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 antimicrobial products
SOP Number: MB-30-00
Date Revised: 12-12-13

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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 1 of8
SOP Number
MB-30-00
Title
Preparation of hard water and other diluents for antimicrobial products
Scope
This SOP describes procedures for the preparation of diluents used to
dilute disinfectant products such as hard water, sterile de-ionized
water and tap water, 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Date SOP withdrawn: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-3 0-00
Date Revised 12-12-13
Page 2 of 8
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
3
8. NON-CONFORMING DATA
3
9.
DATA MANAGEMENT
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
3
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
7
14.
FORMS AND DATA SHEETS
7
15.
REFERENCES
8

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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 3 of 8
1. Definitions
1.	Concentrated disinfectant product = Product that requires a dilution prior
to use.
2.	Product diluent = Sterile liquid used to make product dilutions (e.g. tap
water, de-ionized water or hard water).
3.	Hard water = Water which contains a standardized concentration of
calcium and magnesium ions.
2. Health and
Safety
1. Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Material 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: Disinfectant Product Preparation and Sampling
Procedures and SOP COC-01: Sample Log-in and Tracking of
Disinfectants for procedures on sample handling and storage.
2.	Use hard water within 24 hours of preparation.
3.	Use a Media/Reagent Preparation Sheet for diluents being prepared.
6. Quality Control
For quality control purposes, the required information is documented on the
appropriate record form(s). See section 14.
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.	Sterilization of de-ionized water and tap water must be conducted
according to SOP-QC-13 to prevent the presence of contamination.
3.	If the de-ionized water does not meet the requirements specified in SOP
QC-01 then the water should not be used as a diluent or to prepare a
diluent.
8. Non-conforming
Data
Errors in the preparation of the diluent, if not corrected prior to testing, will
result in a repeat of the study.
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


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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 4 of 8
Materials

11.1 Glassware
a.	Sterile glassware - such as flasks and beakers, to conduct titration
assays.
b.	Sterile volumetric glassware (pipettes, flasks, etc.) - to measure
ingredients used in hard water preparation.
11.2 Equipment
a.	Weigh Balance (weighs up to 5100 g) - to weigh sample containers
prior to and after sample removal.
b.	Serological and micropipettes with tips.
11.3 Hard water
solutions and
reagents
a.	De-ionized water
b.	Hard Water Solution 1. Dissolve 7.94 g MgCh (anhydrous) (or
16.94 g MgCl2'6H20) and 18.50 g CaCh in boiled de-ionized H20,
bring to a volume of 250 mL volumetrically. Filter-sterilize using a
0.2 |im filter unit. Used for the preparation of hard water at various
concentrations.
c.	Hard Water Solution 2. Dissolve 14.01 g NaHCC>3 in boiled de-
ionized H2O; bring to a volume of 250 mL volumetrically. Filter-
sterilize using a 0.2 |im filter unit. Used for the preparation of hard
water at various concentrations.
d.	Hardness Voluette Ampule Standard Solution, 10,000-mg/L as
CaC03 (Hach Company, Catalog No. 218-710). Used in the
accuracy verification of the digital titrator.
11.4 Apparatus
a.	Digital Titrator Kit (Hach Company, Catalog No. 20636-00). For
the titration of the hard water sample and to determine hardness as
CaCC>3 (in parts per million).
i. Kit includes reagents, flask, digital titrator, delivery tube,
and instruction sheet necessary to determine the total water
hardness of the sample. Additional reagents may be
purchased from manufacturer (see section 15 for details and
users manual information).
b.	Various titrating cartridges are available depending on the water
hardness range to be assayed:
i.	Water hardness in the range of 10 - 160 ppm = titration
cartridge of 0.0800 EDTA.
ii.	Water hardness in the range of 200 - 4,000 ppm = titration

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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 5 of 8

cartridge of 0.800 EDTA.
12. Procedure and
Analysis
1.	Per the study sponsor's request, prepare the appropriate diluent used to
dilute the product as stated in section 12.1. If hard water is prescribed as
the diluent, then verify the hardness according to section 12.2. For
products requiring the use of tap water see section 12.3.
2.	Conduct titration of hard water as described in section 12.2.
NOTE: Hard water preparation is conducted as described in Method I of
AO AC standard 960.09., Hard water titration is conducted as described
in Method II of AO AC standard 960.09 (see section 15.1).
12.1 Hard Water (as
CaC03) -
Preparation
a.	Prepare hard water solution 1 volumetrically as in section 11.3, b.
b.	Prepare hard water solution 2 volumetrically as in sectionl 1.3, c.
c.	Add approximately 3A of total deionized water volume to be
prepared to a volumetric flask containing the appropriate amount of
solution 1. Add the appropriate amount of solution 2 to the volumetric
flask. Dilute with deionized water to volume mark. See 12.1, e for
example.
d.	Per 1L: Add 1 mL of hard water solution 1 (for each 100 ppm
hardness desired) and 4 mL of hard water solution 2 to a 1 L
volumetric flask and bring to volume with sterile de-ionized H20.
See example in (e) below:
e.	For example, to prepare 1 L of 400 ppm hard water, add 4 mL hard
water solution 1 and 4 mL hard water solution 2 to a 1 L flask and
bring to volume with sterile de-ionized water.
f.	Determine the pH of the hard water sample. The pH should be
between 7.6 and 8.0 (adjust if necessary).
g.	Filter-sterilize hard water using a 0.2 |im filter unit.

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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 6 of 8
12.2 Hard water
titration
procedure
a.	Using the digital titrator with the appropriate cartridge, verify
hardness of the sterile hard water, in the form of CaC03.
b.	Water hardness should be within -10% to +5% of the target
hardness. For example, an acceptable range of hardness per 400
ppm is 360 to 420 ppm.
c.	When using the digital titrator, follow the digital titrator instructions
for calculating the water hardness (refer to section 15).
d.	Record water hardness results in Media/Reagent Preparation Sheet.
Verify the sterility of the hard water according to SOP MB-10.
12.3 Other Diluents
a.	Sterile de-ionized water. For products requiring dilution with de-
ionized water, an appropriate volume of deionized water is taken
from the port located in room B206. To collect the deionized water,
turn on the Barnstead B-pure Pressure Cartridge System and let run
until the resistivity meter in the cartridge unit reads an acceptable
limit, see QC-01: Quality Assurance of Purified Water for details
on the use and operation of the Barnstead B-pure Pressure Cartridge
System.
i. The Barnstead B-pure Pressure Cartridge System contains
cartridges which raise the quality of the deionized water.
b.	Sterile tap water. For products requiring dilution with tap water,
determine the water hardness of the tap water sample on the day of
use. Collect the tap water sample from room B206 in a clean
container prior to sterilization. Use a digital titrator (see sections
12.2 and 15) to determine the water hardness.
c.	Record water hardness results in Media/Reagent Preparation Sheet.
Verify the sterility of the tap water accordingly to SOP MB-10:
Media and Reagents Preparation and Quality Evaluation.
12.4 Accuracy
verification
(standard
additions
method) for
Hach Kit
a.	On a quarterly basis, determine the accuracy of the digital titrator
using the method found in the Hach kit user's manual.
b.	Accuracy verification procedure:
1.	Prepare a hard water sample and determine the water
hardness as described in section 12.1. Any hard water
sample may be used, for example a 200 ppm or 400 ppm
hard water sample may be used.
2.	Use sample from step 1 for accuracy verification.
3.	Open the standard solution ampule (10,000 mg/L as CaCOs)

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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 7 of 8

4.	Add 0.1 mL of the standard to the titrated sample in step 1.
Swirl to mix.
5.	Titrate the spiked sample to the end point (i.e., when the
titrated sample turns from pink to blue). Write down the
number of digits on the digital titrator display that was used
to reach the end point.
6.	Repeat steps 4 and 5, twice more, for a total of three 0.1 mL
samples of the standard added to the titrated sample.
7.	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 (11 digits of 0.714 M or 56 digits of
0.1428 Mtitrant).
A range of 4 digits is acceptable.
8.	If more or less titrant was used, the problem can be due to
user technique, causing interference.
9.	Conduct the accuracy verification on a quarterly basis.
10.	Record if verification passed or failed on the log sheet (see
section 14).
12.5 Media/Reagent
Preparation
Sheet
a. Record diluent preparation on the Media/Reagent Preparation Sheet
(refer to SOP QC-15, Media Prep and Sterilization Run Numbers).
13. Data Analysis/
Calculations
Hardness may vary by no more than -10% or +5% from the stated value.
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 Hard Water MB-30-00 F1 .xlsx
Solution 1
Media/Reagent Preparation Sheet for Hard Water MB-30-00_F2.xlsx
Solution 2
Example of Media/Reagent Preparation Sheet MB-30-00_F3.xlsx
for 400 ppm Hard Water
Accuracy Verification Quarterly Log Sheet MB-30-00 F4.xlsx

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SOP No. MB-3 0-00
Date Revised 12-12-13
Page 8 of 8
15. References
1.
AO AC Official Method 960.09: Germicidal and Detergent Sanitizing


Action of Disinfectants, Revised First Action 2013.

2.
Hach Digital Titrator Model 16900-01 manual, pages 91-98.

3.
Online Method- Hardness, Total. Method 8213. Hach Company.


DOC316.53.01176. 7thEd. 2012.

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