl 92 ± 17

*95% confidence level, n=3

Data obtained from Ref. 6.

These data are provided for guidance purposes only.

TABLE 4

RECOVERIES OF EXTRACTABLE MERCURY SPECIES SPIKED

INTO NATURAL SOIL MATRIX 1

Mercury spiked species Recovery* (%) HPLC-ICPMS Recovery* (%) Method 7473

HgCl 2 106 ± 15 92.5 ± 0.5

CH 3 HgCl 104 ± 10 95.5 ± 2.5

C 2 H 5 HgCl 92 ± 18 91.0 ± 3.0 HgO NA 93.0 ± 1.0

*95% confidence interval with n = 3

The soil matrix 1 was a soil collected from waste d eposit field and sieved through 60- mesh screen, then heated in a 240 °C oven for one w eek. The mercury residue in the soil matrix was less than 30 ng mercury per g of soil.

Data obtained from Ref. 6. These data are provided for guidance purposes only.

TABLE 5

RECOVERIES OF EXTRACTABLE MERCURY SPECIES SPIKED

INTO NATURAL SOIL MATRIX 2

Mercury spiked species Recovery* (%) HPLC-ICP-MS Recovery* (%) Method

7473 HgCl 2 12 ± 6 Total extractable, 62 ± 8

CH 3 HgCl 89 ± 12

C 2 H 5 HgCl 95 ± 15

*95% confidence interval, n=3 The low recovery of the HgCl 2 is probably due to complexing of the Hg 2+ with components of the sample matrix.Data obtained from Ref. 6.

These data are provided for guidance purposes only.SW-846 Update V 3200 - 26 Revision 1 July 2014 TABLE 6

COMPARISON OF 2.0%HCl+10% ETHANOL EXTRACTION WITH M ETHOD 1311 (TCLP)

FOR EXTRACTABLE MERCURY SPECIES

Reference Material 2.0% HCl + 10% Ethanol extraction a Method 1311 b

BCR 580 1.4 ± 0.4 ppm 3 ± 2 ppb

SRM 2709 40 ± 8 ppb 12 ± 9 ppb

a 95% confidence interval, n=3.

b Method 1311 was scaled down to a 1-gram sample size

Data obtained from Ref. 6. These data are provide d for guidance purposes only.

TABLE 7

RECOVERIES OF EXTRACTABLE MERCURY SPECIES

AFTER SCF SOLID-PHASE EXTRACTION

Eluent CH 3 HgCl C 2 H 5 HgCl HgCl 2 Pass-through (10% Ethanol) <0.5% <0.5 <0.5%

1.0 M HCl + 1.0 M NaCl 96% 99% <0.1%

6 M HCl + saturated NaCl + 0.1%

CuCl 2 ⋅ 2H 2 O <3% <3% 98%

Residue (in SCF) <DL <DL <DL

DL = detection limit (0.01 ng)

Data obtained from Ref. 6. These data are provided for guidance purposes only.

TABLE 8

VALIDATION OF THE METHOD USING BCR 580

Method or (Certified Value) Methyl Mercury as Mercury (ppb) Extractable

Inorganic Mercury (ppm) Nonextractable Mercury (ppm) Total Mercury

(ppm)

HPLC-ICP-MS 73.6 ± 6.3 a 1.4 ± 0.4 b 133 ± 9.6 b 134.5 ± 9.6 c

SCF-Method 7473 78 ± 27 b 0.9 ± 0.3 b 127 ± 7 b 128 ± 7 c (Certified Value) 70.2 ± 3.4 NA NA 132 ± 3

a 95% confidence interval with n = 6

b 95% confidence interval with n = 3

c 95% confidence interval with n = 3 NA = not analyzed

Data obtained from Ref. 6.

These data are provided for guidance purposes only.SW-846 Update V 3200 - 27 Revision 1 July 2014 TABLE 9

VALIDATION OF MICROWAVE-ASSISTED EXTRACTION METHOD

Sample Certified / ‘Made-to’

Value (μg/g) Method

7473 (direct analysis) (μg/g) HPLC-ICP-MS (μg/g)

Hg 2+ CH 3 Hg + Total Hg 2+ CH 3 Hg + Total

Material-1 Inorganic Mercury 4.0 ---- 4.08 ±
0.16 4.26 ± 0.17 ND 4.26 ± 0.17

Organic Mercury ---- 4.0 3.58 ±
0.27 ND 3.81 ± 0.20 3.81 ± 0.20

Mixed Mercury 3.0 3.0 5.73 ±
0.58 3.02 ± 0.06 2.66 ± 0.07 5.68 ± 0.09

Material-2 Inorganic Mercury 6.0 ---- 6.73 ±
1.04 6.06 ± 0.56 ND 6.06 ± 0.56

Organic Mercury ---- 6.0 5.44 ±
0.62 ND 5.94 ± 0.52 5.94 ± 0.52

Standard Reference Materials

SRM 1941a 0.5 ± 0.2 ---- 0.61 ±
0.02 0.67 ± 0.06 ND 0.67 ± 0.06

SRM 2704 1.44 ±

0.07 ---- 1.51 ±
0.05 1.40 ± .08 ND 1.40 ± 0.08

SRM 2709 1.40 ±

0.08 ---- 1.46 ±
0.03 1.28 ± 0.12 ND 1.28 ± 0.12

ND = not detectable (ND = 0.05 ng) Uncertainties are expressed at the 95% confidence l evel with n = 3. Material-1: 100% processed topsoil; and Material-2: a mixture of 75% processed topsoil and

25% Ottawa sand

Data obtained from Ref. 16, 17.

These data are provided for guidance purposes only.SW-846 Update V 3200 - 28 Revision 1 July 2014 TABLE 10

THE DECONVOLUTED CONCENTRATION AND TRANSFORMATION O F MERCURY

SPECIES IN MATERIAL-1 USING SIDMS CALCULATIONS.

Deconvoluted Concentration Interconversion Hg 2+
(μg/g) CH 3 Hg +

(μg/g) Hg 2+ to CH 3 Hg +

(%) CH 3 Hg + to Hg 2+

(%)

DSBE 3.05 ± 0.12 2.69 ± 0.10 1.3 ± 1.5 0.1 ± 1.4

DSAE 2.94 ± 0.07 2.62 ± 0.09 0.8 ± 1.5 0.7 ± 0.6

DSBE = double spiked before extraction. DSAE = doub le spiked after extraction. Uncertainties are expressed at the 95% confidence l evel with n = 3

Data obtained from Ref. 16, 17. These data are provided for guidance purposes only.SW-846 Update V 3200 - 29 Revision 1 July 2014 **TABLE 11 SIDMS ANALYSIS (6800) OF THREE EXTRACTABLE MERCURY**  **SPECIES AND NON**

**EXTRACTABLE MERCURY FROM CRUDE OIL AFTER EPA METHOD 3200 EXTRACTION** 

Sample

Name Extractable Mercury Species,

μg/kg Non-Extractable Mercury

Species, μg/kg Total Mercury*,

μg/kg

Hg 2+ MeHg + EtHg +

Serial #

871833 17.3 ±

3.6 0.8 ±

0.1 10.7 ±

1.1 342.5 ± 46.8 370.2 ± 47.0 (414.1

± 24.7)

Serial #

870987 23.6 ±

3.7 0.5 ±

0.1 7.4 ±

2.2 323.3 ± 30.7 354.8 ± 31.0

(437.8 ± 25.4)

Uncertainties are at 95% CL, n = 12 (3 x 4).

*values in parentheses are total mercury determined by isotope dilution mass spectrometry

(IDMS) of the crude oil after Method 3052 digestion

.

Data obtained from Ref. 26. These data are provided for guidance purposes only.

**TABLE 12**

**MERCURY SPECIATION IN HUMAN HAIR CERTIFIED REFERENC E MATERIAL, IAEA-085, BY**  **HPLC-ICP-MS AND QUANTIFICATION BY EXTERNAL CALIBRAT ION.**  **a THE VALUES ARE**  **MEANS ± 95% CL (n = 4 x 4) b** 

**Extraction procedure Hg**  **2+ (as Hg)**  **(μg/g) CH**  **3 Hg**  **+ (as Hg)** 

**(μg/g) Sum of Species** 

**(μg/g) Mean degree of** 

**Transformation (%)**
**Hg 2+**  **to**

**CH 3**  **Hg +**  **CH 3**  **Hg +**  **to**

**Hg 2+** 

**Method 3200 (ultrasonication)**  **0.33 ± 0.06 20.87 ± 0.37**  **(91 ± 2) 21.21 ± 0.38** 

**(91 ± 2) ----- -----** 

**Method 3200 (ultrasonication) and** 

**Method 6800 1.13 ± 0.25**  **19.80 ± 1.25 (87 ± 6)**  **20.93 ± 1.28**

**(90 ± 6) 9 ± 3 0 ± 1** 

**Method 3200 (MAE) 1.80 ± 0.61 22.67 ± 1.54**  **(99 ± 7) 24.67 ± 1.65** 

**(106 ± 7) ----- -----** 

**Method 3200 (MAE)**

**and Method 6800 0.59 ± 0.22**  **23.65 ± 1.42 (103 ± 6)**  **24.24 ± 1.44**

**(105 ± 6) 4 ± 2 6 ± 1** 

**a Certified total Hg is = 23.2 ± 0.8 μg/g, certified**  **CH 3**  **Hg +**  **is = 22.9 ± 1.0 μg/g and estimated Hg 2+**  **is 0.1 – 0.5 μg/g.**  **b The percent recoveries of total Hg and CH**  **3 Hg**  **+ are indicated in parentheses.** 

Data obtained from Ref. 27. These data are provided for guidance purposes only.SW-846 Update V 3200 - 30 Revision 1 July 2014 **TABLE 13**

**MERCURY SPECIATION IN TUNA FISH CERTIFIED REFERENCE MATERIAL (ERM-CE-464)** 

**BY METHOD 6800 HPLC-ICP-MS AFTER EXTRACTION WITH ME THOD 3200 WITH AND** 

**WITHOUT SPIKING ISOTOPICALLY ENRICHED ANALOGUES.**

**Hg 2+**  **(mg/kg) CH 3**  **Hg +**  **(mg/kg) Sum of Species**  **(mg/kg) Degree of transformation,** 

**mean Hg**  **2+ to** 

**CH 3**  **Hg +**  **CH 3**  **Hg +**  **to**

**Hg 2+** 

**Method 3200 0.06 ± 0.04 3.94 ± 0.12**  **(77 ± 2) 4.00 ± 0.13** 

**(76 ± 2) ------ ------** 

**Method 3200**

**and Method**

**6800 0.11 ± 0.07**  **5.60 ± 0.33 (109 ± 6)**  **5.71 ± 0.34**

**(109 ± 6) 18 ± 4 0.8 ± 0.6** 

**Certified total Hg is 5.24 ± 0.10 mg/kg, certified CH**  **3 Hg**  **+ is 5.12 ± 0.16 mg/kg and estimated Hg**  **2+ is 0.12**  **mg/kg. The percent recoveries of total Hg and CH 3**  **Hg +**  **are indicated in parentheses. Isotopic spiking as described in Method 6800 prior**  **to extraction with Method 3200**

Data obtained from Ref.28. These data are provided for guidance purposes only.SW-846 Update V 3200 - 31 Revision 1 July 2014 FIGURE 1

METHYL MERCURY TRANSFORMATION

Solid line: A methyl mercury standard in acid solu tion before the addition of iron Dash line: A methyl mercury standard in acid solut ion with 500 ppm iron.

NOTE : Transformation observed 8 hr after spiking (HPLC flow rate 0.8 mL/min).

Figure taken from Ref. 6. These data are provided for guidance purposes only.SW-846 Update V 3200 - 32 Revision 1 July 2014 FIGURE 2

Solid line: An ethyl mercury standard in acid solu tion without the addition of iron Dash line: An ethyl mercury standard in acid solut ion with 500 ppm iron.

NOTE : Transformation observed 8 hr after spiking (HPLC flow rate 0.8 mL/min).

Figure taken from Ref. 6. These data are provided for guidance purposes only.SW-846 Update V 3200 - 33 Revision 1 July 2014 METHOD 3200

MERCURY SPECIES FRACTIONATION AND QUANTIFICATION BY MICROWAVE ASSISTED EXTRACTION, SELECTIVE SOLVENT EXTRACTION A ND/OR SOLID PHASE

EXTRACTION

11.8.7 Analysis

11.8.3 Analysis

11.8.7 Analysis

11.8.3 Analysis 11.8.2 Analysis 11.8.6 Analysis 11.8.1 Analysis 11.8.4 Analysis 11.8.1 Analysis

11.2.1.5 or 11.2.2.8

11.7.1.10

Semi-mobile Hg extract 11.7.1.11

Non-mobile Hg residue 11.7.1 Extraction

11.7.2.7

Non-mobile Hg extract Total Hg

11.4 SCF Solid-phase

extraction 11.4.6

Extractable Hg

on SCF disk

11.5.1

Extractable Organic Hg

concentrate 11.5.2

Extractable Inorganic Hg

on SCF disk 11.5.1 Elution

of SCF

11.5.3 Elution

of SCF

11.5.3

Extractable Inorganic Hg

concentrate 11.2.1 Microwave

extraction 11.2.2 Ultrasound

extraction

11.7.2

Extraction 11.2.1.4 or 11.2.2.7

Extractable Hg extract Non-extractable Hg residue

11.8.7 Analysis

11.8.3 Analysis

11.8.7 Analysis

11.8.3 Analysis 11.8.2 Analysis 11.8.6 Analysis 11.8.1 Analysis 11.8.4 Analysis 11.8.1 Analysis

11.2.1.5 or 11.2.2.8

11.7.1.10

Semi-mobile Hg extract 11.7.1.11

Non-mobile Hg residue 11.7.1 Extraction

11.7.2.7

Non-mobile Hg extract Total Hg

11.4 SCF Solid-phase

extraction 11.4.6

Extractable Hg

on SCF disk

11.5.1

Extractable Organic Hg

concentrate 11.5.2

Extractable Inorganic Hg

on SCF disk 11.5.1 Elution

of SCF

11.5.3 Elution

of SCF

11.5.3

Extractable Inorganic Hg

concentrate 11.2.1 Microwave

extraction 11.2.2 Ultrasound

extraction

11.7.2

Extraction 11.2.1.4 or 11.2.2.7

Extractable Hg extract Non-extractable Hg residueSW-846 Update V 3200 - 34 Revision 1 July 2014 Appendix A:

Summary of Revisions to Method 3200 (as compared to previous Revision 0, July 2005)

1. Improved overall method formatting for consisten cy with new SW-846 methods style guidance. The revision number was changed to one a nd the date published to July

2014. 2. Updated and expanded “QUALITY CONTROL” section f or better adherence to current SW-846 method guidelines and for improved alignment with current universal practices for published analytical methods. 3. Additional comment added in Sec. 8.3 concerning holding times.

4. Minor editorial and technical revisions were mad e throughout to improve method clarity.