Modified by David Kelly and Vincent St.Louis. Original version written by Mark L. Olson and John F. De Wild, Wisconsin District Mercury Laboratory, U.S. Geological Survey, Madison, Wisconsin. This protocol has been listed here with permission of the original authors.
1.0 Scope and Application
1.0 Application:
2.0 Summary of Method
3.0 Performance Issues
4.0 Equipment
5.0 Reagents and Standards
6.0 Sampling
7.0 Validation Procedures
8.0 Safety
9.0 Analysis Procedures
10.0 Calculations
11.0 Reporting of Results
12.0 Archiving
13.0 References
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: 0.025 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.
Water samples are distilled to remove potential interferences (Distillation Protocol). 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 20 minutes. Following reaction with NaTEB the distillate is purged with nitrogen gas (N2) for 15 minutes and the MeHg is collected on a Carbotrap after which tubes are dried for 15 minutes. Mercury species are thermally desorbed from the Carbotrap, separated using a gas chromatography (GC) column, reduced using a pyrolytic column, and detected by cold vapor atomic fluorescence spectrometry (CVAFS).
3.1 The distillation procedure (Distillation Protocol) is intended to eliminate interferences from the water sample during the ethylation and analysis procedure.
3.2 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.
4.1 Flow meter(s) capable of maintaining a N2 flow of 250 mL/min.
4.2 Needle valve to shut off N2 flow to bubblers.
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.
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.
4.5 End fittings for Carbotraps are made from heat shrink teflon tubing and solid teflon rod.
4.6 Analytical balance capable of measuring to the nearest 0.1 mg.
4.7 All-plastic pneumatic fixed-volume and variable pipettors in the range of 25 無 to 5 mL.
4.8 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 250oC with a ramp time of 30 seconds.
4.9 The GC column is filled with Chromosorb WAW-DMSC 60/80 mesh (Supelco 2-0152) and kept at 68oC.
4.10 The Pyrolytic column consists of a 7 mm quartz tube filled with quartz wool and heated to 700-800oC by a Nichrome wire coil attached to a transformer. The column breaks down the mercury species to Hg0.
4.11 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.
4.12 The detector analog output is acquired using Varian Star Data Aquisition Workstation hardware and software (Ver. 5.31).
5.1 Reagents: All reagents and/or dry chemicals used to make reagents must be of appropriate purity with respect to mercury. Upon receipt at the laboratory, containers will be marked with the date of receipt and stored appropriately. When reagents are mixed for use, 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 MW starting from RO feedwater. The water is delivered through a final 0.2 uM filter. All water is obtained from a Millipore Gradient A10 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 teflon bottle. Add 27.2 g reagent grade sodium acetate to this solution and dilute gravimetrically with reagent grade water. This solution has an indefinite shelf life.
5.1.3 Ethylating Reagent: Sodium Tetraethyl Borate (NaTEB) is purchased in 1 gram (Strem 11-0575) sealed bottles and kept in the freezer. This reagent is dangerous. Read and understand MSDS and conduct the entire procedure in a fumehood with appropriate protective clothing and gloves. In a 125 mL Teflon bottle, chill 100 mL of reagent grade water to 0oC (until just slushy). Remove a bottle of NaTEB from the freezer, remove the tape seal and rinse outside of bottle with water. Open bottle and pour in about 5 mL of the cold water, recap and shake to dissolve. Pour the NaTEB solution into the 125 mL bottle and shake to mix. Keep on ice for remainder of procedure. Using an Eppendorf repeat pipettor and Combitip, draw up solution and immediately and efficiently dispense 1 mL reagent into 10 2 mL acid washed borosilicate vials with teflon lined caps (Chromatographic Specialties # C85B75562), sealing and freezing each set of 10 vials immediately, until the entire batch has been dispensed. Vials are stored in an amber bag at -200 C with the date of preparation recorded.
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 Stock methyl mercury (approximately 1000 mg/L MeHg as Hg) and secondary stock methyl mercury (approximately 1 mg/L as Hg) are prepared and certified by Alfa custom chemical services (www.alfa.com).
5.2.2 Working concentration MeHg standard (approximately 0.3 ng/L MeHg as Hg) is prepared by serial dilution of the secondary stock in a teflon bottle. 500 uL of glacial acetic acid and 200 uL of HCl is added to the final solution.
5.2.3 New working concentrations must agree within 5% of the previous WC solution, and be calibrated against a vapour standard.
6.1 Samples are preserved by freezing or where this is not possible, by adding Baker Instra-analysed grade HCl (conc) to 0.25% in the field. In the latter case, samples are stored no longer than 2 weeks, dark and refridgerated, prior to analysis. Acid concentration is brought to 1% just prior to distillation and substitutes for the usual distillation reagent acid.
6.2 Sample containers will consist of Teflon bottles cleaned at the laboratory. Teflon equipment is rinsed with tap water, and cleaned by heating in reagent grade HNO3 (conc) at 80oC overnight. After rinsing in reagent grade water, equipment is transferred to a 4 N Baker Instra-analysed HCl bath heated to 65oC overnight. Following a rinse in reagent grade water, bottles are immediately rinsed at least 3 times with reagent grade water, then filled with reagent grade water and brought to 1% with Baker Instra-analysed HCl and capped. Following drying in a Hepa-filtered air flow, bottles are double bagged with the unique etched identifier marked on the outer ziplock bag.
6.3 Distillates must be analyzed within 1 week of distillation.
7.1 Bubbler blanks: A bubbler blank is prepared by dispensing approximately 100 mL of reagent grade water into a bubbler, adding 500 無 of acetate buffer and 100 無 of ethylating reagent. The bubbler blanks are used to measure and correct for bias created during the reaction and analysis processes.
7.1.1 Acceptance criteria: The maximum acceptable absolute concentration for any one bubbler blank must not exceed 5 picograms (pg).
7.1.2 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.
7.2 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 500 無 of acetate buffer and 100 無 of ethylating reagent. Four standards are analyzed at the beginning of a run and their instrument response is used to calculate a mean response factor (RFm) in nanograms per peak area (ng/PA). This response factor must match that calculated using a vapour standard within the acceptance criteria or the analyst must repeat the test and if necessary isolate and correct the problem before proceeding. Standards and bubbler blanks are run alternately during each analysis batch to evaluate instrument stability.
7.2.1 Acceptance criteria:
7.2.1.1 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 = (sPAStd / RFm) x 100
sPAStd = standard deviation among the response, in ng/PA, of the instrument to the standards
7.2.1.2 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 RFm.
7.2.2 Corrective actions:
7.2.2.1 If the %RSD for the initial set of four 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.
7.2.2.2 If at any point during the day the standard analyzed with a batch fails to meet acceptance criteria, a set of standards equivalent to the first set of standards must be analyzed. A RFm will be calculated using this set of standards. If the RFm from this second set of standards agrees within + 10% of the initial RFm, the data will be accepted. If the RFm from the second set of standards fails to agree within + 10% of the initial RFm, the data will be evaluated by the QA officer to decide whether they should be flagged or reanalyzed.
7.3 Distillation blank (DB): A distillation blank is prepared according to Distillation Protocol. The distillation blank is used to measure and correct for bias created during the distillation process.
7.3.1 Acceptance criteria:
7.3.1.1 The maximum acceptable absolute concentration for any one distillation blank (DB) must not exceed 15 picograms. The following formula is used to calculate the absolute DB concentration.
[DB] = (PADB PABB) x RFm
[DB] = absolute concentration of the DB in ng
PADB = peak area of the DB
PABB = mean peak area of the BBs
RFm = mean response factor in ng/PA
7.3.1.2 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 s[DB]) / VS
DDL = daily detection limit
s[DB] = standard deviation among the absolute concentrations for the three DBs
VS = volume of distillate, in liters, analyzed
7.3.2 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.
7.4 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.
7.4.1 Acceptance criteria: The percent recovery of the matrix spike must fall between 75 and 125%. 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
7.4.2 Corrective actions:
7.4.2.1 If the percent recovery for the matrix spike does not fall between 75 and 125% 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.
7.4.2.2 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.
7.5 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.
7.5.1 Acceptance criteria: The relative percent difference between the sample replicates must not exceed 20%.
7.5.2 Corrective actions:
7.5.2.1 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.
7.5.2.2 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.
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.
8.1 Chronic mercury exposure may cause kidney damage, muscle tremors, spasms, personality changes, depression, irritability and nervousness. Reproductive toxicity and teratogenicity is well known, particularly with respect to methyl mercury. Serious exposure could have long term effects on the ability of women or men to have a healthy child. Due to the toxicological and physical properties of Hg, only highly trained personnel using extremely cautionary procedures should handle high concentration standards. It is preferable that high concentration alkyl mercurials be purchased commercially when available. These cautionary measures include use of appropriate gloves and high volume hoods when preparing standards. Medical surveillance testing is required by law in Alberta and in addition to baseline and annual urine testing required for inorganic mercury, users exposed to alkyl mercury compounds including methyl mercury are required to undergo baseline and annual blood monitoring. The testing protocols have been developed between this lab and the University Health Centre, and personnel should take the appropriate cards with them when ordering this test.
8.2 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, the utilization of high volume fume hoods and where this is not possible the use of appropriate full-face cartridge or airline respirators, as follows. Rinsing following nitric acid bath procedures can generate potentially fatal acid mists, against which cartridge-based sorbant respirators are not effective (MSDS # N3660;Mallinckrodt Baker Inc.: Effective Date 7/13/00 Supercedes 6/30/98). A full face shield Scott Ska Pack with an air line supply (compressed air suitable for respiration) and a 5 minute escape bottle is required at this station. Suitable training should be acquired before personnel use this station. In the event of serious contamination, the 5 minute escape bottle can be opened and the airline disconnected. This allows the user 5 minutes to access decontamination equipment out of reach of the airline if necessary. The use, type and location of this equipment should be physically demonstrated as part of this training.
8.3 NaTEB (Sodium tetraethylborate): Skin, eyes, respiratory, CNS toxicity and unknown chronic toxicity. Decomposes to release triethylboron (highly toxic). NEVER handle, use or weigh stock outside a fumehood. Users have reported near instantaneous headaches when a 1% solution was handled briefly outside a fumehood. Disposing of NaTEB: Save NaTEB in the small glass jars and caps bagged in the freezer until you plan to dispose of it. In a fumehood, pour used 1% NaTEB into a 4 L beaker containing tap water and HCl (about 50% v/v). Put the empty teflon vials into another beaker. Rinse the empty vials, caps and glass jars a few times, pouring rinse water into the beaker containing the HCl and NaTEB. Heat the NaTEB and acid mixture to boiling to drive-off triethylboron (which is eventually oxidized to harmless boric acid). Boil-down the mixture to at least half volume, cool, neutralize and dispose of the HCl residue. Rinsed NaTEB vials should be cleaned in a concentrated acid bath before reuse.
9.1 Comments:
9.1.2 Helpful hints:
9.1.2.1 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.
9.1.2.2 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.
9.2 General Description: Refer to section 2.0 for a summary of this method.
9.3 Sample preparation: Samples must be distilled according to Distillation Protocol prior to analysis to remove interferences.
9.4 Initial setup and sample analysis:
9.4.1 Check pressure in Argon tank to verify adequate volume for the day's analyses.
9.4.2 Turn on the pyrolitic column (30 minutes in advance) 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.
9.4.3 Check the temperature of GC column oven to verify it is at 68oC.
9.4.4 Pre-burn the set of eight sample traps.
9.4.5 Thoroughly rinse the bubblers and sparging stoppers with reagent grade water.
9.4.6 Dispense approximately 100 mL of reagent grade water into each of the bubblers.
9.4.7 Pipette appropriate volumes of working standard into bubblers 1,2,3, and 4 respectively. These bubblers represent your standards
9.4.8 Pipette 500 無 of acetate buffer and 100 無 of NaTEB to each of the bubblers. Note: The NaTEB needs to remain at near 0oC. 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. Return to the freezer between bubbler batches. Each 2 mL vial contains enough NaTEB to rinse a pipette tip, fill four bubblers and repeat for the next set. This avoids chronic ethylation failures of larger volumes of this air-, light- and water-sensitive reagent.
9.4.9 Close the 4-way stopcock, swirl the bubblers, and allow to react for 20 minutes.
9.4.10 Attach carbotraps and nitrogen lines, open stopcock and purge bubblers for 15 minutes.
9.4.11 Dry carbotraps in a nitrogen stream for 15 minutes.
9.4.12 Suction bubblers to waste using a teflon line in the fumehood. Fill and rinse bubblers a minimum of 4 times with reagent water, including the spargers.
9.4.13 A record of all distilled sample weights is maintained in spreadsheet form from the initial distillation day. These weights will be used to calculate concentrations.
9.4.14 Repeat for bubbler blanks, distillation blanks, distillation standards, spikes and 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 |
Blank |
|
Samples |
S10 |
S11 |
S12 |
Standard |
BBX = bubbler blank
DBX = distillation blank
SX = sample
9.5 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.
9.5.1 Date of analysis.
9.5.2 Type and date prepared for reagents and standards used.
9.5.3 Name of analyst.
9.5.4 Identification of bubbler contents, volume analyzed, instrument response, and sample trap identification for each analysis performed.
9.5.5 Comments pertaining to special samples run, problem samples, corrective actions taken, and results of any calculations performed to ensure acceptance criteria are being met.
9.6 Shutdown:
9.6.1 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.
9.6.2 Shut the N2 flow off at the tank outlet.
9.6.3 Turn off the transformers for the pyrolytic column and sample trap and turn off sample cooling fan.
9.7 Maintenance records are kept for each Tekran.
9.7.1 Gold traps attached to regulators on the N2 and Ar tanks should be burned clean every time a tank is changed.
9.7.2 Nichrome wire and pyrolysis tube temperature should be checked daily.
9.7.3 Change carbotraps every 4 months.
A Microsoft 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:
10.1 The concentration of MeHg is calculated for the distillate aliquot analyzed and is corrected for the average bubbler blanks, distillation reagent contribution, reagent water if any has been added to sample, and recovery.
10.2 Data validation and evaluation: After the data has been entered into the Microsoft 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.
11.1 Reporting units: Methyl mercury in ng/L as Hg to three significant figures.
11.2 Data transfer: The Microsoft EXCEL spread sheet may be transferred to the customer via e-mail, hard copy, or the internet.
All raw data produced in the laboratory is archived in a filing cabinet located in the laboratory manager's office. Hard copies of Microsoft 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 CD weekly stored in a separate location.
13.1 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
13.2 Deviations from source method and rationale:
13.2.1 Deviations from Horvat et al. are outlined in Olson et al 1997.