¹ Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, March 1983. Available at NTIS as publication number PB84-128677.

² Methods for the Determination of Metals in Environmental Samples. Available at NTIS as publication number PB91-231498, June 1991.

³ Annual Book of ASTM Standards, Vols. 11.01 and 11.02, 1993, American Society for Testing and Materials, 1916 Race Street, Philadelphia, PA 19103.

⁴ 18th edition of Standard Methods for the Examination of Water and Wastewater, 1992, American Public Health Association, American Water Works Association, Water Environmental Federation.

⁵ Techniques of Water Resources Investigations of the U.S. Geological Survey, Book 5, Chapter A-1, Third Edition, 1989. Available at Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402.

⁶ Samples may not be filtered. Samples that contain less than 1 NTU (nephelometeric turbidity unit) and are properly preserved (concentrated nitric acid to pH *2) may be analyzed directly (without digestion) for total metals, otherwise, digestion is required. Turbidity must be measured on the preserved samples just prior to the initiation of metal analysis. When digestion is required, the total recoverable technique as defined in the method must be used; samples cannot be filtered.

⁷ Fluoride in Water and Wastewater. Industrial Method No. 129-71W. Technicon Industrial Systems. Tarrytown, NY, 10591, December 1972.

⁸ Methods for the Determination of Inorganic Substances in Environmental Samples, EPA/600/R/93/100, August 1993. EPA/Environmental Monitoring Systems Laboratory, Cincinnati, OH 45268.

⁹ (Reserved).

¹⁰ Fluoride in Water and Wastewater, Method No. 380-75WE. Technicon Industrial Systems. Tarrytown, NY 10591, February 1976.

¹ The laboratory director must reject any samples, taken for compliance purposes, not meeting these criteria and notify the authority requesting the analysis.

² If HNO₃ cannot be used because of shipping restrictions, immediately ship the sample to the laboratory at ambient temperature. Upon receipts, the sample must be acidified with conc. HNO₃ to pH <2 and held for at least 16 hours before analysis.

³ P = plastic, hard or soft; G = glass, hard or soft.

⁴ In all cases, samples should be analyzed as soon after collection as possible.

⁵ These samples should never be frozen.

⁶ Ascorbic acid should only be used in the presence of residual chlorine.

⁷ Analyze immediately generally means within 15 minutes of sample collection.

¹ The holding time for Heptachor under this method is 7 days.

² Add 10 mg/l HgCL₂ in any drinking water sample that might be expected to exhibit biological degradation of the target pesticides. Samples that have been preserved with HgCl₂ may be disposed of in at least two ways: as a hazardous waste, or by passing over an absorbent column (i.e., Alumina, activated with carbon, etc.) for mercury absorption, with the effluent analyzed periodically for breakthrough. The absorbent would then be disposed of as a hazardous waste. Other techniques may be applicable.

1VAC30-40-320. General laboratory practices.

A. Sterilization procedures.

1. Media, membrane filters and pads shall be removed immediately after completion of sterilization cycle.

2. Membrane filter assemblies shall be sterilized between sample filtration series. A filtration series ends when 30 minutes or longer elapse between individual sample filtrations.

B. Laboratory pure water.

1. Use only satisfactorily tested reagent water from stills or deionization units to prepare media, reagents and dilution/rinse water for microbiological analyses.

2. QC - Test the quality of the lab pure water or have it tested by a certified lab to assure it meets these criteria:

C. Dilution/rinse water.

1. Prepare stock buffer solution according to Standard Methods, 18th Ed., 1992, Part 9050 C.1.a. Autoclave or filter sterilize stock buffer, label and date container and store in refrigerator. Ensure stored stock buffer solution is free of turbidity.

2. Prepare rinse/dilution water by adding 1.25 mL of stock buffer solution and 5 mL of magnesium chloride (MgCl₂) solution to one liter of lab pure water. Make magnesium chloride solution by adding 81.1 g MgCl₂. 6H₂O or 38g of anhydrous MgCl₂ to one liter of lab pure water. Autoclave rinse/dilution water according to Table IV-1 of this chapter.

3. QC - Check each batch of dilution/rinse water for sterility by adding 50 mL of dilution/rinse water to 50 mL of double strength TSB. Incubate at 35° ± 0.5°C for 24 hours and check for growth.

D. Glassware washing.

1. Washing processes shall provide clean glassware with no stains or spotting. Glassware shall be washed in a warm detergent solution and thoroughly rinsed initially in tap water. Use distilled or deionized water for final rinse.

2. QC - Perform the Inhibitory Residue Test (Standard Methods, 18th Ed., 1992, Part 9020 B.3.a.2) ) on the initial use of a washing compound and whenever a different formulation of washing compound, or washing procedure, is used to ensure glassware is free of toxic residue.

E. Media; general requirements.

1. Use dehydrated or ready to use media manufactured commercially. Store dehydrated media in a cool, dry location away from direct sunlight and discard caked or discolored dehydrated media.

2. Date bottles of dehydrated media when received and when opened. Discard dehydrated media six months after opening; if stored in a desiccator from the time of opening, storage is extended to 12 months. Discard dehydrated media that has passed the manufacturer's expiration date. Unopened dehydrated media should be used within two years of date of receipt.

3. QC - Record the date of preparation, type of medium, manufacturer's lot number, sterilization time and temperature, final pH and technician's initials for media prepared in the laboratory. Store prepared media as described in Table IV-3.

4. QC - Check each batch of laboratory-prepared media and each lot number of commercially prepared (ready to use) media before use with a known positive and a known negative culture control. These control organisms can be stock cultures (periodically checked for purity) or commercially available disks impregnated with the organism.

5. Incubate refrigerated broth in culture tubes and bottles with fermentation vials overnight at 35°C before use. Discard tubes and bottles showing growth or bubbles.

6. Check tubes and bottles of broth before use and discard if evaporation exceeds 10% of original volume.

7. QC - For commercially prepared (ready to use) liquid media and agars, record the date received, lot number and pH verification. Discard media by manufacturer's expiration date.

F. Membrane Filter (MF) Media.

Use m-Endo broth or LES Endo agar for the Membrane Filter Test. Ensure that alcohol used in medium rehydration procedure is not denatured.

Prepare medium in a sterile flask and use a boiling water bath or a constantly attended hot plate with a stir bar to bring medium just to the boiling point. Do not boil medium. Do not autoclave medium.

G. Fermentation technique media.

Use lauryl tryptose broth or lauryl sulfate broth in the presumptive test. The appropriate presumptive test medium concentration will vary according to sample volume (10, 20 or 100 mL) in each culture tube/bottle.

Use single strength brilliant green lactose bile (BGLB) broth in the confirmed test. Use LES Endo agar for the completed test. Prepare m-Endo LES agar as described in this subsection.

H. Presence- Absence (P-A) Medium.

Use triple-strength Presence-Absence Broth for the Presence-Absence Test. Autoclave media for 12 minutes at 121°C, leave space between bottles. Do not leave media in theautoclave for more than 30 minutes.

Use single strength brilliant green lactose bile (BGLB) broth in the confirmed test. Use LES Endo agar for the completed test.

I. ONPG-MUG Test Medium.

Use ONPG-MUG Test Medium for the ONPG-MUG Test for total coliform and E. coli. Do not prepare this medium from basic ingredients. Protect medium from light. Do not autoclave medium.

Each lot of ONPG- MUG Test Medium shall be checked before use with stock cultures or commercially available disks impregnated with Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Use sterile distilled water as the test sample and inoculate three tests from each lot of the ONPG-MUG medium with these cultures and incubate at 35° ± 0.5°C for 24-28 hours. The results shall be yellow color with fluorescence with E. coli, yellow color without fluorescence with K. pneumoniae and no color with no fluorescence with P. Aeruginosa.

J. EC Medium.

Use EC Medium to check for fecal coliforms in total coliform positive MF, Fermentation and P-A Tests.

K. EC Medium + MUG.

Use EC Medium + MUG to check for E. coli in total coliform positive MF, Fermentation and P-A Tests. The medium is made up of EC Medium supplemented with 50 ug/ml of 4-methylumbelliferyl-beta-D-glucuronide (MUG). MUG may be added to EC Medium before autoclaving or EC Medium + MUG may be purchased commercially. Use 10 mL of medium in each cluture tube.

Do not use a fermentation vial. Gas production is not relevant to the test and the use of a fermentation vial may cause confusion on test interpretation.

QC - Check uninocultated culture tubes and medium before use with a 365 or 366 nm ultraviolet light to insure they do not fluoresce.

L. Nutrient Agar + MUG.

Use Nutrient Agar + MUG to check for E. coli in total coliform positive MF Tests. The medium is nutrient agar supplemented with 100 ug/ml of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Sertilize agar in 100 ml volumes at 121°C for 15 minutes.

M. Heterotrophic Plate Count Agar.

Temper melted agar to 44-46°C before pouring plates. Hold melted agar no longer than three hours. Do not melt sterile agar more than once.

N. Trypticase Soy Broth. Tryptic Soy Broth or Tryptose Broth.

Use these broths for sterility checks of sample containers, membrane filters and rinse/dilution water. Also use these broths to rehydrate lyophilized disks of control organisms.

O. Trypticase Soy Agar or Tryptic Soy Agar.

Use this agar to prepare slants for growth and storage of control organisms.

1VAC30-40-330. Analytical methodology.

A. Table IV-6 of this chapter describes the EPA-approved methods which are mandatory for microbiological analyses of drinking water. Laboratories shall meet the sampling and analytical methodology requirements incorporated by reference at 1VAC30-40-85 B 3 for microbiology and 1VAC30-40-85 B 5 for alternative test methods.

¹Standard Methods for the Examination of Water and Waste Water, 18th Edition, American Public Health Association, American Waterworks Association, Water Environment Federation, 1992.

²Federal Register, 40 CFR Part 141.

B. Use only the analytical methodology specified in the National Primary Drinking Water Regulations (40 CFR Part 141.21(f)).

B. A laboratory shall be certified for all analytical methods indicated below that it uses. At minimum, the laboratory shall be certified for one total coliform method, one fecal coliform or E. coli method, and the Pour Plate Method for heterotrophic bacteria.

C. Laboratories shall perform a minimum of 20 coliform analyses monthly by each coliform method for which it is certified in order to maintain certification status or qualify for initial certification. The minimum number of coliform analyses (20) may be performed on a variety of water sample types collected from different stages of the water treatment process, raw source water, surface or ground water, as well as drinking water samples collected from a distribution system or private wells.

If any drinking water sample is total coliform-positive, the lab shall analyze that total coliform-positive culture to determine if fecal coliforms are present, except that the lab may test for E. coli in lieu of fecal coliforms. These tests are described in subsections G, H, and I of this section.

Invalidate any sample results that show interference from noncoliform organisms and request another sample from the same sampling point. This interference is generally caused by heterotrophic bacteria and is exhibited by a turbid culture with no gas formation in the presumptive phase of the Fermentation Test, confluent growth without coliforms or TNTC without coliforms in the Membrane Filter Test, a turbid culture bottle without color change in the Presence-Absence Test, or an indeterminate color change in the ONPG-MUG Test.

Public water systems need only determine the presence or absence of total and fecal coliforms; coliform density determination is not required. 100 mL of sample shall be used for each total coliform test.

Incubate cultures within 30 minutes of inoculation.

C. Membrane filter technique.

Shake sample vigorously before analyzing. Sample volume used shall be 100 ± 2.5 mL.

QC - Conduct MF sterility check by filtering 100 mL of sterile rinse water and plating on m-Endo medium at the beginning and the end of each sample filtration series. If sterile controls indicate contamination, reject all data from that series and request immediate resampling of those waters involved in the laboratory error.

QC - Run a 100 mL sterile rinse water blank between every 10 samples if the number of samples in a series exceeds 10.

Invalidate all samples resulting in confluent growth or TNTC (too numerous to count) without evidence of total coliforms. Record as "confluent growth" or "TNTC" and request an additional sample from the same sampling point. Confluent growth is defined as a continuous bacterial growth, without evidence of total coliforms, covering the entire membrane filter. TNTC is defined as greater than 200 colonies on the membrane filter in the absence of detectable coliforms. Do not invalidate the sample when the membrane filter contains at least one total coliform colony.

Typical coliform colonies have a pink to dark-red color with a metallic golden green sheen. Subject all sheen colonies to verification when there are 10 or fewer sheen colonies. When the number of coliform colonies exceeds 10, randomly pick 10 colonies for verification. Alternatively, swab the entire membrane surface and transfer to the verification media.

Verify sheen colonies using single strength LTB and then single strength BGLB broth, or an EPA-approved cytochrome oxidase and beta-galactosidase rapid test procedure.

To verify colonies in LTB and BGLB broth, use a sterile needle, loop, applicator stick or cotton swab. To verify colonies using the rapid test (cytochrome oxidase/beta-galactosidase test), pick isolated colonies using a sterile needle or applicator stick.

QC - If no coliform positive tests result from potable water samples, perform the MF procedure on a known-positive sample each month. Include the verification test for total and fecal coliform (or E. coli).

D. Fermentation Technique.

100 mL of sample shall be used for each presumptive test. Laboratories may use 10 tubes, 5 tubes or a single culture bottle containing lauryl tryptose broth formulated as described in Table IV-5 of this chapter.

Confirm all gas-positive presumptive tubes and bottles in BGLB Broth. The formation of gas in any amount in the fermentation vial of the BGLB broth tube within a 48 ±3 hour incubation time indicates a positive confirmed test.

QC - All presumptive tubes or bottles with turbidity or heavy growth without gas production shall be submitted to the confirmed test to check for the suppression of coliforms. Invalidate all samples which produce a turbid presumptive culture without gas and request an additional sample from the same sampling point, unless total coliforms are detected in the confirmed test.

QC - On a quarterly basis, conduct the completed test on at least 10% of all coliform-positive samples.

QC - If no coliform-positive tests result from potable water samples, perform the fermentation procedure monthly on a known-positive sample. Perform the confirmed test and the completed test on all coliform-positive tubes or bottles. Include the fecal coliform or E. coli test.

E. Presence-Absence (P-A) Coliform Test.

Inoculate 100 mL of sample into P-A culture bottle.

Observe for turbidity and yellow color or turbidity alone after 24 and 48 hours. Confirm yellow cultures in BGLB broth. The presence of gas in the fermentation vial of the BGLB broth tube within a 48 ±3 hour incubation time indicates a positive confirmation test for total coliforms.

QC - Confirm turbid and yellow cultures or turbid cultures with no color change in BGLB broth. Invalidate all samples which produce a turbid culture with no color change and request an additional sample from the same sampling point, unless coliforms are detected in the confirmed test.

QC - On a quarterly basis, conduct the completed test on at least 10% of all coliform-positive samples.

QC - If no coliform positive tests result from potable water samples, perform the P-A Test on a known positive sample at least once a month. Include the confirmed test, the competed test and the fecal coliform or E. coli test.

F. ONPG-MUG Test.

Use 10 tubes, each containing 10 mL of sample, or a single sterile, transparent, nonfluorescent borosilicate glass culture bottle or equivalent bottle containing 100 mL of water sample.

Avoid prolonged exposure of inoculated tests to direct sunlight. Sunlight may hydrolyze indicator compounds and cause false positive results.

Incubate for 24 hours at 35 ±0.5°C. A yellow color indicates the presence of total coliforms.

If yellow color is detected, check for fluorescence in the dark with a 365 or 366 nm UV lamp. Fluorescence indicates the presence of E. coli.

If the color of the ONPG-MUG culture changes during the initial 24-hour incubation period, but is still not as yellow as the comparator, incubate for another four hours. Do not incubate for more than a total of 28 hours.

QC - If, at the end of the additional four-hour incubation period, the color is still not as yellow as the comparator, invalidate the test and request an additional sample from the same sample site.

Laboratories are encouraged to perform parallel testing between the ONPG-MUG Test and another EPA-approved method for total coliforms for at least several months or several seasons to determine the effectiveness of the ONPG-MUG Test on a variety of water submitted for analysis.

G. Fecal Coliform Test.

Use EC Medium for determining whether a total coliform-positive culture contains fecal coliforms.

Laboratories shall conduct fecal coliform analysis in accordance with the following procedures. When the Fermentation Technique or the Presence-Absence (P-A) Test is used to test for total coliforms, gently agitate the positive presumptive fermentation tube or bottle or the positive P-A bottle and transfer the growth with a sterile 3 mm loop or sterile applicator stick into brilliant green lactose bile broth and EC medium to determine the presence of total and fecal coliforms, respectively. Incubate the BGLB broth at 35° ±0.5°C for 24-48 hours and check for gas. Incubate the EC medium at 44.5° ±0.2°C for 24 ±2 hours and check for gas.

When the Membrane Filter Test is used, verify the sheen colonies by one of the following two methods: Swab the entire membrane filter surface with a sterile cotton swab and inoculate the contents of the swab into LTB. Do not leave the swab in the LTB. Alternatively, pick up to ten individual sheen colonies and inoculate into LTB. Gently agitate the inoculated tubes of LTB to insure adequate mixing. Incubate the LTB at 35° ±0.5°C for 24-48 hours. If the LTB tube shows gas within 24-48 hours, transfer by inoculating loop to a tube of BGLB broth and a tube of EC medium. Incubate the BGLB broth at 35° ±0.5°C for 24-48 hours and check for gas. Incubate the EC medium at 44.5° ±0.2°C for 24 ±2 hours and check for gas. The water level of the water bath shall reach the upper level of the medium in the culture tubes. Gas production of any amount in the inner fermentation tube of the EC medium indicates a positive fecal coliform test. The preparation of EC medium is described in Standard Methods, 18th Ed., 1992, Part 9221 E.1.a.. Public water systems need only determine the presence or absence of fecal coliforms; a determination of fecal coliform density is not required.

H. EC Medium + MUG Test (for E. coli).

Use EC Medium supplemented with 50 ug/mL of 4-methylumbelliferyl-beta-D-glucuronide (MUG). The procedure for transferring and incubating a total coliform-positive culture to EC Medium + MUG is the same as that specified in subsection G of this section for transferring and incubating a total coliform-positive culture to EC Medium. After incubation, observe for fluorescence with a 365 or 366 nm ultraviolet light in the dark. A test is positive for E. coli if the medium fluoresces.

I. Nutrient Agar + MUG Test (for E. coli).

This test is used to determine if a total coliform-positive sample, as determined by the Membrane Filter Technique, contains E. coli.

Use Nutrient Agar supplemented with 100 ug/mL of 4-methylumbelli-feryl-beta-D-glucuronide (MUG). Pour agar into 50 mm Petri dishes.

Pick up to 10 coliform colonies for verification in LTB and BGLB as described in subsection C of this section.

Using sterile forceps, transfer the membrane filter containing one or more suspected coliform colonies from the m-Endo medium to the surface of the Nutrient Agar + MUG medium. Incubate plate at 35° ± 0.5°C for four hours and observe for fluorescence using a 365 or 366 nm ultraviolet lamp in the dark. Any amount of fluorescence on a sheen colony is positive for E. coli.

J. ONPG-MUG Test (for E. coli).

See subsection F of this section.

K. Heterotrophic Plate Count (HPC).

Use the pour plate method to determine the HPC for potable water and lab pure water samples. The pour plate method shall be performed as described in Standard Methods, 18th Ed., 1992, Part 9215 B.

QC - Check each flask of HPC agar for sterility by pouring a final control plate. Reject data if controls are contaminated.

1VAC30-40-340. Sample collection, handling and preservation

A. If a laboratory does not collect samples and has no control over sample collection, handling, preservation and identification, the laboratory director must reject any samples not meeting sampling criteria and notify the authority requesting the analyses. QC The laboratory shall have a written sample rejection policy covering those samples that do not meet sampling requirements.

B. Sample collector shall be trained in sampling procedures and, if required, approved by the VDH-DWSE VDH-ODW.

C. Samples shall be representative of the potable water distribution system. Samples collected from Public Water Supplies public water supplies shall be collected in accordance with a Sample Siting Report sample siting report approved by the VDH-DWSE VDH-ODW. Water taps used for sampling are free of aerators, strainers, hose attachments, mixing type faucets and purification devices. Maintain a steady water flow for at least two minutes to clear the service line before sampling. Collect at least a 100 mL sample volume and allow at least ½ 1/2 inch of space in the sample container to facilitate mixing of sample by shaking.

D. Laboratories that collect as well as analyze samples shall ice samples immediately after collection and deliver the samples directly to the laboratory.

E. Holding/travel time between sampling and analysis shall not exceed 30 hours. If the sample is analyzed after 30 hours, the laboratory shall indicate that the data may be invalid because of excessive delay before sample processing. No samples received after 48 hours shall be analyzed.

All samples received in the laboratory shall be analyzed on the day of receipt. In all cases, samples shall be analyzed as soon after collection as possible.

E. The sample container, required preservation, and maximum holding time requirements for sampling and analyzing microbiological contaminants are incorporated by reference at 1VAC30-40-85 B 3.

F. Sample report.

1. Immediately after collection, enter on the sample report form the sample site location, sample type (e.g. regular, repeat, etc.), date and time of collection, free chlorine residual, collector's name and any remarks.

2. Record the date and time of sample arrival at the laboratory and the date and time analysis begins.

1VAC30-40-360. Action response to laboratory results.

A. Immediately notify the appropriate field office of the VDH-DWSE VDH-ODW of any coliform-positive samples from Public Water Supplies public water supplies.

B. All analytical results for compliance shall be reported directly to the VDH-DWSE VDH-ODW as described in 1VAC30-40-40.

C. Repeat sampling shall be initiated on the basis of coliform presence in either the Fermentation Technique confirmed test, unverified MF Test, P-A confirmed test, or ONPG-MUG Test. Data used to determine monthly compliance may be adjusted by using the Fermentation Technique completed test, verified MF Test results or P- A completed test results.

D. Notify the appropriate field office of the VDH-DWSE VDH-ODW when samples from Public Water Supplies public water supplies are invalidated due to interference from noncoliforms.

Part V
Radiochemistry

1VAC30-40-370. Radiochemistry.

A. Laboratories shall meet the sampling and analytical methodology requirements incorporated by reference at 1VAC30-40-85 B 4 for radiochemistry and 1VAC30-40-85 B 5 for alternative testing methods.

B. For radiochemistry certification of laboratories, DGS-DCLS shall require conformance to USEPA "Manual for the Certification of Laboratories Analyzing Drinking Water," EPA-814B-92-002 Chapter VI, Radiochemistry, September 1992. Appropriate revisions of the manual shall become effective upon issuance.