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1.0 Scope and Application:

1.1 Applicable Matrices: This method may be used to determine methyl mercury (MeHg) concentrations in filtered or unfiltered water samples.

1.2 Minimum Reporting Limit: 0.05 ng/L (nanograms per liter).

1.3 Dynamic Range: This method is designed for the measurement of MeHg in the range of 0.025 - 1 ng/L. The upper range may be extended to higher levels with the selection of a smaller sample volume.

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2.0 Summary of Method: Water samples are distilled to remove potential interferences (WDML SOP004). The pH of the distillate is adjusted to 4.9 using acetate buffer. The distillate is then ethylated using sodium tetraethyl borate (NaTEB) and allowed to react for 15 minutes. Following reaction with NaTEB the distillate is purged with nitrogen gas (N₂) for 20 minutes and the MeHg is collected on a Carbotrap. Mercury species are thermally desorbed from the Carbotrap, separated using a gas chromatography (GC) column, reduced using a pyrolytic column, and detected using a cold vapor atomic fluorescence spectrometry (CVAFS).

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3.0 Safety Issues: Before beginning any of the procedures involved in this method, each individual must read and sign the Chemical Hygiene Plan developed for the lab. Specific safety concerns for each chemical can be found in the Material Safety Data Sheets for that chemical - all of which are located in the laboratory.

3.1 Chronic mercury exposure may cause kidney damage, muscle tremors, spasms, personality changes, depression, irritability and nervousness. Due to the toxicological and physical properties of Hg, only highly trained personnel using extremely cautionary procedures should handle high concentration standards. These cautionary measures include use of latex gloves and high volume hoods when preparing standards.

3.2 Strong acid solutions are employed in the cleaning of equipment and preparation of reagents. Proper acid handling techniques should be employed whenever acids are being used. These techniques include the use of acid resistant clothing and the utilization of high volume fume hoods.

3.3 NaTEB is toxic, gives off toxic gases (triethylboron) and is spontaneously combustible. Any NaTEB use should take place in a high volume fume hood. To discard unused portions of ethylating reagent, in a high volume fume hood empty bottles into a large beaker of 6N hydrochloric acid (HCl). Place beaker on a hotplate and boil down to 1/2 volume then discard remaining solution as an acid waste. Triethylboron will bubble off to the air where it is oxidized to harmless boric acid.

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4.0 Sample Preservative, Containers, and Holding Times:

4.1 Samples are preserved by freezing.

4.2 Sample containers will consist of Teflon bottles cleaned at the laboratory. New Teflon bottles are rinsed with tap water, and cleaned by immersing in 4 N trace pure HCl heated to 65°C for at least 48 hour. Immediately following removal from the bath, the bottles are immersed in fresh reagent grade water and rinsed at least 3 times with reagent grade water. Following the rinsing step, each bottle is filled to 25% with 1% trace pure HCl and capped. The exterior of the bottles is allowed to air dry under a mercury-free class 100 laminar flow hood. Dry equipment is double bagged in new zip-type bags with the unique identifier and date cleaned written on the outer bag. After the initial 48 hr. cleaning, only 24 hr. is required.

4.3 Properly preserved samples may be held for 6 months at the laboratory.

4.4 Distillates must be analyzed within 1 week of distillation.

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5.0 Reagents and Standards:

5.1 Reagents: All reagents and/or dry chemicals used to make reagents must be of the highest purity available from the vendor and shown to be low in mercury. Upon receipt at the laboratory, containers will be marked with the date of receipt and stored in the appropriate areas. When reagents are mixed for use in this method, the person who mixes them will initial and date the reagent container.

5.1.1 Reagent water: Ultra pure reagent grade water shown to be > 18 M starting from pre-purified source (distilled, RO, etc.). The water is delivered through a 0.2 uM filter. All water is obtained from a Millipore Academic water purification system.

5.1.2 Acetate buffer: Dispense approximately 50 mL of reagent water and 11.8 mL of glacial acetic acid into a 100 mL mercury clean class A volumetric flask. Add 27.2 g reagent grade sodium acetate to this solution and dilute to mark with reagent grade water. Transfer buffer to 125 mL mercury clean Teflon bottle for storage. This solution has an indefinite shelf life.

5.1.3 Ethylating Reagent: Sodium Tetraethyl Borate (NaBEt₄) is purchased in 1 gram (Strem 11-0575) sealed bottles and kept in the freezer. In a 125 mL Teflon bottle, dissolve 2 g Potassium Hydroxide (KOH) in 100 mL of reagent grade water and chill to 0°C. Remove a bottle of NaBEt₄ from the freezer, remove the tape seal and rinse outside of bottle with water. Open bottle and pour in about 5 mL of the KOH solution, recap and shake to dissolve. Pour the NaBEt₄ solution into the 125 mL bottle and shake to mix. Immediately, the 1% NaBEt₄ in 2% KOH solution is poured into 10 clean 15 mL Teflon vials which are capped and frozen. The date prepared is written on the vial rack. Indefinite storage if kept frozen and in the dark.

5.1.4 Nitrogen (N₂). Ultra high purity grade 5.0 N₂ is used to bubble the Hg species onto the Carbotraps. The N₂ is first passed through a gold bead trap attached to the outlet of the tank to remove any Hg.

5.1.5 Argon (Ar). Ultra high purity grade 5.0 Ar is used as the carrier gas in the analytical system. The Ar is first passed through a gold bead trap attached to the outlet of the tank to remove any Hg.

5.2 Standards: Upon receipt at the laboratory or on the day of preparation, reagent containers should be labeled with the date received or made and the initials of the person preparing them. The stock and substock standards should by stored outside of the clean laboratory to prevent contamination of the entire lab.

5.2.1 Methyl mercury stock solution (1000 mg/L MeHg as Hg): Dispense approximately 750 mL isopropyl alcohol into a 1 L mercury clean class A volumetric flask. Add 1.252 g of reagent grade methyl mercury chloride and dilute to mark with isopropyl alcohol. This solution has an indefinite shelf life when stored in an amber bottle at room temperature. To clean the volumetric flask, first rinse several times with water, fill to approximately 80 % total volume with 30% HCl, place the ground glass stopper on its side over the opening to prevent pressure buildup, and heat to near boiling on a hotplate for 1 hour.

5.2.2 Methyl mercury substock solution (1 mg/L MeHg): Dispense approximately 50 mL of isopropyl alcohol into a 100 mL mercury clean (sec. 5.2.1) class A volumetric flask. This solution has an indefinite shelf life when stored in a Teflon bottle at 4°C .

5.2.3 Methyl mercury working standard (1 ng/mL MeHg): Dispense approximately 50 mL of reagent grade water into a 100 mL mercury clean (sec. 5.2.1) class A volumetric flask. Pipette 100 µL of the substock standard to the volumetric flask and dilute to volume with reagent water. This working standard must be compared to the previous working standard and agree within ± 5%. This solution should be stored in an amber bag at 4°C and prepared every six months.

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6.0 Quality Control:

6.1 Bubbler blanks: A bubbler blank is prepared by dispensing approximately 100 mL of reagent grade water into a bubbler, adding 200 µL of acetate buffer and 100 µL of ethylating reagent. The bubbler blanks are used to measure and correct for bias created during the reaction and analysis processes.

6.1.1 Acceptance criteria: The maximum acceptable absolute concentration for any one bubbler blank must not exceed 5 picograms (pg).

6.1.2 Corrective actions: If the absolute concentration of any one bubbler blank exceeds 5 pg, another set of bubbler blanks should be run to ensure no operator error. If this second set blanks is also out of control the analyst must isolate and correct the problem before continuing.

6.2 Standards: A standard is prepared by dispensing approximately 100 mL of reagent grade water into a bubbler, pipetting a known amount of working standard into the water, adding 200 µL of acetate buffer and 100 µL of ethylating reagent. Three standards of various concentrations are analyzed at the beginning of a run and their instrument response is used to calculate a mean response factor (RF_m) in nanograms per peak area (ng/PA). An additional standard is run at the end of the analysis batch to evaluate instrument stability.

6.2.1 Acceptance criteria:

6.2.1.1 The percent relative standard deviation (%RSD) among the initial group of three standards must fall between 90 and 110%. The %RSD is calculated using the following formula.

%RSD = (PA_Std / RF_m) x 100

PA_Std = standard deviation among the response, in ng/PA, of the instrument to the standards

6.2.1.2 The ratio of concentration (ng) to instrument response (PA) for the standard analyzed at the end of the batch must fall between 90 and 110% of the RF_m.

6.2.2 Corrective actions:

6.2.2.1 If the %RSD for the initial set of three standards falls outside the acceptable range, another set of standards should be run to ensure no operator error. If this second set standards is also out of control the analyst must isolate and correct the problem before continuing.

6.2.2.2 If the standard analyzed at the end of the batch fails to meet acceptance criteria, a set of standards equivalent to the first set of standards must be analyzed. A RF_m will be calculated using this set of standards. If the RF_m from this second set of standards agrees within + 10% of the initial RF_m, the data will be accepted. If the RF_m from the second set of standards fails to agree within + 10% of the initial RF_m, the data will be evaluated by the QA officer to decide whether they should be flagged or reanalyzed.

6.3 Distillation blank (DB): A distillation blank is prepared according to WDML SOP004. The distillation blank is used to measure and correct for bias created during the distillation process.

6.3.1 Acceptance criteria:

6.3.1.1 The maximum acceptable absolute concentration for any one distillation blank must not exceed 15 picograms. The following formula is used to calculate the absolute DB concentration.

[DB] = (PA_DB - PA_BB) x RF_m

[DB] = absolute concentration of the DB in ng

PA_DB = peak area of the DB

PA_BB = mean peak area of the BBs

RF_m = mean response factor in ng/PA

6.3.1.2 A daily detection limit (DDL) is calculated for each sample from the three distillation blanks in a sample batch by the following formula and may not exceed 50 picograms per liter (pg/L).

DDL = (3 x _[DB]) / V_S

DDL = daily detection limit

_[DB] = standard deviation among the absolute concentrations for the three DBs

V_S = volume of distillate, in liters, analyzed

6.3.2 Corrective actions: If the distillation blanks fail to meet either of the acceptance criteria, the entire batch of samples must be distilled and analyzed again.

6.4 Matrix spike: A matrix spike is prepared by adding a known concentration of working standard to a sample prior to distillation and treating like a sample from that point forward. The matrix spike is used to evaluate the efficiency of the distillation and accuracy of the analysis.

6.4.1 Acceptance criteria: The percent recovery of the matrix spike must fall between 80 and 120%. Percent recovery is calculated as follows:

%R = ([MS] - [S]) / [STD]

%R = percent recovery

[MS]= concentration detected in the matrix spike

[S]= concentration in original sample

[STD] = concentration of std added to the original sample

6.4.2 Corrective actions:

6.4.2.1 If the percent recovery for the matrix spike does not fall between 80 and 120% but the sample replicate (sec 6.5) does meet acceptance criteria, a matrix problem may exist. The analyst must identify and attempt to correct the problem and the samples in the batch must be prepared and analyzed again.

6.4.2.2 If the percent recovery for the matrix spike does not fall between 80 and 120% and the sample replicate fails to meet acceptance criteria, a matrix problem may or may not exist. The entire batch of samples must be prepared and analyzed again.

6.5 Sample replicate: A sample replicate is prepared by adding approximately equal amounts of sample to two separate distillation vials. Replicate samples are used to evaluate the precision of the distillation and analysis procedures.

6.5.1 Acceptance criteria: The relative percent difference between the sample replicates must not exceed 20%.

6.5.2 Corrective actions:

6.5.2.1 If the acceptance criteria are not met for the sample replicate but is met for the matrix spike, the sample used as the replicate will be prepared and analyzed again. A %RSD will be calculated using the results from the three analyses on this sample and must not exceed 20%. If the %RSD does exceed 20%, the entire sample set must be prepared and analyzed again.

6.5.2.2 If the acceptance criteria are not met for the sample replicate nor for the matrix spike, the entire sample batch must be prepared and analyzed again.

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7.0 Procedure:

7.1 Comments:

7.1.1 Interferences:

7.1.1.1 The distillation procedure (WDML SOP004) is intended to eliminate interferences from the water sample during the ethylation and analysis procedure.

7.1.1.2 Large amounts of water vapor absorbed onto the carbotraps during bubbling will cause an instrument response in excess of system sensitivity. The response from the water vapor will mask the response from the methyl mercury in the sample, resulting in loss of the sample.

7.1.2 Helpful hints:

7.1.2.1 When working with detection limits in the parts per trillion range, protection of these samples from contamination cannot be over emphasized. The greatest difficulty in low level MeHg analysis is preventing the samples from becoming contaminated. Extreme caution must be used throughout the bottle preparation, collection and distillation procedures to avoid contamination.

7.1.2.2 Extreme caution should be exercised during bubbler rinsing to avoid residual water in the four-way valve. If water is apparent in the valve, use a cotton swab to remove it before allowing flow through the carbotrap.

7.2 General Description: Refer to section 2.0 for a summary of this method.

7.3 Sample preparation: Samples must be distilled according to WDML SOP004 prior to analysis to remove interferences.

7.4 Equipment:

7.4.1 Flow meter(s) capable of maintaining a N₂ flow of 250 mL/min.

7.4.2 Needle valve to shut off N₂ flow to bubblers.

7.4.3 Bubblers are 250 mL Erlenmeyer flask with the standard 24/40 tapered neck. The sparging stopper is fitted with a special four-way valve, this allows the sample to react with the ethylating reagent in a closed environment, then to be purged onto the Carbotrap without opening the flask.

7.4.4 The Carbotraps are constructed of a 7 mm quartz tube, 4" long and with a constriction at 1 1/4" from the outlet end. A quartz wool plug is placed into the inlet end, about 0.2 g (3 cm in the tube) of Carbotrap-graphitized carbon black (Supelco 2-0287) is added and the inlet end is plugged with another piece of quartz wool.

7.4.5 Female end fittings for Carbotraps are made from small pieces of monobarb Teflon tubing.

7.4.6 Heating 1/4"Teflon tubing and sealing one end by pinching with a pliers until cool creates end plugs.

7.4.7 Analytical balance capable of measuring to the nearest 0.1 g.

7.4.8 All-plastic pneumatic fixed-volume and variable pipettors in the range of 25 µL to 5 mL.

7.4.9 A Hewlett Packard model HP3395 integrator, connected to a timer, controls the analytical system. The timer is connected to a transformer that is connected to a Nichrome wire coil wrapped to fit around the Carbotrap. The Carbotrap is heated to 250°C with a ramp time of 30 seconds.

7.4.10 The GC column is filled with Chromosorb WAW-DMSC 60/80 mesh (Supelco 2-0152) and kept at 60°C.

7.4.11 The Pyrolytic column consists of a 7 mm quartz tube filled with quartz wool and heated to 700-800°C by a Nichrome wire coil attached to a transformer. The column breaks down the mercury species to Hg⁰.

7.4.12 The detector is a commercially available Model 2500 CVAFS Mercury Detector from Tekran (Toronto, ON) equipped with a mass flow controller capable of measuring 20 mL/min.

7.4.13 The detector analog output returns to the HP3395 integrator and the peak areas are recorded.

7.5 Initial setup and sample analysis:

7.5.1 Check pressure in Argon tank to verify adequate volume for the day's analyses.

7.5.2 Turn on the pyrolitic column and sample trap transformers, turn on the sample cooling fan, and check the detector to verify the flow rate is set at 20 mL/min and the baseline is near 0.0100.

7.5.3 Check the temperature of GC column oven to verify it is at 60°C.

7.5.4 Start burning the set of eight sample traps: While these traps are being burned proceed with step 7.4.5.

7.5.4.1 Remove the plugs from the ends of the first trap and place it into the analytical train by threading it, with the id number downstream, through the center of the Nichrome wire coil. Center the Nichrome wire over the graphitized carbon black and press start on the integrator.

7.5.4.2 After the 9-minute cycle is complete repeat the steps in 7.4.4.1 for each of the remaining traps.

7.5.4.3 You need to have 4 traps burned before your step 7.6.5 is complete. You have 8 traps and four bubblers. Sample reaction time is 15 minutes and bubbling time is 20 minutes -- the burning (analyzing) of 4 traps takes 36 minutes, you will be reacting, bubbling, and drying a round of samples while burning the previous round. This is the cycle you will follow throughout the day.

7.5.5 Thoroughly rinse the bubblers and sparging stoppers with reagent grade water.

7.5.6 Dispense approximately 100 mL of reagent grade water into each of the bubblers.

7.5.7 Pipette 100, 100, 50, and 25 µL of working standard into bubblers 1,2,3, and 4 respectively. These bubblers represent your standards.

7.5.8 Pipette 200 µL of acetate buffer and 100 µL of NaTEB to each of the bubblers.

Note: The NaTEB needs to remain at near 0°C. It should be removed from the freezer approximately 5 minutes before being added to the bubbler and placed in the dark place to partially thaw.

7.5.9 Tighten the sparging stoppers, ensure the four-way valve is in the closed position, gently swirl the bubblers, and allow to react for 15 minutes. While the samples are reacting connect the N₂ line to the inlet of the four-way valve.

7.5.10 After the reaction time has elapsed, remove the plugs from the ends of the carbotraps, place them onto the outlet of the bubbler with the id numbers downstream, turn the four-way valve to the open position, and allow to bubble for 20 minutes.

7.5.11 After the samples have been bubbled for 20 minutes, turn the four-way valve to the closed position, remove the Carbotraps from the bubbler outlet, remove the N₂ line from the inlet of the four way valve, and place the Carbotrap on the end of the N₂ line with the id numbers downstream. Allow to dry for 7 minutes.

7.5.12 Thoroughly rinse the bubblers and sparging stoppers with reagent grade water.

7.5.13 Dispense approximately 100 mL of reagent grade water into the first three bubblers. Record the full weight of the first distillation blank then add contents to bubbler #4. Record the empty weight of the bottle.

7.5.14 Repeat steps 7.4.8 - 7.4.11. These bubblers represent your bubbler blanks.

7.5.15 While the bubbler blanks are reacting, remove the Carbotraps from the N₂ lines, attach the N₂ lines to the inlets of the four-way valves, and cap both ends of the Carbotraps. Analyze the Carbotraps used to collect the standards as in 7.4.4.

7.5.16 After the blanks have finished bubbling, thoroughly rinse the bubblers and sparging stoppers with reagent grade water and begin analyzing samples according to the table below.

Round	Bubbler 1	Bubbler 2	Bubbler 3	Bubbler 4
Standards	100 pg	100 pg	50 pg	25 pg
Blanks	BB1	BB2	BB3	DB1
Samples	DB2	DB3	S1	S2
Samples	S3	S4	S5	S6
Samples	S7	S8	S9	S10
Samples	S11	S12	S13	25 pg

BBX = bubbler blank

DBX = distillation blank

SX = sample

To analyze a sample, weigh the full receiving vial, pour the entire contents into the bubbler, weigh the empty vial, and proceed as in 7.4.8 - 7.4.11. After reaction and bubbling burn the traps as in 7.4.4.

7.6 Calibration and performance documentation: During the analysis run, the analyst must evaluate the calibration data, bubbler blank values, MS recovery, and RPDs for duplicate analyses to ensure acceptance criteria (sec. 6.0) are being met. The following information must be recorded in the methyl mercury logbook.

7.6.1 Date of analysis.

7.6.2 Type and date prepared for reagents and standards used.

7.6.3 Name of analyst.

7.6.4 Identification of bubbler contents, volume analyzed, instrument response, and sample trap identification for each analysis performed.

7.6.5 Comments pertaining to special samples run, problem samples, corrective actions taken, and results of any calculations performed to ensure acceptance criteria are being met.

7.7 Shutdown:

7.7.1 After all samples and standards have been run, thoroughly rinse the bubblers and sparging stoppers with reagent water. Fill the bubblers completely with reagent water. Replace the sparging stopper into the bubbler with the four way valve in the open position to allow the frit to fill with water. When the frit is filled turn the four-way valve to the closed position. Store the bubbler in the laminar flow hood.

7.7.2 Shut the N₂ flow off at the tank outlet.

7.7.3 Turn off the transformers for the pyrolytic column and sample trap and turn off sample cooling fan.

7.8 Maintenance, maintenance records and Responsibilities

7.8.1 Gold traps attached to regulators on the N₂ and Ar tanks should be burned clean every time a tank is changed.

7.8.2 Nichrome wire temperature should be checked quarterly.

7.8.3 Detector lamp driver voltage should be checked quarterly. If voltage exceeds 12.5, the lamp should be adjusted or replaced according to manufacturers guidelines.

7.8.4 Carbotraps should be repacked fresh, burned clean, and tested every 6 months.

7.9 Calculations: An EXCEL spreadsheet created for this method handles all of the calculations. Data is entered from the benchsheets filled out during the distillation and analysis procedures. A final concentration is calculated as follows:

7.9.1 The concentration of MeHg is calculated for the distillate aliquot analyzed and is corrected for the average distillation and bubbler blanks.

7.9.2 The concentration is calculated for the original sample aliquot distilled by correcting the concentration in the distillate for the percent distilled.

7.10 Data validation and evaluation: After the data has been entered into the EXCEL spreadsheet, someone other than the analyst must verify that no values have been incorrectly entered on either the bench sheets or in the spreadsheet. The data is then evaluated carefully by the QC officer to ensure all data quality objectives have been met for the run and that the data seem reasonable. Data is evaluated as to reasonability if historical data from a site exists.

7.11 Reporting:

7.11.1 Reporting units: Methyl mercury in ng/L as Hg.

7.11.2 Reporting levels and significant figures:

7.11.2.1 Report to the nearest 0.01 ng/L for values less than 10 ng/L.

7.11.2.2 Report to three significant figures for values exceeding 10 ng/L.

7.11.3 Data transfer: After the data has been verified in the EXCEL spread sheet it may be transferred to the customer via e-mail, hard copy, or the internet.

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8.0 Archiving: All raw data produced in the laboratory is archived in a filing cabinet located in the laboratory manager's office. Hard copies of EXCEL spreadsheets and data reports are archived with raw data. All electronic data is archived on the laboratory manager's computer, which is backed up to tape daily.

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9.0 References:

9.1 Method source:

Horvat, M., Liang, L., Bloom, N.S. 1993, Comparison of distillation with other current isolation methods for the determination of methyl mercury compounds in low level environmental samples. Part II. Water. Analytica Chimica Acta. 282: 153-168

Olson, M.L. Cleckner, L.B., Hurley, J.P., Krabbenhoft, D.P., Heelan, T.W. 1997, Resolution of matrix effects on analysis of total and methyl mercury in aqueous samples from the Florida Everglades. Fresenius Journal Analytical Chemistry. 358: 392-396

9.2 Deviations from source method and rationale:

9.2.1 Deviations from Horvat et al are outlined in Olson et al 1997.

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