Final Text
Part I
General Provisions
1VAC30-40-10. Introduction.
The Safe Drinking Water Act (SDWA) of December 16, 1974, mandated the establishment of drinking water regulations. The United States Environmental Protection Agency (USEPA) was authorized to set the national drinking water regulations and oversee the implementation of the SDWA. State governments through their health departments or environmental agencies were to accept the responsibility for the implementation and enforcement of the SDWA'S provisions.
The Virginia Department of Health, Division of Water Supply
Engineering (VDH-DWSE) Office of Drinking Water (VDH-ODW) has
accepted and maintains the primary enforcement responsibility (primacy) under
the SDWA and the requirements of the National Primary Drinking Water
Regulations (NPDWR) 40 CFR 141, 142 and 143 (2009). The regulation at
40 CFR 141.28 requires that all testing for compliance purposes except
turbidity, free chlorine residual, temperature and pH be performed by
laboratories certified by the state.
The Department of General Services, Division of Consolidated
Laboratory Services (DGS-DCLS) has been designated by VDH- DWSE VDH-ODW
as the principal state laboratory. Pursuant to regulation 40 CFR
142.10(b)(3)(i) (2009), DGS-DCLS has established and maintains the state
program for the certification of laboratories conducting analytical
measurements of drinking water contaminants.
This chapter provides the mechanism to assure that laboratories are capable of providing valid data for compliance under the SDWA.
1VAC30-40-20. Definitions.
The following words and terms, when used in this chapter, shall have the following meanings, unless the context already indicates otherwise:
"Analyst" means a chemist, microbiologist, physicist, or technician who actually performs a test. The analyst may carry out the complete test or participate jointly with other analysts. The qualifications an analyst needs depend greatly on functions being performed.
"Certifying team" means experienced DGS-DCLS professionals to perform laboratory on-site evaluations under the SDWA.
"CFR" means Code of Federal Regulations.
"Compliance sample" means any sample required by the Virginia Department of Health to determine that the water quality does not exceed the maximum contaminant level (MCL) for each specified parameter.
"DGS-DCLS" means the Department of General Services-Division of Consolidated Laboratory Services.
"EMSL-LV" means the Environmental Monitoring Systems Laboratory in Las Vegas, Nevada.
"Maximum contaminant level (MCL)" means the maximum permissible level of a contaminant in water which is delivered to the free flowing outlet of the ultimate user of a waterworks.
"Minimum requirements" means criteria which are critical to the generation of valid data. These criteria describe the lowest level of capability at which the analyses can be successfully performed.
"NPDWR" means the National Primary Drinking Water Regulations (40 CFR 141 et seq.) (2009).
"Performance evaluation sample" means annual sample to be analyzed by a laboratory on certain parameters for which certification has been requested or granted. This annual sample is a form of documentation of a laboratory's capabilities in conjunction with on-site inspection evaluations of the laboratory by the certifying team.
"Primary enforcement responsibility (Primacy)" means the primary responsibility for administration and enforcement of primary drinking water regulations and related requirements applicable to public water systems within a state.
"Quality Assurance (QA) Plan" means a written description of a laboratory's quality assurance activities.
"SDWA" means the Safe Drinking Water Act (21 USCS
§ 349; 42 USCS §§ 201, 300f to 300j-9) (42 USC § 300 f et seq.).
"TTHM" means Total Trihalomethanes.
"USEPA" means the United States Environmental Protection Agency.
"VDH-DWSE" "VDH-ODW" means
the Virginia Department of Health-Division of Water Supply Engineering Virginia
Department of Health - Office of Drinking Water.
"Virginia laboratory officer" means the DGS-DCLS coordinator of drinking water laboratory certification activities.
1VAC30-40-30. Public notification for exceeded MCL.
The public notification regulations require that a laboratory
analyzing compliance samples immediately notify the VDH-DWSE VDH-ODW
of all results which exceed an MCL in accordance with Virginia Waterworks
Regulations, 12VAC5-590-530 and 12VAC5-590-540, June 23, 1993; the
Public Notification Final Rule, Federal Register Vol. 52, No. 208, October 28,
1987; and the Public Notification Technical Amendment, Federal Register Vol.
54, No. 72, April 17, 1989.
1VAC30-40-40. Compliance data report.
A. A waterworks with an on-site certified laboratory
shall follow the reporting requirements outlined in Virginia Waterworks
Regulations, VR 355-18-005.09, 2.20 Reporting, June 23, 1993 12VAC5-590-530.
B. A contract laboratory analyzing compliance samples
shall complete the appropriate VDH-DWSE VDH-ODW Sample Input Form
in accordance with the instructions for compliance under the SDWA. The contract
laboratory shall report the analysis result to the VDH-DWSE VDH-ODW
within three days of completion date of sample analysis.
1VAC30-40-85. Incorporation by reference.
A. The sampling, analytical methodology, and laboratory certification requirements of 40 CFR 141 and 143 (2009) are incorporated by reference into this chapter.
B. The specific sampling, analytical methodology, and laboratory certification requirements incorporated by reference are listed below by category:
1. Inorganic chemistry: 40 CFR 141.23, 40 CFR 141.89, and 40 CFR 141.131.
2. Organic chemistry: 40 CFR 141.24 and 40 CFR 141.131.
3. Microbiology: 40 CFR 141.21, 40 CFR 141.74, 40 CFR 141.174, 40 CFR 141.704, and 40 CFR 141.705. 40 CFR 136.3 (a) for e. coli requirements under 40 CFR 141.704.
4. Radiochemistry: 40 CFR 141.25.
5. Alternative testing methods: 40 CFR Part 141, Subpart C, Appendix A.
6. Test methods specified for secondary maximum contaminant levels: 40 CFR 143.4.
C. The exceptions to the requirements for laboratory certification in 40 CFR 141.28, 40 CFR 141.74(a), 40 CFR 141.89(a)(1), 40 CFR 141.131(b)(3), and 40 CFR 141.131(c)(3) are incorporated by reference into this chapter. Laboratory testing for alkalinity, calcium, conductivity, disinfectant residual, orthophosphate, pH, silica, temperature, and turbidity for compliance purposes may be performed by laboratories or persons not certified under this chapter but acceptable to VDH-ODW.
1VAC30-40-100. Evaluation procedure.
A. DGS-DCLS shall notify a laboratory three weeks before the on-site evaluation.
B. During the on-site evaluation, the certifying team shall evaluate the laboratory on its equipment and supplies, general laboratory practices, sample collection, handling and preservation, methodology and quality assurance. A laboratory may be required to analyze an unknown sample or perform analysis on a parameter during the evaluation.
Survey forms may be used as guidelines for complete coverage of the laboratory's activities. Each deviation observed during the laboratory evaluation shall be discussed at the time it is observed. The certifying team shall make an oral report to the laboratory staff at the end of the evaluation.
C. The certifying team shall prepare a narrative and action report for the Virginia laboratory officer. This report shall contain information pertinent to the evaluation. The report shall recommend the parameters in a category for which certification can be granted.
D. DGS-DCLS shall obtain from VDH-DWSE VDH-ODW an
identification number for a newly certified laboratory. DGS-DCLS shall inform VDH-
DSWE VDH-ODW of the certification status of a laboratory.
E. The Virginia laboratory officer shall advise the laboratory within 30 days after the on-site evaluation of its certification status and forward the certifying team's complete report.
F. Each laboratory found to be in noncompliance with this chapter, as indicated in the certifying team report, shall submit documentation of the corrective actions at the time specified by DGS-DCLS.
G. Additional actions toward certification shall be determined based on the specific circumstances.
1VAC30-40-110. Levels of certification.
Certification is granted for individual parameters in a category except for the volatile organic chemicals (VOC's). The VOC's are certified as a group based on the method employed and successful completion of the performance evaluation study.
1. "Certified" means a laboratory that meets the minimum requirements as determined by the certifying team using this chapter. The certification shall be valid for up to three years.
2. "Provisionally certified" means a laboratory which
has deficiencies but can still produce valid data. The laboratory can continue
to report compliance data to VDH-DWSE VDH-ODW. A laboratory shall
be permitted up to six months for correction of deficiencies. The certifying
team may perform an announced or unannounced on-site evaluation to determine
the adequacy of documented corrective actions. The certifying team shall
recommend to the Virginia laboratory officer to upgrade the laboratory's
certification status.
3. "Not certified" means a laboratory that does not meet the minimum requirements as determined by the certifying team using this chapter.
1VAC30-40-130. Maintenance of certified status.
To maintain its certified status, a laboratory shall:
1. Continue to meet the requirements listed in this chapter based on the on-site evaluation.
2. Pass performance evaluation samples on an annual basis (for radiochemistry pass additional two cross-check samples).
3. Perform a minimum of five water analyses for each chemical parameter per month. Refer to 1VAC30-40-330 for the minimum number of microbiology analyses. This shall ensure that the laboratory maintains expertise in the certified categories.
4. Notify DGS-DCLS within 30 days of changes in personnel, equipment or laboratory location which may change the laboratory's analytical capability.
5. Use approved methodology listed in this chapter incorporated
by reference at 1VAC30-40-85.
6. Notify VDH-DWSE VDH-ODW in accordance with
1VAC30-40-30.
1VAC30-40-150. Revocation of certified status.
A laboratory shall be downgraded from certified or provisionally certified to not certified status for:
1. Failure to employ USEPA approved methods incorporated by reference at 1VAC30-40-85.
2. Failure to submit report for the performance evaluation study at the specified time limit unless a waiver is approved by DGS-DCLS.
3. Failure to successfully analyze a parameter that is provisionally certified.
4. Submission of a performance evaluation sample to another laboratory for analysis and reporting the data as its own.
5. Failure to correct identified deficiencies based on an on-site visit.
6. Permitting persons other than qualified personnel to perform and report results for drinking water analysis.
7. Falsification of data or use of other deceptive practices.
8. Failure to notify the VDH-DWSE VDH-ODW in
accordance with 1VAC30-40-30.
1VAC30-40-240. Analytical methodology.
Analytical methods are specified in NPDWR 40 CFR 141 and
143. All procedural steps in the approved methods are considered requirements.
1. Inorganic contaminants. Table III-1 of this chapter shows
the approved methodology for inorganic contaminants.
2. Organic contaminants. Table III-2 of this chapter shows
the approved methodology for organic contaminants.
3. Secondary inoraganic contaminants. Table III-3 of this
chapter shows the approved methodology for secondary inorganic contaminants.
A. Laboratories shall meet the sampling and analytical methodology requirements incorporated by reference at 1VAC30-40-85 B 1 for primary inorganic contaminants, 1VAC30-40-85 B 2 for primary organic contaminants, and 1VAC30-40-85 B 5 for alternative testing methods.
4. Prepackaged kits. B. DPD Colorimetric Test
Kit and FACTS Colorimetric Test Kit are the only acceptable prepackaged
kits for free chlorine residual.
5. C. Measurement for residual disinfectant, turbidity,
pH and temperature need not be made in certified laboratories but may be
performed by any persons acceptable by the VDH-DWSE VDH-ODW. The
following are the critical elements of these tests:
a. 1. Sealed liquid turbidity standards purchased
from the instrument manufacturer shall be calibrated against properly prepared
and diluted formazin or styrene divinylbenzene polymer standards at least every
four months in order to monitor for any eventual deterioration. This
calibration shall be documented. The standards shall be replaced when they do
not fall within 15% of the assigned value of the standard. Solid turbidity
standards composed of plastic, glass, or other materials shall not be used.
b. 2. Calibration interval for color wheels,
sealed ampules, and other visual standards for free chlorine residual at least
every six months. These calibrations shall be documented. By comparing
standards and plotting such a comparison on graph paper, a correction factor
can be derived and applied to all future results obtained on the now calibrated
apparatus.
c. 3. Additional criteria. The following criteria
shall be used by persons for performing free chlorine residual, turbidity, pH
and temperature measurements.
(1) a. Free chlorine residual. Samples shall be
collected in plastic or glass. Samples are not preserved; analyses are made
within 15 minutes. A DPD or FACTS Colorimetric Test Kit, spectrophotometer or
photometer is required.
(2) b. Turbidity. Samples shall be collected in
plastic or glass. Samples are not preserved; analyses are to be made within 15
minutes. Nephelometer is needed with light source for illuminating the sample
and one or more photoelectric detectors with a readout device to indicate the
intensity of light scattered at right angles to the path of the incident light.
Unit may be line/bench or battery/portable operated.
(3) c. pH. Samples shall be collected in plastic
or glass. Samples are not preserved. Analyses are to be made within 15 minutes.
A pH meter is necessary.
(4) d. Temperature. Samples shall be analyzed
immediately. Good A good grade mercury-filled or dial-type
centigrade thermometer, or thermistor are is required.
1VAC30-40-250. Sample collection, handling, and preservation.
A. A written sampling procedure with specified sampling instructions shall be made available to sample collectors. The laboratory shall require strict adherence to correct sampling procedures, complete identification of a sample and prompt transfer of the sample to the laboratory.
B. The collector shall be trained in sampling procedures.
C. The sample needs to be representative of the potable water system. The water tap shall be sampled after maintaining a steady water flow for two or three minutes to clear service line unless otherwise specified by the method, as an example, lead and copper. The tap shall be free of any attachments or water purification devices.
D. The sample report form shall be completed immediately after collection with location, date and time of collection, collector's name, preservative added and any remarks concerning the sample. Indelible ink shall be used.
C. E. The sample container, required preservative
preservation, and maximum holding time requirements for sampling
and analyzing inorganic contaminants are listed in Table III-4 of this
chapter incorporated by reference at 1VAC30-40-85 B 1.
D. F. The sample container, required preservative
preservation, and maximum holding time requirements for sampling
and analyzing organic contaminants are listed in Table III-5 of this
chapter incorporated by reference at 1VAC30-40-85 B 2.
G. The sample container, required preservation, and maximum holding time requirements for alternative test methods are incorporated by reference at 1VAC30-40-85 B 5.
E. H. The laboratory shall reject any sample not
meeting the above criteria and notify the system or individual requesting the
analyses.
1VAC30-40-280. Action response to laboratory results.
When the action response is a designated laboratory responsibility, the laboratory shall notify the proper authority of noncompliance sample results and request resampling from the same sampling point immediately.
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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.
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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.
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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.
Table IV-1 |
|
Material |
Temperature/Time |
Membrane filters and pads |
121°C/10 min. |
Carbohydrate-containing media (except P-A Broth) |
121°C/12-15 min. |
P-A Broth |
121°C/12 min. |
Contaminated materials and discarded tests |
121°C/30 min. |
Membrane filter funnel assemblies (wrapped), sample collection bottles (empty), individual glassware items |
121°C/15 min. |
Dilution water blank (99 mL) |
121°C/15 min. |
Rinse water volumes of 500 mL to 1000 mL |
121°C/30 min. |
Rinse water in excess of 1000 mL |
121°C/time adjusted for volume |
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:
Table IV-2 |
||
Parameter |
Limits |
Frequency |
Conductivity |
Less than 2 Micromho/cm at 25°C |
Monthly |
or |
|
|
Resistivity |
Greater than 0.5 megohms at 25°C |
Monthly |
Heterotrophic Plate Count (Pour Plate) |
Less than 500 CFU/ml |
Monthly |
Total Chlorine Residual |
Nondetectable |
Monthly |
Trace Metals (Pb, Cd, Cr, Cu, Ni, Zn) |
Not greater than 0.05 mg/L per contaminant. |
Annually |
Test for bacteriological quality of reagent water Standard Methods, 18th Ed., 1992, Part 9020 B.3.c.1 |
Ratio 0.8 - 3.0 |
Annually |
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.
Table IV-3 |
|
Media Type |
Maximum Storage |
m-Endo Broth in screw-cap flasks or bottles |
96 hours/4°C |
Poured plates of LES Endo Agar and Nutrient Agar + MUG in sealed plastic bags |
2 weeks/4°C |
LTB,, BGLB, EC Medium, ECMedium + MUG, and TSB in loose-cap tubes |
1 week/4°C |
LTB, BGLB, P-A Broth, EC Medium, EC Medium + MUG and TSB in screw-cap tubesor bottles |
3 months/4°C |
HPC agar in screw-cap flasks or bottles |
2 weeks/4°C |
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.
Table IV-4 |
|
Medium |
pH Range |
Single-Strength LTB |
6.6 - 7.0 |
Double-Strength LTB |
6.5 - 6.9 |
Triple-Strength LTB |
6.4 - 6.8 |
BGLB Broth |
7.0 - 7.4 |
m-Endo Broth and LES Endo Agar |
7.0 - 7.4 |
P-A Broth |
6.6 - 7.0 |
EC Medium and EC Medium + MUG |
6.7 - 7.1 |
Nutrient Agar + MUG |
6.6 - 7.0 |
HPC Agar |
6.8 - 7.2 |
Trypticase Soy Broth and Agar, Tryptic Soy Broth and and Agar, and Tryptose Broth |
7.1 - 7.5 |
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.
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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.
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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.