**SW-846 Update V 3200 - 1 Revision 1**

**October 2012 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** 

**October 2012 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 a** **SW-846 Update V 3200 - 3 Revision 1**

**October 2012 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 “sub**  **speciation.”**

**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 the** **SW-846 Update V 3200 - 4 Revision 1**

**October 2012 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 non**  **extractable 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. A** **SW-846 Update V 3200 - 5 Revision 1**

**October 2012 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 g SW-846 Update V 3200 - 6 Revision 1** 

**October 2012 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 of** **SW-846 Update V 3200 - 7 Revision 1**

**October 2012 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 equivalent SW-846 Update V 3200 - 8 Revision 1** 

**October 2012 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 ically** 

**enriched 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 equivalent SW-846 Update V 3200 - 9 Revision 1** 

**October 2012 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 all** **SW-846 Update V 3200 - 10 Revision 1**

**October 2012 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 re**  **prepared 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 carried** **SW-846 Update V 3200 - 11 Revision 1**

**October 2012 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 project specific 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 non**  **extractable mercury fractions from Sec. 11.8. SW-846 Update V 3200 - 12 Revision 1** 

**October 2012 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 sub**  **speciation 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 4**  ° **C until analysis (within 5 days). This extract contains th**  **e extractable mercury species and can be analyzed directly, concentrated and then ana**  **lyzed or divided for further SW-846 Update V 3200 - 13 Revision 1** 

**October 2012 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 semi**  **mobile 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 fraction** **SW-846 Update V 3200 - 14 Revision 1**

**October 2012 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**

**October 2012 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**

**October 2012 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** 

**October 2012 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 non** 

**mobile 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**

**October 2012 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 g SW-846 Update V 3200 - 19 Revision 1** 

**October 2012 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** 

**October 2012 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** 

**October 2012 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 of** **SW-846 Update V 3200 - 22 Revision 1**

**October 2012 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** 

**October 2012 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 Sub**  **nanograms-per-litre Level by Capillary Gas Chromato graphy after Adsorbent Pre** 

**concentration."** 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**

**October 2012 TABLE 1** 

**EXTRACTABILITY OF MERCURY SPECIES**

**Extractability (%) Species** * **Operationally**

**defined 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** 

**October 2012 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-ICP**

**MS** **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** 

**October 2012 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)**  **Non**

**extractable 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** 

**October 2012 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** 

**October 2012 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**

**October 2012** TABLE 11 SIDMS ANALYSIS (6800) OF THREE EXTRACTABLE MERCURY SPECIES AND NONEXTRACTABLE 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**

**October 2012** 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**

**October 2012 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**

**October 2012 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**

**October 2012 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 residue** **SW-846 Update V 3200 - 34 Revision 1**

**October 2012 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. 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.**