Standard Operating Procedure for Preparation of Hard Water and Other Diluents for Preparation of Antimicrobial Products SOP Number: MB-30-02


US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Preparation of hard water and other diluents for
preparation of antimicrobial products
SOP Number: MB-30-02
Date Revised: 08-21-19

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SOP No. MB-30-02
Date Revised 08-21-19
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SOP Number
MB-30-02
Title
Preparation of hard water and other diluents for preparation of
antimicrobial products
Revisions Made
•	Clarifications made to section 7 (Interferences).
•	Clarifications made to section 11 (Special Apparatus and
Materials) and reagent added to section 11.1.
•	Clarifications made to section 12 (Procedure and Analysis).
•	Added procedure to prepare 200 ppm hard tap water (section 12.3).
•	Added two new forms to section 14 (Example of Media/Reagent
Preparation Sheet for 200 ppm Hard Tap Water and Hard Water
Titration Log Book).
•	Minor editorial changes.

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SOP No. MB-30-02
Date Revised 08-21-19
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SOP Number
MB-30-02
Title
Preparation of hard water and other diluents for preparation of
antimicrobial products
Scope
This SOP describes procedures for the preparation of diluents such
as hard water, sterile de-ionized water, and tap water used to prepare
test substances, and the accuracy verification for the digital titrator.
Application
Procedures are applicable to various types of diluents used in the
preparation of liquid and spray product formulations.


Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:


Data SOP issued:

Controlled copy
number:

Date SOP withdrawn:

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SOP No. MB-30-02
Date Revised 08-21-19
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TABLE OF CONTENTS
Contents	Page Number
1.
DEFINITIONS
3
2.
HEALTH AND SAFETY
3
3.
PERSONNEL QUALIFICATIONS AND TRAINING
3
4.
INSTRUMENT CALIBRATION
3
5.
SAMPLE HANDLING AND STORAGE
3
6.
QUALITY CONTROL
3
7.
INTERFERENCES
4
8. NON-CONFORMING DATA
4
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
9
14.
FORMS AND DATA SHEETS
9
15.
REFERENCES
10

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1. Definitions
1.	Test substance = an antimicrobial formulation used in testing.
2.	Ready-to-use test substance = test substance that requires no activation
or dilution.
3.	Concentrated liquid test substance = liquid or solid test substance that
requires dilution prior to use.
4.	Activation = the combination of a base and an activator to prepare the
final test substance.
5.	Spray test substance = trigger, aerosol, or pump-based test substance.
6.	Test substance diluent = sterile liquid used to make test substance
dilutions (e.g. tap water, de-ionized water, or hard water).
7.	Hard water = water which contains a standardized concentration of
calcium and magnesium ions.
a.	AO AC hard water - may be prepared at various concentrations of
CaC03 (i.e., 100 ppm-500 ppm)
b.	OECD hard water - prepared at a concentration of 375 ppm based
on CaC03 concentration, unless otherwise specified.
c.	Hard tap water - prepared at a concentration of 200 ppm based on
CaC03 concentration, unless otherwise specified.
2. Health and Safety
Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Safety Data Sheet for
hazards associated with specific products.
3. Personnel
Qualifications and
Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOP EQ-03 (weigh balance), QC-01 (QC of water), and QC-19
(pipettes) for details on method and frequency of calibration.
5. Sample Handling
and Storage
1.	Refer to SOP MB-22: Preparation and Sampling Procedures for
Antimicrobial Test Substances and SOP COC-01: Chain of Custody
Procedures for Antimicrobial Samples for procedures on sample
handling and storage.
2.	Use hard water samples within 5 days of preparation.
3.	Use a Media/Reagent Preparation Sheet for preparation of diluents.
6. Quality Control
1.	For quality control purposes, the required information is documented
on the appropriate record form(s), see section 14.
2.	Perform accuracy verification of the digital titrator on a semi-annual
basis. Details provided in section 12.6.

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7. Interferences
1.	Inaccurate water hardness values may occur if the digital titrator is not
properly used and/or does not meet the requirements for accuracy
verification.
2.	Inaccurate results will occur if the delivery tube tip is held above the
solution rather than in the solution (e.g., under the surface) during
sample titration and/or titrator verification procedure.
3.	During the sample titration and titrator verification procedures, verify
that the delivery tube is free of air bubbles and is not misshapen, which
can interfere with the titration process.
4.	During the sample titration and titrator verification procedures, conduct
the verification procedure accurately and without urgency to ensure
titrant is added consistently.
5.	Use clean glass containers for titration samples; do not use plastic
containers.
6.	To prevent contamination, sterilize de-ionized water and tap water
according to SOP QC-13.
7.	Do not use de-ionized water as a diluent or to prepare a diluent if it does
not meet the requirements specified in SOP QC-01.
8. Non-conforming
Data
1.	Errors in the preparation of a diluent will result in an invalid study.
2.	The acceptable range for hard water, expressed as mg/L (ppm) calcium
carbonate (CaC03), is -10% to +5% of the target hardness. Other ranges
of water hardness may be used as appropriate.
9. Data Management
Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
Strict adherence to the protocol is necessary for the validity of the test
results.
11. Special Apparatus
and Materials
1. Reagents.
a.	Sterile water. Use reagent-grade water free of substances that
interfere with analytical methods. Any method of preparation of
reagent-grade water is acceptable provided that the requisite
quality can be met. See Standard Methods for the Examination of
Water and Wastewater and SOP QC-01, Quality Assurance of
Purified Water for details on reagent-grade water.
b.	AO AC hard water solutions
i. Hard Water Solution 1. Dissolve 7.94 g MgCh (anhydrous)
(or 16.94 g MgCh 6H2O) and 18.50 g CaCh in boiled de-
ionized H2O, bring to a volume of 250 mL volumetrically.
Sterilize by membrane filtration. Used for the preparation

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of hard water at various concentrations.
ii. Hard Water Solution 2. Dissolve 14.01 g NaHCCte in
boiled de-ionized H2O; bring to a volume of 250 mL
volumetrically. Sterilize by membrane filtration. Used for
the preparation of hard water at various concentrations.
c.	OECD hard water solutions
i.	Solution A. Dissolve 19.84 g anhydrous magnesium
chloride (or 42.36 g MgCh 6H2O) and 46.24 g anhydrous
calcium chloride (CaCh) in de-ionized water and dilute to
1,000 mL. Sterilize by membrane filtration. Store the
solution in the refrigerator and use for up to one month.
Adjust the recipe for volumes other than 1 L.
ii.	Solution B. Dissolve 35.02 g sodium bicarbonate
(NaHCCte) in water and dilute to 1,000 mL. Sterilize by
membrane filtration. Store the solution in the refrigerator
and use for up to one month. Adjust the recipe for volumes
other than 1 L.
d.	Hardness Voluette Ampule Standard Solution, 10,000-mg/L as
CaCCte (Hach Company, Catalog No. 218-710). Used in the
accuracy verification of the digital titrator.
2. Apparatus
a.	Glassware (e.g., flasks) to conduct titration assays; sterile
volumetric glassware to prepare hard water.
b.	Calibrated weigh balance. To prepare hard water stock solutions.
c.	Digital Titrator Kit (Hach Company, Catalog No. 20636-00). For
the titration of the hard water sample and to determine hardness as
CaCCte in parts per million (ppm).
d.	Titration cartridges. Cartridge concentration is dependent on the
water hardness range (ppm CaCCte) to be assayed
i.	For 10-160 ppm CaCCte use 0.0800 M EDTA titration
cartridge
ii.	For 200-4,000 ppm CaCCte use 0.800 M EDTA titration
cartridge
12. Procedure and
Analysis
1.	AO AC hard water preparation is conducted as described in Method I of
AOAC standard 960.09 (see section 15.1).
2.	OECD hard water preparation is conducted as described in the
Guidance Document on Quantitative Methods for Evaluating the
Activity of Microbicides Used on Hard Non-Porous Surfaces (dated

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June 21, 2013) (see section 15.2).
3.	Titration is conducted as described in Method II of AO AC standard
960.09 (see section 15.1).
4.	Per the study sponsor's request, prepare the appropriate diluent for the
test substance. If hard water is prescribed as the diluent, prepare and
verify the water's hardness according to sections 12.1-12.3. For
products requiring the use of sterile de-ionized water or sterile tap
water, refer to section 12.5.
5.	Verify sterility of all diluents (e.g., hard water, tap water, etc.) prior to
testing.
12.1 Preparation of
AO AC Hard
Water
a.	Per 1 L: Add 1 mL of AOAC Hard Water Solution 1 (for each 100
ppm hardness desired) and 4 mL of AOAC Hard Water Solution 2
to a 1 L volumetric flask and bring to volume with sterile de-
ionized water.
b.	Add approximately 3A of total de-ionized water volume to be
prepared to a volumetric flask containing the appropriate amount of
AOAC Hard Water Solution 1. Add the appropriate amount of AOAC
Hard Water Solution 2 to the volumetric flask. Dilute with de-
ionized water to volume mark.
c.	Example: to prepare 1 L of 400 ppm AOAC hard water, add 4 mL
AOAC Hard Water Solution 1 and 4 mL AOAC Hard Water
Solution 2 to a 1 L flask and bring to volume with sterile de-
ionized water.
d.	Determine the pH of the hard water sample. The pH should be
between 7.6 and 8.0 at room temperature. If necessary, adjust the
pH by using 1 N NaOH or 1 N HC1.
e.	Filter-sterilize the hard water using a 0.2 |im filter unit.
f.	Measure the hardness of the water using a water hardness test kit,
refer to section 12.4.
12.2 Preparation of
OECD Hard
Water
a.	To prepare 1 L of 375 ppm OECD hard water, place 600-700 mL
of de-ionized water in a 1,000 mL volumetric flask and add 6.0
mL of OECD Hard Water Solution A and then 8.0 mL of OECD
Hard Water Solution B.
b.	Mix and add water to the flask to reach 1,000 mL.
c.	Determine the pH of the hard water sample. The pH should be
7.0±0.2 at room temperature. If necessary, adjust the pH by using
1 N NaOH or 1 NHC1.
d.	Filter sterilize the hard water using a 0.2 |im filter unit.

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e. Measure the hardness of the water using a water hardness test kit,
refer to section 12.4.
12.3 Preparation of
Hard Tap Water
Collect municipal tap water sample using pre-cleaned glassware
free of detergents and other potentially interfering substances.
Allow the tap to run 30-60 seconds prior to sample collection.
Record the location and date of sample collection.
To ensure removal of residual chlorine, sterilize tap water by
autoclaving; refer to QC-13.
Determine baseline water hardness using a water hardness test kit
(refer to section 12.4) and document this hardness on the
media/reagent preparation sheet.
If the baseline hardness is below 180 ppm, increase the water
hardness to 200 ppm using the following steps:
i. Calculate the required volume of sterile AO AC Hard Water
Solution 1 according to the equation below. One mL of
AO AC Hard Water Solution 1 should provide
approximately 100 ppm hardness per liter of water:
Required vol. AO AC Hard Water Solution 1 (mL) =
[desired increase in ppm * total volume of tap water (in L)]
100 ppm
li.
ill.
IV.
V.
VI.
Vll.
Add required volume of AO AC Hard Water Solution 1 to a
volumetric flask.
Add approximately 3A of total tap water volume to be
prepared (e.g., approximately 750 mL of sterilized tap
water if total volume of tap water is 1 L) to the volumetric
flask containing the AOAC Hard Water Solution 1.
Add AOAC Hard Water Solution 2 to the volumetric flask
based on the total volume of tap water being prepared (4
mL Solution 2 per 1 L of total volume of tap water).
Dilute with sterilized tap water to reach the desired total
volume of tap water.
Example: to prepare 1 L of 200 ppm hard tap water when
starting with a baseline tap water hardness of 100 ppm, add
1 mL AOAC Hard Water Solution 1 and 4 mL AOAC
Hard Water Solution 2 to a 1 L flask and bring to volume
with sterilized tap water.
Determine the pH of the hard water sample. The pH should
be between 7.6 and 8.0 at room temperature. If necessary,
adjust the pH by using 1 N NaOH or 1 N HC1.

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e.	Filter sterilize the hard tap water using a 0.2 |im filter unit.
f.	Measure the hardness of the water using a water hardness test kit,
refer to section 12.4.
12.4 Hard water
titration
procedure
Measure water hardness on the day the hard water is prepared and
each day it is used.
Using the digital titrator with the appropriate cartridge, verify
water hardness as ppm CaCCte.
Water hardness should be within -10% to +5% of the target
hardness for all hard water samples. For example:
i. An acceptable range of AOAC hard water hardness for 400
ppm is 360 to 420 ppm.
An acceptable range of OECD hard water hardness for 375
ppm is 338-394 ppm.
An acceptable range of hard tap water hardness for 200
ppm is 180-210 ppm.
When using the digital titrator, follow the digital titrator
instructions for calculating the water hardness (refer to section 15).
Record water hardness results in Hard Water Titration Log Book
(see section 14).
li.
in.
12.5 Other Diluents
Sterile de-ionized water. For products requiring dilution with de-
ionized water, sterilize an appropriate volume of de-ionized water
collected from a DI water faucet (available in each laboratory).
Sterile tap water.
i.	For products requiring dilution with tap water, collect the
tap water sample from room B206 in a clean container and
sterilize.
ii.	After sterilizing, measure the hardness of the water using a
water hardness test kit; refer to section 12.4.
12.6 Accuracy
verification
(standard
additions
method) using
CaC03 hard
water samples for
Hach Kit
a.	On a semi-annual basis, determine the accuracy of the digital
titrator using the accuracy verification method found in the Hach
kit user's manual for using CaC03 hard water samples.
Note: The accuracy verification of the digital titrator may be
checked by either the CaC03 hard water method - described in this
SOP, section 12.6b, or by the total chlorine method - described in
SOP EQ-12: Hach's Digital Titrator for Total Chlorine Titration,
section 12.4.
b.	Accuracy verification procedure:

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i.
Prepare a hard water sample and determine the water
hardness as described in section 12.4. Any hard water
sample may be used.

ii.
Use sample from step 12.6b,i for accuracy verification.

iii.
Open the standard solution ampule (10,000 mg/L as
CaC03).

iv.
Add 0.1 mL of the standard to the titrated sample in step
12.6b,i. Swirl to mix.

v.
Titrate the spiked sample to the end point (i.e., when the
titrated sample turns from pink to blue). Record the number
of digits on the digital titrator display that was used to
reach the end point.

vi.
Repeat steps 12.6b,iv and 12.6b,v twice more for a total of
three 0.1 mL samples of the standard added to the titrated
sample.

vii.
Each 0.1 mL of standard that was added will use
approximately 10 digits of the 0.800 M titration cartridge
or approximately 100 digits of the 0.0800 M titration
cartridge to reach the endpoint. A range of ±4 digits is
acceptable.

viii.
Record outcome of accuracy verification procedure on the
log sheet (see section 14).

ix.
If verification procedure fails, repeat steps 12.6b,i-vii.
After the repeat, if the verification procedure continues to
fail, identify the titrator as unusable. Use an alternate
titrator or purchase a new one to ensure titration procedures
are accurate.
13. Data Analysis/
Calculations
None.
14. Forms and Data
Sheets
1. Media/Reagent Preparation Sheets. Sheets are stored separately from
the SOP under the following file names:
Media/Reagent Preparation Sheet for AO AC MB-30-02 Fl.xlsx
Hard Water Solution 1
Media/Reagent Preparation Sheet for AO AC MB-30-02 F2.xlsx
Hard Water Solution 2
Media/Reagent Preparation Sheet for OECD MB-30-02 F3.xlsx
Hard Water Solution A
Media/Reagent Preparation Sheet for OECD MB-30-02 F4.xlsx

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Hard Water Solution B
Example of Media/Reagent Preparation Sheet MB-30-02 F5.xlsx
for AO AC 400 ppm Hard Water
Example of Media/Reagent Preparation Sheet MB-30-02 F6.xlsx
for 200 ppm Hard Tap Water
Accuracy Verification Semi-Annual Log Sheet MB-30-02 F7.xlsx
Hard Water Titration Log Book MB-30-02 F8.docx
15. References
1.	AOAC Official Method 960.09: Germicidal and Detergent Sanitizing
Action of Disinfectants, Revised First Action 2013.
2.	Guidance Document on Quantitative Methods for Evaluating the
Activity of Microbicides Used on Hard Non-Porous Surfaces (June 21,
2013).
3.	Hach Digital Titrator Model 16900-01 manual, pages 139-145.
4.	Online Method - Hardness, Total. Method 8213. Hach Company.
16900-08. 25th Ed. 03/2013.

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