STANDARD OPERATING PROCEDURES FOR ANALYTICAL

METHODS IN AN ENVIRONMENTAL SCIENCE LABORATORY

by
VICTORIA RICHARDS HARKINS, B.A.

A THESIS
IN
CIVIL ENGINEERING
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
IN
CIVIL ENGINEERING

May, 1995
73
.qqS ACKNOWLEDGEMENTS

^f' I would like to express my gratitude to the head of my committee, Dr. Tony R.
Mollhagen, for his wonderfiil sense of humor and "boy secrets." I would also like to thank
Dr. Lloyd Urban and Dr. Ken Rainwater for serving as committee members and their help
in the completion of the final draft Special thanks to Dr. Heyward Ramsey, my graduate
advisor, and Barbi Dickensheet for their guidance throughout the project.
I would also like to thank Brad Thomhill for answering a million questions and
helping me locate the many references needed in the creation of this manual. FinaUy, I
want to thank my husband, David, and my family and friends for keeping me fed and
encouraged.

u
 TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
LISTOFHGURES iv
CHAPTER
L INTRODUCTION 1

1.1 Background Information 1

1.2 Objectives 2
II. METHODS AND MATERL\LS 3
2.1 Resources Used 3
2.2 Materials Compilation 4
2.3 Format 4
2.4 Verification 7
III. RESULTS AND DISCUSSION 9
IV. CONCLUSIONS AND RECOMMENDATIONS 11
4.1 Conclusions 11
4.2 Recommendations 12
REFERENCES 13
APPENDED 14

m
 CHAPTER 1
INTRODUCTION

1.1 Background Information

There are at least two benefits to following the same procedure for any task that
must be undertaken repeatedly. Repetition yields familiarity with the procedure and
subsequentiy economy of action. Familiarity and economy of action first incrementally
reduce the time to complete the task, and second, they incrementaUy reduce error, usually
to predictable levels. In the chemical testing laboratory these are desirable ends, and it is
rare that testing is done in the absence of written instructions. There are many standard
references for chemical analyses avaUable for use in laboratories. Some examples are the
Annual Book of American Society for Testing and Materials (ASTM) Standards, the
official test methods of the Environmental Protection Agency, Standard Methods, produced
by the American PubUc Health Association (among others), instructional manuals provided
by instrument manufacturers, and literature concerning technologies and/or adaptations to
current practices. In standardizing all these methods, certain details are lost when trying to
make the methods general enough for aU laboratories. This is the reason for the
development of Standard Operating Procedures (SOPs) for individual testing faciUties
(Homshuh 1992).

SOPs are recorded, institution-specific standard procedures that personnel can

foUow to ensure data of high quality and integrity. In the environmental
science/engineering arena, SOPs must be generated not only for laboratory analysis, but
also for the reporting of results and coUection, preservation, and transport of samples. The
production of high quaUty data results from systematic consideration of aU potential sources
of error rather than simply the competence of a chemist or the quaUty of the
instrumentation. "If properly developed and foUowed, SOPs insure consistency and good

1
definition for a research program, regardless of who conducts the research" (Homshuh
1992 p. 27). SOPs are written such that minimally trained personnel may perform tasks
without incoiporating high systematic error. Also, for personnel who perform repetitive
tasks, SOPs reduce random error by standardizing the methods used for such analyses.

SOPs provide consistency and uniformity in the production of data, but they can
also be used as a training device (Homshuh 1992). In the case of the Environmental
Science Laboratory (ESL) at Texas Tech University, as weU as many other university
laboratories, employees are usually students and turnover is high, resulting in low
institutional memory. SOPs provide a means of retaining some institutional memory and
providing effective instructions to aid in the training of new personnel.
The manual wiU be available to train students and provide experienced personnel a
reference to use in the case of unexpected errors or questions. A large portion of the
manual wiU be required for four classes that perform testing in the Environmental Sciences
Laboratory. The four classes include two undergraduate classes. Environmental
Engineering Laboratory and Environmental Measurement, and two graduate classes. Water
and Wastewater Analysis and Unit Processes Laboratory.

1.2 Purpose and Objectives

The purpose of this thesis is to create a manual that is as specific as possible to the
ESL concerning instrumentation, detaUed calibration, and procedures. The specific
objectives for creating this manual are to standardize and economize the level of instruction
so that students or people with minimal training can successfiiUy complete aU analyses and
to provide for them a quick reference concerning sample handling, procedures of analysis,
and summary type inforaiation concerning analyses performed in the laboratory.
Provided in the Appendix is a manual that contains aU the methods performed in the
Environmental Science Laboratory.
 CHAPTER 2
METHODS AND MATERIALS

2.1 Resources Used

In the production of this manual, the first step was the identification of resources to
develop background information, procedures for analyses specific to the Environmental
Science Laboratory, and the accuracy that should be expected. The two main texts are
>StflPf1fl^^ Methods for the examination of water and wastewater (hereafter referred to as
Standard Methods) by the American PubUc Health Association and the United States
Environmental Protection Agency approved methods (hereafter, EPA methods). Others
resources cited include manufacturer's operation instmctions and four textbooks including
Chemistry for Environmental Engineering (Sawyer and McCarty 1994), Water Treatment
Plant Design (Sanks 1978), Wastewater Biologv (Water Environment Federation 1990),
and Water Chemistrv (Snoeyink 1980). One other reference used was the 1993 Annual
Book of ASTM Standards section 11 by Water and Environment Technology (American
Society for Testing and Materials 1993). The ASTM standards were used mainly for
procedures not accepted by the Environmental Protection Agency (EPA) such as the
coagulation/flocculation Jar Test widely used by treatment plants for process control. EPA
does not accept this method because it cannot be standardized to quantify accuracy. ASTM
also provides details for some procedures that are not covered in Standard Methods. The
final source used was lecture notes produced by Dr. Tony Mollhagen for his classes in
Water Chemistry, Limnology, Environmental Measurement, and Water and Wastewater
Analysis. The manual is designed for use by students taking this water and wastewater
analysis class, therefore, it would be efficient to have integration of lecture material with the
information provided in the manual.
 2.2 Materials Compilation
The second step in the development of this manual was compilation of germane
portions of the resources into a useful and accessible manual. This included using a text
scanner to copyreferencesinto a word processing program for subsequent editions and
manuaUyretypingseveral of thereferencessuch as manufacturer's instructions that were
difficult to scan due to smaU fonts and/or formats that included manyfiguresor pictures.
Once all the information was collected, editing and organizing was done to create individual
folders for each chemical analysis and qualitative subject After the information was
divided, each method and section was individually created based on the resources
accumulated for that particular subject or analysis. An individual method was prepared for
each procedure performed in the ESL. See Figure 1 for a Ust of the qualitative subjects
included in the manual and Figure 2 for the analytical procedures provided.

2.3 Format
The third step in developing SOPs for the ESL was defining a format to foUow. It
was stated that one of the purposes of this manual was for it to be easy to use and
accessible. In order to accomplish this, a format was chosen so that the subheadings relate
to common topics in different chemical analyses such as interferences andreagentmake-up.
A numbering system was chosen to make the methods easy to follow and to make referrals
within or among methods simple. For example, during a procedure, a statement may read
"Add 2 ml of NaOH (6.8)". The (6.8)refersto thereagentand its recipe already defined
earUer under the subheading 6.0 Reagents.

The format settied upon was a compromise between the formats used by the EPA

and Stt^"^a^<1 Methods. t<>tfl"(1p^^ Methods covers an excess of material, and it can be
 Safety
1.0 Organizing for Safety
2.0 Safety Equipment
3.0 LabOTatory Hazards
4.0 Hazard Management Practices
Laboratory Apparatus and Reagents
1.0 Apparatus
2.0 Reagents
Reagent Grade Water Preparation
1.0 Methods of Preparation
2.0 Reagent Water Quality
Statistics
Data (QuaUty
1.0 Precision
2.0 Bias
3.0 Total Uncertainty
4.0 Checking Correctness of Analyses
5.0 Method Detection Limits
6.0 Method Development and Evaluation
Quality Assurance (QA)
1.0 QA Planning
2.0 QuaUty Control
3.0 C^uaUty Assessment
CoUection and Preservation of Samples
1.0 General Precautions
2.0 Safety Considerations
3.0 Type of Samples
4.0 Collection of Samples/Chain of Custody
5.0 Sampling Methods
6.0 Number of samples
7.0 Quantity
8.0 Preservation

Expression of Results
1.0 Units
2.0 Significant Figures

Figure 1. List of Qualitative Subjects Included in the Manual

A. Acidity R. Oil and Grease
B. Alkalinity S. Oxidation/Reduction Potential
C. Anions T. Petroleum hydrocarbons
D. Boron U.pH
E. BOD V. Phenols
F. Carbon W. Phosphorous
1. Total 1.Total
2. Inorganic 2.0rthophosphate
3. Organic X. Purgeable Aromatics
G. COD Y. Salinity
H. Chlorine Z. SiUca
1. Residual AA. Solids
2. Total 1. Total
I. ChloTophyU a 2. Dissolved and Suspended
J. Coagulant Dosage 3. Fixed and Volatile
K. CoUforms BB. Temperature
L. Conductance CC. Turbidity
M.. Cyanide
N. Dissolved Oxygen
0. Hardness
P. Metals
Q. Nitrogen
1. TKN
2. Amnx)nia
3. Nitrate/Nitrite

Figure 2. List of Analytical Procedures Provided in Manual.

difficult to determine which method or conditions are appUcable to the ESL. For example,

ammonia has six different methods for its determination. Also, the numbering used makes

referrals to previously covered information confusing, such as "see section 4500-

NH3.B.3a" (American PubUc Health Association 1992). However, most Standard

Methods introductions are complete and informative. The EPA format is at the other

extreme from .StanHarH Methods with many instances of insufficient information to

successfuUy complete an analysis with further references. However, the EPA methods

have an effective numbering system that makesreferraleasier, and each method contains

similar headings that aUow for quick access to commonly required information, such as

reagents needed.
 The organization used for analytical procedures contains an introduction that includes
background information, practical appUcations and analysis information. Analysis
information includes scope and appUcation, interferences, reagent make-up, procedure, and
accuracy. Shown in Figure 3 is an outline of the basic information provided in each
method. Some methods have additional headings that are particular to that method such as
comments andrelatedmaterials. In some cases, there are additional, sometimes unique,
steps to the procedure or other appUcations of data produced. For example, in alkalinity,
extra information is providedrelatingalkalinity to calcium carbonate saturation.

Introduction
Scope of AppUcation
Summary of Method
Sample HandUng and Preservation
Interferences
Apparatus
Reagents
Procedure
Calculation
Accuracy
Figure 3. Basic ouUine of methods

2.4 Verification
The final and probably most important step in developing SOPs is verification. The
first portion of the manual was completed in January of 1995 in order to have it avaUable to
students to use for the spring semester Students have used a portion of the manual for
approximately four months to perform the required analyses and as background
information for completing labreports.WhUe performing such tasks, the students have

found and reported errors in procedure and ambiguous instructions, and those have been
corrected. Also, students have been interviewed to coUect their general comments
concerning the utiUty of the manual. Consequentiy, sections have been revised to increase
readabiUty and clarity in analyses the students found difficult to understand. The complete
manual, including the methods not performed by students, is used in the laboratory by lab
personnel. As these methods are used, fiuther verification wiU also be done to correct
mistakes andrewordsections that may be confusing.

In addition to verification done by the students and lab personnel, the manual should
periodicaUy be completelyreviewedto determine that the methods are up-to-date and stiU
reflect current laboratory practices. If changes are made, notice should be made to manual
users and a revised edition should be circulated (Homshuh 1992).

8
 CHAPTER 3
RESULTS AND DISCUSSION

The objective of this thesis was to create a manual that is as specific to the laboratory as
possible conceming instrumentation, detaUed caUbration, and procedures with respect to
the analyses performed in the Environmental Science Laboratory. It is beUeved that this is
the first Standard Operating Procedures for the ESL at Texas Tech University. The
manual was assembled to serve the foUowing purposes: (1) to standardize and reduce the
level of instmction so that students or technicians with minimal training can successfuUy
complete aU analyses and (2) to produce a document that wiU have utiUty for students
completing their educations and weU after their formal education is complete conceming
basic information conceming method specifics.
The objective, the production of a manual, has been completed and is shown in
appendix A. The suitabiUty of the manual for the several appUcations just described are
discussed below.
The first purpose of the manual was to standardize and reduce the level of instruction
so that students or technicians with minimal training can successfuUy complete aU analyses.
At the beginning of the spring semester 1995, two undergraduate classes in Environmental
Engineering Laboratory and one graduate class in Water and Wastewater Analysis were
required to obtain a copy of a portion of the manual to perform the required analyses taught
in these courses. These required analyses did not include those methods needing high
levels of chemical skiUs and/or training. Withrespectto technicians performing the
analyses provided in the manual, most methods were standardized such that the analyses
could be performed with Uttle to no training. However, eight of the analyses reviewed
require training by experienced lab personnel due to the level of instmction required for the
equipment or related safety precautions. See Figure 4 for those analyses considered too
difficult to perform without supervised training.

1. Anions 5. Phosphorous
2. Cyanide a. Total
3. Metals b. Orthophosphate
4. Nitrogen
a. TKN
b. Ammonia
c. Nitrate/Nitrite
Figure 4. Analyses to be pertormed only by trained personnel
The second purpose of the creation of the manual was to provide areferencethat the
students can keep whUefinishingtheir coursework and after their coUege educations are
complete. It is not the objective of the coursework utilizing the ESL to train technicians,
but rather teach the students to be famiUar with procedures performed in laboratories so that
they can effectively supervise anyrelatedwork in this field The manual would serve as a
concise reference to enable the environmental engineer or scientist to be famiUar with
required analyses they may need to acquire. Most useful wiU be the sections on data
quaUty and collection, preservations, transport, and storage of samples. It would also be a
reference containing brief explanations of performed chemical analyses. Instead of
researchers producing their own information conceming the background information and/or
summaries of methods performed, this material can be directiy Ufted out of this manual to
be used in reports, theses, projects, or pubUcations.

10
 CHAPTER 4

CONCLUSIONS AND RECOMMENDATIONS

4.1 Conclusions
The manual shown in appendix A was produced to serve the foUowing needs:
1. to standardize and reduce the level of instmction so that smdents or technicians
with minimal training can successfuUy complete aU analyses, some limitations
apply such that several of the analysesreviewedcan not be developed by an
individual method. These methods require extensive supervised training due to
the level of instmction needed and related safety precautions. No one method
can completely cover aU the intrinsic detaUs that only an experienced technician
can acquire. For example, metal analyses require experienced personnel who
are famiUar with the occurrences and/or reoccuitences of common errors and
know the steps for correction. This can only be accompUshed by experience
using the equipment and
2. to provide for them a quickreferenceconceming sample handUng and
procedures of analysis. Effective supervisors are famiUar with the work they
are proposing. Having this manual as a reference would enable the engineer to
obtain any sampUng requirements and summaries of the analyses to be
performed.

11
 4.2 Recommendations
In order for SOPs toremainuseful, it is necessary that the manual be kept up to
date. The foUowing specific recommendations are offered.

1. It is recommended that if there is a change in the current practice conceming any

of the methods included in the manual,revisionsneed to be done on the current
manual and the up-dated edition redistributed to current users.
2. If new equipment is purchased for analyses not currentiy used in the ESL, new
methods should be produced and reviewed, then integrated into the SOP.
3. When new equipment is purchased for analyses already performed in the lab, it
should be included in the existing procedure as a new altemative for
measurement if the old procedure is stUl viable. In turn, if methods described in
the manual are no longer practiced in the lab, they should be omitted in the
subsequent revisions.
4. The SOP should be integrated into courses using the Environmental Science
Laboratory. Further development and inclusion of the lecture material presented
in the classes needs to be presented. A continuously updated resource
containing aU lecture materials in a semi-text format and the procedures
performed in the ESL would be an ideal result

12
 REFERENCES

American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18thed. American PubUc Health Association, American
Water Works Association, and Water Environment Federation, Washington DC.
American Society for Testing and Materials. (1993). Annual Book of ASTM StanHarHs;.
Vol. 11.02. American Society for Testing and Materials, PhUadelphia, Pa.
Homshuh, M.J. (1992). Good Laboratorv Prayt^'Tf >Stan<lf^rdS American Chemical
Society, Washington, DC.
Snoeyink, V. L.and Jenkins, D. (1980). Water Chemistrv. John WUey & Sons, Inc.,
New York, N.Y.
Sanks, R. L. (1978). Water Treatment Plant Design. Butterworth PubUshers, Stoneham,
Ma.
Sawyer, C. N., and McCarty, P. L. (1994). Chemistry for Environmental Engineering.
4tii ed. McGraw-HUl, Uic, New York, N.Y.
United States Environmental Protection Agency. (1992). Methods for Chemical Analysis
of Water and Wastes. Environmental Monitoring and Support Laboratory, United
States Environmental Protection Agency, Cincinnati, Ohio.
Water Environment Federation. (1990). Wastewater Biology: the Microlife. Special
PubUcation, Water Environment Federation, Alexandria, Virginia, 196 pp.

13
 APPENDIX A

STANDARD OPERATING PROCEDURES FOR

THE ENVIRONMENTAL SCIENCE

LABORATORY AT TEXAS

TECH UNIVERSITY

14
 TABLE OF CONTENTS

Safety 17
Laboratory Apparatus and Reagents 30
Reagent Grade Water 38
Statistics 42
Data (QuaUty 46
C^aUty Assurance 56
CoUection/Preservation 64
Expression of Results 75
Acidity 79
Alkalinity 83
Anions 90
Biochemical Oxygen Demand 102
Boron 110
Total Organic Carbon 112
Chemical Oxygen Demand 116
Chlorine, Total Residual 121
ChlorophyU a 125
Coagulation-Flocculation Jar Test of Water 128
CoUform, Total 133
Conductance 142
Cyanide, Total 148
Oxygen, Dissolved 157
Hardness, Total 162
Metals Introduction 169
Aluminum 185
Antimony 187
Arsenic 189
Barium 191
Cadmium 193
Calcium 195
Chromium 197
Cobalt 199
Copper 201
Iron 203

15
TABLE OF CONTENTS (cont)
Lead 205
Magnesium 207
Manganese 209
Mercury 211
Molybdenum 213
Nickel 215
Potassium 217
Selenium 219
Silver 221
Sodium 223
ThalUum 225
Vanadium 227
Zinc 229
Strontium 231
Nitrogen 233
Nitrogen, Ammonia 235
Nitrogen, Nitrate-Nitrite 239
Nitrogen, Kjeldahl, Total 243
OU and Grease and Total Petroleum Hydrocarbons 247
Oxidation-Reduction Potential of Water 251
pH 257
Phenolics 262
Phosphorous Introduction 266
Phosphorous, Orthophosphate 267
Phosphorous, Total 271
Purgeable Aromatics, Gas Chromatography 275
Salinity 288
Silica, Dissolved 291
Solids Introduction 294
Total SoUds 296
Total Suspended Solids 298
Total Volatile SoUds and Total Fixed SoUds 301
Total VolatUe Suspended SoUds and Total Fixed Suspended SoUds 303
Temperature 305
Turbidity 307

16
 SAFETY
INTRODUCnON
Achievement of a safe and healthy work place is theresponsibiUtyof the institution, the
laboratory manager, the supervisory personnel and, findly, of the laboratory personnel
theinselves. AU laboratory employees must make every effort to protect themselves and
their feUow workers. The labciatory manager shouldreaUzethat accidents have causes,
and therefore can be prevented by a good safety program.

Once an employee or student has become thoroughly famUiar and competent with the new,
safe techniques, the supervisor can be assured that the employee wiU propose other safety
ideas.
1.0 Organizing for Safety
AlthoughresponsibUityfor estabUshing and enforcing a laboratory safety program
ultimately rests with the laboratory dirw:tor, it is desirable, except in smaU laboratories,
to delegate responsibUity to a staff member designated as the safety officer. The safety
officer should be part of the management team to optimize safety training, inspections,
indoctrination of new employees, and acquisition and updating of safety information.
The safety officer should see that personal protective equipment is avaUable and used
appropriately. The officer should make periodic inspections of emergency equipment,
such as fire extinguishers, alarm systems, eye wash, and safety showers; perform
periodic inspections of the laboratory to uncover overlooked hazards; observe that
personnel are foUowing safety rules; and provide reminders for individuals to be aware
of safe practices.

Radiation safety is a specialized part of an overaU safety program. A technicaUy

qualified individual should be designated as the Radiation Safety Officer (RSO). The
RSO ensures that radioactive sources and radiation equipment are used safely and in
accordance with aU pertinent state and federal regulations, and that proper Ucensing
from the appropriate state and/or federal agency is maintained. The Environmental
Science laboratory isreviewedevery 6 months by the Environmental Health and Safety
Department to inspect for any leaks or possible radiation hazards. The Environmental
Health and Safety Department also inspects once a year for occupational safety.

A safety committee should be estabUshed and it must have the support of the laboratory
director. The committees recommendations must be taken seriously and aaed upon
prompUy. Safety committees involve persons with varying expertise in making
decisionsrelatingto safety practices. For example, chemists may have insight into the
handling of hazardous chemicals in a bacteriology section. This assignment can be
educationaUy valuable. It is desirable periodicaUy to rotate members of the safety
committee so that most personnel have an opportunity to become personaUy involved.

Keep good records of all accidents, inspections, training, etc. Preferably use a simple
standardreportform. Thereportshould contain sufficient information to enable the
safety officer, supervisor, and director to determine who was involved, what
happened,-when and where it happened, and what injuries, if any, resulted. The
Occupational Safety and Health Act (OSHA) requires that those accidents causing
major disabiUty be recorded in a log, but it is useful to record all accidents to be able to
evaluate the safety progranL Recording time and nature of each accident may be of

17
 further importance if disabiUty occurs and a Woricers Compensation claim is
appropriate. Maintain a fUe of recommendations of the safety officer or safety
committee, as weU as actions taken by the staff as part of the safety program.
The OSHA "Hazard Communication Standard" or "Right-to Know Regulation"
specifies a method of employee notification about hazixis in the work place. Exposed
laboratory personnel must be under direct supervision andregularobservation of a
technicaUy qualified individual, who must have knowledge of the hazards present,
their healtii effects, andrelatedemergency procedures. The supervisor must educate
laboratory personnel in safe work practices. Personnel have arightto know what
hazardous materials are present, the specific hazards created by those materials, and the
required procedures to protect against these hazards. Laboratory personnel and
students are required to have a detaUed tour of the laboratory andrelatedfaciUties
explaining the safety precautions and measurements to be taken in case of an accident.
Training in safety techniques requires deUberate effort by management In larger
laboratories, safety seminars are important Use educational techniques inclucUng
training films, demonstrations with devices such as fire extinguishers or respirators,
charts listing hazardous chemicals and proper procedures for handling them, and
manuals on safety. Periodically providerefreshertraining. Material Safety Data
Sheets (MSDSs) are provided by chemical manufacturers and are an extremely
important part of the whole approach of informing personnel of hazards in their work
place. These MSDSs should be made avaUable to personnel using hazardous
materials. MSDSs are avaUable to anyone using the laboratory and are located under
the "Right to Know" buUetin board in the main part of the laboratory.

2.0 Safety Equipment

2.1 Laboratory Equipment
2.1.1 Fire extinguishers: There are three general types of fire extinguishers:
2.1.1.1 Water-type extinguishers are usefiil for fires with ordinary
combustibles, such as wood, paper, and rags.
2.1.1.2 Dry-chemical types are effective against most fires, but particularly
those involving flammable Uquids and metals and electrical fires.
2.1.1.3 Carbon dioxide types are useful for smaU fires involving
flammable Uquids and for limited use around electronic
instrumentation and equipment.
Depending upon potential hazards, a laboratoiy may have more than one
type in each room in an easUy accessible location away from the area of
greatest hazard. Halon extinguishers are useful in special areas housing
electronic or computer equipment that might be damaged by conventional
extinguishers. Both halon and carbon dioxide extinguishers create oxygen
deficiencies. The Environmental Science laboratory is equipped with
carbon dioxide type extinguishers. Use aU extinguishers with care. It is
often recommended that multipuipose fire extinguishers (type ABC) be
installed in the laboratory and that hoods containing organic materials be
equipped with halon extinguishers. Be sure that extinguishers are properly
serviced and recharged on a scheduled basis.
2.1.2 Fire blankets: Locate readUy accessible fire blankets close to each
laboratoiy.
2.1.3 Safety showers: Safety showers are an integral part of the laboratory, and

18
 are to be used in accidents involving acids, caustic or other harmful
Uquids, clothing fires, and other emergencies. Locate showers
convenientiy, preferably near a doorway, keep the floor space under them
uncluttered, and testregularly.The safety shower in the ESL is located by
the main door leading into the laboratory.
2.1.4 Eye washes: Remove contact lenses if appUcable. Flush eyes immediately
and thoroughly (15 min) to prevent sight impairment or even blindness
after a chemical splashes into the eye. Splashes to the face are rinsed off
easUy with a four-head eye wash. Some experts claim that the fountain-
Uke stream of waterfroman eye wash tends to drive particulate matter
(glass or metal shards, dirt, grit) into the eye rather than to wash it out. If
this type of accident occurs, consult an eye expert after gendeflushingwith
water. Locate eye washes at or near sinks in a highly visible and central
area awayfromelectrical receptacles. Portable eye washes are located at
the end of each lab bench at a permanent eye is stationed near the main
door below the safety shower. Check eye washes routinely for correct
function; if portable eye washes are used clean them weekly and change the
water to reduce the possibiUty of contamination. The use of eye-wash
botties and some of their problems have been discussed elsewhere.
2.1.5 Safety shields: The most generaUy used type of safety shield protects the
worker from various forms of radiation, such as laser beams and
ultraviolet emissions. Provide conventional chemical hoods with safety
glass and movable doors. Consider shielding when working with glass
vacuum or pressurized systems.
2.1.6 Safety containers: Safety containers are designed to minimize
consequences of an accident or to prevent spread of harmfiil materials.
Use saJfety containers to transport chemicals, especiaUy concentrated acids
and alkaUes. For flammable solvents use safety cans approved by the
Underwriters Laboratories. Glove boxes. Laminar-flow hoods, or
remotely operated confinements for radioactive material or dangerous
pathogens are more compUcated safety containers. Operate these under
negative pressure, with primary fUtration at the exit of gas or air vents
leading to a major ventilation systenL PeriodicaUy check fUter for
efficiency and change and test gloves to prevent accidentalreleaseof
particulates by the pumping action created during use.
2.1.7 Storage faciUties: In storing laboratory materials, it is necessary to know
their properties as weU astiieconsequences of accidents such as spilling,
explosion, or fire. As a general rule, do not store large containers of
reagents in working areas, but use smaUer containers holding only enough
for the days or weeks work. Do not store chemicals that combine to form
explosive, flammable, or dangerous compounds in a manner that would
aUow them to mix if an accident occurred. Store hazardous materials
within a tray or catch bin area. AU hazardous materials are stored in a
locked closet located in the back of the laboratory equipped with safety
devices such as a separate ventUation system andflameproof cabinets.
The poisons are kept separate and locked at aU times.

Storeflammablesolvents in properly vented cabinets approved by the

National Fire Protection Association or in a safetyrefrigerator.Use special
containers forflammablesolvents in quantities of more than 2 L or when
the cumulative amount offlammablesolvents in a room exceeds 8 L.

19
 (Flammable solvents are Uquids with aflashpoint below 60°C and a vapor
pressure exceeding 275 kPa atmospheric at 38°C.) VolatUe or flammable
materials are kept in a cabinet with a separate exhaust system located in the
chemical closet in the back of the laboratory.
2.1.8 Laboratory fiime hoods: Properly designed and operated fume hoods and
biological safety cabinets are essential to a safe laboratory operation. Use
hoods to contain and vent operations with hazardous materials; do not use
them primarily for storage.

Different air-flow and design classifications of fume hoods are avaUable

for different degrees of hazard. Know the air-moving ci^acity of the entire
system; pay particular attention to the air stream exhausted to tiie
community air. Check air flow of hoods on a scheduled basis with an
anemometer. Indicate with a marking pen or tape the sash position
required to obtain an air flow of 30 m/min.
2.1.9 Chemical spiU kits: Provide work and storage areas with chemical spiU kits
obtainedfromcommercial sources or assembled in the laboratory. Use
kits of suitable size to accommodate the quantities of acids, alkaUes, or
solvents being used. Devise and post spill procedures before the need
arises. SpiU fits are avaUable in the chemical closet located at the back of
the laboratory for acids, bases, mercury, and solvents. Absorbent piUows
(pigs) are also avaUable for acids, bases, and solvents. Baking soda is
also avaUable at the ends of each laboratory to use on acid spiUs. The
baking soda neutraUzes the acids to aUow for proper clean-up.

Also avaUable are sand and cat Utter tofirstcontain and then absorb any
possible spiUs. However, it should be noted that the sand or cat Utter did
not change the composition of the spiU and carefiil clean-up procedures
should be used.
2.1.10 Safety waU chart: Post in central location for information on hazardous
laboratory substances. Posted around the laboratory are hazard
classification label signs to quickly alert personnel to any possible hazards
conceming the materials they are working with. Chemicals used in the
laboratory are labeled with colored diamond shaped warning stickers that
detaU its known hazards
2.2 Personal Protective Equipment
Personal protective equipment and materials include such items as laboratory
garments, gloves, shoes, hard hats, glasses, shields, and other safety items used
by an individual. Their selection and use is governed by the particular tasks to be
performed. Such equipment is important for protection but dso serves as a
constantreminderof the need for safety. If it is determined that such are needed,
it is the managers and supervisorsresponsibUityto insure that they are used.
2.2.1 Clothing: Persons! clothing creates a banier between the individual and the
hazard. Employees using radioactive materials, suspected carcinogens,
and pathogenic materials may be required to change from street to
laboratory clothing when entering the work area-and to change again on
leaving. This not only prevents carrying hazardous materials outside the
area but it also permits necessary handling and cleaning of the clothing.
Disposable laboratory clothing should be fire-retardant. Laboratory coats
are avaUable to anyone working in the lab. They are acid resistant
2.2.2 Glovesfiequentiyare important Consult the material safety data sheet for

20
 a solvent or reagent to determine the type of glove to be used. Rubber
gloves may be used when handling hazardous Uquids, leaded gloves for
handling radioactive materials, and surgical gloves for handling pathogenic
material. Insulated gloves are essential for handling hot objects and
extremely cold ones, but do not use asbestos gloves. White cotton gloves
may be used to protect instmments.
2.2.3 Safety shoes are required in laboratories where heavy objects or equipment
is to be moved. Hard hats may be required if there is overhead machinery
in the laboratory.
2.2.4 Safety glasses: Even if the likelihood of accidents-seems smaU, the
consequences of an eye accident may be extremely serious. Require aU
laboratory personnel to wear safety glasses. Most laboratories prohibit use
of contact lenses with safety glasses, but this is an area of controversy and
should be evaluated in each laboratory. Safety glasses protect from
splashes, flying objects, powders, or ultraviolet exposure. However,
ordinary safety glasses do not provide total eye protection. If the work
involves special hazard to the eyes, consider additional protection. For
example, wear lenses with specialfiltersfor glassblowing, welding, laser
work, or exposure to other forms of radiation. When working in a room
with ultraviolet radiation, wear safety glasses with side shields or goggles
with solid side pieces. For certain activities, provide protection for
exposed skin. In working with acid or caustic materials wear a face shield
to protect both the eyes and the face.
2.2.5 Respirators: HaverespiratorsavaUable for emergency situations in which
dangerous gases, fumes, or aerosols may be formed and the room must be
entered before it is thoroughly ventUated. In laboratories using toxic
gases, such as boron trifluoride, chlorine, dimethylamine, ethylene oxide,
fluorine, and hydrogen bromide, providerespirators,preferably Self-
Contained Breathing Apparatus (SCBA), or supplied air.

3.0 Laboratory Hazards

Many laboratory hazards are fairly obvious, but identification of aU likely hazards is
important in developing an effective safety program. Develop a safety plan before any
work is started. This is to provide all personnel working with or near hazardous
materials information about them. Include Material Safety Data Sheets (MSDSs) in the
safety plan and describeroutineand emergency procedures; require that aU personnel
read and sign that they have read and understood the document
To avoid ingestion of toxic or infectious material, prohibit eating, drinking, and
smoking in aU laboratory areas. Post adequate warning signs in the laboratory and
insure that aU containers have clear andreadUyrecognizable labels. Avoidfloorclutter
so that escaperoutesand fire extinguishers are not blocked. Eliminate improper
storage such as precarious shelving or heavy or glass objects overhead.
3.1 Chemical Hazards
Chemical injuries may be external or intemal. External injuriesresultfrom skin
exposure to caustic or corrosive substances such as acids, bases, orreactivesalts.
Take care to prevent accidents, such as splashes and container spiUs.
CoincidentaUy, pay attention to equipment corrosion that ultimately may lead to
safety hazardsfromequipment failure. Intemal injuries may result from toxic or
corrosive effects of substances absorbed by the body.

21
3.1.1 Inorganic acids and bases: Many inorganic acids and bases have
Occupational Safety and Health Personal Exposure Limits and American
Conference of Governmental Industrial Hygienists Threshold Limit Values
(TLV) associated with them. These permissible exposure limits and
threshold limit values indicate the maximum air concentration to which
workers may be exposed. Fumes of these acids and bases are severe eye
and respiratory system irritants. Liquid or soUd acids and bases quickly
can cause severe bums of the skin and eyes. When acids are heated to
increase the rate of digestion of organic materials, they pose a significantly
greater hazard because fiimes are produced and the hot acidreactsvery
quickly with the skin.

Store acids and bases separately in weU-ventUated areas and away from
volatUe orgaiuc and oxidizable materials. The ESL uses a acid storage
cabinet to keep acids separatefromother chemicals. Use containers
(rubber or plastic buckets) to transport acids and bases. Work with strong
acids and bases only in a properly functioning chemical fume hood.
Slowly add acids and bases to water (with constant stirring) to avoid
spattering. If skin contact is made, thoroughly flush the contaminated area
with water, and seek medical attention if irritation persists. Do not rewear
clothing until after it has been cleaned thoroughly. Leather items (e.g.
belts and shoes) wiUretainacids even after rinsing with water and may
cause severe bums if rewom. If eye contact is made, immediately flush
both eyes for a fiiU 15 min at the eye wash and seek medical attention.

Perchloric acidreactsviolentiy or explosively on contact with organic

material. Do not use laboratory fume hoods used with perchloric acid for
organicreagents,particularly volatUe solvents. In addition to these
hazards, perchloric acid produces severe bums when contact is made with
the skin, eyes, orrespiratorytract Preferably provide a dedicated
perchloric acid hood.
The most common injuries suffered with sodium hydroxide are bums of
the skin and eyes. Sodium hydroxide solutions as dUute as 2.5 N can
cause severe eye damage. On dissolution, sodium hydroxide and other
alkalies produce considerable heat (often sufficient to cause boiling).
3.1.2 Metals and inorganic compounds: In general, consider aU laboratory
chemicals hazardous and use only as prescribed. Handle chemicals in a
fume hood, using eye protection and wearing a laboratory coat In case of
accidental skin contact,flushthe affected area thoroughly with water. If
irritation persists, medical examination is advisable.
Some hazards of specific metals and inorganic compounds are as foUows:
Some compounds of arsenic and nickel are highly toxic and may be
carcinogenic. Avoid inhalation, ingestion, and skin contact. Sodium azide
is toxic and reacts with acid to produce still more toxic hydrazoic acid.
When disposed of through a drain it mayreactwith copper and lead
plumbing to form metal azides that are extremely explosive. Azides may
be destroyed by adding a concentrated solution of sodium lUtrite, 1.5 g
NaNQz/ g sodium azide. The extreme toxicity of beryUium and its
compounds isreflectedby its low TLV of 2.() g/m. BeryUium is a

22
 suspected carcinogen in humans. Handle with extreme caution and only in
a laboratory fume hood or glove box. Cyanides are used asreagentsand
may be present in samples. Hydrogen cyanide is a lethal gas. Do not
acidify cyanide solutions except in a closed system or in properly ventUated
hood because hydrogen cyanide may be fonned and Uberated.

Mercury is unusual among the metals in that it is Uquid at room temperature

and has ^preciable vapor pressure. One broken thermometer in a poorly
ventilated room may cause the mercury TLV to be exceeded. Because of
its high volatiUty and toxicity, handle mercury and its compounds very
cautiously and provide a spiU cleanup kit Perchlorate salts are explosive
when mixed with combustible material. They also are severe irritants to
the skin, eyes, andrespiratorysystem. Use care in handling and storing
perchlorates. Sodium borohydride decomposes in water and Uberates
hydrogen, consequentiy creating an explosion hazard. Like many other
inorganic chemicals, it is a strong skin and respiratory system irritant
3.1.3 Orgaiuc solvents and reagents: Most solvents specified in this book have
TLVs for workroom exposure (see Table 1). Many organic reagents,
unlike most organic solvents, do not have TLVs (see Table 2 for those that
do) but this does not mean that they are less hazardous. Some compounds
are suspect carcinogens and should be treated with extreme caution. These
compounds include both solvents andreagentssuch as benzene, carbon
tetrachloride, chloroform, 1,4-dioxane, tetrachloroethylene, and benzidine.
Lists of chemicals with special hazardous characteristics are avaUable frx)m
the Occupational Safety and Health Administration and the National
Institute for Occupational Safety and Health. The Usts of "Regulated
Carcinogens" and of "Chemicals Having Substantial Evidence of
Carcinogenicity" are especiaUy important Determining laboratory
handling procedures on such authoritative Usts decreases the chance for
error.

Solvents used herein faU into several major categories: alcohols,

chlorinated compounds, and hydrocarbons. Exposure to each of these
classes of compounds can have a variety of health effects. Alcohols, in
general, are intoxicants, enable of causing irritation of the mucus
membranes and drowsiness. WhUe diols such as ethylene glycol are
poisonous, triols such as glycerol are not poisonous at aU. Chlorinated
hydrocarbons cause narcosis and damage to the central nervous system and
Uver. Hydrocarbons, like the other two groups, are skin irritants and may
cause dermatitis after prolonged skin exposure. Because of the volatiUty of
these compounds, hazardous vapor concentrations can occur (fire or
explosion hazard). Proper ventilation is essential.

Specific safety precautions for ether are as foUows: Store ether in tightiy-
closed amber botties in an explosion-safe or -proof refrigerator. Store only
with compatible chemicals. Ether is extremelyflammable;eliminate aU
sources of ignition and keep away from heat, sparks, and flames. Ether
causes eye irritation and dermatitis; handle only in a hood and avoid direct
physical contact Ether causes headaches, dizziness, nausea, or
unconsciousness; do not breathe vapors. If a spiU or leak occurs, evacuate
the area, then ventUate and absorb on vermiculite or simUar material. Wear

23
 appropriate OSHA equipment in the spiU area.
Table 1. Threshold Limit Values* for Solvents Specified m
Standard Methods
TLV
Compoundt ppm (v/v)
Acetic acid 10
Acetone 750
Acctonitnle (S) 40
BOTzene(Q 10
n-Butanol (S) 50
r-Butanol 100
Cartxxi disulfide (S) 10
Carbon tetrachloride (CXS) 5
Chlorofom (C) 10
Cyclohexan(Mie (S) 25
Diethyl ether 400
l,4-Dioxane(CXS) 25
Ethanol 1000
Ethyl acetate 400
Ethylene glycol 50
Hexane 50
Isoamyl alcohol 100
Isobutyl alcohol 50
Isopiopyl alcohol 400
Isoprq)yl ether 250
Methanol (S) 200
2-Methoxyethanol (S) 5
Methylene chlcxide (C) 50
Pentane 600
Propanol(S) 200
Pyridine 5
Tetrachloroethylene 50
Toluene 100
Xylene 100
These threshold limit values provide an indication of the maximum
air concratrations to which woikers may be exposed, t (C)
compound is a suspect carcinogen: (S) ref»^ to potential contribution
by skin absorption.

Organicreagentsused in this manual faU into four major categories: acids,

halogenated compounds, dyes and indicators, and pesticides. Most organic
acids have irritant properties. They are predominantiy soUdsfromwhich
aerosols may be produced. Dyes and indicators also present an aerosol
problem. Handle pesticides with caution because they are poisons and
avoid contact with the skin. Wear gloves and protective clothing.The
chlorinated compounds present much the same hazards as the chlorinated
solvents (narcosis and damage to the central nervous system and Uver).
Proper labeling of the compound, including a date for disposal based on the
manufacturers recommendation, permits tracking chemical usage and
disposal of outdated chemicals.

24
 Table 2. Threshold Limit Values for Reagents Specified in
Standard Methods.
Compound TLV
2-Aminoethanol 3 ppm (v/v)
Benzidine (CXS)* —
Benzyl chloride Ippm (v/v)
Chlorobenzene 75 ppm (v/v)
2-Chloro-6-(trichloromethyl) pyridine 10 mgAn^
Diethanolamine 3 ppm (v/v)
N^thalcnc 10 ppm (v^)
Oxalic acid 1 mg/mJ
Phenol 5 ppm (v/v)

* (Q ccxnpound is a suspected carcinogen; (S) refers to potratial

contribution by skin absorption.

3.2 Biological Hazards

Water laboratory safety also includes microbiological hazards. Pathogenic
microorganisms may produce human disease by accidental ingestion, inoculation
or injection or other means of cutaneous penetration. Good laboratory safety
techniques wiU control these agents. Primary dangers associated with
microbiological hazards involve hand-noouth contact whUe handling contaminated
laboratory materials and aerosols created by inoculating, pipetting, centrifuging,
or blending of samples or cultures.
Do not mix dUutions by blowing airtiirougha pipet into a microbiological culture.
When working with grossly polluted samples, such as sewage or high-density
microbial ciUtures, use a pipetting device attached to the mouth end of the pipet to
prevent accidental ingestion. Never pipet by mouth. Even for drinking water
samples mouth pipetting is inadvisable. Because untreated waters may contain
waterbome pathogens, discard aU used pipets into ajar containing disinfectant
solution for decontamination before glassware washing. Do not place discarded;
pipets on table tops, on laboratory carts, or in sinks without adequate
decontamination, (^ternary ammonium compounds that include a compatible
detergent, or solutions of sodium hypochlorite are satisfactory disinfectants for
pipet discard jars. Use highest concentrations recommended for these commercial
products provided that this concentration does not cause a loss of markings or
fogging of pipets. Replace disinfectant solution in the discard container daUy.
SteriUze contaminated materials (cultures, samples, used glassware, serological
discards, etc.) by autoclaving before throwing them away or processing for reuse.
Shattering of culture-containing tubes during centrifugation also produces
microbiological aerosols. Use leakproof blenders and keep themtightiycovered
during operation. Inserting a hot loop into aflaskof broth ciUture creates a
serious hazard due to aerosolized microorganisms. Using electric heater
incineration for steriUzing inoculating loops or needles may be desirable, but avoid
possible electrical shock that could occur by touching the loop to the inside of the
heater core.

Good personal hygiene practices are important in the control of contact exposures.

25
 Frequentiy disinfect hands and working surfaces. Provide drinking water outside
the laboratory, preferably from a foot-operated drinking fountain. Suggest
immuiuzation of laboratory staff against tetanus and possibly typhoid or,other
appropriate infectious agents, depending on nature of the work. Eliminate fUes
and other insects to prevent contamination of sterile equipment, media, samples,
and bacterial cultures and to prevent spread of infectious organisms to personnel
via this vector. InstaU screens in aU windows and outer doors if there is no air-
conditioning, and spray periodicaUy with pesticides along toe-stripping, sink and
storage cabinet areas, and utiUty service channels. Because laboratories also may
include a chemistry section that analyzes waters for pesticides, apply these
carefiiUy within the immediate areas of microbiological activity.
3.3 Radiation Hazards
AU persons are exposed to ionizing radiation. The average annual radiation dose
to the whole body from cosmic, terrestrial, and intemal sources, medical and
dental X-rays, etc., is about 185 mrems/year. Prevent unnecessary continuous or
intermittent occupational exposure, and prevent accidents that mayresultin
dangerous radiation exposure. In laboratories. X-rays, ultraviolet light, and
radioactive materialrepresenthazards that must be minimized.

Provide a Radiation Safety Manual, a Handbook of Laboratory Safety, or similar

manual to aU persons working with radioactive materials or radiation-producing
machines. This manual should discuss procedure for obtaining authorization to
use, order, handle, and store radionucUdes. It also should include procedures for
safe handling of unsealed radioactive material and procedures to foUow in ease of
radiation accidents, for decontamination, for personnel monitoring, for laboratory
moiutoring, and for disposal of radioactive materials.
3.3.1 Raidioactive materials: RadionucUdes are used in laboratories to develop
and evaluate analytical methods, to prepare counting standards, and to
caUbrate detectors and counting instruments. Sealed sources, such as the
nickel 63 detector ceU used in electron capture gas chromatograph units,
are comnwn. Instmct aU persons dealing with radioactive materials about
associated health hazards. EstabUsh proper procedures and incorporate
radiation safety measures to minimize exposure and accident.
3.3.2 Radiation-producing machines: Minimize hazardsfromdevices, such as X-
ray diffraction apparatus or electron microscope, by adhering strictiy to
manufacturers operating manual and procedures outiined in the referenced
safety manuals.
3.3.3 Ultraviolet radiation (UV): UV is usedfrequentiy.With properly
constmcted and operated instruments, it is not a significant hazard but can
be harmful when used for controlUng microorganisms in laboratory rooms
or for SteriUzing objects. Provide proper shielding,rememberingthat
shiny metal surfaces reflect this energy; shut off UV lamps when not in
use. Post warning signs and instaU indicator lights to serve as a constant
reminder when IW lamps are on. Wear safety glasses or goggles with
soUd side pieces whenever there is a possibiUty of exposure to UV
radiation.
3.4 Physical Hazards
3.4.1 Electrical: Conform electrical wiring, connections, and ^paratus to the
latest National Electrical Code. Fire, explosion, power outages, and
electrical shocks are all serious hazards that mayresultfrom incorrect use
of electrical devices. Ground aU electrical equipment or use double

26
 insulation. Use ground fault circuit breakers to the maximum extent
possible. Do not locate electrical receptacles inside fume hoods, do not use
equipment with frayed cords or cracked insulation nor spark-producing
electrical equipment near volatUeflammablesolvents. Use approved safety
refrigerators. Disconnect electrical equipmentfromthe power supply
before service orrepairis attempted and never bypass safety interlocks.
Equipmentrepairby employees not thoroughly acquainted with electrical
principles may present particularly dangerous situations.
3.4.2 Mecharucal: ShSeld or guard drive belts, puUeys, chain drivers, rotating
shafts, and other types of mecharucal power transmission apparatus.
Laboratory equipment requiring this guarding includes vacuum pumps,
mixers, blenders, and grinders. Shield portable power tools used in
laboratories. Guard eqiupment such as centrifiiges, which have high-
speed revolving parts, against fly-aways. Securely fasten equipment that
has a tendency to vibrate (e.g., centrifuges and air compressors) to prevent
the tendency to "walk", and locate awayfrombotties and other items that
may fall from shelves or benches because of the vibration.
3.4.3 Compressed gases: Gas cylinders may explode or "rocket" if improperly
handled Leaking cylinders may present an explosion hazard if tiie
contents areflammable,they are an obvious health hazard if the contents
are toxic, and they may lead to death by suffocation if the contents are inert
gases. OSHAregulationsgovem use and storage of compressed gases.
Transfer gas cylinders oiUy by carts, hand tmcks, or doUies. Secure gas
cylinders properly during storage, transport, and use and leave valve safety
cover on cyUnders during storage and transport Avoid the use of adapters
or couplers with compressed gas. Permanentiy identify cylinder contents.

4.0 Hazard Management Practices

4.1 Monitoring
The estabUshment of policies, practices, procedures, and methods to prevent
exposure of laboratory personnel to hazardous materials is only part of an
effective safety program. Simultaneous estabUshment of a monitoring or feedback
system is essential to insure that the protective features acmaUy work.
4.1.1 Chemical monitors: Devices capable of direct measurement of
concentration in a persons breathing zone have been described. Use either
active devices requiring a pump to pull air across the cell or passive
monitors thatrelyon dUfusion. These devices can measure inorgaiuc as
well as organic compounds with the appropriate sorbent. Particulates can
be coUectad on afilterusing an active device.
4.1.2 Biohazard monitors: These are an essential part of microbiological
monitoring. Include a pre-employment physical examination accomparued
by hematological-serological and other testsrelatedto exposure. Provide
for subsequent annual examinations consisting of serological tests,
biochemical fiinction studies, and chest X-rays. Vaccination and other
prophylactic measures are reqiured. Archive serum specimens for future
reference as necessary. Continual enforcement of basic laboratory safety
mles completes the monitoring program.
4.1.3 Radiochemical monitors: These include wipes, portable survey
instmments, and air samples. Multiple monitors usuaUy are required.
Measure extemal radiation exposure with personnel dosimeters, preferably

27
 die film dosimeter for measuring accumulated radiation over a period of
time. Pocket ionization chambers, thermoluminescent dosimeters, and
thimble chambers can be used to supplement the film dosimeter.

Whole body or gamma spectrometry radiation detectors determine the

presence of radioactive substances in the body, but these instruments are
expensive and require specialized training. Body waste also can be
monitored for radionuclides.

In addition to personnel monitoring, carry out general area monitoring.

Analyze aU equipment and suppUes that have been in contact with
radioactive substances. Use dosimetric and wipe tests. Thin-windowed
GM-counters are suitable for wipe samples and for monitoring skin and
clothing. An alpha scintiUation monitor is needed to detect alpha emitters.
4.2 Disposal of Wastes
General considerations: Stringent requirements exist for the disposal of wastes
involving potential criminal and civU UabiUty on both organizations and
individuals. Specifics vary by state and local area and are subject to change.
Licenses and/or permits may be required; consult local and state authorities.
A plan for the safe disposal of chemical and biological substances used in the
laboratory is important and should be discussed with the laboratory supervisor
and, if appropriate, with the safety coordinator. InstaU convenient coUection
systems, use properly labeled containers, provide fire-protected storage, and
provide a special separate storage area for hazardous or highly toxic materials.
Use metal safety cans for storing waste solvents and segregating incompatible
materials. Consider using special containers for extremely hazardous or highly
toxic wastes and special packing procedures to avoid breakage or damage to the
container in transportation. Store incompatible hazardous materials separately.

Waste disposal methods include incineration, burial, evaporation, neutralization,

chemicalreaction,special treatments, and use of commercial disposal speciaUsts.
Safety information including the method of choice for waste disposal of specific
inorganic and organic compounds is presented in a series of Material Safety Data
Sheets (MSDSs).
Sometimes it may be permissible to dispose of some hazardous chemicals down
the drain at certain concentrations but only with written permission of the local
wastewater authority on a chemical-by-chemical basis.
4.2.1 Chemical wastes—Used solvents can be distiUed and recovered for reuse.
Noncombustible solvents can be evaporated if vapors do not create an
environmental problem. SmaU quantities offlammablesolvents and
chemicals can be burned on the ground, in shaUow metal containers, or in
incinerators, provided that this does not violate airregulations.Neutralize
acidic and basic materials before final disposal. Many soluble, nontoxic
materials can be diluted carefuUy into a sewer system if it is certain that
they wiU not harm the plumbing system or environment. Whenever
possible, convert hazardous materials by chemicalreactionor other
processes to itmocuous compounds before disposal. If this is not feasible,
engage a commercial disposal speciaUst to dispose of hazardous or highly
toxic materials off the premises. Dispose of nonretumable gas cylinders

28
 witii the help of qualified persoimel only.
4.2.2 Biological wastes—Sterilize aU infectious or toxic substances, and aU
contaminated equipment or apparatus before washing, storage, or disposal.
Autoclaving is the steriUzation method of choice. GeneraUy, heat in an
autoclave under a pressure of 103 kPa to achieve a chamber temperature of
at least 121°C for a minimum of 15 min. Measure time after the material
being sterilized reaches 121°C. If materials are to be autoclaved in plastic
bags, add water to the contents to insure wet heat Dry heat and chemical
treatment also have been used for sterilizing non-plastic items. After
SteriUzation, the wastes can be handled safely by conventional disposal
systems. CoUect contaminated combustible wastes and aiumal carcasses in
impermeable containers for disposal by incineration.
4.2.3 Radiochemical wastes—GeneraUzed (Usposal criteria for radioactive wastes
have been developed by the National Committee on Radiation Protection
and Measurements. These recommendations have been given official
status by pubUcation in the Federal Register. Two general phUosophies
govem the disposal of radioactive wastes: (a) dUution and dispersion to
reduce the concentration of radionucUdes by carrier dUution or dUution in a
receiving medium and (b) concentration and confinement, usuaUy
involving reduction in waste volume with subsequent storage for decay
purposes.
Airbome wastes can be treated by either method. VentUation includes
dischargefromhooded operations to the atmosphere. Typical radioactive
gases include iodine, krypton, and xenon. Iodine can beremovedby
scmbbing or byreactionwith stiver nitrate. Noble gases can be removed
by adsorption; standard techniques can be used for particulates. DUution
methods are suitable for Uquids with low activity. Uitermediate levels may
be treated by various physical-chemical processes to separate the waste into
a nonradioactive portion that can be disposed of by dUution and a high-
activity portion to be stored. SoUd wastes may consist of equipment,
glassware, and other materials. When possible, decontaminate these
materials and reuse. Decontamination usually results in a Uquid waste.
Combustible materials that cannot be decontaminated often are burned with
special precautions; permits for bunting may be required. Use storage for
decay or permanent storage for treatingradioactivewastes when
alternatives are not avaUable.
4.2.4 Other special wastes—^Federalregulationsconcemed with polychlorinated
biphenyls (PCBs), dioxins, and asbestos require special attention for
wastes containing these materials.

29
 LABORATORY APPARATUS AND REAGENTS
INTRODUCTION
This section contains a discussion of requirements for laboratory apparatus and reagents
that are common to many of the methods presented in the manual. This section discusses
the general considerations that laboratory persoimel and students should be famiUar with
such as basic types of sample containers or glassware and preparation of common reagents
(such as H2SO4 and NaOH). Requirements that are more or less specific to the particular
determination being performed are described under the methods to which they apply. In
addition, observe the general considerations presented in this section. The requirements of
radiological, baaeriological, biological and toxicity/bioassay methods tend to differ in
many respeas from those of chemical and physical tests.

1.0 Apparatus

1.1 Containers
For general laboratory use, the most suitable material for containers is resistant
borosiUcate glass. Special glassware is avaUable with characteristics such as high
resistance to alkali attack, low boron content, or exclusion of tight. Choose
stoppers, caps, and plugs toresistthe attack of material contained in the vessel.
Metal screw caps are a poor choice for samples that wiU cause them to corrode
readily. Glass stoppers are unsatisfactory for strongly alkaline Uqitids because of
their tendency to stick fast. Rubber stoppers are exceUent for alkaline Uquids but
unacceptable for orgaiuc solvents, in which they sweU or disintegrate, or trace
metal solutions, which may be contaminated by them. Use
polytetrafluoroethylene (TTE or PTFE) for burets that contain strongly alkaline
Uquids. When appropriate, use other materials such as porcelain, nickel, iron,
platinum, stainless steel and high siUca glass.

CoUect and store samples in botties made of borosiUcate glass, hard mbber,
plastic, or other inert material as appropriate for specific analyses (Table 1).
Forrelativelyshort storage periods, or for constituents that are not affected by
storage in soft glass, such as calcium, magnesium, sulfate, chloride, and perhaps
others, the 2.5-L acid bottie "bell closure" is satisfactory. This closure holds a
glass or polyethylene disk against the ground-glass surface or a polyethylene
insert on the bottie lip and insures adequate protection. If part of tiie sample is to
be analyzed later for siUca, sodium, or other substances that would be affected by
prolonged storage in soft glass, transfer it to a smaU plastic bottie, while leaving
theremainderof the sample in the soft-glass bottie.

CarefuUy clean sample botties before each use. Rinse glass botties, except those
to be used for chromium or manganese analyses, either with a cleaning mixture
made by adding 1 L concentrated H2SO4 slowly, with stirring, to 35 mL saturated
sodium dichromate solution, or with 2% KMn04 in 5% KOH solution foUowed
by an oxaUc acid solution. Commercial alternates also are avaUable. Rinse with
other concentrated acids toremoveinorganic matter. Detergents are exceUent
cleansers for many purposes; use either detergents or concentrated HCl for
cleaning hard-mbber and plastic botties. After the botties have been cleaned, rinse
thoroughly withreagent-gradewater.

30
 For shipment, pack botties in wooden, metal, plastic or heavyfiberboardcases,
with a separate compartment for each bottie. Line boxes with insulating material
to protectfrombreakage. Samples stored in plastic botties need no protection
against breakage by impact or through fi:eezing.

Minimumi Maximum Storage/

Container Sample Preservation
Determination Material Size(mL') Regulatory*Recommended
Acidity P. G(B) 100 Refrig.24 h/14 d
Alkalinity P, G 200 Refrig.24 h/14 d
BCD P,G 1000 Refrig.6 h/48 h
Boron P 100 None required28 d/6 m
Bromide P,G — None required 28 d/28d
Carbon, Tot. Org. G 100 Analyze immed.; orrefrig.and add HCl to
pH<2 7 d/28 d
Carbon dioxide P.G 100 Analyze immed. stat/N.S.
CCD P, G 100 Analyze ASAP, or add H2SO4 to pH<2
7 d/28 d
Chlorine, Residual P, G 500 Analyze iminediatelyO.5 h/stat
Chlorine dioxide P.G 500 Analyze immediatelyO.5 h/N.S.
ChlorophyU P, G 500 30 d in dark30 d/N.S.
Color P,G 500 Refrigerate48 h/48 h
Conductivity P,G 500 Refrigerate28 d/28 d
Cyanide
Total P,G 500 Add NaOH to pH>12, refrig. in dark
24 h/14 d; 24 h if S2-
Chlor. amenable P,G 500 Add 100 mg NaiSiOs/Lstat/M d;
24 h if Sulfide present
Fluoride P 300 None required 28 d/28 d
Hardness P.G 100 Add HNO3 to pH<26 months/6 months
Iodine P.G 500 Analyze immediatelyO.5 h/N.S.
Metals, general P(A), G(A) — For diss, metalsfilterimmed., add HNO3 to
pH<26 m/6 m
Chromium VI P(A), G(A) 300 Refiigerate24 h/24 h
Mercury P(A), G(A) 500 Add HNO3 to pH<2, 4°C, refrig.28 d/28 d
Nitrogen
Ammonia P.G 500 Analyze ASAP or add H2SO4 to pH<2,
refrig.7 d/28 d
Nitrate P.G 100 Analyze as soon as possible or refrig.
48 h/48 h (28 d chlor)
Nitrate + nitrite P, G 200 Add H2SO4 to pH<2, refrig.none/28 d
Nitrite P, G 100 Analyze ASAP orrefrig.none/48h
Total Kjeldahl P.G 500 Refrig.; add H2SO4 to pH<27 d/28 d

(cont.)

31
Table 1. Summary of Sampling and Preservation Requirements (Cont)
Minimum Maximum Storage/
Container Sample Preservation
Determination Material Size(mL) Regulatory*Recommended

Odor G 500 Analyze ASAP, refrig.6 h/N.S

OU and Grease G, w-m 1000 Add H2SO4 to pH<:2, refiig.28 d/28 d
Org. Compounds
Pesticides G(S),TFE- — Refrig.; add 1000 mg ascorbic acid/L if
resid.7 d/7 d untU
lined cap chlorine present extraction; 40 d after
Phenols P, G 500 Refiig.; add H2S04 to pH<2*/28 d
Purgeables G, TFE- 50 Refrig.; add HQ to pH < 2; add 1000 mg
7d/14d
lined cap ascorbic acid/L ifresid.chlorine present
Oxygen, Dissolved G, BOD bottie
Electrode 300 Analyze immediatelyO.5 h/stat
Winkler 300 Titration may be delayed after acidification
8h/8h
Ozone G 1000 Analyze mimediatelyO.5 h/N.S.
pH P. G — Analyze immediately2 h/stat
Phosphate G(A) 100 For diss, phosphate fUter immed.
refrigerate48 h/N.S.
Salinity G, wax seal 240 Analyze immediately or use wax seal
6 months/N.S.
SiUca P — Refrigerate, do notfreeze28 d/28 d
Sludge Digester Gas G, gas — N.S.
bottie
SoUds P, G — Refiig. 7 d/2-7 d
Sulfate P, G — Refrig. 28 d/28 d
100 Refrig.e, add 2 drops 2N zmc acetate/100
SuUide P, G mL,28 d/7 d
add NaOH to pH > 9
Taste G 500 Analyze ASAP, refrig.24 h/N.S.
Temperature P.G — Analyze immediatelystat/stat
Turbidity P.G — Analyze same day; store in dark up to 24
h,24 h/48 h
refrig.

* See mdividual assays for additional detaUs. For determinations not Usted, use glass or
plastic contamers, preferablyrefrigerateduring storage and analyze as soon as possible.
Refiigerate = storage at 4° C mtiiedark. P = plastic (polyetiiylene or equivalent): G =
glass; G(A) or P(A) + rmsed witii 1 + 1 HNCSa; G(B) = glass, borosiUcate; G(S) = glass,
rinsed witii organic solvents; N.S. = not stated mreferences;stat = no storage aUowed,
analyze immediately
1.2 Volumetric Glassware
CaUbrate volumetric glassware or obtain a certificate of accuracy from a competent
laboratory, volumetric glassware is caUbrated either "to contain" (TC) or "to
deUver" (TD). Glassware designed "to deUver" wiU do so witii accuracy only

32
 when the inner surface is so scrupulously clean that water wets it immediately and
forms a uniform film upon emptying. Whenever possible, use borosiUcate
glassware. For accurate work use Class A volumetric glassware.

CarefuUy measure weights and volumes in preparing standard solutions and

caUbration curves. Observe simUar precautions in measuring sample volumes.
Use volumetric pipets or burets where the volume is designated to two decimal
places (X.OO mL) in the text Use volumetric flasks where specified and where
the volume is given as 1000 mL rather than 1 L.
1.3 Nessler Tubes
Unless otherwise indicated use "taU"-form nessler tubes made ofresistantglass
and selected from uniformly drawn tubing. The glass should be clear and
colorless and the tube bottoms should be plane-paraUel. When the tubes are fiUed
with Uquid and viewedfix)mthe top with atightsource beneath, there should be
no dark spots nor any lens-like distortion of the transmitted tight: The tops of the
tubes should beflat,preferably fire-poUshed; and smooth enough to permit cover
sUps to be cemented on for sealing. Nessler mbes with standard-taper clear glass
tops are avaUable commerciaUy. Graduation marks should completely encircle the
tubes.

The 100-mL tubes should have a total length of approximately 375 mm. Their
inside diameter should approximate 20 mm and the outside diameter 24 mm. The
graduation mark should be as near as possible to 300 mm above the inside
bottom. Tubes sold in sets should be of such uniformity that this distance does
not vary more than 6 mm. (Sets are avaUable commerciaUy in which the
maximum difference between tubes is not more than 2 mm.). A graduation mark
at 50 mL is permissible.
The 50-mL mbes should have a total length of about 300 mm. Their inside
diameter should approximate 17 mm and the outside diameter 21 mm. The
graduation mark on the tube should be as near as possible to 225 mm above the
inside bottom. Tubes sold in sets should be of such uniformity that this distance
does not vary more than 6 mm. (Sets are avaUable commercially in which the
maximum difference between tubes is not more than 1.5 mm.). A graduation
mark at 25 mL is permissible.

2.0 Reagents
2.1 Reagent QuaUty
Use only the best quality chenticalreagentseven though this instmction is not
repeated in the description of a particular metiiod. Order chemicals for which the
American Chemical Society has pubUshed specifications in the "ACS grade."
Order other chenucals as "analyticalreagentgrade" or "spectral grade orgaiuc
solvents."
Unfortunately, many commercial dyes for which the ACS grade has not been
estabUshed faU to meet exacting analytical requirements because of variations in
the color response of different lots. In such cases, use dyes certified by the
Biological Stain Commission.

33
 Where neitiier an ACS grade nor a certified Biological Stain Commission dye is
available, purify the soUd dye through recrystaUization.
The foUowing standard substances, each bottie of which is accompanied by a
certificate of analysis, are issued by the National Instimte of Standards and
Technology (NIST), Department of Commerce, Washmgton, D.C., for tiie
purpose of standardizing analytical solutions:
Acidimetric:
84j: Acid potassium phthalate
350a: Benzoic acid
Qxidimetric:
40h: Sodium oxalate
83d: Arsenic trioxide
136e: Potassium dichromate
Buffer:
185f: Potassium hydrogen phthalate
1861c: Potassium dihydrogen phosphate
186nc: Disodium hydrogen phosphate
187c: Borax
188: Potassium hydrogen tartrate
189a: Potassium tetroxalate
191a: Sodium bicarbonate
192a: Sodium carbonate

Many hundreds of other standards issued by NIST are described in Special

PubUcation 260.
A successful dithizone test demandsreagentsof the highest purity. Chloroform
and carbon tetrachloride are avaUable in a grade declared to be suitable for
dithizone methods. Select reagents of this quaUty for the dithizone methods
described in this manual.
Water-soluble sodium salts of the common indicators usuaUy are recommended
for indicator preparation because of their general avaUabiUty and reasonable cost.
When alcohol or ethyl alcohol is specified for preparing such indicators as
phenolphthalein, use 95% ethyl alcohol. When isopropyl alcohol is specified use
a simUar grade.
Certain organic reagents are somewhat unstable upon exposure to the atmosphere.
If the stability of a chemical, is limited or unknown, purchase small lots at
frequent intervals.
Dry all anhydrousreagentchemicals required for preparation of standard
caUbration solutions andtitrantsin an oven at 105 to 110°C for at least 1 to 2 h
and preferably overnight After cooling to room temperature in an efficient
desiccator, promptiy weigh the proper amount for dissolution. Shoitid a different
drying temperature be necessary, tlus is specified for the particular chemical. For
hydrated salts, substitute mUder drying in an efficient desiccator for oven-drying.
2.2 Common Acid and AlkaU Solutions

34
2.2.1 Concentration units used: Reagent concentrations are expressed in terms of
normaUty, molarity, and additive volumes.

A normal solution (N) contains one gram equivalent weight of solute per
titer of solution.

A molar solution (M) contains one gram molecular weight of solute per titer
of solution.

In additive volumes (a + b), thefirstnumber, a,refersto the volume of

concentratedreagent;the second number, b, refers to the volume of
distUled water required for dUution. Thus, "1-1-9 HCl" denotes that 1
volume of concentrated HCl is to be dUuted witii 9 volumes of distUled
water.

To make a solution of exact normaUty from a chemical that cannot be

measured as a primary standard, prepare arelativelyconcentrated stock
solution and then make an exact dUution to the desired strength.
Altematively, make a solution of sUghtiy higher concentration than that
desired, standardize, and make suitable adjustments in concentration by
dUution; or, use the solution as first standaixiized and modify the calculation
factor. This last procedure is useful especiaUy for solutions that slowly
change strength and must berestandardizedfrequentiy—^for example,
sodium thiosulfate solution. Adjustment to exact normaUty specified is
desirable when a laboratory makes a large number of determinations with
one standard solution.

Determinations are in accord with the instmctions in this manual as long as

normality of a standard solution does notresultin atitrationvolume so
small as to preclude accurate measurement or so large as to cause abnormal
dUution of thereactionmixture, and as long as the solution is standardized
properly and the calculations are made properly.
2.2.2 Preparation and dUution of solutions: If a solution of exact normaUty is to
be prepared by dissolving a weighed amount of a primary standard or by
dUuting a stronger solution, bring it up to exact volume in a volumetric
flask.
Accurately prepare stock and standard solutions prescribed for colorimetric
determinations in volumetric flasks. Where concentration does not need to
be exact, mix the concentrated solution or soUd with measured amounts of
water, using graduated cylinders for these measurements. There is usuaUy
a significant change of volume when strong solutions are mixed, resulting
in a total volume less than the sum of volumes used. For approximate
dUutions, volume changes are negligible when concentrations of 6N or less
are dUuted.

Mix thoroughly and completely when making dUutions. One of the most
common sources of error in analyses using standard solutions dUuted in
volumetric flasks is faUure to attain complete mixing.
2.2.3 Storage of solutions: Some standardized solutions alter slowly because of
chemical or biological changes. The practical life, requiredfrequencyof

35
 standardization, or storage precautions are indicated for such standards.
Others, such as dUute HCl, are nonreactive. Yet their strength, too, may
change by evaporation that is not prevented by a glass stopper. Changes in
temperatures cause a bottie to "breathe," and aUow some evi^ration.

Do not consider a standard vaUd for more than a year unless it is

restandardized. It is vaUd for that length of time only if conditions
miitimize evaporation and it has been demonstrated previously that
preservation techniques are adequate. If the bottie is opened often or it is
much less than half fuU, significant evaporation occurs in a few months.
Verify concentration of standard solutions that have been stored.
Use glass botties of chemicaUyresistantglass except where glass is
incompatible (e.g., siUca solutions). For standard solutions that do not
react with rubber or neoprene, use stoppers of these materials, because they
can, if properlyfitted,prevent evjq)oration as long as the bottie is closed.
Screw-cap bottles also are effective. If the cap has a gasket of reasonably
resistant material, permissible usage wiU be about the same as for mbber
stoppers.
2.2.4 Hydrochloric and sulfiiric acid as alternatives: DUute standardized H2SO4
and HCl are caUed for in various procedures. Often these solutions are
interchangeable. Where one is mentioned, the other may be used if it is
known to make no difference.
2.2.5 Preparation: Although instmctions usually describe preparation of 1 L of
solution, prepare smaUer or larger volumes as needed. Instmctions calling
for the preparation of 100 mL usually involve either short-lifereagentsor
those used in smaU amounts.
A safe general mle is to add more concentrated acid or alkaU to water, with
stirring, in a vessel that can withstand thermal shock, and then to dUute to
final volume after cooling to room temperature.
2.2.6 Uniform reagent concentrations: An attempt has been made to estabUsh a
number of uniform common acid and base concentrations that wiU serve for
adjustment of pH of samples before color development or final titration.
The foUowing acid concentrations are recommended for general laboratory
use: the concentrated reagent of commerce, 6N, IN, O.IN, and 0.02N.
See Table 1 below for directions for preparing these acid concentrations.
The required 15N, 6N, and IN NaOH solutions, and 5N, 3N, and 0.2N
NH4OH solutions shown in Table 2.

36
Table 1. Preparation of Uniform Acid solutions
Desired Component Hydrochloric Sulfuric Nitric Acid
Acid (HQ) Acid (H2SO4) (HNO3)
NormaUty of concentrated reagent 11-12 36 15-16
Volume (ml) of concentrated reagentto
prepare 1 L of:
18 N solution 500(1+1)
6N solution 500(1+1) 167(1+5) 380
IN solution 83(1+11) 28 64
0.1 N solution 8.3 2.8 6.4
Volume (ml) of 6Nreagentto
prepare IL of O.IN soln 17 17 17
Volume (ml) of INreagentto
prepare IL of 0.02 N soln 20 20 20

Table 2. Preparation of Uniform Sodium Hydroxide Solutions

Required Required
Weight of Volume of
NormaUty NaOH to prepare 15NNaOHto
of 1000 ml of prepare 1000 ml
NaOH solution of solution
Solution g ml
6 240 400
1 40 67
0.1 4 6.7

Preparation of ammonium hydroxide solutions, NH4OH: Prepare 5 N, 3N,

and 0.2N NH4OH solutions by diluting 333 ml, 200 ml, and 13 ml,
respectively, of concentrated reagent to 1000 ml with distiUed water

37
 REAGENT-GRADE WATER
INTRODUCnON
One of the most important aspects of analysis is the preparation ofreagent-gradewater to
be used for dUution ofreagentsand for blank analysis. Reagent-grade water covers a range
from Type I with no detectable concentration of the conq)ound or element to be analyzed at
the detection limit of the analytical method to Type EI for washing and quaUtative analysis
(see Table 1), Reagent-grade water should befreeof substancestiiatmterfere witii
analytical methods. The quaUty of water required isrelateddirectiy to the analysis being
made. Requirements for water quaUty may differ for organic, inorganic, and biological
constituents depending on the use(s) for which the water is intend^.
Any method of preparation ofreagent-gradewater is acceptable provided that the requisite
quaUty can be met Improperly maintained systems may add contaminants. Reverse
osmosis, distiUation, and deionization in various combinations aU can produce reagent-
grade water when used in the proper arrangement UltrafUtration and/or ultraviolet
treatment also may be used as part of tiie process. Table 2 Usts commonly avaUable
processes for water purification and major classes of contaminants removed by
purification.

Table 1. Reagent Water Specifications

Quality Parameter Type I Type II Type III

Bacteria, CFUAnL <10t <1000 NS

pH NS* NS 5-8
Resistivity, megohm-cm at 25*'C >10 >1 0.1
Conductivity, nmho/cm at 25*0 <0.1 1 10
Si02, mg/L <0.05 <0.1 <1
Particulate matter^ 0.22 Jim filter NS NS
Organic contaminants^ Activated carfoon§ NS NS

• NS = not specified.
t Preferably bactOTa-firee.
t Process specification; not measured by end user.
§ Pretreatment, and possibly past-treatment for cCTtain uses.

1.0 Metiiods for Preparation of Reagent-Grade Water

1.1 DistiUation
Prepare laboratory-grade distiUed water by distilling water from a stiU of aU-
borosiUcate glass, fused quartz, tin, ortitanium.To remove ammonia distiU from
an acid solution. Remove CO2 by boiling the water for 15 min and cooling
rapidly to room temperature; exclude atmospheric CO2 by using a mbe containing
soda lime or a commerciaUy avaUable COi-removing agent
Boiling the water may add other impurities by leaching impuritiesfromthe
container. Pretreat feed water and provide periodic maintenance to minimize scale
formation within the stiU. Pretreatment may be required where the feed water
contains significant concentrations of calcium, magnesium, and bicarbonate ions;
it may involve demineralization via reverse osmosis or ion exchange. Resistivity

38
 of distiUed water (Type II) should be > 1.0 megohm-cm at 25°C and for Type I,
10 megohm-cm. Measurements are more accurately made with in-line cells.
1.2 Reverse Osmosis
Reverse osmosis is a process in which water is forced under pressure through a
semipermeable membraneremovinga portion of dissolved constituents and
suspended impurities. Product water quaUty depends on feed water quaUty.

Select thereverseosmosis membrane naodule appropriate to the characteristics of

the feed water. Obtainrejectiondata for contaminants in the feed water at the
operating pressure to be used in preparingreagent-gradewater. Set overaU water
production to make the most economical use of water without compromising the
final quaUty of the permeate. Selection of spiral-wound or hoUow fiber
configurations depends on folding potential of the feed water. Regardless of
configuration used, pretreatment may be required to minimize membrane fouling
with coUoids or particulates and to minimize introduction of chlorine, iron, and
other oxidizing compounds that may degradereverseosmosis membranes.
Periodic flushing of the membrane modules is necessary.
1.3 Ion Exchange
Prepare deionized water by passing feed water through a mixed bed ion
exchanger, consisting of strong anion and strong cationresinsmixed together.
When Ae system does not run continuously, recirculate product water through
ion-exchange bed. Resistivity for Type I water should be 10 megohm-cm (in-
Une) at 25°C.
Use separate anion and cationresinbeds in appUcations whereresinregeneration
is economicaUy attractive. In such instances, position the anion exchanger
downstream of the cation exchanger toremoveleachatesfromthe cation resin.
Proper bed sizing is critical to the performance of the resins. In particular, set the
length-to-diameterratioof the bed in accordance with the maximum process flow
rate to ensure that optimal face velocities are not exceeded and that sufficient
residence time is provided.
In appUcations where tiie feedwater has significant quantities of organic matter,
remove organics to minimize potential fouling of theresins;Possible
pretreatments include prefUtration, distiUation,reverseosmosis, or adsorption.
1.4 Adsorption
Adsorption is generaUy used toremovechlorine and organic impurities. It is
accompUshed typicaUy with granular activated carbon. Efficiency of orgaitics
removal depends on the nature of the organic contaminants, the physical
characteristics of tiie activated carbon, andtiieoperating conditions. In general,
organics adsorption efficiency is inversely proportional to solubUity and may be
inadequate for theremovalof low-molecular-weight, polar compounds.
Performance differences among activated carbons are attributable to the use of
different raw materials and activation procedures. Select the ^propriate activated
carbon witiiregardto these differences. Even witii optimum activated carbon,
proper performance wiU not be attained unless the column is sized to give
required face velocity andresidencetime attiiemaximum processflowrate.
Use of activated carbon may adversely affect resistivity. This effea may be
controUed by use of reverse osmosis, mixed resins, or special adsorbents. To
achieve the lowest level of organic contamination, use mixtures of poUshing

39
 resins with special carbons in conjunction with additional treatment steps, such as
reverse osmosis, natural carbons, ultraviolet oxidation, or ultrafUtration.
2.0 Reagent Water QuaUty
2.1 QuaUty Guidelines
Several guidelines forreagentwater quaUty, based on contaminant levels, are
available (see Table l)P-3 Methods and uses Usted below are based on National
Committee for CUnical Laboratory Standards (NCCLS) guideUnes.
Use Type I water in test methods requiring minimum interference and bias and
maximum precision. Type n water is intended to provide the user with water in
whichtiiepresence of bacteria can be tolerated. It is used fortiiepreparation of
reagents, dyes, or staining. Type HI water may be used for glassware washing,
preliminary rinsing of glassware, and as feedwater for production of higher-graide
waters.

Type Ireagentwater, having a minimumresistivityof 10 megohms-cm, 25°C (in

tine), typically is prepared by distiUation, deionization, orreverseosmosis
treatment of feedwater foUowed by poUshing with a mixed-bed deionizer and
passage through a 0.2-nm-pore membrane filter. Altematively treat by reverse
osmosis foUowed by carbon adsorption and deionization. Determine quaUty at
the time of production. Mixed bed deionizers typicaUy add smaU amounts of
organic matter to water, especiaUy if the beds are fresh. Resistivity of Type I
water should be >10 megohm-cm at 25°C, measured in-Une. Resistivity
measurements wiU not detect organics or non-ionized contaminants, nor wiU they
provide an accurate assessment of ionic contaminants at the microgram-per-Uter
level. Thus, make separate measurements of contaminants such as TOC, Si02,
and bacterial counts.
Type n water typicaUy is produced by distiUation or deionization. Resistivity
shoitid be >1 megohm-cm at 25°C. Observe the same precautions on
measurements of other contaminants.
Type in water should have a minimumresistivityof 0.1 megohm-cm.
Other contaminants in reagent water are Usted in Table 1.
The pH of Type I or Type n water cannot be measured accurately without
contaminating the water. Measure other constituents as required for individual
tests.
Type I water cannot be stored without significant degradation; produce it
continuously and use it immediately after processing. Type n water may be
stored, but keep storage to a minimum and provide quality consistent with the
intended use. Store only in materials that protect the waterfromcontamination,
such as TFE and glass for organics analysis or plastics for metals. Store Type HI
water in materials that protect the water from contamination.

40
 Table 2. Water Purification Processes.
Maior Classes of Contaminants »
Dissolved Dissolved Dissolved Pyrogens/
Processes Ion. Solids lon.Gases Organics Particulates Bacteria
Endotoxins

Distillation G-Et P G E E E
Deicxiization E E P P P P
Reverse osmosis Gt P G E E E
Caibon adscMption P P§ G-EQ P P P
Filtration P P P E E P
Ultrafiltration P P G# E E E
Ultraviolet oxidation P P G-EO P G« P

Permission to use this table fh)m C3-A2, Vol. 11, No. 13, Aug. 1991, •'Preparation and Testing of
Reagent Watw in the Clinical Laboratory - Second Edition" has been granted by the National Committee
fa* Clinical Lalxxatcxy Standards. The complete current standard may be obtained from National
Committee for Clinical Laboratory Standards, 771 E. Lancaster Ave., Villanova, Pa., 19085.

* E = Excellent (a^)able of complete or near totalremoval),G = Good (capable ofremovinglarge

percentages), P = Poor (littie or no removal).
t Resistivity of water purified by distillation is anOTderof magnitude less than water produced by
deionization, due mainly to the presence of CO2 and sometimes H2S, NH3, and other ionized gases if
present in the feedwater.
$ Resistivity of dissolved ionized solids dq)ends on original feedwater resistivity.
§ Activated carbon removes chlcxine by adsorption,
n When used in ccxnbination with other purification processes, special grades of activated carbon and other
synthetic adsorbents exhibit excellent capabilities forremovingorganic contaminants. Their use,
however, is targeted toward specific ccnnpounds and applications.
# Ultrafilters have demonstrated usefulness in reducing specific feedwater organic contaminants based on the
rated molecular weight cut-off of the membrane.
0 185 nm ultraviolet oxidation (batch process systems) is effective inremovingtrace organic contaminants
when used as post-treatment. Feedwater makeup plays a critical role in the performance of these batch
processors.
00 254 nm UV sterilizers, while not physically removing bacteria, may have bactericidal or bacteriostatic
capabilities limited by intensity, contact time, and flow rate.

41
 Statistics
1.0 Normal Distribution
If a measurement is repeated many times under essentiaUy identical conditions, the
resitits of each measurement, x, wiU be distributed randomly about a mean value
(arithmetic average), because of uncontroUable or experimental error. If an infinite
number of such measurements were to be accumulated, the individual values would be
distributed in a curve simUar to those shown in Figure 1. The curve at left iUustrates
the Gaussian or normal distribution, which is described precisely by the mean, p., and
the standard deviation, a.
1.1 Average
The mean, or average, of the distribution is simply the sum of aU values divided
by the number of values so summed, i.e., p = ZiXi/ n. Because no
measurements arerepeatedan infinite number of times, an estimate of the mean is
made, using the same summation procedure but with n = to a fiitite number of_
repeated measurements (10, or 20, or...). This estimate of p, is denoted by x.
1.2 Standard Deviation
The standard deviation of the normal distribution is defined as:
a=[I(;c-^l)2/nll/2.
Again, the analyst can only estimate the standard deviation because the number of
observations made isfinitethe estimate of a is denoted by s and is calculated as
foUows:
S = [I(JC-^)2/«.l]l/2.

1.3 Central Tendencies

The standard deviation fixes the width, or spread, of the normal distribution, and
also includes a fixed fraction of the values making up the curve. For example,
68.27% of the measurements Ue between p ± 1 a, 95.45% between p ± 2 a„
and 99.70% between p ± 3 a,. It is sufficientiy accurate to state that 95% of the
values are within ± 2 a and 99% within ± 3 a. When values are assigned to the ±
a multiples, they are confidence Umits. For example, 10 ± 4 indicates that the
confidence limits are 6 and 14, whUe valuesfrom6 to 14representthe confidence
interval.
Another useful statistic is the standard error of tiie mean, a ^, which is the
standaid deviation divided by the square root of the number of values, or:

This is an estimate of the accuracy of the mean and impUes that another sample
from the same population would have a mean within some multiple of this.
Mititiples of this statistic include the samefiractionof the values for a stated
above. In practice, a relatively smaU number of average values is avaUable, so the
confidence intervals of the mean are expressed as:
x± tsl yn

42
 where t has the foUowing values for 95% confidence intervals:

n t n /
2 12.71 5 2.78
3 4.56 10 116
4 3.18 oo 1.96

The use of t compensates for the tendency of a smaU number of values to

underestimate uncertainty. For n > 15, it is common to use « = 2 to estimate the
95% confidence interval.

StiU another statistic is therelativestandard deviation (RSD), also known as the

coefficient of variation (CV), which commonly is expressed as a percentage. This
statistic is calculated as 100 a/p and its estimate is 100 s/x (standard deviation
divided by the mean and multipUed by 100). This statistic normaUzes the standard
deviation to the mean of the parameter being measured. It is sometimes used to
make direct comparisons among analyses that include a wide range of
concentrations. For example, if analyses at low concentrations yield aresultof 10
±1.5 mgA- and at high concentrations 100 ± 8 mg/L, the standard deviations do
not appear comparable. However, therelativestandard deviations are 100
(1.5/10) = 15% and 100 (8/100) = 8%, which mdicates tiie lesser variation
obtained at the higher concentration. SimUarly, this statistic is employed to
compare different methods of measuring the same parameter or even comparisons
of theresititsof testingrepUcatedsamples at different laboratories.

In a normal distribution (a bell shaped curve), the mean, median, and mode are aU
the same. The mode is the most frequent value obtained through a series of
analyses, and the median is the middle value in a ranked series of the values
obtained.
1.4 Lx)g-Normal Distribution
In many cases theresultsobtained from analysis of environmental samples wiU
not be normally distributed, i.e., a graph of the data wUl be obviously skewed, as
shown atrightin Figure 2, with the mode, median, and mean being distinctiy
different. To obtain a nearly normal distribution, convert theresultsto logarithms
and then calculate x and s. The antUogarithms of these tw£ values are estimates of
the geometric mean and the geometric standard deviation, Xg and Sg.
1.5 Test for OutUers/Rejection of Data
Quite often in a series of measurements one or more of theresititswiU differ
greatiy from the other values. TheoreticaUy, noresultshould be rejected, because
it may indicate either a faulty techitique that casts doubt on allresultsor it may be a
tme variant in the distribution. In practice,rejecttheresultof any analysis in
which a known error has occurred. In environmental studies, extremely high and
low concentrations of contaminants may indicate the existence of areas with
problems or areas with no contamination, so they should not be rejected
arbitrarily.
An objective test for outUers has been described. If a set of data is ordered from
low to high: XL, X2 . . . XH and the average and standard deviation are
calculated, then suspected high or low outUers can be tested by the foUowing
procedure. First, calculate the statistic T:

43
 T = (XH - x)/s for a high value, or
T = (x - XL)/S for a low value.

Second, compare the value of t with the valuefromTable 1 for either a 5% or 1%

level of significance. If the calculated T is larger than the table value for the
number of measurements, n, then the XH, or XL is an outUer at that level of
significance.

Table 1. Critical Values for 5% and 1% tests of

Discordance for a Single OutUer in a Normal Sample

Number of Critical Value

Measurements 5% 1%

3 1.15 1.15
4 1.46 1.49
5 1.67 1.75
6 1.82 1.94
7 1.94 2.10
8 2.03 2.22
9 2.11 2.32
10 2.18 2.41
12 2.29 2.55
14 2.37 2.66
15 2.41 2.71
16 2.44 2.75
18 2.50 2.82
20 2.56 2.88
30 2.74 3.10
40 2.87 3.24
50 2.96 3.34
60 3.03 3.41
100 3.21 3.60
120 3.27 3.66

44
 NORMAL DISTRIBUTION

RANGE

Figure 1. Examples of Normal Distribution.

NON-NORMAL DISTRIBUTION

a.
Hi
m
D
Z

RANGE

Figure 2. Examples of Non-normal Distribution.

45
 DATA QUALITY
1.0 Precision
Precision is a measure of the closeness with which multiple analyses of a given sample
agree with each other. Assess precision by repUcate analyses, by repeated analyses of
a stable standard, or by analysis of known additions to samples. Precision is specified
by the standard deviation of the results. If overaU precision of a study is desired,
analyze dupUcate samples. This latter precision includes the random errors involved in
sampling as weU as the errors in sample preparation and analysis. In most cases, lack
of precision is caused by human error.
When only a fewrepUcationsare used, for example, dupUcate sample analysis or
dupUcate extract analysis, tiie range ofresults,R, is nearly as efficient as the standard
deviation because the two measures differ by a constant (1.128s = R for dupUcates,
1.693s = R for tripUcates).
Precision is defined as the standard deviation of a sample group. It is computed by the
foUowing formula: SD = V Y (Deviations'^
n-1
where: Deviation = mean - observed value
n = number of observations
2.0 Bias
Bias is a measure of systematic error. It has two components, one due to the method,
and the other to a laboratory's use of the method. The bias of a method is measured
best by a laboratory intercomparison study in which the difference between the grand
average and the known (or tme) value is tiie method bias. The laboratory bias is the
difference between the laboratory average recovery and the tme value and is, therefore,
a combination of the two biases. Bias is considered to be systematic error. Systematic
error isrepeatableor constant enx)r from physical or chemical limitations of method
and/or improper caUbration or calculation. Assess the laboratory bias by measuring
the recovery of known additions or by analyzing dupUcate samples andretainingthe
sign of the differences when calculating the average difference. From this value,
subtract the method biasfroman intercomparison study to determine the bias due to
the laboratory practices as it interprets the method.

Bias is calculated by: (Tme value-mean value) *100

Tme value
The principal indicators of data quaUty are its bias and precision, which, when
combined, express its accuracy. Therelationshipamong these terms is shown in
Figure 1. Of the four possible outcomes, only the condition of low bias and high
precision is accurate.

3.0 Total Uncertainty

Total uncertainty is some appropriate combination of the random and systematic
uncertainties in a given measurement system. The random uncertainties are assessed
by calculating precision; they are ascertained statisticaUy. The systematic uncertainties
are the biases and those random uncertainties that cannot be evaluated statisticaUy.
However, bias can be estimated only from a thorough knowledge of aU steps in the
measurement process using the judgment of an experienced analyst Therefore, there

46
 is considerable vagueness in the assessment Because the biases can be assessed, it is
customary to consider only them in evaluating total uncertainty. These biases can
occur at several points in the system, for example, in weighing the sample, in the result
produced by the analytical instrument, in changes in quaUty of reagents or in
incomplete extraction.

However, for ease of calculation it has been accepted to calculate bias as shown in the
Data QuaUty section of this manual. Total uncertainty is thus determined by taking
both the lack of precision and bias in percentages and subtract from 1(X) to determine
the total uncertainty wit 1(X)% being equal totiietme value. The value of the analysis
is thenreportedwith such percentage to demonstrate the confidence in the value. An
example is a value of 5 mg/L witii 95% accuracy states that there is a possible 5% error
in the valuefix)meither bias or lack of precision.

Accuracy = tme value - total error (total uncertainty)

Other methods of calculating total uncertainty may be more accurate and representative
of the tme error. One such method uses uncertainties that have been assessed by the
standard deviation (Sx) and biases represented by bi, these quantities may be combined
in quadratic form to calculate uncertainty:

Ux =(sx^ + 1/3 Ibi2)i'2

GeneraUy, the instmmental and extraction biases (B) are the largest and the equation
simplifies to:

Ux = (SJP + B2)1I2

To express the uncertainty in the form of confidence levels, assume that the bias is
known with Uttie error and add it to higher confidence levels of the variance; for
example, for 95% confidence level:

Ux(95%) = (2s^ + B2)m

If the concentration of the constituent is within a narrow range, use the concentration
units for B and Sx, otherwise, use the coefficient of variation for Sx and the fractional
form of B.
4.0 Checking Correctoess of Analyses
The foUowing procedures for checking correcmess of analyses are appUcable
specificaUy to water samples for whichrelativelycomplete analyses are made. These
include pH, conductivity, total dissolved soUds (TDS), and major anionic and cationic
constituents that are indications of general water quaUty.
The checks described do not require additional laboratory analyses. Three of the
checks require calculation of the total dissolved soUds and conductivity from measured
constituents. Sum concentrations (in milUgrams pertiter)of constiments to calculate
the total dissolved soUds are as follows:

Total dissolved soUds = 0.6 (aUcaUnity) -i- Na + K + Ca + Mg -H CI -h SO4 + SiOa +

47
N03 + F

Calculate electrical conductivityfromthe equation:

G = )X:-(kiX + k2) (Cp'2

where:

G = conductivity of salt solution,

C = concentration of salt solution,
A = equivalent conductance or salt solution at infinite dilution,
ki,k2- constants forrelaxationor ion cloud effect and electrophoretic effect
relative to ion mobiUty.
4.1 Anion-CIation Balance
The anion and cation sums, when expressed as miUiequivalents pertiter,must
balance because aU potable waters are electricaUy neutral. The test is based on the
percentage difference defined as foUows:
% difference = l(X)[(Zcations - Zanions) -^ (Zcations + Zanions)!
and the criteria for acceptance are as foUows:

Anion Sum Acceptable

(meq/L) Difference

0-3.0 ± 0.2 meq/L

3.0-10.0 ±2%
10.0-800 ± 2-5%

4.2 Measured TDS = Calculated TDS

The measured total dissolved soUds concentration should be higher than the
calculated one because a significant contributor may not be included in the
calculation. If the measured value is less than the calculated one, the higher ion
sum and measured value are suspect; the sample should be reanalyzed. If the
measured soUds concentration is more than 20% higher than the calculated one,
the low ion sum is suspect and selected constituents should be reanalyzed. The
acceptable ratio is as foUows:
1.0 < (measured TDS -i- calculated TDS) < 1.2

4.3 Measured EC (conductivity) = Calculated EC

If the calculatai conductivity is higher than the measured value,reanalyzethe
higher ion sum. If the calculated EC is less than the measured one, reanalyze the
lower ion sum. The acceptable ratio is as foUows:
0.9 < (calculated EC + measured EC) < 1.1

4.4 Measured EC and ion Sums

Both the anion and cation sums should be Vioo of the measured EC value. If

48
 either of the two sums does not meet this criterion, that sum is suspect, reanalyze
the sample. The acceptable criteria are as foUows:

1(X) X anion (or cation) sum, meq/L = (0.9-1.1) EC

4.5 Calculated TDS to EC Ratio
If tiie ratio of calculated TDS to conductivity faUs below 0.55, tiie lower ion sum
is suspect;reanalyzeit. K the ratio is above 0.7, the higher-ion sum is suspect;
reanalyze it. Ifreanalysiscauses no change in the lower ion sum, an unmeasured
constiment, such as ammonia or nitrite, may be present at a significant
concentration. If poorly dissociated calcium and sulfate ions are present, the TDS
may be as high as 0.8 times the EC. The acceptable criterion is as foUows:

calculated TDS + conductivity = 0.55-0.7

4.6 Measured TDS to EC Ratio
The acceptable criteria for this ratio arefrom0.55 to 0.7. If the ratio of TDS to
EC is outside these limits, measured TDS or measured conductivity is suspect,
reanalyze.
5.0 Method Detection Limits
Current practice identifies several detection limits, each of which has a defined
purpose. These are the instmment detection limit (IDL), the lower limit of detection
(LLD), the method detection Umit (MDL), and the limit of quantitation (L0(2).
OccasionaUy the instmment detection limit is used as a guide for determining the MDL.
Therelationshipamong these limits is approximately IDL: LLD : MDL: L(5Q- PQL =
1:2:4:10:50.

5.1 Determining Detection Limits

5.1.1 IDL (instrument detection limit): An operating analytical instrument usuaUy
produces a signal (noise) even when no sample is present or when a blank
is being analyzed. Because any QA program requiresfrequentanalysis of
blanks, the mean and standard deviation become weU known; the blank
signal becomes very precise, i.e., the Gaussian curve of the blank
distribution becomes very narrow. The IDL is the constituent concentration
that produces a signal greater than three standard deviations of the mean
noise level or that can be determined by injecting a standard to produce a
signal that is five times the signal-to-noise ratio. The IDL is useful for
estimating the constituent concentration or amount in an extract needed to
produce a signal to permit calculating an estimated method detection limit
5.1.2 LLD Gower limit of detection): The LLD is the amount of constituent that
produces a signal sufficientiy large that 99% of the trials with that amount
wiU produce a detectable signal. Determine the LLD by multiple injections
of a standard at near zero concentration (concentration no greater that five
times the IDL). Determine the standard deviation by the usual method. To
reduce the probabiUty of a Type I error (false detection) to 5%, multiply s
by 1.645 from a cumulative normal probabUity table. Also, toreducetiie
probabiUty of a Type n error (false nondetection) to 5%, double this
amount to 3.290. As an example, if 20 determinations of a low level
standard yielded a standard deviation of 6 pg/L, the LLD is 3.29 x 6 = 20

49
 5.1.3 MDL (metiiod detection Umit): The MDL differsfromthe LLD in tiiat
samples containing the constituent of interest are processed through the
complete analytic^ metiiod. The method detection limit is greatertiianthe
LLD because of extraction efficiency and extract concentration factors. The
MDL can be achieved by experienced analysts operating weU-caUbrated
instmments on a non-routine basis. For example, to determine the MDL,
add a constituent toreagentwater, or to the matrix of interest, to make a
concentration near the estimated MDL. Analyze seven portions of this
solution and calculate tiie standard deviation (s). From a table of the one-
sided t distribution, selecttiievalue of t for 7 - 1 = 6 degrees of freedom
and attiie99% level; this value is 3.14. The product 3.14 times s is tiie
desired MDL.
5.1.4 LOQ (limit of quantification): Although the LOQ is useful within a
laboratory, the practical quantitation Imtit (PQL) has been proposal as the
lowest level achievable among laboratories within specified limits during
routine laboratory operations. The PQL is significant because different
laboratories wiU produce different MDLs even though using the same
analytical procedures, instruments, and sample matrices. 'Die PQL is about
fivetimesthe MDL andrepresentsa practical androutinelyachievable
detection limit with arelativelygood certainty that anyreportedvalue is
reUable.
5.1.5 PQL (Practical (Quantitation Limit): 5 to 10timestiieMDL. The PQL takes
into account matrix effects and is used widely byregularatoryagencies.
6.0 Method Development and Evaluation
Although standard methods are avaUablefrommany nationally recognized sources,
there may be occasions when they cannot be used or when no standard method exists
for a particular constituent or characteristic. Therefore, method development may be
required. Method development is the set of experimental procedures devised for
measuring a known amount of a constiment in various matrices, in the case of
chemical analyses, or a known characteristic (e.g., biological or toxicological) of
various matrices.
Whether an entirely new method is developed by acceptedresearchprocedures or an
existing method is modified to meet special requirements, vaUdation by a three-step
process is required: determination of single-operator precision and bias, analysis of
independentiy prepared unknown samples, and determination of method mggedness,
and when possible equivalency testing.

6.1 Single-Operator Charaaeristics

This part of the vaUdation procedure requires determining the method detection
limit (MDL) as in the Method Detection Lintit section of this manual (Data QuaUty
5.0), the bias of the method, i.e the systematic error of tiie method, and the
precision obtainable by a single operator, i.e., the random error introduced in
using the method. To make Aese determinations, analyze at least 7 but preferably
10 or more portions of a standard at each of seventi concentrations in each matrix
that may be used.
Use one concentration at, or sUghtiy above, the MDL and onerelativelyhigh so
that the range of concentrations for which the method is appUcable can be
specified. The determination should also include the analysis of a blank for each
of the matrices under evaluation.

50
 The use of several concentrations to determine bias and precision wiUrevealthe
form of therelationshipbetween these method characteristics and the
concentration of the substance, the characteristic toxicity of the substance, or the
biological factor of interest. Thisrelationshipmay be constant, linear, or non-
linear and is a si^iificant characteristic of the method that should be explained
clearly. Calculation of precision and bias for a single concentration in a single
matrix is shown in the foUowing table ofresultsfix)meightrepUcateanalyses of a
standard with a known concentration of 1.30 mg/L.

Result Difference Squared

mg/L (-1.30) Difference

1.23 -0.07 0.0049

1.21 -0.09 0.0081
1.30 0.0 0.0
1.59 0.29 0.0841
1.57 0.27 0.0729
1.21 -0.09 0.0081
1.53 0.23 0.0529
1.25 -0.05 0.005
Sum 0.49 0.2335

The bias is 0.49/8 = 0.06 mg/L and the precision is the square root of 0.2335/(8-
1) or 0.18 mg/L (note that this is simUar to the calculation for standard deviation).
Bias can also be calculated using the formulas given in the Data QuaUty section of
this manual ((Tme value - mean value) / Tme value = (1.3- 1.36) / 1.3 = 0.05).
The matrix effects (usuaUy interferences) may be manifested by changes in the
detection limit, linearity, bias, and precision with different matrices or with
changes in the nature of the same matrix (e.g. high or low organic matter in
sediments, low or high redox potential in groundwater, or high or low chloride in
a surface water.
6.2 Analysis of Unknown Samples
This step in the method validation procedure requires analysis of independentiy
prepared standards where the value is unknown to the analyst Analyze each
unlaiown in repUcate by following the standard operating procedure for the
method. The mean amount recovered should be within three standard deviations
(s) of the mean value of the standard but preferably within 2 s.
Obtain the unknowns from other personnel in the analyst's laboratory using either
purchased analytical-grade reagents or standards avaUable from National Institute
of Standards and Technology (NIST), EPA, or other suitable sources. If
avaUable for the particular constituent, performance evaluation samples from
EPA-Cincinnati are particularly usefiti.
6.3 Method Ruggedness
A test of the mggedness, i.e., stability of theresuUproduced when steps in tiie
metiiod are vari^ istiiefinalvaUdation step. It is especiaUy important to
determine this characteristic of a method if it is to be proposed as a standard or
reference method. A properly conducted mggedness test wiU point out tiiose
procedural steps in whichrigoris critical and those in which some leeway is
permissible.

51
 The Association of Official Analytical Chemists has suggested a method for this
test in which eight separate analyses can be used to determine the effect of varying
seven different steps in an analytical procedure. To iUustrate, suppose the effect
of changing the following factors is to be determined:

Factor Nominal Variation

Mixing time 10 min 12 min
Portion size 5g lOg
Acid concentration IM I.IM
Heat to lOO^C 95''C
Hold heat for 5 min 10 min
Stirring yes no
pH adjust 6.0 6.5

To make the determination, denote the nominal factors by capital letters A through
G and the variations by the corresponding lower-case letters. Then set up a table
of the factors as follows:
If combination 1 is analyzed, the result wUl be s. If combination 2 is analyzed,
theresultwiU be t, and so on until all eight combinations have been analyzed. To
determine the effect of varying a factor, find the fourresultswhere the factor was
nominal (aU caps) and the four where it was varied (all lower case) and compare
the averages of the two groups. For example, to compare the effect of changing
C to c, use results (s + u H- w -t- y)/4 and (t -t- v -i- x -i- z)/4. Calculate aU seven
pairs to get seven differences, which can then be ranked to reveal those with a
significant effect on the results. If there is no outstanding difference, calculate the
average and standard deviation of the eightresultss through z. The standard
deviation is a reaUstic estimate of the precision of the method. This design tests
main effects, not interactions.
6.4 Equivalency Testing
After a new method has been validated by the procedures Usted above, it may be
pmdent to test the method for equivalency to standard methods, unless none exist.
This requires analysis of a minimum of three concentrations by the altemate and
by the standard method. If the range of concentration is very broad, test more
concentrations. Once an initial set of analyses (five or more) has been made at
each chosen concentration, apply the foUowing statistical steps.
6.4.1 Test the distribution of data for normaUty and transform the data if
necessary (See the Statistics section of this manual).
6.4.2 Select an appropriate sample size based on an estimate of the standard
deviation
6.4.3 Test the variances of the two,methods using the F-ratio statistic.
6.4.4 Test the average values of the two methods using a Student t statistic.
An explanation of each of these steps with additional techniques and examples has
been pubUshed. Because the number of analyses can be very large, the
calculations become complex and famUiarity with basic statistics is necessary. A
Ustmg of standard,reference,and equivalent methods for water analysis is
avaUable.
6.5 Collaborative Testing
Once a new or modified method has been developed and vaUdated it is appropriate
to determine whether the method shoitid be made a standard metiiod. The

52
procedure to convert a method to standard status is tiie coUaborative test In tiiis
test, a number of laboratories use the standard operating procedure to analyze a
select number of samples to determine the methods bias and precision as would
occur in normal practice.

In planning for a coUaborative test, consider the foUowing factors: a precisely

written standard operating procedure, tiie number of variables to be tested, tiie
number of levels to be tested, and the number ofrepUcatesrequired. Because
method precision is estimated by the standard deviation, which itself is the result
of many sources of variation, the variables that affect it must be tested. These
may include the laboratory, operator, apparams, and concentration range.
6.5.1 Variables. Test at least the following variables:
6.5.1.1 Laboratory: Involve at least three different laboratories, although
more are desirable to provide a better estimate of the standard
deviation.
6.5.1.2 Apparatus: Because model and manufacturer differences can be
sources of error, analyze at least two repUcates of each
concentration per laboratory.
6.5.1.3 Operators: To determine overaU precision, involve at least six
analysts with not more than twofromeach laboratory.
6.5.1.4 Levels: Iftiiemethod development has indicated that the relative
standard deviation is constant, test three levels covering the
range of the method. If it is not constant, use more Levels
spread uniformly over the operating range.
6.5.1.5 If matrix effects are suspected, condua the test in each medium
for which the method was developed. If this is not feasible, use
appropriate grades ofreagentwater as long as this is stipulated in
theresultingstatement of method characteristics.
6.5.2 Number of RepUcates
Calculate the number ofrepUcatesafter the number of variables to be tested
has been determined by using the formula:
r > 1 + (30/P)
where:
r - number ofrepUcatesand
P = the product of several variables.
The minimum number ofreplicatesis two. As an example, if three levels
of a substance are to be analyzed by single operators in six laboratories on
a single apparatus, then P is calculated as follows:
/> = 3 x l x 6 x l = 18
and the number ofrepUcatesis
r>l+(30/18)>2.7orr = 3
6.5.4 Ulustrative CoUaborative Test
Send each of five laboratories four concentrations of a compound (4.3,
11.6,23.4, and 32.7 mg/L) with instmctions to analyze in tripUcate, using
the SOP provided. Tabulateresultsas shown in the table below (the
results for only one concentration are shown). Because there are no
obviously aberrant values (use the method in the Statistics section to reject
outUers), use aU the data.

53
Calculate the average and standard deviation for each laboratory. Use aU
15resultsto calculate a grand average and standard deviation. The
difference between the average of each laboratory and the grand average
reveals any significant bias, such astiiatshown for Laboratories 1 and 3.
The difference between the grand average and the known value is the
metiiod bias, e.g., 33.0 - 32.7 = 0.3 m ^ or 0.9%. The relative standard
deviation of tiie grand average (1.5 mgA.) is 4.50%, which is tiie metiiod
precision, and the s for each laboratory is the single-operator precision.

Deviation
Result Experimental From From Grand
Lab. mg/L 1+s Known Average

1 32.7
35.2 34.7 ± 1.8 2.0 1.7
36.3
2 32.6
33.7 33.3 ± 0.6 0.6 0.3
33.6
3 30.6
30.6 31.2 ± 1.0 -1.5 -1.8
32.4
4 32.6
32.5 33.0 ± 0.8 0.3 0
33.9
5 32.4
33.4 32.6 ± 0.8 -0.1 -0.4
32.9
(Ix)/n = 33 1=1.3 I = -0.2
s=1.5

As noted in the table, the sum of the deviationsfromthe known value for
the laboratories was 1.3, so the average deviation (bias) was 1.3/5 = 0.26,
rounded to 0.3, which is the same as the difference between the grand
average and the known value.

For aU four unknowns in this test, the percentage results indicate

(increasmg bias and decreasing precision as the concentration decreases.
Therefore, to describe the method in a formal statement the precision would
be given by a straight tine with the formula y = mx + b, where y is the
relative standard deviation, m is the slope of the line, x is the concentration,
and b is therelativestandard deviation at concentration = 0. The values
found from the coUalx)rative test are shown in the foUowing table.

54
 Known Amount CV
Amount Found (% Standard Bias
mg/L mg/L DeviaticMi) %
4.3 4.8 12.5 11.5
11.6 12.2 10.2 5.6
23.4 23.8 5.4 1.9
32.7 33 4.5 0.9

Theseresultsindicate that the method is acceptable. However,

concentrations of less than about 10 mg/L require greater care in analysis.

TARGET ANALOGY FOR

EXPRESSIONS OF ACCURACY
MODERATE UNCERTAINTY
(= MODERATE ACCURACY)
HIGH P RECISION
(= LOW RANDOM ERROR)
HIGH BIAS
(= HIGH SYSTEMATIC ERROR)
HIGH U NCERTAINTY
(= LOW AOCURAOr)
LOW P RECISION
(= HIGH RANDOM ERROR)
HIGH BIAS
(= HIGH SYSTEMATIC ERROR)

LOWU NCERTAINTY MODERATE U NCERTAINTY

(= HIGH AOCURAQT) (= MODERATE ACCURACY)
HIGH P RECISION LOWP REQSION
(= LOW RANDOM ERROR) (= HIGH RANDOM ERROR)
LOW BAS LOW BIAS
(= LOW SYSTEMATIC ERROR) (= LOW SYSTEMATIC ERROR)

Figure 1. Relationship between Bias and Precision.

55
 QUALITY ASSURANCE
QuaUty assurance (QA) is a set of operating principles that, if strictiy foUowed during
sample coUection and analysis, wUl produce data of known and defensible quaUty. That is,
the accuracy of the analyticalresuUcan be stated with a high level of confidence. Included
in quaUty assurance are quaUty assurance planning, quality control, and quaUty assessment.
1.0 QuaUty Assurance Planning
A QA plan includes the following: cover sheet with plan approval signatures, staff
organization andresponsibiUties,sample control and documentation procedures,
standard operating procedure (SOP) for each analytical metiiod, analyst training
requirements, equipment preventive maintenance procedures, caUbration
procedures, corrective actions, intemal quaUty control activities, performance
audits, data assessment procedures for bias and precision, and data reduction,
vaUdation, and reporting.
The cover sheet with approval signatures indicates that the plan has been reviewed
and judged suitable, and that the organization andresponsibiUtiessection outlines
the chain-of-command and assigns specific functions to each person involved.
Sample control and documentation procedures permit tracing a sample and its
derivatives through aU steps from coUection to analysis and display of results.
Documentation always is important but is especiaUy so when chain-of-custody
requirements arc imposed.

Equipment preventive maintenance procedures are required. A strict preventive

maintenance program wUl reduce instrument malfunctions, maintain caUbration, and
reduce downtime. CaUbration procedures, corrective actions, intemal quality
control activities, performance audits, and data assessments for bias and precision
are discussed in (^ality Control (2.0) and Quality Assessment (3.0).
Data reduction, vaUdation, andreportingare the final features of a QA program.
Thereadingobtained from an analytical instrument must be adjusted for such
factors as instrument efficiency, extraction efficiency, sample size, and background
value, before it becomes a useful result. The QA plan specifies the correction
factors to be applied as weU as the steps to be foUowed in validating the result.
Reportresultsin standard units of mass, volume, or concentration. Use a
prescribed method forreportingresults below the method detection limit.
Accompany each result or set of results by a statement of uncertainty.

2.0 QuaUty Control

The Environmental Science Laboratory (ESL) uses both intemal and extemal C^aUty
Control (QC) procedures. The intemal QC includes: certification of operator
competence, recovery of known additions, analysis ofreagentblanks, calibration with
standards, analysis ofrepUcates,and maintenance of control charts. Extemal C^aUty
control includes purchasing standards from an outside source with guaranteed analyses
or different laboratories analyzing the same samples.
2.1 Certification of Operator Competence
Before an analyst is permitted to doreportablework, competence in making the
analysis is to be demonstrated. Reqmrements vary, but for most inorganic and
organic chemical analyses, demonstration of acceptable single-operator precision

56
 and bias is sufficient Determination of single-operator precision and bias
requires the preparation of a minimum of fourrepUcatesof an independentiy-
prepared check sample having a concentration between 5 and 50 times tiie method
detection Umit (MDL) for tiie analysis in tiie ESL. General Umits for acceptable
work are shown in Table 1.
2.2 Recovery of Known Additions
The use the recovery of known additions is part of theregularanalytical protocol.
Known additions are used to verify the absence of matrix effects. Whenever a
new matrix type is to be analyzed, verification of the amount of interference is
undertaken. Where dupUcates are not appUcable, for example, and when the
constituent of interest is absent or suspected to be absent, recovery of known
additions for 10% of the samples are made. Where dupUcates also are being
analyzed, the sum of the dupUcates and known additions must equal at least 10%
of the number of samples. The known addition is made between 5 and 50 times
the MDL or between 1 and 10 times the ambient level, whichever is greater. A
known addition is not made above the demonstrated linear range of the method.
Concentrated additions solutions are used so volume change in sample is
negUgible. See Table 1 for acceptable limits.

Table 1. Acceptance Limits for DupUcate Samples and Known Additions to Water and
Wastewater.
Recovery of Precision of Precision of
Known Low-Level High-Level
Additions* DupUcates*t DupUcates*t$
Analysis % ±% ±%
Metals 80-120 25 10
VolatUe organics 70-130 40 20
VolatUe gases 50-150 50 30
Base/neutrals 70-130 40 20
Acids 60-140 40 20
Anions 80-120 25 10
Nutrients 80-120 25 10
Other inorgaitics 80-120 25 10
Total organic carbon 80-120 25 10
Total organic halogens 80-120 25 15
Herbicides 40-100 40 20
Organochlorine pesticides 50-140 40 20
Captan 20-130 40 20
Endosulfans 25-140 40 20
Endrin aldehyde 25-140 40 20
Organophosphoms pesticides 50-200 40 20
Trichlorophon 20-200 40 20
Triazine pesticides 50-200 40 20
Clarbamate pesticides 50-150 40 20
* Acklitions calculated as % of the known addition recovered, duplicates calculated as the difference as a
percentage of the mean [KXXxi - X2/x]. t Low-level refers to concentrations less than 20 times the MDL.
High-level refers to concentration greater than 20 times the MDL.
t Also acceptance limits for independent laboratory control standards and certification of operator
competence.

57
2.3 Analysis of Reagent Blanks
Reagent blanks are analyzed whenever new reagents are used, or as often as
required in specific methods. A minimum of 10% of the sample load are analyzed
as reagent blanks. This procedure monitors purity of reagents and the overaU
procedural blank. A reagent blank is analyzed after any sample with a
concentration greater than that of the highest standard or on any sample that might
result in carryoverfix)mone sample totiienext. Analysis of blanks test for
contamination during sampling and testing. Blanks are protection against "false
positives" which are Type I errors statingtiiata constituent is present when it
actuaUy is not. See figure 3 for an example of a false positive.
2.4 ClaUbration with Standards
As a minimum, three different dUutions of the standard are measured when an
analysis is initiated. The standard curve is verified at least daily by analyzing one
or more standards within the linear range, or as specified in the incUvidual
method. Reportable analyticalresultsare only those within the range of the
standard dUutions used. Values above the highest standard are not reported
unless: (1) a prior demonstration of a greater linear range has been made; (2)
instmment parameters have not been changed; and (3) the value is less than 1.5
times the highest standard. The lowest reportable value is the MDL, provided that
the lowest standard is less than 10 times the MDL. If a blank is subtracted, the
result is reported even if it is negative. The use of standards as quaUty controls
within a test is protection against "false negatives" which are Type II errors stating
that a constituent is absent when it is actually present Seefigure3 for an
example of a false negative.
2.5 Analysis of RepUcates
When most samples have measurable levels of the constituent being determined,
analysis of dupUcate samples is an effective means for assessing precision. At
least 10% of the samples are analyzed in duplicate. DupUcates and known
additions are analyzal in matricesrepresentativeof the samples being testes. See
Table 1 for acceptable limits for duplicate analyses.
2.6 Control Charts
Control charts are essential instmments for quality control. There are three types
of control charts commonly used in laboratories: a means chart for
standards—^laboratory control standards (LCS) or calibration check standards
(CCS); a means chart for background orreagentblank results; and a range chart
forrepUcateanalyses. The charts are essential instmments for quaUty control.
Each type of chart is described below.
2.6.1 Means chart. The means chart for standards is constmcted from the
average and standard deviation of a standard. It includes upper and lower
wanting levels (WL) and upper and lower control levels (CL). (Dommon
practice is to use ± 2s and ± 3s limits for the WL and CL, respectively,
where srepresentsstandard deviation. These values are derived from
stated values for standardreferencematerials, if used fortiielaboratory
control standard (LCS) or caUbration check standard (CCS), or from
repUcate analyses of a CCS. The chart can be set up by using either the
calculated values for mean and standard deviation or by using percentages.
Percentage is necessary when the concentration varies. A chart is
constmcted for each analytical instrument. Results are entered on the chart
each time the LCS or CCS is analyzed.
2.6.2 Range chart. The range chart is used for evaluation of dupUcate analyses.
If the standard deviation of the method is known, use the factors from

58
 Table 2 to constmct the central line and warning and control limits as in
Figure 1. Perfect agreement between dupUcatesresultsin no difference
when tiie values are subtracted, so tiie base tine ontiiechart is zero. The
standard deviation is converted to the range so that the analyst need only
subtract the tworesultsto plot the value on the control chart. The mean
range is computed as:

R=D2S
the control limit as

CL = R ±3s(R) = D4R

and the warning limit as

WL = R ±2s(R) = R±2/3 (D^ - J)
where
D2 = factor to convert s to the range (1.128 for dupUcates, as given in
Table 2),
s(R) = standard deviation of the range, and
D4 = factor to convert mean range to 3s(R) (3.267 for dupUcates, as given
in Table 2)

. 10-
CL
R^NGEOF
DUPLICATES, WL
mg/L 5_

Date

Figure 1. DupUcate Analyses of a standard

More commoitiy, the range can be expressed as a function of the relative

standard deviation (coefficient of variation). NormaUze the range by
dividing by the average. Determine the mean range for the pairs analyzed

59
 by

R = (lRi)/n

and the variance (square of the standard deviation) as

sR2 = (lRi2 - riR2)/(n - 1)

Then draw lines on the chart at R" + 2SR and R + 3SR and, for each
dupUcate analysis, calculate normalized range and center theresulton the
chart Figure 2 is an example of such a chart.

. 0.6
NORMAUZED
VALUE OF
Range 0.4- CL

WL
0.2- R

0
Date or Sample Sequence

Figure 2. Range Chart of variable ranges

2.6.3 Chart analvses. If the warning limits (WL) are at the 95% level, 1 out of
20 points, on the average, would exceed that limit, whereas only 1 out of
1(X) would exceed tiie control limits (CL). Take the foUowing actions:
Control Umit—^If one measurement exceeds a CL, repeat the analysis
immediately. If therepeatis witiiin the CL, continue analyses. If it
exceeds the CL, discontinue analyses and cortect the problem-
Warning limit: If two oftiireesuccessive points exceed a WL, analyze
anotiier sample. If the next point is less than the WL, continue analyses; if
next point exceeds the WL, discontinue analyses and correct the problem.

Standard deviation: If four of five successive points exceed 1 5, or are in

decreasing or increasing order, analyze another sample. If the next point is
less than 1 s, or changes tiie order, continue analyses; otherwise
discontinue analyses and correct the problem.
Central line: If six successive samples are above the central tine, analyze

60
 anotiier sample. If the next point is below the central tine, continue
analyses; if the next point is on the same side, discontinue analyses and
correct problem. The above considerations apply when the conditions are
either above or below the central line, but not on both sides, e.g., four of
five values must exceed either + 1 s or - 1 s. After correcting the problem,
reanalyze half the samplestestedbetween the last in-control measurement
and the out-of-control one.

Another important function of control charts is assessment of

improvements in method precision. In the means and range charts, if
measurements never or rarely exceedtiieWL, recalculatetiieWL and CL
using the 20 most recent data points. Trends in precision can be detected
sooner if running averages of 20 are kept on a daUy basis.
2.7 Analysis of ExtemaUy SuppUed Standards
As a minimum, extemaUy supplied standards are analyzed whenever analysis of
known additions does not result in acceptable recovery or once each day, whichever is
more frequent. Laboratory control standards are used with concentrations between 5
and 50 times the MDL or near sample ambient levels, whichever is greater. When
possible, certified reference materials are used as laboratory control standards. See
Table 1 for acceptable limits for high-level dupUcates. Extemal quaUty control samples
are used in the same manner asreagentblanks, standards, and dupUcates. Using
extemaUy provided standards allows for determination of any errors that may be
produced in sample or standard preparation, sample storage, or sample transportation
that may not be detected by intemal standards treated in the same manner.

Table 2. Factors for Computing Lines on Range Control Charts

Number of Factors for Factors for
Observations Central Line Control Limits
n (D2) (D4)
—2 rns TI^—
3 1.693 2.575
4 2.059 2.282
5 2.326 2.115
6 2.534 2.004
Rosentrin, M. and A.S. Golden (1964)

61
 Standard Blank Sample
Injection & Injection & Injection &
Solvent Front Solvent Front Solvent Front

Standard Analyte

Normal
Positive

Normal
Negative

False
Positive

False
Negative

Figure 3. False Positives and Negatives

62
3.0 C^aUty Assessment
QuaUty assessment is the process of using extemal and intemal quaUty control measures to
determine the quaUty of the data product by the labcratory. It includes such items as
performance evaluation samples, laboratory intercomparison samples, and performance audits
as weU as the intemal QC described in Section 1020B. They are appUed to test the recovery,
bias, precision, detection limit, and adherence to standard operating procedure requirements.
3.1 Performance Evaluation Samples
Use samples with known amounts of the constiment of interest suppUed by an
outside agency or blind additions prepared independentiy within the laboratory to
determine recovery achieved by an analyst In general, method uncertainty wiU have
been estabUshed beforehand; acceptable recovery faUs within the estabUshed
uncertainty. For example, if the acceptable range of recovery for a substance is 85 to
115%, then the analyst is expected to achieve a recovery within that range on aU
performance evaluation samples.
Commercial and governmental programs supply samples containing various
constituents in various matrices. A good quality assessment program requires
participation in periodic laboratory intercomparison studies. Adjust frequency of
participation to the quaUty of theresultsproduced by the analysts being tested. For
routine procedures, quarterly analyses are reasonable. Official agencies conducting
such smdies are Usted l)elow.
3.2 Performance audits
Make oitiy unscheduled performance audits using a check Ust made to document the
manner in which a sample is treated from time of receipt to final reporting of the
result The goal is to detect any deviations from the standard operating procedure so
that corrective action can be taken. A recommended format witii a few iititial items in
the check Ust is shown in Table 3.

Table 3. Audit of a SoU Analysis Procedure.

Procedure Comment Remarks
I. Sample entered into logbook yes lab number assigned

2. Sample weighed yes dry weight

3. Drying pnxedure followed no maintenance of oven not dcHie

4 Balance a. calibrated yes once per year

b. Cleaned and zero adjusted yes weddy

5. Sample ground yes to pass 50 mesh

6. BaU mtil cleaned yes should be after each sample

63
 COLLECTION AND PRESERVATION OF
SAMPLES
INTRODUCTION
It is an old axiom that theresultof any testing method can be no better than the sample on
which it is performed. Neither, the care taken by the analyst or theresolutionof the
instrument has any value if the sample was not coUected in a manner or at a time to
represent the parameters under study, or the sample was incorrectiy preserved or stored.
It is not practical to specify detaUed procedures fortiiecoUection of aU samples here
because of the varied purposes and analytical procedures. More detaUed information
appears in connection with specific methods. This section presents general considerations
appUcable priiiiarily to chemical analyses. See appropriate sections for samples to be used
in toxicitytestingand microbiological or biological examination.
The objective of sampling is to coUect a portion of material smaU enough in volume to be
transported convenientiy and handled in the laboratory whUe stiU accurately representing
the material being sampled. This objective implies that therelativeproportions or
concentrations of aU pertinent components wiU be the same in the samples as in the material
being sampled, and that the sample wiU be handled in such a way that no significant
changes in composition occur before the tests are made.

A sample may be presented to the laboratory for specific determinations with the coUector
takingresponsibiUty,for its vaUdity. Often, in water and wastewater work, the laboratory
conducts or prescribes the sampling program, which is determined in consultation with the
user of the test results. Such consultation is essential to insure selecting samples and
analytical methods that provide a tme basis for answering the questions that prompted the
sampUng.

1.0 General Precautions.

Obtain a sample that meets the requirements of the sampling program and handle it in
such a way that it does not deteriorate or become contaminated before itreachesthe
laboratory. Before filling, rinse sample bottie two or three times with the water being
coUected uitiess the bovlt contains a preservative or dechlorinating agent Depending
on determinations to be performed, fUl container full (most orgaitics determinations) or
leave space for aeration, mixing etc. (microbiological analyses). For samples that wiU
be shipped, preferably leave an air space of about 1% of container capacity to aUow for
thermal expansion.
Special precautions are necessary for samples containing organic compounds and trace
metals. Because many constituents may be present at concentrations of micrograms
per liter, they may be totaUy or partiaUy lost if proper sampling and preservation
procedures are not foUowed.
Representative samples of some sources can be obtained only by making composites
of samples coUected over a period of time or at many different sampling points. The
detaUs of coUection vary so much witii local conditionstiiatno specific
recommendations would be uitiversaUy applicable. Sometimes it is more informative

64
to analyze numerous separate samples instead of one composite so as not to obscure
maxima and minima.

Sample carefuUy to insure that analyticalresultsrepresentthe actual sample

composition. Important factors affectingresultsaretiiepresence of suspended matter
or turbidity, the method chosen for itsremoval,and the physical and chemical changes
brought about by storage or aeration. Particular care is required when processing
(grinding, blending, sieving,filtering)samples to be analyzed for trace constituents,
especiaUy metals and organic compounds. Some deteraunations, particularly of lead,
can be invaUdated by contaminationfi-omsuch processing. Treat each sample
indiyiduaUy withregardto the substances to be determinai the amount and nature of
turbidity present, and other conditions that may influence the results
It is impractical to give directions covering aU conditions, and die choice of technique
for coUecting a homogeneous sample must be left to the analyst's judgement. In
general, separate any significant amount of suspended matter by decantation,
centrifugation, or an ^propriate fUtration procedure. Often a sUght turbidity can be
tolerated if experience shows that it wiU cause no interference in gravimetric or
volumetric tests and that its influence can be corrected in colorimetric tests, where it
has potentiaUy the greatest interfering effect. Whenrelevant,state whether or not the
sample has been fUtered. To measure the total amount of a constituent, do not remove
suspended soUds but treat them appropriately.

Make a record of every sample coUected and identify every bottie, preferably by
attaching an appropriately inscribed tag or label. Record sufficient information to
provide positive sample identification at a later date, including the name of the sample
coUector, the date, hour, and exact location, the watertemperature,and any other data
that may be needed for correlation, such as weather conditions, water level, stream
flow, post-sampling, handling, etc. Provide space on the label for the initials of those
assuming sample custody and for thetimeand date of transfer. Fix sampling points
by detailed description, by maps, or with the aid of stakes, buoys, or landmarks in a
manner that wiU permit their identification by other persons withoutreUanceon
memory or personal guidance. Particularly when sampleresultsare expected to be
involved in Utigation, use formal "chain-of-custody" procedures, which trace sample
history from coUection to final reporting.

Cool hot samples collected under pressure whUe they are stiU under pressure. Before
coUecting samples from distribution systems,flushlines sufficientiy to insure that the
sample isrepresentativeof the supply, taking into account the diameter and length of
the pipe to beflushedand the velocity of flow.
CoUect samples from weUs oitiy after the weU has been pumped sufficientiy to insure
that the samplerepresentsthe groundwater source. Sometimes it wiU be necessary to
pump at a specified rate to achieve a characteristic drawdown, if this determines the
zones from which tiie weU is suppUed. Record pumping rate and drawdown.

When samples are collected from ariveror stream, observedresultsmay vary with
deptii, stream flow, and distance from shore and from one shore to the otiier. If
equipment is avaUable, take an integrated sample from top to bottom in the middle of
the stream orfromside to side at noid-depth, in such a way that tiie sample is integrated

65
 according to flow. If only a grab or cateh sample can be coUected, take it in the middle
of the stream and at mid-depth.

Lakes and reservoirs are subject to considerable variationsfix>mnormal causes such as

seasonal-stratification, rainfaU, mnoff, and wind. Choose location, depth, and
frequency of sampling depending on local conditions and the puipose of the
investigation. Avoid surface scum.

For certain constituents,sampling location is extremely important Avoid areas of

excessive turbulence because of potential loss of volatile constituents and of potential
presence of toxic vapors. Avoid sampling at weirs because such locationstendto
favorretrievalof Ughter-than-water, immiscible compounds. GeneraUy, coUect
samples beneath the surface in quiescent areas. If composite samples are required,
take care that sample constituents are not lost during compositing because of improper
handling of portions being pooled. For example, casual dumping together of portions
rather than addition to the composite through a submerged siphon can cause
unnecessary volatilization. When necessaryrefiigeratethe composited portions to
minimize volatilization.

Use onlyrepresentativesamples (or those conforming to a sampling program) for

examination. The great variety of conditions under which coUections must be made
makes it impossible to prescribe a fixed procedure. In general, take into account the
tests or analyses to be made and the purpose for which theresultsare needed.
2.0 Safety Considerations
Because sample constituents can be toxic,take adequate precautions during sampling
and sample handling. Toxic substances can enter through the skin and, in the case of
vapors, through the lungs. Inadvertent ingestion can occur via direct contact with
foods or by adsorption of vapors onto foods. Precautions may be limited to wearing
gloves or may include coveralls, aprons, or other protective apparel. Always wear eye
protection. When toxic vqwrs might be present, sample only in weU-ventUated areas
or use arespiratoror self-contained breatiiing apparams. In a laboratory, open sample
containers in a fume hood. Never have food near samples or sampUng locations
always wash hands thoroughly before handling food.

Ifflammableorgaiuc compounds may be present, take adequate precautions. Prohibit

smoking near samples, sampUng locations, and in the laboratory. Keep sparks,
flames, and excessive heat sources away from samples and sampling locations. Avoid
buUdup offlammablevapors in arefrigeratorstoring samples because electrical arcing
at contacts oftiietiiermostat,the door activatedtightswitch, or otiier electrical
components may trigger a fire or explosion. Ifflammablecompounds are suspected or
known to be present and samples are to berefrigerated,use only specially designed
explosion-prc>of refrigerators. When in doubt as to the level of safety precautions
needed, consult an appropriately trained, industrial hygienist. Samples with
radioactive contaminants require other safety considerations consult a health physicist

3.0 Types of Samples

3.1 Grab or cateh samples strictiy speaking, a sample coUected at a particulartimeand
place canrepresentonly the composition of the source at that time and place.

66
 However, when a source is known to be fairly constant composition over a
considerable period of time or over substantial distances in all directions, then the
sample may be said torepresenta longer time period or a larger volume, or both,
than the specific point at which it was coUected In such circumstances, some
sources may be represented quite weU by single grab samples. Examples are
some water suppUes, some surface waters and rarely, some wastewater streams.

When a source is known to vary with time, grab samples coUected at suitable
intervals and analyzed separately can document the extent, frequency, and
duration of these variations. Choose sampling intervals on the basis of the
frequency with which changes may be expected, which may varyfromas Uttie as
5 min to as long as 1 hour or more. Seasonal variations in natural systems may
necessitate sampling over months. When the source composition varies in space
rather than time, collect samplesfromappropriate locations.

Use great care in sampling wastewater sludges, sludgebanks, and muds. No

definite procedure can be given, but take every possible precaution to obtain a
representative sample or one conforming to a sampling program.
3.2 Composite samples: In most cases, the term composite samplerefersto a mixture
of grab samples coUected at the same sampling point at different times.
Sometimes thetermcomposite is used to distinguish this type of sample from
others. Time-composite samples are most use^l for observing average
concentrations that wiU be used, for example,in calculating the loading or the
efficiency of a wastewater treatment plant As an altemative to the separate
analysis of a large number of samples, foUowed by computation of average and
totalresultscomposite samplesrepresenta substantial saving in laboratory effort
and expense. For these purposes, a composite sample representing a 24-h period
is considered standard for most determinations. Under certain circumstances,
however, a composite sample representing one shift, or a shortertimeperiod, or a
complete cycle of a periodic operation, may be preferable. To evaluate the effects
of special, variable, or irregular discharges and operations, coUect composite
samplesrepresentingthe period during which such discharges occur.

For determining components or characteristics subject to significant and

unavoidable changes on storage, do not use composite samples. Make such
determinations on individual samples as soon as possible after coUection and
preferably at the sampling point Analyses for all dissolved gases, residual
chlorine, soluble sulfide,temperature,and-pH are examples of this type of
determination. Changes in such components as dissolved oxygen or carbon
dioxide, pH, or temperature may produce secondary changes in certain inorganic
constiments such as iron,manganese, alkalinity, or hardness. Use time-com5X)site
samples only for determining components that can be denaonstrated to remain
unchanged under the conditions of sample coUection and preservation.

Take individual portions in a wide-mouth bottie having a diameter of at least 35

mm at the mouth and a capacity of at least 120 mL. CoUect these portions every
hour—in some cases every half hour or even every 5 min and mix at the end of
the sampling period or combine in a single bottie as coUected. If preservatives are
used, add them to the sample bottie initially so that aU portions of the composite
are preserved as soon as coUected. Analysis of individual samples sometimes
may be necessary.

67
 It is desirable, and often essential, to combine individual samples in volumes
proportional to flow. A final sample volume of 2 to 3 L is sufficient for sewage,
effluents, and wastes.

Automatic sampling devices are avaUable however, do not use them unless the
sample is preserved as described below. Clean sampUng devices, including
botties, daily to eliminate biological growths and other deposits.
3.3 Integrated samples: For certain purposes, the information is provided best by
analyzing mixtures of grab samples coUectedfromdifferent points
simultaneously, or as nearly as possible. Such mixtures sometimes are caUed
integrated samples. An example of the need for such sampUng occurs in ariveror
stream that varies in composition across its width and depth. To evaluate average
composition or total loading, use a mixture samplesrepresentingvarious points in
the cross-section, in proportion to their relative flows. The need for integrated
samples also may exist if combined treatment is proposed for several separate
wastewater streams, the interaction of which may have significant effect on
treatabiUty or even on composition. Mathematical prediction of the interactions
may be inaccurate or inqwssible andtestinga suitable integrated sample may
provide more useful information.

Both natural and artificial lakes show variations of composition with both depth
and horizontal location. However, under many conditions, neither total nor
averageresultsare especially significant; local variations are more important In
such cases, examine san5)les separately rather than integrate them.
Preparation of integrated samples usuaUy requires special equipment to coUect a
sample from a known depth without contaminating it with overlying water.
Knowledge of the volume, movement, and composition of the various parts of the
water being sampled usuaUy is required. Therefore, collecting integrated samples
is a compUcated and specialized process that cannot be described in detaU.
4.0 CoUection of Samples/Chain-of-Custody Procedures
It is essential to ensure sample integrity from coUection to datareporting.This
includes the abiUty to trace possession and handling of the sample from the time of
coUection through analysis and final disposition. This is referred to as chain-of-
custody and is important intiieevent of Utigation involving theresultsof testing.
WhUe Utigation is not involved, chain-of-custody procedures are useful for routine
control of sample flow.
A sample is considered to be under a person's custody if it is ui tiie individual's
physical possession, mtiieindividual's sight, secured in a tamper-proof way by tiiat
individual, or is secured in an arearestrictedto autiiorized personnel. The foUowmg
procedures summarize the major aspects of chain-of-custody. More detaUed
discussions are avaUable.
4.1 Sample labels: Use labels to prevent sample misidentification. Gummed paper
labels or tags generaUy are adequate. Include at leasttiiefoUowing information:
sample number, name of coUector, date and tune of coUection, and place of
coUection. Affix labels to sample containers before or attiietimeof sampling.
FiU label out with waterproof ink at time of coUection.

68
 4.2 Sample seals: Use sample seals to detect unauthorized tampering with samples up
to the time of analysis. Use gummed paper seals that include, at least, the
foUowing information: sample number (identical with number on sample label),
coUectors name, and date and time of sampling. Plastic shrink seals also may be
used Attach seal in such a way that it is necessary to break it to open the sample
container. Affix seal to container before sample leaves custody of sampling
personnel.
4.3 Field log book: Record aU information pertinent to a field survey or sampling in a
bound log book. As a minimum, include the foUowing in the log book: purpose
of sampUng, location of sampling point, name and address of field contact,
producer of material being sampled and address, if different from location, and
type of sample. If sample is wastewater, identify process producing waste
stream. Also provide suspected sample composition, including concentrations,
number and volume of samples taken, description of sampling point and sampling
method, date andtimeof coUection, coUectors sample identification number(s),
sample distribution and how transported, references such as maps or photographs
of the sampling site, field observations and measurements, and signatures of
personnel responsible for observations. Because sampling situations vary widely
no general rule can be given as to the information to be entered in the log book. It
is desirable to record s&icient information so that one could reconstmct the
sampUng withoutreUanceon the coUector's memory. Protect the log book and
keep it in a safe place.
4.4 Chain-of-custody record: FiU out a chain-of-custody record to accompany each
sample or group of samples. The record includes the foUowing information:
sample number, signature of coUector, date, time, and address of coUection,
sample type, signatures of persons involved in the chain-of-custody, and inclusive
dates of possession.
4.5 Sample analysis request sheet: The sample analysis request sheet accompanies
samples to the labwatory. The collector completes thefieldportion of such a form
that includes most of the pertinent information noted intiielog book. The
laboratory portion of such a form is to be completed by laboratory personnel and
includes: name of person receiving tiie sample, laboratory sample number, date of
sample receipt, and determinations to be paformed.
4.6 Sample delivery to laboratory: DeUver sample to laboratory as soon as practicable.
Accompany sample witii chain-of-custody record and a sample analysis request
sheet. DeUver sample to sample custodian.
4.7 Receipt and logging of sample: Intiielaboratory, the sample custodian receives
the sample and inspects its condition and seal, reconcUes label information and
seal against tiie chain-of-custody record, assigns a laboratory number, logs
sample m tiie laboratory log book, and stores it in a secured storage room or
cabinet untU it is assigned to an analyst.
4 8 Assignment of sample for analysis: The laboratory supervisor usuaUy assigns tiie
sample for analysis. Once in tiie laboratory, tiie supervisor or analyst is
responsible for tiie samples care and custody.
5.0 SampUng Metiiods:
5.1 Manual sampUng: Manual sampUng involves no equipment but may be unduly
costiy andtime-consumingforroutineor large-scale sampUng programs.
5 2 Automatic sampUng: Automatic samplers can eUminate human errors in manual
sampUng, can reduce labor costs, may provide tiie means for more frequent

69
 sampling, and are used increasingly. Be sure that the automatic sampler does not
contaminate the sample. For example, plastic components may be incompatible
with certain organic compounds that are soluble the plastic parts. If sample
constiments are generally known, contact the manufacturer of an automatic
samplerregardingpotential incompatibiUty of plastic components. Manual
sampling with a glass container and in accordance with appropriate safety
procedures may be best Program an automatic sampler in accordance with
sampling needs. Carefully mateh pump speeds and mbing sizes to the type of
sample to be taken.
5.3 Sample Containers: The type of sample container used is of utmost importance.
Containers typicaUy are made of plastic or glass, but one material may be
preferred over the other. For example, siUca and sodium may be leached from
glass but not plastic, and trace levels of metals may sorb onto the waUs of glass
containers. For samples containing organics, avoid plastic containers except those
made offluorinatedpolymers such as polytetrafluoroethylene (TFE).
From samples containing volatUe organics some compounds may dissolve into the
waUs of plastic containers or such compounds may even leach substances from
the plastic. Container failure due to breakdown of the plastic is possible. Some
organics are compatible with certain plastics (see manufacturer's Uterature).
However, even if compatibUity is assured, recognize that the waUs of a plastic
container can be porous to volatile organics. Glass containers generaUy are
preferred with volatUe organics. Container caps, typicaUy plastic, also can be a
problem with organics. Use foU or TFE liners. Semm vials with TFE-Uned
mbber or plastic septa are useful. See Table 1 for recommended containers.

6.0 Number of Samples

Given the random variations in both an analytical procedure and the occurrence of a
constiment at a point of sampUng, a single sample may be insufficient for a desired
level of uncertainty. If an overaU standard deviation is known, the required number of
samples may be estabUshed by the foUowing relationship:
N>(ts-i-U)2
where
N - number of samples,
t = Student t statistic for a given confidence level
s = overaU standard deviation, and
U = acceptable level of uncertainty.
7.0 (Quantity
Collect a 2 L sample for most physical and chemical analyses. For certain
determinations, larger samples may be necessary. Table 1 shows the volumes
ordinarily required for analyses. Do not use tiie same sample for chemical (organic
and inorganic), bacteriological, and microscopic examinations because methods of
coUecting and handling are different

8.0 Sample Preservation

Complete and unequivocal preservation of samples, whether domestic wastewater,
industrial wastes, or natural waters, is a practical impossibUity. Regardless of tiie

70
sample nature, complete stabiUty for every constituent never can be achieved At l)est,
preservation techniques onlyretardchentical and biological changes that inevitably
continue after sample coUection.

8.1 Sample Storage before Analysis

8.1.1 Nature of sample changes: Some determinations are more likely than others
to be affected by sample storage before analysis. Certain cations are
subject to loss by adsorption on, or ion exchange with, the waUs of glass
containers. These include aluminum, cadmium, chrontium, copper, iron,
lead, manganese, stiver, and zinc, which are best coUected in a separate
clean bottie and acidified with nitric acid to a pH below 2.0 to minimize
precipitation and adsorption on container walls.
Temperature changes quickly; pH may change significantiy in a matter of
minutes, dissolved gases (oxygen, carbon dioxide) may be lost. Determine
temperature, pH, and dissolved gases in the field. With changes in the pH-
alkalinity-carbon dioxide balance, calcium carbonate may precipitate and
cause a decrease in the values for calcium and for total hardness.
Iron and manganese are readUy soluble in their lower oxidation states but
relatively insoluble in their higher oxidation states, therefore, these cations
may precipitate or they may dissolve from a sediment, depending on the
redox potential of the sample. Microbiological activity may be responsible
for changes in the nitrate-nitrite-ammonia content, for decreases in phenol
concentration and in BOD, or for reducing sulfate to sulfide. Residual
chlorine is reduced to chloride. Sulfide, sulfite, fertous iron, iodide, and
cyanide may be lost through oxidation. Color, odor, and turbidity may
increase, decrease, or change in quaUty. Sodium, siUca, and boron may be
leached from the glass container. Hexavalent chromium may be reduced to
chromic ion.

Biological changes taking place in a sample may change the oxidation state
of some constiments. Soluble constituents may be converted to organicaUy
bound materials in ceU stmctures, or cell lysis mayresultin release of
cellular material into solution. The well-laiown nitrogen and phosphoms
cycles are examples of biological influences on sample composition.
Zero head-space is important in preservation of samples with volatUe
organics. Avoid loss of volatile materials by coUecting sample in a
completely fiUed container. Achieve.titis by overfilling bottie before
capping or seating. Semm vials with septum caps are particularly useful in
that a sample portion for analysis can be taken through the cap by using a
syringe.
8.2 Time Interval
Time interval between coUection and analysis: In general, the shorter the time that
elapses between coUection of a sample and its analysis, the morereUablewiU be
the analytical results. For certain constiments and physical values, immediate
analysis in the field is required. For composited samples it is common practice to
use the time at the end of composite coUection as the sample coUection time.

71
 Minimum Maximum Storage
Sample Recommended/
Determination Container Size (mL) Preservation Regulatory*
Acidity P. G(B) 100 Refiig.24 h/14 d
Alkalinity P,G 200 Refiig.24 h/14 d
BOD P,G 1000 Refiig.6 h/48 h
Boron P 100 None required28 d/6 m
Bromide P,G — None reqmred 28 d/28 d
Carbon, Tot. Org. G 100 Analyze immed.; orrefrig.and add HCl to
pH<2 7 d/28 d
Carbon dioxide P,G 100 Analyze immed stat/N.S.
COD P,G 100 Analyze ASAP, or add H2SO4 to pH<2
7 d/28 d
Chlorine, Residual P.G 500 Analyze immediatelyO.5 h/stat
Chlorine dioxide P,G 500 Analyze immediatelyO.5 h/N.S.
ChlorophyU P,G 500 30 d in dark30 d/N.S.
Color P, G 500 Refiigerate48 h/48 h
Conductivity P.G 500 Refrigerate28 d/28 d
Cyanide
Total P.G 500 Add NaOH to pH>12, refrig. ui dark
24 h/14 d; 24 h if S2-
Chlor. amenable P, G 500 Add 100 mg Na2S2CVLstat/14 d;
24 h if Sulfide present
Fluoride P 300 None required 28 d/28 d
Hardness P.G 100 Add HNO3 to pH<26 months/6 months
Iodine P.G 500 Analyze immediatelyO.5 h/N.S.
Metals, general P(A), G(A) — For (tiss. metals fUter immed., add HNO3 to
pH<26 m/6 m
Chromium VI P(A), G(A) 300 Refrigerate24 h/24 h
Mercury P(A), G(A) 500 Add HNO3 to pH<2, 4°C, refrig.28 d/28 d
Nitrogen
4^

Ammonia P, G 500 Analyze ASAP or add H2SO4 to pH<2,

refrig.7 d/28 d
Nitrate P.G 100 Analyze as soon as possible or refrig.
48 h/48 h (28 d chlor)
Nitrate + nitrite P, G 200 Add H2SO4 to pH<2, refrig.none/28 d
Nitrite P.G 100 Analyze ASAP orrefiig.none/48h
Total Kjeldahl P.G 500 Refrig.; add H2SO4 to pH<27 d/28 d

(cont.)

72
Table 1. Summary of SampUng and Preservation Requuements (Cont.)
Minimum Maximum Storage
-^ . . ^ . Sample Recommended/
Detemimation Contamer Size (mL) Preservation Regulatory^
Odor G 500 Analyze ASAP, refrig.6 h/N.S
OU and Grease G, w-m 1000 Add H2SO4 to pH<2, refiig.28 d/28 d
Org. Compounds
Pesticides G(S), TFE- Refiig.; add 1000 mg ascorbic acid/L if
resid7 d/7 d until
Uned cap chlorine present extraction; 40 d after
Phenols P.G 500 Refrig.; add H2S04 to pH<2*/28 d
Purgeables G, TFE- 50 Refrig.; add HCl to pH < 2; add 1000 mg
7d/14d
Uned cap ascorbic acid/L if resid chlorine present
Oxygen, Dissolved G, BOD bottie
Electrode 300 Analyze immediatelyO.5 h/stat
Winkler 300 Titration may be delayed after acidification
Ozone 8h/8h
G 1000 Analyze immediatelyO.5 h/N.S.
pH P. G Analyze immediately2 h/stat
Phosphate G(A) 100 For diss, phosphatefilterimmed.
refrigerate48 h/N.S.
Salinity G, wax seal 240 Analyze immediately or use wax seal
6 months/N.S.
SiUca P Refrigerate, do notfreeze28 d/28 d
Sludge Digester Gas G, gas N.S.
bottie
SoUds P.G Refiig. 7 d/2-7 d
Sulfate P.G Refrig. 28 d/28 d
Sulfide P.G 100 Refrig.e, add 2 drops 2N zinc acetate/100
mL,28 d/7 d
add NaOH to pH> 9
Taste G 500 Analyze ASAP, refiig.24 h/N.S.
Temperature P.G Analyze immediatelystat/stat
Turbidity P.G Analyze same day; store in dark up to 24
h,24 h/48 h
refrig.

* See individual assays for additional details. For determinations not Usted, use glass or
plastic containers, preferablyrefiigerateduring storage and analyze as soon as possible.
Refiigerate = storage at 4° C in the dark. P = plastic (polyethylene or equivalent): G =
glass; G(A) or P(A) +rinsedwitii 1 + 1 HNCJs; G(B) = glass, borosUicate; G(S) = glass,
rinsed with organic solvents; N.S. = not stated inreferences;stat = no storage allowed,
analyze immediately

73
 It is impossible to state exactiy how much elapsedtimemay be aUowed between
sample coUection and analysis. This depends ontiiecharacter of tiie sample, tiie
analyses to be made, andtiieconditions of storage. Changes caused by growtii of
microorganisms are greatiyretardedby keeping tiie sample intiiedark and at a
lowtemperature.Whentiieuiterval between sample coUection and analysis is
long enough to produce changes in eitiiertiieconcentration ortiiephysical state of
the constiment to be measured, foUow the preservation practices given in Table 1.
Record time elapsed between sampling and analysis, and which preservative, if
any, was added.
8.3 Preservation Techniques
To minimize the potential for volatization or biodegradation between sampling and
analysis, keep samples as cool as possible witiiout freezmg. Preferably pack
samples in cmshed or cubed ice or commercial ice substitutes before shipment
except for plant or animal tissue. Avoid using dry ice or freezing samples in
glass containers because ice expands and wiU break the glass. Furthermore,
freezing wiU separate dissolved and suspended matterfromtiiewater matrix so
that, upon thawing, the sample must bereintegratedby thorough mixing. Dry ice
also may effect a pH change in samples since it is soUd CO2. Keep composite
samples cool with ice or atrefrigerationsystem set at 4°C during compositing.
Analyze samples as quickly as possible on arrival at the laboratory. If immediate
analysis is not possible, storage at 4°C is recommended for most samples.
Use chemical preservatives only when they are shown not to interfere with the
analysis being made. When they are used, add them to the sample bottie initiaUy
so,that all sample portions are preserved as soon as coUected. No single method
of preservation is entirely satisfactory; choose the preservative with due regard to
the determinations to be made. Because a preservation method for one
determination may interfere with another one, samples for multiple determinations
may need to be spUt and preserved separately. AU methods of preservation may
be inadequate when applied to suspended matter. Because formaldehyde affects
so many analyses, do not use it.
Methods of preservation arerelativelylimited and are intended generally to retard
biological action,retardhydrolysis of chemical compounds and complexes, and
reduce volatiUty of constiments. Preservation methods are Umited to pH control,
chemical addition, the use of amber and, opaque botties,refrigeration,fUtration,
and freezing. Table 1tistspreservation methods by constituent.
The foregoing discussion is by no means exhaustive and comprehensive. Clearly
it is impossible to prescribe absolute mles for preventing all possible changes.
Additional advice wUl be found intiiediscussions under individual
determinations, but to a large degree the dependabiUty of an analytical
determination rests on the experience and good judgment of the person coUecting
the sample.

74
 Expression of Results
1.0 Units
This manual uses tiie International System of Units (SI) and chemical and physical
results are expressed ui mUUgrams pertiter(mg/L). Record only tiie significant
figures. If concentrations generaUy are lesstiian1 mgA^, it may be more convenient to
expressresultsin micrograms pertiterOig/L). Use ^ig/L when concentrations are less
than 0.1 mg/L.

Express concentrations greatertiian10 000 mg/L ui percent, 1% being equal to 10 000

mg/L, when tiie specific gravity is 1.00. In soUd samples and Uquid wastes of high
specific gravity, make a correction iftiieresultsare expressed as parts per miUion
(ppm) percent by weight:

ppm by weight =mg/L+ specific gravity

% by weight = mg/L + (10 000 x specific gravity)

In such cases, if theresultis given as milUgrams pertiter,state specific gravity.

The unit equivalents per miUion (epm), or the identical and less ambiguous term
milUgram-equivalents pertiter,or miUiequivalents pertiter(me/L or sometimes
meq/L), can be valuable for making water treatment calculations and checking analyses
by anion-cation balances.
Table 1 presents factors for converting concentrations of common ions from
milUgrams pertiterto miUiequivalents pertiter,and vice versa. The teim
milUequivalent used in this tablerepresentsO.CiOl of an equivalent weight The
equivalent weight, in turn, is defined as the weight of the ion (sum of the atomic
weights of the atoms making up the ion) divided by the number of charges normaUy
associated with the particular ion. The factors for convertingresultsfrommilUgrams
pertiterto miUiequivalents pertiterwere computed by dividing the ion charge by
weight of the ion. Conversely, factors for convertingresultsfrommtiUequivalents per
titer to milUgrams pertiterwere calculated by dividing the weight of the ion by the ion
charge.

2.0 Significant Figures

2.1 Reporting Requirements
To avoid ambiguity inreportingresultsor in presenting directions for a procedure,
it is the custom to use "significantfigures."All digits in areportedresuUare
expected to be known definitely, except for the last digit, which may be in doubt
Such a number is said to contain only significant figures. If more than a single
doubtful digit is carried, the extra digit or digits are not significant If an andytical
result is reported as "75.6 mg/L," the analyst should be quite certain of the "75,"
but may be uncertain as to whether the ".6" should be .5 or .7, or even .4 or .8,
because of unavoidable uncertainty in the analytical procedure. If the standard
deviation were known from previous work to be ± 2 mg/L, the analyst would
have, or should have, roundai off theresultto "76 mgA^" beforereportingit. On
the other hand, if the method were so good that aresultof "75.61 mg/L" could

75
 have been conscientiouslyreported,tiientiieanalyst should not haveroundedit
off to 75.6.

Report only suchfiguresas are justified by tiie accuracy of tiie work. Do not
foUow the aU-too-common practice of requuingtiiatquantities Usted in a column
have tiie same number offigurestotiierightoftiiededmal point
2.2 Rounding Off & F-
Round off by dropping digitstiiatare not significant. If tiie digit 6,7, 8, or 9 is
dropped, uicrease preceding digit by one unit; if tiie digit 0,1,2, 3, or 4 is
dropped, do not alter preceduig digit. If tiie digit 5 is dropped,roundoff
preceding digit totiienearest even number:tiius2.25 becomes 2.2 and 2.35
becomes 2.4.
2.3 Ambiguous Zeros
The digit 0 may record a measured value of zero or it may serve merely as a spacer
to locate the decimal point. Iftiieresultof a sulfate determination isreportedas
420 mg/L, the report recipient may be in doubt whether the zero is significant or
not, because the zero cannot be deleted. If an analyst calculates a total residue of
1146 mg/L, butreaUzesthat the 4 is somewhat doubtful andtiiattherefore tiie 6
has no significance, the answer should beroundedoff to 1150 mg/L and so
reported but here, too, thereportrecipient wiU not know whether the zero is
significant. Although the number could be expressed as a power of 10 (e.g., 11.5
X 102 or 1.15 X 103), this form is not used generaUy because it would not be
consistent with the normal expression ofresultsand might be confusing. In most
other cases, there wtil be no doubt as to the sense in which the digit 0 is used. It
is obvious that the zeros are significant in such numbers as 104 and 40.08. In a
number written as 5.000, it is understood that aU the zeros are significant, or else
the number could have been rounded off to 5.(X), 5.0, or 5, whichever was
appropriate. Whenever the zero is ambiguous, it is advisable to accompany the
result with an estimate of its uncertainty.
Sometimes, significant zeros are dropped without good cause. If a buret is read
as "23.60 mL," it should be so recorded, and not as "23.6 mL." The first number
indicates that the analyst took the trouble to estimate the second decimal place;
"23.6 mL" would indicate arathercareless reading of the buret.
2.4 Standard Deviation
If a calculation yields as aresult"1476 mg/L" with a standard deviation estimated
as ± 40 mg/L, report it as 1480 ± 40 mg/L. However, if the standard deviation is
estimated as ± 100 mg/Lroundoff tiie answer stiU further addreportas 15(X) ±
100 mg/ L. By this device, ambiguity is avoided and thereportrecipient can teU
that the zeros are only spacers. Even if the problem of ambiguous zeros is not
present, showing the standard deviation is helpful in that it provides an estimate of
retiabitity.
2.5 Calculations
As a practical operatuig rule,roundoff theresultof a calculation in which several
numbers are multiptied or divided to as few significant figures as are present in the
factor witii the fewest significant figures. Suppose thattiiefoUowing calculations
must be made to obtaintiieresultof an analysis:

(56 X 0.003 462 x 43.22) + 1.684

76
A ten-place calculator yields an answer of "4.975 740 998." Round off titis
number to "5.0" because one of the measurements that entered into the calculation,
56, has only two significant figures. It was unnecessary to measure the other
three factors to four significantfiguresbecause tfie "56" is tiie "weakest link in tiie
chain" and limits accuracy of the answer. If the other factors were measured to
oitiy three, instead of four, significant figures, the answer would not suffer and
the labor rrtight be less.

When numbers are added or subtracted, the number that has the fewest decimal
places, not necessarily the fewest significantfigures,puts the lintit on the number
of places that justifiably may be carried in the sum or difference. Thus the sum
0.0072
12.02
4.0078
25.9
4886
4927.9350
must be rounded off to "4928," no decimals, because one of the addends, 4886,
has no decimal places. Notice that another addend, 25.9, has only three significant
figures and yet it does not set a lintit to the number of significantfiguresin the
answer.
The preceding discussion is necessarily oversimptified. Thereaderisreferredto
mathematical texts for more detailed discussion.

77
Table 1. Conversion Factors (MiUigrams per Liter—MtiUequivalents per liter).
Factors are based on ion charge and not on redoxreactionstiiatmay be possible
for certain of these ions. Cations and anions are Usted separately in alphabetical
order.
me/L = mg/L = me/L = mg/L =
Cation mg/L X me/L X Anion mg/L X me/Lx

A13+ 0.1112 8.994 BO2- 0.023 36 42.81

B3+ 0.277 5 3.603 Br 0.012 52 79.90
Ba2+ 0.014 56 68.67 ci- 0.028 21 35.45
Ca2+ 0.049 90 20.04 CO32- 0.033 33 30.00
Cr3+ 0.057 70 17.33 0042- 0.017 24 58.00
F- 0.052 64 19.00
Cu2+ 0.031 47 31.77 HCO3- 0.016 39 61.02
Fe2+ 0.035 81 27.92 HPO42- 0.020 84 47.99
Fe3+ 0.053 72 18.62 H2PO4- 0.010 31 96.99
H+ 0.992 2 1.008 HS- 0.030 24 33.07
K+ 0.025 58 39.10 HSO3- 0.012 34 81.07
HSO4- 0.010 30 97.07
Li+ 0.144 1 6.941 I- 0.007 880 126.9
Mg2+ 0.082 29 12.15 NO2- 0.021 74 46.01
Mn2+ 0.036 40 27.47 NO3- 0.016 13 62.00
Mn4+ 0.072 81 13.73 OH- 0.058 80 17.01
Na+ 0.043 50 22.99 PO43 0.031 59 31.66
NH4+ 0.055 44 18.04 S2- 0.062 38 16.03
Pb2+ 0.009 653 103.6 51032- 0.026 29 38.04
Sr2+ 0.022 83 43.81 SO32- 0.024 98 40.03
Zn2+ 0.030 59 32.69 SO42- 0.020 82 48.03

78
METHOD #: 305.1 Approved for NPDES (Technical Revision 1974)
TITLE: Acidity (Titrimetric)
ANALYTE:
Acidity

INSTRUMENTATION: Titration
INTRODUCTION
Acidity of a water is its quantitative capacity to react witii a strong base to a designated pH
The measured value may vary significantiy witii tiie end-point pH used in tiie
determination. Acidity is a measure of an aggregate property of water and can be
interpreted m terms of specific substances only whentiiechemical composition of tiie
sample is known. Strong mmeral acids, weak acids such as carbonic and acetic, and
hydrolyzuig salts such as iron or aluminum sulfates may contribute totiiemeasured acidity
accordmg to tiie metiiod of determmation. Acids contribute to corrosiveness and influence
chemical reaction rates, chentical speciation, and biological processes. The measurement
alsoreflectsa change intiiequaUty of tiie source water.

A normal source of acidity in natural waters is carbon dioxide. It may enter surface waters
by absoiptionfromtiieatmosphere, but only whentiiepartial pressure of carbon dioxide in
tiie water is lesstiiantiiepartial pressure of tiie carbon dioxide in tiie atmosphere, in
accordance with Henry's law. Carbon dioxide may also be produced tii waters tiirough
biological oxidation of organic matter, particularly in poUuted water. In such cases, if
photosyntiietic activity istimited,tiiepartial pressure of carbon dioxide ui the water may
exceedtiiatof the atmosphere and carbon dioxide wiU escapefromthe Uquid Thus it may
be concluded that surface waters are constantiy absorbing or giving up carbon dioxide to
maintain an equiUbrium witii the atmosphere. The amounttiiatcan exist at equiUbrium is
very smaU because of the low partial pressure of carbon dioxide in the atmosphere.

Ground-waters and watersfromthe hypoUmnion of stratified lakes and reservou^ often

contain considerable amounts of carbon dioxide. This concentration results from bacterial
oxidation of organic matter with which the water has been in contact, and under these
conditions, the carbon dioxide is not free to escape to the atmosphere. Carbon dioxide is
an end product of both aerobic and anaerobic bacterial oxidation: therefore its concentration
is not Umited by the amount of dissolved oxygen originally present. It is not uncommon to
encounter ground-waters with 30 to 50 mg/I of carbon dioxide. This is particularly tme of
waters that have percolated through sotis that do not contain enough calcium or magnesium
carbonate to neutralize the carbon dioxide through formation of bicarbonates .

CO2 + CaCOs + H2O —> Ca(HC03)2

Acidity is oftittieconcern from a sanitary or pubUc health viewpoint Carbon dioxide is

present in malt and carbonated beverages in concentrations greatiy in excess of any
concentrations known in natural waters, and no deleterious effects due to the carbon
dioxide have been recognized. In these cases, the excess CO2 is maintained in solution by
pressure and lowtemperature.Waters that contain mineral acidity are so unpalatable that
problemsrelatedto human consumption are nonexistent. Acid waters are of concern
because of their cortosive characteristics and the expense involved inremovingor

79
controlUng tiie corrosion-producing substances. The corrosive factor in most waters is
carbon dioxide, but in many industrial wastes it is mineral acidity.

Where biological processes of treatment are used,tiiepH must orduiarUy be maintauied

witiiin tiie range of 6 to 9.5. This criterion often requires adjustment of pH to favorable
levels, and calculation of tiie amount of chemicals needed is based upon acidity values in
most cases.

The principle of acidity depends on hydrogen ions present in a sample as aresultof

dissociation or hydrolysis of solutes whichreactwitii additions of standard aUcati. Acidity
thus depends on the end-point pH or indicator used The constmction of atitrationcurve
by recording sample pH after successive smaU measured additions oftitrantpermits
identification of inflection points and buffering capacity, if any, and allows the acidity to be
determined withrespectto any pH of interest In thetitrationof a single acidic species, as
in the standardization ofreagents,the most accurate end point is obtainedfromthe
inflection point of atitrationcurve. The inflection point is the pH at which curvature
changesfix)mconvex to concave or vice versa.

Because accurate identification of inflection points may be difficult or impossible in

buffered or complex mixtures, thetitrationin such cases is carried to an arbitrary end-point
pH based on practical considerations. Forroutinecontroltitrationsorrapidpreliminary
estimates of acidity, the color change of an indicator may be used for the end point
Samples of industrial wastes, acid mine drainage, or other solutions that contain
appreciable amounts of hydrolyzable metal ions such as iron, aluminum, or manganese are
treated with hydrogen peroxide to ensure oxidation of any reduced forms of polyvalent
cations, and boUed to hasten hydrolysis. Acidityresultsmay be highly variable if this
procedure is not foUowed exactiy.

IdeaUy, the end point of the aciditytin^tionshould correspond to the stoichiometric

equivalence point for neutralization of acids present. The pH at the equivalence point wiU
depend on the sample, the choice among multiple inflection points, and the intended use of
the data.
1.0 Scope and AppUcation

1.1 This method is applicable to surface waters, sewage and industrial wastes,
particularly mine drainage and receiving streams, and other waters containing
ferrous iron or other polyvalent cations in a reduced state.
1.2 The method covers the rangefromapproximately 10 mg/L acidity to
approximately 1(XX) mg/L as CaCOs, using a 50 mL sample.

2.0 Summary of Method

2.1 The pH of the sample is determined and a measured amount of standard acid is
added, as needed, to lower the pH to 4 or less. Hydrogen peroxide is added, the
solution boiled for several minutes, cooled, andtitratedelectrometricaUy with
standard alkali to pH 8.2.

3.0 Definitions

80
 3.1 This method measures the mineral acidity of a sample plus the acidity resulting
from oxidation and hydrolysis of polyvalent cations, including salts of iron and
aluminum.

4.0 Interferences

4.1 Suspended matter present in the sample, or precipitates formed during the titration
may cause a sluggish electrode response. This may be offset by aUowing a 15-20
second pause between additions oftitrantor by slow drop wise addition of titrant
as the endpoint pH is approached.
4.2 Dissolved gases contributing to acidity or aUcalinity, such as CO2, hydrogen
sulfide, or ammonia, may be lost or gained during sampling, storage, or titration.
Minimize such effects bytitratingto the end point promptiy after opening sairq)le
container, avoiding vigorous shaking or mixing, protecting sample from the
atmosphere duringtitration,and letting sample become no warmer than it was at
coUection.

5.0 Apparatus

5.1 Use any commercial pH meter that can bereadto 0.05 pH unit. Standardize and
caUbrate according to the pH method given in this manual. Pay special attention to
temperature compensation and electrode care. If automatic temperature
compensation is not provided,titrateat 25 ± 5°C.
5.2 Magnetic stirrer
5.3 Standardtitrationequipment
6.0 Reagents
6.1 Hydrogen peroxide (H2O2, 30% solution).
6.2 Standard sodium hydroxide: O.IN: Add 4 g NaOH to ICXX) ml of deionized
water.
6.3 Sodium Hydroxide Solution, 0.02 N: Add 200 ml of 0.1 N (6.2) uito 1000 ml of
deionized water.
6.4 Standard sulfuric acid, 0.02 N: Add 0.5656 ml concentrated sulfuric acid to
1000 mlreagent^ ^ e water.

7.0 Sampling and Storage

7.1 CoUect samples in polyethylene or borosiUcate glass botties and store at a low
temperature. FUl botties completely and cap tightiy. Because waste samples may
be subject to microbial action and to loss or gain of CC)2 or other gases when
exposed to air, analyze samples witiiout delay, preferably witiiin 1 day. If
biological activity is suspected analyze within 6 h. Avoid sample agitation and
prolonged exposure to air.

8.0 Procedure
8.1 Pipet 50 mL of tiie sample into a 250 mL beaker.
8.2 Measure the pH of tiie sample. If tiie pH is above 4.0, add standard sulfuric acid
(6.3) in 5.0 mL increments to lowertiiepH to 4.0 or less. If tiie initial pH of tiie
sample is lesstiian4.0, tiie uicremental addition of sulfuric acid is not required

81
 8.3 Add 5 drops of hydrogen peroxide (6.1).
8.4 Heat tiie sample to bolting and continue botiing for 2 to 4 ntinutes. In some
instances, tiie concentration of ferrous iron in a sample is such that an additional
amount of hydrogen peroxide and a sUghtiy longer botiing time may be required
8.5 Cool tiie sample to roomtemperatureandtitrateelectrometricaUy witii standard
sodium hydroxide (6.3) to pH 8.2.
9.0 Calculations

9.1 Acidity, as mg/L CaCOs = [(A * B)-(C * D)] * 50,000

ml of sample
where:

A = volume of standard sodium hydroxide used in titration

B = normaUty of standard sodium hydroxide
C = volume of standard sulfuric acid used to reduce pH to 4 or less
D = normaUty of standard sulfuric acid
9.2 If it is desired toreportacidity in miUiequivalents pertiter,thereportedvalues as
CaCOs are divided by 50, as foUows:

Acidity as meq/L = mg/L CaCOs

50
10.0 Accuracy

10.1 On a roundrobinconducted by ASTM on 4 acid mine waters, including

concentrations up to 2000 mg/ L, the precision was found to be ± 10 mg/L.
References
United States Environmental Protection Agency. (1992). Methods for Chemical Analvsis
of Water and Wastes. Method #305.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.
Sawyer, C. N., and McCarty, P. L. (1994). Chemistrv for Environmental Engineering.
4tii ed. McGraw-HiU, Inc., New York, N.Y., 464-466.
American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed. Method 2310. American PubUc Health Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 2-23.

82
METHOD #: 310.1 Approved for NPDES (Editorial Revision 1978)
TITLE: AUcaUnity (Titrimetric, pH 4.5)
ANALYTE:
Alkalinity

INSTRUMENTATION: Titration
INTRODUCTION
AUcalinity of a water is its acid-neutraUzing capacity. It is tiie sum of aUtiietitratablebases.
AlkaUrtity is also interpreted to tiie lowering of pH values. Thus substances which form
buffers (weak acids andtiieu*conjugate salts) contribute to aUcalinity. The measured value
may vary significantiy with the end-point pH used. AlkaUnity is a measure of an aggregate
property of water and can be interpreted intermsof specific substances only when the
chemical composition of the sample is known. Included below are discussions of the
compounds of alkaUnity and its contribution to calcium carbonate CaCOs saturation.

Alkalinity is significant in many uses and treatments of natural waters and wastewaters.
Because the alkalinity of many surface waters is primarily a function of carbonate,
bicarbonate, and hydroxide content, it is usually taken as an indication of the sums of
concentrations of tiiese constituents. Bicarbonatesrepresentthe major form of alkalinity,
since they are formed in considerable amounts from the action of carbon dioxide upon basic
materials in the soU.

These inorganic carbon species are also components of the most important buffer system in
natural and most process waters, the carbonate buffer system (carbonic acid-bicarbonate-
carbonate). In addition, a few organic acids that are quiteresistantto biological oxidation
for example, humic acid, form salts that add to the alkalinity of natural waters. In poUuted
or anaerobic waters, salts of weak acids such as acetic, propionic, and hydrosuUuric may
be produced and would also contribute to alkalinity. In other cases, ammonia or
hydroxides may make a contribution to the total alkalinity of a water. The measured values
also may include contributions from borates, phosphates, siUcates, or other bases if these
are present.
AlkaUrtity in excess of alkaline earth metal concentrations is significant in determining the
suitabitity of a water for irrigation (see a discussion of sodium absorption ratio, SAR).
AlkaUnity measurements are used in the interpretation and control of water and wastewater
treatment processes. As theratesof most chemical and biological processes are pH
dependent, substances which moderate pH are important. Raw domestic wastewater has
an aUcalinity less than, or only sUghtiy greatertiian,tiiatof tiie water supply. Properly
operating anaerobic digesters typicaUy have supernatant aUcalinities in the range of 2000 to
40(X) mg calcium carbonate (CaC03)/L.

Total aUcalinity is usuaUy determined bytitrationdirectiy to a single end point. However

the concentrations of inorganic carbon species can be estimates by step wisetitrationto a
pH 8.3 tiien to 4.5. Traditionally color pH indicators were used, phenolphtiialem for 8.3
and for 4.5 Bromcresol green or a mixed bromcresol green- metiiyl red indicator to
determine total aUcalinity. The assumption istiiatat pH 8.3,tiiereare equal concentrations
of carbonate and bicarbonate (equivalence). The results obtained fromtiiephenolphthaleui
and total alkalinity determinations offer a means for stoichiometric classification of the tiiree

83
principal forms of aUcalinity present ui many waters. The classification ascribestiieentire
alkalinity to bicarbonate, carbonate, and hydroxide, and assumes tiie absence of otiier
(weak) inorganic or organic acids, such as siUcic, phosphoric, and boric acids. It further
presupposes the incompatibiUty of hydroxide and bicarbonate aUcalinities. Because tiie
calculations are made on a stoichiometric basis, ion concentrations in the strictest sense are
notrepresentedin theresults,which may differ significantiy from acmal concentrations
especiaUy at pH > 10. Phenolphthaleui alkalirtity is determined in the same procedure as
total alkaUnity described in section 7.0. Accor(ting totitisscheme:

1) Carbonate (CO32-) alkalirtity is present when phenolphthalein alkalirtity is

not zero but is less than total alkalirtity.

2) Hydroxide (OH) alkalirtity is present if phenolphthalein alkalirtity is more

than half the total alkalirtity.
3) Bicarbonate (HCO3-) alkalinity is present if phenolphthalein alkalirtity is
less than half the total alkalirtity.
Theserelationshipsmay be calculated by the foUowing scheme, where P is phenolphthalein
alkalirtity and T is total alkalirtity):
Select the smaUer value of P or (T - P). Then, carbonate alkaUnity equals twice the
smaller value. When the smaller value is P, the balance (T - 2P) is bicarbonate.
When the smaUer value is (T - P), the balance (2P - T) is hydroxide. AU results are
expressed as CaCOa. The mathematical conversion of theresultsis shown in Table
2320:11. (A modification of Table 2320:11tiiatis more accurate when P = 0.5 T has
been proposed.
This value of 8.3 corresponds to the equivalence point for the conversion of carbonate ion
to bicarbonate ion:
CO32- + H+ <—> HCO3-

The use of a pH of about 4.5 for tiie end point for tiie second step oftiietittation
corresponds approximately to the equivalence point for the conversion of bicarbonate ion to
carbortic acid:
HCO3- + H+ <—> H2CO3

Total AUcaUnity is used bytitratingto a pH of 4.5 witiiout stopping at any intermediate pH

values. Total aUcaluiity is used when specific concentrations of carbonate species is not
required.
As far as is known, tiie aUcaUnity of a water has Uttie pubUc healtii significance. Highly
aUcaline waters are usuaUy unpalatable, and consumerstendto seek otiier supplies.
ChemicaUy treated waters sometimes have rather high pH values which have met with
some objection on the part of consumers. Fortiiesereasons,standards are sometUnes
estabUshed on chemicaUy treated waters. Such standards,relatingto phenolphtiialeui and
total and excess aUcaUnity, are too detailed to summarize here. Natural waters witii high

84
aUcaUnity have cortespondmg high CO2 levels, and when temperatures are also high, tiiey
are commonly eutrophic.

1.0 Scope and AppUcation (The information presented is based on calculating only total
alkalirtity).

1.1 This metiiod is appUcable to drutidng, surface, and saUne waters, domestic and
industrial wastes.
1.2 The method is suitable for aU concentration ranges of aUcaUnity; however,
appropriate aUquots should be used to avoid atitrationvolume greatertiian50 mL
because of dUution of the sample by the titrant.
1.3 Automatedtitrimetricanalysis is equivalent
1.4 Hydroxyl ions present m a sample as aresultof dissociation or hydrolysis of
solutesreactwitii additions of standard acid. AUcaUnitytiiusdepends ontiieend-
point pH used.
1.5 For samples of low aUcaUnity Gesstiian20 mg CaCOs/L) use an extrapolation
technique based on the near proportionaUty of concentration of hydrogen ions to
excess oftitrantbeyond the equivalence point The amount of standard acid
required to reduce pH exactiy 0.30 pH unit is measured carefully. Because this
change ui pH corresponds to an exact doubting of tiie hydrogen ion
concentt^tion, a simple extrapolation can be made to the equivalence point
1.6 Alkalinity can be used for estimations of calcium carbonate saturation. See
section 9.0.
2.0 Summary of Method

2.1 An unaltered sample istitratedto an electrometricaUy determined end point of pH

4.5. The sample must not be fUtered, dUuted, concentrated, or altered in any
way.

3.0 Comments
3.1 The sample should berefrigeratedat 4°C and run as soon as practical. Do not
open sample bottie before analysis.
3.2 Substances, such as salts of weak organic and inorganic acids present in large •
amounts, may cause interference intiieelectrometric pH measurements. This can
occur in digestors and highly eutrophic natural water.
3.3 For samples having high concentrations of mineral acids, such as rxtine wastes
and associated receiving waters,titrateto an electrometric endpoint of pH 3.9.
3.4 OU and grease, by coating the pH electrode, may also interfere, causing sluggish
response.
3.5 Soaps, oUy matter, suspended solids, or precipitates may coat the glass electrode
and cause a sluggish response. AUow additional time betweentitrantadditions to
let electrode come to equiUbrium or clean the electrodes occasionaUy. Do not
filter, dUute, concentrate, or alter sample.

4.0 Apparams
4.1 pH meter or electricaUy operatedtitratorthat uses a glass electrode and can be
read to 0.05 pH urtits. Standardize and caUbrate according totiiepH method in

85
 this rnanual. If automatic temperature compensation is not provided make
titration at 25 ± 2°C.
4.2 Use an appropriate sized vessel to keep tiie air space above tiie solution at a
minimum.
4.3 Magnetic stirrer, pipets, flasks and otiier standard laboratory equipment
4.4 Burets, Pyrex 50, 25 and 10 mL.
5.0 Reagents

5.1 Sodium carbonate solution, approximately 0.05 N: Place 2.5 ± 0.2 g (to nearest
mg) Na2C03 (dried at 250°C for 4 hours and cooled in desiccator) into a 1 titer
volumetric flask and dUute to the mark.
5.2 Standard acid (sulfuric or hydrochloric), 0.1 N: DUute 3.0 mL concentrated
H2SO4 or 8.3 mL concentrated HCl to 1 titer witii distiUed water. Standaixtize
versus 40.0 mL of 0.05 N Na2C03 solution witii about 60 mL distiUed water by
titrating potentiometrically to pH of about 5. Lift electrode and rinse into beaker.
BoU solution gentiy for 3-5 minutes under a wateh glass cover. Cool to room
temperature. Rinse cover glass into beaker. Continue titration to the pH
inflection point. Calculate normaUty using: N = A * B/ 53.00 * C where:
A = g Na2C(>3 weighed into 1 titer
B = mL Na2C03 solution taken for titration
C = mL acid used to inflection point
1 mL 0.1000 N solution = 5.00 mg CaC03.
Standardization is done to check the accuracy of the prepared normaUty acid
against a known amount of Na2C03.
5.3 Standard acid (sulfuric or hydrochloric), 0.0202 N: Place 0.5656 ml of
concentrated Sulfiiric acid in 1(XX) nti distiUed water. Standardize by
potentiometric titration of 15.0 mL 0.05 N Na2C03 solution as above. 1 ml = 1
mg CaC03

6.0 Procedure
6.1 Sample size
6.1.1 Use a sufficientiy large volume of titrant ( > 20 mL in a 50 mL buret) to
obtain good precision whUe keeping volume low enough to pemtit sharp
end point.
6.1.2 For < 10(X) mg CaC03 /L use 0.02 N titrant, use a 25 ml sample
6.1.3 For > 1000 mgCaC03/L use O.IN titrant
6.1.4 A pretiminary titration is helpful
6.2 Potentiometric titration
6.2.1 Place sample in flask by pipetting with pipet tip near bottom of flask.
6.2.2 Measure pH of sample
6.2.3 Add standard acid 0.1 ml at a time (5.2 orj5.3), being careful to stir
thoroughly but gentiy to aUow needle to obtain equUibrium.
6.2.4 Titrate to pH 4.5. Titrate to the end-point pH without recording
intermediate pH values and without undue delay urtiess specified
otherwise. Note: 5374 smdents are required to create a buffer curve, so
the intermediate values need to be reconied As the end point is
approached make smaUer additions of acid and be sure tiiat pH equiUbrium
is reached before adding more titrant. Record volume of titrant

86
 6.3 Potentiometrictitrationof low alkalirtity
6.3.1 For aUcaUnity of <20 mg/Ltittate100-200 mL as above (6.2) ustiig a 10
mL nticroburet and 0.02 N acid solution (5.3).
6.3.2 Stoptitrationat pH in range of 4.3-4.7, record volume and exact pH.
Very carefuUy addtitrantto lower pH exactiy 0.3 pH units and record
volume.
7.0 Calculations

7.1 Potentiometrictitrationto pH 4.5

AUcaUnity, mg/L CaC03= A * N * 50,000 / mL of sample
where:
A = mL standard acid ^__^_^^____
N = normality of standard acid |see6.1.2above|
Report pH of end point used as foUows: "The alkalinity to pH = mg CaCChJL"
and indicate clearly if this pH corresponds to an inflection point of the titration
curve.
7.2 Potentiometrictitrationof low alkalinity:
Total aUcaUnity, mg/L CaC03 = (2B - C) * N * 50,000 / mL of sample
where:
B = mLtittantto first recorded pH
C = total mLtitranttoreachpH 0.3 units lower
N = normality of acid |see 6.1 above
8.0 Accuracy
8.1 In a single laboratory (EMSL) using surface water samples at an average
concentration of 122 mg CaCOs/L, the standard deviation was ±3.
8.2 Sodium carbonate solutions equivalent to 0 and 5 mg CaCQs/L were analyzed by
laboratories acconiing to the procedure of 6.2.4. The standard deviations were 8
and 5 mg/L, respectively, witii negtigible bias.
8.3 Four laboratories analyzed six samples having total aUcalinities of about 1000 mg
CaCQs/L and containing variousratiosof carbonate/bicarbonate by the
procedures of botii phenolphtiialein and 6.2.4. The pooled standard deviation
was 40 mg/L, witii negligible difference between the procedures.
9.0 Total AUcaUnity and its relationship with Calcium Carbonate Saturation
When measuring total aUcalinity, the predominant species measured carbonate species, such
as calcium carbonate. The presence of CaC03 has many influences m water such as water
quaUty and its corrosiveness capabUities. Calcium carbonate (CaC03) saturation indices
commonly are used to evaluate tiie scale-fonmng and scale-dissolvuig tendencies of water.
Assessuig tiiese tendencies is useful in corrosion control programs and ui preventing
CaCOs scaling in pipmg and equipment such as industrial heat exchangers or domestic
water heaters.
Waters oversaturated witiirespectto CaCOs tend to precipitate CaC03. Waters
undersaturated witii respect to CaC03 tend to dissolve CaCOs. Saturated waters, i.e.,
waters in equUibrium witii CaCOs, have neitiier CaCOs-precipitating nor CaOb

87
dissolvmg tendencies. Saturationrepresentstiiedividuig tine between "precipitation Ukely"
and "precipitation not likely."

Several water quaUty characteristics must be measured to calculate tiie CaC03 samration
indices described here. Minimum reqmrements are total aUcalinity, total calcium, pH, and
temperature. The ionic strengtii also must be calculated or estimated from conductivity or
total dissolved soUds measurements. Measure pH at the system water temperamre using a
temperature-compensated pH meter. If pH is measured at a differenttemperature,for
example in the laboratory, correct the measured pH. In measuring pH and alkalirtity,
minimize CO2 exchange between sample and atmosphere. IdeaUy, seal the sample from the
atmosphere during measurements; at a mirtimum, avoid vigorous stirring of unsealed
samples.

The economic losses caused by corrosion of water systems are very large. Hudson and
Gticreas cited the strUdng example of a 55 mgd (2.41 m3/sec) water distribution system
under attack from a corrosive water. They estimated that the distribution system
(composed ofreinforcedconcrete, asbestos-cement and cement-lined cast iron pipe) was
losing 5(X) short tons (453 metric tons) of piping annuaUy as CaC03. Over a period of five
years, the carrying capacity of the system had declined severely, as indicated by a decUne in
the Hazen-WilUams coefficientfix)m130 to 80. In addition, large concrete storage tanks
had corroded to the point that for safety reasons they could only be half fiUed They
estimated that the nation's economic loss to deterioration of distribution systems is $375
miUion annuaUy, but that this could be avoided if corrosive waters were stabiUzed v^ith
ortiy $27 miUion worth of time.

The majority of treatments involve the placement of inert films at the solid-water interface.
In simplesttermsthe fUms prevent contact between the water and the soUd surface, and the
corrosive process cannot proceed. The film may be mechanically appUed (for example,
coal tar enamel or cement linings), derivedfromthe deposition of chemicals (for example,
polyphosphates, siUcates or calcium carbonate), or formedfromthe products of the
corrosivereactionitself. Combinations of methods may be used—^for example chentical
deposition upon a mechanicaUy applied liner to cover holes or "hoUdays" in the liner.
Chemical conditioiting in thisrespectmeans the conditioning of waters so that CaC03 can
be precipitated from them.

References
American Pubtic Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed Method 2320. American PubUc Health Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 2-25.
Sawyer, C. N., and McCarty, P. L (1994). Chemistrv for Envu-onmental Engineering.
4tiied. McGraw-HiU, Inc., New York, N.Y., 471.

United States Envuonmental Protection Agency. (1992). Metiiods for Chentical Analvsis
of Water and Wastes. Metiiod #310.1. Environmental Monitoring and Support
Laboratory, United States Envuonmental Protection Agency, Cuicuinati, Ohio.

88
American PubUc Healtii Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed Method 2330. American PubUc Healtii Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 2-28.

89
METHOD #: 300.0 Recommended for Approval for NPDES (November 1991)
TITLE: The Determination Of Inorganic Anions In Water By Ion
Chromatography

mSTRUMENTATION: IC
ESTTROpUCnON:
Determination of the common anions such as bromide, chloride,fluoride,nitrate, rtitrite,
phosphate, and sulfate often is desirable to charaaerize a water and/or to assess the need
for specific treatment Although conventional colorimetric, electrometric, or titrimetric
methods are avaUable for determining individual artions, only ion chromatography provides
a single instmmental technique that may be used for theirrapid,sequential measurement.
Ion chromatography eliminates the need to use hazardousreagentsand it effectively
distuiguishes among tiie haUdes (Bi^, C1-, and F-) andtiieoxy- ions (PO4-. SO42-. or
NO2-, NO3-).

The principle of ion chromatography is based on a water sample injected into a stream of
carbonate-bicarbonate eluent and passed through a series of ion exchangers. The artions of
interest are separated on the basis of their relative affinities for a low capacity, strongly
basic artion exchanger (guard and separator columns). The separated anions are directed
through a hoUowfibercation exchanger membrane (fiber suppressor) or micromembrane
suppressor bathed in continuously flowing strongly acid solution (regenerant solution). In
the suppressor the separated anions are converted to their highly conductive acid forms and
the carbonate-bicarbonate eluent is converted to weakly conductive carbonic acid. The
separated anions in their acid forms are measured by conductivity. They are identified on
the basis ofretentiontime as compared to standards. Quantitation is by measurement of
peak area or peak height

1.0 Scope and Application

1.1 This method covers the determination of the foUowing inorganic anions.
Bronude
Chloride
Fluoride
Nitrate-N
Nitrite-N
Ortho-Phosphate-P
Sulfate
1.2 The matrices appUcable are shown below:
1.2.1 Drinking water, surface water, mixed domestic and industrial wastewaters,
groundwater, reagent waters, sotids (after extraction 2.3).
1.3 This metiiod is restricted to use by or under the supervision of analysts
experienced in the operation of ion chromatograph.
2.0 Summary of Method
2.1 A smaU volume of sample, typicaUy 2 to 3 mL, is introduced into an ion
chromatograph. The anions of interest are separated and measured, using a
system comprised of a guard column, separator column, suppressor device, and
conductivity detector.

90
 2.2 The multilaboratory rangetestedfor each anion is as follows in mg/L:

Bromide 0.63- 21.0

Chloride 0.78 - 26.0
Huoride 0.26- 8.49
Nitrate-N 0.42- 14.0
Nitrite-N 0.36- 12.0
Ortho-P 0.69- 23.1
Sulfate 2.85 - 95.0
2.3 In order to use this metiiod for soUds an extraction procedure must be performed
(See 9.7).
3.0 Interferences

3.1 Interferences can be caused by substances with retention times that are similar to
and overlap those of the artion of interest. Large amounts of an artion can
interfere with the peak resolution of an adjacent artion. Sample dUution and/or
spiking can be used to solve most interference problems.
3.2 The water dip or negative peak that elutes near, and can interfere with, the fluoride
peak can usually be eUminated by the addition of the equivalent of 1 mL of
concentrated eluent (5.3 100 X (tiie solution diluted to 200 ml rathertiian2 titers))
to 100 mL of each standard and sample.
3.3 Method interferences may be caused by contaminants in the reagent water,
reagents, glassware, and other sample processing apparatus that lead to discrete
artifacts or elevated baseUne in ion chromatograms.
3.4 Samples that contain particles larger than 0.45 nticrons and reagent solutions that
contain particles larger than 0.20 nticrons require fUtration to prevent damage to
instrument columns and flow systems.
3.5 Any anion that is notretainedby the column or ortiy sUghtiy retained wiU elute in
the area offluorideand interfere. Known coelution is caused by carbonate and
other smaU orgaitic anions. At concentrations offluorideabove 1.5 mg/L this
interference may not be significant, however, it is the responsibUity of the user to
generate precision and accuracy information in each sample matrix.
3.6 The acetate anion elutes early during the chromatographic run. The retention
times of the anions also seem to differ when large amounts of acetate are present
Therefore, this method is not recommended for leachates of soUd samples when
acetic acid is used for pH adjustment
3.7 The quantitation of unretained peaks should be avoided, such as low molecular
weight organic acids (formate, acetate, propionate, etc.) which are conductive and
coelute with or nearfluorideand would bias thefluoridequantitation in some
drinking and most waste waters.

4.0 Apparatus and Materials

4.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
4.2 Ion chromatograph: Analytical system complete witii ion chromatograph and aU
required accessories including syringes, analytical columns, compressed gasses
and detectors.

91
 4.2.1 Anion guard column: A protector of the separator column. If omitted from
the system theretentiontimes wiU be shorter. UsuaUy packed with a
substrate the same as that in the separator column.
4.2.2 Artion separator column.
4.2.3 Artion suppressor device: such as a Dionex Artion MicroMembrane
Suppressor (P/N 37106).
4.2.4 Detector - Conductivity ceU: approximately 1.25 pL intemal volume,
(Dionex, or equivalent).
4.3 Chromatography Software if avaUable, such as PE Nelson Turbochrom 4.0 or the
Dionex AI-450 Data Chromatography Software. Systems using a stripchart
recorder and integrator or other computer based data system may achieve
approximately the same MDL's.
5.0 Reagents and Consumable Materials
5.1 Sample botties: Glass or polyethylene of sufficient volume to aUow repUcate
analyses of artions of interest
5.2 Reagent water: DistiUed or deionized water,freeof the anions of interest Water
should contain particles no larger than 0.20 nticrons.
5.3 Eluent solution: Sodium bicarbonate 1.7 mM, sodium carbonate 1.8 mM.
Dissolve 0.2856 g sodium bicarbonate (NaHC03) and 0.3816 g of sodium
carbonate (Na2C03) in reagent water (5.2) and dilute to 2 titers.
5.4 Regeneration solution (MicroMembrane Suppressor): Sulfuric acid 0.025 N.
DUute 2.8 mL cone, sulfuric acid (H2SO4) to 4titerswithreagentwater.
5.5 Stock standard solutions, ICXX) mg/L (1 mg/mL): Stock standard solutions may
be purchased as certified solutions or preparedfromACS reagent grade materials
(dried at 105°C for 30 min.) as Usted below.
5.5.1 Bromide (Br) 1000 mg/L: Dissolve 1.2876 g sodium bromide (NaBr) m
reagent water and dUute to 1 titer.
5.5.2 Chloride (CI) 1000 mg/L: Dissolve 1.6485 g sodium chloride (NaCl) in
reagent water and dUute to 1 titer.
5.5.3 Fluoride (F-) 1000 mg/L: Dissolve 2.2100 g sodiumfluoride(NaF) ui
reagent water and dUute to 1 titer.
5.5.4 Nitrate (NO3- -N) 1000 mg/L: Dissolve 6.0679 g sodium nitrate (NaN03)
in reagent water and dUute to 1 titer.
5.5.5 Nitrite (NO2- -N) 1000 mg/L: Dissolve 4.9257 g sodium nitrite (NaN02)
in reagent water and dUute to 1 titer.
5.5.6 Phosphate (HPO4 2- -P) 1000 mg/ L: Dissolve 4.3937 g potassium
phosphate, monobasic (KH2PO4) inreagentwater and dilute to 1 liter.
5.5.7 Sulfate (SO42) 1000mg/L: Dissolve 1.8141 gpotassium sutfate (K2SO4)
in reagent water and dUute to 1 titer.
Note: StabUity of standards: Stock standards are stable for at least one
montii when stored at 4°C. Dtiute working standards should be prepared
weekly, except those that contain rtitrite and phosphate should be prepared
fresh daUy.
6.0 Sample CoUection, Preservation and Storage
6.1 Samples should be coUected mtiioroughlyclean glass or polyetiiylene botties.

92
 6.2 Sample preservation and holding times for the anionstiiatcan be determined by
this method are as foUows:
Analyte Preservation Holding Time
Bromide None requu-ed 28 days
Chloride None required 28 days
Fluoride None requu-ed 28 days
Nitrate-N
chlorinated Cool to 4°C 28 days
nonchlorinated cone. H2SO4 14 days
pH<2
Nitrite-N Cool to 4°C 48 hours
o-Phosphate-P Cool to 4°C 48 hours
Sulfate Cool to 4°C 28 days
6.3 The method of preservation and the holding time for samples analyzed by this
method are detemtined by the artions of interest. In a given sample, the artion that
requires the most preservation treatment and the shortest holding time wiU
determine the preservation treatment It is recommended that aU samples be
cooled to 4°C and held no longertiian28 days.
7.0 CaUbration and Standardization
7.1 For each analyte of interest, prepare a blank and caUbration standards at a
minimum of three concentration levels by adding accurately measured volumes of
one or more stock standards to a volumetricflaskand dUuting to volume with
reagent water. If a sample analyte concentration exceeds the calibration range the
sample may be dUuted to fall within the range. If this is not possible then three
new calibration concentrations must be chosen, two of which must bracket the
concentration of the sample analyte of interest. Each attenuation range of the
instrument used to analyze a sample must be catibrated individually.
7.2 Using injections of 0.1 to 1.0 mL (determined by injection loop volume) of each
caUbration standard, tabulate peak height or arearesponsesagainst the
concentration. The results are used to prepare a caUbration curve for each analyte.
During this procedure,retentiontimesmust be recorded
7.3 The caUbration curve must be verified on each working day, or whenever the
anion eluent is changed, and after every 20 samples. If theresponseor retention
time for any analyte variesfromthe expected values by more than ± 10%, the test
must be repeated, using fresh caUbration standards. If the results are stiU more
than ±10%, a new calibration curve must be prepared fortiiatanalyte.
7.4 NonUnearresponsecanresultwhen the separator column capacity is exceeded
(overloading). Theresponseof the detector totiiesample when dUuted 1:1, and
when not dUuted, should be compared If the calculatedresponsesare the same,
samples of this total anionic concentration need not be dUutol.
7.5 Remove sample particulates, if any, byfilteruigtiux)ugha prewashed 0.2 pm pore
diameter membrane filter. Inject sample and compare againstretentiontimes and
caUbration curves obtained in 7.2 and 7.3.
7.7 Software is now avaUable to calculate peak areas andretentiontimes on-Une and
compare against unknowns.
9.0 Procedure

93
 9.1 Tables 1 summarizestiierecommendedoperating conditions fortiieion
chromatograph. Included in this table are estimatedretentiontimestiiatcan be
achieved by this metiiod The actualretentiontimes must be determined witii
standards at the time of analysis.
9.2 Check system caUbration daily and, if required, recaUbrate.
9.3 Load and mject a fixed amount of weU ntixed sample. Flush mjection loop
thoroughly, using each new sample. Use tiie same size loop for standards and
samples. Recordtiieresultingpeak size in area or peak height units. An
automated constant volume injection system may also be used
9.4 The width oftiieretentiontime window used to make identifications should be
based upon measurements of actual retention time variations of standards over the
course of a day. Three times the standard deviation of aretentiontime can be
used to calculate a suggested window size for each analyte. However, the
experience of the analyst should weigh heavUy in the interpretation of
chromatograms.
9.5 If the response for the peak exceeds the working range of the system, dUute the
sample with an appropriate amount ofreagentwater and reanalyze.
9.6 If theresultingchromatogram fatis to produce adequate resolution, or if
identification of specific anions is questionable, fortify the sample with an
appropriate amount of standard and reanalyze.
Note: Retention time is inversely proportional to concentration. Nitrate and
sulfate exhibit the greatest amount of change, although aU artions are affected to
some degree. In some cases this peak migration may produce poorresolutionor
identification. Thus, it is important to select concentrations of standards that
approximate expected concentrations of unknown analytes.
9.7 The foUowing extraction should be used for soUd materials. Add an amount of
reagent water equal totentimes the weight of dry soUd material taken as a sample.
This slurry is mixed together fortenminutes using a magnetic stirring device.
FUter theresultingslurry before injecting using a 0.45 pm membrane type filter.
This can be the type that attaches directiy to the end of the syringe. Care should
be taken to show that good recovery and identification of peaks is obtained with
the users matrix through the use of spikes.

10.0 Calculation
10.1P*repare separate caUbration curves for each anion of interest by plotting peak size
in area, or peak height units of standards against concentration values. Compute
sample concentration by comparing sample peakresponsewith the standard
curve.
10.2 Reportresultsin mg/L.
10.3 Report
NOr as N, NO3- as N, and HPO4 2- as P
10.4 If it is desirable to express these as concentrations of NO3, NO2, and PO4, the
conversions are:
C=H*F*D
where:
C = mg anion/L
H = p ^ height or area
F =responsefactor = concentration of standard/height or area
D = dilution factor for tiiose samples requiring dUution

94
11.0 Accuracy - Metiiod Detection Lunit

11.1 The method detection limit (MDL) is defined as the mirtimum concentration of a
substance that can be measured andreportedwitii 99% confidencetiiattiievalue
is above zero. The MDL concentrations Usted in Table 1 were obtained using
reagent waters.
11.2 Single operator accuracy and precision forreagent,drinking and surface water,
and mixed domestic and industrial wastewater are listed in Table 2.
11.3 Multiple laboratory accuracy and precision data forreagent,drinking and waste
water are given for each anion in tables 3 through 9. Datafromnineteen
laboratories were used for this data.
11.4 Some of the bias statements, for example chloride and sulfate, may be
misleading due to spiking smaU increments of the anion into large naturaUy
occurring concentrations of the same artion.
11.5 In order to verify that standards have been prepared correctiy a reference
standard check should be performed using a standard of known concentration
prepared by an independent source.

95
Table 1. Chromatographic Conditions and Detection Limits In Reagent Water
Retention MDL
Analyte Peak # (*) Time (min) (mgA.)
Fluoride 1 1.2 0.01
Chloride 2 1.7 0.02
Nitrite-N 3 2.0 0.004
Bromide 4 2.9 0.01
Nitrate-N 5 3.2 0.002
o-PhosphateJ-P6 5.4 0.003
Sulfate 7 6.9 0.02
Standard ConditicMis:
Columns: as specified in 4.2.2
Detector, as specified in 4.2.4
Pump Rate: 2.0 mlVmin.
Eluent: as specified in 5.3
Sample Loop: 50 \xL

96
 Number Mean
Standard
A 1
Sample Spike of Recovery Deviation
Analyte Type (mg/L) RepUcates % (mg/L)
Bromide RW 5.0 7 99 0.08
DW 5.0 7 105 0.10
SW 5.0 7 95 0.13
WW 5.0 7 105 0.34
GW 5.0 7 92 0.34
SD 2.0 7 82 0.06
Chloride RW 20.0 7 96 0.35
DW 20.0 7 108 1.19
SW 10.0 7 86 0.33
WW 20.0 7 101 5.2
GW 20.0 7 114 1.3
SD 20.0 7 90 0.32
Fluoride RW 2.0 7 91 0.05
DW 1.0 7 92 0.06
SW 1.0 7 73 0.05
WW 1.0 7 87 0.07
GW 0.4 7 95 0.07
SD 5.0 7 101 0.35
Nitrate-N RW 10.0 7 103 0.21
DW 10.0 7 104 0.27
SW 10.0 7 93 0.17
WW 10.0 7 101 0.82
GW 10.0 7 97 0.47
SD 10.0 7 82 0.28
Nitrite-N RW 10.0 7 97 0.14
DW 10.0 7 121 0.25
SW 5.0 7 92 0.14
WW 5.0 7 91 0.50
GW 10.0 7 96 0.35
SD 2.0 7 98 0.08
o-Phosphate-P RW 10.0 7 99 0.17
DW 10.0 7 99 0.26
SW 10.0 7 98 0.22
WW 10.0 7 106 0.85
GW 10.0 7 95 0.33

Sulfate RW 20.0 7 99 0.40

DW 50.0 7 105 3.35
SW 40.0 7 95 1.7
WW 40.0 7 102 6.4
•
GW 40.0 7 112 3.2

97
RW = Reagent Water WW = Mixed Domestic and Industrial Wastewater DW = Drinking Water
GW = Groundwater SW = Surface Water SD = USEPA ac Solid (Shale)
Table 3. Determination of Bias for Fluoride
Water Am't Added Am't Found S(t) S(o) Bias
mg/L mgA. %
Reagent U.Zb 0.25 0.08 0.11 -3.8
0.34 0.29 0.11 -14.7
2.12 2.12 0.07 0.12 0.0
2.55 2.48 0.14 -2.7
6.79 6.76 0.20 0.19 -0.4
8.49 8.46 0.30 -0.4
Drinking 0.26 0.24 0.08 0.05 -57.0
0.34 0.34 0.11 0.0
2.12 2.09 0.18 0.06 -1.4
2.55 2.55 0.16 0.0
6.79 6.84 0.54 0.25 +0.7
8.49 8.37 0.75 -1.4
Waste 0.26 0.25 0.15 0.06 -3.8
0.34 0.32 0.08 -5.9
2.12 2.13 0.22 0.15 -K).5
2.55 2.48 0.16 -2.7
6.79 6.65 0.41 0.20 -2.1
8.49 8.27 0.36 -2.6

Table 4. Determination of Bias for Chloride

Water Am't Added Am't Found S(t) S(o) Bias
mg/L mg/L %
Reagent 0.78 0.79 0.17 0.29 +1.3
1.04 1.12 0.46 +57.0
6.50 6.31 0.27 0.14 -2.9
580.0 7.76 0.39 -0.5
20.8 20.7 0.54 0.62 -0.5
26.0 25.9 0.58 -0.4

Drinking 0.78 0.54 0.35 0.20 -30.8

1.04 0.51 0.38 -51.0
6.50 5.24 1.35 1.48 -19.4
580.0 6.02 1.90 -22.8
20.8 20.0 2.26 1.14 -3.8
26.0 24.0 2.65 -57.0

Waste 0.78 0.43 0.32 0.39 -44.9

1.04 0.65 0.48 -355.0
6.50 4.59 1.82 0.83 -29.4
580.0 5.45 2.02 -30.1
20.8 18.3 2.4 11.57 -11.8
26.0 23.0 2.50 -11.5

98
Table 5. Determination of Bias for Nitrite - Nitrogen
Water Am't Added Am't Found S(t) S(o) Bias
mg/L m ^ %
Reagent U.36 U.!57 0.04 0.04 +2.8
0.48 0.48 0.06 0.0
3.00 3.18 0.12 0.06 +6.0
3.60 3.83 0.12 +6.4
9.60 9.84 0.36 0.26 +2.5
12.0 12.1 0.27 +0.6
Drinking 0.36 0.30 0.13 0.03 -16.7
0.48 0.40 0.14 -16.7
3.00 3.02 0.23 0.12 40.7
3.60 3.62 0.22 +0.6
9.60 9.59 0.44 0.28 -0.1
12.0 11.6 0.59 -3.1
Waste 0.36 0.34 0.06 0.04 -5.6
0.48 0.46 0.07 -4.2
3.00 3.18 0.13 0.10 +6.0
3.60 3.76 0.18 +4.4
9.60 9.74 0.49 0.26 +1.5
12.0 12.0 0.56 +0.3

Table 6. Determination of Bias for Brontide

Water Am't Added Am't Found S(t) S(o) Bias
mg/L mg/L %
"Reagent 0.63 0.69 0.11 0.05 +9.5
0.84 0.85 0.12 +1.2
5.24 5.21 0.22 0.21 -0.6
6.29 6.17 0.35 -1.9
16.8 17.1 0.70 0.36 +1.6
21.0 21.3 0.93 +1.5

Drinking 0.63 0.63 0.13 0.04 0.0

0.84 0.81 0.13 -3.6
5.24 5.11 0.23 0.13 -2.5
6.29 6.18 0.30 -1.7
16.8 17.0 0.55 0.57 +0.9
21.0 20.9 0.65 -0.4

Waste 0.63 0.63 0.15 0.09 0.0

0.84 0.85 0.15 +1.2
5.24 5.23 0.36 0.11 -0.2
6.29 6.27 0.46 -0.3
16.8 16.6 0.69 0.43 -1.0
21.0 21.1 0.63 +0.3

99
Table 7 Determination of Bias for Nitrate - Nitrogen
Water Am't Added Am't Found S(t) S(o) Bias
mg/L mg/L %
Reagent U!42 U!4T um—0152—mr\
0.56 0.56 0.06 0.0
3.51 3.34 0.15 0.08 -4.8
4.21 4.05 0.28 -3.8
11.2 11.1 0.47 0.34 -1.1
14.0 14.4 0.61 +2.6
Drinking 0.42 0.46 0.08 0.03 +9.5
0.56 0.58 0.09 +3.6
3.51 3.45 0.27 0.10 -1.7
4.21 4.21 0.38 0.0
11.2 11.5 0.50 0.48 +2.3
14.0 14.2 0.70 +1.6
Waste 0.42 0.36 0.07 0.06 -14.6
0.56 0.40 0.16 -28.6
3.51 3.19 0.31 0.07 -9.1
4.21 3.84 0.28 -8.8
11.2 10.9 0.35 0.51 -3.0
14.0 14.1 0.74 +0.4

Table 8. Determination of Bias for Ortho-Phosphatefe

Water Am't Added Am't Found S(t) S(o) Bias

mg/L mg/L %
Reagent 0.69 0.69 0.06 0.06 0.0
0.92 0.98 0.15 +6.5
5.72 0.36 0.18 -0.9
6.92 6.78 0.42 -2.0
18.4 18.8 1.04 0.63 +2.1
23.1 23.2 0.35 +0.4
Drinking 0.69 0.70 0.17 0.17 +1.4
0.92 0.96 0.20 +4.3
5.77 5.43 0.52 0.40 -5.9
6.92 6.29 0.72 -9.1
18.4 18.0 0.68 0.59 -2.2
23.1 22.6 1.07 -2.0
Waste 0.68 0.64 0.26 0.09 -52.0
0.92 0.82 0.28 -10.9
5.77 5.18 0.66 0.34 -10.2
6.92 6.24 0.74 -9.8
18.4 17.6 2.08 1.27 -4.1
23.1 22.4 0.87 -3.0

100
 Table 9. Determination of Bias for Sulfate
Water Am't Added Am't Found S(t) S(o) Bias
mg/L mg/L %
Reagent 2.85 2.83 0.32 0.52 -0.7
3.80 3.83 0.92 +0.8
23.8 24.0 1.67 0.68 +0.8
28.5 28.5 1.56 -0.1
76.0 76.8 3.42 2.33 +1.1
95.0 95.7 3.59 +0.7

Drinking 2.85 1.12 0.37 0.41 -60.7

3.80 2.26 0.97 -40.3
23.8 21.8 1.26 0.51 -8.4
28.5 25.9 2.48 -9.1
76.0 74.5 4.63 2.70 -2.0
95.0 92.3 5.19 -2.8
Waste 2.85 1.89 0.24 -33.7
3.80 2.10 1.25 -44.7
23.8 20.3 3.19 0.58 -14.7
28.5 24.5 3.24 -14.0
76.0 71.4 5.65 3.39 -6.1
95.0 90.3 6.80 -5.0

References
United States Environmental Protection Agency. (1992). Methods for Chemical Analysis
of Water and Wastes. Method #300.0. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.
American Pubtic Health Association. (1992). Standard Metiiods for the Examination of
Water and Wastewater. 18th ed. Method 4010. American Public Health Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 1-1.

101
METHOD #: 405.1 Approved for NPDES (Editorial Revision 1974)
TITLE: Biochemical Oxygen Demand (5 Days, 20°C)
ANALYTE:
Biological Oxygen Demand, BOD

rS[STRUMENTATION: Probe
DSTTRODUCnON
The biochemical oxygen demand (BOD) determination is an empuical test in which
standardized laboratory procedures are used to determine the relative oxygen requirements
of waste-waters, effluents, and polluted waters. The test has its widest appUcation in
measuring waste loadings to treatment plants and in evaluating the BOD-removal efficiency
of such treatment systems. Thetestmeasures the oxygen utilized during a specified
incubation period for the biochemical degradation of orgaitic material (carbonaceous
demand) and the oxygen used to oxidize inorganic material such as sulfides and ferrous
iron. It also may measure the oxygen used to oxidize reduced forms of nitrogen
(nitrogenous demand) unless their oxidation is prevented by an inhibitor. The seeding and
dUution procedures provide an estimate of the BOD at pH 6.5 to 7.5.
Although ortiy the 5-d BOD (BOD5) is described here, many variations of oxygen demand
measurements exist. These include using shorter and longer incubation periods, tests to
determineratesof oxygen uptake, and continuous oxygen-uptake measurements by
respirometric techrtiques. Altemative seeding, dUution, and incubation conditions can be
chosen to mintic receiving-water conditions, thereby providing an estimate of the
environmental effects of wastewaters and effluents.

1.0 Scope and Application

1.1 The biochemical oxygen demand (BOD) test is used for determining the relative
oxygen requirements of municipal and industrial wastewaters. AppUcation of the
test to orgartic waste discharges allows calculation of the effect of the discharges
on the oxygen resources of the receiving water. Data from BOD tests are used for
the development of engineering criteria for the design of wastewater treaunent
plants.
1.2 The BOD test is an empirical bioassay-type procedure which measures tiie
dissolved oxygen consumed by microbial life while assimilating and oxidizuig the
organic matter present The standard test conditions include dark incubation at
20°C for a specified time period (often 5 days). The actual environmental
conditions oftemperature,biological population, water movement, sunUght, and
oxygen concentration cannot be accurately reproduced in the laboratory. Results
obtained must take into accounttiieabove factors when relatuig BOD results to
stream oxygen demands. Dissolved oxygen is measured initially and after
incubation, andtiieBOD is computedfiromtiiedifference between utitial and final
DO. Because tiie initial DO is determined immediately aftertiiedUution is made,
all oxygen uptake, includingtiiatoccurring during the first 15 min, is mcluded in
the BOD measurement
1.3 Carbonaceous Versus Nitrogenous BOD. Oxidation of reduced forms of
nitrogen, mediated by microorganisms, exerts nitrogenous demand. Nitrogenous
demand historicaUy has been considered an mterference intiiedetermination of

102
 BOD, as clearly evidenced bytiieinclusion of ammonia in the dUution water. The
interference from nitrogenous demand can now be prevented by an inhibitory
chemical. If an inhibituig chemical is not used, the oxygen demand measured is
the sum of carbonaceous and nitrogenous demands.

The extent of oxidation of nitrogenous compounds during the 5-d incubation

period depends on the presence of microorganisms enable of carrying out this
oxidation. Such organisms usuaUy are not present in raw sewage or primary
effluent in sufficient numbers to oxidize significant quantities of reduced nitrogen
forms in the 5-d BOD test Many biological treatment plant effluents contain
significant numbers of nitrifying organisms. Because oxidation of nitrogenous
compounds can occur in such samples, inhibition of nitrification is recommended
for samples of secondary effluent, for samples seeded with secondary effluent,
and for samples of poUuted waters.
Report results as CBOD5 when inhibiting the rtitrogenous oxygen demand. When
rtitrification is not inhibited, report results as BOD5.

2.0 Summary of Method

2.1 The sample of waste, or an appropriate dUution, is incubated for 5 days at 20°C in
the dark. The reduction in dissolved oxygen concentration during the incubation
period yields a measure of the biochemical oxygen demand.

3.0 Comments
3.1 Determination of dissolved oxygen in the BODtestmay be made by use of either
the Modified Winkler witii FuU-Bottie Technique ortiieProbe Metiiod in titis
manual.
3.2 Additional information relating to oxygen demanding characteristics of
wastewaters can be gained by applying the Total Orgartic Carbon and Chentical
Oxygen Demand tests (also found intitismanual).
3.3 Dtiution Requirements. The BOD concentration in most wastewaters exceeds the
concentration of dissolved oxygen (DO) available in an air-saturated sample.
Therefore, it is necessary to dilute tiie sample before incubation to bring the
oxygen demand and supply into appropriate balance. Because bacterial growth
requires nutrients such as nitrogen, phosphoms, and trace metals, these are added
to the dUution water, which is buffered to ensure that the pH of the incubated
sample remains in a range suitable for baaerial growtii. Complete stabtiization of
a sample may require a period of incubation too long for practical purposes;
therefore, 5 d has been accepted as tiie standard incubation period.
3.4 Samples for BOD analysis may degrade significantiy during storage between
coUection and analysis, resulting in low BOD values. Minimize reduction of BOD
by analyzing sample promptiy or by cooling it to near-freezingtemperatureduring
storage. However, even at low temperature, keep holding time to a minimum.
Warm chUled samples to 20°C before analysis.
3.4.1 Grab samples: If analysis is begun witiiin 2 h of coUection, cold storage
is unnecessary. If analysis is not started within 2 h of sample coUection.
keep sample at or below 4°Cfiromtiietimeof coUection. Begm analysis
wititin 6 h of coUection; whentiiisis not possible because tiie sampUng
site is distantfiromtiielaboratory, store at or below 4°C and report lengtii

103
 andtemperatureof storage with the results. In no case start analysis more
than 24 h after grab sample coUection. When samples are to be used for
regulatory purposes make every effort to deUver samples for analysis
within 6 h of coUection.
3.4.2 Composite samples: Keep samples at or below 4°C during compositing.
Lintit compositing period to 24 h. Use the same criteria as for storage of
grab samples, starting the measurement of holding time from end of
compositing period. State storagetimeand conditions as part of the
results.
4.0 Apparams

4.1 Incubation botties, 300-mL capacity. Clean botties with a detergent, rinse
thoroughly, and drain before use. As a precaution against drawing air into the
dUution bottie during incubation, use a water-seal. Obtain satisfactory water seals
by inverting botties in a water bath or by adding water to theflaredmouth of
special BOD botties. AU the ESL botties are equipped withflaredmouths for
water seals. Place a paper or plastic cup or foU cap overflaredmouth of tx)ttie to
reduce evaporation of the water seal during incubation.
4.2 Air incubator or water bath, thermostatically controUed at 20 ± 1°C. Exclude aU
tight to prevent possibiUty of photosynthetic production of DO.

5.0 Reagents
5.1 Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g
Na2HP04-7H20, and 1.7 g NH4CI ui about 500 mL distiUed water and dUute to 1
L. The pH should be 7.2 without further adjustment Discard reagent (or any of
the following reagents) if there is any sign of biological growth in the stock bottie.
Commercial buffers are also avaUable.
5.2 Magnesium sulfate solution: Dissolve 22.5 g MgS04-7H20 in distiUed water and
dUute to 1 L.
5.3 Calcium chloride solution: Dissolve 27.5 g CaCh in distiUed water and dUute to 1
L.
5.4 Ferric chloride solution: Dissolve 0.25 g FeCl3-6H20 in distiUed water and dUute
to 1 L.
5.5 Acid and aUcaU solutions, IN, for neutralization of caustic or acidic waste
samples.
5.5.1 Acid: Slowly and whUe stirring, add 28 mL concentrated sulfuric acid to
distiUed water. Dilute to 1 L.
5.5.2 AUcaU: Dissolve 40 g sodium hydroxide in distiUed water. DUute to 1 L.
5.6 Sodium sulfite solution: Dissolve 1.575 g Na2S03 in 1000 mL distiUed water.
This solution is not stable; prepare daily.
5.7 Nitrification inhibitor, 2-chloro-6-(trichloro metiiyl) pyridine.
5.8 Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade
glutamic acid at 103°C for 1 h. Add 150 mg glucose and 150 mg glutamic acid to
distiUed water and dtiute to 1 L. Prepare fresh immediately before use.
5.9 Ammonium chloride solution: Dissolve 1.15 g NH4CI in about 500 mL distiUed
water, adjust pH to 7.2 witii NaOH solution, and dtiute to 1 L. Solution contains
0.3 mg N/mL.

104
6.0 Procedure

6.1 Preparation of dUution water: Place desired volume of water ui a suitable bottie
and add 1 mL each of phosphate buffer, MgS04, CaCl2, and FeCls solutions/L of
water or add tiie contents of one buffer piUow. Before use bring dUution water
temperature to 20°C. Saturate witii DO by shaking ui a partiaUy fUled bottie or by
aeratmg with organic-fireefilteredau-. Altematively, store in cotton-plugged
botties long enough for water to become saturated witii DO. IntiieESL, oxygen
saturated dUution water is maintained in a large aerated vessel located at the end of
a lab bench. Protect water quaUty by using clean glassware, mbing, and tx)tties.
6.2 DUution water check: Usetitisprocedure as aroughcheck on quaUty of dUution
water.
If the oxygen depletion of a candidate water exceeds 0.2 mg/L, obtain a
satisfactory water by improving purification orfromanother source.
Altematively, if nitrification inhibition is used, store the dUution water, seeded as
prescribed below, in a darkened room at room temperature untU the oxygen uptake
is sufficientiy reduced to meettiiedUution-water check criteria. Check quality of
stored dUution water on use, but do not add seed to. dilution water stored for
quaUtv improvement. Storage is not recommended when BOD5 are to be
determined without rtitrification inhibition because rtitrifying organisms may
develop during storage. Check stored dilution water to determine whether
sufficient ammonia remains after storage. If not, add ammonium chloride solution
to provide a total of 0.45 mg ammonia/L as nitrogen. If dilution water has not
been stored for quaUty improvement, add sufficient seeding material to produce a
DO uptake of 0.05 to 0.1 mg/L in 5 d at 20°C. Uicubate a BOD bottie fuU of
dUution water for 5 d at 20°C. Determine initial and final DO as in sections 6.7
and 6.10. The DO uptake in 5 d at 20°C should not be more than 0.2 mg/L and
preferably not more than 0.1 mg/L.
6.3 Glucose-glutamic acid check: Because the BOD test is a bioassay its resitits can be
influenced greatiy by the presence of toxicants or by use of a poor seeding
material. DistiUed waters frequentiy are contaminated with copper, some sewage
seeds are relatively inactive. Low results always are obtained with such seeds and
waters. Periodically check dUution water quaUty, seed effectiveness, and
analytical technique by making BOD measurements on pure organic compounds
and samples with known additions. In general, for BOD determinations not
requiring an adapted seed, use a mixture of 150 mg glucose/L and 150 mg
glutamic acid/L as a "standard" check solution. Glucose has an exceptionaUy high
and variable oxidation rate but when it is used with glutantic acid, the oxidation
rate is stabtiized and is simUar to that obtained witii many municipal wastes.
Altematively, if a particular wastewater contains an identifiable major constituent
that contributes totiieBOD, use this compound ui place of tiie glucose-glutamic
acid. Determine the 5-d 20°C BOD of a 2% dUution of the glucose-glutamic acid
standard check solution using the techniques outlined in 6.4.
6.4 Seeding:
6.4.1 Seed source: It is necessary to have present a population of
nticroorgartisms capable of oxidizing the biodegradable orgartic matter in
the sample. Domestic wastewater, unchlorinated or otherwise-
undisuifected effluents from biological waste treatment plants, and surface
waters receiving wastewater discharges contain satisfactory nucrobial
populations. Some samples do not contain a sufficient nticrobial
population (for example, some untreated uidustrial wastes, disinfected

105
 wastes, high-temperature wastes, or wastes witii extreme pH values).
For such wastes seed tiie dUution water by adding a population of
microorganisms.
6.4.1.1 The preferred seed is effluentftioma biological treatment system
processuig tiie waste. Wheretitisis not avaUable, use
supematantfromdomestic wastewater after settiing at room
temperature for at least 1 h but no longertiian36 h. When
effluentfrorna biological treatment process is used, inhibition of
rtitrification is recommended.
6.4.1.2 Some samples may contain materials not degraded at normal rates
by tiie nncroorganisms ui settied domestic wastewater. Seed
such samples witii an adapted microbial population obtained firom
the undisinfected effluent of a biological process treating the
waste. Intiieabsence of such a faciUty, obtain seedfromthe
receiving water below (preferably 3 to 8 km)tiiepoint of
discharge. When such seed sources also are not avatiable,
develop an adapted seed in the laboratory by continuously
aerating a sample of settied domestic wastewater and adding
smaU daUy increments of waste.
6.4.1.3 Optionally use a soU suspension or activated sludge, or a
commercial seed preparation to obtain the initial microbial
population. Determine the existence of a satisfactory population
by testing the performance of the seed in BODtestson the
sample. BOD values that increase with time of adaptation to a
steady high value indicate successful seed adaptation.
6.4.2 Seed control: Determine BOD of the seeding material as for any other
sample. This is the seed control. From the value of the seed control and a
knowledge of the seeding material dilution (in the dUution water)
determine seed DO uptake. Ideally, make dUutions of seed such that the
largest quantity results in at least 50% DO depletion. A plot of DO
depletion, in niUUgrams pertiter,versus mUlUiters seed should present a
straighttinefor which the slope indicates DO depletion per milUUter of
seed. The DO-axis intercept is oxygen depletion caused bytiiedUution
water and should be less than 0.1 mg/L. To determine a sample DO
uptake subtract seed DO uptakefix)mtotal DO uptake. The IX) uptake of
seeded dUution water should be between 0.6 and 1.0 mg/L.
6.5 Sample pretreatment:
6.5.1 Samples containing caustic alkalinity or acidity: NeutraUze samples to pH
6.5 to 7.5 with a solution of sulfuric acid (H2SO4) or sodium hydroxide
(NaOH) of such strength that the quantity of reagent does not dilute the
sample by more than 0.5%. The pH of seeded dilution water should not
be affected by the lowest sample dUution.
6.5.2 Samples containing residual chlorine compounds: If possible, avoid
samples containing residual chlorine by sampling ahead of chlorination
processes. Chlorine is a disinfectant and is toxic, at some level, to
microorgartisms. If the sample has been chlorinated but no detectable
chlorine residual is present, seed the dtiution water. If residual chlorine is
present, dechlorinate sample and seed the dUution water (^ 6.6). Do not
test chlorinated/ dechlorinated samples without seeding the dUution water.
In some samples chlorine wiU dissipate within 1 to 2 h of standing in the
tight This often occurs during sample transport and handling. For

106
 samples in which chlorine residual does not dissipate in a reasonably short
tune, destroy chlorine residual by adding Na2S03 solution. Determine
reqmred volume of Na2S03 solution on a 100- to 1000-mL portion of
neutraUzed sample by adding 10 mL of 1 + 1 acetic acid or 1 + 50 H2SO4,
10 tnL potassium iodide (KI) solution (10 g/100 mL) per 1000 mL
portion, andtitratingwith Na2S03 solution to the starch-iodine end point
for residual. Add to neutralized sample the relative volume of Na2S03
solution determined by tiie above test, mix, and after 10 to 20 min check
sample for residual chlorine. (NOTE: Excess Na2S03 exerts an oxygen
demand and reacts slowly with certain orgartic chloramine compounds that
may be present in chlorinated samples.)
6.5.3 Samples containing other toxic substances: Certain industrial wastes, for
example, plating wastes, contain toxic metals. Such samples often requUe
special study and treatment.
6.5.4 Samples supersaturated witii DO—Samples containing moretiian9 mg
DO/ L at 20°C may be encountered in cold waters or in water where
photosynthesis occurs. To prevent loss of oxygen during incubation of
such samples, reduce DO to saturation at 20°C by bringing sample to
about 20°C in partiaUyfiUedbottie whUe agitating by vigorous shaking or
by aerating with clean,filteredcompressed air.
6.5.5 Sampletemperatureadjustment: Bring samples to 20 ±1°C before making
dUutions.
6.5.6 Nitrification inhibition: If nitrification inhibition is desired add 3 mg 2-
chloro-6-(trichloro methyl) pyridine (TCMP) to each 300-mL bottie before
capping or add sufficient amounts to the dilution water to make a final
concentration of 10 mg/L. (NOTE: Pure TCMP may dissolve slowly and
canfloaton top of the sample. Some commercial formulations dissolve
more readily but are not 1(X)% TCMP; adjust dosage accordingly.)
Samples that may require rtitrification inhibition include, but are not
limited to, biologicaUy treated effluents, samples seeded with biologicaUy
treated effluents, andriverwaters. Note the use of nitrogen inhibition in
reporting results.
6.6 DUution technique: DUutionstiiatresult in a residual DO of at least 1 mg/L and a
DO uptake of at least 2 mg/L after 5 d incubation produce the most reUable resuUs.
Make several dilutions (usually 3-5) of prepared sample to obtain DO uptake in
this range. Experience with a particular sample wtil permit use of a smaller
number of dUutions. A morerapidanalysis, such as COD, may be correlated
approximately witii BOD and serve as a guide in selecting dtiutions. In tiie
absence of prior knowledge, use the foUowing dilutions: 0.0 to 1.0% for strong
industrial wastes, 1 to 5% forrawand settied wastewater, 5 to 25% for
biologically treated effluent, and 25 to 100% for pollutedriverwaters.

7.0 Procedure
7.1 The ESL normally makestiireerepUcate sets of eachtiireedilutions and blank.
CE 5374, CE 3171, and ENVE 3203 wiU not do repUcates. The amount of waste
added to each dUution is estimated as foUows:
7.1.1A COD test is run ontfiesample. The amount of waste is calculated by
ml of sample added to BOD botties = 5.0 mg/L (desired DO level) * 300 ml in BOD bottie / COD value
This value gives you the ntiddle value to be used. The otiier dtiutions are half and

107
 double this in addition to the blank.
7.2 Dtiutions prepared du^tiy ui BOD botties—Usuig a wide-tip volumetric pipet,
addtiiedesu^ sample volume (7.1.1) to individual BOD botties of known
capacity. Add appropriate amounts of seed material (0.6 ml) and buffer
solution (contents of one buffer pillow) to tiie individual BOD botties and to
the blank. FiU botties with enough dUution water, seeded U* necessary, so tiiat
insertion of stopper wiU displace aU air leaving no bubbles.! For dtiutions greater
tiian 1:100 make a primary dtiution in a graduated cyUnderbefore making final
dUution in the bottie. Stoppertightiyand water-seal. Rinse DO electrode between
determinations to prevent cross-contamination of samples.
7.3 Determination of irutial DO: If the sample contains materials that reactrapidlywitii
DO, determine utitial DO unmediately afterfiUingBOD bottie witii dUuted sample.
Ifrapidirtitial DO uptake is insigrtificant, thetimeperiod between preparing
dUution and measuring initial DO is not critical. Determine Dissolved Oxygen in
accordance with the DO metiiod provided in this manual.
7.4 Dtiution water blartic: Use a dUution water blank as aroughcheck on quaUty of
unseeded dilution water and cleartiiness of incubation botties. Together with each
batch of samples incubate a bottie of unseeded dUution water. Determine irtitial
and final DO as in sections 7.2 and 7.5. The DO uptake should no more than 0.2
mg/L and preferably not more than 0.1 mg/L.
7.5 Incubation: Incubate at 20°C± 1°C BOD botties contairting desired dUutions, seed
controls, dUution water blanks, and glucose-glutanuc acid checks. Water-seal
botties as described in section 6.6.1.
7.6 Determination of final DO: After 5 d incubation determine DO in sample dtiutions,
blanks, and checks as in section 7.2.
8.0 Calculations
8.1 When dUution water is not seeded:
BOD5, mg/L = (Di - D2)/P

8.2 When dUution water is seeded:

BOD5, mg/L = ((Di - D2) - (Bi - B2)f)/P

Where:
Di = DO of dUuted sample immediately after preparation, mg/L,
D2 = DO of diluted sample after 5 d incubation at 20°C, mg/L,
P = decimal volumetric fraction of sample used (ml of waste in BOD
bottie / ml in BOD bottie (300))
Bi = DO of seed control before incubation, mg/L,
B2 = DO of seed control after incubation mg/L, and
f = ratio of seed in dUuted sample to seed in seed control = (% seed
in diluted sample)/(% seed in seed control). In our case, this is
1.
If seed material is added directiy to sample or to seed control botties:
f = (volume of seed ui dUuted sample)/(volume of seed in seed control)
8.3 Report results as CBOD5 if nitrification is inhibited.

108
 8.4 If more than one sample dUution meetstiiecriteria of a residual DO of at least 1
nag/L and a DO depletion of at least 2 mg/L and there is no evidence of toxicity at
higher sample concentrations or the existence of an obvious anomaly, average
results in the acceptable range.
8.5 In these calculations, do not make corrections for DO uptake by the dUution water
blank during incubation. This correction is unnecessary if dUution water meets
the blartic criteria stipulated above. Iftiiedilution water does not meet tiiese
criteria, proper corrections are difficult and results become questionable.
9.0 Accuracy

9.1 Eighty-six analysts in fifty-eight laboratories analyzed natural water samples plus
an exact increment of biodegradable organic compounds. At a mean value of 2.1
and 175 mg/L BOD, tiie standard deviation was ± 0.7 and ± 26 mg/L,
respectively (EPA Method Research Study 3).
9.2 There is no acceptable procedure for determining the accuracy of the BOD test.
References
United States Environmental Protection Agency. (1992). Methods for Chemical Analvsis
of Water and Wastes. Method #405.1. Environmental Monitoring and Support
Laboratory, Urtited States Environmental Protection Agency, Cincinnati, Ohio.
American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed. Method 5210. American PubUc Healtii Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 5-1.

109
METHOD #: 212.3 Approved for NPDES (Issued 1974)

TITLE: Boron (Colorimetric, Carmine)

ANALYTE:
Boron, B

INSTRUMENTATION: HACH 2000/DR

INTRODUCTION:
Although it is an element essential for plant growth, boron in excess of 2.0 mg/L in
irrigation water is deleterious to certain plants. Some plants may also be adversely affected
by concentrations as low as 1.0 mg/L (or even less in commercial green houses). Drinking
waters rarely contain more than 1 mg B/L and generally less than 0.1 mg/L, concentrations
considered innocuous for human consumption. Boron may occur naturaUy in some waters
or may find its way into a water course through cleaning compounds and industrial waste
effluents. Seawater contains approximately 5 mg B/L, and this element is found in saUne
esmaries in association with other seawater salts. The ingestion of large amount of boron
can affect the central nervous system. Protracted ingestion may result in a clinical
syndrome known as borism.
1.0 Scope and Application
1.1 This carmine method optimum range on undiluted or unconcentrated samples is
0.5 to 14 mg/L of boron.
1.3 This method is appUcable to drinking, and surface waters, domestic and industrial
wastes.
2.0 Summary of Method
2.1 Boron is determined by its reaction with carminic acid in the presence of sulfuric
acid to produce a reddish to bluish color. The amount of color is directiy
proportional to the boron concentration.

3.0 Interferences
3.1 The ions commonly found in water and wastewater do not interfere.

4.0 Apparams and Materials

4.1 Hach 2000/DR meter
4.2 Boro Ver 3 Boron Reagent Powder Ptilows
4.3 Sulfuric Acid
4.4 Water, deionized
5.0 Procedure
5.1 OntiieHACH 2000/DR meter, enter metiiod 40 and dial tiie wavelengtii to 605
nm. Press Read/Enter.
5.2 Measure 75 ml of Sulfuric acid using a 100 ml graduated cyUnder mto a 300 ml
erlenmeyer flask.

110
 5.3 Add tiie contents of one Boro Ver 3 Reagent powder piUow to tiie flask. Swirl to
mix.
5.4 Accurately pipet 2.0 ml of deionized water into a 125 erlenmeyerflask(the
blartic).
5.5 Accurately pipet 2.0 ml of sample uito anotiier 125 ml erienmeyer flask.
5.6 Add 35 ml of the Boro Ver 3/sulfuric acid reagent solution to each erlenmeyer
flask. Swirl to mix completely.
5.7 Press Shift Timer. A 25 minute reaction period wiU begin. When the timer beeps
the display wiU show mg/L B.
5.8 Pour 25 nti of eachflaskinto sample ceUs. Place tiie blank intotiieceU holder and
press Zero.
5.9 Place the prepared sample into the ceU holder and press Read/Enter.
6.0 Accuracy

6.1 In a single laboratory, using a standard solution of 10 mg/L boron and one
representative of reagent with DR/2000, a single operator obtained a standard
deviation of ± 0.20 mg/L boron.
References

American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed. Method 4500-b. American PubUc Health
Association, American Water Works Association, and Water Environment Federation,
Washmgton DC, 4-18.
Hach Company. (1992). DR/2000 Spectrophotometer Handbook. Metiiod 8015. Hach
Company, Loveland, Co.
United States Environmental Protection Agency. (1992). Metiiods for Chemical Analysis
of Water and Wastes. Method #212.3. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

Ill
METHOD #415.2 Gssued December 1982)
TITLE: Organic Carbon, Total (Combustion or Oxidation)
ANALYTE:
Total Organic Carbon, TOC
DSfSTRUMENTATION: Carbon Analyzer
INTRODUCTION
The organic carbon in water and wastewater is composed of a variety of organic
compounds in various oxidation states. Some of these carbon compounds can be oxidized
further by biological or chentical processes, and the biochemical oxygen demand (BOD)
and chentical oxygen demand (COD) may be used to characterizetiiesefractions.The
presence of organic carbontiiatdoes not respond to eithertiieBOD or CODtestmakes
them unsuitable fortiiemeasurement of total orgartic carbon. Total orgartic carbon (TOC)
is a more convenient and direct expression of total orgartic content than either BOD or
COD, but does not provide the same kind of information. If a repeatable empirical
relationship is estabUshed between TOC and BOD or COD,tiienTOC can be used to
estimate tiie accompanying BOD or COD. This relationship must be established
independentiy for each set of matrix conditions, such as various points in the n-eatment
process. Unlike BOD or COD, TOC is independent of the oxidation state of the orgartic
matter and does not measure other organicaUy bound elements, such as rtitrogen and
hydrogen, and inorganics that can contribute to the oxygen demand measured by BOD and
COD. TOC measurement does not replace BOD and COD testing.
There are sixfractionsof Total Carbon. The methods and instruments used in measuring
TOC analyze fractions of total carbon (TC) and measure TOC by two or more
determinations. These fractions of total carbon are defined as: inorganic carbon (IC)—the
carbonate, bicarbonate, and dissolved CO2; total organic carbon (TOC)—aU carbon atoms
covalentiy bonded in organic molecules; dissolved orgartic carbon (DOC)—the fraction of
TOC that passes through a 0.45-pm-pore-diam fUter, particulate organic carbon
(POC)—also referred to as nondissolved organic carbo; thefractionof TOC retained by a
0. 5 pm fUter: volatile organic carbon (VCX!)—also referred to as purgeable orgartic
carbon, thefractionof TOC removedfroman aqueous solution by gas stripping under
specified conditions; and nonpurgeable orgartic carbon (NPOC)—thefractionof TOC not
removed by gas stripping.

To detemune the quantity of organically bound carbon, the organic molecules must be
broken down to single carbon units and converted to a single molecular form that wiU be
measured quantitatively. TOC metiiods utUize heat and oxygen, utoviolet irradiation,
chentical oxidants, or combinations of these oxidants to convert orgartic carbon to carbon
dioxide (CO2). The CO2 may be measured directiy by a nondispersive infrared analyzer, it
may be reduced to metiiane and measured witii aflameionization detector, or CO2. may be
titrated chemicaUy.
In most water samples, the IC fraction is many times greater thantiieTOC fraction.
EUminating or con^nsating for IC interferences requires multiple determinations to
measure tme TOC. IC mterference can be eUminated by acidifying samples to pH 2 or less
to convert IC species to CO2, or IC interference may be compensated for by separately
measuring total carbon (TC) and inorganic carbon. By using persulfate-uluaviolet

112
oxidation TC and IC are measured separately andtiieinterferences nonnal caused by IC are
eUmmated. The difference between tiie TC and IC is TOC.
1.0 Scope and AppUcation

1.1 This method mcludes tiie measurement of organic carbon ui drinking, surface and
saline waters, domestic and uidustrial wastes. Exclusions are noted under
Defirtitions and Interferences.
1.2 The metiiod is most appUcable to measurement of organic carbon above 1 mg/L.
2.0 Summary of Method

2.1 Organic carbon in a sample is converted to carbon dioxide (CO2) by catalytic

combustion or wet chentical oxidation. The CO2 formed can be measured directiy
by an infrared detector or converted to methane (CH4) and measured by a flame
ionization detector. The amount of CO2 or CH4 is dti-ectiy proportional to tiie
concentration of carbonaceous material in the sample.
3.0 Defirtitions

3.1 The carbonaceous analyzer measures aU of the carbon in a sample. Because of

various properties of carbon-containing compounds in liquid samples, preliminary
treatment of the sample prior to analysis dictates the definition of tiie carbon as it
is measured. Forms of carbon that are measured by the method are:
A) soluble, nonvolatUe orgartic carbon; for instance, natural sugars
B) soluble, volatile organic carbon; for instance, mercaptans.
C) insoluble, partiaUy volatUe carbon; for instance, oUs.
D) insoluble, particulate carbonaceous materials, for instance;
cellulose fibers.
E) soluble or insoluble carbonaceous materials adsorbed or entrapped on
insoluble inorgartic suspended matter, for instance, oily matter
adsorbed on sUt particles.
3.2 The final usefuhiess of the carbon measurement is in assessing the potential
oxygen demanding load of organic material on a receiving stream. This statement
applies whether the carbon measurement is made on a sewage plant effluent,
industrial waste, or on water taken directiyfromthe stream. Iii this light,
carbonate and bicarbonate carbon are not a part of the oxygen demand in the
stream and therefore should be discounted in the final calculation or removed prior
to analysis. The manner of preliminary treatment of the sample and instmment
settings defines the types of carbon which are measured. Instmment
manufacturer's instmctions should be foUowed.

4.0 Sample Handling and Preservation

4.1 Sampling and storage of samples in glass botties is preferable. Sampling and
storage in plastic botties such as conventional polyethylene and containers is
permissible if it is estabUshedtiiatthe containers do not contribute contaminating
organics to the samples.
NOTE 1: A brief study performed intiieEPA Laboratory indicatedtiiatdistiUed
water stored in new one quart containers did not show any increase in organic
carbon after two weeks exposure.

113
 4.2 Because of the possibUity of oxidation or bacterial decomposition of some
^n?^!lf''!? f^'^^u' "T?^^'' *^ ^^P^ ^^ ^ ^ ^tween coUection of samples
^^l^n^ ^ ^ ^ ' ' ' '^'''."^ ^ ^^P^ ^° ^ mimmum Also, samples should be kept
cool (4 C) and protectedfiiomsunUght and atmospheric oxygen
4.3 In mstances where analysis cannot be perfonned wititin two hours (2 hours) fix)m
tune of sampUng, tiie sample is acidified (pH < 2) witii HCl or H2SO4
5.0 Interferences

5.1 Carboriate and bicarix)nate carbon represent an interference undertiietenns of titis

<o ^- ™"^^ ^ removed or accounted for uitiiefinalcalculation.
5.2 This procedure is applicable only to homogeneous samples which can be mjected
mto tiie apparams reproducible by means of a microUter type syringe or pipette.
The opemngs of tiie syringe or pipette Umittiiemaxunum size of particles which
may be mcluded intiiesample.
6.0 Apparams

6.1 Apparams for blending or homogenizing samples: GeneraUy, a Waring-type

blender is satisfactory.
6.2 Total Organic Carbon Analyzer. The ESL usestfieShimadzu brand analyzer.
7.0 Reagents

7.1 DistiUed water used in preparation of standards and for dUution of samples should
be ultra pure to reduce the carbon concentration of tiie blank. Carbon dioxide-
free, double disttiled water is recommended. Ion exchanged waters are not
recommended because oftiiepossibiUties of contamination witii organic materials
from the resins.
7.2 Potassium hydrogen phtiialate, stock solution, 1000 mg carbon/liter: Dissolve
0.2128 g of potassium hydrogen phtiialate (Primary Standard Grade) ui distiUed
water and dilute to 1(X).0 mL.
NOTE 2: Sodium oxalate and acetic acid are not recommended as stock solutions.
7.3 Potassium hydrogen phthalate, standard solutions: Prepare standard solutions
from the stock solution by dtiution witii distiUed water.
7.4 Carbonate-bicarbonate, stock solution, 1000 mg carbon/Uter: Weigh 0.3500 g of
sodium bicarbonate and 0.4418 g of sodium carbonate and transfer botii to the
same 100 mL volumetric flask. Dissolve with distiUed water.
7.5 Carbonate-bicarbonate, standard solution: Prepare a series of standards sintilar to
step 7.3.
NOTE 3: This standard is not required by some instmments.
7.6 Blank solution: Use the same distiUed water (or simUar quaUty water) used for
the preparation of the standard solutions.
8.0 Procedure

8.1 AUow at least 30 minutes warm-up time. Leave instmment console on

continuously when in daUy use, except for the ultraviolet light source, which
should be turned off when not in use for more than a few hours.
8.2 Analyze the standards 7.3 in order to check the linearity of the instrument at least
once each day. |From the main menu, press 2. Sample measurement. On this

114
 screen, you are aUowed the input of any information conceming the sample: its
number, dUution, etc. This page is skipped. From this screen, press Fl to
go to the sampUng instmctions. FoUow the instmctions on the screen: Set the
sample container on tiie shelf, wipe the inlet mbe witii a Kim wipe, and uisert it
mto tiie sarnpling mbe. Press START. The fu^t result given wiU be total carbon.
It wiU continue to do repUcates iintti a satisfactory relative standard deviation is
determined. The second result wiU be inorgartic carbon. The analyzer wiU
automaticaUy take the difference of the two and produce the total organic carbon
present Once the cycle is complete (tiie screen wiU blink COMPLETE), press
Fl to go back to the sample measurement screen.
8.3 Analyze the samples and system blank. Follow the same instmctions given 8.2.
Note: If the sample contains suspended matter, fUtration is necessary to avoid
clogging of the machine.

9.0 Calculations

9.1 The values are read off the final digital readout in pg/L. The system blank reading
obtained in 8.3 must be subtiacted from aU reagent distUled water, standard and
sample readings.

10.0 Accuracy
10.1 Twenty-eight analysts in twenty-one laboratories analyzed distiUed water
solutions contairting exact increments of oxidizable orgartic compounds, with the
foUowing results:

Increment as Precision as Accuracy as

TOC Standard Deviation Bias, Bias,
mg/Uter TOC,mg/liter mg/liter %
4.9 3.93 + 15.27 +0.75
107 832 + 1.01 +1.08
(FWPCA Metiiod Smdy 3, Demand Analyses)
References
United States EnvUonmental Protection Agency. (1992). Metfiods for Chemical Analvsis
of Water and Wastes. Metiiod #415.2. Envux)nmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

American PubUc Healtii Association. (1992). Standard Metiiods for tiie Examination of
Water and Wastewater. 18tiied. Metiiod 5310. American Public Healtii Assoaation,
American Water Works Association, and Water Environment Federation, Washington
DC, 5-10.

115
METHOD #: 410.4 Penduig Approval for Sect 304(h), CWA (Issued 1978)

TITLE: Chentical Oxygen Demand (Colorimetric, Automated; Manual)

ANALYTE:
Chemical Oxygen Demand, COD
INSTRUMENTATION: Spectrophotometer, Autoanalyzer
INTRODUCTION
Oxygen demand is an important parameter for determining the effect of orgartic poUutants
on receiving waters. As microorganisms in the environment consume these materials,
oxygen is depleted from the water. This can have an adverse effect on fish and plant life.
There are three main methods of measuring oxygen demand: directiy by Biochemical
Oxygen Demand (BOD) and/or Chemical Oxygen Demand (COD) and indirectiy by Total
Orgartic Carbon (TOC). BOD, because it uses nticroorgartisms for oxidation, gives the
closest picture of the processes happening in natural waters. Thetestloses some of its
value, though, because of the length of time—five days—it takes to get results. The BOD
test also is inadequate as an indicator of orgartic poUution when used with industrial
wastewater contairting heavy metal ions and cyanides. Microorgartisms become poisoned
by toxic substances and are unable to oxidize wastes.

Unlike BOD, the two other methods (TOC and COD) do not employ biological processes.
TOC utilizes a strong oxidizing agent under controUed conditions to measure the total
amount of organic material in a sample. Thistestassumes that aU orgartic matter in the
sample wtil decompose, and that aU the decomposition wiU consume O2 and produce CO2
(which is the parameter being measured). Results may not be as accurate as COD or BOD
in predicting environmental oxygen demand because oxygen demands may differ between
compounds with the same numter of organic carbons intiieirstmctures. What usuaUy is
needed is the amount of oxygen required to completely oxidize the carbon and hydrogen
present in the orgaitic matter. The difference in oxygen demand between two compounds
which have the same amount of organic carbon can be seen in the foUowing equations
which show the oxidation of oxaUc acid and ethanol:

oxalic acid C2H2O4 + 1/2 O2 2CO2 + H2O

etiianol C2H6O + 3 O2 2CO2 + 3H2O

Each molecule of ethanol uses up six times as much oxygen as an equivalent amount of
oxaUc acid and thus would have a much greater effect ontiiedissolved oxygen present in a
receiving water.
The chemical oxygen demand (COD) is used as a measure of the oxygen equivalent of tiie
organic matter content of a sample that is susceptible to oxidation by a strong chemical
oxidant The dichromate reflux metiiod is preferred over procedures using otiier oxidants
because of superior oxidizing abUity, applicabiUty to a wide variety of samples, and ease of
manipulation. Oxidation of most organic compounds is 95 to 100% oftiietiieoretical
value.

116
1.0 Scope and AppUcation

1.1 This metiiod covers the detennination of COD in surface waters, domestic and
industrial wastes.
1.2 The appUcable range oftiieAutoAnalyzer metiiod is 3-900 mg/L, for tiie
spectrophotometer range, 20-900 mgA.; and HACH metiiod 0-15,000 mg/L.
2.0 Summary of Method

2.1 There are three metiiods to detemtine COD uitiieESL, aU usmg essentiaUy tiie
same metiiod; Auto Analyzer, Spectrophotometer, andtiieHACH DR/2000
Spectrophotometer. The HACH procedure is the preferred method because it
deUvers the quickest results.
2.2 Sample, blanks and standards in sealed mbes are heated in an oven or block
digester in the presence of dichromate at 150°C. After two hours, the mbes are
removedfromthe oven or digester, cooled and measured spectrophotometricaUy
at 6(X) nm or 620 nm on the HACH spectrophotometer.
3.0 Sample Handling and Preservation
3.1 CoUect the samples in glass botties if possible. Use of plastic containers is
permissible if it is known that no orgartic contaminants wiU not be contributed by
the containers.
3.2 Samples should be analyzed as soon as possible, or if storage is required they
must be preserved with sulfuric acid to a pH < 2 and maintained at 4°C untU
analysis. Analysis should be within 28 days.
3.3 If there is large amounts of settieable soUds, samples should be homogertized in a
blender before an aliquot is taken.

4.0 Interferences
4.1 Chlorides are quantitatively oxidized by dichromate and represent a positive
interference. Mercuric sulfate is added to the digestion mbes to complex the
chlorides.
4.2 Volattie straight-chain aUphatic compounds are not oxidized to any appreciable
extent. This faUure occurs partly because volattie organics are present in the
vapor space and do not come in contact with the oxidizing Uquid. Straight-chain
aUphatic compounds are oxidized more effectively when stiver sulfate (Ag2S04)
is added as a catalyst. However, Ag2S04 reacts witii chloride, bromide, and
iodide to prxxiuce precipitatestiiatare oxidized only partially. The difficulties
caused by the presence oftfiehaUdes can be overcome largely, though not
completely, by complexing with mercuric sulfate (HgS04) before the refluxing
procedure. Although 1 g HgS04 is specified for 50 mL sample, a lesser amount
may be used where sample chloride concentration is known to be less than 2000
mg/L, as long as a 10:1ratioof HgS04:Cl- is maintained. Do not use tiie test for
samples containing moretfian2000 mg C1-/L. Techniques designed to measure
COD in saline waters are avaUable.
4.3 Nitrite (NO2-) exerts a COD of 1.1 mg (h/mg NO2 -N. Because concentrations
of NO2 in waters rarely exceed 1 or 2 mg NO2 -N/L, tiie interference is
considered insignificant and usuaUy is ignored. To elimmate a significant

117
 mterference due to NO2-, add 10 mg sulfamic acid for each mg NO2-N present in
tiie sample volume used; addtfiesame amount of sulfamic acid totiiereflux vessel
containing the distiUed water blank.
4.4 Reduced uiorgartic species such as ferrous uion, sulfide, manganous manganese,
etc., are oxidized quantitatively under the test conditions. For samples contaimng
significant levels of these species, stoichiometric oxidation can be assumed from
known initial concentration of the uiterfering species and corrections can be made
to the COD value obtained.
5.0 Apparams
5.1 Drying oven or|block digester, 150"^
5.2 Coming culmre mbes, 16 x 100 mm or 25 x 150 mm with Teflon Uned screw cap
5.3 Spectrophotometer, Technicon AutoAnalyzer, or HACH DR/2000 Direct
Reading Spectrophotometer.
6.0 Reagents
6.1 Digestion solution: Add 10.2 g K2Cr207,167 mL concentrated. H2SO4 and 33.3
g HgS04 to 500 mL of distiUed water, cool and dUute to 1 titer.
6.2 Catalyst solution: Add 22 g Ag2S04 to a 4.09 kg bottie of concentrated. H2SO4.
Stir untU dissolved.
6.3 Sampler wash solution: Add 500 mL of concentrated H2SO4 to 500 mL of
distilled water.
6.4 Stock potassium acid phthalate: Dissolve 0.850 g in 800 mL of distiUed water and
dtiute to 1titer.1 mL = 1 mg COD
6.4.1 Prepare a series of standard solutions that cover the expected sample
concentrations by diluting appropriate volumes of the stock standard.
6.5 There are commerciaUy avaUable COD tubes that already have the digestion and
catalyst solutions prepared.
7.0 Procedure
7.1 Wash all culture mbes and screw caps with 20% H2SO4 before their first use to
prevent contamination. Trace contamination may be removed from the mbes by
igniting them in a muffle oven at 5(X)°C for 1 hour.
7.2 Technicon Auto Analyzer
7.2.1 Add 2.5 mL of sample to the 16 x 100 mm mbes.
7.2.2 Add 1. 5 mL of digestion solution (6.1) and ntix.
7.2.3 Add 3.5 mL of catalyst solution (6.2) carefully down the side of the culture
mbe.
7.2.4 Captightiyand shake to mix layers.
7.2.5 Process standards and blanks exactiy as the samples.
7.2.6 Place ui oven or block digester at 150°C for two hours.
7.2.7 Cool, and place standards in sampler in order of decreasing concentration.
Complete fiUing sampler tray with unknown samples.
7.2.8 Measure color intensity on AutoAnalyzer at 6(X) nm.
7.3 Spectrophotometer
7.3.1 The following procedure may be used if a larger sample is destied or a
spectrophotometer is used in place of an AutoAnalyzer.

118
 7.3.2 Add 10 mL of sample to 25 x 150 mm culture mbe.
7.3.3 Add 6 mL of digestion solution (6.1) and mix.
7.3.4 Add 14 mL of catalyst solution (6.2) down tiie side of culmre mbe.
7.3.5 Captightiyand shake to mix layers.
7.3.6 Place in oven or block digester at 150°C for 2 hours.
7.3.7 Cool, aUow any precipitate to settie and measure intensity in
spectrophotometer at 600 nm. Use only optically matehed culmre mbes or
a single ceU for spectrophotometric measurement.
7.4 HACH DR/2000
7.4.1 Turn Spectrophotometer
on COD '
reactor to preheat to 150°C.
7.4.2 Use Program # 434,435, or 436 COD based ontiierange of values
expected. The ESL uses 435 HR.
7.4.3 Choose which range of commerciaUy prepared vials needed based on
expected COD values expected.
7.4.3.1 0-150 m ^ , low range
7.4.3.2 0-1500 mg/L, mid range
7.4.3.3 0-15,000 mg/L, high range
7.4.4 Place two milUUters of sample into vial, cap tightiy. Place two milliUters of
reagent water into a vial to be your quality blank.
NOTE: Use only exact volumes. A larger sample wiU dtiute the acid
concentration and lower the boiling point This wiU increase pressure and
could shatter the vial or cap.
7.4.5 Place blank and samples in digester at 150°C. Leave the samples in the
digester for two minutes. If after two minutes, the solution is green, the
sample is too high and dUution is required.
7.4.6 After two minutes, heat at 150°C for two hours.
7.4.6 Remove from the digester and aUow mbes to cool 10 minutes.
7.4.7 Read vials on HACH DR/2(XX) spectrophotometer. Picture instmctions are
provided.
8.0 Calculation
8.1 Prepare a standard curve by plotting peak height or percent transntittance against
known concend-ations of standards.
8.2 Compute concentration of samples by comparing sample response to standard
curve.
8.3 If using HACH DR/2000 values are obtained duectiy.
9.0 Accuracy
9.1 Forty-eight synthetic samples containing potassium hydrogen phthalate and NaQ
were tested byfivelaboratories. At an average COD of 193 mg O2/L in the
absence of chloride, the standard deviation was ± 17 mg O2/L (coefficient of
variation 8.7%). At an average COD of 212 mg O2/L and 100 mg C1-/L, tiie
standard deviation was ± 20 mg O2/L (coefficient of variation, 9.6%)

119
References

United States Environmental Protection Agency. (1992). Metiiods for Chemical Analvsis
of Water and Wastes. Method #410.4. Environmental Monitoring and Support
Laboratory, Urtited States Environmental Protection Agency, Cincinnati, Ohio.
Hach Company. (1992). DR/2Q0Q Spectrophotometer Handbook. Metiiod 8000. Hach
Company, Loveland, Co.
American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18tiied. Metiiod 5220. American PubUc Healtii Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 5-6.

120
METHOD #: 330.1 Approved for NPDES (Editorial Revision 1978)
TITLE: Chloruie, Total Residual (Titrimetric, Amperometric)
ANALYTE:
Chlorine, CI

INSTRUMENTATION: Titration
INTRODUCnON
The chlorination of water suppUes and polluted waters serves primarily to destroy or
deactivate disease-producing microorganisms. A secondary benefit, particularly in treating
drinking water, is tiie overall improvement in water quaUty resulting from the reaction of
chlorine with ammonia, iron, manganese, sulfide, and some orgartic substances.
Chlorination may produce adverse effects. Taste and odor characteristics of phenols and
other organic compounds present in a water supply may be intensified. PotentiaUy
carcinogertic chloro-orgartic compounds such as chloroform may be formed- Combined
chlorine formed on chlorination of ammonia- or amine-bearing waters adversely affects
some aquatic life. To fulfiU the primary purpose of chlorination and to minimize any
adverse effects, it is essential that proper testing procedures be used with a foreknowledge
of the limitations of the analytical determination.

Chlorine applied to water in its molecular or hypochlorite form initiaUy undergoes

hydrolysis to form free chlorine consisting of aqueous molecular chlorine, hypochlorous
acid, and hypochlorite ion. The relative proportion of thesefreechlorine forms is pH- and
temperature-dependent. At the pH of most waters, hypochlorous acid and hypochlorite ion
wiU predontinate.
Free chlorine reacts readtiy with ammortia and certain nitrogenous compounds to form
combined chlorine. With ammortia, chlorine reacts to form the chloramines:
monochloramine, dichloramine, and nitrogen trichloride. The presence and concentrations
of these combined forms depend chiefly on pH, temperature, initial chlorine-to-nitrogen
ratio, absolute chlorine demand, and reaction time. Bothfreeand combined chlorine may
be present simultaneously. Combined chlorine in water suppties may be formed in the
treatment ofrawwaters containing ammortia or by the addition of ammortia or ammoitium
salts. Chlorinated wastewater effluents, as weU as certain chlorinated industrial effluents,
normaUy contain only combined chlorine. HistoricaUytiieprincipal analytical problem has
been to distinguish between fiee and combined forms of chlorine.

1.0 Scope and Application

1.1 The amperometrictitrationmetiiod is appUcable to aU types of waters and wastes
that do not contain a substantial amount of orgartic matter.

2.0 Summary of Metiiod

2.1 Chlorine (hypochlorite ion, hypochlorous acid) and chloramines
stoichiometricaUy Uberate iodinefrompotassium iodide at pH 4 or less.

121
 2.2 The iodine istitratedwitii standard reducing agent such as sodiumtitiosulfateor
phenylarsine oxide usuig an amperometer to determinetfieend pouit.
2.3 The results are calculated as mg/L CI eventfioughtiieacmal measurement is of
total oxidizing power because chlorine istfiedominant oxidizing agent present
2.4 Steps can be done in a mannertiiatfree chlorine (6.3) is determined uitiieprocess
of determining total chlorine (6.4).
3.0 Interferences

3.1 Manganese, rtitrite and iron do not interfere.

3.2 Stirring can lower chlorine values by volatilization.
3.3 If dUution is necessary, it must be done witii disttiled water which is free of
chlorine, chlorine-demand and ammonia.
3.4 Copper and stiver poison the electrode.
3.5 Exposure to sunUght or other strong tight or agitation wiU accelerate the
reduction of chlorine. Therefore, start chlorine determinations as soon as
possible and do not store samples for chlorine determination..
4.0 Apparams

4.1 An amperometer consisting of a microammeter with necessary electrical

accessories, a ceU unit with a salt bridge, reference electrode and an agitator:
CommerciaUy avaUable. If the entire system (includingtiu-antdeUvery system) is
to be used, make sure that the volume read off the pipet or buret is really being
deUvered to the sample ceU. Reservoir type system sometimes back up,
producing false reacUngs.
4.2 A microburet, 0-2 mL or 0-10 nti, depending on required precision, accuracy and
range.
5.0 Reagents
5.1 Phenylarsine oxide solution (0.(X)564 N), commercially available, WaUace and
Tieman or equivalent. Standardize with potassium bUodate (5.8, 5.9).
5.2 Potassium Iodide, KI, crystals.
5.3 Potassium Iodide Solution: Dissolve 50 g KI in freshly boiled and ccx)led distUled
water and dUute to 1 liter. Store in colored, glass-stoppered bottie in refrigerator.
Discard when yellow color develops.
5.4 CommerciaUy available starch indicators such as thyodene or equivalent may be
used.
5.5 Acetate buffer solution (pH 4): Dissolve 146 g anhydrous NaC2H302 or 243 g
NaC2H302-3H20 in 400 mL disttiled water, add 480 g concentrated acetic acid
and dtiute to 1titerwith distiUed water.
5.6 Sulfuric Acid (1:4): Slowly add 200 mL H2SO4 (sp. gr. 1.84) to 800 mL of
distilled water.
5.7 pH Buffer 7
5.8 Potassium btiodate (0.1N): Dissolve 3.249 g potassium bUodate, previously
dried 2 hours at 103°C, in distilled water and dUute to 1.0 titers. Store in a glass
stoppered bottie.
5.9 Potassium btiodate (0.005 N): DUute 50 mL of 0.1 N potassium bUodate (5.8) to
1-titer ui a volumetric flask. Store in a glass stoppered bottie.

122
 5.10 Standardization of ^ N phenylarsuie oxide (PAO): Dissolve approximately
7AI r .?^ ^ ^^'^^ ''^ 100 to 150 mL distiUed water, add 10 mL H2SO4 solution
(5.6) followed by 20 mL 0.005 N potassium btiodate solution (5.9). Place ui
dark for 5 muiutes; dtiute to 300 mL andtitratewitii 0.00564 N phenylarsine
oxide solution (5.1) to a pale straw color. Add a smaU scoop of indicator (5.4).
Wait untti homogeneous blue color develops and continuetiietitrationdrop by
drop imttitiiecolor disappears. Run ui dupUcate. DupUcate detenninations
should agree wititin ±0.05 mL.
NPAO = 20 * o.nns
mLPAO
Adjust PAO solution if necessary and recheck.
6.0 Procedure

6.1 Place 200 mL of sample uitiiesample container. This volume is convenient

becausetiieburet reading in mUUliters is equivalent to mg/L CI. Up to 2 mg/L is
reUablytitratedtitisway. Smaller sample aUquots dUuted to 200 mL are used for
concentrations greatertiian2 mg/L. The constmction oftiieceU and electrode
component usually reqmre 200 mL of sample.
6.2 Free Chlorine
6.2.1 Add 1 ml of pH buffer 7 to produce a pH of 6.5 to 7.5.
6.2.2 Place sample ontitratorstand and tumtitratorto FREE.
6.2.3 Let the needle stabtiize. Tid^te witii standard phenylarsine oxide titrant
(5.1), observing current changes on nticroammeter. Addtitrantin
progressively smaUer increments untti aU needle movement ceases. Make
successive buret readings when needle action becomes sluggish, signaUng
approach of end point Subtract last very small incrementtfiatcauses no
needle response because of over-titration. Altematively, use a system
involving continuous current measurements and determine end point
mathematicaUy. Tum thetitratorOFF.
6.4 Total Chlorine Do not Refill Buret.
6.4.1 Add 1.0 mL KI solution (5.3).
6.4.1 Add 1.0 mL acetate buffer (5.5).
6.4.3 Return sample totitratorstand and tum to TOTAL.
6.4.4 Titrate with 0.(X)564 N PAO (5.1). As each increment is added the needle
deflects toward rest. When the needle no longer deflects subo^ct the last
drop added from the buret reading to obtain the mg/L CI. Less and/or
slower deflection signals that the end point is near. Tum thetitratorOFF
and replace storage beaker to thetitratorstand.
7.0 Calculations
7.1 For 0.(X)564 N PAO and a 2(X) mL sample there are no calculations. The buret
reading is in mg/L. The last increment, when the needle does not deflect toward
rest, must be subtracted. 1 ml oftitrant= 1 mg/L CI
7.2 When a different volume of sample is used calculate as foUows:
mg/L CI as Ch = A*200
ml of sample

123
8.0 Accuracy

8.1 Moretiian20 laboratories analyzed prepared samples of 0.64 and 1.83 mg/L total
CI. The relative standard deviations were 24.8% and 12.5%respectivelyand tfie
relative errors were 8.5% and 8.8% respectively. In a single operator, single
laboratory situation the foUowingresuUswere obtained.

Sample Average Stand Dev. Rel. Stand. Dev.

Matrix mg/L mg/L %
DistiUed Water 038 002 6T
3.50 0.01 0.2
Drinking Water 0.97 0.03 2.6
River Water 0.57 0.02 3.0
Domestic Sewage 0.41 003 6.9

References

Urtited States Environmental Protection Agency. (1992). Methods for Chemical Analysis
of Water and Wastes. Method #330.1. Environmental Monitoring and Support
Laboratory, Urtited States Environmental Protection Agency, Cincinnati, Ohio.
American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed. Method 4500. American PubUc Health Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 4-42.

124
TITLE: ChlorophyU a

DSfSTRUMENTATION: Fluorometer
INTRODUCTION
The concentration of photosynthetic pigments is used extensively to estimate phytoplankton
biomass,and indirectiy the trophic state of the waterbody. AU green plants contain
ChlorophyU a, which constimtes approximately 1 to 2% of tiie dry weight of planktonic
algae. Other pigments that occur in phytoplankton include chlorophyUs b and c,
xanthophyUs, phycobUins, and carotenes. The important chlorophyU degradation products
found in the aquatic environment are the chlorophylUdes, pheophorbides, and pheophytins.
Therelativeabundance of the materials on a cmde scale indicates whether algal population
are in growth or death phases. The presence or the various photosynthetic pigments is
used, among other features, to separate the major algal groups.
The three methods for determining chlorophyU a in phytoplankton are the
spectrophotometric, thefluorometric,and the high-performance Uquid chromatographic
(HPLC) techrtiques. Fluorometry is more sensitive than spectrophotometry, requires less
sample, and can be used for in-vivo measurements. These optical methods can
significantiy under- or overestimate chlorophyU a concentrations, in part because of the
overlap of tiie absorption andfluorescencebands of co-occurring accessory pigments and
chlorophyll degradation products. HPLC is a useful method for quantifying photosynthetic
pigments including chlorophyll a, accessory pigments (e.g., chlorophylls b and c), and
chlorophyU degradation products (chlorophylUdes, pheophorbides, and pheophytins).
Pigment distribution is useful for quantitative assessment of phytoplankton commuitity
composition and zooplankton grazing activity. This method covers the use of fluorometric
determination of ChlorophyU a.

1.0 Scope and Application

1.1 This method covers the determination of Chlorophyll a in surface waters.

2.0 Summary of Method

2.1 A water sample is fUtered to obtain algae cells. The ceUs are ground with the fUter
which is made of glassfibersto disintegratetiieceUs and organeUes containing
pigments. Pigments are extracted witii a solvent. The solvent is concentrated,
and the amount of chlorophyU present is detemtined using a fluorometer.

3.0 Sample Handling and Preservation

3.1 Conduct work with chlorophyU extracts in subduedtightto avoid photo-
degradation. Use opaque containers or wrap them with aluminum foti.
3.2 Samples taken fix)m water having pH 7 or higher may be placed in airtight plastic
bags and storedfrozenfor 3 weeks. Samples from acidic water must be
processed promptiy to prevent chlorophyU degradation.

4.0 Interferences
4.1 Pheophorbide a and pheophytin a, two common degradation products of
chlorophyU a can uiterfere witii tiie determination of chlorophyll a because tiiey

125
 absorbtightandfluorescem tiie sameregionof tiie spectrum as does chlorophyU
a. If tiiese pheopigments are present, significant errors in chlorophyU a values wtil
result.
4.2 Pheopigments can be measured eitiier by spectrophotometry orfluorometry,but
m maruie andfreshwaterenvironmentstfiefluorometricmetiiod is unreUable
when chlorophyU b co-occurs. Upon acidification of chlorophyll b,tiieresulting
fluorescence emission of pheophytin b coincident witiitfiatof pheophytin a, tiius
produdng underestunation and overestimation of chlorophyU a and
pheopigments, respectively.
5.0 Apparams

5.1 Tissue grinder: SuccessfuUy macerating glassfiberfUters uitissuegruiders witii

gruiding mbe and pestie of conical design may be difficult. Preferably use
round-bottom grinding mbes witii a matching pestie having grooves intiieTFE
tip. These are provided
5.2 CUrtical centrifuge.
5.3 Centrifuge mbes, 15-mL graduated, screw-cap.
5.4 FUtration equipment, fUters, glassfiberor membrane (0.45pm porosity, 47-mm
diam); vacuum source; solvent-resistant disposablefilterassembly, 1.0 pm pore
size; 10-mL solvent-resistant syringe.
5.5 Fluorometer, equipped with a high-intensity F4T.5 blue lamp, photomultipUer
mbe R-446 (red-sensitive), sUding window orifices 1 x, 3 x, 10 x, and 30 x, and
filters fortightemission (CS2-64) and excitation (CS-5-60). A high-sensitivity
door is preferable.
6.0 Reagents

6.1 Saturated magnesium carbonate solution: Add 1.0 g finely powdered MgCOs to
100 mL distUled water.
6.2 Aqueous acetone solution: Mix 90 parts acetone (reagent grade BP 56°C) with 10
parts saturated magnesium carbonate solution (6.1).

7.0 Procedure
7.1 The pigments are extracted from the plankton concentrate with aqueous acetone
The ease with which the chlorophylls areremovedfrom the cells varies
considerably with different algae. To achieve consistentiy the complete extraction
of the pigments, dismpt the cells mechanicaUy with atissuegrinder. Glass fiber
fUters are preferred forremovingalgaefromwater. The glassfibersassist in
breaking the cells during grinding, larger volumes of water can be fUtered, and
no precipitate forms after acidification. Inert membrane fUters such as polyester
fUters may be used where these factors are irrelevant
7.2 Concentrate sample by centrifuging or filtering as soon as possible after
coUection. If processing must be delayed, hold samples on ice or at 4°C and
protect from exposure to tight Use opaque botties because even brief exposure to
tight during storage wiU alter chlorophyll values. Use glassware and cuvettes that
are clean and acid-free.
7.3 Place sample in atissuegrinder and cover with 2 to3mL 90% aqueous acetone
solution, and macerate at 500 rpm for 1 min. Use TFE/glass grinder for a glass-
fiber fUter and glass/glass grinder for a membrane fUter. The ESL has a manuaUy

126
 operated grinder. Grind untti the fUter is m solution.
7.4 Tratisfer sample to a screw-c^ centrifuge mbe and rinse grinder witii a few
milUUters 90% aqueous acetone and add the rinse to the extraction slurry. Adjust
total volume to a constant level, 5 to 10 mL, with 90% aqueous acetone. Use
solvent sparingly and avoid excessive dUution of pigments. Steep samples at least
2 h at 4°C intiiedark.
7.5 Clarify by fUtering through a solvent-resistant disposable fUter (to minimize
retention of extract infilterandfilterholder, force 1 to 2 mL airtiux)ughtfiefUter
after the extract), or by centrifuging in closed mbes for 20 min at 500 g
(convert to rpm). Decant clarified extract into a clean, caUbrated 15-mL screw-cap
centrifuge mbe and measure total volume.
7.6 CaUbratefluorometerwith a chlorophyU solution of known concentration as
foUows: Prepare chlorophyU extract and analyze spectrophotometricaUy.
Prepare serial dUutions of the extract to provide concentrations of approximately
2, 6, 20, and 60pg chlorophyU a / L.
7.7 Measure samplefluorescenceat sensitivity settings that wiU provide a mid scale
reading. (Avoid using the 1 x window because of quenching effects.) Convert
fluorescencereadingsto concentrations of chlorophyU a by multiplying the
readings by the appropriate caUbration factor.

8.0 ResuUs

8.1 ChlorophyU a,
pg/L= [F * exn-act volume, ml (From 7.5)] / Volume of Sample,ml
where:
F= Fluorometry reading
Reference
American PubUc Healtii Association. (1992). Standard Metiiods fortfieExamination of
Water and Wastewater. 18tiied. Metiiod 10200 H. American Pubtic Healtii
Association, American Water Works Association, and Water Environment Federation,
Washmgton DC, 10-17.

127
TITLE: Standard Practice for Coagulation-Rocculation Jar Test of Water
INTRODUCTION
Coagulation is a process for combuting smaU particles into larger aggregates. It is an
essential component of accepted water treatment practice in which coagulation,
sedimentation and fUtration processes are combined in series toremoveparticulates from
water. It is very irnportant to recognizetiiattitisconventional treatment system does much
more. Concentrations of poUutants (inorganic, nonUvmg organic, and biological) are in
most mstances much higher m suspended soUdstfianmtiiewaters witii which tiiese solids
are associated. Hence, water treatment fortfieremovalof particulates also accompUshes
tfie removal of many harmful substancesfromwater. Some examples foUow.

Clays are a major portion of namral "turbidity" in raw water suppUes. Witiitfiepossible
exception of asbestos, tiiey are not duectiy responsible for harmful effects on human
healtii. There is some possibiUtytiiattfieycan exert important healtfi effects by adsorption,
transport andreleaseof inorganic and organic toxic substances, vimses and bacteria.
Direct evidence for theremovalof clays from water suppUes using coagulation is lacking,
since clay particles are not detected du-ectiy inroutinewater analysis. However, laboratory
smdies are numerous. For example. Black and Hannah and Packham have demonstrated
effective coagulation of kaoUrtite, montmorUlonite and otiier clay suspensions using alum
under conditions simUar to those found in potable water treatment

Epidemiological smdies have shown that occupational exposure to asbestos dust can lead to
cancers in the gastrointestinal tract This has led to the suggestion that asbestos particles in
water suppUes can cause simUar problems. Results of pilot-scale coagulation and fUtration
experiments for theremovalof asbestosfibersfromtfieDuluth water supply have been
reported by Black and Veatch and Logsdon and Symons. Two types of asbestos fibers
were found, amphibole and chrysotUe. The amphibolefiberswere larger and probably
negatively charged; the chrysotilefiberswere smaUer and may have been positively
charged. The majority of the particles of both minerals were less than 2 pm in length. The
amphibolefiberswere easilyremovedby coagulation andfiltration,while considerable
difficulties were encountered inremovingthe chrysotUe fibers. Since conventional fUters
are able toremovevery smaU particles, and since conventional pretreatment chemistry in
coagulation is designed for negative particles, it is plausible that changes in coagulant
addition would provide goodremovalsof positively charged chrysottie particles.

Coagulation is effective in theremovalof color and many other organic macromolecules

and particulates from water. Here again, laboratory smdies provide the bulk of the
avaUable evidence. HaU and Packham have smdied the coagulation of huntic and fulvic
acids by iron (HI) and aluminum (HI) salts. These orgartic materials have natural origins in
the decay of plant materials and are ubiquitous in natural waters. These investigators found
that humic acids werereadUyremovedwhereas a significant fraction of the fulvic acids
were notremovedby either coagulant SimUarresititshave been reported by others,
including Black and WilUams and Rook. The abiUty of conventional coagulation processes
to provide forremovalof humic substances is impcMtant in a new perspective. These
substances have been shown to bereactantsin the formation of chloroform and other
halomethanes by chlorine during disinfection. Since humic substances can be removed
directiy by coagulation and may also be adsorbed on clays that are also effectively treated
by coagulation, it is reasonable to conclude that coagulation as conventionally practiced or
with minor modifications can provide significant decreases in haloform production from
chlorination.

128
Large microorganisms mcludmg algae and amoebic cysts are readUyremovedby
coagulation and fUtration. Bacterialremovalsof99% are also achievable. Moretiian98%
of poUovfrus type 1 wasremovedby conventional coagulation and fUo^tion. Several
recent smdies have showntiiatbaaeria and viral agents are attached to organic and
inorganic particulates. Hence,removaloftiieseparticulates by conventional coagulation
and fUtration is a major component of effective treatment fortiieremovalof patfiogens.

FuiaUy, many toxic organic substances such as PCB and DDT and also many inorganic
toxic materials are adsorbed on naturaUy occurring inorganic and organic particulates.
Removal of particulates wUl also provideremovalof tiiese hazardous substances.
PROCESS DESCRIPTION
A coagulation process has two separate and distinct components or steps. First, particles in
tfie water must be treated chemically to maketfiem"stic^" or unstable. This uivolves tfie
addition of one or more chemicals inrapidmix tanks. Second, these destabiUzed particles
must be brought into contact with each other so that aggregation can occur. This is done by
gentie stirring of the water inflocculationtanks. If eitiier one of these steps is not properly
designed, the coagulation process wUl not function, i.e., aggregation wUl not occur.
The water to be treated is brought to the r^id mixing tank where destabilizing chenucals
are added; vigorous mixing occurs for a short time, normaUy 1 min or less. UsuaUy one
mixing tank is used, although occasionaUy two tanks may be used in series when two
coagulants requiring separate addition are employed. DestabiUzation is fast and essentiaUy
complete after this rapid mixing. The water and its destabilized particles are then
introduced into theflocculationtank, where gentiefluidmotion brings the particles into
contact so that aggregates can form. This gentie mixing is usually done mecharticaUy,
although hydraulic mixing is sometimes employed using baffled tanks. Detention times of
1 hr are typical. Therapidmixing andflocculationtanks together bring about aggregation,
and comprise the coagulation process. No materials are removed from the water in these
tanks. In fact, materials are added in the form of coagulant chemicals. Sotids are removed
in subsequent settiing andfiltrationfaciUties. These soUd-Uquid separation processes must
remove the particles present in the originalrawwater and the chemicals added to bring
about coagulation. These sotids leave the treatment system in sludgefiromthe settling tanks
and in backwash water from the fUters. Disposal of these water treatment plant wastes is a
problem in itself. Here it is important to notetiiatthe characteristics of tiiese wastes (sotids
concentration, quantity, dewatering capabiUty) are functions not only of therawwater
supply but also of the materials used as coagulants .

1.0 Scope and AppUcation

1.1 This practice covers a general procedure for the evaluation of a treatment to reduce
dissolved, suspended, coUoidal, and nonsettieable matterfix)mwater by chemical
coagulation-flocculation, foUowed by gravity settUng. The procedure may be
used to evaluate color, turbidity, and hardness reduction.
1.2 The practice provides a systematic evaluation of the variables normally
encountered in the coagulation-flocculation process.
1.3 This standard does not purport to address the safety problems associated witfi its
use. It istfieresponsibiUty of tfie user oftftisstandard to estabUsh appropriate
safety and healtii practices and determinetfies^pUcabUity ofregulatoryUntitations
prior to use.

129
2.0 Summary of Practice

2.1 The coagulation-flocculation test is carried out to detemtinetiiechemicals,

dosages, and conditions requued to achieve optimum results. The primary
variables to be uivestigated usuigtiierecommended practice include, but are not
limited to:
2.1.1 Chemical additives,
2.1.2 pH,
2.1.3 Temperature,and
2.1.4 Order of addition and mixing conditions.
3.0 Significance and Use

3.1 This practice permits the evaluation of various coagulants and coagulant aids used
in the treatment of water and waste water for the same water and the same
experimental conditions.
3.2 The effects of concentration of the coagulants and coagulant aids and their order
of addition can also be evaluated by this practice.
3.3 This test can not be used for EPA reporting processes but may be used in day to
day in-plant operations.
4.0 Interferences
4.1 Temperature Change (During Test): Thermal or convection currents may occur,
interfering with the settling of coagulated particles. This can be prevented by
temperature control.
4.2 Gas Release (During Test): Flotation of coagulated floe may occur due to gas
bubble formation caused by mechanical agitator,temperatureincrease or chemical
reaction.
4.3 Testing-Period: Biological activity or other factors may alter the coagulation
characteristics of water upon prolonged standing. For this reason the period
between sampling and testing should be kept to a minimum, with the time being
recorded.
5.0 Apparams
5.1 Multiple Stirrer: A multiposition stirrer witfi continuous speed variation from
about 20 to 150 rpm should be used. The stirring paddles should be oftightgage
corrosionresistantmaterial aU of the same configuration and size. An iUuminated
base is useful to observe the floe formation. Precautionary measures should be
taken to avoid heat being imparted bytiieUlumination system which may
counteract normal settling.
5.2 Jars (or Beakers), all of tiie same size and shape; 2000-mL Griffin beakers may
be used (lOOO-tnL recommended minimum size).
5.3 Reagent Rack, a means of introducing each solution to aU jars simultaneously.
There should be at least onerackfor each test solution or suspension.

6.0 Reagents
6.1 Purity of Reagents: Reagent grade chemicals shaU be used in all tests. Unless

130
 otiierwise indicated, it is intended tiiat aU reagents shall conform to tiie
specifications of tiie Comntittee on Analytical Reagents oftfieAmerican Chemical
Society, where such specifications are avatiable. Otiier grades may be used,
provided it is first ascertained tiiat tiie reagent is of sufficientiy high purity to
permit its use witiiout lessening tiie accuracy of tiie determination.
6.2 Purity of Water: Reagent grade water.
6.3 The foUowing chemicals and additives are typical of tiiose used for test solutions
and suspensions. The latter, witii tiie exception of coagulant aids, may be
prepared daUy by mixing chemicals with water to a concentration of 10 (±0.1)
g/L (1.0 mL of test solution or suspension when added to 1 L of sample is
equivalent to 10 mg/L):
Prime Coagulants
AIum[Al2(S04)3 • 8 H2O] Fen-ous Sulfate(FeS04 • 7H2O)
Ferric sulfate [Fe2(S04)3 • XH2O] Magnesium Carbonate(MgC03 •3H2O)
Femic chloride (FeCIs • 6H20) Sodium Aluminate(NaA102)
Coagulant Aids
Activated siUca
Artiortic
Catiortic Polyelectrolytes
Weighting Agents
Bentottite
Kaolin
Other clays and minerals
MisceUaneous
Activated carbon (powdered)
6.4 Coagulant Aids. There are numerous commercially avaUable coagulant aids or
polyelectrolytes. AU polyelectrolytes are classified aniortic, cationic or nortionic,
depending upon their composition. These aids may have the abiUty to produce
large, tough, easily-settied floe when used alone or in conjunction with inorgartic
coagulants. A smaU dosage (under I mg/L) may pemtit a reduction in the dosage
of, or complete elimination of, the coagulant In the latter case, the polyelectrolyte
would be considered the prime coagulant rather than a coagulant aid. Aids come
in powdered and Uquid form. Powdered aids should be prepared as 0.1%
solutions with appropriate aUquots to provide proper dosage. Always add
powdered aids to the dissolving water rather than the reverse, and add slowly to
the shoulder of a vortex created by stirring. If a vortex is not formed, tiie dry
powder wiU merely collect on the surface of the water in gummy masses and
become very difficult to dissolve. Dissolving time may vary from several minutes
to several hours. Suggested manufacturers' procedures for wetting, dissolving,
and storing should be followed when avaUable. Liquid forms can be readUy
prepared to the above strength without difficulty.

7.0 Sampling
7.1 CoUect tiie water sample at least 6 uiches under tiie surface by plunging tiie
container mouth down into tiie water and turning the moutfi towards tfie current ar
by dragging the container slowly horizontal. (Hare should be taken not to disturb
the bottom of the water source or along tfie sides so as not to stir up any settied

131
 soUds. This would create erroneous errors.
8.0 Procedure

8.1 Measure equal volumes (2000 mL) of sample mto each oftiiejars. As many
sample portions may be used astiiereare positions ontiiemultiple stirrer (6 jars).
Locate beakers sotiiattiiepaddles are off-center, but cleartiiebeaker waU by
about 6.4 mm (1/4 m.). Record tiie sample temperature attfiestart of the test.
8.2 Test chemicals are added incrementaUy to each jar. The ESL uses pipets provided
to incrementaUy add 1 ml to each jar (e.g. jar 1 receives 1 ml, jar 4 receives 4 ml,
and so on.)
8.3 Starttiiemultiple stirrer operating attiie"flash mix" speed of approximately 120
rpm. Add the test solution or suspensions, at predetermined dosage levels and
sequence. Flash ntix for approximately 1 min after the additions of chemicals.
Record the flash mix time and speed (rpm).
8.4 Reduce the speed to 30 rpm to keepfloeparticles uniformly suspended
tim>ughouttiie"slow mix" period. Slow mix for 20 min. Recordtiietimefor tfie
first visible floe formation. Every 5 min (during the slow ntix period), record
relative floe size and mixer speed (rpm). If coagulant aids are used, mixing speed
is critical because excessive stirringtendsto break up earlyfloeformation and
may redisperse the aid.
8.5 After the slow mix period, withdraw the paddles and observe settiing of floe
particles. Record the time required for the bulk of the particles to settie. In most
cases thistimewtil be that required for the particles to settie to the bottom of the
beaker; however, in some cases there may be interfering convection currents. If
so, the recorded settiing time should be that at which the unsettied or residual
particles appear to be moving equaUy upward and downward.
8.6 After 15 nun of settiing, record the appearance offloeon the beaker bottom.
Record the sample temperature. By means of a pipet or siphon, withdraw an
adequate sample volume (75 ml) of supematant Uquor from the jar at a point one
half of the depth of the sample, to conduct color, turbidity, pH and other
required analyses.
8.7 Repeat steps 8.1 through 8.6 untti aU pertinent variables have been evaluated.
8.8 The times given in 8.3, 8.4, and 8.6 are only suggestions.
9.0 ReproducibUity
9.1 It is recognized thatreproducibUityofresultsis important. To demonstrate
reproducibUity, the so-called 3 and 3 procedure is suggested. In this procedure,
dupUcate sets of 3 jars each are treated simultaneously with the same chemical
dosages in jars 1, 2, and 3 and 4, 5, and 6.
References
Sanks, R. L. (1978). Water Treatment Plant Design. Butterworth PubUshers, Stoneham,
Ma., 283.
American Society for Testing and Materials. (1992). Annual Book of ASTM Standards.
Vol. 11.02. Metiiod D 2035. American Society for Testing and Materials,
PhUadelphia, Pa., 722.

132
TITLE: CoUform Bacteria, Total
INTRODUCTION
CoUform bacteria, especiaUy tiie fecal coUforms, are natural, normaUy harmless mhabitants
of tiie mtestines of aU warm-blooded animals mcluding humans. CoUforms coexist m fecal
material witii patiiogens or disease-causing organisms such as certaui bacteria, vuaises, and
protozoa. Altfiough coUform bacteria are most abundant ui fecal material,tiieymay also be
found in soU and on vegetable matter.

CoUforms are highly concentrated in wastewater and generaUy sparse or not present in
otiier habitats. Because oftftiscorrelation between coUforms and wastewater, the presence
of coUform bacteria in water is considered an indication of contamination. Fecal
contaminated water can transmit bacterial diseases such as typhoid fever, dysentery,
gastroenteritis, and cholera; viral diseases such as hepatitis and poUo; and mtestinal
parasites such as Giardia, Cryptosporidium, and Entamoeba.

One of tfie most smdied species and, perhaps most famiUar, is Escherichia coli. which
occurs in the gut (colon) of man. They are gram negative, aerobic and facultative
anaerobic, non spore forming, rod-shaped tecteria. Other bacterial species that share these
characteristics or are like E._£2U, are said to be coUforms. CoUform bacteria are usually
associated with fecal material, but some species thrive on certain types of vegetation, in
soils, and in some industrial processes. These species, like E. coU. are formed only in the
gut of warm blooded vertebrate (mammals, bird).

Total coUforms are by definition more abundant—^by about three to five times—than fecal
coUforms, though thisratiovaries widely. Because of their greater abundance, total
coUforms provide a morerepresentativemeasure of wastewater poUution than do fecal
coUforms, by permitting the detection of even stight amounts of contamination. This
sensitivity is useful when evaluating clean waters, such as sheU-fishing areas and drinking
water. Natural, non-wastewater sources of total cotiforms, such as artimal sources and
decaying vegetation, may unfortunately cause a sample to be classified as wastewater-
contaminated because of a positive testresultcreated by coUforms from non-wastewater
sources. This possibiUty of false positives also exists to a lesser extent with the fecal
coUforms, which have a few non-fecal sources. The fecal coUforms are more closely
associated with fecal discharges than are total coUforms. Also, they are less subject to
regrowth and are less common in unpoUuted areas than total coUforms.

Fecal coliform:fecal streptococcus ratios can help to identify sources of poUution. Ratios
greater than 4.4 indicate fecal poUution from human sources;ratiosless than 0.7 indicate
nonhuman sources; andratiosbetween indicate a mixture of human and artimal sources.
Because of therapiddie-off of fecal streptococci outside of the animal host, theseratiosare
ortiy valid within 24 hours foUowing the discharge.

1.0 Scope and AppUcation

1.1 This method is applicable to drinking and surface waters.

2.0 Summary of Method

2.1 Total CoUforms are measured by using membranes tofilterout bacteria which are
then grown on an enriched medium for measurement.

133
3.0 Conunents

3.1 WhUe indicators are very useful in assessing the overaU microbiological poUution
of natural waters, no incticator species is perfect GeneraUy, a high concentration
of an indicator species suggests high concentrations of pathogens. Exceptions to
this are that pathogens may be abundant when indicators are not, and vice versa.
These exceptions may resultfromnatural effects, water poUution, or measurement
interferences, and can cause water to be falsely classified.
3.2 A false or erroneous classification as "uncontaminated" canresultwhen coUform
concentrations are low, but pathogens are high. This error occurs because some
pathogens die off slowly in water, but coUforms die off rapidly. This
phenomenon makes coliforms a valid indicator ortiy for recentiy-contaminated
water. Other conditions that may alter the expectedrelationshipsbetween coUform
and pathogen levels include the abiUty of some pathogens (for example. Vibrio
spp.) to multiply in natural waters, epidentics within a population that have caused
pathogen levels to be unexpectedly high when coUform levels are normal, or
laboratory testing difficulties giving erroneously low or high coUform test results.
Underestimated concentrations of coUforms mayresultfrom the poor growth of
coUfOTm cultures caused by the presence of certain non-coUform species, heavy
metals, or chlorine.
4.0 Interferences
4.1 Coliforms die off rapidly in water. With a half-life of about 15 hours, very few
coliforms wiU survive more than 3 days after entering the receiving waters.
Because of thisrapiddie-off, coliformstendto berestrictedin distribution to the
vicirtity of the discharge source. In contrast, someresistantbacteria, vimses, and
protozoa may survive much longer than coUforms in receiving waters. Therefore,
these pathogens may be present in waters that no longer contain any Uving
coliforms and may travel downstream to endanger distant areas long after the
coliforms have died off.
4.2 Suspended sotids can also interfere with testing procedures by causing abnormal
colony growth and by clogging membrane filters.
4.3 Ultraviolet radiation (sunUght) and elevatedtemperamresboth serve to increase, the
die-offrateof coUform bacteria,
4.4 Other factors, such as saUnity, nutrients, heavy metal concentrations, and
predation may also affect coUform survival.
5.0 Sampling and Preservation

5.1 Sampling Procedures

5.1.1 When the sample is coUected, leave ample air space intfiebottie (at least 2.5
cm) to faciUtate mixing by shaking, before examination. CoUect samples
that arerepresentativeof tiie water being tested,flushor disinfect sample
ports, and use aseptic techniques to avoid sample contamination. Keep
sampUng bottie closed until it is to be fiUed. Remove stopper and cap as a
unit; do not contaminate inner surface of stopper or cap and neck of bottie.
FiU container witiioutrinsing,replacestopper or cap immediately, and if
used, secure hood around neck of bottie.

134
 5.1.1.1 Manual sampUng: Take samples from ariver,stream, lake, or
reservoir by holding tiie bottie near its base uitfiehand and
plunging it, neck downward, below tiie surface. Tum bottie untti
neck points sUghtiy upward and moutii is directed toward tiie
current If there is no current, as in the case of a reservoir, create a
currerit artificially by pushing bottie forward horizontaUy m a
direction awayfix)mtfiehand. When samplingfroma boat, obtain
samples from upstream side of boat If it is not possible to coUect
samples from these situations in this way, attach a weight to base of
bottie and lower it into the water. In any case, take care to avoid
contact witii bank or stream bed; otiierwise, water fouling may
occur.
5.1.1.2 Sampling apparams: Special apparams that permits mechanical
removal of bottie stopper below water surface is required to coUect
samples from depths of a lake or reservoir. Various types of deep
sampling devices are avaUable. The most common istfieZoBeU J-Z
sampler, which uses a sterUe 350-mL bottie and a mbber stopper
through which a piece of glass mbing has been passed. This mbing
is connected to another piece of glass mbing by a mbber connecting
hose. The unit is mounted on a metal frame containing a cable and a
messenger. When the messenger is released, it strikes the glass
mbing at a point that has been sUghtiy weakened by a fUe mark. The
glass mbe is broken by the messenger and the tension set up by the
mbber connecting hose isreleasedand the mbing swims to the side.
Water is sucked into the bottie as a consequence of the partial
vacuum created by seating the urtit at time of autoclaving.
Commercial adaptations of this sampler and others are avatiable.
5.1.2 Potable water: If the water sample is to be takenfroma disuibution-system
tap without attachments, select a tap that is supplying water from a service
pipe directiy connected with the main, and is not, for example, served from
a cistem or storage tank. Open tap fully and let water mn to waste for 2 or
3 min, or for a time sufficient to permit clearing the service line. Reduce
water flow to permit filling bottie without splashing. K tap cleanliness is
questionable, apply a solution of sodium hypochlorite (100 mg NaOCl/L) to
faucet before sampling; let water mn for additional 2 to 3 nun after
treatment. Do not samplefi:omleaking t^s that aUow water to flow over
the outside oftfietap. In samplingfix)ma mixing faucetremovefaucet
attachments such as screen or splash guard, mn hot water for 2 min, then
cold water for 2 to 3 min, and coUect sample as indicated above. If the
sample is to be taken from a wellfittedwith a hand pump, pump water to
waste for about 5 min before coUecting sample. If the well is equipped with
a mechartical pump collect samplefroma tap on the discharge. If there is no
pumping machinery, coUect a sample directiyfiromthe weU by means of a
Sterilized bottiefittedwith a weight attiiebase; take care to avoid
contantinating samples by any surface scum. In drinking water evaluation,
collect samples offirtishedwater andfromdistribution sites selected to
assure systematic coverage during each month. CarefuUy chcx)se
distribution system sample locations to include dead-end sections to
demonstrate bacteriological quaUty throughout the network and to ensure
that locaUzed contamination does not occur through cross-connections,
breaks in tiie distribution lines, or reduction in positive pressure. Sample

135
 locations may be pubtic sites (poUce andfirestations, government office
buUdings, schools, bus and train stations, auports, community parks),
commercial estabUshments (restaurants, gas stations, office butidings.
industrial plants), privateresidences(sin^eresidences,apartment
buildings, and town house complexes), and special sampUng stations buUt
into the distribution network. EstabUsh sampling program in consultation
with state and local healtii authorities.
5.1.3 Raw water Supply: In coUecting samples directiy from ariver,stream,
lake, reservoU, spring, or shaUow weU. Obtain samples representative of
the water that is the source of supply to consumers. It is undesirable to take
samples too neartiiebank or too farfromtiiepouit of drawoff, or at a depth
above or below the point of drawoff.
5.1.4 Surface waters: Stream smdies may be short-term, high intensity efforts.
Select bacteriological sampling locations to include a Iwseline location
upstreamfromthe study area, industrial and municipal waste outfalls into
the main strearn smdy area, tributaries except those witfi a flow less than
10% of the main stream, intake points for murticipal or industrial water
faciUties, downstream samples based on stream flow time and downstream
recreational areas. Dispersion of wastewaters into the receiving stream may
necessitate preliminary cross-section smdies to determine completeness of
nuxing. Where a tributary stream is involved, select the sampling point near
the confluence with the main stream. Samples may be coUectedfroma boat
or from bridges near critical smdy points. Choose sampUng frequency to be
reflective of stream or water body conditions. For example, to evaluate
waste discharges, sample every 4 to 6 h and advance thetimeover a 7 to 10
-d period. To monitor stream and lake water quaUty estabUsh sampling
locations at critical sites. Sampling frequency may be seasonal for
recreational waters, daily for water supply intakes, hourly where waste
treatment control is erratic and effiuents are discharged into shellfish
harvesting areas, or even continuous.
5.1.5 Bathing teaches: SampUng locations for recreational areas should reflect
water quaUty within the entire recreational zone. Include sites from
upstream peripheral areas and locations adjacent to drains or natural
contours that would discharge storm water collections or septic wastes.
Collect samples in the swimming areafix)ma uniform depth of
approximately 1 meter. Consider sediment sampling of the water-beach
(soil) interface because of exposure of young chUdren at the water's edge.
To obtain baseline data on marine and estuarine bathing water quaUty
include sampUng at low, high, and ebb tides. Relate sampUng frequency
directiy to the p ^ bathing period, which generaUy occurs in the aftemoon.
Preferably, collect daily samples during the recogrSzed bathing season;
minimum sampling includes Friday, Saturday, Sunday, and hoUdays.
When limiting sampling to days of peak recreational use, preferably coUect a
sample in the morning and the aftemoon. Correlate bacteriological data with
turbidity levels or rainfall over the watershed to make rapid assessment of
water quality changes.
5.1.6 Sediments and sludges: The bacteriology of bottom sediments is important
in water supplyreservoirs,in lakes,rivers,and coastal waters used for
recreational purposes, and ui sheUfish-growingwaters. Sediments may
provide a stable index of tiie general quality of tiie overiyuig water,
particularly wheretiiereis great variabtiity in its bacteriological quaUty.

136
 SampUngfrequencymreservou^and lakes may be related more to seasonal
changes in watertemperaturesand stormwater runoff. Bottom sediment
changes uiriverand esmarine waters may be more erratic, beuig uifluenced
by stormwater mnoff, increased flow velocities, and sudden changes in tiie
quaUty of effluent discharges. Bacteriological exantination of sludges from
water and waste water treatment processes is desirable to determuie tfie
impact oftiieirdisposal uito receiving waters, ocean dumpuig, or burial in
landfiU operations. Sludge monitoring also may indicate the effectiveness
of wastewater treatment processes.
5.2 Sample Volume
5.2.1 An ideal sample volume wiU yield about 50 coUform colonies and not more
than 200 colonies of aU types on a membrane-fUter surface. Analyze
drinking waters byfilteruig100 to 1000 mL, or by fUteringrepUcatesmaUer
sample volumes such as dupUcate 50-mL or fourrepUcatesof 25-mL
portions. Analyze other waters byfilteringtiireedifferent volumes (dUuted
or undUuted), depending on the expected bacterial density. When less than
20 mL of sample (dUuted or undUuted) is to befiltered,add approximately
10 mL sterUe dtiution water to the funnel before fUtration. This increase in
water volume aids in uniform dispersion of the bacterial suspension over the
entire effectivefilteringsurface. See Table I for recommended sample
sizes.
6.0 Apparatus

6.1 Sample botties: Appropriate containers for fecal coUform samples may be of glass
or autoclavable nontoxic polypropylene. They are usuaUy 125 or 250 mL (4 or 8
oz) in volume, have secure stoppers or screw caps, and are free of chipped,
scratched, or etched surfaces. Before use, the botties must be carefully cleaned
and steriUzed. Prior to steriUzation, dechlorinating or chelating agents are added
to the bottie, if necessary. Disposable pre-steriUzed containers are also avaUable
for sample coUection.
6.2 Containers for culture medium: Use clean borosiUcate glassflaskspresterilized to
reduce bacterial contamination. Any size or shape offlaskmay be used, but
erlenmeyer flasks with metal caps, metal foU covers, or screw caps provide for
adequate mixing of the medium contained and are convenient for storage.
6.3 Culture dishes: Use sterile borosiUcate glass or disposable plastic petri dishes, 60
X 15 mm, 50 X 12 mm, or other appropriate size. Wrap convenient numbers of
clean, glass culture dishes in metal foil if sterilized by dry heat, or suitable heavy
wrapping paper when autoclaved. Incubate loose-Udded glass and disposable
plastic culture dishes intightiyclosed containers with wet paper or clotii to prevent
moisture evaporation withresultantdrying of medium and to maintain a humid
environment for optimum colony development Disposable plastic dishes that are
tight-fitting and meet the specifications noted above are avaUable commerciaUy and
are used widely.
6.4 FUtration units: Thefilter-holdingassembly (constmcted of glass, autoclavable
plastic, porcelain, or stainless steel) consists of a seantiess funnel fastened to a
base by a locking device or held in place by magnetic force. The design should
permit the membranefilterto be held securely ontiieporous plate of the
receptacle without mechanical damage and aUow allfluidto pass through the
membrane during filtration. Separately wrap the two parts of tfie assembly in
heavy wrapping paper, steriUze by autoclaving, and store untU use. Altematively,

137
 treat previously cleaned unwrapped parts by ultraviolet radiation fortfieinitial
stertiization before use uitiietestprocedure, or before reusuig units during a
Wtration senes. Field umts may be sanitized by igniting alcohol or immersing ui
botimg water for 5 mm. Do not ignite plastic parts. SterUe, disposable field units
may be used.
6.5 Membrane filter: Use membranefilterswitii aratedpore diameter suchtiiattiiere
is cor^leteretentionof coUfonn bacteria. Use onlytiiosefiltermembranes tiiat
have been found,tiuroughadequate quaUty controltestingand certification by tiie
tnanufacmrer, to exhibit: fitil retention of tiie organisms to be cultivated, stabUity
m use,fi-eedomfiromchemical extractablestfiatmay inhibit bacterial growtfi and
development, a satisfactory speed offiltration(wititin 5 min, no significant
mfluence on medium pH (beyond ± 0.2 units), and no uicrease in number of
confluent colonies or spreaders compared to control membrane filters. Use
membranes grid mariced in such a mannertfiatbacterial growtii is neitiier utiiibited
nor stimulated alongtfiegridtineswhentiiemembranes witii entrapped bacteria
are uicubated on a suitable medium. Preferably usefreshstocks of membr^e
filters and if necessary store them in an environment without extremes of
temperature and humidity. Obtain no more than a year's supply at any one time.
Preferably use presteriUzed membrane fUters for which the manufacmrer has
certified that the sterilization technique has neitiier induced toxicity nor altered the
chemical or physical properties of the membrane. If membranes are sterilized in
tiie laboratory, autoclave for 10 min at 121°C. Attiieend of tiie sterilization
period, let the steam escaperapidlyto minimize accumulation of water of
condensation on filters. Commonly used are Whatman 934-AH 55 mm filters.
6.6 Forceps: Smooth-tipped, witiiout cormgations on tiie inner sides of the tips.
Sterilize before use by dipping in 95% etiiyl or absolute metiiyl alcohol and
flaming.
6.7 Incubators: Use incubators to provide atemperatureof 35 ± 0.5°C and to maintain
a high level of humidity (approximately 90%relativehumidity).
6.8 Microscope andtightsource: To determine colony counts on membrane fUters,
use a magrtification of 10 to 15 diameters and a cool whitefluorescenttightsource
adjusted to give maximum sheen discernment. Optimally use a binocular wide
field dissecting nticroscope. Do not use a microscope iUuminator with optical
system fortightconcentration from an incandescenttightsource for discerning
coUform colonies on Endo-type media.
6.9 The need for uniformity dictates the use of dehydrated media.
7.0 Procedure
7.1 Prepare Medium lot which is commerciaUy avaUable. Commonly used is DIFCO,
Bacto m ENDO AGAR LES. Suspend 51 grams in 1titerdistiUed or deionized
water containing 20 ml ethanol and heat to botiing to dissolve completely. Cool to
45-50°C. Dispense 4 ml amounts into lower halves of 50 - 60 mm Petri dishes
and allow to solidify.
7.2 Select a sample size based on 5.2. The ESL uses dilutions of:
30 ml sample / 70 ml DI water
5 nti sample / 95 ml of DI water
1 nti sample / 99 ml of DI water
7.3 Insert a sterUe rinse water sample (100 mL) after fUtration of a series of 10
samples to check for possible cross contamination or contaminated rinse water.
Incubate the control membrane culture under the same conditions as the sample.

138
 MUTt: Use stertieftlu^tionunits at me beginnmg of eachfiltrationseries as a
minimum precaution to avoid accidental contamuiation. A fUtration series is
considered to be intermpted when an interval of 30 min or longer elapses between
sample filtrations. After such intermption, treat any further sample fUtration as a
newfiltrationseries and steriUze aU membranefilterholders in use. CE 5374,
3171, and ENVE 3203 do a blank before doing tiie samples.
7.4 Upon completion offinalrinseand the fUtration process disengage vacuum,
unlock andremovefunnel, immediatelyremovemembrane fUter witfi sterile
forceps, and place it on selected medium with arollingmotion to avoid entrapment
of air.
7.5 FUter sample: Using sterile forceps, place a sterile membrane fUter (grid side up)
over porous plate of receptacle. CarefuUy place matched funnel unit over
receptacle and lock it m place. FUter sample under partial vacuum. Witii fUter stUl
in place,rinsefunnel by fUtering three 2()- to 30-mL portions of sterile dUution
water. Mark the top of the corresponding petri dishes with dUution amounts.
7.6 Decontaminate this equipment between successive functions by using an
ultraviolet (UV) steriUzer for 2 min, flowing steam, or botiing water for 5 min.
Do not expose membranefilterculture preparations to random UV radiation leaks
that might emanate from the sterilization cabinet Eye protection is recommended;
either safety glasses or prescription-ground glasses afford adequate eye protection
against stray radiationfroma UV sterilization cabinet that is nottight-tightduring
the exposure interval. Clean UV mberegularlyand check it periodicaUy for
effectiveness to insure that it wiU produce a 99.9% bacterial kiU in a 2-min
exposure.
7.7 Place prepared fUter directiy on agar as described in preceding section and
uicubate for 22 to 24 h at 35 ± 0.5°C.

8.0 Calculation

8.1 To determine colony counts on membrane fUters, use a low-power (10 to 15

magrtifications) binocular wide-field dissecting nticroscope or other optical device,
with a cool whitefluorescenttightsource directed from above withraysas nearly
perpendicular as possible to the plane of the filter. The typical coUform colony
has a pink to dark-red color with a metaUic surface sheen. The sheen area may
vary in size from a smaU pinhead to complete coverage of the colony surface.
Atypical coUform colonies can be dark red or nucleated without sheen. Colonies
that lack sheen rnay be pink, red, white, or colorless and are considered to be
nonconforms. I The total count ot colonies (coltibrm and noncoltiorm) on Endo-
type medium has no consistentrelationshipto the total number of bacteria present
in the original sample. However, a high count of nonconform colonies may
interfere with the maximum development of coUforms. Refrigerating cultures
(after 22 h incubation) with high densities of nonconform colonies for 0.5 to 1 h
before counting may deter spread of confluence while aiding sheen discernment
Anaerobic mcubation at 35°C for 24 h for some groundwater samples may
suppress nonconform colonies but must be carefully evaluated to insure no loss of
conform recovery. Samples of disinfected water or wastewater effluent may
include stressed organisms that growrelativelyslowly and produce maximum
sheen in 22 to 24 h. Organismsfromundisinfected sources may produce sheen at
16 to 18 h, and the sheen subsequentiy may fade after 24 to 30 h.
8.2 Conform verification: Typical sheen colonies may be produced occasionally by
nonconform organisms. Atypical colonies (dark red or nucleated colonies witiiout

139
 sheen) occasionally may be coUforms. Verification of botii colony types is
advisable. Verify by atestfor lactose fennentation or by using altemative
procedures mvolvmg eitiier a rapid (4 h)testof two key biochemicalreactionsor a
multi-test system for speciation.
8.2.1 Lactose fermentation: Verify aU typical and atypical coUfonn colonies
mcluded intfiedirect count or a mimmum of five such colonies firom
drinkmg water samples by transferring growtii from each colony to lauryl
tryptose brotii; incubate at 35 + 0.5°C for 48 h. Gas formed ui lauryl
tryptose brotii and confirmed in brUUant green lactose brotii wititin 48 h
verifies the colony as a coUform
8.2.2 Altemative coUform verifications: Applytfiisaltemative coUform
verification procedure to isolated colonies on the membranefilterculmre.
If a mixed culture is suspected or if colony separation is less than 2 mm,
streak the growth to M-Endo medium to assure culture purity or submit the
mixed growth to the fermentation mbe method.
8.2.3 Rapidtest:Arapidverification of colonies utilizestestreactionsfor
cytochrome oxidase (CO) and, galactosidase (ONPG). Cotiform reactions
are CO negative and ONPG positive wititin 4 h incubation of mbe culture
or micro (spot) test procedure.
8.3 AU bacteria that produce a red colony witii a metalUc sheen witiiui 24 h mcubation
at 35°C on an Endo-type medium are considered members of the coUform group.
The sheen may covertiieentu^ colony or may appear only in a central area or on
the periphery. The coUform group thus defined is based on the production of
aldehydesfiromfermentation of lactose. Whtie this biochemical characteristic is
part of the metaboUc pathway of gas production in the multiple-mbe test, some
variations in degree of metaUic sheen development may be absent among coUform
strains. However, this sUght difference in indicator definition is not considered
critical to change its pubtic health significance, particularly if suitable smdies have
been conducted to estabtish therelationshipbetweenresultsobtained by the
membrane fUter and those obtained by the standard tube dUution procedure.
8.4 Compute the count, using membranefilterswith 20 to 80 coUform colonies and
not more than 2(X) colonies of aU types per membrane, by the foUowing equation:
conform colonies counted x 100 .
(Total) conform colonies/l(X) mL = mL sample filtered

For verified coUform counts, adjust the initial count based upon the positive
verification percentage andreportas "verified coUform count per 100 mL."
Percentage verified coUfornis = number of verified colonies X 1(X)
total number of coUform colonies
subjected to verification

9.0 Accuracy
9.1 The Table below Ulustrates some 95% confidence limits. These values assume
that bacteria are distributed randorrtiy and follow a Poisson distribution. For
results with counts, c, greater than 20 organisms, calculate the approximate 95%
confidence lintits using the foUowing normal distribution equations:
Upper lintit = c + 2 Lower lintit = c - 2

140
 95% CONFIDENCE LIMITS FOR MEMBRANE FILTER
COLIFORM RESULTS USDSfG 100-ML SAMPLE
Number of CoUform
Colorties Counted

95% Confidence Lintits

Lower Upper Lower Upper
0.0 3.7 4.7 18.4
Ol 5.6 5.4 19.7
02 7.2 6.2 21.0
06 8.8 6.9 22.3
1.0 102 7.7 23.5
1.6 11.7 8.4 24.8
2.2 13. 1 9.4 26.0
2.8 14.4 10.7 28.4
3.4 15.8 11.5 29.6
4.0 17.1 12.2 30.8

TABLE 1. SUGGESTED SAMPLE VOLUMES FOR MEMBRANE FILTER

TOTAL COLIFORM TEST

Volume (X) To Be FUtered mL

Water Source 100 50 10 1 01 0.01 0.001 0.0001

Drinking water X
Swimming pools X
WeUs, springs X X X
Lakes, reservoirs X X X
Water supply intake X X X
Bathing beaches X X X
River water X X X X
Chlorinated sewage X X X
Raw sewage X X X X

References
Water Envuronment Federation. (1990). Wa5;tewater Binlngv: the Microlife. Special
PubUcation, Water Envu-onment Federation, Alexandria, Virginia, 196 pp.
American PubUc Healtii Association (1992). Standard Methods fortiieExamination of
Water and Wastewater. 18tiied. Metiiod 9222. American PubUc Healtfi Association,
American Water Works Association, and Water Envuronment Federation, Washington
DC, 9-53.

141
METHOD #: 120.1 Approved for NPDES (Editorial Revision 1982)
TITLE: Conductance (Specific Conductance, pmhos at 25°C)
ANALYTE:
Conductance

INSTRUMENTATION: Conductivity Meter

DSTTRODUCnON
Thetermconductance is atermwhichrefersto the abUity of materials to carry an electric
current. Liquids which carry an electric current are generaUyreferredto as electrolytic
conductors. The flow of currenttiiroughelectrolytic conductors is accompUshed by the
tnovement of electric charges, positive and negative ions, when the Uquid is under the
influence of an electrical field. The conductance of a Uquid can be defined by its electrical
properties, theratioof current to voltage between any two points within the tiquid. As the
two points move closer togetiier or further aparttfiisvalue would change. To have useful
mearting for analytical purposes, a dimension needs to be given to the measurement; i.e.,
the physical parameters of the measurement
By defirting the physical parameters of the measurement, a standard measure is cn^ated.
This standard measure is referred to as specific conductance or conductivity. It is defined
as the reciprocal of theresistancein ohms, measured between the opposing faces of 1 cm
cube of Uquid at a specific temperature. The urtits used to define conductivity are: 1/ohm =
1 mho =1000 mmhos = 1,(XX),000 pmhos. S.I. units may be used in place of mhos; 1
mho =1 Siemen (S).

In theory then, a conductivity measuring ceU is formed by two 1 cm square surfaces spaced
1 cm apart CeUs of different physical configuration are characterized by their cell constant,
K. K is a function of the electrode areas, the distance between the electrodes and the
electrical field pattem between the electrodes. The theoretical ceU just described has a cell
constant of K = 1.0. Often for considerations having to do with sample volume or space, a
ceU's physical configuration is designed differentiy. Cells with constants of 1.0 cm-i or
greater normally have smaU, widely spaced electrodes. CeUs with constants of 0.1 cm-i or
less normaUy have large closely spaced electrodes.
Since K is a "factor** whichreflectsa particular ceU's physical configuration, it must be
multiptied by the observed conductance to obtain the acmal conductivity reading. For
example, for an observedreadingof 200 mhos using a ceU witii K = 0. 1 cm-i, the
conductivity value is 200 x 0.1 = 20 pmhos.
Solutions witii low conductivity, up to 1-2 mmhos, are best measured witii ceUs having a
ceU constant of K = 0.1 cm-i. CeUs witii K = 1.0 cm-i are best used for solutions witii
conductivity of > 1 mho to 100 mmhos. CeUs witii K = 10.0 cm-i are best used for
solutions with conductivity of 10 pmhos to 2 mhos.
In a simptified approach,tfieceU constant is defined astfieratiooftfiedistance between tfie
electrodes, d, totiieelectrrode area, A. This however neglects tiie existence of a fringe-field
effect, which effects tfie electrode area by tiie amount AR Therefore K = d/ (A + AR).
Because it is normaUy unpossible to measuretfiefringe-field effect and the amount of A~ to

142
calculatetfieceU constant, K,tfieactual K of a specific ceU is detemtined by a comparison
measurement of a standard solution of known conductivity (e.g., KCl, 0.01 mol/1).

The conductivity of a solution witii a specific electrolyte concenti^tion wiU change witfi a
change in temperature. By convention, the conductivity of a solution istfiatwhich it
exhibits at 25°C. A measurement made at 25'*Ctiiereforeneeds no compensation.
Measurements made at any othertemperatureneed compensation. Temperature coefficients
are different for different solutions. Some examples are: Ultra pure water 4.55 % / "C,
Salt (NaCl) 2.12, 5% NaOH 1.72, DUute Ammonia 1.88, and 10% HCl 1.32.

DistiUed water produced m a laboratory generaUy has a conductivity intiierange 0.5 to 3

pmhos/cm. The conductivity increases shortiy after exposure to both air and the water
container because of the accumulation of dissolved gasses and the dissolution of the vessel.
The conductivity of potable waters in the United States ranges generaUy from 50 to 15(X)
pmhos/cm. The conductivity of domestic wastewaters may be near that of the local water
supply, although some industrial wastes have conductivities above 10,(XX) pmhos/cm.
Conductivity instmments are used in pipetines, channels, flowing streams, and lakes and
can be incorporated in multiple-parameter monitoring stations using recorders.

Most problems in obtaining good data with conductivity mortitoring equipment are related
to electrode fouling and to inadequate sample circulation. Conductivities greater than 10
OOO to 50 (XX) pmho/cm or less than about 10 pmho/cm may be difficult to measure with
usual measurement electronics and ceU c^acitance.
Laboratory conductivity measurements are used to:
1) Estabtish degree of mineralization to assess the effect of the total concentration of
ions on chentical equiUbria, physiological effect (chiefly osmotic) on plants or
animals, corrosion rates, etc.
2) Assess degree of mineraUzation of distiUed and deionized water.
3) Evaluate variations in dissolved mineral concentration of raw water or
wastewater. Minor seasonal variations found inreservoirwaters contrast sharply
with the daUyflucmationsin some pollutedriverwaters. Wastewater containing
significant trade wastes also may show a considerable daUy variation.

4) Estimate sample size to be used for common chemical determinations and to check
results of a chemical analysis.
5) Determine amount of ionic reagent needed in certain precipitation and
neutralization reactions,tfieend point being denoted by a change in slope of tfie
curve resultingfromplotting conductivity against buret readings.
6) Estimate total dissolved solids (mg/L) in a sample by multiplyuig conductivity (in
micromhos per centimeter) by an empirical factor. This factor may vary firom
0.55 to 0.9, depending on the soluble components of the water and on the
temperamre of measurement. Relatively high factors may be required for saUne or
boUer waters, whereas lower factors may apply where considerable hydroxide or
free acid is present. Eventiioughsample evaporation results intfiechange of

143
 bicarbonate to carbonatetfieempirical factor is derived for a comparatively
constant water supply by dividing dissolved sotids by conductivity.
Conductivity is one of several criteria used to evaluate water for its many uses in a
chemistry laboratory. There aretiireespecification levels forreagentwater. Types I, H,
and m. These levels rangefiromType I witii no detectable analytes to Type HI which is
suitable for washmg and quaUtative analysis. The conductivities for each are as foUows:
Type I < 0 1 pmho/cm at 25°C; Type fl = 1; and Type HI = 10 These levels are achieved
by subntitting water to different purification technologies such as distillation, deionization,
carbon absorption, and several others.
1.0 Scope and AppUcation

1.1 This method is applicable to drinking, surface, and saline water, domestic and
industrial wastes and acid rain (atmospheric deposition).
2.0 Summary of Method

2.1 The specific conductance of a sample is measured by use of a self-contained

conductivity meter, Wheatstone bridge-type, or equivalent
2.2 Samples are preferable analyzed at 25°C. If not,temperaturecorrections are made
andresultsreported at 25°C.
3.0 Comments

3.1 Instmment must be standardized with KQ solution before daUy use.

3.2 Conductivity cell must be kept clean.
3.3 Field measurements with comparable instruments are reliable.
3.4 Temperature variations and correctionsrepresentthe largest source of potential
error.
4.0 Sample Handling and Preservation
4.1 Analyses can be performed either in the field or laboratory.
4.2 If analysis is not completed within 24 hours of sample coUection, sample should
be fUtered through a 0.45 nticronfilterand stored at 4°C. FUter and apparams
must be washed with high quaUty distiUed water and pre-rinsed with sample
before use.

5.0 Apparams
5.1 Conductivity Meter, preferably with temperature compensation
15.1.1 YSI Model J2|
5.1.2 Orion Model 160
5.1.3 If the conductivity meter does not havetemperamrecompensation, use
correction factors in Section 9.
5.1.4 Conductivity can also be determined by measuringresistanceusing a multi
tester and the procedure outUned in section 8.1.

144
6.0 Reagents

6.1 Conductivity water: Prepared reagent-grade water. The conductivity should be

smaU compared totiievalue being measured.
6.2 Stantod potassium chloride solution, KCl, O.OIOOM. Dissolve 745.6 mg
anhydrous KCl m conductivity water and dtiute to 1000 mL in a class A
volumetnc flask at 25°C. This is tiie standardreferencesolution which at 25°C
has a conductivity of 1412 pmhos/cm. It is satisfactory for most samples when
the ceU has a constant between 1 and 2 cm-i. Store m a glass-stoppered
borosiUcate glass bottie.
7.0 CeU CaUbration

7.1 The analyst should use tiie standard potassium chloride solution (6.2) and Table I
to check tfie accuracy oftfieceU constant and conductivity bridge.
8.0 Procedure

8.1 Measure by Resistance

8.1.1 AUow samples to come to room temperature (23 to 2TC), if possible.
8.1.2 Determine the temperamre of samples withui 0.5°C. Iftiietemperatureof
the samples is not 25°C, maketemperaturecorrection in accordance witfi tfie
instmction in Section 9 to convertreadingto 25°C.
8.1.3 Determination of ceU constant: Rinse conductivity ceU with at least three
portions of O.OIM KCl solution. Adjust temperamre of a fourth portion to
25.0 ± 0.1°C. If a conductivity meter displays resistance, R, ohms,
measure resistance of this portion and note temperature. Compute ceU
constant, C:

C (cm-i) = (OOOl 412)(RKCI)[1 + 0.019(t - 25)]

where:
RKCI = measuredresistance,ohms, and
t = observed temperature, °C.
Rinse ceU with one or more portions of sample. Adjust temperature of a final
portion to about 25°C. Measure sampleresistanceand note temperature to ±0.1 °C
8.2 Conductivity meters often indicate conductivity directiy. Commercial probes
commonly contain a temperature sensor. With such instmments,rinseprobe three
times with O.OIOOM KCl (6.2), as above.
8.2.1 YSI model 32 "
8.2.1.1 Place approximately 50 ml of sample in a 1(X) ml beaker. Place
conductivity probe in sample and stir briefly.
8.2.1.2 Rotatetemperaturecompensation control to desired % / oc. Some
common values are given intfieintroduction. The temperature
compensation has already been set and does not need to be
adjusted.
8.2.1.3 Tum meter function knob from OFF to TEMPERATURE and wait
for a stable reading. Tum the knob two spaces to
CONDUCTANCE.

145
 8.2.1.4 Rotate range switeh to the lowest range which wtil give a reading.
If a "1" appears, the conductivity is highertiiantfiatrange. If a
"u" appears,tiieconductivity is lower. NOTE: On tiie 0.1 to 2
mho range, allow extra time for stabtiization.
8.2.2 Model 160 (Orion)
8.2.2.1 Selection of range
8.2.2.1.1 Manual Range Selection: Switch meter OFF, switch
meter ON whUe pressing the "DOWN" scroti key at tiie
same time. Wait approximately 2 seconds for LCD
display segment test completion. Now, by successive
depressions of the S/cm key, you select the several
conductivity ranges of the meter. Start with the lowest
range. An OFL display indicates that your value is out
of range (OVERFLOW).
8.2.2.1.2 Automatic Range Selection. You can switch from
manual to automatic measuring range selection as
follows: Switeh meter OFF, switch meter ON whUe
pressing the "UP" scroti key at the same time. Wait for
LCD display segmenttestcompletion. The meter is
now set fortiieAUTORANGING mode.

9.0 Calculation
9.1 Thesetemperaturecorrections are based on the standard KCl solution.
9.1.1 If the temperature of the sample is below 25°C, add 2% of thereadingper
degree.
9.1.2If the temperature is above 25°C, subtract 2% oftfiereadingper degree.
9.2 Reportresultsas Specific Conductance, mhos or pmho / cm at 25*^^. ~

10.0 Accuracy
10.1 In a single laboratory (EMSL) using surface water samples witii an average
conductivity of 536 umhos/cm at 25°C,tiiestandard deviation was ± 6.

Table I: Temperature of OOIM KCl

versus conductivity values.

Conductivity 0.01 M KCl

°C pmhos/cm
21 1305
22 1332
23 1359
24 1386
25 1413
26 1441
27 1468
28 1496

References

146
Orion Research Incorporated. (1990). Model 160 Conductivity Meter Instruction Manual.
Model 160. Orion Research Inc., Laboratory Products Group, The Scrafft Center,
Boston, Ma.
American PubUc Health Association. (1992). Standard Methods for the Exantination of
Water and Wastewater. 18tiied. Method 2510. American Pubtic Health Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 2-44.
Urtited States Environmental Protection Agency. (1992). Metfiods for Chemical Analvsis
of Water and Wastes. Method #120.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincirmati, Oliio.

147
METHOD #: 335.2 Approved for NPDES (Technical Revision 1980)
TITLE: Cyanide, Total (Titrimetric; Spectrophotometric)
ANALYTE:
Cyanide, CN

INSTRUMENTATION: Spectrophotometer
INTRODUCTION
"Cyanide"refersto aU of the CN groups in cyartide compoundstfiatcan be determined as
the cyartide ion, CN-, by the methods used. The cyartide compounds ui which cyanide can
be obtained as CN are classed as simple and complex cyartides.
Sunple cyanides are represented bytfieformula A(CN)x, where A is an aUcaU (sodium,
potassium, ammonium) or a metal, and x, the valence of A, is tfie number of CN groups.
In aqueous solutions of sunple alkali cyanides, the CN group is present as CN and
molecular HCN, theratiodepending on pH and the dissociation constant for molecular
HCN (pKa = 9.2). In most natural waters HCN greatiy predominates. In solutions of
sirnple metal cyartides, the CN group may occur also in the form of complex metal-cyartide
artions of varying stabiUty. Many simple metal cyartides are sparingly soluble or almost
insoluble [CuCN, AgCN, Zn(CN)], but they form a variety of highly soluble, complex
metal cyartides in the presence of alkaU cyartides.
Complex cyartides have a variety of formulae, but the alkaU-metalUc cyartides normaUy can
berepresentedby AyM(CN)x. In this formula, Arepresentsthe alkaU present y times, M
the heavy metal (ferrous and ferric iron, cadntium, copper, rtickel, stiver, zinc, or others),
and X the number of CN groups; x is equal to the valence of A taken y times plus that of the
heavy metal. Irtitial dissociation of each of these soluble, alkaU-metallic, complex cyartides
yields an artion that is the radical M(CN)xy-. This may dissociate further, depending on
several factors, with the Uberation of CN- and consequent formation of HCN.
The great toxicity to aquatic life of molecular HCN is weU known; it is formed in solutions
of cyanide by hydrolyticreactionof CN with water. The toxicity of CN- is less than that of
HCN; it usually is unimportant because most of thefreecyartide (CN group present as CN
or as HCN) exists as HCN, as the pH of most natural waters is substantially lower than the
pKa for molecular HCN (9.2). The toxicity to fish of most tested solutions of complex
cyartides is attributable mainly to the HCNresultuigfromdissociation of the complexes.
Analytical distinction between HCN and other cyartide species in solutions of complex
cyanides is possible. Evidence has suggested one way of effectively ueating waters witii
fish life and the presence of HCN is tiie addition of a nickel solution. For exaniple, at pH
7.5, a solution of nickel has an HCN level of 0.08 mg /L and a solution contauting no
nickel has a level of 1.1 mg/L. Therefore, tfie addition of nickel to a waterresourcehas a
profound effect on its toxicity).

Cyanides gain access to the water environment through the discharge of rinse waters firom
plating operations andfromrefineryand coal-coking wastewaters. The cyartide ion has a
relatively short half-Ufe because it can serve as a source of energy for aerobic bacteria,
provided the concentration is kept below its toxic threshold to them. For this reason, it
should be of Uttie concem where biological treatment systems are employed uitfied^atment

148
of municipal wastes or where several days detention has occurred in namral waters. The
standard of 0.2 mg/1 undoubtedly is included to protect against mdustries witii direa
discharges to natural waters.

1.0 Scope and Application

1.1 This metiiod is applicable totiiedetermination of cyanide in drinking, surface and

saline waters, domestic and industrial wastes.
1.2 Thetitrationprocedure using stiver rtitrate v^dth
p-dimethylamino-benzal-rhodanine indicator is used for measuring concentrations
of cyanide exceeding 1 mg/L (0.25 mg/250 mL of absorbing tiquid).
1.3 The colorimetric procedure is used for concentrations below 1 mg/L of cyanide
and is sensitive to about 0.02 mg/L.
1.4 This inethod is restricted to use by or under the supervision of analysts
experienced in the operation of hazardous materials.
2.0 Summary of Method

2.1 The cyartide as hydrocyartic acid (HCN) isreleasedfiromcyanide complexes by

means of, areflux-distiUationoperation and absorbed in a scmbber containing
sodium hydroxide solution. The cyartide ion in the absorbing solution is then
determined by volumetrictitrationor colorimeUicaUy.
2.2 In the colorimeuic measurement the cyanide is converted to cyanogen chloride,
CNCl, by reaction with chloramine-T at a pH less than 8 without hydrolyzing to
the cyanate. After thereactionis complete, color is formed on the addition of
pyridine-pyrazolone or pyridine-barbituric acid reagent The absorbance isreadat
620 nm when using pyridine- pyrazolone or 578 nm for pyridine-barbituric acid.
To obtain colors of comparable intensity, it is essential to have the same salt
content in both the sample and the standards.
2.3 Thetitrimetricmeasurement uses standard solution of stiver nitrate to ticate
cyartide in the presence of a stiver sensitive indicator.
3.0 Sample HandUng and Preservation
3.1 The sample should be coUected in plastic or glass botties of 1titeror larger size.
AU botties must be thoroughly cleansed andtiioroughlyrinsedtoremovesoluble
materialfiromcontainers.
3.2 Oxidizing agents such as chlorine decompose most of the cyanides. Test a drop
of the sample with potassium iodide-starch test paper (KI-starch paper); a blue
color indicates the need for treatment Add ascorbic acid, a few crystals at a time,
untU a drop of sample produces no color on the indicator paper. TTien add an
additional 0.06 g of ascorbic acid for eachtiterof sample volume.
3.3 Samples must be preserved witii 2 mL of 10 N sodium hydroxide pertiterof
sample (pH > or = 12) attfietime of coUection.
3.4 Samples should be analyzed asrapidlyas possible after coUection. If storage is
requu^, tfie samples should be stored in arefrigeratoror in an ice chest fiUed
with water and ice to maintaintemperatureat 4°C.

149
4.0 Interferences

4.1 Interferences are elimuiated or reduced by usingtfiedistUlation procedure

described in Procedure 7.1, 7.2 and 7.3.
4.2 Sulfides adversely affect the colorimetric andtitrationprocedures. Samples that
contain hydrogen sulfide, metal sulfides or otiier compounds that may produce
hydrogen suUide during the distUlation should be distiUed bytfieoptional
procedure described in Procedure 7.2.
4.3 Fatty acids wiU distUl and form soaps undertiiealkalinetitrationconditions,
malang the end point almost impossible to detect.
4.3.1 Acidify the sample with acetic acid (1 + 9) to pH 6.0 to 7.0. Caution:
This operation must be performed ui the hood and the sample left there
untU it can be made alkaline again after the extraction has been performed.
4.3.2 Extract with iso-octane, hexane, or chloroform (preference in order
named) with a solvent volume equal to 20% of the sample volume. One
extraction is usuaUy adequate to reduce the fatty acids below the
interference level. Avoid multiple extractions or a long contact time at low
pH in order to keep the loss of HCN at a minimum. When the extraction
is completed, imrrlediately raise the pH of the sample to above 12 with
NaOH solution.
4.4 Highresultsmay be obtained for samples that contain nitrate and/or rtitrite.
During the disttilation nitrate and rtitrite wiU form rtitrous acid which wiU react
with some organic compounds to form oximes. These compounds formed wiU
decompose under test conditions to generate HCN. The interference of rtitrate and
rtitrite is eliminated by pretreatment with sulfantic acid.

5.0 Apparams
5.1 Reflux distiUation apparams such as shown in figure 1 or 2. The boiling flask
should be of 1 liter size with irtiet mbe and provision for condenser. The gas
absorber may be a Fisher-MUUgan scmbber.
5.2 Microburet, 5.0 mL (for titration).
5.3 Spectrophotometer suitable for measurements at 578 nm or 620 nm with a 1.0 cm
ceU or larger.
5.4 Reflux distiUation apparams for sulfide removal. The boiUng flask same as 6.1.
The sulfide scmbber may be a Wheaton Bubber #709682 witii 29/42 joints, size
100 mL. The air inlet mbe should not be fritted. The cyanide absorption vessel
should be identical to tiie sulfide scmbber. The air inlet mbe should be fritted.
5.5 Flow meter, such as Lab Crest with stainless steel float (Fisher 11-164-50).

6.0 Reagents
6.1 Sodium hydroxide solution, 1.25 N: Dissolve 50 g of NaOH in distiUed water
and dtiute to 1titerwitfi distUled water.
6.2 Lead acetate: Dissolve 30 g of Pb (C2H302)-3H20 ui 950 mL of distiUed water.
Adjust the pH to 4.5 with acetic acid. DUute to 1 titer.
6.3 Sodium Hydroxide Solution 0.25 N: Dissolve 10 g of NaOH ui distiUed water
and dtiute to 1titerwitfi distUled water.
6.4 Sulfuric acid; 18N: Slowly add 500 mL of concentrated H2SO4 to 500 mL of
distiUed water.

150
 6.5 Sodiumdihydrogenphosphate, 1 M: Dissolve 138 gof NaH2P04H20 in 1 titer
of distUled water. Refrigerate this solution.
6.6 Stock cyanide solution: Dissolve 2.51 g of KCN and 2 g KOH in 900 mL of
distiUed water. Standardize witfi 0.0192 N AgN03. DUute to appropriate
concentration so that 1 mL = 1 mg CN.
6.7 Standard cyanide solution, intermediate: DUute 100.0 mL of stock (ImL = 1 mg
CN) to 1000 mL v^itfi distiUed water (1 mL = 100.0 pg).
6.8 Working standard cyanide solution: PreparefreshdaUy by dUuting 100.0 mL of
intermediate cyanide solution to 1000 mL with distiUed water and store in a glass
stoppered bottie. 1 mL = 10.0 g CN.
6.9 Standard stiver nitrate solution, 0.0192 N: Prepare by cmshing approximately 5
g AgNOs crystals and dryuig to constant weight at 40°C. Weigh out 3.2647 g of
dried AgNO^, dissolve in distUled water, and dtiute to 1000 mL (1 mL = 1 mg
CN).
6.10 Rhodanine indicator: Dissolve 20 mg of p-dimethyl-amino-benzalrhodanine in
1(X) mL of acetone.
6.11 Chloramine T solution: Dissolve 1.0 g of white, water soluble Chloramine T in
100 mL of distiUed water and refrigerate unttireadyto use. Prepare fresh daily.
6.12 Color Reagent:One of the foUowing may be used:
6.12.1 Pyridine-Barbituric Acid Reagent: Place 15 g of barbimric acid in a 250
niL volumetric flask and add just enough distUled water to wash the sides
of the flask and wet the barbituric acid. Add 75 mL of pyridine and mix.
Add 15 mL of cone. HCl, mix, and cool to room temperature. DUute to
250 mL with distiUed water and mix. Thisreagentis stable for
approximately six months if stored in a cool, dark place.
6.12.2 Pyridine-pyrazolone solution:
6.12.2.1 3-Methyl-1 -phenyl-2-pyrazolin-5-onereagent,saturated
solution: Add 0.25 g of 3-methyl-l-phenyl-2-pyrazoUn-5-one
to 50 mL of distiUed water, heat to 60°C witii stirring. Cool to
room temperature.
6.12.2.2 3,3'Dimetiiyl-l, r-diphenyl-[4,4'-bi-2 pyrazoline]-5,5'dione
(bispyrazolone): Dissolve 0.01 g of bispyrazolone in 10 mL of
pyridine.
6.12.2.3 Pour solution (6.12.2.1) through non-acid-washedfilterpaper.
CoUect the fUtrate. Through the same fUter paper pour solution
(6.12.2.2) coUecting thefiltratein the same container as fUtrate
from (6.12.2.1). Mix until thefilu-atesare homogeneous. The
mixedreagentdevelops a pink color but this does not affect the
color production with cyanide if used within 24 hours of
preparation.
6.13 Magnesium chloride solution: Weigh 510 g of MgCl2-6H20 into a 1000 mL
flask, dissolve and dtiute to 1 liter witfi distiUed water.
6.14 Sulfantic acid, NH2SO3H.

7.0 Procedure
7.1 For samples without sulfide.
7.1.1 Place 500 mL of sample, or an aliquot dUuted to 500 mL ui tiie 1 titer
botiing flask. Pipet 50 mL of sodium hydroxide (6.1) uito tiie absorbing
mbe. Iftfieapparams in Figure 1 is used, add distiUed water until tfie

151
 spiral is covered. Connect tfie botiing flask, condenser, absorber, and trap
m tiie train. (Figure 1 or 2).
7.1.2 Start a slow stream of aU enteringtfieboUuigflaskby adjusting tiie
vacuum source. Adjust the vacuum so that approximately two bubbles of
air per second enters tiie boUuigflasktiuroughtiieau- utiet mbe. Prroceed
to 7.4.
7.2 For samples that contain sulfide.
7.2.1 Place 500 mL of sample, or an aUquot dUuted to 500 mL in tfie 1 Uter
boiUng flask. Pipet 50 nJ^ of sodium hydroxide (6.1) to tiie absorbing
mbe. Add 25 mL of lead acetate (6.2) totfiesulfide scmbber. Connect
the botiing flask, condenser, scmbber and absorber m the train. The flow
meter is connected totfieoutiet mbe oftiiecyanide absorber.
7.2.2 Start a stream of air entering the boiling flask by adjusting the vacuum
source. Adjust the vacuum so that approximately 1.5titersper minute
enterstfiebotiingflasktiiroughthe air utiet mbe. The bubbleratemay not
remain constant while heat is being appUed to the flask. It may be
necessary toreadjustthe airrateoccasionaUy. Proceed to 7.4.
7.3 If samples contain NO3, and / or NO2, add 2 g of sulfamic acid solution (6.14)
after the air rate is set through the air irtiet mbe. Mix for 3 minutes prior to
addition of H2SO4.
7.4 Slowly add 50 mL 18 N sulfuric acid (6.4) through the air inlet ml)e. Rinse the
mbe with disttiled water and aUow the airflow to mix theflaskcontents for 3 min.
Pour 20 mL of magnesium chloride (6.13) into the air irtiet and wash down with a
stream of water.
7.5 Heat the solution to boiUng. Reflux for one hour. Tum off heat and continue the
airflow for at least 15 minutes. After cooling the botiingflask,disconnect
absorber and close off the vacuum source.
7.6 Drain the solutionfromthe absorber into a 250 mL volumetric flask. Wash the
absorber with distiUed water and add the washings to the flask. DUute to the mark
with distUled water.
7.7 Withdraw 50 mL or less of the solution from theflaskand transfer to a 1(X) mL
volumetric flask. If less than 50 mL is taken, dtiute to 50 mL with 0.25 N
sodium hydroxide solution (6.3). Add 16.0 mL of sodium phosphate solution
(6.5) and ntix.
7.7.1 Pyridine-barbituric acid metiiod: Add 2 mL of chloramine T (6.11) and
mix. See Note 1. After 1 to 2 minutes, add 5 mL of pyridine-barbituric
acid solution (6.12.1) and ntix. Dilute to mark with ctistiUed water and mix
again. AUow 8 minutes for color development thenreadabsorbance at
578 nm in a 1 cm ceU within 15 minutes.
7.7.2 Pyridine-pyrazolene method: Add 0.5 mL of chloramine T (6.11) and
mix. See Note 1 and 2 below. After 1 to 2 minutes add 5 mL of
pyridine-pyrazolone solution (6.12.2) and mix. DUute to mark with
distiUed water and mix again. After 40 minutesreadabsorbance at 620
nm in a 1 cm ceU.
NOTE 1: Some distiUates may contain compounds that have a chlorine
demand. One minute after the addition of chloramine T,testfor residual
chlorine with Kl-starch paper. If the test is negative, add an additional 0.5
mL of chloramine T. After one minute, recheck tiie sample.
NOTE 2: Moretfian05. mL of chloramine T wiU prevent the color from
developing with pyridine-pyrazolone.

152
 7.8 Standard curve for samples without sulfide.
7.8.1 Prepare a series of standards by pipeting suitable volumes of standard
solution (6.8) uito 250 mL volumetric flasks. To each standard add 50
mL of 1.25 N sodium hydroxide and dilute to 250 mL witii distiUed water.
Prepare as follows:

ML of Working Standard Solution Cone, g CN

(1 mL = lOpg CN) per 250 mL
0 BLANK
1.0 10
2.0 20
5.0 50
10.0 100
15.0 150
20.0 200

7.8.2 It is not imperative that aU standards be distiUed in the same manner as the
samples. It is recommended that at least two standards (a high and low) be
distiUed and compared to simtiar values on the curve to insure that the
distillation techrtique isreUable.If distUled standards do not agree within
±10% of the undistiUed standards the analyst should find the cause of the
apparent error before proceeding.
7.8.3 P^pare a standard curve by plotting absorbance of standard versus
cyartide concentrations.
7.8.4 To check the efficiency of the sample distiUation, add an increment of
cyanidefiromeither the intermediate standard (6.7) or the working
standard (6.8) to 5(X) mL of sample to insure a level of 20 g/L. ftoceed
with the analysis as in Procedure (7.1.1).
7.9 Standard curve for samples with sulfide.
7.9.1 It is imperative that aU standards be distiUed in the same manner as the
samples. Standards distUled by this metiiod wiU give a linear curve, but
as the concentration increases, the recovery decreases. It is recommended
that at least 3 standards be distiUed.
7.9.2 Prepare a standard curve by plotting absorbance of standard versus
cyanide concentrations.
7.10 Titrimetric method.
7.10.1 If the sample contains more than 1 mg/L of CN, transfertfiedistiUate or a
suitable aliquot dUuted to 250 ml, to a 500 mL Erlenmeyer flask. Add 10-
12 drops of the benzalrhodanine indicator.
7.10.2 Titrate with standard stiver nitrate to the first change in colorfromyeUow
to brownish-pink. Titrate a distUled water blank using the same amount of
sodium hydroxide and indicator as in the sample.
7.10.3 The analyst should famiUarize himself witfi the end point of the titration
and the amount of indicator to be used before actuaUytitratingthe
samples.

8.0 Calculation
8.1 Iftfiecolorimetric procedure is used, calculatetfiecyanide, ui pg/L, uitfieoriginal
sample as foUows:

153
 CN, ng/L = (A * 1,000) * (50)
5- T
where:
A = pg CN readfromstandard curve
B = mL of original sample for distiUation
C = mL taken for colorimetric analysis
8.2 Usuig thetitrimetricprocedure, calculate concentration of CN as foUows:
CN, mg/L = (A - B ) * 1,000 250
mLorig sample X mL of aUquot titrated
where:
A = volume of AgNOs fortitrationof sample.
B = volume of AgNOs fortitrationof blank.

9.0 Accuracy

9.1 In a single laboratory (EMSL), using mixed industrial and domestic waste
samples at concentrations of 0.06, 0.13, 0.28 and 0.62 mg/L CN, the standard
deviations were ±0.005, ±0.007, ±0.031 and ±0.094, respectively.
9.2 In a single laboratory (EMSL), using mixed industrial and domestic waste
samples at concentrations of 0.28 and 0.62 mg/L CN, recoveries were 85% and
102%, respectively.
References

Jenkins, D. and Snoeyink, V. L. (1980). Water Chemistry. John Wtiey & Sons, Inc.,
New York, N.Y., 222.
Sawyer, C. N., and McCarty, P. L. (1994). Chentistry for Environmental Engineering.
4tii ed. McGraw-Hill, Inc., New York, N.Y., 637.
American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18tiied. Method 45(X). American PubUc Health Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 4-18.
United States Envuronmental Protection Agency. (1992). Metfiods for Chentical Analysis
of Water and Wastes. Metiiod #335.2. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincumati, Ohio.

154
ALLIIIN CONDENSER --CONNECTING TUBING

AIR INLET TUDE

SUCTION

ONE LITER
BOILING FLASK
GAS ADSORBER

FIGURE 1

CYANIDE DISTILLATION APPARATUS

155
 COOLING WATER

TO LOW VACUUM
SOURCE
INLET TUBEv

-- ADSORBER

DISTILLING FLASK

HEATER-*-

FIGURE 2
CYANIDE DISTILLATION APPARATUS

156
METHOD #: 360.1 Approved for NPDES Gssued 1971)
TITLE: Oxygen, Dissolved (Membrane Electrode)
ANALYTE:
Oxygen, O2

INSTRUMENTATION: Probe
E^miODUcnoN
Dissolved oxygen (DO) levels ui natural and wastewaters depend ontiiephysical,
chentical, and biochemical activities uitfiewater body. The analysis for DO is a keytestui
water poUutiori and waste treatment process control. Dissolved Oxygen controls the rate
and extent of simple chemical oxidationreactions,the kinds and abundances of aquatic
biota, and water quaUty. Concentrations are affected by temperature (inc temp, decDO),
other dissolved materials, pressure (inc. press, inc DO), oxygenation processes, and
oxygen demand.

Two methods for DO analysis are avatiable: the Winkler or iodometric method and its
modifications and the electrometric method using membrane elecdxxies. The iodometric
method is atitrimetricprocedure based on the oxidizing property of DO whUe the
membrane electrode procedure is based on therateof diffusion of molecitiar oxygen across
a membrane. The choice of procedure depends on the interferences present, the accuracy
desired, and, in some cases, convenience or expedience.

Various modifications of the iodometric metiiod have been developed to eliminate or

minimize effects of interferences; nevertheless, the method still is inappUcable to a variety
of industrial and domestic wastewaters. Moreover, the iodometric method (wet chemistry
method) is not suited for field testing and cannot be adapted eastiy for continuous
monitoring or for DO detemtinations in sim.
On the other hand, membrane electrodes provide an exceUent method for DO analysis in
polluted waters, highly colored waters, and strong waste effluents. They are recommended
for use especiaUy under conditions that are unfavorable for use of the iodometric method,
or when that test and its modifications are subject to serious errors caused by interferences.
Membrane electrodes are commerciaUy avaUable in some variety. In aU these instruments
tfie "diffusion current" is Unearly proportional to the concentration of molecular oxygen.
The current can be converted easUy to concentration units (e.g., miUigrams pertiter)by a
number of calibration procedures. Field and laboratory measurements arereUablewitfi
properly caUbrated instruments andresultscan bereturnedinrealtune.

1.0 Scope and Application

1.1 The probe method for dissolved oxygen is recommended for tiiose samples
contauiuig materials which uiterfere withtfiemodified Winkler procedure such as
sulfite,titiosulfate,polythionate, mercaptans, free chlorine or hypochlorite,
orgartic substances readtiy hydrolyzed in alkaline solutions, fiee iodine, intense
color or turbidity and biological floes.
1.2 The probe metiiod is recommended as a substitute fortfiemodified Wutider
procedure in monitoring of streams, lakes, outfalls, ete., where it is desu-ed to

157
 obtaui a continuous record of tiie dissolved oxygen content oftfiewater under
observation.
1.3 The probe metiiod may be used as a substimte fortiiemodified Winkler procedure
in BOD determinations
2.0 Summary of Method

2.1 The most common instrumental probes for determination of dissolved oxygen in
water are dependent upon electrochemical reactions. Under steady-state
conditions,tfiecurrent or potential can be correlated witfi DO concentrations.
Interfacial dynamics attfieprobe-sample interface are a factor ui probe response
and a significant degree of interfacial turbulence is necessary. For precision
performance, turbulence should be constant.
3.0 Sample Handting and Preservation

3.1 Where possible, coUect the sample in a 3(X) ml BOD incubation bottie.
3.2 At time of sampting, the sample temperature should be recorded as precisely as
possible.
3.3 Do not delay the determination of dissolved oxygen in samples having an
appreciable iodine demand or containing ferrous iron.
4.0 Interferences
4.1 Dissolved orgartic materials are not known to interfere in the output from
dissolved oxygen probes.
4.2 Dissolved inorgartic salts are a factor in the performance of dissolved oxygen
probe.
4.2.1 Probes with membranes respond to partial pressure of oxygen which in
tum is a function of dissolved inorganic salts. Conversion factors for
seawater and brackish waters may be calculated from dissolved oxygen
saturation versus salinity data. Conversion factors for specific inorgartic
salts may be developed experimentaUy. Broad variations in the kinds and
concentrations of salts in samples can make the use of a membrane probe
difficitiL
4.3 Reactive compounds can interfere with the output ortiieperformance of dissolved
oxygen probes.
4.3.1 Reactive gases which pass throughtiiemembrane probes may interfere.
For example, chlorine wiU depolarizetfiecatfiode and cause a high
probe-output. Long-term exposures to chlorine wtil coat the anode with
the chloride of the anode metal and eventuaUy desensitize the probe.
AUcaline samples in which free chlorine does not exist wtil not uiterfere.
Hydrogen sulfide ynH interfere witfi membrane probes iftiieappUed
potential is greater than the half-wave potential oftfiesulfide ion. If tfie
appUed potential is lesstiianthe half-wave potential, an uiterfering reaction
wtil not occur, but coatuig of the anode with the sulfide of the anode metal
can take place.
4.4 Dissolved oxygen probes aretemperaturesensitive, and temperature
compensation is normaUy provided by the manufacturer. Membrane probes have
atemperaturecoefficient of 4 to 6 percent C dependent upontiiemembrane
employed.

158
5.0 Apparams
5.1 YeUow Springs Instrument (YSI) Model 59 or anotiier equivalent dissolved
oxygen probe.
6.0 CaUbration:
6.1 Three separate caUbration techrtiques are discussed here: caUbration in air,
catibration in air-saturated water, and caUbration by Winkler titration. Choose the
one technique which bestfitsyour application. Calibration in air is the sunplest
and most accurate method of caUbration.
6.1.1 To caUbrate in air.
6.1.1.1 Place a prepared probe in air at 1(X)%relativehumidity. To
achieve this, BOD probes can be placed in a BOD bottie with
approximately 1" of water.
6.1.1.2 Switch to CALIBRATE. The display wiU read:
CALIBRATE IN PERCENT?
6.1.1.3 Select percent calibration by pressing CONFIRM. A display
similar to the one below wiU be shown.
ENTER CAL VALUE
LAST = 96.4%

6.1.1.4 Using t and i , adjust the number totfiecalibration value

determined from the altimde or pressure chart on the back of the
Model 59. SKIP setstiievalue to 100.0%; and shows tiie
foUowing display:
ENTER CAL VALUE
SAT. = 100%
you may wish to do this to bringtiiesetting quickly to a value
near the one you want, then continue with T and i . Pressing
SKIP again setstiievalue to 0.0%. Pressing SKIP atitirdtime
brings it back to the last calibration value. The % caUbration
values used by the ESL is 88.5% because of the
elevation of Lubbock.
6 1 1 5 When you have adjustedtiiereadingtotfiedesu:ed value Get's say
you chose 98.0%), press CONHRM. The instmment wtil
display tiie legend "Please wait" for a few seconds. Then it wtil
display:
CALIBRATED TO
98.0%
6.1.1.6 You are nowreadyto measure dissolved oxygen.
6.1.i (!:atibration in Au--Saturated Water [ [^ [ ] ~
6.1.2.1 A second altemative for caUbration is to caUbrate m au--saturated
water. This procedure istfiesame astiieprocedure for caUbration
in air excepttfiattiiecalibration medium is sauirated water ratfier
tfian saturated air. To caUbrate ui air-sattirated water:
6.1.2.2 AU-saturate a volume of water (300 to 500 mL) by aerating for at
least 15 minutes at arelativelyconstant temperamre.

159
 6.1.2.3 Place the probe intfiesample and provide adequate stirring (at
least 1 foot per second). Switch to O2-TEMP and observe
temperature and oxygen readings for stabiUty. It may take 5
minutes fortfietemperature to come to equiUbrium If you have
just turnedtiieinstrument on, aUow 15 ntinutes fortfiesystem to
equiUbrate.
6.1.2.4 Switeh to CALIBRATE, select percent caUbration,tiienadjust tfie
number intfiedisplay totfiecorrect value based on current
barometric pressure or altimde and press CONFIRM.
6.1.3 CaUbration by Wutider Titration
6.1.3.1 A third altemative for caUbration is to caUbrate to a known oxygen
value detennined by a Winklertitration.Winklertitrationis not
performed in this manual, but information conceming this titration
techrtique can be found in Standard Methods.
6.2 Fresh water: For unpolluted samples where interfering substances are absent,
caUbrate in the test solution or distiUed water, whichever is more convenient
6.3 Salt water: CaUbrate directiy with samples of seawater or waters having a
constant salt concentration in excess of 1000 mg/L.
6.4 Fresh water containing pollutants or interfering substances: CaUbrate with
distiUed water because erroneous results occur with the sample.
6.5 Salt water containing poUutants or interfering substances: Calibrate with a sample
of clean water containing the same salt content as the sample. Add a concentrated
potassium chloride (KQ) solution to distiUol water to produce the same specific
conductance (See conductivity in this manual) as that in the sample. For poUuted
ocean waters, calibrate with a sample of unpolluted seawater.
6.6 Estuary water containing varying quantities of salt: CaUbrate with a sample of
uncontaminated seawater or distilled or tap water. Detemtine sample chloride or
salt concentration and revise caUbration to account for change of oxygen solubiUty
in the esmary water.

7.0 Procedure
7.1 WhUe the selector switeh is set to 02-Ten^, place the prepared probe in the
sample to be measured.
7.2 AUow 3-5 minutes for temperature equiUbration.
7.3 Begin stirring at least 30 seconds before taking readings.
7.4 Observe reacUngs for stabiUty and record measurements.

8.0 Calcitiation
8.1 Recorded measiu^ments are direct values.

9.0 Accuracy
9.1 Manufacturer's specification claim 0.1 mg/LrepeatabUity(precision) witii ± 1 %
bias.

References
American Pubtic Healtii Association. (1992). Standard Metiiods fortfieExamination of
Water and Wastewater. 18tiied. Metiiod 4500. American PubUc Healtii Association,

160
 Americ^a. Water Works Association, and Water Environn^nt Federation. Washington

YeUow Springs Instmment Co., Inc YSI MOHPI SO T^JCO^I ^ rx.

^M. YSI Model 59. YeUow g g ^ i ^ l ^ ^ ^ e r g E V ^ ^ ^ ^ ^ ^ ^ ^ ^

161
 METHOD #: 130.2 Approved for NPDES (Editorial Revision 1978,1982)
TITLE: Hardness, Total (mg/L as CaCCb) (Titrimetric, EDTA)
ANALYTE:
Hardness, Total (mg/L as CaCOs)
INSTRUMENTATION: Titration
INTRODUCTION
Ori^aUy, water hardness was understood to be a measure of tiie capacity of water to
precipitate soap. Hardness is caused by polyvalent cations. Such ions are capable of
reactmgwitii soap to form precipitates and witii certain anions present intfiewater, to form
scale. 1 he pnncipal hardness-causuig cations are calcium, magnesium, strontium, ferrous
uon, and mariganous ions. These cations, plus tiie most important anions witfi which tiiey
are associated, are shown m Table 1 uitiieorder oftiieirrelative abundance m natural
waters. Aluminum and ferric ions are sometimes considered as contributing to tiie
hardness of water. However,tiieirsolubUity is so Umited attiiepH values of namral
waterstiiationic concend^tions are negUgible

The hardness ui water is derived largelyfi-omcontact witiitfiesoU and rock formations

Ram water as it faUs upontiieearth is incapable of dissolvuig tiie tremendous amounts of
soUds found m many namral waters. The abiUty to dissolve is gamed mtiiesoU where
carbon dioxide is released by bacterial action. The soti water becomes highly charged witii
carbori dioxide, which, exists in equiUbrium witii carbonic acid. Undertfielow pH
conditions that develop, basic materials, particularly limestone formations, are dissolved.
Suice Umestone is not pure carbonate but includes impurities such as sulfates, chlorides,
and sUicates, tiiese materials become exposed totiiesolvent action of tiie water as tfie
carbonates are dissolved, and they pass into solution too.
In general, hard waters originate in areas where the topsoti istfiickand Umestone
formations are present. Soft waters originate in areas where the topsoti is thin and
limestone formations are sparse or absent.

Table 1 Principal cations causing hardness in water

and the major artions associated with them
Cations causing hardness Artions

Ca2+ HCO3-
Mg2+ SO42-
Sr2+ Cl-
Fe2+ NO3-
Mn2+ Si032-

When hardness numerically is greater than the sum of carbonate and bicarbonate alkalinity,
that amount of hardness equivalent to the total alkalinity is caUed "carbonate hardness"; the
amount of hardness in excess of this is caUed "noncarbonate hardness." When the hardness
numericaUy is equal to or less than the sum of carbonate and bicarbonate alkalinity, aU
hardness is carbonate hardness and noncarbonate hardness is absent The hardness may

162
range ftom zero to hundreds of rrtilUgrams per Uter, depending on the source and n-eatment
to which the water has been subjected.

The hardness of waters varies considerablyfi-omplace to place. In general, surface waters

are softer than ground-waters. The hardness of waterreflectsthe nature of the geological
formations with which it has been in contact The softest waters are found in the New
England, South Atiantic, and Pacific Northwest states. Iowa, Illinois, Indiana, Arizona,
New Mexico, and the Great Plains states have the hardest waters.
Waters are commortiy classified in terms of the degree of hardness, as foUows:

mg/1 Degree of hardness

0-75 Soft
75-150 Moderately hard
150-300 Hard
300 up Very hard

There are two methods to determine hardness: Metiiod 1, hardness by calculation is

appticable to all waters and yields the higher accuracy. If a mineral analysis is performed,
hardness by calculation can bereported.Metiiod 2,tfieEDTAtitrationmethod measures
the calcium and magnesium ions and may be appUed with appropriate modification to any
kind of water. The procedure described affords a means ofrapidanalysis.

METHOD 1: ,r i
Calculation of tfie hardness caused by each ion is performed by use oftfiegeneral formula
Hardness (ui mg/1) as CaCOs = M2+ (in mg/1) x (50)
eq wt of M2+
where M2+representsany divalent metalUc ion.
Example: Calculatetfiehardness of a water with tfie
foUowing analysis:
mg/1 jngA

Na+—20 CI—-W
Ca2+—15 SO42—16
Mg2+_10 NO3—I
Sr2+—2 Alkalinity—50

Only tfie divalent cations, Ca2+, Mg^+, and Sr2+ cause

hardness:

Cation Equiv.wt. Hardness, mg/1 as CaCQs

Ca2+ 20.0 (15)(50)/(20.0)= 37.5

Mg2+ 12.2 (10)(50)/(12.2)= 41.0
Sr2+ 43.8 (2)(50)/(43.8)=^
Total Hardness = 80.8

163
METHOD 2:
Ethylenediaminetetraacetic acid and its sodium salts (abbreviated EDTA) form a chelated
soluble complex when added to a solution of certain metal cations. If a smaU amount of a
dye such as Eriochrome Black T or Calmagite is added to an aqueous solution containing
calcium and magnesium ions at a pH of 10.0 ± 0.1, the solution becomes wine red. If
EDTA is added as atitrant,the calcium and magnesium wiU be complexed, and when aU of
the magnesium and calcium has been complexwi the solution tumsfromwine red to blue,
marking the end point of thetitration.Magnesium ion must be present to yield a
satisfactory end point. To insure this, a srnaU amount of complexometricaUy neutral
magnesium salt of EDTA is added to the buffer, this automatic;aUy introduces sufficient
magnesium and obviates the need for a blank correction.

The sharpness of the end point increases with increasing pH. However, the pH carmot be
increased indefirtitely because of the danger of precipitating calcium carbonate, CaCOs, or
magnesium hydroxide, Mg(OH)2, and because the dye changes color at high pH values.
The specified pH of 10.0 ± 0.1 is a satisfactory comprontise. A Untit of 5 min is set for the
duration of thetitrationto minimize thetendencytoward CaCOs precipitation.

1.0 Scope and AppUcation. The EDTA Titration is consideredfromhere on.

1.1 This method is appUcable to drinking, surface, and saUne waters, domestic and
industrial wastes.
1.2 The method is suitable for aU concentration ranges of hardness; however, in order
to avoid largetitrationvolumes, use a sample aliquot containing not more than 25
mg CaCOs.
1.3 Automatedtitrationmay be used.
2.0 Summary of Method
2.1 Calcium and magnesium ions in the sample are sequestered upon the addition of
disodium etiiylenediaminetetraacetate(NaiEDTA). The end pouit oftiiereaction
is detected by means of Eriochrome Black T uidicator, which has a red color ui tfie
presence of calcium and magnesium and a blue color when the cations are
sequestered.
3.0 Sample Handling and Preservation
3.1 Cool to 4°C, analyze as soon as possible; or add HNO3 to pH < 2,tiienanalyze
within 6 months.

4.0 Comments
4 1 Excessive amounts of heavy metals can uiterfere,tiiussome process waters can
yield suspect values. This is usually overcome by complexuig tfie metals witii
4 1.1 Routine addition of sodium cyanide solution (Caution: deadly poison) to
prevent potential metaUic mterference is recommended. When higher
concentrations of heavy metals are present, detemtine calcium and
magnesium by a non-EDTA metfiods such as measurement of Ca2+ and

164
 Mg2+ by metals determinations (See Metals uitftismanual) and obtaui
hardness by calculation.
4.2 Suspended or coUoidal organic matter also may mterfere withtiieend pomt.
Elirninate this interference by evaporating the sample to dryness on a steam bath
and heatuig in a muffle furnace at 550°C unttitfieorganic matter is completely
oxidized. Dissolvetfieresiduein 20 mL 1 N hydrochloric acid (HCl), neutralize
to pH 7 with 1 N sodium hydroxide (NaOH), make up to 50 mL witfi distiUed
water, and cool to roomtemperatureand continue according to the general
procedure.
4.3 Titration Precautions: Conducttitrationsat or near normal roomtemperature.The
color change becomes impracticaUy slow as the sample approaches freezing
temperature. Indicator decomposition becomes a problem in hot water. The
specified pH may produce an environment conducive to CaCOs precipitation.
Although thetitrantslowly redissolves such precipitates, a drifting end point often
yields low results. Completion of thetittationwithin 5 min minimizes tiie
tendency for CaCOs to precipitate. The foUowing three methods also reduce the
precipitation loss.

1) DUute sample with distUled water to reduce CaCOs concentration. This

simple expedient has been incorporated in the procedure. If precipitation
occurs at this dilution of 1 + 1, use modification 2) or 3). Using too small a
sample contributes systematic error due to the biuet-reading error.

2) If the approximate hardness is known or is determined by a preliminary

titration add, 90% or more oftitrantto sample, before adjusting pH with
buffer.
3) Acidify sample and stu- for 2 min to expel CO2 before pH adjustment.
Determine alkalirtity to indicate amount of acid to be added

5.0 Apparams
5.1 Standard laboratorytitrimetricequipment

6.0 Reagents
6.1 Buffer solution
6.1.1 If magnesium EDTA is avatiable: Dissolve; 16.9 g NH4CI (Ammonium
chloride) in 143 mL concentrated- NH4OH (Ammonium hydroxide) in a
250 mL volumetric, add 1.25 g of magnesium salt of EDTA and dtiute to
the mark with distilled water. Then go to 6.1.3.
6.1.2 If magnesium EDTA is unavaUable: Dissolve 1.179 g disodium EDTA
(disodium salt of ethylenediaminetetraacetic acid dihydrate, analytical
reagent grade) and 780 mg MgS04*7 H2O magnesium sulfate (or 644 mg
MgCl2*6 H2O magnesium chloride) in 50 mL distiUed water. Add tfiis
solution to a 250 mL volumetric flask contaimng 16.9 g NH4CI and 143 mL
concentrated. NH4OH witii ntixuig and dUute totiiemark witii distiUed
water.
6.1.3 Store m atightiystoppered plastic bottie to prevent loss of ammonia; stable
for approxunately one month. Dispense witii bulb operated pipet Discard

165
 when 1 or 2 mL added to sample faUs to produce a pH of 10.0 ± 0.1 at end
point of titration.
6.1.4 Commercially avaUable "odorless buffers" which are more stable, may be
used.
6.2 Inhibitors: For most waters inhibitors are not necessary. If interfering ions are
present use one of the foUowing:
6.2.1 Inhibitor I: Adjust samples to pH 6 or higher witii buffer or 0.1 N NaOH.
Add 250 mg sodium cyanide (NaCN) in powder form. Add sufficient
buffer to adjust to pH 10.0 ± 0.1 (CAUTION: NaCN is extremely
poisonous. Take extra precautions in its use. Flush solutions containing
this inhibitor down the drain with large quantities of water after insuring
that no acid is present to Uberate volatUe poisonous hydrogen cyanide.)
6.2.2 Inhibitor II: Dissolve 5.0 g Na2S*9 H2O sodium sulfide nonahydrate or
3.7 g Na2S*5 H2O m 100 mL distiUed water. Exclude au- witii tightiy
fitted mbber stopper. This gives sulfide precipitates which may obscure
the end point if large quantities of heavy metis are present IDeteriorates
rapidly through air oxidation.
6.2.3 Inhibitor HI: Dissolve 4.5 g hydroxylamine hydrochloride in 1(X) mL of
95% ethanol or isopropanol.
6.2.4 MgCDTA: Magnesium salt of 1,2-cyclohexanediaminetetraaceticacid.
Add 250 mg per 1(X) ml sample and dissolve completely before adding
buffer solution. Use this complexing agent to avoid using toxic or
odorous inhibitors when uiterfering are present in concentrations that
affect the end point but wiU not contribute significantiy to the hardness
value.
6.3 Indicator: Use a commerciaUy avaUable indicator such as Calmagite indicator
(MaUinckrodt) or one of the formulations described below (6.3.1-6.3.3):
6.3.1 Mix 0.5 g Eriochrome Black T with 4.5 g hydroxylantine hydrochloride.
Dissolve in 1(X) mL of 95% ethanol or isopropanol.
6.3.2 Eriochrome Black T: Sodium salt of l-(hydroxy-2-naphthylazo)-5-rtitro-2-
napthol-4-siilfonic acid; No 203 in the Color Index. Dissolve 0.5 g dye in
100 g 2,2',2"-nitrilotriethanol (also called triethyanolantine) or 2-
methoxymethanol (also caUed ethylene glycol monomethyl ether). Add 2
drops per 50 ml solution to betitrated.Adjust volume if necessary
6.3.3 Mix togetiier 0.5 g Eriochrome Black T and 100 g NaCl.
6.3.4 Cahnagite: l-(l-hydroxy-4-methyl-2-phenylazo)-2-naphtol-4-sulfonic
acid. This is stable in aqueous solutions and produces the same color
change as Eriochrome Black T, with a sharper end pouit. Dissolve 0.10 g
Calmagite in 1(X) mL distiUed water. Use 1 mL per 50 mL solution to be
titrated. Adjust volume if necessary.
6.4 Standard EDTAtiu-ant,0.02 N: Place 3.723 g analyticalreagentgrade disodium
ethylenediaminetetraacetatedihydrate in a 1titervolumetricflaskand dtiute to the
mark witii distiUed water. Check witii standard calcium solution (6.4.1) by
titration (6.4.5). Store in polyetiiylene. Check periodically because of gradual
deterioration.
6.4.1 Standard calcium solution 0.02 N: Place l.CjOO g anhydrous calcium
carbonate (primary standard low in metals) in a 5(X) mL flask. Add, a
Uttie at a time, 1 + 1 HCl (6.4.2) until aU of tiie CaCOs has dissolved
Add 200 mL distUled water. BoU for a few minutes to expel CO2. Cool.
Add a few drops of methyl red indicator (6.4.3) and adjust to uitermediate

166
 orange color by adding 3 N NH4OH (6.4.4) or 1 + 1 HCl (6.4.2) as
required (Juantitatively transfer to a 1 Uter volumetricflaskand dUute to
mark with distiUed water.
6.4.2 Hydrochloric acid solution, 1 + 1.
6.4.3 Metiiyl red uidicator: Dissolve 0.10 g metiiyl red ui distiUed water ui a 100
mL volumetric flask and dtiute to the mark.
6.4.4 Ammonium hydroxide solution, 3 N: Dtiute 210 mL of concentrated
NH4OH to 1 Uter witfi distilled water.
6.4.5 Standardizationtitrationprocedure: Place 10.0 mL standard calcium
solution (6.4.1) in vessel contaimng about 50 mL distiUed water. Add 1
mL buffer solution (6.1). Add 1-2 droips uidicator (6.3) or smaU scoop of
dry uidicator (6.3.3). Titrate slowly with contuiuous stirring untti the last
reddish tuige disappears,adding last few drops at 3-5 second intervals. At
end point the color is blue. Totaltitrationduration should be 5 minutes
from the time of buffer addition.
N of EDTA = 0 2
mL of EDTA
6.5 Ammonium Hydroxide, 1 N: DUute 70 mL of concentrated. NH4OH to 1 Uter
with distiUed water.
7.0 Procedure
7.1 Pretreatment
7.1.1 For drinking waters, surface waters, saline waters, and dUutions thereof,
no pretreatment steps are necessary. Proceed to 7.2.
7.1.2 For most wastewaters, and highly poUuted waters, the sample must be
digested as given in the Metals Methods section of this manual.
Following this digestion, proceed to 7.2.
7.2 Titration of sample-normal to high hardness (5-150 mg/L):
7.2.1 Sample should require <15 mL EDTAtittant(6.4) andtitrationshoitid be
completed within 5 minutes of buffer addition.
7.2.2 Place 25.0 mL sample intitrationvessels, neutraUze with 1 N ammonium
hydroxide (6. 5) and dilute to about 50 mL. Samples used in class
do not require neutralization.
7.2.3 Add 1 to|2mHbuffer solution (6.1).
7.2.4 If end pomt is not sharp (as detemtined by practice mn) add inhibitor at
this point (see 7.4).
7.2.5 |Add 4 to 6 drops indicator solution](6.3.1 or 6.3.2) or smaU scoop of
dried powder indicator formulation (6.3.3).
7.2.6 Titrate slowly with continuous stirring with standard EDTAtitrant(6.4)
until last reddish tint disappears. Solution is normaUy blue at end point.
7.2.7 Daytightor a dayUghtfluorescentlamp is recommended highly because
orxtinary incandescenttightstend to produce a reddishtingein the blue at
the end pomt.
7.3 Titration of sample- high to very high hardness (greater tiian 150 mg/L)
7.3.1 Sample should require <15 mL EDTAtio-ant(6.4) andtitrationshould be
completed within 5 minutes of buffer addition.
7.3.2 Place 15.0 mL sample uititrationvessels, neutraUze witfi 1 N ammonium
hydroxide (6. 5) and dUute to about 50 mL.
7.3.3 Perform steps 7.2.3 to 7.2.7.

167
 7.4 Titration of sample-low hardness Qess than 5 mg/L)
7.4.1 Use a larger sample (100 rnL)
7.4.2 Use proportionately larger amounts of buffer, inhibitor and indicator.
7.4.3 Use a microburet and mn a blartic using redistilled, distiUed or deionized
water.
7.5 To correct for interferences:
7.5.1 Some metal ions interfere by causing fading or indistinct end points.
Irtitibitors reduce this for 25.0 mL samples dUuted to 50 mL.
7.5.2 Inhibitor I: At step 7.2.4 add 250 mg NaCN. Add sufficient buffer to
achieve Ph 10.0 ± 0.1 to offset alkalinityresultingfrom hydrolysis of
sodium cyartide.
7.5.3 Inhibitor U: At step 7.2.4 add 1 mL of utitibitor H (6.2.2)
7.5.4 Inhibitor HI: At step 7.2.4 add 1 mL of utitibitor m (6.2.3).

8.0 Calculations: Hardness (EDTA) as

mgCaC03/L= (A x N x 50.000^
mL sample
where:
A = mL EDTAtitrant(6.4)
N = normaUty of EDTA titrant
ml sample = sample volume before dUution
9.0 Reporting results
9.1 Whenreportinghardness statetfiemethod used for example hardness (calc.) or
hardness (EDTA).
10.0 Accuracy
10.1 In a suigle laboratory (EMSL), usuig surface water samples at an average
concentration of 194 mg CaCOs, tiie standard deviation was ± 3.
10.2 A syntiietic unknown sample containuig 610 mg/L total hardness as CaCOs
contributed by 108 mg/L Ca and 82 mg/L Mg, andtfiefoUowuig supplementary
substances: 3.1 mg/L K, 19.9 mg/L Na, 241 mg/L chloride, 0.25 mg/L nitrite N,
1.1 mg/L nitrate N, 259 mg/L sulfate, and 42.5 mg/L total aUcaUnity (contributed
by NaHCC^) m distilled water was analyzed in 56 laboratories bytiieEDTA
titrunetric metiiod witfi arelativestandard deviation of 2.9% and arelativeerror
(bias) of 0.8%.

References
American Pubtic Healtii Association. (1992). Standard Metiiods fortfieExamination of
y/fltpr and Wastewater. 18th ed. Metiiod 2340. American Public Healtfi Assoaation,
American Water Works Association, and Water Environment Federation, Washmgton
DC, 2-35.
Sawyer, C.N., and McCarty, P. L. (1994). rheTpif>try fQf EnvirQPmgntal EngJngcrinS-
4tii ed. McGraw-HUl, Inc., New York, N.Y., 485.

United States Envux)nmental Protection Agency (1992). Mgthyds for Chgn4gal Analysis of
Water and Wastes. Metiiod #130.2. Envux)nmental Monitonng arid Support
Latwratory United States Envux)nmental Protection Agency, Cmcmnati, Ohio.

168
TITLE: Atomic Absorption Methods
INTRODUCTION:
The effects of metals in water and wastewater rangefrombeneficialtfuoughtroublesome to
dangerously toxic. Some metals are essential, others may adversely affect water
consumers, wastewater treatment systems, and receiving waters. There are drinking water
standards for Ba, Cu, Ni, Zn, F, CN (not a metal), and for tiie "heavy metals" such as Cd,
Pb, As, Se, Cr, and Ag. Otiier important metals are Na, K, Ca, and Mg.
In the detertnination of individual metals, there is not one methcxi that can be foUowed
Therefore, in this manual, an overview wiU be made and the instmctions for eqitipment
operation wiU be presented The individual metal procedures wiU then be presented with
theu-respectiverequirements in the overaU operation withreferralback to the main
procedures.

Metals may be determined satisfactorily by atomic absorption, inductively coupled plasma,

or, with somewhat less precision and sensitivity, colorimetric methcxis. The absorption
methods include flame and electrothermal techiiiques. Flame methods generaUy are
appUcable at mcxierate concentration levels in clean and complex-matrix systems.
Electrothermal methods generaUy can increase sensitivity if matrix effects are not severe.
Matrix modifiers may compensate for some matrix effects. Inductively coupled plasma
(ICP) techrtiques are appUcable over a broad linear range and are especiaUy sensitive for
refractory elements. In general, detection lintits for ICP methcxis are higher than those for
electrothermal methcxis. Colorimetric methcxis for some inctividuals are applicable when
interferences are known to be within the capacity of the particular methcxi. This manual
covers atomic absorption. Preliminary treatment of samples often required Appropriate
pretreatment methods are described for each type of analysis. The fractions of metals that
can be measured are:

1) Dissolved metals: Those constituents (metals) of an unacidified sampletiiatpass

through a 0.45-m membrane fUter.
2) Suspended metals: Those constituents (metals) of an unacidified sampletiiatare
retained by a 0.45-m membrane fUter.
3) Total metals: The concentration of metals detemtined on an unfiltered sample after
vigorous (Ugestion, or the sum of the concentrations of metals in both (Ussolved
and suspended fractions.
4) Acid-extractable metals: The concentration of metals ui solution after treatment of an
unfiltered sample witii hot dtiute mineral acid.

1.0 Summary of Metficxi

1.1 Atontic absorption spectrometryresemblesemissionflamephotometry intiiata
sample is aspirated into aflameand atomized. The major difference is that ui
flame photometry tiie amount oftightemitted is measiied, where ui atontic
absorption spectrometry atightbeam is directedtfux)ughtfieflame,uito a
monochromator, and onto a detectortiiatmeasures tiie amount oftightabsorbed
bytfieatomized element ui tiie flame. For some metals, atomic absorption
exhibits supericjr sensitivity overflameentission. Because each metal has its own

169
 characteristic absorption wavelengtii, a source lamp composed oftfiatelement is
used;tiusmakestfiemetiiodrelativelyfrieefix)mspectral or radiation
interferences. The amount of energy attiiecharacteristic wavelengtii absort)ed in
theflameis proportional totfieconcentration oftiieelement uitfiesample over a
lumted concentration range.

2.0 Sampling and Sample Preservation

2.1 Before coUecting a sample, decide what fiction is to be analyzed (dissolved

suspended, total, or acid-extractable). This decision wtil detemtine ui part
whethertfiesample is acidified witfi or witfiout fUtration andtfietype of digestion
reqmred. Serious errors may be introduced duruig sampUng and storage because
of contantination from sampUng device, faUure toremoveresidues of previous
samples from sample container, and loss of metals by adsorption on and/or
precipitation ui sample container caused by faUure to acicUfytfiesample property.
2.2 Sample Containers. The best sample containers are made of quartz or TFE.
Becausetfiesecontainers are expensive,tfiepreferred sample container is made of
polypropylene or Unear polyetfiylene witfi a polyetiiylene cap. BorosUicate glass
containers also may be used, but avoid soft glass containers for samples
contairting metals in the microgram-per-Uter range. Store samples for
determination of stiver intight-absorbingcontainers. Use only containers and
filters that have been acid rinsed.
2.3 Preservation. Preserve samples immediately after sampUng by acidifying witfi
concentrated nitric acid (HNO3) to pH <2. FUter samples for dissolved metals
before preserving. Usually 1.5 mL concentrated HNO3/L sample (or 3 mL 1 + 1
HNO3 /L sample) is sufficient for short-term preservation. For samples with high
buffer capacity, increase amount of acid (5 mL may be requued for some alkaline
or highly buffered samples). Use commercially available high-purity acid or
prepare high-purity acid by subboUing cUstUlation of acid. After acidifying
sample, preferably stcjre it in arefrigeratorat approximately 4°C to prevent change
in volume due to evaporation. Under these conctitions, samples with metal
concentrations of several mUUgrams per Uter are stable for up to 6 months (except
mercury, for which the Umit is 5 weeks). For nticrogram per-Uter metal levels,
analyze samples as scx)n as possible after sample coUection. Altematively,
preserve samples for mercury analysis by adcUng 2 mL/L 20% (w/v) K2Cr207
solution (prepared in 1 + 1 I^Os). Store in arefrigeratornot contaminated with
mercury. (CAUTION: Mercury concentrations may incn^ase in samples stored in
plastic bottles in mercury-contaminated laboratories.)

3.0 Sources of Contamination

3.1 Avoid intrcxiucing contaminating metals from containers, distiUed water, cjr
membrane filters. Some plastic caps or cap liners may uitroduce metal
contamination; for example, zinc has been found in black bakeUte-type screw caps
as well as in many mbber and plastic products, and cadmium has been found in
plastic pipet tips. Lead is a ubiquitous contaminant in urban air and dust
3.2 Contaminant Removal. Thoroughly clean sample containers witfi a metal-free
non-ionic detergent solution, rinse with tap water, soak in acid, and then rinse
with metal-free water. For quartz, TFE, or glass materials, use 1 + 1 HNO3, 1 +
1 HCl, or aquaregia(3 parts concentrated HCl + 1 part concentrated HNO3) for

170
 soakuig. For plastic material, use 1 + 1 HNO3 or 1 + 1 HCl. Retiable soakuig
conditions are 24 h at 70°C. Chrontic acid or chromium-free substinites may be
used to remove organic deposits from contamers, butrinsecontainers tiioroughly
with water to remove traces of chrontium Do not use chromic acid for plastic
containers or if chromium is to be determined. Always use metal-free water in
analysis and reagent preparation (see 10.1.2.3). Intiiesemetfiods, tfie word
"water" means metal-free water.
3.3 Airbome Contantinants. For analysis of microgram-per-Uter concentrations of
metals, airbome contantinants in the form of volattie compoimds, dust, scx)t, and
aerosols present in laboratory air may become significant To avoid
contamination use "clean laboratory" faciUties such as commerciaUy available
laminar-flow clean-air benches or custom-designed work stations and analyze
blanks thatreflectthe complete procedure.

4.0 Pretreatment of Samples

4.1 Samples containing particitiates or orgartic material generaUy require pretteatment

before analysis. "Total metals" includes aU metals, inorgarticaUy and organicaUy
bound, both dissolved and particulate. Colorless, transparent samples primarily
drinking water) containing a turbidity of <1 NTU, no odor, and suigle phase may
be analyzed directiy by atontic absorption spectroscopy or inductively coupled
plasma spectroscopy for total metals without cUgestion. For further verification or
if changes in existing matrices are encx)untered, compare ctigested and unctigested
samples to ensure comparable results. On coUection, acidify such samples to pH
<2 with concentrated nitric acid (HNO3, see 5.1) and analyze directiy. Digest aU
other samples before determining total metals. To analyze for ctissolved metals,
fUter sample, acidifyfiltrate,and analyze ctirectiy. To determine suspended
metals,filtersample, ctigestfilterand the material on it, and analyze. To
determine acid-extractable metals, extract metals as incticated below and analyze
extract This section describes general pretreatment for samples in which metals
are to be determined accorcting to procedures for the incUvidual metals described
here-in.
4.2 Take care not to introduce metals into samples during preliminary treatment
During pretreatment avoid contact with mbber, metal-based paints. Cigarette
smoke, paper tissues, and aU metal products including those made of stairtiess
steel, galvanized metal, and brass. Conventional fume hocxis can contribute
significantiy to sample contamination, particularly during acid cUgestion in open
containers. Plastic pipettipsoften are contaminated with copper, iron, zinc, and
cadmium; before use soak in 2 N HCl or HNO3 for several days andrinsewitii
deionized water. Check reagent-grade acids used for preservation, extraction, and
cUgestion for purity. If excessive metal concentrations are found, purify tiie acids
by distUlation or use ultra-pure acids. Carry blankstfuoughaU digestion and
fUtration Steps and applytfienecessary corrections totfieresults.
4.3 If dissolved or suspended metals are to be determined, fUter sample attimeof
collection using a preconditioned plastic fUtering device witfi eitfier vacuum or
pressure, contauting afiltersupport of plastic or TFE,tfux)ugha prewashed
ungridded 0.4 to 0.45-m-pore-diam membranefilter(polycarbonate or cellulose
acetate). Before use,filtera blank consisting ofreagentwater to msure freedom
fix)m contamination. Precondition fUter and fUter device by ruisuig witfi 50 mL
deionized water. IftfiefUter blank contains significant metals concentrations.

171
 soak membranefiltersui approxunately 0.5 N HCl or 1 + 1 HNO3
(recommended for electrotiiermal analysis) and rinse witii water before use.
NOTE: Different filters display different sorption and fUtration characteristics; for
trace analysis,testfilterto verify complete recovery of metals. If fUter is to be
digested for suspended metals, record sample volume fUtered and mclude a- filter
in determination of blank. Before fUtering, centrifuge highly mrbid samples ui
acid-washed TFE or high density plastic tubes to reduce loading on fUters.
Stirred, pressurefilterunits foul less readtiy than vacuum fUters;filterat a
pressure of 70 to 130 kPa. Afterfiltrationacidifyfiltrateto pH 2 witii
concentrated HNO3 and analyze directiy. If a precipitate forms on acidification,
digest acidified fUtrate before analysis usmg Metiiod E. Retaui fUter and digest it
for direct determination of suspended metals.
CAUTION: Do not use perchloric acid to ctigest membrane fUters.
4.4 Preliminary Treatment for Acid-Extractable Metals
4.4.1 Extractable metals are Ughtiy adsorbed on particulate material. Because
some san^le cUgestion may be unavoidable userigicUyconttx)Ued
conctitions to obtain meaningful andreproducibleresults.Maintain
constant sample volume, acid volume, and contact time. Express results as
extractable metals and specify extraction conctitions. At coUection, acidify
entire sample witfi 5 mL concentrated HNOj/L sample. To prepare
sample, mix weU, transfer 1(X) mL to a beaker orflask,and add 5 mL 1 +
1 high-purity HCl. Heat 15 min on a steam bath. Ftiter through a
membrane fUter, adjust fUtrate volume to 1(X) mL with water, and analyze.
4.5 Preliminary Digestion for Metals
4.5.1 Toreduceinterference by orgartic matter and to convert metal asscx:iated
with particitiates to a form (usuaUy the free metal that can be determined by
atontic absorption spectrometry or inductively-coupled plasma
spectroscopy) use one of the cUgestion techniques presented below. Use
the leastrigorouscUgestion methcxi required to provide complete and
consistent recovery compatible with the analytical methcxi and the metal
being analyzed. Nitric acid wiU digest most samples adequately. Nitrate is
an acceptable matrix for bothflameand electrotiiermal atontic absorption.
Some samples may require adcUtion of perchloric, hydrochloric, or sulfuric
acid for complete digestion. These acicis may interfere in the analysis of
some metals and aU provide a pcx)rer matrix for electrothermal analysis.
Confirm metal recovery for each cUgestion and analytical prc«edure used.
As a general rule, HNO3 alone is adequate for clean samples or easUy
oxicUzed materials; HNO3-H2SO4 or HNO3-HCI digestion is adequate for
readUy oxidizable organic matter; HNO3-HCIO4 or HNO3-HCIO4-HF
digestion is necessary for difficult-to-oxidize organic matter or ntinerals.
If large amounts of organic matter are present, c&y ashing the sample before
acid treatment wtil facititate digestion.
4.5.2 DUute samples witfi Ag concentrations greatertfian1 mg/L to contam less
than 1 mg/L Ag for flame atomic absorption metfiods and 25 g/L or less for
electrothermal analysis.
4.6 Prepare solid samples or Uquid sludges witfi high solids contents on a weight
basis. Mix sample and transfer a suitable amount (typically 1 g of a sludge witfi
15% total soUds) to a pre-weighed digestion vessel. Reweigh and calculate
weight of sample. Proceed witfi one of the digestion techniques presented below.
Reportresultson wet- or dry-weight basis as foUows:

172
 Metal concentration, mg/kg (wet-weight basis) = A*B / g sample

Metal concentration, mg/kg (dry-weight basis) = (A*B / g sample) * 100 /D

where: A = concentration of metal in digested solution, mg/L,
B = final volume of digested solution, mL, and
D = total sotids, %

Always prepare acid blanks for each type of digestion performed Experience
iricUcates that a blank made witiitfiesame acids and subjected totfiesame
digestion procedure astfiesample can correct for unpurities present in acids, in
reagent water, or on glassware.
5.0 Digestion Methcxis

5.1 Nitric Acid Digestion

5.1.1 Apparatus
5.1.1.1 Hotplate.
5.1.1.2 Conical (erlenmeyer) flasks, 125-mL, or Griffin beakers, 150 mL,
acid-washed and rinsed with water.
5.1.2 Reagents
5.1.2.1 Nitric acid, HNO3, concentrated
5.1.3 Pnxjedure
5.1.3.1 Mix sample and transfer a suitable volume (50 to 100 mL) to a
125-mL corticalflaskor beaker. Add 5 mL concentrated HNO3
and a few boiUng chips, glass beads, or Hengar granules. Bring
to a slow boU and evaporate on a hot plate to the lowest volume
possible (about 10 to 20 mL) before precipitation occurs.
Continue heating and adding concentrated HNO3 as necessary
until cUgestion is complete as shown by a light-colored, clear
solution. Do not let sample dry during cUgestion.
5.1.3.2 Wash downflaskor beaker walls witii water and thenfilterif
necessary. Transfer fUtrate to a lOO-mL voliunetric flask with two
5-mL portions of water, adding theserinsingsto the volumetric
flask. Cool, cUlute to mark, and mix thoroughly. Take portions of
this solution for required metal determinations.
5.2 Nitric Acid-Hydrochloric Acid Digestion
5.2.1 Apparatus
5.2.1.1 Hotplate.
5.2.1.2 Conical (erlenmeyer) flasks, 125-mL, or Griffin beakers, 150 mL,
acid-washed and rinsed with water.
5.2.2 Reagents
5.2.2.1 Nitric acid, HNO3, concentrated
5.2.2.2 Hydrcx:hloric acid, 1 + 1.
5.2.3 Procedure
5.2.3.1 Total HNOs/HQ: Transfer a measured volume of weU mixed,
acid-preserved sample appropriate for the expected metals
concentrations to aflaskor beaker. Add 3 mL concenu-ated
HNO3. Place flask or beaker on a hot plate and cautiously

173
 evaporate to lesstfian5 mL, making certaintiiatsample does not
boti andtfiatno area oftfiebottom of tiie container is aUowed to go
dry. Ccx)l and add 5 mL concentrated HNO3. Cover contamer
witii a watch glass andreturnto hot plate. Increase temperature of
hot plate sotiiata gentierefluxaction occurs. Continue heating,
adding additional acid as necessary, until digestion is complete
(generaUy mdicated whentfiedigestate is ujit in color or does not
change m appearance witii continued refluxuig). Evaporate to less
tiian 5 mL and cool. Add 10 mL 1 -»-1 HCl and 15 mL water per
100 mL anticipated final volume. Heat for an additional 15 nun to
dissolve any precipitate or residue. Cool, wash down beaker
waUs and watch glass witii water, andfiltertoremovemsoluble
material that could clog the nebuUzer. Altematively cenuifuge or
let settie ovemight Adjust to a predetemtined volume based on
the expected metals concentrations.
5.2.3.1 Recoverable HNO3/HCI: Fortftislessrigorousdigestion
procedure, transfer a measured volume of weU-mixed, acid-
preserved sample to aflaskor beaker. Add 2 mL 1 + 1 HNO3 and
10 mL 1 + 1 HCl and heat on a steam bath or hot plate untti
volume has been reduced to near 25 mL, making certain sample
does not boti. Ccx)l and fUter toremoveinsoluble material or
altematively centrifuge or let settie ovemight Adjust volume to
1(X) mL and mix.
6.0 Interferences

6.1 Chentical interference: Many metals can be determined by direct aspiration of

sample into an air-acetylene flame. The most troublesome type of interference is
temied "chemical" andresultsfromthe lack of absorption by atoms bound in
molecular combination in the flame. This can occur when theflameis not hot
enough to cUssociate the molecules or when the cUsscx:iated atom is oxictized
immecUately to a compound that wiU not cUssociate further at the flame
temperature. Such interferences may be reduced or elintinated by adding specific
elements or compounds to the sample solution. For example; the interference of
phosphate in the magnesium determination can be overcome by adcUng
lanthanum. SimUarly, intrcxiuction of calcium eliminates siUca interference in the
detemtination of manganese. However, silicon and metals such as aluntinum,
barium, berylUum, and vanadium require the higher-temperature, nitrous oxide-
acetylene flame to cUsscx:iate their molecules. The rtitrous oxide-acetylene flame
also can be useful in mirtimizing certain types of chemical interferences
encountered in the air-acetylene flame. For example, the interference caused by
high concentrations of phosphate in the determination of calcium in the air-
acetylene flame does not cxx:ur in the rtitrous oxide-acetylene flame.
6.2 Brines and seawater can be analyzed by direct aspiration but sample dtiution is
recommended. Aspiration of solutions containing high concentrations of
cUssolved sotids often results in solids buUdup on the bumer head. This requires
frequent shutdown of theflameand clearting of the bumer head. Preferably use
background correction when analyzing waters that contain in excess of 1% sotids.
especiaUy when the primaryresonancetineof the element of interest is below 240
nm Make morefrequentrecovery checks when analyzing brines and seawater to
insure accurateresititsin these concentrated and complex matrices.

174
6.3 Banum and otiier metals ionize intfieflame,tiierebyreducing tiie ground state
(potentiaUy absorbmg) population. The addition of an excess of a cation (sodium,
potassium, or lititium) havuig a sintilar or lower ionization potential wUl overcome
titis problem. The wavelengtii of maxunum absorption for arsenic is 193.7 nm
and for selenium 196.0 nm—wavelengtiis at whichtiieair-acetylene flame
absorbs intensely.
6.4 Backgroimd correction: Molecular absorption andtightscattering caused by soUd
particles m the flame can cause ertoneously high absorption valuesresultingui
positive errors. When such phenomena occur, use background correction to
obtaui accurate values. Use any one of tiuee types of background correction:
continuum-source, Zeeman, or Smitfi-Hieftje correction.
6.4.1 Continuum-source background correction—^A continuum source
background corrector utilizes either a hydrogen-fiUed hollow cathode lamp
with a metal cathcxie or a deuterium arc lamp. When both the line source
hollow-cathcxie lamp and the continuum source are placed in the same
optical path and are times-shared, the broadband backgroundfrx)mthe
elemental signal is subtracted electronicaUy, and theresultantsignal wiU be
background-compensated Bothtiiehydrogen-fiUed hoUow-catfiode lamp
and deuterium arc lamp have lower intensities than either the line source
hollow cathcxie lamp or elec^trodeless discharge lamps. To obtain a vaUd
correction, mateh the intensities oftfiecontinuum source with the line
source hollow-cathcxie or electrodeless discharge lamp. The matching may
result in lowering the intensity of the line source or increasing the sUt width;
these measures have the cUsadvantage of raising the detection limit and
possibly causing non-Unearity of the caUbration curve. Background
correction using a continuum source corrector is susceptible to interference
from other absorbing lines in the spectral bandwidth. Miscorrection cxx:urs
from significant atontic absorption of the continuum source racUation by
elements other than that being determined When a line source hoUow-
cathcxie lamp is used without background cortection, the presence of an
absorbing tine from another element in the spectral bandwidth wiU not
cause an interference urtiess it overlaps the line of interest. Continuum-
source background correction wiU notremovedirect absorption spectral
overlap, where an element otiier than that being determined is capable of
absorbing the line racUation of the element under smdy.
6.4.2 Zeeman background correction—^This correction is based on the principle
that a magnetic field spUts the spectral line into two linearly polarized Ught
beams parallel and perpencUcular to the magnetic field One is called the pi
(n) component and the other the sigma (a) component. These two Ught
beams have exactiy the same wavelength and differ only in the plane of
polarization. The n line wiU be absorbed by botfi the atoms of the element
of interest and by the l)ackground caused by broadband absorption and Ught
scattering of the sample matrix. The line wiU be absorl)ed only by the
background Zeeman background cortection provides accurate background
correction at much higher absorption levels than is possible with continuum
source background correction systems. It also vutuaUy eUntinates the
possibiUty of errorfromstmctiired background. Because no additional
Ught sources are required,tfieaUgnment and intensity Urnitations
encountered using continuum sources are eliminated. Disadvantages of the
Zeeman method mclude reduced sensitivity for some elements, reduced

175
 Unear range, and a "roUover" effect wherebytfieabsorbance of some
elements begins to decrease at high concentrations, resulting in a two-sided
caUbration curve.
6.4.3 Srnitfi-Hieftje background cortection—^This correction is based on tiie
principletfiatabsorbance measured for a specific element is reduced as tfie
current totfiehoUow catiiode lamp is uicreased whtie absorption of
nonspecific absorbmg substancesremamsidentical at aU curtent levels.
When this metiiod is appUed, the absorbance at a high-current mode is
subtracted from the absorbance at a low-current mode. Under these
conditions, any absorbance due to nonspecific background is subtracted out
and cortected for. Sntith-Hieftje background correction provides a number
of advantages over continuum-source correction. Accurate ccjrrection at
higher absorbance levels is possible and errorfix)mstmctured background
is virtuaUy eliminated In some cases, spectral interferences also can be
eliminated The usefulness of Sntith-Hieftje background correction with
electrcxieless cUscharge lamps has not yet been established
7.0 Sensitivity, Detection Limits, and Optimum Concentration Ranges

7.1 The sensitivity of flame atomic absorption spectrometry is defined as the metal
concentration that produces an absorption of 1% (an absorbanc^e of approximately
0.0044). The instmment detection Untit is defined here as the concentration that
prcxiuces absorption equivalent to twice the magrtimde of the background
flucmation. Sensitivity and detection lintits vary with the instmment, the element
determined the complexity of the matrix, and the techrtique selected The
optimum concentration range usuaUy startsfromthe concenttation of several times
the sensitivity and extends to the concentration at which the caUbration curve starts
to flatten. To achieve best results, use concenu-ations of samples and standards
within the optimum concentration range of the spectrometer.

8.0 Apparams
8.1 Atomic absorption specnx)meter, consisting of a Ught source emitting the line
spectrum of an element (hoUow-catiicxie lamp or electrodeless discharge lamp), a
device for vaporizing the sample (usuaUy aflame),a means of isolatuig an
absorption line (moncx^hromator orfilterand adjustable slit), and a photoelectric
detector with its associated electronic amplifying and measuring equipment
8.2 Bumer: The most common type of bumer is a prentix, which introduces tiie spray
into a condensing chamber for removal of large droplets. The bumer may be
fitted witfi a conventional head contauiuig a smgle slot; atfu-ee-slotBoting head,
which may be preferred for direct aspiration witii an air-acetyleneflame;or a
special head for use with nitrous oxide and acetylene.
8.3 Readout: Most uistruments are equipped with eitfier a digital or nuU meter readout
mechanism. Most mcxlem mstmments are equipped with microprocessors
capable of uitegrating absorption signals over time and linearizingtiiecaUbration
curve at high concentrations.
8.4 Lamps: Use eitiier a hoUow-catfiode lamp or an electrodeless discharge lamp
(EDL). Use one lamp for each element beuig measured Multi-element hoUow-
catfiode lamps generaUy provide lower sensitivitytiiansingle-element lamps.
EDL's take a longer tune to warm up and stabtiize.

176
 8.5 Pressure-reducing valves: MaUitaui suppUes of fuel and oxidant at pressures
somewhat higher than the controUed operatuig pressure of the instmment by using
suitable reducing valves. Use a separate reducuig valve for each gas.
8.6 Vent: Place a vent about 15 to 30 cm abovetiiebumer toremovefumes and
vapors from the flarne. This precaution protects laboratory personnel from toxic
vapors, protects the instrumentfromcortosive vapors, and prevents flame
stabitityfrx)mbeing affected by rcx)m drafts. A damper or variable-speed blower
is desirable for mcxiulating air flow and preventingflamecUsturbance.

9.0 (QuaUty Assurance/Quality Control

9.1 Analyze a blank between sample or standardreadingsto verify baseline stabiUty.
Re-zero when necessary.
9.2 To one sample out of every ten (or one sample from each group of samples if less
than ten are being analyzed) add a known amount of the metal of interest and
reanalyze to confirm recovery. The amount of metal added should be
approximately equal to the amount found If littie metal is present add an amount
close to the ntidcUe of the linear range of the test Recovery of added metal should
be between 85 and 115%.
9.3 Analyze an adcUtional standard solution after every ten samples or with each bateh
of samples, whichever is less, to confirm that thetestis in control.
10.0 Procedures. For some metals there is more than one methcxi of analysis. The
method chosen shouldreflecttiiedesired detection Umit Electrotiiermal Atomic
Absorption Spectix)metric method (10.4) offers much lower detection limits than
Direct Air-Acetylene Flame metiiod (10.1). However, some metals can only be
detemtined by one method.
10.1 Duect Air-Acetylene Flame Metfiod
10.1.1 This method is appUcable totfiedetemtination of antimony, bismutii,
cadmium, calcium, cesium, chromium, cobalt, copper, gold, uicUum,
u-on, lead, Uthium, magnesium, manganese, nickel, palladium,
platinum, potassium, rhcxUum, mthenium, stiver, sodium, strontium,
thallium, tin, and zinc.
10.1.2 Reagents . ,, ^,
10.1.2.1 Air, cleaned and dried through a suitable fUter toremoveoti,
water, and other foreign substances. The source may be a
compressor or commerciaUy bottied gas.
10.1.2.2 Acetylene, standard commercial grade. Acetone, which
always is present in acetylene cylinders, can be prevented
fix)m entermg and damaguigtfiebumer head by replacing a
cyUnder when its pressure has faUen to 689 kPa (100 psi)
acetylene.
10.1.2.3 Metal-free water: Use metal-free water for preparing aU
reagents and caUbration standards and as dilution water.
Prepare metal-free water by deionizing tap water and/or by
usuig one of the foUowing prcx:esses, depending on tiie
metal concentration ui the sample: single disttilation,
redistUlation, or sub-boUuig. Always check deionized or
distiUed water to determine whether the element of interest is
present ui trace amounts. (NOTE: If tiie source water

177
 contauis Hg or other volatUe metals, suigle- or redistiUed
water may not be suitable fcjr trace analysis because these
metals distiU over with the distiUed water. In such cases,
use sub-boiUng to prepare metal-free water).
10.1.2.4 Nitric Acid, HNO3 concentrated.
10.1.2.5 Standard metal solutions: Prepare a series of standard metal
solutions in the optimum concentration range by appropriate
dUution of the following stock metal solutions with water
containing 1.5 mL concentrated HNO3/L. Thoroughly dry
reagents before use. In general, usereagentsof the highest
purity. Fc5r hydrates, use fresh reagents. See incUvidual
metal methcxis for recipes.
10.1.3 The basic steps in the procedure wiU be presented Note: An Air-
acetylene bumer head needs to be placed in the instrument over the
flame source before the instmment is turned on. The bumer head is
lcx:ated behind the flame shield. Usingtfiemetal handles, twist the
head on or off.
10.1.3.1 Tum on the instmment using the red power button to the
right of the screen.
10.1.3.2 Allow a 5 minute warm-up time.
10.1.3.3 Press Mcxiify Program to change an already existing
program. All metals have a basic program.
10.1.3.4 Choose the metal of uiterest by inputting its # and press
Recall Program.
10.1.3.5 Press Index, and go to 6. Optimization by pressing 6 and
new page.
10.1.3.6 Move the Vertical adjusmient knob to 15
10.1.3.7 InstaU a hoUow-catiiode lamp fortiiedesired metal in tfie
instmment androughlyset the wavelength ctial according to
the metal being analyzed Set stit widtii according to the
element being measured
10.1.3.8 Optimize wavelengtii by adjustmg wavelengtfi dial and lamp
untti optimum energy gain is obtained Do this by watching
the scale shown on the screen, ff it goes off scale press
Rescale. Adjust toreachtfiemaximum widtii. Press
Rescale again. Press Optimize Signal.
10.1.3.9 Press Instrument Zero. Tumtfievertical adjustment knob
slowly up untti a small band shows on the screen.
10.1.3.10 Make sure aspUation mbe is ui DI water.
10.1.3.11 Tum ontfiegas and aU. The valves are to the left of tfie
instmment.
NOTE: The gas used, eitfier acetylene or nitrous, depends
on the metal being detemtined See individual metal
metiiods for tiie appropriate gas to be used
10.1.3.12 Press Ignite and hold until aflamestarts. Sometimes, this
has to berepeatedif the machine has been off for a period
of time.
10.1.3.13 Use 3 dUutions of standards based metal recipes given m
their respective methods.
10.1.3.14 Press Index, and go to Calibration.

178
 10.1.2.15 Aspirate a blank for a couple of ntinutes. Press Read. If
tiie blank is too high, use tiie SoUi Type button to go back
to tiie Blank. Press Read agam.
10.1.3.16 Do for all tiuee standards. The mstmment wUl
automaticaUy prepare a caUbration curve, unlesstfieUis an
error ui caUbration. It wiU prompt iftiierehas been an
ertor. If there is an error, check standards and repeat
caUbration.
10.1.3.17 Press Analytical Results and analyze samples.
10.1.4 Calculations
10.1.4.1 Calculate concentration of each metal ion, in nticrograms
per Uter for trace elements, and in milUgrams per Uter for
more common metals, byreferringtotfieappropriate
caUbration curve prepared. Altematively, read
concentration directiy from the instrumentreadoutif the
instrument is so equipped. If the sample has been dUuted,
multiply by the q)propriate cUlution factor.
10.2 Du-ect NiUDus Oxide-Acetylene Rame Metfiod
10.2.1 This methcxi is appUcable to the determination of aluminum, barium,
beryllium, molybdenum, osmium, rhenium, sUicon, thorium, titanium,
and vanadium.
10.2.2 Reagents
10.2.2.1 AU: See 10.1.2.1.
10.2.2.2 Acetylene: See 10.1.2.2.
10.2.2.3 Metal-free water: See 10.1.2.3.
10.2.2.4 Nitric acid, HNO3, concentrated
10.2.2.5 Nitrous oxide, commercially available cylinders. Fit rtitrous
oxide cyUnder with a special non-freezableregulatoror
wrap a heating coU around an orctinaryregulatorto prevent
flashback at the bumer caused by reduction in nitrous oxide
flow through a frozen regulator.
10.2.2.6 The prcx^ure is the same as 10.1.3 except that a rtittous
bumer head is placed in the instmment and rtitrous oxide is
used a the gas source.
10.2.8 (Halculations, Calculate concentration of each metal ion in milUgrams
per Uter. Altematively,readthe concentration directiy from the
instmment readout if the instmment is so equipped. Uf sample has been
cUluted, multiply by the appropriate dtiution factor.
10.3 Cold-Vapor Atomic Absorption Spectrometric Methcxi
10.3.1 This metficxi is applicable totfiedetermination of mercury.
10.3.2 Apparatus. When possible, dedicate glassware for use in Hg analysis.
Avoid using glassware previously exposed to high levels of Hg, such
as tiiose used in COD, TKN, or CI analysis.
10.3.2.1 Atontic absorption spectrometer, mercury haUow cathode
lamp, and cold vapor accessory unit
10.3.2.2 Absorption ceU, a glass tube approximately 2.5 cm in
cUameter.
10.3.2.3 CeU support: Strap ceU to theflatnitrous-oxide bumer head.
10.3.2.4 Connecting mbuig, glass mbuig to pass mercury vapor
frx)m reactionflaskto absorption ceU and to interconnect aU

179
 otiier components. Clear vinyl plastic mbing may be
substituted for glass.
10.3.3 Reagents
10.3.3.1 Metal-free water, see 10.1.2.3.
10.3.3.2 Stock mercury solution: Dissolve 1.354 g mercuric
chloride, HgCh, ui about 70 mL water, add 10 mL
concenu-ated HNCV3, and dtiute to 1000 mL witii water to
give 1000 pg/L Hg.
10.3.3.3 Standard mercury solutions: Prepare a series of standard
mercury solutions containing 0 to 5 \ig/L by appropriate
dilution of stcx^k mercury solution with water containing 10
mL concentrated HNO3/L. Prepare standards daUy.
10.3.3.4 Nitric acid, HNO3, concentrated
10.3.3.5 Potassium permanganate solution: Dissolve 50 g KMn04 in
water and dilute to 1 L.
10.3.3.6 Potassium persulfate solution: Dissolve 50 g K2S2O8 in
water and cUlute to 1 L.
10.3.3.7 ScxUum chloride-hydroxylamine sulfate solution: Dissolve
120 g NaCl and 120 g (NH20H)2 H2SO4 in water and
cUlute to 1 L. A 10% hydroxylantine hydrochloride solution
may be substituted for the hydroxylantine sulfate.
10.3.3.8 Stannous ion (Sn2+) solution: Use either stannous chlcjride
(10.3.3.8.1), or stannous sulfate (10.3.3.8.2), to prepare
this solution containing about 7.0 g Sn2+/100 mL.
10.3.3.8.1 Dissolve 10 g SnCh in water containuig 20
mL concentrated HCl and dtiute to 100 mL.
10.3.3.8.2Dissolve 11 g SnS04 ui water containing 7
mL concentrated H2SO4 and cUlute to 100
mL. Both solutions decompose with aging.
If a suspension forms, stir reagent
continuously during use. Reagent volume is
sufficient to prcx:ess about 20 samples; adjust
volumes prepared to accommodate number of
samples prcx^essed.
10.3.3.9 Sulfuric acid, H2SO4, concentrated. 0.5 N, dUute 14 ml
concentrated H2SO4 in water and dilute to 1 Uter.
10.3.4 Procedure
10.3.4.1 The prcx:edure is sintilar to 10.1.3 with adjusttnents
made for the cold vapor accessory unit. Set wavelength
to 253.7 nm.
10.3.4.2 Placetfieabsorption ceU mtotiieceU support (metal piece
that fits over the nitrous bumer head. Install absc«ption
ceU. Alignment wtil betfiesame as Optimization m
10.1.3.8 except that optunization wiU betfux)ughthe
absorption ceU. Connect asscx:iated equipment to
absorption ceU witfi vinyl plastic tubing attached to cold
vapor generator accessory unit Tum on gasses and air.
AUow aU to flow continuously. Tum on the cold vapor
generatcjr accessory unit and aUow it to run for 5
minutes.

180
 1 rx o >! ^ ! ? ^ ? - ^"orcscent Ughting may uicrease baseUne noise.
IU.3.4.2 Standardization: Transfer 100 mL of each of tiie 1.0,
2.0, and 5.0 pg/L Hg standard solutions and a blank of
100 mL water to 250-mL erlenmeyerreactionflasks.
Add 5 mL concenu-ated H2SO4 and 2.5 mL concenu-ated
HNO3 to each flask. Add 15 mL KMn04 solution to
each flask and let stand at least 15 mm. Add 8 mL
K2S2O8 solution to eachflaskand heat for 2 h m a water
bath at 95°C. Cbol to rcx)m temperature.
10.3.4.3 Treating eachflaskindividuaUy, add enough NaCl-
hydroxylamine sulfate solution to reduce excess
KMn04, then add 5 mL SnCh or SnS04 solution and
immediately attachflaskto aeration apparatus. As Hg is
volatilized and carried into the absorption ceU,
absorbance wiU increase to a maximum within a few
seconds. As soon as reccjrder returns approximately to
the base line,removestopperfromreactionflask,and
replace with aflaskcontaining water. Flush system for
a few seconds and run the next standard in the same
manner. Constmct a standard curve by plotting peak
height versus micrograms Hg.
10.3.4.4 Analysis of samples: Transfer 1(X) mL sample or portion
dUuted to 1(X) mL containing not more than 5.0 pg Hg/L
to a reaction flask. Treat as in 10.3.4.3. Seawater,
brines, and effluents high in chlorides require as much
as an additional 25 mL KMn04 solution. During
oxidation step, chlorides are converted to free chlorine,
which absorbs at 253 nnL Remove aUfreechlorine
before the Hg is reduced and swept into the ceU by using
an excess (25 mL) of hydroxylantine sulfate reagent.
Removefreechlorine by sparging sample gentiy with air
or rtitrogen after adding hydroxylamine reducing
solution. Use a separate mbe and frit to avoid carryover
ofresidualstannous chloride, which could cause
reduction and loss of mercury.
10.3.5 Calculation
10.3.5.1 Detemtine peak height of samplefromrecorder chart and
read mercury value from standard curve prepared
accorcting to 10.3.4.3.
10.4 Electrotiiermal Atomic Absorption Spectrometric Metfiod
10.4.1 This methcxi is suitable for determination of micro quantities of
aluntinum, antimony, arsenic, barium, berylUum, cadmium,
chromium, cobalt, copper, iron, lead, manganese, molybdenum,
nickel, selenium stiver, and tin. Electrothermal atomic absorption
spectroscopy is based ontfiesame principle as direct flame
atomization but an electricaUy heated atomizer or graphite fumace
replaces the standard bumer head A cUscrete sample volume is
dispensed mto the graphite sample mbe (or cup). TypicaUy,
detemtinations are made by heatingtfiesample uitfueeor more
stages. FUst, a low current heats the mbe to drytfiesample. The

181
 second, or charring stage, destroys organic matter and volatiUzes
other matrix components at an uitermediate temperature. FmaUy, a
high current heats tiie mbe to uicandescence anci, m an uiert
atmosphere, atomizestfieelement beuig detemtined. Additional
stagesfrequentiyare added to aid ui drymg and charring, and to
clean and CCK)1 the mbe between samples. The resultant ground-state
atomic vapor absorbs moncx:hromatic radiationfrx)mtiiesource. A
phc)toelec;tric detector measurestfiemtensity of d-ansmitted radiation,
which is inversely proportional totfiequantity of ground-state atoms
in the optical path over a limited range. Because the amounts
analyzed are very minute, background cortection is necessary.
10.4.2 Apparams
10.4.2.1 Atomic absorption spectrometer. The instrument must
have background ccjrrection c^abiUty.
10.4.2.2 Source lamps such as 8.4.
10.4.2.3 Graphite fumace: Use an electricaUy heated device with
electrortic control circuitry designed to carry a graphite
mbe or cup through a heating program that provides
sufficient thermal energy to atomize the elements of
interest. Fumace heat controUers with only three heating
steps are adequate ortiy forfreshwaters with low
dissolved solids content. For salt waters, brines, and
other complex matrices, use a fumace controUer with up
to seven individuaUy progranuned heating steps. Fit the
ftmiace mtotiiesample compartment oftiiespecttx)meter
in place of the conventional bumer assembly or use
commerciaUy avaUable equipment specifically for trace
metal determination. Use argon as a purge gas to
minimize oxidation of the fumace mbe and to prevent the
formation of metaUic oxides. Use graphite mbes with
Lvov platforms to minintize interferences and to
improve sensitivity. See each incUvidual methcxi.
10.4.3.4 Readout.
10.4.3.5 Sample cUspensers: Use microUter pipets (5 to 100 pL)
or an automatic sampUng device designed for the specific
instmment.
10.4.3.6 Vent.
10.4.3.7 Ccx)ling water supply: Ccx)l with tap waterflowingat 1
to 4 L/min or use a recirculating ccx)ling device.
10.4.4 Reagents
10.4.4.1 Metal-free water, see 10.1.2.3.
10.4.4..2 Nitric acid, HNO3,1 + 1 and concenu-ated.
10.4.4..3 Matrix modifiers:
10.4.4.3.1 Ammonium nitrate, 10% (w/v): Dissolve
100 g NH4NO3 ui water. DUute to 1000
mL with water.
10.4.4.3.2 Ammonium phosphate, 40%: Dissolve 40 g
(NH4)2HP04 m water. Dilute to 100 mL
with water.

182
 10.4.4.3.3 Calcium nitrate, 20 000 mg CaA.: Dissolve
11.8 g Ca(N03)2*4H20 in water. DUute to
100 mL with water.
10.4.4.3.4 Nickel nitrate, 10 000 mg Ni/L: Dissolve
49.56 g Ni(N03)2*6H20 ui water. DUute
to 10(X) mL with water.
10.4.4.3.5 Phosphoric acid, 10% (v/v): DUute 10 mL
concentrated H3PO4 to 100 mL witii water.
10.4.4.4 Stock metal solutions.
10.4.5 Prcx:edure
10.4.5.1 Sample pretreatment: Before analysis, pretreat aU
samples as mcUcated below. Rinse all glassware witii 1
+ 1 HNO3 and water. Carry out cUgestion procedures in
a clean, dust-free laboratory area to avoid sample
contamination. Fcjr cUgestion of trace aluminum, use
polypropylene or TFE utensUs to avoid leachable
aluminum from glassware.
10.4.5.2 Instrument operation: Tum on the power in the
foUowing order: 1. Spectrophotometer, 2. Zeeman
lamp source. Printer, and Computer.
10.4.5.3 Tum on water circulator undertiiecomputer.
10.4.5.4 Tum on Argon gas lcx:ated to the left of the Atomic
Absorption instrument.
10.4.5.5 Disconnect water hose, and move sampler to the holer to
the left on top of the machine. PuU the atomizer out of
the way of the Ught source.
10.4.5.6 On the computer, select which metal to mn. The
machine wiU automaticaUy lcx:ate the correct lamp source
to use. However, make sure it is in the case.
10.4.5.7 Maximize the signal by turning the top knob on the back
of thetightsource first and then the bottom. Press
Rescale (Fl).
10.4.5.8 Place either graphite mbe or graphite mbe in the atomizer
depending on which metal is being analyzed See metal
methcxis.
10.4.5.9 Replace atomizer, sampler tray, and water drain mbe.
10.4.5.10 Use vertical adjustment knob to maxintize signal.
10.4.5.11 Instrument caUbration: Prepare standards for instrument
caUbration. Some metal analyses, the instrument wiU
dtiute the stcx:k standard immecUately or it may be
necessary to make up the individual standards. Check
each metal program for which one appUes. Prepare
standards fresh daily.
10.4.5.8 P*repare a blank and at least three caUbration standards in
the appropriate concentration range for correlating
element concentration and instrument response. Match
the matrix of the standard solutions totfioseof the
samples as closely as possible. In most cases, this
simply requires matehing the acid background of the
samples. For seawater or brines, however, use the
metal-free matrix as the standard solution dUuent. In

183
 addition, addtiiesame concentration of matrix modifier
(if required for sample analysis) totfiestandard
solutions.
10.4.5.10 Sample analysis: Analyze aU samples at least m
dupUcate or untti reproducibleresultsare obtainedA
variation of 5-10% is considered acceptable
reproducibUity. AveragerepUcatevalues.
10.4.6 Calculations
10.4.6.1 Direct detemtination:
pg metaVL = C * F
where:
C = metal concentration as read directiy from the
instmment orfromthe calibration curve, pg/L., and
F = dUution factor.
11.0 Accuracy is presented with each metal method.
References

American PubUc Health Association (1992). Standard Metiiods for the Exantination of
Water and Wastewater. 18th ed. Method 3(XX), American Pubtic Healtfi Asscxtiation,
American Water Works Asscx:iation, and Water Environment Federation, Washington
DC, 1-1.

184
METHOD #: 202.1 and #202.2 Approved for NPDES Gssued 1978)
TITLE: Aluntinum (AA, Fumace Technique)
ANALYTE:
Aluminum, Al

DSfSTRUMENTATION: AA

Fumace: C^tunum Concentration Range: 20-200 pg/L

Detection Lmtit: 3 g/L
Duect air Optimum Concentration Range: 5-50 mg/L
DetecticHi Limit: 0.1 mg/L

1.0 Preparation of Standard Solution

1.1 Stock solution: Place 1.0 g aluminum metal, 15 ml of concentrated HCL and 5 ml
of HNO3 m a beaker. Warm gentiy until dissolved Transfer to 1 Uter.
1.2 For metiiod 10.1, Dissolve 95 g of potassium chloride (KCl) m 1000 ml of
deionized water. To each 100 ml of standard and sample, add 2 ml of potassium
chloride solution.
1.3 Prepare dUutions of the stcx:k solution to be used as caUbration standards at the
time of analysis.
1.4 The calibration standard should be diluted to contain 0.5% (v/v) HNO3.
1.5 For metiicxi 10.3 use 10.4.4.3.3 as a matrix mcxUfier if needed.
2.0 San^le Preservation

2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atontic
Absorption Methcxis section of this manual.
3.0 Sanq)le Preparation
3.1 Prepare as desc^ribed under section 5.0 of the Atontic Absorption Methcxis.
Sample solutions for analysis should contain 0.5% (v/v) HNO3.

4.0 Instmment Parameters

4.1 Fumace
4.1.1 Drying Time and Temp: 30 sec-125°C.
4.1.2 Ashing Time and Temp: 30 sec-1300°C.
4.1.3 Atomizuig Time and Temp: 10 sec-2700°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.6 Wavelength: 309.3 nm, andtiieuse a L'vov platform is recommended to
incniease sensitivity as in 10.4.2.3.
4.2 Direct air
4.2.1 Aluminum HaUow catfiode lamp
4.2.2 Wavelengtii: 309.3 nm, sUt widtii: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant Nitrous Oxide

185
5.0 Analysis Procedure

5.1 Fortiieanalysis procedure andtiiecalculation, see 10.2 or 10.4 oftfieAtomic

Absorption Methcxis section of this manual.
Notes
1. Background cortection may be reqmred if tiie sample contains high dissolved
sotids.
2. It has beenreportedthat chloride ion andtiiatnitrogen used as a purge gas
suppress tiie aluminum signal. Therefore, tiie use of haUde acids and nio-ogen
as a purge gas should be avoided.
6.0 Accuracy

6.1 Six synthetic concenu-ates containuig varying levels of aluminum were added to
natural water samples. The statistical results were as below:

Standard
Number Tme Values Mean Values Deviation Accuracy as
of Labs gA. g/L gA. % Bias
38 1205 1281 299 6.3
38 1004 1003 391 -0.1
37 500 463 202 -7.4
37 625 582 272 -6.8
22 35 96 108 175
21 15 109 168 626

References
United States Environmental Protection Agency. (1992). Methods for Chemical Analysis
of Water and Wastes. Methcxi #202.1 and #202.2. Environmental Monitoring and
Support Labcjratory, United States Environmental Protection Agency, Cincinnati, Ohio.

186
Mhinuu w: jm.i and #ZU4.2 Approved for NPDES Gssued 1978)
TITLE: Antimony (AA, Fumace Technique)
ANALYTE:
Antimony, Sb

DSfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 20-300 g/L

Detection Limit: 3 g/L
Direct AJr. Optimum Concentration Range: l-40mg/L
Detection Lintit: 0.2 mg/L

1.0 Preparation of Standard Solution

1.1 Stock solution: Antimony: Dissolve 0.2669 g K(SbO)C4H406 (antimony

potassium tartrate) m water, add 10 mL 1 -t-1 HCl and dtiute to 1000 mL witii
water; 1.00 mL = 100 pg Sb or dissolve 2.7426 K(SbO)C4H406 in deionized
water, and dilute to 1 Uter. 1 ml = 1 mg Sb (1000 mg/L).
1.2 Prepare dUutions of the stock solution to be used as caUbration standards at tiie
time of analysis.
1.3 The calibration standard should be diluted to contain 0.2% (v/v) HN03.
1.4 In method 10.4, use 10.4.4.3.4 as a matrix modifier if needed.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 The pnxedures for preparation of the sample as given in part 5.0 of the Atomic
Absorption Methcxis section of this manud should be foUowed including the
adcUtion of sufficient 1:1 HCl to cUssolve the cUgestedresiduefor the analysis of
suspended or total antimony. The sample solutions used for analysis should
contain 2% (v/v) HN03.

4.0 Instmment Parameters

4 1 Fumace
4.1.1 Drying Time and Temp: 30 sec-125°C.
4.1.2 Ashuig Tune and Temp: 30 sec-800°C.
4.1.3 Atomizuig Time and Temp: 10 sec-2700°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.5 Wavelengtii: 217.6 nm
4.2 DuectAu-
4.2.1 Antimony HaUow Catiiode Lamp
4.2.2 Wavelengtii: 217.6 nm, sUt widtii: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant Air

187
 4.2.5 Type ofFlame: Fuel lean
5.0 Analysis Procedure

5.1 For tiie analysis procedure andtiiecalculation, see 10.1 or 10.4 of tiie Atomic
Absorption Methods section of this manual.
5.2 Notes
5.2.1 The use of background correction is recorrunended.
5.2.2 Nitrogen may also be used as the purge gas.
6.0 Precision and Accuracy

6.1 A single laboratory (EMSL), using a mixed industrial-domestic waste effluent at

concentrations of 5.0 and 15 mg Sb/ L, the standard deviations were ± 0.08 and
±0.1, respectively. Recoveries at these levels were 96% and 97%, respectively.
References
United States Environmental Protection Agency. (1992). Methcxis for Chemical Analvsis
of Water and Wastes. Methcxi #204.1 and #204.2. Environmental Mortitoring and
Support Laboratory, United States Environmental Protection Agency, Cincirmati,
Ohio.

188
METHOD #: 206.2 Approved for NPDES and SDWA Gssued 1978)
TITLE: Arsenic (AA, Fumace Technique
ANALYTE:
Arsenic, As

DSfSTRUMENTATION: AA

Optimum Concentration Range: 5-100 g/L

Detection Limit: 1 g/L

1.0 Preparation of Standard Solution

1.1 Stock solution: Dissolve 1.320 g of arsenic trioxide, AS2O3 (analytical reagent
grade) ui 100 mL of deionized distiUed water containing 4 g NaOR Acidify tfie
solution witfi 20 mL concentrated. HN03 and dtiute to 1 Uter. 1 mL = 1 mg As
(1000 mg/L).
1.2 Nickel Nitrate Solution, 5%: Dissolve 24.780 g of ACSreagentgrade
Ni(N03)2-6H20 in deionized cUstiUed water and make up to IQOmL.
1.3 Nickel Nitrate Solution, 1%: Dtiute 20 mL oftfie5% nickel niu-ate to 100 mL witfi
deionized cUstiUed water.
1.4 Working Arsenic Solution: Prepare dUutions of the stcx^k solution to be used as
caUbration standards at the time of analysis. Withdraw appropriate aUquots of the
stcx:k solution, add 1 mL of concentrated. HNO3,2mL of 30% H2O2 and 2niL of
the 5% nickel rtitrate solution. DUute to 100 mL with deionized distiUed water.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 or 4.0 of the Atomic
Absorption Methods section of this manual.
3.0 Sample Preparation
3.1 Transfer 1(X) mL of well-mixed sample to a 250 mL Griffin beaker, add 2 mL of
30% H2O2 and sufficient concentrated HNO3 toresultin an acid concentration of
l%(v/v). Heat for 1 hour at 95°C or untiltiievolume is sUghtiy lesstfian50 mL.
3.2 Cool and bruig back to 50 mL witii deionized distilled water.
3.3 Pipet 5 mL oftftisdigested solution into a 10-mL volumetric flask, add 1 mL of
the 1% nickel niu-ate solution and dilute to 10 mL witfi deionized distiUed water.
The sample is nowreadyfor injection intotfiefumace.
NOTE: If solubiUzation or digestion is not requUed, adjusttfieHNO3
concenti-ation of the sample to 1% (v/v) and add 2 mL of 30% H2O2 and 2 mL of
5% nickel nitrate to each 100 mL of sample. The volume oftfiecaUbration
standard should be adjusted with deionized distiUed water to mateh the volume
change of the sample.
4.0 Instmment Parameters (General)
4.1 Drymg Tune and Temp: 30 sec-125°C.

189
 4.2 Ashmg Tune and Temp: 30 sec-1100°C.
4.3 Atomizuig Time and Temp: 10 sec-27o6°C.
4.4 Purge Gas Atmosphere: ii-gon
4.5 Wavelengtii: 193.7 nm, andtiieuse of a LVov platform is recommended for
increased sensitivity as in 10.4.2.3.
5.0 Analysis Procedure

5.1 Fortfieanalysis procedure andtiiecalculation, see 10.4 of tiie Atomic Absorption

Metiiods section oftitismanual. The use of background correction is requUed.
6.0 Precision and Accuracy

6.1 In a single laboratory (EMSL), using a mixed mdustrial-domestic waste effluent

containing 15 g/L and spUced witfi concenti^tions of 2,10 and 25 g/L, recoveries
of 85%, 90% and 88% were obtainedrespectively.Therelativestandard deviation
at tiiese concentrations levels were ± 8.8%, ± 8.2%, ± 5.4% and ± 8.7%,
respectively.
6.2 In a single laboratory (EMSL), using Cincinnati, Ohio tap water spiked at
concentrations of 20, 50 and 100 g As/L, tiie standard deviations were ± 0.7, ±
1.1 and ± 1.6 respectively. Recoveries attfieselevels were 105%, 106% and
101%, respectively.
References
United States Environmental Protection Agency. (1992). Metficxls for Chemical Analysis
of Water and Wastes. Method #206.2. Environmental Mortitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

190
METHOD #: 208.1 and #208.2 Approved for NPDES and SDWA (Issued 1978)
TITLE: Barium (AA, Fumace Techrtique)
ANALYTE:
Barium, Ba

INSTRUMENTATION: AA

Fumace: Optimum Concenu-ation Range: 10-200 g/L

Detection Limit: 2 g/L
Direct air: Optimum concentration Range: 1-20 mg/L
Detection Limit: 0.1 mg/L
1.0 Preparation of Standard Solution

1.1 Stock solution: Dissolve 1.7787 g Barium chloride (BaCl2*2H20) m deionized

water and dtiute to 1 liter. 1 ml = 1 mg Ba (lOOO mg/L). To each standard and
sample add 2 ml of KCl solution. See Aluminum 1.2.
1.2 Prepare dtiutions of the stcx:k solution to be used as caUbration standards at the
time of analysis.
1.3 The calibration standard should be cUluted to contain 0.5% (v/v) HNO3.

2.0 Sample Preservation

2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under 5.0. Sample solutions for analysis should contain 0.5
HNO3.
4.0 Instmment Parameters

4.1 Fumace
4.1.1 Drying Tune and Temp: 30 sec-125°C.
4.1.2 Ashmg Tune and Temp: 30 sec-1200°C.
4.1.3 Atomizuig Tune and Temp: 10 sec-2800°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.5 Wavelengtii: 553.6 nm
4.2 Duect Air
4.2.1 Barium haUow catfiode lamp
4.2.2 Wavelengtii: 553.6 nm, sUt widtii: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant Nitrous Oxide
4.2.5 Fuel rich
5.0 Analysis Procedure
5.1 For tiie analysis procedure andtiiecalculation, see 10.2 or 10.4 oftfieAtontic

191
 Absorption Methcxis section of this manual.
5.2 Notes
5.2.1 The use of halide acid should be avoided
5.2.2 Because of possible chemical mteraction, nitrogen should not be used as a
purge gas.

6.0 Accuracy

6.1 In a single laboratcjry (EMSL), using Qncinnati, Ohio tap water spiked at
concentrations of 500 and 1000 g Ba/L, tiie standard deviations were ± 2.5 and ±
2.2 g, respectively. Recoveries at these levels were 96% and 102%, respectively
A dUution of 1:1() was required to bring the spikes within the analytical range of
the methcxi.

References

United States Environmental Protection Agency. (1992). Methcxis for Chemical Analvsis
of Water and Wastes. Method #208.1 and #208.2. Envuonmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincirmati,
Ohio.

192
METHOD #: 213.1 and #213.2 Approved for NPDES and SDWA (Editorial Revision
1974)

TITLE: Cadntium (AA, Direct Aspuation)

ANALYTE:
Cadmium, Cd

DSfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 0.5-10 pg/L

Detection Limit: 0.1 pg/L
Direct air: Optunum Concenttation Range: 0.05-2 mg/L using a wavelength of 228.8 nm
Detection Lintit: 0.005 mg/L
1.0 Preparation of Standard Solution
1.1 Stock Solution: CarefuUy weigh 2.282 g of cadntium sulfate (3CdS04*8H20,
analyticalreagentgrade) and dissolve in deionized cUstiUed water make up to 1
Uter with deionized cUstiUed water. 1 mL = 1 mg Cd (1(XX) mg/L).
1.2 Prepare dtiutions of the stcx^k solution to be used as caUbration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid and at the same concentration as wiUresultin the sample to be analyzed
either directiy or after prcx:essing.
1.3 In 10.4, use 10.4.4.3.2 as a matrix mcxUfier if needed.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methods section of this manual.

3.0 Sample Preparation

3.1 The procedures for preparation of the sample as given in part 5.0 oftiieAtomic
Absorption Metiiods section oftitismanual have been found to be satisfactory.

4.0 Instmmental Parameters

4.1 Fumace
4.1.1 Drying tune and temp: 30sec-125°C
4.1.2 Ashmgtimeand temp: 30sec-500°C
4.1.3 Atomizuigtimeand temp: 10sec-1900°C
4.1.4 Purge gas Atmosphere: Argon
4.1.5 Wavelengtii: 228.8 nm
4.2 Duect air
4.2.1 Cadmium hoUow catiiode lamp
4.2.2 Wavelengtii: 228.8 nm, slit widtii: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant Air
4.2.5 Type offlame:Oxidizuig

193
5.0 Analysis Procedure

5.1 For analysis procedure and calculation, see 10.1 or 10.4 of tiie Atomic
Absorption Metiicxis section of this manual.

6.0 Accuracy

6.1 An interlaboratory smdy on trace metal analyses by atontic absorption was

conducted by the C^ality Assurance and Laboratory Evaluation Branch of EMSL.
Six synthetic concentrates containing varying levels of aluminum, cacimium,
chromium, copper, iron, manganese, lead and zinc were added to natural water
samples. The statisticalresultsfor cadmium were as foUows:

Standard
Number Tme Values Mean Value Deviation Accuracy as
of Labs g/Uter g/Uter g/Uter % Bias«

74 71 70 21 -2.2
73 78 74 18 -5.7
63 14 16.8 11.0 19.8
68 18 18.3 10.3 1.9
55 1.4 3.3 5.0 135
51 2.8 2.9 2.8 4.7

References
United States Envux)nmental Protection Agency. (1992). Metfiods for Chentical Analysis
of Water and Wastes. Method #213.1 and #213.2. EnvUonmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincumati,
Ohio.

194
METHOD #: 215.1 Approved for NPDES (Editorial Revision 1974)
TITLE: Calcium (AA, DUect AspUation)
ANALYTE:
Calcium, Ca

DSfSTRUMENTATION: AA

Optimum Concentration Range: 0.2-7 mg/L usuig a wavelengtii of 422.7 nm

Detection Limit: 0.01 mg/L

1.0 Preparation of Standard Solution

1.1 Stock Solution: Suspend 1.250 g of CaCOa (analytical reagent grade), dried at
180°C for 1 hour before weighing, in deionized distiUed water and dissolve
cautiously witii a ntinimum of dtiute HCl. DUute to 1000 mL witfi deionized
distiUed water. 1 mL = 0.5 mg Ca (500 mg/L).
1.2 Lantfianum chloride solution: Dissolve 29 g of La2Q3, slowly and m smaU
portions, in 250 mL concentrated HCl (Caution: Reaction is violent) and dtiute to
5(X) mL with deionized cUstiUed water.
1.3 Prepare dUutions of the stcx^k calcium solutions to be used as caUbration standards
at the time of analysis. To each 10 mL volume of caUbration standard and sample
alike add 1.0 mL of the lanthanum chloride solution, i.e., 20 mL of standard or
sample -i- 2 mL LaCls = 22 mL.

2. Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 oftfieAtomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 For the analysis of total calcium in domestic and industrial effluents, the
procedures for the determination of total metals as given in part 5.0 of the Atomic
Absorption Methcxis section of this manual have been found to be satisfactory.
3.2 For ambient waters, arepresentativeaUquot of a weU-mixed sample may be used
directiy for analysis. If suspended solids are present in sufficient amounts to clog
the nebulizer, tiie sample may be aUowed to settie andtfiesupematant Uquid
analyzed directiy.

4.0 Instrumental Parameters

4.1 Calcium hollow catfiode lamp
4.2 Wavelengtfi: 422.7 nm, sUt widtii: 0.5nm or wavelengtii: 239.9 nm , stit widtii:
0.2 nm
4.3 Fuel: Acetylene
4.4 Oxidant Air
4.5 Type offlame:Reducmg
5.0 Analysis Procedure

195
 5.1 For analysis procedure and calculation, see 10.1 oftfieAtomic Absorption
Methcxis section of this manual.
5.2 Notes
5.2.1 Phosphate, sulfate and aluminum interfere but are masked by the addition of
lanthanuriL Since low calcium valuesresultif the pH of the sample is above
7, both standards and samples are prepared in dtiute hydrochloric acid
solution. Concentrations of magnesium greater than 1000 mg/L also cause
low calcium values. Concentrations of up to 500 mg/L each of socUum,
potassium and rtitrate cause no interference.
5.2.2 Aniortic chentical interferences can be expected if lanthanum is not used in
samples and standards.
5.2.3 The rtitrous oxide-acetyleneflamewiU provide two to five times greater
sensitivity andfi-eedomfrom chentical interferences. Ionization interferences
should be controUed by adding a large amount of alkaU to the sample and
standards. The analysis appears to befreefromchemical suppressions in
the rtitrous oxide-acetylene flame. (Atomic Absorption Newsletter 14,29
[1975]).
5.2.4 The 239.9 nm line may also be used This line has arelativesensitivity of
120.
5.2.5 The EDTAtittimeuicmetfiod may also be used (Standard Methods, 14tfi
Edition, p 189 or EPA metiiod 215.2).
6.0 Precision and Accuracy
6.1 In a single laboratory (EMSL), usuig distiUed water spUced at concentrations of
9.0 and 36 mgCa/L, the standard deviations were ± 0.3 and ± 0.6, respectively.
Recoveries at both these levels were 99%.

References
United States Envux)nmental Protection Agency. (1992). Mgthods for Chgmical Analysis
of Water and Wastes. Metfiod #215.1. EnvUonmental Monitoring and Support
Laboratory, United States EnvUonmental Protection Agency, Cuicinnati, Ohio.

196
NffiTOOD #: 218.1 and #218.2 Approved for NPDES and SDWA (Editorial Rev. 1974,
197 o)

TITLE: Chromium (AA, DUect Aspiration)

ANALYTE:
Chromium, Cr
INfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 5-100 pg/L

Detection Limit: 1 pg/L
Direct aU: Optimum Concentration Range: 0.5-10 mg/L usmg a wavelengtii of 357.9 nm
Detection Lunit: 0.05 mg/L

1.0 Preparation of Standard Solution

1.1 Stock Solution: Dissolve 1.923 g of chromium dioxide (Cr03, reagent grade) in
deionized cUstiUed water. When solution is complete, acicUfy with recUstiUed
HN03 and dtiute to 1titerwitfi deionized disttiled water. 1 mL = 1 mg Cr (1000
mg/L).
1.2 Prepare dtiutions of the stcx^k solution to be used as caUbration standards at the
time of analysis. The c^aUbration standards should be prepared using the same type
of acid and at the same concentration as wiUresultin the sample to be analyzed
either directiy or after processing.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atontic
Absorption Methods section of this manual.
3.0 Sample Preparation
3.1 The prcx:edures for preparation of the sample as given in part 5.0 of the Atomic
Absorption Methcxis section of this manual have been found to be satisfactory.
4.0 Instrumental Parameters
4 1 Furnace
4.1.1 Drymgtimeand temp: 30 sec-125°C
4.1.2 Ashmgtimeandtemp:30 sec-1000°C
4.1.3 Atomizuigtimeandtemp:10 sec-2700°C
4.1.4 Purge gas: Argon
4.1.5 Wavelengtii: 357.9 nm
4.2 Direct air
4.2.1 Chromium hoUow cathode lamp
4.2.2 Wavelengtii: 357.9 nm, 520.8 nm, sUt widtii: 0.2
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: Nitrous oxide
4.2.5 Type offlame: Fuel rich

197
5.0 Analysis Procedure

5.1 For analysis procedure and calculation, see 10.1 or 10.4 of tiie Atontic Abson)tion
Methcxis secmon of this manual.
5.2 Notes
5.2.1 The foUowing wavelengths may also be used:
359.3 nm Relative Sensitivity 1.4
425.4 nm Relative Sensitivity 2
427.5 nm Relative Sensitivity 3
428.9 nm Relative Sensitivity 4
5.2.2 The fuelrichair-acetyleneflameprovides greater sensitivity but is subject to
chemical and matrix mterference from iron, nickel, and other metals. If tfie
analysis is performed m a leanflametfiemterference can be lessened but tfie
sensitivity wiU also be reduced
5.2.3 The suppression of botii Cr (EI) and Cr (VI) absorption by most uiterfering
ions in fuelrichair-acetyleneflamesis reportecUy conttoUed by the addition
of 1% ammonium bifluoride m 0.2% sodium suUate [Talanta 20,631
(1973)]. A 1% oxine solution is alsoreportedto be useful.
5.2.4 For levels of chrontium between 50 and 200 g/L wheretfieau--acetylene
flame can not be used or for levels below 50 g/L, use 10.4.
6.0 Precision and Accuracy
6.1 An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the C^ality Assurance and Laboratory Evaluation Branch of EMSL.
Six synthetic concentrates containing varying levels of aluminum, cadmium,
chromium, copper, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for chromium were as follows:

Standard
Number Tme Values Mean Value Deviation Accuracy as
of Labs g/Uter g/Uter g/Uter % Bias

74 370 353 105 -4.5

76 407 380 128 -6.5
72 74 72 29 -3.1
70 93 84 35 -10.2
47 7.4 10.2 7.8 37.7
47 15.0 16.0 9.0 6.8

References
United States Envux)nmental Protection Agency. (1992). Metfiods for Chemical Analvsis
of Water and Wastes. Metfiod #218.1 and #218.2. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincinnati,
Ohio.

198
METHOD #: 219.1 and #219.2 Approved for NPDES (Editorial Revision 1978)
TITLE: Cobalt (AA, Direct Aspkation)
ANALYTE:
Cobalt, Co

DSfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 5-l(X) pg/L

Detection Umit: 1 pg/L
Direct ak: Optimum Concenn^tion Range: 0.5-5 mg/L usmg a wavelengtii of 240.7 nm
Detection Limit: 0.05 mg/L

1.0 Preparation of Standard Solution

1.1 Stock Solution: Dissolve 4.307 g of cobaltous chloride, CoC12*6H20 (analytical

reagent grade), in deionized distiUed water. Add 10 mL of concenuated nitric acid
and dUute to 1 Uter with deionized cUstiUed water. 1 mL = 1 mg Co (1000 mg/L).
1.2 Prepare dUutions of the stcx:k cobalt solution to be used as calibration standards at
the time of analysis. The calibration standards should be prepared using the same
type of acid and at the same concentration as wiUresultin the sample to be
analyzed either directiy or after prcx^essing.
1.3 In methcxi 10.4, use 10.4.4.3.2 or ascorbic acid as a matrix mcxUfier if needed.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 of the Atontic Absorption
Methcxis section of this manual.
3.0 Sample Preparation
3.1 The procedures for preparation of the sample as given in parts 4.0 and 5.0 of the
Atomic Absorption Methods section of this manual have Been found to be
satisfactory.
4.0 Instrumental Parameters

4.1 Fumace
4.1.1 Dryingtimeandtemp:30 sec-125°C
4.1.2 Ashuigtimeandtemp:30 sec-900°C
4.1.3 Atomizuigtimeandtemp:10 sec-2700 *^C
4.1.4 Purge gas atmosphere: Argon
4.1.5 Wavelengtii: 240.7 nm
4.2 Direct air
4.2.1 Cobalt hoUow catficxie lamp
4.2.2 Wavelengtii: 240.7 nm, 346.6 nm, 391 nm, stit widtii: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: AU
4.2.5 Type ofFlame: Oxidizing

199
5.0 Analysis Procedure

5.1 For analysis procedure and calculation, see 10.1 or 10.4 oftfieAtomic
Absorption Metiiods section oftfiismanual. For levels of cobalt below 100 g/L,
10.4 is recommended.

6.0 Precision and Accuracy

6.1 In a single laboratory (EMSL), using a mixed industrial-domestic waste effluent

at concentrations of 0.20,1.0 and 5.0 mg Co/L,tfiestandard deviations were±
0.013,± 0.01 and± 0.05, respectively. Recoveries at these levels were 98%,
98%, and 97%, respectively.

References
Urtited States Environmental Protection Agency. (1992). Methcxis for Chentical Analysis
of Water and Wastes. Methcxi #219.1 and #219.2. Environmental Monitoring and
Support Lalx)ratory, United States Environmental Protection Agency, Cincirmati,
Ohio.

200
METHOD #: 220.1 and #220.2 Approved for NPDES Gssued 1978)
TITLE: Copper (AA, Fumace Technique)
ANALYTE:
Copper, Cu

D^STRUMENTATION: AA

FURNACE: Optunum Concenu-ation Range: 5-100 g/L

Detection Limit: 1 g/L
DIRECT AIR: Optimum Concenu-ation Range: 0.2-5 mg/L
Detection Lintit: 0.02 mg/L

1.0 Preparation of Standard Solution

1.1 Stock solution: Dissolve 0.100 g copper metal in 2 mLconcenfratedHNOs, add

10.0 mL concenu-ated HNO3 and dilute to 1000 mL witii water; 1.00 mL = 100
PgCu.
1.2 Prepare dUutions oftfiestock solution to be used as calibration standards at tfie
time of analysis.
1.3 The caUbration standard should be dUuted to contain 0.5% (v/v) HN03.
1.4 In method 10.4, use 10.4.4.3.1 or ascorbic acid as a matrix modifier if needed.
2.0 Sample Preservation

2.1 For sample handling and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation

3.1 Prepare as described under 5.0. Sample solutions for analysis should contain 0.5
(v/v) HNO3.

4.0 Instmment Parameters

4.1 Fumace
4.1.1 Drymg Time and Temp: 30 sec-125°C.
4.1.2 Ashing Tune and Temp: 30 sec-900°C.
4.1.3 Atomizuig Tune and Temp: 10 sec-2700°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.5 Wavelengtii: 324.7 nm
4.2 Du-ect air
4.2.1 Copper HaUow cathcxie lamp
4.2.2 Wavelength: 324.7 nm, sUt widtii: 0.5 nm, or wavelength: 222.6 nm, slit
width: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: Au-
4.2.5 Type of Flame: Oxidizing
5.0 Analysis Procedure

201
 5.1 For tiie analysis procedure andtiiecalculation, see 10.1 or 10.4 oftfieAtontic
Absorption Methcxis section of this manual.
5.2 Notes
5.2.1 Background correction may be reqmred if the sample contams high
cUssolved soUds.
5.2.2 Nitrogen may also be used as the purge gas.
6.0 Accuracy

6.1 Six synthetic concentrates containing various levels of copper were added to
natural water samples. The statistical results for copper are as foUows:

Standard
Number Tme Values Mean Value Deviation Accuracy as
of Labs UgA. Hg/L Itg/L % Bias
91 302 305 56 0.9
92 332 324 56 -2.4
86 60 64 23 7.0
84 75 76 22 1.3
66 7.5 9.7 6.1 29.7
66 12 13.9 9.7 15.5

References
United States Environmental Protection Agency. (1992). Methods for Chemical Analvsis
of Water and Wastes. Methcxi #220.1 and #220.2. EnvUonmental Monitoring and
Support Laboratory, United States Envu-onmental Protection Agency, Cmcmnati, Ohio.

202
METHOD #: 236.1 and #236.2 Approved for NPDES (Editorial Revision 1974,1978)
TITLE: Iron (AA, Duect AspUation)
ANALYTE:
Iron, Fe
DSfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 5-l(X) pg/L

Detection Limit: 1 pg/L
Optimum Concentration Range: 0.3-5 mg/L using a wavelengtfi of 248.3 nm
Detection Limit: 0.03 mg/L
1.0 Preparation of Standard Solution

1.1 Stcx:k Solution: Carefully weigh l.(XX) g of pure iron wire (analytical reagent
grade) and cUssolve in 5 mL redistilled HN03, warming if necessary. When
solution is complete make up to 1 liter with deionized distiUed water. 1 mL = 1 mg
Fe (1000 mg/L).
1.2 Prepare dtiutions of the stcx:k solution to be used as caUbration standards at the
time of analysis. The caUbration stanciards should be prepared usmgtfiesame type
of acid and at the same concentration as wiUresultin the sample to be analyzed
either ciirectiy or after pnxessing.
1.3 In methcxi 10.4, use 10.4.4.3.1 as a matrix mcxUfier if needed.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methcxis section of this manual.

3.0 Sample Preparation

3.1 The procedures for preparation of tiie sample as given in part 5.0 oftfieAtontic
Absorption Methods section of this manual have been found to be satisfactory.

4.0 Instrumental Parameters

4.1 Fumace
4.1.1 Drymgtimeandtemp:30 sec-125°C
4.1.2 Ashmg time andtemp:30 sec-1000°C
4.1.3 Atontizmgtimeandtemp:10 sec-2700°C
4.1.4 Purge gas atoiosphere: Argon
4.1.5 Wavelengtii: 248.3 nm
4.2 DUect air
4.2.1 Iron hoUow catiiode lamp
4.2.2 Wavelengtii: 248.3 nm, 372.0 nm, sUt widtii: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: Air
4.2.5 Type offlame:Oxidizuig

203
5.0 Analysis Procedure

5.1 For analysis pnx:edure and calculation, see 10.1 or 10.4 of die Atomic Absorption
Methcxis section of this manual.
5.2 Notes
5.2.1 The foUowing lines may also be used:
248.8 nm Relative Sensitivity 2
271.9 nm Relative Sensitivity 4
302.1 nm Relative Sensitivity 5
252.7 nm Relative Sensitivity 6
372.0 nm Relative Sensitivity 10
5.2.2 For concenu-ations of Uon below 0.05 mg/L, Section 10.4 is recommended
6.0 Precision and Accuracy

6.1 An interlaboratory smdy on n^ce metal analyses by atontic absorption was

conducted by tiie C^ality Assurance and Laboratory Evaluation Branch of EMSL.
Six synthetic concentrates containing varying levels of aluminum, cadntium,
chromium, copper, iron, manganese, lead and zinc were added to natural water
samples. The statisticalresultsfor iron were as follows:

Standard
Number Tme Values Mean Value Deviation Accuracy as
of Labs g/Uter g/Uter g/Uter % Bias
82 840 855 173 1.8
85 700 680 178 -2.8
78 350 348 131 -0.5
79 438 435 183 -0.7
57 24 58 69 141
54 10 48 69 382

References
United States EnvUonmental Protection Agency. (1992). Metfiods for Chemical Analvsis
ofWater and Wastes. Method #236.1 and #236.2. EnvUonmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cmcmnati, Ohio.

204
METHOD #: 239.1 and #239.2 Approved for NPDES and SDWA (Issued 1978)
TITLE: Lead (AA, Fumace Technique)
ANALYTE:
Lead,Pb

D^STRUMENTATION: AA

Fumace: Optunum Concentration Range: 5-100 g/L

Detection Limit: 1 g/L
DUect ak: Optimum Concentration Range: 1-20 mg/L usmg a wavelengtii of 283.3 nm
Detection Limit: 0.1 mg/L
1.0 Preparation of Standard Solution

1.1 Stock solution: Dissolve 0.1598 g lead nio^te, Pb(N03)2, Ui a ntinimum amount
of 1 -J-1 HNO3, add 10 mL concentrated HNO3, and dilute to 1000 mL witii
water; 1.00 mL = 100 pg Pb or dissolve 1.599 g of lead nio-ate, Pb(N03)2 m
deionized water, add 10 nti redistiUed HNO3 to afinalvolume of 1 Uter. 1 ml = 1
mgPb (1000 mg/L)
1.2 For method 10.4 use either Lanthanum Nitrate Solution: Dissolve 58.64 g of ACS
reagent grade La203 in 100 mL concentrated. HNO3 and cUlute to 1(XX) rnL with
deiortized cUstiUed water. 1 mL = 50 mg La or 10.4.4.3.1 as matrix mcxUfiers.
1.3 Working Lead Solution: Prepare cUlutions of the stcx^k lead solution to be used as
caUbration standards at the time of analysis. Each caUbration stanciard should
contain 0.5% (v/v) HNO3. To each 100 mL of dUuted standard add 10 mL of tiie
lanthanum rtittate solution.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 oftfieAtomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under 5.0. Sample solutions for analysis should contain
0.5% (v/v) HN03.
3.2 To each 1(X) mL of prepared sample solution add 10 mL oftfielanthanum niu-ate
solution.

4.0 Instmment Parameters

4.1 Fumace
4.1.1 Drying Tune and Temp: 30 sec-125°C.
4.1.2 Ashuig Time and Temp: 30 sec-500°C.
4.1.3 Atomizuig Tune and Temp: 10 sec-2700°C.
4.1.4 Purge Gas Aunosphere: Argon
4.1.5 Wavelength: 283.3 nm, and the use of a L*vov platform is recommended to
increase sensitivity as in 10.4.2.3.
205
 4.2 Duect air
4.2.1 Lead haUow catiiode lamp
4.2.2 Wavelengtii: 283.3 nm, 261.4 nm, sUt widtii: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: AU
4.2.5 Type offlame:Oxidizing
5.0 Analysis Prcx:edure

5.1 Fortfieanalysis procedure mtiiecalculation see 10.1 or 10.4 oftiieAtomic

Absorption Methcxis section oftitismanual.
5.2 Notes
5.2.1 The use of background cortection is recommended.
5.2.2 Greater sensitivity can be achieved usmgtfie217.0 nmtine,but tfie
optimum concentration range is reduced. The use of a lead electtodeless
cUscharge lamp attiiislower wavelengtii has been found to be
advantageous. Also a lower atomization temperamre (2400°Q may be
preferted.
5.2.3 To suppress sulfate interference (up to 1500 ppm) lanthanum is added as tfie
nitrate to both samples and calibration standards. (Atontic Absorption
Newsletter Vol. 15, No. 3, p 71, May-June 1976.)
5.2.4 Since glassware contamination is a severe problem in lead analysis, all
glassware should be cleaned immecUately prior to use, and once cleaned,
should not be open to the atoiosphere except when necessary.
6.0 Precision and Accuracy
6.1 In a single laboratory (EMSL), using Cincinnati, Ohio tap water spiked at
concentrations of 25, 50, and 100 g Pb/L, the standard deviations were ± 1.3, ±
1.6, and ± 3.7,respectively.Recoveries at these levels were 88%, 92%, and 95%
respectively.
References
United States EnvUonmental Protection Agency. (1992). Metiiods for Chentical Analvsis
of Water and Wastes. Methcxi #239.1 and #239.2. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincinnati,
Ohio.

206
METHOD #: 242.1 Approved for NPDES (Editorial Revision 1974,1978)
TITLE: Magnesium (AA, Duect AspUation)
ANALYTE:
Magnesium, Mg

D^STRUMENTATION: AA

Optimum Concenu-ation Range: 0.02-0:5 mg/L usmg a wavelengtii of 285.2 nm

Sensiuvity: 0.007 mg/L
Detection Lintit: 0.(X)1 mg/L

1.0 Preparation of Stanciard Solution

1.1 Stock Solution: Dissolve 0.829 g of magnesium oxide, MgO (analytical reagent
grade), Ui 10 mL of redistiUed HN03 and dtiute to 1 Uter witfi deionized distilled
water. 1 mL = 0.50 mg Mg (500 mg/L).
1.2 Lantfianum chloride solution: Dissolve 29 g of La203, slowly and Ui smaU
portions Ui 250 mL concentrated. HCl, (Caution: Reaction is violent), and dtiute
to 5(X) mL with deionized disttiled water.
1.3 Prepare dtiutions of the stock magnesium solution to be used as caUbration
standards at thetimeof analysis. To each 10 mL volume of calibration stanciard
and sample alUce add 1.0 mL of the lantiianum chloride solution, i.e., 20 mL of
standard or sample + 2 mL LaC13 = 22 mL.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 For the analysis of total magnesium in domestic and industrial effluents, tfie
procedures for the determination of total metals as given in part 5.0 of the Atomic
Abscnption Methcxis section of this manual have been found to be satisfactory.
3.2 For ambient waters, arepresentativeaUquot of a weU-mixed sample may be used
directiy for analysis. If suspended sotids are present in sufficient amounts to clog
the nebulizer, the sample may be aUowed to settie and the supematant tiquid
analyzed directiy.
3.3 Samples should be preserved with (1:1) niuic acid to a pH of 2 at the time of
coUec^tion.
4.0 Instmmental Parameters
4.1 Magnesium hoUow cathcxie lamp
4.2 Wavelength: 285.2 nm, 0.5 nm or wavelength: 202.6 nm, sUt widtii: 1.0 nm
4.3 Fuel: Acetylene
4.4 OxiciantAir
4.5 Type offlame: Oxidizing

207
5.0 Analysis Procedure

5.1 For the analysis pnxedure in the calculation see 10.1 of the Atontic Absorption
Methods sec^tion of this manual.

Notes
1. The interference caused by aluminum at concenu^tions greater than 2 mg/L is
masked by adcUtion of lanthanum. Sodium, potassium and calcium cause no
interference at concentrations less than AOO mg/L.
2. The following line may also be used: 202.5 nm Relative Sensitivity 25
3. To cover the range of magnesium values normaUy observed in surface waters
(0.1-20 mgA-), U is suggested that eitfiertfie202.5 nm line be used ortfiebumer
head be rotated A 90°rotationoftiiebumer head wiU produce approxunately one-
eight the normal sensitivity.
4. The gravimetric metiiod may also be used (Standard Metfiods, 14tfi EcUtion, p
221).
5.0 Accuracy
5.1 In a single laboratory (EMSL), usmg distiUed water spUced at concenu-ations of
2.1 and 8.2 mg Mgyl tiie standard deviations were ± 0.1 and ± 0.2, respectively.
Recoveries at both of these levels were 1(X)%.

References
United States EnvUonmental Protection Agency. (1992). M^^thods for Chemical Analysis
^fWatPT and Wastes. Metfiod #242.1. EnvUonmental Monitonng and Support
Laboratory, United States EnvUonmental Protection Agency, Cmcmnati, Ohio.

208
METHOD #: 243.1 and #243.2 Approved for NPDES Gssued 1978)
TITLE: Manganese (AA, Fumace Technique)
ANALYTE:
Manganese, Mn

INSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 1-30 g/L

Detection Limit: 0.2 g/L
DUect aU: Optimum Concentration Range: 0.1-3 mg/L
Detection Limit: 0.01 mg/L

1.0 Preparation of Standard Solution

1.1 Stock solution: Dissolve 0.1000 g manganese metal in 10 mL concenuated HQ

ntixed witii 1 mL concenu-ated HNC^. Dtiute to 1000 mL witfi water, 1.00 mL =
100 pg Mn.
1.2 Prepare dUutions of tiie stock solution to be used as calibration standards at the
time of analysis.
1.3 The caUbration standard should be dUuted to contain 0.5% (v/v) HN03.
1.4 In method 10.4, use ascorbic acid or 10.4.4.3.1 as matrix modifiers as needed.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 oftiieAtomic
Absorption Methods section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under 5.0. Sample solutions fcjr analysis should contain
0.5% (v/v) HN03.

4.0 Instmment Parameters

4.1 Fumace
4.1.1 Drying Tune and Temp: 30 sec-125°C.
4.1.2 Ashing Time and Temp: 30 sec-1000°C.
4.1.3 Atomizuig Tune and Temp: 10 sec-2700°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.5 Wavelengtii: 279.5 nm
4.2 Direct aU
4.2.1 Manganese haUow catfiode lamp
4.2.2 Wavelengtii: 279.5 nm, 403.1 nm, 321.7 nm, sUt widtii: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: aU
4.2.5 Type offlame:Oxidizuig
5.0 Analysis Pnxjedure

209
 5.1 For tiie analysis procedure andtiiecalculation, see 10.1 or 10.4 oftfieAtomic
Absorption Methcxis section of this manual.
5.2 Notes
5.2.1 The use of background cortection is recommended.
5.2.2 Nitrogen may also be used as the purge gas.
6.0 Accuracy

6.1 Six synthetic concentrates containing various levels of manganese were added to
natui^ water samples. The statistical results for manganese are as foUows:

Standard
Number Tme values Mean values Deviation Accuracy as
of Labs UgA. Hg/L Hg/L % Bias

77 426 432 70 1.5

78 469 474 97 1.2
71 84 86 26 2.1
70 106 104 31 -2.1
55 11 21 27 93
55 17 21 20 22

References
United States EnvUonmental Protection Agency. (1992). Metiiods for Chemical Analysis
of Water and Wastes. Metiiod #243.1 and #243.2. Environmental Monitoring and
Support Laboratory, United States EnvUonmental Protection Agency, Cuicinnati, Ohio.

210
METHOD #: 245.1 Approved for NPDES and SDWA (Issued 1974)
TITLE: Mercury (Manual Cold Vapor Technique)
ANALYTE:
Mercury, Hg
INSTRUMENTATION: AA
1.0 Scope and Application

1.1 This methcxi is appUcable to drinking, surface, and saUne waters, domestic and
industrial wastes.
1.2 In addition to inorganic forms of mercury, orgartic mercurial may also be present
These organo-mercury compounds wiU notrespondto the cold vapor atomic
absorption technique urtiess they are first broken down and converted to mercuric
ions. Potassium permanganate oxicUzes many of these compounds, but recent
smcUes have shown that a number of organic mercurial, including phenyl mercuric
acetate and methyl mercuric chloride, are only partiaUy oxiciized by this reagent.
Potassium persulfate has been found to give approximately 1(X)% recovery when
used as the oxiciant with these compounds. Therefore, a persulfate oxidation step
foUowing the adcUtion of the permanganate has been included to insure that
organo-mercury compounds, if present, wtil be oxiciized to the mercuric ion
before measurement A heat step is reqitired for methyl mercuric chloride when
present in or spiked to a natural system. For cUstUled water the heat step is not
necessary.
1.3 The range of the methcxi may be varied through instmment anci/or recorder
expansion. Using a 1(X) mL sample, a detection lintit of 0.2 pg Hg/L can be
achieved; concentrations below this level should bereportedas <0.2.
2.0 Summary of Methcxi
2.1 TheflamelessAA procedure is a physical methcxi based on the absorption of
racUation at 253.7 nm by mercury vapor. The mercury is reduced to the elemental
state and aeratedfiromsolution in a closed system. The mercury vapor passes
through a ceU positioned in the light path of an atomic absorption
spectrophotometer. Absorbance (peak height) is measured as a function of
mercury concentration and recorded in the usual manner.

3.0 Sample HancUing and Preservation

3.1 Until more conclusive data are obtained, samples should be preserved by
aciciification with rtitric acid to a pH of 2 or lower immecUately at thetimeof
coUection. If only cUssolved mercury is to be detemtined, the sample should be
fUtered through an all glass apparams before the acid is added Fcjr total mercury
thefiltrationis ontitted. See section 10.3.4.2.

4.0 Interference
4.1 Possible interference from sulfide is etimmated by the addition of potassium
permanganate. Concentrations as high as 20 mg/L of sulfide as sodium sulfide do

211
 not interfere with the recovery of added inorgartic mercury from cUstUled water.
4.2 Q)pper has also been reported to mterfere; however, copper concenu-ations as
high as 10 mg/L had no effect on recovery of mercury from spiked samples.
4.3 Sea waters, brines and industrial effluents high in chlorides reqmre additional
permanganate (as much as 25 ml). Duringtfieoxidation step, chlorides are
converted to free chlorine which wiU also absorb racUation of 253 nm Care must
be taken to assure that free chlorine is absent before the mercury is reduced and
swept into the ceU. This may be accompUshed by using an excess of
hydroxylamine sulfate reagent (25 ml). In addition,tiiedead aU space mtfieBOD
bottie must be purged before the addition of stannous sulfate. Both inorganic and
organic mercury spikes have been quantitatively recovered from sea water using
this techrtique.
4.4 Interferencefix)mcertain volatile orgartic materials which wiU absorb at this
wavelength is also possible.
5.0 Analysis Procedure

5.1 See Section 10.3 of the Atomic Absorption Methcxis for the cold-vapor methcxi
instmctions.
6.0 Precision and Accuracy

6.1 In a single laboratory (EMSL), using an Ohio River composite sample

with a background mercury concentration of 0.35 g/L, spiked with concentrations
of 1.0, 3.0 and 4.0 g/L, tiie standard deviations were ± 0.14, ± 0.10 and ± 0.08,
respectively. Stanciard deviation at the 0.35 level was ± 0.16. Percent recoveries
at tiie three levels were 89, 87, and 87%, respectively.
6.2 In a joint EPA/ASTM interlaboratory smdy of the cold vapor techrtique for total
mercury in water, increments of organic and inorgartic mercury were added to
natural waters. Recoveries were determined by difference. A statistical summary
of this smdy follows:

Standard
Number Tme Values Mean Value Deviation Accuracy as
of Labs g/Uter g/Uter g/Uter %Bias

76 0.21 0.349 0.276 66

80 0.27 0.414 0.279 53
82 0.51 0.674 0.541 32
77 0.60 0.709 0.390 18
82 3.4 3.41 1.49 0.34
79 4.1 3.81 1.12 -7.1
79 8.8 8.77 3.69 -0.4
78 9.6 9.10 3.57 -5.2

References
United States Environmental Protection Agency. (1992). Methcxis for Chemical Analysis
of Water and Wastes. Method #245.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

212
METHOD #: 246.1 and #246.2 Approved for NPDES Gssued 1978)
TITLE: Molybdenum (AA, Fumace Technique)
ANALYTE:
Molybdenum, Mo

INSTRUMENTATION: AA

Fumace: Optimum Concenu^tion Range: 3-60 g/L

Detection Limit: 1 g/L
Direct aU: Optunum Concenu^tion Range: 1-40 mg/L
Detection Lintit: 0.1 mg/L
1.0 Preparation of Stanciard Solution

1.1 StCKk solution: Dissolve 1.840 g of ammonium molybdate (NH4)6Mo7024-4H20

in deionized water and cUlute to 1 Uter. 1 nti =1 mg/L (lOOOmg/L).
1.2 Prepare dUutions of the stcxk solution to be used as calibration standards at the
time of analysis.
1.3 The caUbration standard should be dtiuted to contain 0.5% (v/v) HN03.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 of the Atontic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under 5.0. Sample solutions for analysis should contain
0.5% (v/v) HN03.
4.0 Instmment Parameters
4.1 Fumace
4.1.1 Drymg Time and Temp: 30 sec-125°C.
4.1.2 Ashmg Tune and Temp: 30 sec-1400°C.
4.1.3 Atomizing Tune and Temp: 15 sec-2800°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.5 Wavelength: 313.3 nm
4.2 Direct aU
4.2.1 Molybdenum haUow cathode lamp
4.2.2 Wavelengtii: 313.3 nm, stit widtii: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: Nitrous oxide
4.2.5 Type offlame:Fuel rich

5.0 Analysis Procedure

5.1 For the analysis procedure and the calculation, see 10.2 or 10.4 of the Atontic
Absorption Metiiods section of this manual.

213
 5.2 Notes
5.2.1 Background correction may be reqmred if the sample contains high
cUssolved sotids.
5.2.2 The use of nitrogen as a purge gas with methcxi 10.4 is not recommended.
6.0 Accuracy

6.1 In a smgle laboratory (EMSL), using a mixed indusuial-domestic waste effluent at

concentrations of 0.30,1.5, and 7.5 Mo/L, tiie stanciard deviations were ± 0.(X)7,
± 0.02 and ± 0.07, respectively. Recoveries at these levels were 1(X) %, 96%,
and 95 %, respectively.
References
United States Environmental Protection Agency. (1992). Methods for Chemical Analvsis
of Water and Wastes. Method #246.1 and #246.2. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincirmati,
Ohio.

214
METHOD #: 249.1 and #249.2 Approved for NPDES (Editorial Revision 1978)
TITLE: Nickel (AA, Duect AspUation)
ANALYTE:
Nickel, Ni

DSFSTRUMENTATION: AA
Fumace: Optimum Concentration Range: 5-50 pg/L
Detection Limit: 1 pg/L
Direct aU: Optunum Concenu^tion Range: 0.3-5 mg/L using a wavelengtii of 232.0 nm
Detection Lintit: 0.04 mg/L
1.0 Preparation of Standard Solution

1.1 Stock Solution: Dissolve 4.953 g of nickel nitrate, Ni(N03)2-6H20 (analytical

reagent grade) in deionized cUstiUed water. Add 10 mL of concentrated rtitric acid
and dtiute to 1 Uter with deionized cUstiUed water. 1 mL = 1 mg Ni (1(XX) mg/L).
1.2 Prepare dUutions of the stock rtickel solution to be used as caUbration stanciards at
the time of analysis. The calibration standards should be prepared using the same
type of acid and at the same concentration as wiUresultin the sample to be
analyzed either directiy or after prcx^ssing.
1.3 In methcxi 10.4, use 10.4.4.3.2 as a matrix mcxUfier if needed.
2.0 Sample Preservation
2.1 For sample handling and preservation, see part 2.0 of the Atomic Absorption
Methcxis section of this manual.
3.0 Sample Preparation
3.1 The procedures for preparation of the sample as given in parts 4.0 and 5.0 of the
Atomic Absorption Methcxis section of this manual have been found to be
satisfactory.

4.0 Instrumental Parameters

4.1 Fumace
4.1.1 Drying tUne andtemp:30 sec -125°C
4.1.2 AshUigtimeandtemp:30 sec -800°C
4.1.3 Atomizingtimeandtemp:10 sec -2700°C
4.1.4 Purge gas atmosphere: Argon
4.1.5 Wavelengtii: 232.0 nm
4.2 DUect aU
4.2.1 Nickel hoUow cathode lamp
4.2.2 Wavelengtii: 232.0 nm, 341.5 nm, sUt width: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: Air
4.2.5 Type ofFlame: Oxidizuig

215
5.0 Analysis Procedure

5.1 For analysis procedure and calculation, see 10.1 or 10.4 oftfieAtontic Absorption
Methods sec:tion of this manual.
5.2 Notes
5.2.1 For levels of nickel below 100 g/L, 10.4 is recommended.
5.2.2 The heptoxime methcxi may also be used (Stanciard Methods, 14th EcUtion,
p 232).
6.0 Interferences

6.1 The 352.4 nm wavelength is less susceptible to spectral interference and may be
used The caUbration curve is more linear at this wavelength; however, there is
some loss of sensitivity.
7.0 Precision and Accuracy
7.1 In a single laboratory (EMSL), using a ntixed indusuial-domestic waste effluent at
concentrations of 0.20, 1.0 and 5.0 mg Ni/ L , the standard deviations were ±
0.011 , ±0.02 and ± 0.04, respectively. Recoveries at these levels were 100%,
97% and 93%, respectively.
References
United States Environmental Protection Agency. (1992). Methods for Chemical Analysis
of Water and Wastes. Methcxi #249.1 and #249.2. EnvUonmental Mortitoring and
Support Laboratory, United States Environmental Protection Agency, Cincirmati,
Ohio.

216
METHOD #: 258.1 Approved for NPDES (Editorial Revision 1974)
TITLE: Potassium (AA, DUect AspUation)
ANALYTE:
Potassium, K

DSfSTRUMENTATION: AA

Optimum Concenu-ation Range: 0.1-2 mg/L using a wavelengtii of 766.5 nm

Sensitivity: 0.04 mg/L
Detection Lintit: 0.01 mg/L

1.0 Preparation of Standard Solution

1.1 Stock Solution: Dissolve 0.1907 g of KCl (analyticalreagentgrade), dried at

110°C, in deionized distiUed water and make up to 1 Uter. 1 mL = 0.10 mg K (100
mg/L).
1.2 Prepare dUutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid and at the same concentration as wiUresultin the sample to be analyzed
either ciirectiy or after pnxessing.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methods section of this manual.
3.0 Sample Preparation
3.1 For the analysis of total potassium in domestic and industrial effluents, the
procedures for the determination of total metals as given in part 5.0 of the Atontic
Absorption Methods section of this manual have been found to be satisfactory.
3.2 For ambient waters, a representative aUquot of a weU ntixed sample may also be
used dUectiy for analysis. If suspended sotids are present in sufficient amounts to
clog the nebulizer, the sample may be aUowed to settie and the supematant tiquid
analyzed ciirectiy.

4.0 Instmmental Parameters

4.1 Potassium hoUow cathode lamp
4.2 Wavelength: 766.5 nm, 769.9 nm, sUt widtii: 1.0 nm
4.3 Fuel: Acetylene
4.4 Oxidant AU
4.5 Type offlame: Stightiy oxidizuig
5.0 Analysis Procedure
5.1 For the analysis procedure and the calculation, see 10.1 of the Atontic Absorption
Methcxis section of this manual.

217
 Notes
1. In aU-acetylene or other hightemperatureflames(> 2800°C), potassium can
experience partial iortization which indirectiy affec:ts abscjrption sensitivity. The
presence of other alkaU salts in the sample can reduce this ionization and thereby
enhance analyticalresults.The ionization suppressive effect of scxUum is smaU if
theratioof Na to K is under 10. Any enhancement due to socUum can be stabtiized
by adcUng excess scxUum (1(XX) g/rnL) to both sample and standard solutions. If
more stringent control of ionization is required the adcUtion of cesium should be
considered. Reagent blanks should be analyzed to correct for potassium impurities
in the buffer stcx:k.
2. The 404.4 nm line may also be used. This line has a relative sensitivity of 500.
3. To cover the range of potassium values normaUy observed in surface waters
(0.1-20 mg/L), U is suggested that the bumer head berotatedA 90 degree rotation
of the bumer head provides approximately one-eighth the normal sensitivity.

6.0 Precision and Accuracy

6.1 In a single laboratory (EMSL), usmg cUstiUed water samples spiked at
concentrations of 1.6 and 6.3 mg KyL. The standard deviations were ± 0.2 and ±
0.5,respectively.Recoveries at these levels were 103% and 102%, respectively.

References
United States Environmental Protection Agency. (1992). Metfiods for Chemical Analvsis
of Water and Wastes. Metficxi #258.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincirmati, Ohio.

218
METHOD #: 270.2 Approved for NPDES and SDWA (Issued 1978)
TITLE: Selenium (AA, Fumace Technique)
ANALYTE:
Selertium, Se

INSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 5-100 g/L

Detection Lintit: 2 pg/L
1.0 Preparation of Standard Solution
1.1 StcKk Selertium Solution: Dissolve 0.3453 g of selenous acid (actual assay 94.6%
H2Se03) in deionized cUstiUed water and make up to 200 mL. 1 mL = 1 mg Se
(1000 mg/L).
1.2. Nickel Nitrate Solution, 5%: Dissolve 24.780 g of ACSreagentgrade
Ni(N03)2-6H20 in deionized cUstiUed water and make up to 100 mL.
1.3 Nickel Nitrate Solution, 1%: Dilute 20 mL oftfie5% nickel niu-ate to 100 mL witfi
deionized cUstiUed water.
1.4 Worldng Selertium Solution: Prepare dtiutions of the stcx;k solution to be used as
caUbration standards at the time of analysis. Withdraw appropriate aUquots of the
stock solution, add 1 mL of concentrated. HNO3, 2 mL of 30% H2O2 and 2 mL
of the 5% nickel nitrate solution. Dtiute to 1(X) rriL with deionized cUstiUed water.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 of the Atontic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 Transfer 100 mL of well-ntixed sample to a 250 mL GriffUi beaker, add 2 mL of
30% H2O2 and sufficient concentrated HNO3 toresultin an acid concentration of
1 %(v/v). Heat for 1 hour at 95°C or until the volume is sUghtiy less than 50 mL.
3.2 Ccx)l and bring back to 50 mL with deionized cUstUled water.
3.3 Pipet 5 mL of this cUgested solution into a 10-mL volumetric flask, add 1 mL of
the 1% nickel niu-ate solution and dilute to 10 mL with deionized distUled water.
The sample is nowreadyfor injection into the fumace. NOTE: If solubiUzation or
cUgestion is not requued adjust the HNO3 concenu-ation of tfie sample to 1% (v/v)
and add 2 mL of 30% H2O2 and 2 mL of 5% nickel nitrate to each 100 mL of
sample. The volume of the calibration standard should be adjusted with deionized
cUstiUed water to matehtfievolume change of the sample.

4.0 Instmment Parameters

4.1 Drying tUne andtemperature:30 sec-125°C
4.2 Charringtimeandtemperature:30 sec-1200°C
4.3 Atontizing time andtemperature:10 sec-27(X)°C

219
 4.4 Purge Gas Atmosphere: Argon
4.5 Wavelengtii: 196.0 nm.
5.0 Analysis Procedure

5.1 For the analysis procedure and the calculation see 10.4 oftfieAtontic Absorption
Methcxis sec:tion of this manual.
Notes
1. The use of background correction is recommended.
2. Selenium analysis suffers interferencefix)mchlorides (> 800 mg/L) and suUate (>
200 mg/L). For the analysis of industrial effluents and samples witfi concenu-ations
of sulfate from 2(X) to 2000 mg/L, botfi samples and standards should be prepared
to contain 1% rtickel.
6.0 Accuracy

6.1 Using a sewage treatment plant effluent contammg < 2 g/L and spiked with a
concentration of 20 g/L, a recovery of 99% was obtained
6.2 Using a series of indusuial waste effluents spUced at a 50 g/L level, recoveries
ranged from 94 to 112%.
6.3 Using a 0.1% nickel nitrate solution as a synthetic matrix with selenium
concenu-ations of 5,10, 20,40, 50, and 100 gA-,relativestandard deviations of
14.2, 11.6, 9.3, 7.2, 6.4 and 4.1%, respectively, were obtained at the 95%
confidence level.
6.4 In a single laboratcjry (EMSL), using Cincirmati, Ohio tap water spiked at
concentrations of 5,10, and 20 g Se/L, the standard deviations were ± 0.6, ± 0.4,
and ± 0.5, respectively. Recoveries at these levels were 92%, 98%, and 100%,
respectively.
References
United States Environmental Protection Agency. (1992). Metiiods for Chentical Analysis
of Water and Wastes. Methcxi #270.2. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

220
METHOD #: 272.1 and #272.2 Approved for NPDES and SDWA (Issued 1978)
TITLE: Stiver (AA, Fumace Techrtique)
ANALYTE:
Stiver, Ag

DSfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 1-25 g/L

Detection LUnit: 0.2 g/L
Direct aU: (Optimum Concentration Range: 0.1-4 mg/L
Detection Limit: 0.06 mg/L
1.0 Preparation of Stanciard Solution

1.1 Stock Solution: Dissolve 0.1575 g silver nitiate, AgNOs, Ui 100 mL water, add
10 mL concenttated HNO3, and make up to 1000 mL; 1.00 mL = 100 pg Ag or
Dissolve 1.575 g of AgNOsin deionized water, add 10 ml of concenuated HNO3
and make up to 1 Uter. 1 ml = 1 mg Ag( lOOOmg Ag/L).
1.2 Prepare dtiutions of the stcx:k solution to be used as calibration standards at the
time of analysis.
1.3 The caUbration standard should be dtiuted to contain 0.5% (v/v) HN03.
1.4 In methcxi 10.4, use 10.4.4.3.2 as a matrix mcxUfier if needed.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 of the Atontic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under 5.0. Sample solutions for analysis should contain
0.5% (v/v) HN03.
4.0 Instmment Parameters
4.1 Fumace
4.1.1 Drymg Time and Temp: 30 sec- 125°C.
4.1.2 Ashing Time and Temp: 30 sec-400°C.
4.1.3 Atomizuig TUne and Temp: 10 sec-2700°C.
4.1.4 Purge Gas Atmosphere: Argon
4.1.5 Wavelengtii: 328.1 nm
4.2 Direct aU
4.2.1 Stiver haUow catiiode lamp
4.2.2 Wavelength: 328.1 nm, stit width: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: AU
4.2.5 Type offlame:Oxidizing
5.0 Analysis Prcx:edure

221
 5.1 For the analysis procedure and the calculation, see 10.1 or 10.4 of tiie Atomic
Absorption Methods section of this manual.
5.2 Notes
5.2.1 Background correction may be reqmred if the sample contains high
cUssolved sotids.
5.2.2 The use of haUde acids should be avoided.
6.0 Accuracy:

6.1 In a smgle laboratory (EMSL), USing Cincumati Ohio tap water spUced at
concentrations of 25, 50, and 75 g AgA-, the standard deviations were ± 0.4, ±
0.7, and ± 0.9,respectively.Recoveries at these levels were 94%, 1(X)% anci
104%, respectively.
Reference
Urtited States Environmental Protection Agency. (1992). Metiicxis for Chemical Analvsis
of Water and Wastes. Method #272.1 and #272.2. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincinnati,
Ohio.

222
METHOD #: 273.1 Approved for NPDES (Editorial Revision 1974)
TITLE: Sodium (AA, DUect AspUation)
ANALYTE:
ScxUum, Na

INSTRUMENTATION: AA

DUect aU: Optimum Concenu-ation Range: 0.03-1 mg/L usmg a wavelengtii of 589.6 nm
Detection Umit: 0.002 mg/L

1.0 Preparation of Standard Solutions

1.1 Stock Solution: Dissolve 2.542 g of NaCl (analyticalreagentgrade), dried at

140°C in deionized disttiled water and make up to 1 Uter. 1 mL = 1 mg Na (1000
mg/1).
1.2 Prepare dtiutions of the stcx:k solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid and at the same concentration as wiUresultin the sample to be analyzed
either directiy or after pnxessing.
2.0 Sample Preservation

2.1 For sample handUng and preservation see part 2.0 and 4.0 of the Atomic
Absorption Method section of this manual.
3.0 Sample Preparation
3.1 Fcjr the analysis of total scxUum in domestic and industrial effluents, the
procedures for the determination of total metals as given in part 5.0 of the Atontic
Absorption Methcxis section of this manual have been found to be satisfactory.
3.2 For ambient waters arepresentativealiquot of a weU-mixed sample may be used
dUectiy for analysis. If suspended solids are present in sufficient amounts to clog
the nebuUzer, the sample may be aUowed to settie and the supematant Uquid
analyzed directiy.

4.0 Instmmental Parameters (General)

4.1 ScxUum hoUow cathcxie lamp
4.2 Wavelength: 589.6 nm, 330.2 nm, stit widtii: 0.5 nm
4.3 Fuel: Acetylene
4.4 Oxiciant AU
4.5 Type offlame: Oxidizing
5.0 Analysis Prcx:edure
5.1 For the analysis prcx:edure and the calculation, see 10.1 of the Atontic Absorption
Methcxis section of this manual.
Notes
1. The 330.2 nmresonancelUie of scxUum, which has arelativesensitivity of 185,

223
 provides a convenient way to avoid the need to cUlute more concentrated solutions
of socUum.
2. L<>w-temperatureflamesincrease sensitivity by reducing the extent of ionization of
this easUy ionized metal. Ionization may also be controUed by aciding potassium
(10(X) mg/L) to both standards and samples.
3. Theflamephotomeuic methcxi may also be used (Stanciard Methcxis, 14th EcUtion,
p. 250).
6.0 Accuracy

6.1 In a single laboratory (EMSL), using cUstiUed water samples spiked at levels of
8.2 and 52 mg Na/L, the stanciard deviations were ±0.1 and ± 0.8, respectively.
Recoveries at these levels were 102% and 100%.
Reference

Urtited States Environmental Protection Agency. (1992). Methcxis for Chemical Analvsis
of Water and Wastes. Methcxi #273.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cmcmnati, Ohio.

224
METHOD #: 279.1 and #279.2 Approved for NPDES (Technical Revision 1978)
TITLE: ThalUum (AA, DUect AspUation)
ANALYTE:
ThalUum, Th
INSTRUMENTATION: AA

Fumace: OptUnum Concentration Range: 5-100 pg/L

Detection Lkitit: 1 pg/L
DUect aU: Optimum Concentration Range: 1-20 mg/L usmg a wavelength of 276.8nm
Detection Limit: 0.1 mg/L
1.0 Preparation of Standard Solution
1.1 Stcx:k Solution: Dissolve 1.303 g of thalUum niu-ate, TINO3 (analytic reagent
grade) in deiortized cUstiUed water. Add 10 mL of concentrated, rtitric acid and
dtiute to 1 liter witfi deionized cUstUled water. 1 mL = 1 mg Tl(1000mg/L).
1.2 Prepare cUlutions of the stcx:k thaltium solution to he used as calibration stanciards
at the time of analysis. The caUbration standarcis should he prepared using rtiuic
acnd and at the same concentration as wiUresultin the sample to he analyzed either
dUectiy or after prcx:essing.
2.0 Sample Preservation
2.1 The sample handling and preservations, see part 2.0 of the Atomic Absorption
Methcxis section of this manual.
3.0 Sample Preparation
3.1 The procedures for preparation of the sample as given in parts 4.0 and 5.0 of the
Atontic Absorption Methcxis section of this manual have been found to be
satisfactory if HCl is ontitted.

4.0 Instmmental Parameters

4.1 Fumace
4.1.1 Drying time andtemp:30 sec-125°C
4.1.2 Ashing time andtemp:30 sec -4(X)°C
4.1.3 Atomizing time andtemp:10 sec -2400°C
4.1.4 Purge gas atmosphere: Argon
4.1.5 Wavelengtii: 276.8 nm
4.2 Direct aU
4.2.1 ThalUum hollow catfiode lamp
4.2.2 Wavelengtii: 276.8 nm, sUt widtii: 0.5 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: AU
4.2.5 Type offlame:Oxidizing

225
5.0 Analysis Procedure

5.1 For the analysis procedure andtiiecalculation, see 10.1 or 10.4 of tiie Atontic
Abscjrption Methods section of this manual. For concenu-ations of thaUium below
0.2 mg/L, 10.4 is recommended.
6.0 Accuracy

6.1 In a single laboratory (EMSL), using a mixed industrial-domestic waste

effluent at concenu-ations of 0.60, 3.0 and 15 mg Tl/L,tfiestandard
deviations were ± 0.018, ± 0.05 and ± 0.2, respectively. Recoveries at
these levels were 100%, 98% and 98%, respectively.
Reference

United States Environmental Protection Agency. (1992). Methcxis for Chentical Analvsis
of Water and Wastes. Metiiod #279.1 and #279.2. EnvUonmental Monitoring and
Support Laboratory, United States EnvUonmental Protection Agency, Cincinnati,
Ohio.

226
METHOD #: 286.1 and #286.2 Approved for NPDES Gssued 1978)
TITLE: Vanadium (AA, Fumace Technique)
ANALYTE:
VanacUum, V
DSfSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 10-200 g/L

Detection Limit: 4 g/L
DUect aU: OptUnum Concentration Range: 2-100 mg/L
Detection Lintit: 0.2 mg/L
1.0 Preparation of Standard Solution
1.1 Stock solution: Dissolve 1.7854 g vanacUum pentoxide, V2O5 in 10 ml of
concentrated nitric acid and dtiute to 1 Uter with deiortized cUstiUed water. 1 ml= 1
mg/L (lOOOmg/L).
1.2 Prepare dUutions of the stock solution to be used as calibration standards at the
time of analysis. These solutions are also to be used for "standard adcUtions".
1.3 The caUbration standard should be dtiuted to contain 0.5% (v/v) HN03.
2.0 Sample Preservation
2.1 For sample handUng and preservation, see part 2.0 and 4.0 of the Atomic
Absorption Methcxis section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under 5.0. Sample solution for analysis should contain 0.5%
(v/v) HNO3.

4.0 Instmment Parameters

4.1 Fumace
4.1.1 Drymg Time and Temp: 30 sec-125°C.
4.1.2 Ashing Time and Temp: 30 sec-1400°C.
4.1.3 Atontizmg TUne and Temp: 15 sec-2800°C.
4.1.4 Purge Gas Atmosphere: Argon.
4.1.5 Wavelength: 318.4 nm.
4.2 Direct aU
4.2.1 Vanadium haUow catfiode lamp
4.2.2 Wavelengtii: 318.4 nm, sUt widtii: 0.2 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: Nitrous oxide
5.2.5 Type offlame: Fuel rich
5.0 Analysis Procedure
5.1 Fortfieanalysis procedure andtiiecalculation, see 10.2 of the Atomic Absorption

227
 Metiiods section of this manual.
5.2 Notes
5.2.1 Background correc^tion may be reqiured if the sample contains high
cUssolved sotids.
5.2.2 Because of possible chemical interaction, rtitrogen should not be used as the
purge gas.
6.0 Accuracy

6.1 In a single laboratory (EMSL), using a mixed indusuial-domestic waste effluent

spiked at concentrations of 2.0. 10, and 50 mg V/L, the standard deviations ware
± 0.1, ±0.1 and ±0.2 respectively. Recoveries at these levels were 1(X)%, 95%,
and 97%, respectively.
Reference
United States Environmental Protection Agency. (1992). Methods for Chemical Analvsis
of Water and Wastes. Method #286.1 and #286.2. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincinnati,
Ohio.

228
METHOD #: 289.1 and #289.2 Approved for NPDES (Editorial Revision 1974)
TITLE: Zinc (AA, Direct Aspiration)
ANALYTE:
Zinc, 21n

INSTRUMENTATION: AA

Fumace: Optimum Concentration Range: 0.2-4 pg/L

Detec^tion Limit: 0.05 pg/L
DUect aU: Optimum Concend-ation Range: 0.05-1 mg/L usmg a wavelengtfi of 213.9 nm
Detection LUnit: 0.(X)5mg/L
1.0 Preparation of Standard Solution

1.1 Stcx:k Solution: CarefuUy weigh l.CX) g of zinc metal (analyticalreagentgrade)

and dissolve cautiously Ui 10 mL HN03. When solution is complete make up to 1
Uter with deionized distilled water. 1 mL = 1 mg Zn (1000 mg/L).
1.2 Prepare dUutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid and at the same concentration as wiUresultin the sample to be analyzed
either directiy or after pnxessing.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 of the Atomic Absorption
Methcxis section of this manual.
3.0 Sample Preparation
3.1 The procedures for preparation of the sample as given in parts 4.0 and 5.0 of the
Atomic Absorption Methcxis section of this manual have been found to be
satisfactory.
4.0 Instmmental Parameters
4.1 Fumace
4.1.1 Dryingtimeand temp: 30 sec- 125°C
4.1.2 Ashmg time andtemp:30 sec-400°C
4.1.3 Atomizing tUne and temp: 10 sec-2500°C
4.1.4 Purge gas atmosphere: Argon
4.1.5 Wavelengtii: 213.9 nm
4.2 Direct aU
4.2.1 Zinc hoUow cathcxie lamp
4.2.2 Wavelengtii: 213.9 nm, 307.6 nm, stit widtii: 1.0 nm
4.2.3 Fuel: Acetylene
4.2.4 Oxidant: AU
4.2.5 Type offlame:Oxidizing

5.0 Analysis Procedure

229
 5.1 For tiie analysis procedure andtfiecalculation, see 10.1 or 10.4 oftfieAtontic
Absorption Methcxis section of this manual.
5.2 Notes
5.2.1 High levels of sUicon may interfere.
5.2.2 The aU-acetylene flame absorbs about 25% oftfieenergy attiie213.9 nm
line.
5.2.3 The sensitivity may be increased by the use of low-temperature flames.
5.2.4 Some sample container cap liners can be a source of zinc contamination.
To circumvent or avoid this problem, the use of polypropylene caps is
recommended.
5.2.5 The cUthizone colorimetric methcxi may also be used (Stanciard Methcxis,
14tfi Edition, p 265).
5.2.6 For concentrations of zUic below 0.01 mg/L, method 10.4 is
recommended.
6.0 Accuracy
6.1 An interlaboratory study on trace metal analyses by atontic absorption was
conducted by the C^ality Assurance and Laboratory Evaluation Branch of EMSL.
Six synthetic concentrates containing varying levels of aluminum, cadntium,
chromium, copper, Uon, manganese, lead and zinc were added to natural water
samples. The statisticalresultsfor zinc were as foUows:

Standard
Number Tme Values Mean Value Deviation Accuracy as
of Labs g/liter g/liter g/Uter % Bias

86 281 284 97 1.2

89 310 308 114 -0.7
82 56 62 28 11.3
81 70 75 28 6.6
62 7 22 26 206
61 11 17 18 56.6

References
United States Environmental Protection Agency. (1992). Metiicxis for Chemical Analysis
of Water and Wastes. Methcxi #289.1 and #289.2. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

230
METHOD #: 3500 Sr

TITLE: Strontium (AA, Fumace Technique)

ANALYTE:
Strontium, Sr

INSTRUMENTATION: AA
Detection Lintit: 0.2 mg/L
1.0 Preparation of Standard Solution
1.1 Stcx:k solution: Strontium: Suspend 0.1685 g SrCOs in water and cUssolve
cautiously with a minimum amount of 1 + 1 HNO3. Add 10.0 mL concentrated
HNO3 and dtiute to 1000 mL witfi water: 1 mL = 100 ^g Sr.
1.2 Prepare cUlutions of the stcx^k solution to be used as caUbration stanciards at the
time of analysis.
1.3 The calibration standard should be diluted to contain 0.5% (v/v) HN03.
2.0 Sample Preservation
2.1 For sample hancUing and preservation, see part 2.0 and 4.0 of the Atontic
Abscjrption Methods section of this manual.
3.0 Sample Preparation
3.1 Prepare as described under section 5.0 of the Atontic Absorption Methods.
Sample solutions for analysis should contain 0.5% (v/v) HNO3.

4.0 Instrument Parameters

4.1 Strontium hollow cathcxie lamp
4.2 Wavelength: 460.7 nm, slit widtii: 0.5 nm
4.3 Fuel: Acetylene
4.4 Oxidant: nitrous oxide
4.5 Type offlame:Strongly oxidizing
5.0 Analysis Procedure
5.1 Fcjr the analysis prcx:edure and the calculation, see 10.1 of the Atontic Absorption
Methcxis section of this manual.
5.2 Notes
5.2.1 A very low pH can prcxiuce interferences.
6.0 Precision and Accuracy
6.1 Strontium concentrations in the range 12.0 to 16.0 mg/L can be determined with
an accuracy within ± 1 to 2 mg/L.

231
Reference
American PubUc Healtii Association (1992). Standard Metfiods for the. Examination of
Water and Wastewater. 18tfied Metfiod 3111. American PubUc Health
Asscx:iation, American Water Works Asscx:iation, and Water EnvUonment Federation,
Washington DC, 3-13.

232
TITLE: Nitrogen

INTRODUCTION
In waters and wastewaters tiie forms of niu-ogen of greatest Uiterest are, Ui order of
decreasing oxidation state, niu-ate, nidite, ammonia, and organic nitrogen. AUtiieseforms
of nidx>gen, as weU as nitrogen gas (N2), are biochemicaUy interconvertible and are
components of the nitrogen cycle. They are of Uiterest for many reasons.
Organic nitrogen is defined functionaUy as cjrganicaUy bound nitrogen in the trinegative
oxidation state. It does not mclude aU orgartic rtitrogen compoimds. AnalyticaUy, organic
nitrogen and ammonia can be determined together and have been referred to as "Total
Kjeldahl nitrogen,(TKN)" atermtfiatreflectstfietechnique used UitiieUdetemtination. To
determine orgaitic rtitrogen alone, a separate analysis of ammonia must be undertaken and
theresultsubductedfix)mTKN. Organic nitrogen includes such natural materials as
proteins and peptides, nucleic acids and urea, and numerous synthetic orgaitic materials.
Typical orgartic rtitrogen concentrations varyfinoma few hundred micrograms per Uter in
some lakes to more than 20 mg/L inrawsewage.

Total oxiciized rtitrogen is the sum of niu-ate and nitrite niux)gen. Nitrate generaUy cxxurs in
trace quantities in surface water but may attain high levels in some groundwater. In
excessive amounts, it contributes to the illness known as methemoglobinemia in infants. A
lintit of 10 mg rtitrate as nitrogen/L has been imposed on cirinking water to prevent this
cUsorder. Nitrate is found only in small amounts in fresh domestic wastewater but in the
effluent of rtitrifying biological treatment plants rtitrate may be found in concentrations of
up to 30 mg rtiu-ate as nitrogen/L. It is an essential nutrient for many photosynthetic
autotrophs and in some cases has been identified as the growth-Untiting nuuient.
Nitrite is as intermecUate oxidation state of nitrogen, both the oxidation of ammonia to
rtittate and in the reduction nitrate. Such oxiciation and reduction may cxx:ur in wastewater
u-eatment plants, water distribution systems, and natural water rtitrite can enter a water
supply system through its use a corrosion inhibitor in industrial pnxess water. Nitrite is
the acmal etiologic agent of methemoglobinemia. Nitrous acid w is formed from nitrite in
acicUc solution, can react with second amines (RR'NH) to form nitrosamines (RR'N-NO),
many of which are known to be carcinogens. The toxicologic significant of rtitrosation
reactions in vivo and in the natural environment is the subject of much current concem and
research.

Ammonia is present naturaUy Ui surface and wastewaters concentration generaUy is low in

groundwaters because it acisorbs to soU particles and clays and is not leachedreacUlyfirom
sc It is prcxiuced largely by deamination of orgartic nitrogen-contairting compounds and by
hydrolysis of urea. The oxidation of ammonia to nitrite and nitrite to niu-ate is faciUtated by
bacteria,respectivelyNitrosomonas and Nitrobacter. At some water treatment plants,
ammortia is added to react with chlorine to form a combined chlorine residual.

In the chlorination of wastewater effluents containing ammonia, virtuaUy nofreeresidual

chlorine is obtained untti ammonia has been oxidized. Rather,tfiechlorine reacts ammonia
to form mono- and cUchloramines. Ammortia concentrations encountered in water vary
from less than 10 pg ammonia nitrogen/L in some natural surface and groundwaters to
mcjre than 30 mg/L in some wastewaters.

233
In this manual, orgartic rtitrogen isreferredto as organic N, nitrate niu-ogen as NO3-N,
nitrite nitrogen as NO2-N, ammonia nitrogen as NH3-N. The metfiocis to determine
ammonia, rtitrate-nitrite, and Total Kjeldahl Nitrogen for the determination of organic
rtitrogen wiU be presented.

References
American PubUc Health Asscx:iation. (1992). Standard Methods fc?r the Examination of
Water and Wastewater. 18tiied. Metiiod 45(X). American PubUc Healtii Association,
American Water Works Asscxiation, and Water Environment Federation, Washington
DC, 4-75.

234
METHOD #: 350.1 Approved for NPDES (Editorial Revision 1978)
TITLE: Nitrogen, Ammonia (ColorUnetric, Automated Phenate)
ANALYTE:
Nitrogen, N
Ammortia, NH3

INSTRUMENTATION: Autoanalyzer
1.0 Scope and Application

1.1 This methcxi covers the determination of ammortia in cirinking, surface, and saline
waters, domestic and industrial wastes in the range of 0.03 to 3.0 mg/L NH3 as
N. This range is for photometric measurements macie at 600nm. Approximately
120 samples per hour can be analyzed
1.2 This method is restricted to use by or under the supervision of analysts
experienced in the operation of an autoanalyzer.
2.0 Summary of Methcxi
2.1 The automated procedure for the determination of ammortia utilizes the Berthelot
Reaction, in which the formation of a blue colored compound believed to be
closelyrelatedto Uidophenol cx:curs when the solution of an ammortium salt is
added to scxUum phenoxide, foUowed by the adcUtion of scxUum hypcx:hlorite. A
solution of EDTA is acided to the sample stream to eliminate the prectipitation of
the hydroxides of calcium and magnesium. ScxUum nitropmsside is acided to
intensify the blue color.

3.0 Sample HandUng and Preservation

3.1 Preservation by addition of 2 mL cone. H2SO4 pertiterand refrigeration at 4°C.

4.0 Interferences
4.1 Calcium and magnesium ions may be present in concentration sufficient to cause
precipitation problems during analysis. A 5% EDTA solution is used to prevent
the precipitation of calcium and magnesium ionsfiromriverwater and indusuial
waste. For sea water a socUum potassium tartrate solution is used.
4.2 Sample turbidity and color may interfere witfi this method. TurbicUty must be
removed by fUtration prior to analysis. Sample color that absorbs in the
photometric range used wiU also interfere.

5.0 Apparams
5.1 TRAACS 800 AutoAnalyzer Unit consisting of:
5.1.1 Sampler.
5.1.2 Manifold
5.1.3 Proportioning pump.
5.1.4 Heating bath with double delay coU
5.1.5 Colorimeter

235
 5.1.6 Recorder.
5.1.7 Digital printer
6.0 Reagents

6.1 DistiUed water: Special precaution must be taken to insure tiiat distiUed water is
free of ammonia. Such water is prepared by passage of cUstiUed water through an
ion exchange column comprised of a mixture of both strongly acidic cation and
strongly basic anion exchange resins. Theregenerationof the ion exchange
colunm should be carried out according to the insuuction of the manufacturer.
NOTE 1: AU solutions must be made using ammonia-free water.
6.2 Sulfuric acid 5N: AU scmbber solution. Carefully acid 139 mL of cone. suUuric
acid to ^proximately 5(X) mL of ammonia-free distiUed water. Cool to room
temperature and dilute to 1titerwith ammonia-finee distUled water.
6.3 AlkaUne Phenol: Usmg a 1 Uter Erlenmeyerflask,dissolve 83 g phenol in 800 mL
of cUstUled water. In smaU increments, cautiously add with agitation, 96 g of
NaOH, 50% w/w. Perioctically cool flask under water faucet. When ccx)l, cUlute
to 1 Uter with distiUed water. Stable for two weeks.
6.4 ScxUum hypochlorite solution: Dilute 86 mL of a bleach solution containing
5.25% NaOCl (such as "Clorox") to 100 mL witii distiUed water. AvaUable
chlorine level should approximate 2 to 3%. Since "Clorox" is a proprietary
prcxiuct, its formulation is subject to change. The analyst mustremainalert to
detecting any variation in this prcxiuct significant to its use in this pnxedure. Due
to the instabitity of this prcxiuct, storage over an extended period should be
avoided.
6.5 DiscxUum ethylenecUamine-teU-aacetate (EDTA) (5%): Dissolve approximately 1.0
g 50% w/w socUum hydroxide and 41 g of cUsocUum EDTA in about 8(X) nti of DI
water. DUute to 1 liter. Add 3 ml of Brij-35 and mix weU.
6.6 ScxUum nitropmsside: Dissolve 1.1 g of sodium ititropmsside in 1 Uter of
cUstiUed water.
6.7 Standard Solution A: In one liter volumetricflaskcontaining about 8(X) ml of DI
water and dissolve 1 nti of chloroforriL Add 0.4717 of ammonium sulfate and
swUl to cUssolve. DUute to one Uter and mix thoroughly.
6.9 Standard solution B: Dtiute 20.0 mL of standard solution A (6.7) to 1(X).0 mL
with distiUed water. Prepare fresh daily.
6.10 Using standard solutions A and B, prepare the foUowing standards in 1(X) mL
volumetric flasks (preparefreshdaily):

236
 NHB-N, mg/L mL Standard Solution
Solution B
0.4 2.0
0.8 4.0
1.2 6.0
1.6 8.0
2.0 10.0
3.0 15.0
Solution A
0.20 0.2
0.40 0.4
1.20 1.20
1.60 1.60
2.0 2.0

7.1 Prcxedure
7.1 Since the intensity of the color used to quantify the concentration is pH
dependent, the acid concentration of the wash water and the standard ammortia
solutions should approximate that of the samples. For example, if the samples
have been preserved with 2 mL cone. HiSO^/Uter, the wash water and stanciards
should also contain 2 mL cone. H2S04/Uter.
7.2 For a working range of 0.03 to 3.00 mg NH3-N/L.
7.3 The basic prcxedure to run the TRACCS automated procedures are presented.
However, ortiy persoimel trained by the lab manager should perform these
analyses.
7.3.1 Tum on the computer, this starts the pump operating automaticaUy.
7.3.2 Press CR (Chart and Run)
7.3.3 Press F4 and enter OPl (operate pump 1) with aU the mbes in acid wash
solution. Wait 5-10 ntinutes.
7.3.4 Press EDIT. Recalltiiemetiiod:
NH3EPA
7.3.5 Press F5 twice.
7.3.6 Change the standarcis and sample protcx:ol appropriate to the dtiution of
standards (using either solution A or B, see 6.10).
7.3.7 Press F6 to save
7.3.8 Prepare samples for the tray (see 4.2) andreagentsneeded. Place
appropriate tubes into theUrespectivereagents.Different for each test
9 Water 6 Sample
3 EDTA 7 Phenate
5 Nitropmsside 4 Hypochlorite
8 Water
7.3.9 Press F2QuU
7.3.10 Press BG to begin a base and gain. If unsuccessful. Press CBl 70 to
enforce a base of 70 and CGI 13 to enforce a gain. Remn the Base and
Gain.
7.3.11 Press F8 to prepare for mn: channel 1, 85%, and speed 30

237
 7.3.12 Press F2, main menu.
7.3.13 Quit
7.3.14 Press CR, Press F7: chart speed 60, fUe name (name of analyte mn and
date), operator name. Start.
7.3.15 At the end of the run, from the main menu, print out aU the results.
7.3.16 Take out aU the mbes and place them back into the acid wash solution.
7.3.17 Press CR F4 command. Press HPl to wash quickly for 5 minutes.
7.3.18 Press F4, QP 1, to quU pump.
7.3.19 Place aUtfiembes mto DI water. Tum offtfiecomputer.
8.0 Calculations

8.1 Prepare appropriate stanciard curve derived from prcx^essing ammonia standards
through nianifold Compute concentration of samples by comparing sample peak
heights with standard curve. If done on the computer, calculations are exact and
results are directiy obtained
9.0 Accuracy

9.1 In a single laboratory (EMSL), using surface water samples at concentrations of

1.41, 0.77, 0.59 and 0.43 mg NH3-N/L, tiie standard deviation was ± 0.005.
9.2 In a single laboratory (EMSL), using surface water samples at concentrations of
0.16 and 1.44 mg NH3-N/L, recoveries were 107% and 99%, respectively.

References
Bran + Luebbe Analyzing Technologies, Inc. (1987). Operation Manual for the TRAACS
8(X) System. Industrial Method No. 780-86T. Bran + Luebbe Analyzing
Technologies, Inc., Elmsford, N.Y.
Urtited States Environmental Protection Agency. (1992). Metfiods for Chentical Analvsis
of Water and Wastes. Methcxi #350.1. Environmental Monitoring and Support
Laboratory, United States EnvUonmental Protection Agency, Cincinnati, Ohio.

238
METHOD #: 353.1 Approved for NPDES and SDWA (Reissued w/ Rev. 1978)
TITLE: Nitrogen, Nitrate-Niuite (Colorimeuic, Automated, HydrazUie
Reduction)
ANALYTE:
Nitrogen, N
Nitrate, NO3
Nidite, NO2

INSTRUMENTATION: Autoanalyzer
1.0 Scope and AppUcation

1.1 This method is appUcable to cirinking and surface water, and domestic and
iridustrial wastes. The appUcable range of tiiis metiiod is 0.02-2 mg/L
rtitrate-nitrite nitrogen. Approximately 120 samples per hour can be analyzed
1.2 This method is restricted to use by or under the supervision of analysts
experienced in the operation of ion chromatograph.
2.0 Summary of Method
2.1 The automated prcx:edure for the determination of niuate utilizes the reaction
whereby nitrate is reduced to nitrite by an alkaline solution of hydrazine suUate
containing a copper catalyst The stream is then treated with suUartilaminde under
acicUc conctitions to form a soluble dye which is measured colorimetrically. The
final product measuredrepresentsthe rtitrite ion originally present plus that
formed from the rtitrate. Chloride, sulfide, ferric ion and phosphate ions
interfere.

In order to determine rtitrate levels, the rtitrite alone must be subtractedfiromthe

total (rtitrate + rtitrite). This can be achieved by substituting cUstiUed water for the
copper, hydrazine and NaOHtineson the martifold A separate calibration curve
should be determined for nitrate plus nitrite and for nitrite alone.

3.0 Sample HandUng and Preservation

3.1 Analysis should be made as scx)n as possible. If analysis can be made within 24
hours, samples should be preserved byrefrigerationat 4°C. When samples must
be stored for more than 24 hours, they should be preserved with 2 mL of sulfuric
acid (H2SO4) per Uter and refrigerated.
3.2 Sample turbicUty may interfere with this methcxi Turbididty must beremovedby
fUtration prior to analysis.

4.0 Interferences
4.1 Sample color that absorbs in the photometric range used for analysis wiU
interfere.
4.2 The apparent NO3 and NO2 concentrations varied ±10 percent with
concentrations of sulfide ion up to 10 mg/L.

239
5.0 Apparatus

5.1 TRAACS 800 AutoAnalyzer Unit consisting of:

5.1.1 Sampler.
5.1.2 Manifold
5.1.3 Proportioning pump.
5.1.4 HeatUig batii witii double delay coU
5.1.5 Colorimeter
5.1.6 Recorder.
5.1.7 Digital printer
6.0 Reagents

6.1 Color developUigreagent:To approxUnately 500 mL of distiUed water add 200

mL concenu-ated phosphoric acid (sp. gr. 1.834), 10 g suU^anilamide
(H2NC6H4SO2NH2) foUowed by 0.8 g N (1-Naphtiiyl)
etfiylenediamUie-dUiydrochloride. Dilute tiie solution to 1titerwitii distiUed water
and store Ui a dark bottie mtiierefrigerator.This solution is stable for
approximately 1 month.
6.2 Copper sulfate stock solution: Dissolve 3.0 g of copper sulfate (CuS04-5H20) Ui
cUstiUed water and dilute to 1 Uter.
6.3 Hydrazine sulfate stock solution: Dissolve 30.0 g of hydrazine sulfate
(N2H4H2SO4) in 900 mL of distiUed water and dilute to 1 Uter. This solution is
stable for approximately 6 months.
6.4 Brij-35, a surfactant to increase the mobiUty of the solution in the tubing.
6.5 Chloroform used as a preservative
6.6 Sodium hydroxide: Dtiute 14.4 g 50% w/w Sodium hydroxide m about 800 ml of
DI water. Add 2 ml of Brij-35 and ntix weU.
6.7 WorkUigreductor Solution: Dilute 30 ml of stock hydrazUie sulfate (6.3) to about
900 ml witfi DI water. Add 10 nti of stock cupric sulfate solution (6.2). Dtiute
to one Uter with DI water. Store in an amber container and stable for one month.
To test the working reductor solution, mn botii a 2.0 mg/L standard of nitrite and
rtitrate. If the apparent nitrate concentration is much lower than the the rtitrite
concentrations, adjust the working reductor solution by 1 nti of stock hycUaiUie
solution (6.3) until the two standards are equal.
6.8 Stock niu-ate solution (lOOmg/L NO3-N): Dissolve 0.7218 g of KNO3, oven
dried at 100-105°C for 2 hours, Ui distUled water and dilute to 1 liter. Add 1 mL
chloroform (6.5) as a preservative. Stable for 6 months.
6.10 Stock nitrite solution (100 mg/L NO2-N): Dissolve 0.6072 g KNO2 Ui 500 mL of
cUstiUed water and cUlute to 1 Uter. Preserve witfi 2 mL of chloroform (6.5) and
keep under refrigeration.
6.12 Using the stcx:k niu-ate solution (6.8), prepare the following standards Ui 1(X) mL
volumetric flasks. At least one rtitrite standard should be compared to a nitrate
standard at the concenu-ation to verify the efficiency of the reduction.

Cone, mg NO3-N/L mL of stcx^k solution/100 mL

05 O
1.0 1.0
2.0 2.0

240
7.0 Procedure

7.1 The basic procedure to mntfieTRACCS automated procedures are presented.

However, only personnel d^ned bytfielab manager should perform tiiese
analyses.
7.1.1 Tum ontiiecomputer,tfiisstartstfiepump operatUig automatically.
7.1.2 Press CR (Chart and Run)
7.1.3 Press F4 and enter OP 1 (operate pump 1) with aUtfiembes Ui acid wash
solution. Wait 5-10 ntinutes.
7.1.4 Press EDIT. RecaUtfiemetfiod:
NO3EPA
7.1.5 Press F5 twice.
7.1.6 Change the standards and sample protocol appropriate to the standards
(see 6.11).
7.1.7 Press F6 to save
7.1.8 Prepare samples fortfietray (see 3.1) and reagents needed Place
appropriate tubes into theUrespectivereagents.
9 Water 3 NaOH
6 Sample 7 Working Reductor Solution
5 DI water 4 DI water
8 Color reagent
7.1.9 Press F2 Quit
7.1.10 Press BG to begin a base and gain. If unsuccessful. Press CB1 70 to
enforce a base of 70 and CGI 13 to enforce a gain. Remn the Base and
Gain.
7.1.11 Press F8 to prepare for mn: channel 1, 85%, and speed 30
7.1.12 Press F2, main menu.
7.1.13 C^tit
7.1.14 Press CR, Press F7: chart speed 60, file name (name of analyte mn and
date), operator name. Start.
7.1.15 At the end of the run, from the main menu, print out aU the results.
7.1.16 Take out aU the mbes and place them back into the acid wash solution.
7.1.17 Press CR F4 command, Press HPl to wash quickly for 5 minutes.
7.1.18 Press F4, QP 1, to quit pump.
7.1.19 Place aU the mbes into DI water. Tum off the computer.
7.2 Run a 2.0 mg/L NO3-N and a 2.0 mg/L NO2-N standard through the system to
check for 1(X)% reduction of nitrate to nioite. The two peaks should be of equal
height.
8.0 Calculation
8.1 Prepare a standard curve by plotting peak heights of processed standards against
known concentrations. Compute concentrations of samples by comparing sample
peak heights with the standard curve.

9.0 Accuracy
9.1 In a single laboratory using cirinking water, surface water and industrial waste at
concentrations of 0.39, 1.15, 1.76 and 4.75 pg NO3-N/L, tiie standard deviations
were ± 0.02, ± 0.01, ± 0.02, and ± 0.03, respectively. In a single laboratory

241
 usmg drinking water at concentrations of 0.75 and 2.97 the recoveries were 99%
and 101%.
References
United States EnvUonmental Protection Agency. (1992). Metfiods for Chemical Analvsis
of Water and Wastes. Method #353.1. EnvUonmental Monitoring and Support
Laboratory, Urtited States EnvUonmental Protection Agency, Cincirmati, Ohio.
Bran-I-Luebbe AnalyzUig Technologies, Inc. (1987). Operation Manual fortfieTRAACS
800 Svstem. Industrial Metfiod No. 782-86T. Bran + Luebbe AnalyzUig
Technologies, Inc., Elmsford, N.Y.

242
METHOD #: 351.2 PendUig Approval for NPDES Gssued 1978)

TITLE: Nitrogen, Kjeldahl, Total (Colorimetric, Semi-Automated Block

Digester, AAA)
ANALYTE:
Nitrogen, N

INSTRUMENTATION: Autoanalyzer
1.0 Scope and AppUcation

1.1 This methcxi covers the determination of total Kjeldahl rtitrogen in cirinking and
surface waters, domestic and industrial wastes. The procedure converts niu-ogen
components of biological origin such as amino acids, proteins and peptides to
ammonia, but may not convert the nitrogenous compounds of some industrial
wastes such as antines, rtitro compounds, hydrazones, oximes, semicarbazones
and somerefractorytertiaryamines. The appUcable range of this method is 0.04
to 2 mg/L TKN. The upper range may be extended with sample cUlution.
1.2 This methcxi is restricted to use by or under the supervision of analysts
experienced in the operation of ion chromatograph.
2.0 Summary of Methcxi
2.1 The sample is heated in the presence of sulfuric acid, K2SO4 and HgS04 for two
and one half hours. Theresidueis ccx)led dtiuted to 25 mL and placed on the
AutoAnalyzer for ammonia determination. This digested sample may also be used
for Total phosphoms determination.
3.0 Defirtitions
3.1 Total Kjeldahl nitrogen is defined as the sum of free-ammonia and orgartic
rtitrogen compounds which are converted to ammonium sulfate (NH4)2S04,
under the conditions of cUgestion described below.
3.2 Organic Kjeldahl nitrogen is defined as the cUfference obtained by subtracting the
free-ammonia value (Ammonia, this manual) from the Total Kjeldahl nitrogen
value.

4.0 Sample HandUng and Preservation

4.1 Samples may be preserved by adcUtion of 2 mL of cone H2SO4 per Uter and
stored at 4°C. Even when preserved intfiismanner, conversion of orgartic
rtitrogen to ammortia may cx^cur. Therefore, samples should be analyzed as soon
as possible.
4.2 Brij-35 is a surfactant used to ease flow of the solutions within the analyzer tubes.
However, Brij-35 contains phosphoms as an impurity. Cleanse system weU if
phosphcjms is to be measured after a chemistry that uses Brij-35. If a precipitate
forms aftertfieaddition of saticylate, Unmediately stoptiieproportionUig pump
andflushtiiecoils witfi water usmg a syringe. Precipitation of saUcyUc acid is
caused by a low pH. BeforerestartUigthe system, check the concenu-ation of the
sulfiiric acid solutions and/or the working buffer solution.

243
5.0 Apparams

5.1 TRAACS 800 AutoAnalyzer Unit consisting of:

5.1.1 Sampler.
5.1.2 Manifold
5.1.3 Proportioning pump.
5.1.4 Heating bath with double delay coti
5.1.5 Colorimeter
5.1.6 Recorder.
5.1.7 Digital printer
5.2 Chemware TFE (Teflon boiUng stones), Markson Science, Inc., Box 767,
DeUnar, CA 92014)
5.3 Block Digester-40
6.0 Reagents

6.1 Mercuric SuUate: Dissolve 8 g red mercuric oxide (HgO) Ui about 75 ml of 10%
sulfuric acid and stU until dissolved Dilute to 100 ml witfi 10% sulfuric acid and
mix thoroughly.
6.2 Digestion Solution: (SuUuric acid-mercuric sulfate potassium sulfate solution):
Dissolve 133 g of K2SO4 Ui 700 mL of distiUed water and 200 mL of cone
H2SO4. Add 25 mL of mercuric sulfate solution and cUlute to 1 Uter.
6.3 Sulfuric Acid Solution: Add 80 mL of concenu-ated sulfiiric acid to 2000 mL of
ammoniafreecUstiUed water.
6.4 Stcx^k ScxUum Hydroxide: Dissolve 400 g of scxUum hydroxide 50% w/w in 900
mL of ammonia-free cUstiUed water and cUlute to 1 Uter.
6.5 Stcx:k ScxUum Potassium Tartrate Solution: Dissolve 200 g scxUum
potassium-tartrate in about 800 mL of ammonia-free cUstUled water and dtiute to 1
Uter.
6.6 Stcx:k Buffer Solution: Dissolve 134.0 g of scxUum phosphate, cUbasic
(Na2HP04) in about 8(X) mL of ammoniafreewater. Acid 40 g of scxUum
hycUoxide 50% w/w and dtiute to 1 Uter.
6.7 Working Buffer Solution: Combine thereagentsin the stated order, acid 250 mL
of stcx:k scxUum potassium tartrate solution (6.5) to 200 mL of stcx:k buffer
solution (6.6) and mix. Add 2(X) mL socUum hydroxide solution (6.4) and cUlute
to 1 Uter.
6.8 ScxUum Salicylate/SocUum Nitropmsside Solution: Dissolve 175 g of scxUum
salicylate and 0.35 g of socUum nitropmsside (ScxUum rtiu-oferricyande cUhycirate)
in about 600 mL of ammonia-firee water and cUlute to 1 Uter.
6.9 ScxUum Hypochlorite Solution: Dilute 7.0 mL scxUum hypcx:hlorite solution
(clorox) to 1(X) mL with ammonia free cUstUled water.
6.10 Stcx:k Solution A (2000 ppm): Dissolve 0.9434 g of ammonium sulfate in about
60 ml of DI water. DUute to 100 ml and ntix tiioroughly.
6.11 Stock Solution B(200ppm): Dilute 10 ml of Stock solution A mto 100 ml of
cUstiUed water.
6.11 Standards are prepared as foUows per 1(X) ml cUstiUed water:

244
 mg/LN ml of stcx:k soln B
2.5 5
2 4
1.5 3
1 2
0.5 1
50 50
Note: Standarcis are to be digested just as aretfiesamples.
7.0 Digestion PrcK:edure
7.1 To 20 of sample, add 5 mL of cUgestion solution (6.2) and mix (use a vortex
ntixer).
7.2 Add (4-8) Teflon boiUng stones (5.3). Too many boiUng chips wtil cause the
sample to boti over.
7.3 With Block Digester in manual mode set low and hightemperatureat 2(X)°C and
preheat unit to 2(X)°C. Place mbes in cUgester and switch to automatic mode. Set
lowtemperaturetuner for 1 hour. Reset high temperature to 380°C and set timer
for 2 1/2 hours.
7.4 AUow the sample to cool for 5 minutes and add 20 mL with ammonia-free water.
The mbes are ccx)l enough to cUlute when the white acid fumes have cUssipated
and the upper half of the tube is ccx)l enough to hancUe comfortably. The tubes
should not be aUowed to ccx)l to the point of K2SO4. The samples arereadyfor
analysis.
Colorimetric Analysis
7.5 The basic procedure to mn the TRACCS automated pnxedures are presented.
However, only personnel trained by the lab manager should perform these
analyses.
7.5.1 Tum on the computer, this starts the pump operating automatically.
7.5.2 Press CR (Chart and Run)
7.5.3 Press F4 and enter OPl (operate pump 1) with all the tubes in acid wash
solution. Wait 5-10 minutes.
7.5.4 Press EDIT. RecaUtfiemetfiod:
TKNEPA
7.5.5 Press F5 twice.
7.5.6 Change the standards and sample protcx:ol appropriate to the standards
(see 6.11) with standards in decreasing order with dtiution stanciard last
7.5.7 Press F6 to save
7.5.8 Prepare samples for tiie tray (see 3.1) andreagentsneeded Place
appropriate tubes into theU respective reagents. * See 7.6.
9 Acid water 3 Buffer
6 Sample 7 Salicylate nitropmsside (last in / last out)
5 Hypcx:hlorite 4 DI water
8 DI water
7.5.9 Press F2 Quit
7.5.10 Press BG to begin a base and gain. If unsuccessful. Press CBl 70 to
enforce a base of 70 and CGI 13 to enforce a gain. Remn the Base and
Gain.
7.5.11 Press F8 to prepare for mn: channel 1, 85%, and speed 30
7.5.12 Press F2, main menu.
7.5.13 (Juit

245
 7.5.14 Press CR, Press F7: chart speed 60, file name (name of analyte mn and
date), operator name. Start. See 7.6.
7.5.15 At the end of the run, from the main menu, print out aU the results.
7.5.16 Take out aUtiietubes and placetiiemback mto the acid wash solution.
7.5.17 Press CR F4 command. Press HPl to wash quickly for 5 ntinutes.
7.5.18 Press F4, QP 1, to quU pump.
7.5.19 Place aU tubes in DI water and tum off computer.
7.6 When reagents have been pumpUig for at least five ntinutes, place tiie salicylate
line in itsrespectivecontainer and allow the system to equiUbrate. If a precipitate
forms after the addition of saticylate, the pH is too low. Immediately stop the
proportioning pump andflushthe cotis with water using a syringe. Before
restarting the system, checktfieconcenu-ation of the sulfuric acid solutions and/or
the working buffer solution.
7.7 To prevent precipitation of scxUum salicylate in the waste tray, which can clog the
tray outiet, keep the niuogen flow cell pump tube and the rtiu-ogen Colorimeter
"To Waste" mbe separate from all other lines or keep tap waterflowingin the
waste tray.
8.0 Calculations

8.1 Prepare standard curve by plotting peak heights of processed standards against
concentration values. Compute concenu-ations by comparing sample peak heights
with standard curve.
9.0 Accuracy
9.1 In a single laboratory (EMSL), using sewage samples of concentrations of 1.2,
2.6, and 1.7 mg N/L, the precision was ± 0.07, ± 0.03 and ±0.15, respectively.
9.2 In a single laboratory (EMSL), using sewage samples of concentrations of 4.7
and 8.74 mg N/L, the recoveries were 99 and 99%, respectively.

References
Bran + Luebbe Analyzing Technologies, Inc. (1987). Operation Manual fortiieTRAACS
800 System. Industrial methcxi No. 786-86T. Bran + Luebbe AnalyzUig
Technologies, Inc., Elmsford, N.Y.
United States EnvUonmental Protection Agency. (1992). Metfiods for Chentical Analvsis
of Water and Wastes. Methcxi #351.2. EnvUonmental Monitoring and Support
Laboratory, United States EnvUonmental Protection Agency, Cincinnati, Ohio.

246
METHOD #: 413.2 (Editorial Revision 1978)

TITLE: Oti And Grease and Total Petroleum Hydrocarbons (Specnx)photometric, Infrared)
ANALYTE:
OU, Grease

INSTRUMENTATION: IR
INTRODUCTION:
In the determination of oti and grease, an absolute quantity of a specific substance is not
measured. Rather, groups of substances with similar physical characteristics are
determinai quantitatively on the basis oftiieUcommon solubiUty Ui an organic exttacting
solvent "OU and grease" is defined as any material recovered as a substance soluble in the
solvent It includes otiier material exu-acted by the solvent from an acidified sample (such
as sulfur compounds, certain organic dyes, and chlorophyU) and not volatilized during the
test.
Certain constiments measured by the oti and grease analysis may influence wastewater
treatmerit systems. If present in excessive amounts, they may interfere with aerobic and
anaerobic Itiological processes and lead to decreased wastewater treatment efficiency.
When cUscharged in wastewater or treated effluents, they may cause surface films and
shoreline deposits leacUng to envUonmental degradation.
A knowledge of the quantity of oil and grease present is helpful in proper design and
operation of wastewater treaunent systems and also may caU attention to certain treaunent
difficulties.
In the absence of specially mocUfied industrial products, oil and grease is composed
primarily of fatty matterfromartimal and vegetable sources andfromhydrcx^arbons of
petroleum origin. A knowledge of therelativecomposition of a sample ntiitimizes the
ciifficulty in determining the major source of the material and simplifies the correction of oti
and grease problems in wastewater treaunent plant operation and stteam poUution
abatement
The determination of Total Petroleum Hydrcx:arbons follows this same methcxi but the
sample is in contact with siUca gel to remove polar substances such as fatty acids.

1.0 Scope and Application

1.1 This methcxi includes the measurement offluorcx:arbon-l13 (Freon) exu-actable
matter from surface and saUne waters, indusuial and domestic wastes. It is
a5)pUcable to the detemtination of hycirocarbons, vegetable oils, animal fats,
waxes, soaps, greases and related matter.
1.2 The method is appUcable to measurement of most Ught petroleum fuels, although
loss of about haU of any gasoline present during the exttaction manipulations can
be expected.
1.3 Tlie methcxi covers the rangefinom0.2 to 1(XX) mg/L of extractable material.
1.4 WhUe this method can be used to obtain an estUnate oftfieoti and grease that
would be measured gravimetrically, in many cases the estimate more accurately
describes the parameter, as it wiU measure volatiles more effectively and is not

247
 susceptible to interferences such as exuactable suUur. It can be used witii the
Petroleum Hycirocarbon procedure to obtain an oti and grease value and a
petroleum hycUcxjarbon value on tiie same sample.
2.0 Summary of Method

2.1 The sample is acidified to a low pH (< 2) and cxti-acted witfi fluorocarbon-113.
The oti and grease is determined by comparison of the irtirared absorbance of the
sample exu-act with stanciards.
3.0 Sampling and Storage

3.1 Arepresentativesample of 1 Uter volume should be coUected in a glass bottie. If

analysis is to be delayed for more than a few hours, the sample is preserved by
the addition of 5 mL HCl (6.1) attfietimeof coUection andrefrigeratedat 4°C.
3.2 Because losses of grease wiU cxx:ur on sampling equipment, the coUection of a
composite sample is impractical. Individual portions collected at prescribed time
intervals must be analyzed separately to obtain the average concentration over an
extended pericxi.
4.0 Apparams
4.1 Separatory funnel, 2(XX) ml, with Teflon stopccx:k.
4.2 IrUrared spectrophotometer, scarming. Non-scanning insd-uments may also be
used but can be subject to positive interferences in complex chentical
wastewaters.
4.3 CeUs, 10 rmn, 50 mm, and 100 mm path length, scxUum chloride or infrared
grade glass. The ESL uses a 10 mm path length.
4.4 FUter paper, Whatman No. 40,11 cm.
5.0 Reagents
5.1 Hycirochloric acid, 1:1. Mix equal volumes of concend-ated HCl and distiUed
water.
5.2 Fluorxx:arbon-113, (l,l,2-dichloro-l,2,2-difluoroetiiane), b.p. 48°C.
5.3 ScxUum sulfate, anhycirous crystal.
5.4 CaUbration mixtures:
5.4.1 Reference oU: Pipet 15.0 mL n-hexadecane, 15.0 mL iscxKtane, and 10.0
mL chlorobenzene into a 50 mL glass stoppered bottie. Maintain the
integrity of the mixture by keepUig stoppered except when witfidrawUig
aUquots.
5.4.2 Stock standard: Pipet 1.0 noLreferenceoti (5.4.1) into a tared 200 mL
volumetric flask and immediately stopper. Weigh and dtiute to volume
withfluorcx:arbon-113.The concenttation of this stcx:k is determined using
tfie measured weight For example, if tfie 1.0 ml ofreferenceoti weighs 5
g than the concend-ation is 5 g / 2(X) ml = 25,000 mg/L.
5.4.3 Wcjrking standards: Pipet appropriate volumes of stcx;k standard (5.4.2)
mto 100 mL volumedic flasks according totfiecell patii lengtii to be used.
Dtiute to volume witiifluorocarbon-113.Calculate concenttation of
standards from the stock stanciard.
5.5 Sitica gel, 100 to 200 mesh, which has been deactivated witii 2% water.

248
6.0 Procedure

6.1 Mark the sample bottie at the water meniscus for later determination of sample
volume. If the sample was not acidified attimeof coUection, add 5 mL
hydrochloric acid 66.1) totfiesample bottle. After ntixingtfiesample, check tfie
pH by touching pH-sensitive paper to the cap to insure that the pH is 2 or lower.
Add more acid if necessary.
6.2 Pour the sample into a separatory funnel.
6.3 Add 30 mLfluorocarbon-113(5.2) totfiesample bottie androtatetiiebottie to
rinse the sides. Transfer the solvent into the separatory funnel. Exd-act by
shaking vigorously for 2 ntinutes. AUow the layers to separate.
6.4 FUter the solvent layer into a 100 mL volumetric flask through a funnel containing
solvent-moistenedfilterpaper.
NOTE: An emulsion that faUs to cUssipate can be broken by pouring about 1 g
scxUum sulfate (5.3) into thefilterpaper cone and slowly cUairting the emulsion
through the salt AdcUtional 1 g portions can be added to the cone as required.
6.5 Repeat (6.3 and 6.4) twice more with 30 mL portions of fresh solvent, combining
aU solvent in the volumetric flask.
6.6 Rinse thetipof the separatory funnel,filterpaper, and the furmel with a total of
5-10 mLfluorocarbon-113and collect therinsingsin the flask. DUute the extract
to 1(X) nti, and stopper the flask.
6.7 Select appropriate working standarcis and ceU path length accorcting to the
foUowing table of approximate working ranges:

Patfi length Range

10 mm 2-40 mg
50 mm 0.4-8 mg
100 mm 0.1-4 mg

6.8 Determination of Oti and Grease: Scan standarcis and samples from 32(X) cm-1 to
2700 cm-1 withfluonxarbon-113in the reference beam andrecordtheresultson
absorbance paper. The absorbances of samples and standards are measured by
constmcting a sttaight baseline over the range of the scan and measuring the
absorbance oftfiepeak maximum at 2930 cm-i and subdactUig the baseline
absorbance at that point Iftfieabsorbance exceeds 0.8 for a sample, select a
shorter patfilengtfi or dtiute as requUed.
6.9 Use a calibration plot of absorbance vs. mg oil preparedfromtiiestandards to
determine the mg oU in the sample solution.
6.10 Detemtination of Total Pettoleum Hydrocarbons: Remove sufficient exd-act from
the 100 nti volumedic flask to lowertfielevel of the Uquid to about 20 mm below
tfie base oftfieneck of tfie flask. Add about 3 g of stiica gel totfieUquid and
insert a magnetic stirring bar.
6.10.1 Placetfieflaskon a magnetic stirrer and stir the extract for 10 min at a rate
sufficient to cause continuous convection of the sitica gel but not so great
as to cause splashing or a vortex down to the stirring bar.
6.10.2 AUow tiie stiica gel to settle completely.
6.10.3 Measure the Uiftared absorbance oftfiedeated exd-act mtfiesame manner
that was used in 6.8.

249
7.0 Calculation

7.1 Oti and Grease

7.1.1 dtiuted: mg/L total oti and grease = (R * D) / V where: R = oti Ui solution,
determined from calibration plot, in milUgrams. D = extract dtiution factor,
if used y = volume of sample, determined byrefilUngsample bottie to
caUbration line and correcting for acid addition if necessary, Ui titers.
7.1.2 Undiluted: Read dUectiy off of caUbration plot prepared usmg standards.
7.2 Total Pedoleum Hydrcx:arbons
7.2.1 DUuted: mg/L Total Pedt)leum Hydrocarbons = (R * D) / V where: R
=amount of petroleum hycUocarbons in 100 ml of deated extract,
determined from caUbration plot, in miUigrams. D = extract dtiution factor,
if used V = volume of sample, detemtined by refilling sample bottie to
caUbration line and correcting for acid adcUtion if necessary, in titers.
7.2.2 UndUuted: Read ciirectiy off of caUbration plot prepared using stanciards.
8.0 Accuracy

8.1 The two oU and grease methcxis mtftismanual weretestedby a single laboratory
(EMSL) on sewage. This methcxi determined the oti and grease level in the
sewage to be 17.5 mg/L. When 1 Uter portions of the sewage were dosed with
14.0 mg of a mixture of #2 fiiel oti and Wesson oti, the recovery was 99% with a
standard deviation of ± 1.4 mg/L.

References
United States Environmental Protection Agency. (1992). Metfiods for Chemical Analysis
of Water and Wastes. Method #413.2. EnvUonmental Monitoring and Support
Laboratory, United States EnvUonmental Protection Agency, Cincirmati, Ohio.
American PubUc Healtii Association. (1992). Standard Methods fortfieExamination of
Water and Wastewater. 18th ed Metiiod 5520. American PubUc Healtfi Association,
American Water Works Asscx^iation, and Water Environment Federation, Washington
DC, 5-24.

250
TITLE: Standard Practice for Oxidation-Reduction Potential of Water
INSTRUMENTATION: pH Meter
INTRODUCTION
Oxidation and reduction (redox)reactionsmediate the behavior of many chentical
constituents in drinking, process, and wastewaters as weU as most aquatic compartments of
the environment Thereactivitiesand mobiUties of important elements in biological
systems (e.g., Fe, S, N, and C), as well as tiiose of a number of otiier metaUic elements,
depend strongly on redox conditions. Reactions involving both electtons and protons are
pH- and Eh-ciependent; therefore, chenticalreactionsin aqueous mecUa often can be
charaaerized by pH and Eh together with the activity of cUssolved chentical species. LUce
pH, Ehrepresentsan intensity factor. It does not characterizetfiecapacity (i.e., poise) of
the system for oxidation or reduction.
The potential difference measured in a solution between an inert incticator elecdcxie and the
standard hydrogen elecd-ode should not be equated to Eh, a thermodynamic property, of the
solution. The assumption of a reversible chemical equtiibrium, fast electrode kinetics, and
the lack of interferingreactionsat the elec:trode surface are essential for such an
interpretation. These conctitions rarely, if ever, are met in natural water.
Thus, although measurement of Eh in water isrelativelystraightforward many factors limit
the interpretation of these values. These factors include irreversiblereactions,electrode
poisoning, the presence of multiple redox couples, very smaU exchange currents, and inert
redox couples. Eh values measured in thefieldcortelate pcx)rly with Eh values calculated
from the redox cx)uples present. Nevertheless, measurement of redox potential, when
properly perfcjrmed and interpreted, is useful in developing a more complete understanding
of water chemistry.
1.0 Scope and Application
1.1 TTiis practice covers the apparatus and prcx:edure for elec:trometric measurement of
oxidation-reduction potential (ORP) in water. It does not deal with the maimer in
which the solutions are prepared, the theoretical interpretation of the
oxidation-reduction potential, or the estabtishment of a stanciard
oxidation-reduction potential for any given system. The practice described has
been designed for theroutineand process measurement of oxidation-reduction
potential.

2.0 Summary of Practice

2.1 This is a practice designed to measuretfieORP which is defined as the
electromotive force between a noble metal electrode and areferenceelecuxxie
when immersed in a solution. The practice describes the elecdonic equipment
available to make the measurement and describes how to determine the sensitivity
oftfieelecti-odes as well astfiecaUbration of equipment to solutions havUig a
known potential. The ORP electrcxies are inert and measure theratioof the
activities oftfieoxidized totfiereduced species Ui the process reactions.

3.0 Interferences

251
 3.1 The ORP electtodesreUablymeasured ORP Ui nearly aU aqueous solutions and Ui
general are not subject to solution mterferencefiromcolor, Uirbidity, coUoidal
matter, and suspended matter.
3.2 The ORP of an aqueous solution is sensitive to change Uitemperatureof tfie
solution, but temperature correction is rarely done due to its minimal effect and
complex reactions. Temperature corrections are usuaUy applied only when it is
desUed to relatetfieORP totiieactivity of an ion Uitfiesolutions.
3.3 The ORP of an aqueous solution is sensitive to pH variations when the
oxidation-reductionreactioninvolves either hydrogen or hydroxyl ions. The ORP
gerieraUy tends to increase with an increase in hydrogen ions and to decrease with
an increase in hycUoxyl ions during such reactions.
3.4 Reproducible oxidation-reduction potentials cannot be obtained for chemical
systenis that are notreversible.The measurement of end pomt potential Ui
oxidation-reductiontittationis sometimes of this type.
3.5 If the metalUc portion of the ORP elecdxxie is sponge-like, materials absorbed
from solutions may not be washed away, even by repeated rinsings. In such
cases, the electrode may exhibit a memory effect, particularly U it is desired to
detect arelativelylow concentration of a particular species immecUately after a
measurement has been made in arelativelyconcenttated solution. A brightiy
poUshed metal electrcxie surface is required for accurate measurements.
3.6 The ORPresititingfrom interactions among several chemical systems present in
mixed solutions may not be assignable to any single chemical.
4.0 Apparatus
4.1 Meter: Most laboratory pH meters can be used for measurements of ORP by
substitution of an appropriate set of electrodes and meter scale. The choice wtil
depend on the accuracy desired in the determination.
4.1.1 Most process pH meters can be used for measurement of ORP by
substitution of an appropriate set of elec^trodes and meter scale. These
instmments are generally much more mgged than those which are used for
very accurate measurements in the laboratory. UsuaUy, these more mgged
instmments produceresultsthat are somewhat less accurate and precise
than those obtainedfromlaboratory insdiiments. Each of these analyzers is
satisfactory for prcx:ess ORP measurements. The choice of analyzer is
generally based on how closely the characteristics of the analyzer match the
requUements of the application. Typical factors which may be considered
include, for example, the types of signals which the analyzer can prcxiuce
to drive extemal devices, and the span ranges avaUable.
4.2 Reference Electtcxie: A calomel, stiver-silver chloride, or other reference electrode
of constant potential shaU be used If a saturated calomel elecd-ode is used, some
potassium chloride crystals shall be contained in the saturated potassium chloride
solution. If thereferenceelectrode is of theflowingjunction type, the design of
the electrode shaU aUow for each measurement a fresh Uquid junction to be fcjrmed
between the solution of potassium chloride and the stanciard or the test solution.
The elecdxxie design shil also aUow d^ces of solution to be washed from the
outer surfaces of the elecdodes. To ensure the desUed slow outward flow of the
reference-electrode solution, the solution pressure Uiside the Uquid junction should
be somewhat Ui excess of that outsidetfiejunction. In nonpressurized
appUcations this requUement can be met by maintainUig the Uiside solution level
higher than the outside solution level. If thereferenceelectrode is of tiie

252
 nonflowUig junction type,tiieseoutward flow and pressurization considerations
shaU not apply.
4.3 Oxidation-Reduction Elecdxxie: A noble metal is used Uitfieconsttuction of
oxiciation-reduction elecdwies. The most common metals employed are:
platinum, gold, and stiver. U is Unportant to select a metaltiiatis not attacked by
the test solution. The consdnction oftiieelecdxxie shaU be suchtfiatonly the noble
metal comes Ui contact witfitfietestsolution. The area oftfienoble metal Ui
contact with the test solution should be approximately 1 cm2.
4.4 Electrcxie Assembly: A conventional electrode holder cjr support can be employed
for laboratory rneasurements. Many different styles of elecuxxie holders are
suitable for various process appUcations such as measurements in an open tank,
prcxiess pipe line, pressure vessel, or a high pressure sample line.
5.0 Reagents and Materials

5.1 Aqua Regia: MUc 1 volume of concenttated niuic acid (HNO3, sp gr 1.42) witfi 3
volumes of concendated hydrochloric acid (HCL, sp gr 1.1 8). It is
recommended that ortiy enough solution be prepared for immecUate requirements.
5.2 Buffer Standard Salts:
5.2.1 Phtiialate Reference Buffer Solution (pH= 4.00 at 25°C): Dissolve 10.12 g
of potassium hycirogen phthalate (KHC8H4O4) in water and dtiute to IL.
5.2.2 Phosphate Reference Buffer Solution (pH= 6.86 at 25°C): Dissolve 3.39 g
of potassium ciihycUogen phosphate (KH2PO4) and 3.53 g of anhycUous
discxUum hydrogen phosphate (Na2HP04) in water and cUlute to 1 L.
5.3 Chrontic Acid Qeaning Solution:Dissolve about 5 g of potassium cUchromate
(K2Cr207) in 500 mL of concentrated sulfuric acid (H2SO4, sp gr 1.84).
5.4 Detergent: Use any commercially avatiable "low-suds" Uquid or solid detergent.
5.5 Nitric Acid (1+1): Mix equal volumes of concentrated rtitric acid (HNO3, sp gr
1.42) and water.
5.6 Redox Standard Solution; Ferrous-Ferric Reference Solution: Dissolve 39.21 g of
ferrous ammonium sulfate (Fe(NH4)2-(S04)2-6H20), 48.22 g of ferric
ammonium sitifate (FeNH4(S04)2*12H20) and 56.2 mL of sulfuric acid (H2SO4,
sp gr 1.84) in water and dtiute to 1 L. It is necessary to prepare the solution using
reagent grade chemicals that have an assay confining them to be within 1% of the
nominal composition. The solution should be stored in a closed glass or plastic
container.
5.6.1 The fertous-ferricreferencesolution is a reasonably stable solution with a
measurable oxiciation-reduction potential.
5.7 Redox Reference QuinhycUone Solutions: Mix 1 L of pH 4 buffer solution, (see
7.4.1), with 10 g of quirihydrone. Mix 1 L of pH 7 buffer solution, (see 7.4.2),
with 10 g of quinhydrone. Be sure that excess quinhycUone is used in each
solution so that soUd crystals are always present. Thesereferencesolutions are
stable for only 8 hours.

6.0 SampUng
6.1 Do not store samples, analyze on coUection. MinUnize both atmospheric contact
and delay in analysis.

7.0 Preparation

253
 7.1 Elecdxxie Treadnent: Iftfieassembly is m Uitermittent use, the Unmersible ends of
the elecdxxie should be kept in water between measurements. Covertfiejunctions
and fiU-holes of reference elecdxxies to reduce evaporation during prolonged
storage.
7.2 ORP Electrode Cleaning: Remove daces of foreign matter. Immerse tiie
oxidation-reduction elecdxxie Ui warm aquaregia(70°C) and aUow to stand for a
pericxi of about 1 ntin. This solution dissolves the noble metal as weU as any
foreign matter sotiiattfieelecdxxie should not be aUowed to stand Ui it longer than
the time specified. The above treatment in aqua regia may also be used cautiously
to recondition an elecdxxie that has become unreUable in its operation. It is also
possible to cleantfieelecdxxie in HNO3 (1+1). Warmtfiesolution and electrode
gradually to boiling. Maintain U just below the boiling point for about 5 min and
then aUow the solution and electrode to ccx>l. Wash the electrcxie in water several
times. It is desirable to clean the electrode daUy. An altemative cleaning prcx:edure
is to immerse the elecdxxie at roomtemperaturein chrontic acnd cleaning mixture
and then rinse fUst with dtiute hydrochloric acid and then thoroughly with water.
Pretiminary clearting with a detergent sometimes is desUable toremoveoUy
residues. A ntild abrasive can be used to remove some particulate matter. In these
cleaning operations particular care must be exercised to protect the glass-metal
sealsfromsudden changes of temperature, which might crack them.

8.0 CaUbration and Standardization

8.1 Before using electtonic type meters, tum them on and aUow them to warm up
thoroughly. Set the scale or range to the desired miUivolt level expected in the test
solution.
8.2 Verify the sensitivity of the electrcxies by noting the change in milUvolt reading
when the pH of thetestsolution is altered. The ORP will increase when the pH of
the test solution decreases and the ORP wtil decrease if the test solution pH is
increased. Place the sample in a clean glass beaker and agitate the sample. Insert
the electrodes and note the ORP or rrtilUvolt reacting. Add a smaU amount of a
dtiute NaOH solution and note the value of the ORP. If the ORP cirops sharply
when the caustic is acided, the electrcxies are sensitive and operating properly. If
the ORP does not respond as above when the caustic is added the electrxxies
should be cleaned as described in 7.2 and the above prcx:edure repeated.
8.3 Checking Response Electrodes to the Standard Redox Solutions (sec 5.6 and 5.7):
Wash the metal andreferenceelecdxxie and the sample cup or container with three
changes of water or by means of aflowingstreamfroma wash bottie. FiU the
sample container with fresh redox standard solution and immerse the electrodes.
Tum the range switch to the proper range and engage the operating button. Adjust
the asymmetry conttol to the nuUivoU potential of the standard redox solution.
Without changing the setting of the asymmedy potential knob,repeatthe above
pnxedure untti two successive insdiiment readings are constant. The readings
should not ciifferfromthe miUivolt value of the standard redox solution by more
than 10 mV.
8.3.1 It usuaUy suffices to check the sensitivity oftfieelecdxxies since the
important feature is the change of potential asrelatedto the concentration of
the oxiciant or reductant present The actual numerical value of the potential
wiU vary depending on the constituents present in the water.
8.4 Indirect CaUbration and Standardization:

254
 8.4.1 Employ this prcx:edure when it is not convenient or practical toremovethe
electrodes from theflowUigstream or container Ui whichtfieORP is bemg
determined. Use of a laboratory ORP meter or an adcUtional analyzer is
rcquUed.
8.4.2 Verify the sensitivity oftfielaboratory ORP meter or adcUtional process
analyzer in accordance with 8.2.
8.4.3 CoUect a grab sampletfiatis representative oftfiematerialtfiatis Ui contact
witfi the electtodes of the analyzertfiatis to be standardized. If a
submersion-style electrode chamber is Ui use, coUecttfiesample from the
discharge oftfiechamber. Immediately dansport the sample to tfie
laboratory ORP meter or additional prcx^ess analyzer and measure the ORP.
It is absolutely essential thattfiesample berepresentativeof the solution m
contac:t with the elecdxxies oftfieanalyzer l)eing adjusted and that the
integrity of the sample be maintained untti its ORP has been measured. In
particular, the temperature of the sample must remain constant Then adjust
the standarcUzation control on the pnxess analyzer being caUbrated until the
reacting corresponds to the ORP of the sample. Repeat the prcx^edure
described above until two successivereadingsare obtained that differ by no
moretfian10 mV. This procedure cannot be employed iftfieORP of tfie
solution being tested isfluctuatingby more than 10 mV at the tUne of
StandarcUzation.
8.4.4 The sensitivity of the electrcxies can also be verified by a determination of
the concentration of the oxidants or reductants in a grab sample coUected in
accordance with 6.1.
8.4.5 It is necessary to determine the conditions required in each incUvidual
system to use this methcxi of verifying electtode sensitivity. For example,
the chlorineresidualdetermination can be used to verify the sensitivity of
an ORP electrode system used to control an alkaline chlorination cyanide
destmction system.

9.0 Procedure
9.1 The oxidation-reduction potential (redox) methcxi entaUs using the 920 A mcxiel
pH meter by Orion. On the meter, insert the electtcxie into input 1 or 2.
Identify the electrcxie type as redox by pressing the 2nd, then electrode key.
Select 25, redox and press yes. Press the mode key until mV is cUsplayed.
Measurements wtil be made directiyromtfiemeter.
9.2 After the assembly has been checked for sensitivity (9.2) cjr stanciardized as
described in 9.4, wash the electrodes with three changes of water cjr by means
of a flowing stteam from a wash bottie. Place the sample in a clean glass
beaker or sample cup and insert the electtcxies. Provide adequate agitation
throughout the measurement period. ReadtfiemiUivolt potential of the solution
aUowing sufficient time fortfiesystem to stabtiize. Measure successive
portions of the sample unttireacUngson two successive portions cUffer by no
more than 10 mV. A systemtfiatis very slow to stabiUze probably wtil not
yield a meaningful ORP.
9.3 The ORP value is cUsplayed continuously and can be noted at any specific time.

10.0 Calculation
10.1 If tiie meter is caUbrated m mUlivolts, read the oxidation-reduction potential

255
 dUectiy fromtfiemeter scale. This ORP potential isrelatedtotfiereference
electrode use in the measurement
10.2 Calculatetfieoxidation-reduction potential of tiie sample, Ui mtiUvolts, referred
to the hydrogen scale as follows:

Eh = Eobs + Eref

where:
Eh = oxidation-reduction potentialreferredto the hydrogen scale, mV,
Eobs = observed oxidation-reduction potential of the noble metal-reference
elecdxxie employed, mV, and
Eref. = oxidation-reduction potential of thereferenceelectrode asrelatedto the
hydrogen elecdxxie, mV.
11.0 Report

11.1 Report the oxidation-reduction potential to the nearest 10 mV, Uiterpolating the
meter scale as required When considered appropriate, thetemperatureat which
the measurement was made, the elecdx)de system employed, and the pH at the
time of measurement, may also be reported
12.0 Precision and Bias
12.1 The ESL uses a Type in meter with a range of 0-±1400mv, precision of ± 0.2,
and accuracy of ± 0.7.
References
American Society for Testing and Materials. (1993). Annual Bcx)k of ASTM Standards.
Vol.11.01. Method D 1498. American Scxiiety for Testing and Materials, Philadelphia,
Pa, 319.
Orion Research Incorporated (1990). Bench Top Ph/ISE meter Instmction Manual.
Mcxiel 920A. Orion Research Inc. Laboratory Products Group, Boston, Ma.
American PubUc Health Association. (1992). Standard Methcxis for the Examination of
Water and Wastewater. 18th ed. Methcxi 2580. American Public Health Asscxnation,
American Water Works Asscxnation, and Water EnvUonment Federation, Washmgton
DC, 2-6.

256
METHOD #: 150.1 Approved for NPDES (Editorial Revision 1978,1982)
TITLE: pH (Electtomedic)
ANALYTE:
pH
INSTRUMENTATION: pH Meter
INTRODUCTION:
Measurement of pH is one of the most important andfrequentiyusedtestsin water
chemistry. PracticaUy every phase of water supply and wastewatertteatment,e.g., acid
base neutralization, water softening, precipitation, coagulation, cUsinfection, and corrosion
control, is pH-dependent. pH is used in alkalinity and carbon dioxide measurements and
many other acid-base equilibria. At a giventemperaturethe intensity of the acicUc or basic
character of a solution is indicated by pH or hydrogen ion activity. Alkalirtity and acicUty
are the acid- and base-neuttaUzing capacities of a water and usually are expressed as
miUigrams CaC03 per liter. Buffer capacity is the amount of strong acid or base, usuaUy
expressed in moles per Uter, need to change the pH value of a 1 L sample by 1 unit. pH as
defined by Sorenson is - log [H+]; U is the "intensity" factor of acicUty. Pure water is very
sUghtiy ionized and at equiUtdum the ion prcxiuct is:

[H+][OH-] = Kw = 1.01 X 10-14 at 25°C (1)

and
[H+] = [OH] = 1.005x10-7

where
[H+] = activity of hydrogen ions, moles/L,
[OH] = activity of hydroxyl ions, moles/L, and
Kw = ion prcxiuct of water.
Because of ionic Uiteractions in aU but very dtiute solutions, U is necessary to use the
"activUy" of an ion and not Us molar concend-ation. Use of the term pH assumestiiattiie
activity of tiie hydrogen ion, an-h is bemg considered. The approximate equivalence to
molarity, [H+] can be presumed only Ui very dtiute solutions (ionic sdength <0.1).
A logaritiunic scale is convenient for expressUig a wide range of ionic activities. Equation
1 Ui logaritfimic form and corrected toreflectactivity is:
(- loglO aH*) + (- loglO aoH-) = 14 (2)

or
pH + pOH = pKw

where:

257
 pH = loglOaH+ and
pOH = loglO aoH-
Equation 2 states that as pH increases pOH decreases cortespondingly and vice versa
because pKw is constant for a giventemperature.At 25°C, pH 7.0 is neutral,tfieactivities
of the hydrogen and hydroxyl ions are equal, and each cortesponds to an approximate
activity of 10-7 molesA-. The neutral point is temperature-dependent and is pH 7.5 at 0°C
andpH6.5at60°C.
The pH value of a highly dtiute solution is approximately the same as the negative cornmon
logarithm of the hycirogen ion concentration. Natural waters usuaUy have pH values in the
range of 4 to 9, and most are sUghtiy basic because of the presence of bicarbonates and
carbonates of the alkaU and alkaline earth metals. Seefigure1 below for some common pH
values.

lemon blood ammonia

battery juice
beer baking bleach
acid vinegar soda
_L
pH 0r T2 1 — r T8 T" 10
T" 1
12 14

most rivers
& lakes

Figure 1. Common pH values

1.0 Scope and AppUcation

1.1 This metfiod is applicable to drinking, surface, anci saUne waters, domestic and
Uidusdial wastes and acid rain (aunospheric deposition).

2.0 Summary of Method

2 1 The pH of a sample is detemtined elecdx)medically using eitiier a glass elecdxxie
Ui combUiation witfi areferencepotential or a combUiation elecdxxie.

3.0 Sample Handling and Preservation

3.1 Samples should be analyzed as soon as possible, preferably Uitfiefieldattiietime
3 2 High^urity waters and waters not at equUibrium witiitfieadnosphere are subject
* to changes when exposed totfieadnosphere,tiiereforethe sample contamers
should be filled completely and kept sealed prior to analysis. Loss or gain of

258
 dissolved gasses, for example CO2, CI2, and H2S can change tiie pH over time
(see also 8.4 and 8.5 below).
4.0 Interferences

4.1 The glass elecdxxie, Ui general, is not subject to solution mterferencesfromcolor,

turbidity, coUoidal matter, oxidants, reductants or high salUiity.
4.2 ScxUum error at pH levels greatertfian10 can be reduced cjr eliminated by using a
"low scxUum error" electrcxie.
4.3 Coatings of oUy material or particulate matter can impaU elecdxxie response.
These coatings can usuaUy beremovedby gende wiping or detergent washing,
followed by distUled water rinsing. An additional d-eadnent with hydrochloric
acid (1 nti of HCl + 9 nti of disttiled water) may be necessary toremoveany
remairting fUm.
4.4 Temperature effects on the electrometiic measurement of pH arise from two
sources. Thefirstis caused by the change in elecdxxie output at various
temperatures. This interference can be condx)Ued with insdximents having
temperature compensation or by caUbrating the electrcxie-instrument system at the
temperature of the samples. The second source is the change of pH inherent in
the sample at varioustemperatures.This error is sample dependent and carmot be
controUed, it should therefore be noted byreportingboth the pH and temperature
at thetimeof analysis.
5.0 Apparatus
5.1 pH Meter-laboratory orfieldmodel. Pcx:ket pH meters cannot be caUbrated and
are subjec^t to some temperature and concentration errors. They are useful when
accuracy only to ± 0.2-0.5 pH units is desUed Accuracy to 0.1 pH units requUes
instmments withtemperaturecompensation and caUbration c^apabiUty. A wide
variety of instmments are commercially avaUable with various specifications and
optional equipment, such as Orion model 950 A.
5.2 Magnetic stirrer anci Teflon-coated stirring bar.
5.3 Thermometer ortemperaturesensor for automatic compensation.

6.0 Reagents
6.1 Standard buffer solutions.
6.1.1 CommerciaUy avaUable in liquid form. Care should be taken for expUation
dates.
6.1.2 Commercially avaUable in powder form such as pHycirion buffers, mix one
capsule per 1(X) nti of deionized water.
7.0 CaUbration. Each insdnment/elecd-ode system must be calibrated at a minUnum of two
points that brackettiieexpected pH oftfiesamples and are approxUnatelytfueepH
units or nK)re apart. CaUbration buffer solutions are usuaUy available at pH 4, 7, and
10. For example, if tiie expected pH is to be above 7, use buffer solutions of 7 and
10.
7.1 Auto caUbration for Orion model 950 A. The pH meter automaticaUy recognizes
the buffer solutions.
7.1.1 Press CALIBRATE ontfiepH meter pad. The screen wtil ask "How many

259
 buffers", enter 2 and press YES.
7.1.2 Place pH 7 buffer Ui a smaU beaker (approximately 50 ml) witii stir bar and
stU gentiy. Insert pH elecdxKie.
7.1.3 Wait for a stable pH reading.
7.1.4 Once stable, the meter automaticaUy recognizes the value. Press yes.
7.1.5 If during auto caUbration, the shown value is ± 0.5 pH urtitsfromcortect
value, the correct value can be entered and continue as manual caUbration
(7.2).
7.1.6 Place either the pH 4 or 10 buffer solution m another small beaker and do
the same as 7.1.3. Once caUbration is complete, the scmeen wiU show
"RDY".
7.2 Manual c:aUbration
7.2.1 Same as 7.1.1 and 7.1.2.
7.2.2 When prompted enter value of buffer and press yes.
7.2.3 Do for all buffers.

8.0 Prcx:edure

8.1 StanciarcUze the meter and elecd-cxie system as outlined in Section 7.0.
8.2 Place the sample or buffer solution in a clean glass beaker using a sufficient
volume to cover the sensing elements of the electrodes and to give adequate
clearance fortiiemagnetic stining bar.
8.2.1 If field measurements are being made the electrodes may be immersed
ciirectiy in the sample sdeam to an adequate depth and moved m a manner to
insure sufficient sample movement across the electrode sensing element as
incticated by drift free (< 0.1 pH) readings.
8.3 If the sample temperature differs by more than 2°C from the buffer solution the
measured pH values must be ccjrrected. The ESL instruments are equipped with
automatic compensators that electronically adjust fortemperaturecUfferences.
8.4 After rinsing and gentiy wiping the elecdxxies, immerse them into the sample
beaker or sample stream and stU at a constant rate to provide homogeneity and
suspension of solids. Rate of stirring should minimize the aU d-ansferrateat the
aU water interface of the sample. Note and record sample pH and temperature.
Repeat measurement on successive volumes of sample untti values differ by less
than 0.1 pH units. Two or three volume changes are usuaUy sufficient.
8.5 For acidrainsamples U is most importanttfiattfiemagnetic stirrer is not used
Instead, swUl the sample gentiy for a few seconds after the Uidxxiuction of the
elecu-ode(s). AUowtiieelecdxxie(s)tfieequtiibrate. The aU-water mterface
should not be disduted whtie measurement is bemg made. If tiie sample is not in
equiUbrium witii tiie adnosphere, pH values wtil change astiiedissolved gases are
eitiier absorbed or desorbed Record sample pH and temperature.

9.0 Maintenance
9.1 Storage. The elecdxxie should be stored Ui ~ pH 7 solution (distiUed water) when
not in use.
9.2 The elecdode should berinsedof any salt build up witii distiUed water and wiped
with a Kim wipe.
9.3 Thereferencechamber should be drained andfiUedwitii fresh solution
(commerciaUy provided) once a week.

260
10.0 Calculation

10.1 pH meters read dUecUy Ui pH units. Report pH to tfie nearest 0.1 unit and
temperamre to tfie nearest degree C. Note: Use only two significant
figures when recording pH values.
11.0 Accuracy

10.1 By careful use of a laboratory pH meter with gcxxi electrcxies, a precision of ±

0.()2 pH unit and bias of ± 0.05 pH unit can be achieved. However, ± 0.1 pH
urtit represents the Umit of accuracy under normal conctitions, especiaUy for
measurement of water and poorly buffered solutions. For this reason, report pH
values to the nearest 0.1 pH unit. A synthetic sample of a Clark and Lubs buffer
solution of pH 7.3 was analyzed electromedicaUy by 30 laboratories with a
standard deviation of ± 0.13 pH unit
10.2 In a single laboratory (EMSL), using surface water samples at an average pH of
7.7, the standard deviation was ±0.1.
References
Orion Research Incorporated (1991). Triode pH Electrode Instmction Manual. Model
91-57BN. Orion Research Inc., Laboratory Products Group, Boston, Ma.
Orion Research Incorporated (1990). Bench Top Ph/ISE meter Instmction Manual.
Mcxiel 920A. Orion Research Inc., Laboratory Prcxiucts Group, Boston, Ma.
United States EnvUonmental Protection Agency. (1992). Metfiods for Chemical Analvsis
of Water and Wastes. Metficxi #150.1. EnvUonmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincumati, Ohio.
American PubUc Health Association. (1992). Standard Methods for tfie Examination of
Water and Wastewater. 18tfied. Method 4500. American Public Health Asscx:iation,
American Water Works Asscx^iation, and Water EnvUonment Federation, Washington
DC, 4-65.

261
METHOD #: 420.1 Approved for NPDES (Editorial Revision 1978)

TITLE: Phenolics (Specdx)photomedic, Manual 4-AAP Witfi DistiUation)

ANALYTE:
PhenoUcs

INSTRUMENTATION: Spectrophotometer
INTRODUCTION
Phenols, defined as hydroxy derivatives of benzene and its condensed nuclei, may occur
in dotnestic and indusdial wastewaters, natural waters, and potable water suppUes.
Chlorination of such waters may produce odorous and objectionable-tasting chlcjrophenols.
Phenol removal processes in water u-eattnent mclude superchlorination, chlorUie dioxide or
chloramine deatment, ozonation, and activated carbon adsorption.
Phenol concentrations in water are of concem because of the wide variety of human health
and environmental problems that have been linked to theU presence in the environment.
Among these are several types of cancer, central nervous system disorders, adverse
reprcxiuctive outeomes (e.g. bUth defects), and a range of specific cUsorders in humans and
other species.
1.0 Scope and Application

1.1 This methcxi is applicable to the analysis of drinking, surface and saline waters,
domestic and industrial wastes.
1.2 The method is capable of measuring phenoUc materials at the 5 pg/L level when
the colored end product is extracted and concend^ted in a solvent phase using
phenol as a standard
1.3 The methcxi is capable of measuring phenoUc materials that contain more than 50
pg/L in the aqueous phase (without solvent extraction) using phenol as a standard.
1.4 It is not possible to use this method to differentiate between different kinds of
phenols.
2.0 Summary of Metiicxi
2.1 PhenoUc materialsreactwith 4-aniinoantipyrine Ui the presence of potassium
ferricyanide at a pH of 10, to form a stable recicUsh-brown colored antipyrine dye.
The amount of color produced is a function of the concend-ation of phenoUc
material.

3.0 Comments
3.1 For most samples a prelimUiary distiUation is requUed toremoveuiterfering
materials.
3.2 Colorresponseof phenolic materials witfi 4-ammo antipyrine is not tiie same for
aU compounds. Because phenoUc type wastes usually contain a variety of
phenols, it is not possible to duplicate a mixture of phenols to be used as a
standard Fortiiisreason phenol has been selected as a standard and any color
prxxiuced by thereactionof other phenolic compoimds is reported as phenol. This

262
 value wiU representtfieminimum concend-ation of phenoUc compounds present Ui
the sample.

4.0 Sample HandUng and Preservation

4.1 Biological degradation is Uihibited bytfieaddition of 1 g/L of copper sulfate to tfie

sample and acidification to a pH of lesstfian4 with phosphoric acid. The sample
should be kept at 4°C and analyzed witftin 24 hours after coUection.
5.0 Interference

5.1 Interferences from sulfur con^unds are eliminated by acidifying the sample to a
pH of less than 4 with H3PO4 and aeratmg briefly by stUring and adding CUSO4.
5.2 OxidizUig agents such as chlorine, detected by the Uberation of icxUne upon
acicUfication in the presence of potassium icxUde, areremovedimmecUately after
sampUng by the adcUtion of an excess of ferrous ammonium sulfate (7.10). If
chlorine is notremoved,the phenoUc compounds may be partiaUy oxidized and
theresultsmay be low.
6.0 Apparams
6.1 DistiUation apparams, aU glass consisting of a 1 Uter pyrex cUstUUng apparams
with Graham condenser (Coming No. 3360 or equivalent).
6.2 pH meter.
6.3 Spectrophotometer, for use at 460 or 510 nm.
6.4 Funnels.
6.5 Filter paper.
6.6 Membrane filters.
6.7 Separatory funnels, 500 or 1,(XX) mL.
6.8 Nessler mbes, short or long form.

7.0 Reagents
7.1 Phosphoric acid solution, 1 + 9: DUute 10 mL of 85% H3PO4 to 100 mL witii
cUstiUed water.
7.2 Copper suUate solution: Dissolve 100 g CUSO4-5H2O Ui distUled water and dtiute
to 1 Uter.
7.3 Buffer solution: Dissolve 16.9 g NH4CI Ui 143 mL cone. NH4OH and dtiute to
250 mL witfi distUled water. Two mL should adjust 100 mL of distiUate to pH
10.
7.4 4-Ammoantipyrine solution (4AAP): Dissolve 2 g of 4AAP Ui distiUed water and
dtiute to 100 mL.
7.5 Potassium ferricyanide solution: Dissolve 8 g of K3Fe(CN)6 in disttiled water and
dtiute to 100 mL.
7.6 Stock phenol solution: Dissolve 1.0 g phenol Ui freshly botied and cooled distiUed
water and cUlute to 1 liter. 1 mL = 1 mg phenol.
7.7 WorkUig solution A: Dilute 10 mL stock phenol solution to 1 Uter witfi distUled
water. mL = 10 pg phenol.
7.8 Working solution B: Dilute 100 mL of workUig solution A to 1000 mL witii
distiUed water. 1 mL = 1 pg phenol.

263
 7.9 Chlorofonn, CHCb
7.10 Ferrous ammonium sulfate: Dissolve 1.1 g ferrous ammonium suUate Ui 500
mL disdUed water contaUting 1 mL concend-ated H2SO4 and dilute to 1titerwitfi
freshly boiled and cooled distiUed water.
8.0 Prcx:edure
8.1 DistiUation
8.1.1 Measure 500 mL sample mto a beaker. LowertfiepH to approximately 4
witii 1 + 9 H3PO4 (7.1), add 5 mL CUSO4 solution (7.2) andttansferto tiie
distiUation apparams. Omit adding H2PO4 and CUSO4 if sample was
preserved as described in 4.1.
8.1.2 DistiU 450 mL of sample, stop the distUlation, and when boiling ceases add
50 mL of warm distiUed water totfieflaskandresumedistiUation untti 500
mL have been coUected
8.1.3 If the cUstUlate is turbid,filterthrough a prewashed membrane fUter
(rinsedwith type Ireagentgrade water as defined Ui the Reagent-Grade
Water section in this manual).
8.2 Chloroform exdaction methcxi
8.2.1 Using working solution B (7.8), prepare tiie foUowing standards.
Standards may be prepared by pipetting the requUed volumes into the
separatory funnels and dUutUig to 5(X) mL witii distiUed water.

mL of working solution B Concentration pg/L

0.0 0.0
3.0 6.0
5.0 10.0
10.0 20.0
20.0 40.0
25.0 50.0

8.2.2 Place 500 mL of cUstUlate or an aUquot dtiuted to 5(X) mL in a separatory

funnel. The sample should not contain more than 25 pg phenol.
8.2.3 To sample and standards add 10 mL of buffer solution (7.3) and mix. The
pH should be 10 ±0.2.
8.2.4 Add 3.0 mL aminoantipyrine solution (7.4) and mix.
8.2.5 Add 3.0 mL potassium ferricyanide solution (7.5) and mix.
8.2.6 After three ntinutes, exd-act witfi 25 mL of chloroform (7.9). Shake the
separatory funnel at least 10times,let CHCI3 settie, shake agam 10 times
and let chloroform settie again. Vent chloroform fumes into hood.
8.2.7 Ftiter chloroform extracts throughfilterpaper. Do not acki more
chloroform. Carry outfiltrationin a hood. Dispose of chloroform in
environmentaUy acceptable manner.
8.2.8 Read the absorbance of the samples and stanciards against the blank at 460
nm.

264
9.0 Calculation

9.1 Prepare a standard curve by plottUigtfieabsorbance value of standards versus tfie

corresponding phenol concenu-ations.
9.2 Obtain concend-ation value of sample dUectiyfix)mstandard curve.
10.0 Accuracy

10.1 Using the exttaction procedure for concend-ation of color, six laboratories
analyzed samples at concentrations of 9.6,48.3, and 93.5 pg/L. Standard
deviations were ± 0.99, ± 3.1 and ± 4.2 g/L, respectively.
10.2 Using the dUect photometric procedure, six laboratories analyzed samples at
concend-ations of 4.7,48.2 and 97.0 mg/L. Standard deviations were ± 0.18, ±
0.48 and ± 1.58 mg/L, respectively.
References

Sawyer, C. N., and McCarty, P. L. (1994). Chemisd-v for Environmental Engineering.

4tii ed. McGraw-Hill, Inc., New York, N.Y., 309.
United States EnvUonmental Protection Agency. (1992). Metiiods for Chemical Analysis
of Water and Wastes. Method #420.1. Environmental Monitoring and Suppcjrt
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.
American PubUc Health Association. (1992). Standard Methods for the Examination of
Water and Wastewater. 18th ed. Methcxi 5530. American PubUc Health Asscx:iation,
American Water Works Asscxdation, and Water Environment Federation, Washington
DC, 5-30.

265
TITLE: Phosphorous
INTRODUCTION
This is tiie Uidxxiuction fortiiefoUowUig metiiods: Orthophosphate and Phosporous, Total.
Phosphoms cxjcurs Ui natural waters and in wastewaters aUnost solely as phosphates.
These are classified as orthophosphates, condensed phosphates (pyro-, meta-, and otiier
polyphosphates), and organicaUy bound phosphates. Phosphorous is not very abundant
but when it is present, it occurs in solution, Ui particles or deditus, or Uitfiebodies of
aquatic organisms. Phosphorous is veryreactive,tiiereforeit bUids quickly. Also, unlike
otfier rnacronudients (CHNOPS)tiiereis no gaseous phase to help phosphorous witfi
recUsdibution to areas of low occmrence. For these reasons, cUssolved phosphorous in
standing natural waters wiU usuaUy be lesstiianeitfiertfiesedUnents oftfiesame waterlxxiy
or stream cUaining to it

These forms of phosphate arisefix)ma variety of sources. SmaU amounts of certain

condensed phosphates are added to some water supplies during treatment. Larger
quantities C)f the same compounds may be added when the water is used for laundering or
other cleaning, because these materials are major constituents of many commercial cleaning
preparations. Phosphates are used extensively in the u-eadnent of botier waters.
Orthophosphates appUed to agricultural cjrresidentialcultivated land as fertilizers are carried
into surface waters with storm mnoff and to a lesser extent with melting snow. Organic
phosphates are formed primarily by biological pnxesses. They are condibuted to sewage
by body wastes aU food residues, and also may be formed from orthophosphates in
biological treatment processes or by receiving water biota.

Phosphoms is essential to the growth of orgartisms and can be the nutrient that lintits the
primary prcxiuctivity of a bcxiy of water. In instances where phosphate is a growth-limiting
nutrient, the cUscharge ofrawor treated wastewater, agricultural drainage, or certain
industrial wastes totiiatwater may stimulate the growth of photosynthetic aquatic micnx)-
and mac:ro-organisms in nuisance quantities.
Phosphates also occur in bottom secUments and in biological sludges, both as precipitated
inorganic forms and incorpcjrated into organic compounds.
Phosphoms analyses embcxiy two general procedural steps: (a) conversion of the
phosphoms form of interest to dissolved orthophosphate, and (b) colorimeuic
determination of cUssolved orthophosphate.
Because phosphoms may cxxur in combination with organic matter, a cUgestion methcxi to
determine total phosphoms must be able to oxidize orgartic matter effectively to release
phosphoms as orthophosphate. After digestion, Uberated orthophosphate is detemtined

References
American PubUc Healtii Association. (1992). Standard Metiiods fortfieExamination of
Water and Wastewater. 18th ed Metficxi 4500. American PubUc Healtfi Asscx:iation,
American Water Works Association, and Water Environment Federation, Washington
DC, 4-108.

266
METHOD #: 365.1 Approved for NPDES, CWA (Ed. Rev. 1974, 1978)
TITLE: Phosphorous, Ortiiophosphate (Automated, Ascorbic Acid)
ANALYTE:
Phosphoms, P

INSTRUMENTATION: Autoanalyzer
1.0 Scope and AppUcation

1.1 These methcxis cover the determination of specified forms of phosphoms in

drinking, surface and saline waters, and domestic and indusdial wastes.
1.2 The methcxis are based on reactions that are specific for the orthophosphate ion.
These forms are defined in Section 4.
1.2.1 Except for in depth and detatied stucUes, the most commonly measured
fc>mis are phosphoms and cUssolved phosphoms, and orthophosphate and
cUssolved orthophosphate. Hycirolyzable phosphoms is normally foimd
only in sewage-type samples. Insoluble forms of phosphoms are
detemtined by calculation.
1.3 The methcxis are usable in the 0.04-2.0 mg P/L range. Approximately 120
samples per hour can be analyzed.
1.4 This metficxi isrestrictedto use by or under the supervision of analysts
experienced in the operation of ion chromatograph.
2.0 Summary of Method
2.1 Ammortium molybdate and antimony potassium tartratereactin an acid mecUum
with cUlute solutions of phosphoms to form an antimony-phospho-molybdate
complex. This complex is reduced to an intensely blue-colored complex by
ascorbic acid. The color is proportional to the phosphoms concentration.
2.2 Ortiy orthophosphate forms a blue color in this test Polyphosphates (and some
organic phosphoms compounds) may be converted to the orthophosphate form
by manual sulfuric acid hydrolysis. Organic phosphoms compounds may be
converted to the orthophosphate form by manual persulfate digestion. The
developed color is measured automatically on the AutoAnalyzer.

3.0 Sample HandUng and Preservation

3.1 If bentfiic deposits are present Uitfiearea bemg sampled, great care should be
taken not to include these deposits because oftfiepotential concenttation of
phosphate.
3.2 Sample containers may be of plastic material, such as containers, or of Pyrex
glass.
3.3 & the analysis cannot be performed the same day of coUection, the sample should
be preserved bytfieadcUtion of 2 mL concend-ated H2SO4 pertiterand
refrigeration at 4°C.
4.0 Interferences
4.1 No mterference is caused by copper, Uon, or siUcate at concend-ations many times

267
 greatertiiantfieUreportedconcendation Ui sea water. However, high Uon
concend-ations can cause precipitation of and subsequent loss of phosphoms.
4.2 The salt error for samples rangUig from 5 to 20% salt content was found to be
less than
1 %.
4.3 Arsenate is determined simUarly to phosphoms and should be considered when
present in concentrations higher than phosphoms. However, at concentrations
found in sea water, it does not interfere.
4.4 Samplettu-biditymust beremovedby fUd-ation prior to analysis for
Orthophosphate. Samples for total or total hydrolyzable phosphoms should be
fUtered only after digestion. Sample color that absorbs mtfiephotometric range
used for analysis wiU also interfere.
6.0 Apparatus

6.1 TRAACS 800 AutoAnalyzer Unit consisting of:

6.1.1 Sampler.
6.1.2 Mamfold
6.1.3 Proportioning pump.
6.1.4 Heating bath with double delay coti
6.1.5 Colorimeter
6.1.6 Recorder.
6.1.7 Digital printer
7.0 Reagents
7.1 Sulfuric acid solution, 5 N: Slowly add 70 mL of concend-ated. H2SO4 to
approximately 4(X) mL of cUstiUed water. Ccx)l to rcx)mtemperatureand dtiute to
500 mL with distUled water.
7.2 Ascorbic acid: Dissolve 10 g of ascorbic acid in 1(X) mL of cUstiUed water. The
solution is stable for about a week if prepared with water containing no more than
trace amounts of heavy metals and stored at 4°C. Store in an amber container.
7.3 Aerosol-22, surfactant
7.4 Dissolve 4.3 g of ammonium molybdate and 0.12 g of antimony potassium
tartrate in about 800 nti of DI water. Cautiously, with swUUng, slowly add 27 nti
of suUuric acid Ccx)l to rcx)m temperature, dilute to one liter with DI water and
mix thoroughly. Acid 7 nti of Aerosol-22. Thisreagentis stable for about one
month without added surfactant After Aerosol-22 is added, stabiUty is about one
week.
7.5 Stock phosphoms solution: Dissolve 0.4393 g of pre-dried (105°C for 1 hour)
KH2PO4 Ui distilled water and dilute to 1000 mL. 1.0 mL = 0.1 mg P.
7.6 Standard phosphoms solution: DUute 100.0 mL of stock solution (7.4) to 10(X)
mL witii distiUed water. 1.0 mL = 0.01 mg P.
7.7 Standard phosphoms solution: DUute 100.0 mL of standard solution (7.5) to
1000 mL witii distilled water. 1.0 mL = 0.001 mg P.
7.8 Prepare a series of standards by cUlutUig suitable volumes of standard solutions
(7.5) and (7.6) to 100.0 mL witfi distilled water. The foUowing dilutions are
suggested:

268
 mL of Standard
Concend-ated.,
Phosphoms Solution (1 l>i v^^ p/|

0.0 0.00
2.0 0.02
5.0 0.05
mo.
mL of Standard
Concend-ated.
Phosnhonis Solution (7.6^ mgP/L
2.0 0.20
5.0 0.50
8.0 0.80
10.0 1.00
8.0 Prcx:edure

8.3 The basic prcx:edure to mn the TRACCS automated procedures are presented.
However, only personnel trained by the lab manager should perform these
analyses.
8.3.1 Tum on the computer, this starts the pump operating automaticaUy.
8.3.2 Press CR (Chart and Run)
8.3.3 Press F4 and enter OPl (operate pump 1) with all the tubes in acid wash
solution. Wait 5-10 minutes.
8.3.4 Press EDIT. RecaUtiiemetiiod:
PO4EPA
8.3.5 Press F5 twice.
8.3.6 Change the standards and sample protcx:ol appropriate to the dtiution of
standards (see 7.8).
8.3.7 Press F6 to save
8.3.8 Prepare samples for the tray (see 4.4) and reagents needed. Place
appropriate tubes into theUrespectivereagents.
9 Water 3 Molybdate
6 Sample 7 Ascorbic Acid
5,4,8 Water
8.3.9 Press F2 QuU
8.3.10 Press BG to begin a base and gain. If unsuccessful, Press CBl 70 to
enforce a base of 70 and CGI 13 to enforce a gain. RemntfieBase and
Gain.
8.3.11 Press F8 to prepare for mn: channel 1, 85%, and speed 30
8.3.12 Press F2, main menu.
8.3.13 C^t
8.3.14 Press CR, Press F7: chart speed 60, file name (name of analyte run and
date), operator name. Start.
8.3.15 Attfieend of the mn, fromtfiemain menu, print out aUtfieresults.
8!3! 16 Take out aUtfiembes and placetfiemback mtotfieacid wash solution,
8!3! 17 Press CR F4 command. Press HPl to wash quickly for 5 ntinutes.
8.3.18 Press F4, QP 1, to quU pump.
8.3.19 Place all the dibes mto DI water. Tum off the con:^)uter.

269
9.0 Calculation

9.1 Prepare a standard curve by plotting peak heights of processed standards against
known concentrations. Compute concend-ations of samples by comparing
sample peak heights witfi standard curve. Any sample whose computed value is
lesstfian5%ofitsUnmecUate predecessor must be remn. CalcuUitions are done
automaticaUy on the computer, andtfieresults are duect
10.0 Accuracy

10.1 Six laboratories participatUig m an EPA Metfiod Study, analyzed four natural
water samples contaUting exact mcrements of orthophosphate, witfi tfie
foUowing results:

Increment as Precision as Accuracy as

orthophosphate Standard Deviation Bias, Bias,
mg P/Uter mg P/Uter % mg P/Uter
0.04 0.019 + 16.7 +0.007
0.04 0.014 - 8.3 -0.003
0.29 0.087 -15.5 -0.05
0.30 0.066 -12.8 -0.04

10.2 In a single laboratory (EMSL), using surface water samples at concentrations

of 0.04, 0.19, 0.35, and 0.84 mg P/L, standard deviations were ± 0.005, ±
0.000, ± 0.003, and ± 0.000, respectively.
10.3 In a single laboratory (EMSL), using surface water samples at concend-ations
of 0.07 and 0.76 mg P/L, recoveries were 99% and 100%, respectively.

References
Bran + Luebbe Analyzing Technologies, Inc. (1987). Operation Manual fortfieTRAACS
800 Svstem. Indusdial metiiod No. 781-86T. Bran + Luebbe Analyzing
Technologies, Inc., Elmsford, N.Y.
United States Environmental Protection Agency. (1992). Metfiods for Chemical Analysis
of Water and Wastes. Metfiod #365.1. EnvUonmental Monitoring and Support
Laboratcjry, United States Environmental Protection Agency, Cincinnati, Ohio.

270
METHOD #: 365.4 PendUig Approval for NPDES, CWA (Issued 1974)

TITLE: Phosphorous, Total (Colorimedic, Automated, Block Digester AA II)

ANALYTE:
Total Phosphoms, P

INSTRUMENTATION: Autoanalyzer
1.0 Scope and AppUcation

1.1 This methcxi covers the determination of total phosphoms in drirtidng water,
surface water and domestic and Uidustrial wastes. The appUcable range of this
methcxi is 0.1 to 5 mg P/L.
1.2 This method is restricted to use by or under the supervision of analysts
experienced in the operation of an autoanalyzer.
2.0 Summary of Method

2.1 The sample is heated intiiepresence of sulfuric acid, K2SO4 and HgS04 for two
and one haU hours. Theresidueis cooled, dtiuted to 25 mL and placed on the
AutoAnalyzer for Total phosphoms determmation.
3.0 Sample HandUng and Preservation
3.1 Sample containers may be of plastic material, such as a cubitainer, or of Pyrex
glass.
3.2 ff the analysis cannot be performed the day of coUection, the sample should be
preserved by the adcUtion of 2 mL of concentrated H2SO4 per liter and
refrigeration at 4°C.
4.0 Apparams
4.1 TRAACS 800 AutoAnalyzer Unit consisting of:
4.1.1 Sampler.
4.1.2 Manifold
4.1.3 Proportioning pump.
4.1.4 Heating bath with double delay coU
4.1.5 Colorimeter
4.1.6 Recorder.
4.1.7 Digital printer
4.2 Block Digester BD-40
4.3 Autoanalyzer

5.0 Reagents
5.1 Mercuric sulfate: Dissolve 8 g red mercuric oxide (HgO) Ui 50 mL of 1:4 sulfiiric
acid (10 concend-ated H2SO4:40 mL distiUed water) and dilute to 100 mL witii
cUstiUed water.
5.2 Digestion solution: (Sulfuric acid-mercuric sulfate-potassium sulfate solution):
Dissolve 133 g of K2SO4 Ui 6(X) mL of distiUed water and 200 mL of cone.

271
 H2SO4. Add 25 mL of mercuric sulfate solution (5.1) and dtiute to 1 Uter.
5.3 Sulfuric acid solution (0.72 N): Add 80 mL of concend-ated sulfuric acid to 1800
of cUstiUed water, mix and dtiute to 2 titers.
5.4 Molybdate/antUnony solution: Dissolve 11 g of ammonium molybdate and 0.25 g
of antimony potassium tartrate in about 8(X) mL of cUstiUed water and dtiute to 1
Uter. Transfer to a Ughtresistantcontainer.
5.5 Ascorbic acid solution: Dissolve 154 g of ascorbic acid in about 800 mL of
cUstUled water and dtiute to 1titer.Transfer to a Ughtresistantcontamer. Stable
for at least five ciays.
5.6 Aerosol-22, surfactant
5.7 Acid/salt dUuent: Dissolve 6.3 g of Sodium chloride in about 800 nti of DI water.
Add 20 ml of sulfuric acid and dilute to one Uter. Add 7 ml of Aerosol-22 (5.6)
and mix. This reagent is stable only without added surfactant. After Aerosol-22
is added, stabiUty is about one week.
5.8 Stock Solution (2.0 mg/L P) Dissolve 0.8788 g of Potassium dihydrogen
phosphate (KH2PO4) in about 60 ml of DI water. Dtiute to 1(X) ml and mix
thoroughly.
5.9 Standards are prepared in 1(X) nti of cUstiUed water.

Cone, mg/L P mL of stCKk solution/1(X) mL

2.0 i.O
1.8 1.8
1.5 1.5
1.2 1.2
0.8 0.8
0.5 0.5

6.0 Digestion Procedure

6.1 To 20 or 25 mL of sample, add 5 mL of cUgestion solution and mix. (Use a
vortex mixer).
6.2 Add 4-8 Teflon boiling chips. Too many boiUng chips wiU cause tiie sample to
boil over.
6.3 Witfi Block Digester Ui manual mode set low and hightemperamreat 200°C and
preheat unit to 200°C. Place tubes Ui digester and switch to automatic mode. Set
lowtemperaturetimerfor 1 hour. Reset high temperature to 380°C and set tuner
for 21/2 hours.
6.4 Ccx)l sample and dtiute to 25 mL witii distiUed water, ff TKN is also to be
detemtinedtfiesample should be diluted witfi ammonia-free water Type I (see tfie
Reagent-Grade Water section oftfiismanual).
Colorimedic Analysis
6.5 Once tiie sample have been digested,tfiesamples arereadyfor analysis.
6.6 The basic procedure to mntfieTRACCS automated procedures are presented.
However, only personnel d-ained bytfielab manager should perform tiiese
analvses
6 6.1 Tum on the computer,tftisstartstfiepump operating automaticaUy.
6'.6!2 Press CR (Chart and Run) ,. -^ „ u u • •.. u
6.6.3 Press F4 and enter OPl (operate pump 1) witii alltfietubes m acid wash
solution. Wait 5-10 ntinutes.
6.6.4 Press EDIT. RecaUtiiemetiiod:

272
 TPEPA
6.6.5 Press F5 twice.
6.6.6 Change the standards and sample protocol appropriate to the standarxis
(see 5.9).
6.6.7 Press F6 to save
6.6.8 Prepare samples for tfie tray (see 3.0) and reagents needed Place
appropriate tubes into tfieU respective reagents. Excluding the
molybdate/antimony tine, place aU reagent tines UitfieUrespective
containers:
9 Acid water 3 acid/salt dUuent
6 sample 7 Molybdate (last in / first out)
5 Ascorbic acid 4 DI water
8 DI water
When reagents have been pumping for at least five minutes, place tfie
molybdate/antimony line in its container and aUow the system to
equtiibrate.
6.6.9 Press F2 Quit
6.6.10 Press BG to begin a base and gain. If unsuccessful. Press CBl 70 to
enforce a base of 70 and CGI 13 to enforce a gain. Remn the Base and
Gain.
6.6.11 Press F8 to prepare for mn: channel 1, 85%, and speed 30
6.6.12 Press F2, main menu.
6.6.13 C^tit
6.6.14 Press CR, Press F7: chart speed 60, file name (name of analyte mn and
date), operator name. Start.
6.6.15 At the end of the run, from the main menu, print out aU the results.
6.6.16 Take out aU the mbes and place them back into the acid wash solution.
6.6.17 Press CR F4 command. Press HPl to wash quickly for 5 minutes.
6.6.18 Press F4, QP 1, to quit pump.
6.6.19 Place aU the mbes into DI water. Tum off the computer.
7.0 Calculations
7.1 Prepare a standard curve by plotting peak heights of processed standards against
concentration values. Compute concentrations by comparing sample peak heights
with the standard curve. If done on the computer, calculations are done and
results are ciirectiy obtained

8.0 Accuracy
8.1 In a single laboratory (EMSL) using sewage sample containing total P at levels of
0.23, 1.33, and 2.0, the precision was ± 0.01, ± 0.04, and ± 0.06, respectively.
8.2 In a single laboratory (EMSL) using sewage samples of concentration 1.84 and
1.89, tfie recoveries were 95 and 98%, respectively.

References
Bran-I-Luebbe Analyzing Technologies, Inc. (1987). Operation Manual for tiie TRAACS
800 Svstem. Indusdial metfiod No. 787-86T. Bran-h Luebbe AnalyzUig
Technologies, Inc., Elmsford, N.Y.

273
United States EnvUonmental Protection Agency. (1992). Metiicxis for Chemical Analysis
of Water and Wastes. Methcxi #365.4. EnvUonmental Monitoring and Support
Laboratory, United States EnvUonmental Protection Agency, CUicinnati, Ohio.

274
METHOD #: 602 (July 1991)
TITLE: Purgeable Aromatics
INSTRUMENTATION: GC
INTRODUCTION
A number of aromatic and certain aUphatic hydrocarbons have been detected in ground and
surface waters. The origins of these compounds Ui water supplies are frequentiy unknown
and often condX)versial, but in the case of surface waters the sources usually are fuel and/or
oU spiUs. Hydrocarbon contamination of ground-waters has been attributed to leaking
underground fuel storage tanks, Uidusdial wastes, landfills, underground Geachfield)
cUsposal of domestic and trade wastes, and in some cases, illegal cUscharges. Toxicological
smcUes have linked at least some of these compouncis, for example, benzene, to adverse
human health effects.

1.0 Scope and AppUcation

1.1 This methcxi covers the determination of various purgeable aromatics. The
foUowing parameters may be determined by this methcxi:
ANALYTE: Benzene, Chlorobenzene, 1,2-Dichlorobenzene, 1,3-
Dichlorobenzene, 1,4-Dichlorobenzene, Etfiylbenzene, and Toluene.
1.2 This is a purge and trap gas chromatographic (GC) method appUcable to the
determination of the compoundstistedabove in municipal and indusuial
cUscharges. When this methcxi is used to analyze unfarniUar samples for any or
all of the compouncis above, compound identifications should be supported by at
least one additional quaUtative techrtique such as the use of a confirmation column
that produces the same concentrations but at different detention times. Also after
the use of two columns, mass spectrophotometry can be used to confirm results.
1.3 The methcxi detection lintit (MDL, defined in Section 12.1) for each parameter is
Usted in Table 1. The MDL for a specific wastewater may differfromthose
Usted, depending upon the nature of interferences in the sample matrix.
1.4 Any mcxUfication of this method, beyond those expressly permitted, shaU be
considered as a major mocUfication subject to appUcation and approval of altemate
test prcx:ediires.
1.5 This methcxi is resdicted to use by or under the supervision of analysts
experienced in the operation of a purge andttapsystem and a gas chromatograph
and in the interpretation of gas chromatograms. Each analyst must demonstrate
the abiUty to generate acceptableresultswitfi this methcxi using the procedure
described in Section 8.2.

2.0 Summary of Method

2.1 An inert gas is bubbled through a 5 mL water sample contained in a speciaUy
designed purging chamber at ambienttemperature.The aromatics are efficientiy
transfertcd from the aqueous phase totiievapor phase. The vapor is swept
through a sorbent d-ap where the aromatics are d-apped After purging is
completed, the d-ap is heated and backfiushed witfi tfie Uiert gas to desorb tfie
aromatics onto a gas chromatographic column. The gas chromatograph is
temperature programmed to separate the aromatics which are then detected with a
flame ionization detector.

275
 2.2 The metiiod provides an optional gas chromatographic columntiiatmay be helpful
inresolvingtiiecompounds of Uiterest from mterferencestiiatmay occur.
3.0 Interferences

3.1 In^urities intfiepurge gas and organic compounds outgassUig fromtfieplumbUig

ahead of the trap account fortfiemajority of contamination problems. The
analytical system must be demonsdated to befreefix>mcontamination under tfie
conditions of the analysis by mnning laboratoryreagentblanks as described Ui
Section 8.1.3. The use of non-Teflon plastic mbUig, non-Teflontiu^adsealants,
or flow condx)llers with mbber components in the purge and trap system should
be avoided.
3.2 Samples can be contaminated by cUffusion of volatile organics through the septum
seal mto the sample during shipment and storage. A field reagent blank prepared
from reagent water and carried through the sampling and handling protcx:ol can
serve as a check on such contamination.
3.3 Contamination by carry-over can occur whenever high level and low level
samples are sequent analyzed To reduce carry-over,tfiepurging device and
sample syringe must be rinsed with reagent water between sample analyses.
Whenever an unusuaUy concentrated sample is encountered, it should be followed
by an analysis ofreagentwater to check for cross contamination. For samples
containing large amounts of water-soluble materials, suspended soUds, high
boiling compounds or high aromatic levels, it may be necessary to wash the
purging device with a detergent solution,rinseit v^th cUstiUed water, and then dry
it in an oven at 105°C between analyses. The d-ap and other parts of the system
are also subject to contamination; therefore,frequentbakeout and purging of the
entire system may be requUed.
4.0 Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined: however, each chentical compound should be treated as a
potential health hazard. From this viewpoint, exposure to these chemicals must
be reduced to the lowest possible level by whatever means avatiable. The
laboratory isresponsiblefor maintaining a current awareness ftie of OSHA
regulationsregardingthe safe hancUing of the chemicals specified in this methcxi.
AreferencefUe of material data handling sheets should also be made avaUable to
aU personnel involved in the chentical analysis.
4.2 The foUowing parameters covered by this methcxi have been tentatively classified
as known or suspected, human or mammaUan carcUiogens: benzene and 1,4-
dichlorobenzene. Primary standards of these toxic compounds should be
prepared in a hcxxi. A NIOSH/MESA approved toxic gasrespiratorshould be
wom when the analyst hancUes high concentrations of these toxic compouncis.

5.0 Apparams and Materials

5.1 SamplUig equipment, for discrete sampUng.
5.1.1 Vial: 25-mL capacity cjr larger (ESL uses 40 ml), equipped with a screw
cap with a hole intfiecenter (Pierce #13075 or equivalent). Detergent
wash,rinsewith tap and cUstilled water, and dry at 105°C before use.
5.1.2 Sepmm: Teflon-fa^ siUcone Offeree #12722 or equivalent). Detergent

276
 wash,rinsewitii tap and distiUed water, and dry at 105°C for 1 h before
use.
5.2 Purge and d-^ system: The purge and trap system consists of three separate
pieces of equipment: A purgUig device, d-ap, and desorber. Several complete
systems are now commercially avaUable.
5.2.1 The purging device must be designed to accept 5-mL samples with a water
column at least 3 cm deep. The gaseous head space betweentfiewater
column and the trap must have a total volume of lesstfian15 mL. The
purge gas must passtfux)ughthe water column as finely divided bubbles
with a cUameter of less than 3 mm at the origin. The purge gas must be
introduced no more than 5 mm from the base of the water colunm.
5.2.2 The trap must be at least 25 cm long and have an inside diameter of at least
0.105 Ui.
5.2.2.1 The trap is packed with 1 cm of methyl siUcone-coated packing
(Section 6.4.2) and 23 cm of 2.6-cUphenylene oxide polymer
(Section 6.4.1) as shown in Figure 2. lliis trap was used to
develop the methcxi performance statements in Sec:tion 12.
5.2.3 The desorber must be capable ofrapidlyheating the trap to 180°C. The
polyrner section of thettapshould not be heated higher than 180°C and the
remaining sections should not exceed 2(X)°C.
5.2.4 The purge and trap system may be assembled as a separate unit or be
coupled to a gas chromatograph.
5.3 Gas chromatograph: An analytical system complete with a temperature
programmable gas chromatograph suitable for on-colunm injection and aU
requUed accessories including syringes, analytical columns, gases, detector, and
strip-chart recorder. A data system is recommended for measuring peak areas.
The ESL uses Turbichrom 4 by Perkin-Elmers.
5.3.1 Colunm : 6 ft long x 0.082 in. ID stainless steel or glass, packed with 5%
SP1200 and 1.75% Bentone-34 on Supelcoport (100/120 mesh) or
equivalent This column was used to developtfiemethcxi performance
statements in Section 12. GuideUnes for the use of altemate colunm
pacldngs are provided in Section 10.1.
5.3.2 Detector: Flame ionization detector. Guidelines for the use of altemate
detectors are provided in Section 10.1.
5.4 Syringes: 5-niL glass hypcxiemtic with Luerlok tip (two each), if appUcable to the
purging device.
5.5 Micro syringes: 25-pL, 0.006 in. ID needle.
5.6 Syringe valve: 2-way, witfi Luer ends (tfu-ee each).
5.7 Bottie: 15-mL, screw-cap, witii Teflon cap lUier.
5.8 Balance: Analytical, capable of accurately weighUig 0.0001 g

6.0 Reagents
6.1 Reagent water: Reagent water is defined as a water in which an interfcrant is not
observed at the MDL oftiieparameters of interest. This istermedtype I Reagent
water quaUty. More Uiformation aboutreagentquaUty water can be found in the
Reagent-Grade water section in this manual.
6.1.1 Reagent water can be generated by passing tap watertfux)ugha carbon
filter bed containing about 1 lb of activated carbon (Ftid-asorb-300, Calgon
Corp.,or equivalent).
6.1.2 A water purification system (MUUpore Super-Q or equivalenO may be

277
 used to generate reagent water.
6.1.3 Reagent water may also be prepared by boUUig water for 15 mUi.
Subsequentiy, whtie maUitaUiUigtfietemperamreat go 90°C, bubble
contaminant-free inert gastfux)ughtfiewater for 1 h. Whtie stiU hot,
transfer the water to a nartow mouth screw-cap bottie and seal witii a
Teflon-lined septum and cap.
6.2 SocUum thiosulfate: (ACS) Granular.
6.3 Hydrochloric acid (1 + 1)-Add 50 mL of concendated HCl (ACS) to 50 mL of
reagent water.
6.4 Trap Materials:
6.4.1 2,6-Diphenylene oxide polymer: Tenax, (60/80 mesh), chromatographic
grade or equivalent.
6.4.2 Metiiyl siUcone packUig: 3% OV-1 on Chromosorb-W (60/80 mesh) or
equivalent.
6.5 Methanol: Pesticide quaUty or equivalent
6.6 Stock stanciard solutions: Stock stanciard solutions may be preparedfrompure
stanciard materials or purchased as certified solutions. Prepare stcx:k standard
solutions in methanol using assayed d liquids. Because of the toxicity of benzene
and 1,4-cUchlorobenzene, primary dUutions of these materials should be prepared
in a hocxi. A NIOSH/MESA approved toxic gas respUator should be used when
the analyst handles high concend-ations of such materials. The NIOSH/MESA
gasrespUatoris a seU-contained breathing apparams (SCBA) to provide a supply
of aU whtie working with the toxic compounds.
6.6.1 Place about 9.8 mL of methanol into a 10-mL ground glass stoppered
volumetric flask. AUow theflaskto stand, unstoppered, for about 10 ntin
or untti all alcohol-wetted surfaces have dried Weigh theflaskto the
nearest 0.1 mg.
6.6.2 Using a 1(X) ^£L syringe, immecUately add two or more drops of assayed
reference material to theflask,then reweigh. Be sure that the drops fall
directiy into the alcohol without contacting the neck of the flask.
6.6.3 Reweigh, dilute to volume, stopper, then mix by inverting the flask
several times. Calculate the concentration in pg/1 from the net gain in
weight. When compound purity is assayed to be 96% or greater, the
weight can be used without correction to calculate the concenu-ation of the
stock standard. Commercially prepared stock standards can be used at
any concentration if they are certified by the manufacturer or by an
independent source.
6.6.4 Transfer the stCKk standard solution into a Teflon-sealed screw-cap bottie.
Store at 4°C and protect from Ught
6.6.5 All stanciards must be replaced after one month, or sooner if comparison
with check standards incUcates a problem.
6.7 Secondary dilution standards: Usmg stock standard solutions, prepare secondary
dtiution stanciards in metfianol that contain the compounds of interest, either
singly or mixed together. The secondary cUlution standarcis should be prepared at
concentrations such thattfieaqueous caUbration standards pepared in Sections
7.3.1 or 7.4.1 will bracket the worldng range of the analytical system. Secondary
solution standarcis must be stored witfi zero headspace and should be checked
frequentiy for signs of degradation or evaporation, especially just prior to
preparing calibration standards from them.
6.8 Quality control check sample concentrate: See Section 8.2.1.

278
7.0 CaUbration

7.1 Assemble a purge and d^p systemtfiatmeetstfiespecifications Ui Section 5.2.

Condition tiie trap ovemight at 180°C by backflushUig witfi an mert gas flow of at
least 20 mL/min. Condition the d-ap for 10 min once datiy prior to use.
7.2 Connecttfiepurge and trap system to a gas chromatograph. The gas
chromatograph must be operated usingtemperatureandflowrateconditions
equivalent totfiosegiven Ui Table 1. CaUbratetfiepurge and d-ap-gas
chromatographic system using eitfiertiieextemal standard technique (Section 7.3)
ortfieintemal standard technique (Section 7.4).
7.3 Extemal stanciard caUbration procolure:
7.3.1 Prepare caUbration stanciards at a minimum of three concend^tion levels
for each parameter by carefuUy adding 20.0 pL of one or more secondary
diliition standards to 100, 500, or 1000 mL ofreagentwater. A 25-pL
syringe with a 0.006 in. ID neecUe should be used fortfiisoperation. One
of the extemal standards should be at a concentration near, but above, the
MDL (Table 1) and the other concentrations should correspond to the
expected range of concentrations found inrealsamples or should define
the working range of the detector. These aqueous stanciards must be
preparedfreshdaily.
7.3.2 Analyze each caUbration stanciard according to Section 10, and tabulate
peak height or arearesponsesversus the cx)ncend'ation in the stanciard.
Theresultscan be used to prepare a calibration curve for each compound
Altematively, if theratioof response to concentration (calibration factor) is
a constant over the worldng range (<10%relativestanciard deviation.
RSD). Linearity through the origin can be assumed and the average ratio
or calibration factor can be used in place of a calibration curve.
7.4 Intemal standard caUbration prcx;edure: To use this approach, the analyst must
select one or more intemal standards that are sintilar in analytical behavior to the
compounds of interest The analyst must further demonstrate that the
measurement of the intemal standard is not affected by method or madix
interferences. Because of these Untitations, no intemal standard can be suggested
that is applicable to all samples. The compound, a,a,a,-trifluorotoluene,
recommended as a siurogate spiking compound in Section 8.7 has been used
successfuUy as an intemal standard
7.4.1 Prepare caUbration standards at a minimum of three concentration levels
fcjr each parameter of interest as described in Section 7.3.1.
7.4.2 Prepare a spiking solution containing each of the intemal stanciards using
the pnxedures described in Section 6.6 and 6.7. It is recommended that
the secondary dtiution standard be prepared at a concenu-ation of 15
pg/mL of each intemal standard compound. The adctition of 101 of this
stanciard to 5.0 mL of sample or caUh-ation standard would be equivalent
to 30 g/L.
7.4.3 Analyze each calibration standard according to Section 10, adding 10 pL
of intemal standard spUdng solution dUectiy to the syringe (Section 10.4).
Tabulate peak height or arearesponsesagainst concenu-ation for each
compound and intemal standard, and calculateresponsefactors (RF) for
each compound using Equation 1.
Equation 1.
RF= [A(s)l [C(is)] / [A(is)l [C(s)l
where: A(s) = Response for the parameter to be measured A(is) =

279
 Response for tfie intemal standard. C(is) = Concend^tion of tfie Uitemal
standard C(s) = Concend-ation of tfie parameter to be measured
If tfie RF value over tfie workUig range is a constant (<10% RSD), tfie
RF can be assumed to be invariant and tfie average RF can be used for
calculations. Altematively tfie results can be used to plot a caUbration
curve of response ratios, A(s)/A(is), vs. RF.
7.5 The working caUbration curve, caUbration factor, or RF must be verified on each
working day by tfie measurement of a Cy: check sample.
7.5.1 Prepare tfie QC check sample as described Ui Section 8.2.2.
7.5.2 Analyze tfie QC check sample accordmg to Section 10.
7.5.3 For each parameter, compare the response (Q) with the corresponcUng
caUbration acceptance criteria found Ui Table 2. If tfie responses for aU
parameters of interest fall within the designated ranges, analysis of actual
samples can begin. If any incUvidual Q faUs outside the range, a new
caUbration curve. caUbration factor, or RF must be prepared for tiiat
parameter according to Section 7.3 or 7.4.

8.0 QuaUty Condx)l

8.1 Each laboratory that uses this method is required to operate a formal quaUty
control program. The minimum requirements of this program consist of an initial
demonstration of laboratory capabUity and an ongoing analysis of spiked samples
to evaluate and document ciata quality. The laboratory must maintain records to
document the quaUty of data that is generated Ongoing data quaUty checks are
compared with estabUshed performance criteria to detemune if the results of
analyses meet the performance characteristics of the method. When results of
sample spikes incUcate atypical method performance, a quaUty conux)l check
stanciard must be analyzed to confirm that the measurements were performed in an
in-control mcxie of operation.
8.1.1 The analyst must make an initial, one-time, demonstration of the abtiity to
generate acceptable accuracy anci precision with this method This ability
is estabUshed as described in Section 8.2.
8.1.2 In recognition of advances that are occurring in chromatography, the
analyst is permitted certain options (detatied In Section 10.1) to improve
the separations or lower the cost of measurements. Each time such a
mociification is made to the method, the analyst is requUed to repeat the
prcxedure in Section 8.2.
8.1.3 Each day. the analyst must analyze areagentwater blank to demonstrate
that interferences from tfie analytical system are under conu-ol.
8.1.4 The laboratory must on an ongoing basis, spike and analyze a minimum of
10% of aU samples to monitor and evaluate laboratory data quality. This
prcxedure is described in Section 8.3.
8.1.5 The laboratory must on an ongoing basis, demonsttatetfu-oughtiie
analyses of quality condol check standards that the operation of the
measurement system is in control. This pnxedure is described in Section
8.4. The frequency of tfie check standard analyses is equivalent to 10% of
aU samples analyzed but may be reduced if spike recoveries from samples
(Section 8.3) meet all specUied quaUty condol criteria.
8.1.6. The laboratcjry must maintain performance records to dcxument the quaUty
of data that is generated. This prcx^edure is described in Section 8.5.
8.2 To establish tfie abiUty to generate acceptable accuracy and precision, the analyst

280
 must perform the following operations.
8.2.1 A quaUty condol ((JC) check sample concendate is requUed contaUting
each parameter of mterest at a concendmion of 10 pg/mL in metfianol.
The QC check sample concend-ate must be obtaUied from tfie U.S.
Envux)nmental Protection Agency, EnvUx>nmental Monitoring and Support
Laboratory m Cmcinnati, Ohio, if avaUable. If not availablefix)mtfiat
source, tfie QC check sample concend-ate must be obtaUiedfix)manotfier
extemal source. If not availablefix)meitfier source above, tfie QC check
sample concend-ate must be prepared by tfie labcjratory using stock
standards prepared Uidependentf yfix)mtfioseused for caUbration.
8.2.2 Prepare a QC check sample to contain 20 pg/L of each parameter by
addUig 200 pL of QC check sample concend-ate to 100 mL of reagent
water.
8.2.3 Analyze four 5-mL aUquots of tfie weU-mixed QC check sample accordmg
to Section 10.
8.2.4 Calculate the average recovery (X) Ui pg/L and the standard deviation of
the recovery (s) in g/L, for each parameter of interest using the four
results.
8.2.5 Fcjr each parameter compare s and X with the ccjrresponding acceptance
criteria for precision and accuracy, respectively, found in Table 2. If s
and X for all parameters of mterest meet tfie acceptance criteria, the system
performance is acceptable and analysis of acmal samples can begin. If any
incUvidual s exceeds the precision Untit or any incUvidual X faUs outside
the range for accuracy, the system performance is unacceptable for that
parameter.
Note: The large number of parameters in Table 2 present a substantial
probabtiity that one or more wiU faU at least one of the acceptance criteria
when all parameters are analyzed
8.2.6 When one or more of the parameters tested faU at least one of the
acceptance criteria, the analyst must prcx:eed according to Section 8.2.6.1
or 8.2.6.2.
8.2.6.1 Lcx:ate and correct tfie source of the problem andrepeatthe test
for all parameters of interest beginning with Section 8.2.3.
8.2.6.2 Beginnmg with Section 8.2.3, repeat tfie test only for those
parameters that faUed to meet criteria. Repeated fatiure, however,
wtil confirm a general problem with the measurement system. If
this cxxurs, Icxate and correct the source of the problem and
repeat the test for aU compounds of interest beginning with
Section 8.2.3.
8.3 The laboratory must, on an ongouig basis, spike at least 10% of the samples from
each sample site being monitored to assess accuracy. For laboratories analyzing
one to ten samples per month, at least one spiked sample per month is required.
8.3.1 The concenu-ation of the spUce in tfie sample should be detemtined as
foUows:
8.3.1.1 ff, as m compliance monitoring, the concend-ation of a specific
parameter in the sample is being checked against a regulatory
concentration Umit, the spike should be attiiatlintit or 1 to 5
times highertfiantfiebackground concenu-ation determined in
Section 8.3.2, whichever concend-ation would be larger.
8.3.1.2 If the concenttation of a specific parameter Ui the sample is not
t)eing checked against a Untit specific totfiatparameter, tfie sptice

281
 should be at 20 pg/L or 1 to 5 times higher tiian tiie background
concendation detemtined in Section 8.3.2, whichever
concen&ation would be larger.
8.3.2 Analyze oiie 5-niL sample aUquot to determine tfie background
concend-ation (B) of each parameter. If necessary, prepare a new QC
check sample concenttate (Section 8.2.1) appropriate for tfie background
concend-ations in the sample. SpUce a second 5-niL sample aUquot witii 10
1 of the QC check sample concendate and analyze it to determine the
concend-ation after spUdng (A) of each parameter. Calculate each percent
recovery (P) as 100(A-B)%/T, where T is tiie known due value of tiie
spike.
8.3.3 Compare tfie percent recovery (P) for each parameter witfi tfie
ccaresponding QC acceptance criteria found in Table 2. These acceptance
criteria were calculated to include an allowance for error in measurement
of both the background and spike concentrations, assunting a spike to
background ratio of 5:1. This error wiU be accounted for to the extent that
the analyst's spike to background ratio approaches 5:1. If spiking was
performed at concendation lower than 20 g/L, the analyst must use either
the QC acceptance criteria in Table 2, or optional QC acceptance criteria
calculated for the specific spike concentration. To calculate optional
acceptance criteria for the recovery of a parameter: (1) Calculate accuracy
(X') using the equation in Table 3. substimting the spike concentration (T)
for C; (2)calculate overaU precision (S') using the equation in Table 3,
substituting X' for X; (3) calculate the range for recovery at the spike
concend-ation as (100 X/T) + 2.44(100 S7 T)%,(7)
8.3.4 If any incUvidual P faUs outside the designated range for recovery, that
parameter has faUed the acceptance criteria. A check standard containing
each parameter that faUed the criteria must be analyzed as described in
Section 8.4.
8.4 If any parameter fatis the acceptance criteria for recovery in Section 8.3, a QC
check stanciard containing each parameter that faUed must be prepared and
analyzed
Note: The fiequency for tfie required analysis of a QC check stanciard wiU depend
upon the number of parameters being simultaneously tested, the complexity of the
sample matrix, and the performance of the laboratory.
8.4.1 Prepare the C^C check standard by addUig 10 pL of ( ^ check sample
concentrate (Sections 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC
check standard needs only to contain the parameters that fatied criteria in
the test in Section 8.3.
8.4.2 Analyze the QC check stanciard to determine the concendation measured
(A) of each parameter. Calculate each percent recovery (P(s)) as 1(X)
(A/T)%, where T is the tme value of the standard concentration.
8.4.3 Compare the percent recovery (P(s)) for each parameter witfi tfie
corresponding QC acceptance criteria found in Table 2. Only parameters
that failed tfie test Ui Section 8.3 need to be compared with these criteria.
If the recovery of any such parameter faUs outside the designated range,
the laboratory performance fortfiatparameter is judged to be out of
cond-ol, and tfie problem must be immecUately identified and corrected
The analytical result fortfiatparameter Ui the unspUced sample is suspect
and may not be reported forregulatorycompUance purposes.
8.5 As part of the (JC program for tfie laboratory, metiicxi accuracy for wastewater

282
 samples must be assessed and records must be maUitained. After tiie analysis of
five spUced wastewater samples as m Section 8.3, calculate tiie average percent
recovery (?) andtfiestandard deviation oftfiepercent recovery (sp). Express tfie
accuracy assessment as a percent recovery Uiterval from P-2s(p), to P+2s(p). If
P=90% and S(p)=10%, for example, the accuracy interval is expressed as 70-
110%. Update the accuracy assessment for each parameter on aregularbasis
(e.g. after each five totennew accuracy measurements).
8.6 It is recommended that the laboratory adopt adcUtional quaUty assurance practices
for use with this method The specific practices that are most prcxiuctive depjend
upon the needs of the laboratory and the nature of the samples. Field dupUcates
may be analyzed to assess the precision of tiie environmental measurements.
When doubt exists over the identification of a peak on the chromatogram,
confirmatory techrtiques such as gas chromatography with a cUssimUar colunm,
specific element detector, or mass spectrometer must be used Whenever
possible, the laboratory should analyze standardreferencematerials and
participate inrelevantperformance evaluation smdies.
8.7 The analyst should monitor both the performance of the analytical system and the
effectiveness of the methcxi in dealing with each sample matrix by spiking each
sample, standard, and reagent water blank with surrogate compounds (e.g. a, a,
a-trifluorotoluene) recommended to encompass the range of the temperature
program used in tUis methcxL From stcxk standard solutions prepared as in
Section 6.6, add a volume to give 750 pg of each surrogate to 45 mL of reagent
water contained in a 50-mL volumetricflask,mix and cUlute to volume for a
concendation of 15 mg/1. Add 101 of this surrogate spUdng solution directiy mto
the 5-niL syringe with every sample andreferencestandard analyzed Prepare a
fresh surrogate spiking solution on a weekly basis. If the intemal standard
caUbration prcxedure is being used, the surrogate compounds may be added
ciirectiy to the intemal standard spiking solution (Section 7.4.2).
9.0 Sample CoUection, Preservation, and HancUing
9.1 The samples must be iced orrefrigeratedfromthe time of coUection untti analysis.
If the sample contains free or combUied chlorine, add scxUum thiosulfate
preservative (10 mg/40 mL is sufficient for up to 5 ppm CI) totiieempty sample
bottie just prior to shippUig to the sampling site. EPA Method 330.4 or 330.5
may be used for measurement ofresidualchlorine. Fieldtestkits are avaUable for
this purpose.
9.2 CoUect about 500 mL of sample in a clean container. AdjusttfiepH of the sample
to about 2 by adding 1 + 1 HCl while stining. FiU the sample bottie in such a
maimer that no aU bubbles pass through the sample as the bottie is being filled
Seal tiie bottie sotfiatno aU bubbles are enttappai m it Maintaintfiehermetic
seal on tiie sample bottle untiltimeof analysis.
9.3 AU samples must be analyzed witftin 14 days of coUection.

10.0 Procedure
10.1 Table 1 surmnarizestfierecommended operatUig conditions fc)rtfiegas
chromatogr^h. Included intftistable are estimatedretentiontimes and MDL tiiat
can be achieved under these conditions. Otfier packed columns, chromatographic
conditions, or detectors may be used iftfierequUements of Section 8.2 are met.
10.2 CaUbratetfiesystem datiy as described Ui Section 7.

283
10.3 Adjusttfiepurge gas (nid-ogen or heUum)flowrateto 40 mL/mUi. Attachtfiedap
Utiet to the purging device, and settiiepurge and trap system to purge. Open tfie
syringe valve located ontiiepurging device sample Uidoduction needle.
10.4 AUow the sample to come to ambienttemperamreprior to indoducing it to tfie
syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe
valve. Open the sample bottie (or standard) and carefuUy pour the sample into the
syringe barrel to just short of overflowing. Replace the syringe plunger and
compress the sample. Open the syringe valve and vent anyresidualaU whtie
adjusting the sample volume to 5.0 mL. Since this process of taking an aUquot
destroys the vaUdity of the sample for future analysis, the analyst shouldfiUa
second syringe at this time to protect agaUist possible loss of data. Add 10.0 pi of
the surtogate spUdng solution (Section 8.7) and 10.0 pi oftfieinternal standard
spUdng solution (Section 7.4.2), if applicable, throughtiievalve bore, then close
the valve.
10.5 Attach the syringe-syringe valve assembly totfiesyringe valve on the purging
device. Open the syringe valves and inject the sample into the purging chamber.
10.6 Close both valves and purge the sample for 12.0 ±0.1 ntin at ambient
temperature.
10.7 After the 12-nun purge time, cUsconnect the purging devicefromthe trap. Dry the
trap by maintaining a flow of 40 mlV ntin of dry purge gas through it for 6 min.
If tiie purging device has no provision for bypassing the purger for this step, a
ciry purger should be inserted into the device to minimize moisture in the gas.
Attach the trap to the chromatograph, adjust the purge and trap system to the
desorb mode, and begin totemperatureprogram the gas chromatograph.
Intrcxiuce the trapped materials to the GC column byrapicUyheating the tr^ to
180°C whtie baclrflushing the trap v^th an inert gas between 20 and 60 mL/min
for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be
used as a secondary trap by ccx)Ung it to 30°C (subambienttemperature,if poor
peak geometry and randomretentiontime problems persist) instead of the initial
programtemperatureof 50°C.
10.8 Whtie the trap is being desorbed into the gas chromatograph column, empty the
purging chamber using the sample intrcxiuction syringe. Wash the chamber with
two 5-niLflushesofreagentwater.
10.9 After desorbing the sample for 4 min, recondition the d-ap byreturningthe purge
andttapsystem to the purge mode. Wait 15 s, then close the syringe valve on the
purging device to begin gas flow throughtfietrap. The traptemperatureshould
be maintained at 180°C. After approximately 7 mm, dim off thettapheater and
open the syringe valve to stoptfiegasflowtiiroughtiied-ap. Whentfiettapis
ccx)l, the next sample can be analyzed
10.10 IdentUy the parameters mtfiesample by comparingtfieretentiontUnes of tfie
peaks intfiesample chromatogram with those oftfiepeaks in stanciard
chromatograms. The widtii oftfieretentiontimewindow used to make
identifications should be based upon measurements of actualretentiontUne
variations of standards over the course of a day. Threetimesthe stanciard
deviation of aretentiontime for a compound can be used to calculate a suggested
window size; however,tfieexperience oftfieanalyst should weigh heavUy Ui tfie
interpretation of chromatograms.
10.11 Iftiieresponsefor a peak exceedstfieworkUig range of tfie system, prepare a
solution of tfie sample witfi reagent water fromtfieaUquot mtfiesecond syringe
and reanalyze.

284
11.0 Calculations

11.1 Determine tiie concenu-ation of mdividual compounds Uitfiesample.

11.1.1 Iftfieextemal standard calibration procedure is used, calculate tiie
concentration oftfieparameter bemg measured fromtfiepeak response
usmgtfiecaUbration curve or caUbration factor detennUied m Section
11.1.2 IftfieUiternal standard calibration procedure is used, calculate tfie
concend-ation intiiesample usmgtiieresponsefactor (RF) detemtined Ui
Secdon 7.4.3 and Equation 2.
Equation 2.
Concend-ation [g/Ll= [A(s)l [C(is)] / [A(is)] [RF]
where: A(s) = Response fortiieparameter to be measured. A(is) = Response
^^ *^ internal standard. C(is) = Concend-ation of tiie Uitemal standard.
11.2 Report results m pg/L witiiout correction for recovery data. AU QC data obtaUied
should be reported witfi tiie sample results.
12.0 Method Performance

12.1 The metiiod detection Umit (MDL) is defined astiientinimum concend-ation of a

substance that can be measured andreportedwitii 99% confidencetfiattfievalue is
above zero. The MDL concend-ations listed in Table 1 were obtained usmg
reagent water. SimUar results were achieved usingrepresentativewastewaters.
The MDL actuaUy achieved m a given analysis wiU vary depending on instrument
sensitivity and matrix effects.
12.2 This methcxi has been demonstrated to be appUcable for the concend-ation range
from the MDL to KKX) x MDL. Direct aqueous injection techniques should be
used to measure concend-ation levels above 1(XX) x MDL.
12.3 This method was tested by 20 laboratories using reagent water, drinking water,
surface water, and three industrial wastewaters spik^ at six concentrations over
the range 2.1 to 550 pg/L. Single operator precision, overall precision, and
methcxi accuracy were found to be directiyrelatedto the concenu-ation of the
parameter and essentiaUy independent of the sample mauix. Linear equations to
describe theserelationshipsare presented in Table 3.
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND
METHOD DETECTION LIMITS
Retention time Methcxi
Parameter (ntin) Detection limit
Column (UgA.)
Benzene 3.33 0.2
Toluene 5.75 0.2
Ethylbenzene 8.25 0.2
Chlorobenzene 9.17 0.2
1,4-Dichlorobenzene 16.8 0.3
1,3-Dichlorobenzene 18.2 0.4
11,2-Dichlorobenzene 25.9 0.4
Column 1 condiuons: Supelcoport (100/120 mesh) coated with 5% SP-
1200/1.75% Bentone-34 packed in a 6 ft X 0.085 in. ID stainless steel column
with helium carrier gas a 36 mL/min flow rate.

285
 TABLE 2: CALIBRATION AND QC ACCEPTANCE CRITERIA
Range for Limit for Range for X Range
Parameter Q(pgA.) Q(pgA.) (Hg/L) for P J»(s)(%)
Benzene 15.4-24.6 4.1 10.0-27.9 39-150
Chl(xx)benzene 16.1-23.9 3.5 12.7-25.4 55-135
1,2-Dichlorobenzene 13.6-26.4 5.8 10.6-27.6 37-154
13-I>ichl(xobaizene 14.5-25.5 5.0 12.8-25.5 50-141
1,4-Dichlorobenzene 13.9-26.1 5.5 11.6-25.5 42-143
Ethylbenzene 12.6-27.4 6.7 0.0-28.2 32-160
Toluene 15.5-24.5 4.0 11.2-27.7 46-148
Q = Concentration measure in QC check sample, in g/L (Section 7.5.3). s =
Standard deviation of four recovery measurements, in g/L (Section 8.2.4). X =
Average recovery for four recovery measurements, in g/L (Section 8.2.4). P(s)P=
Percent recovery measured (Section 8.3.2, Section 8.4.2). (a) Criteria were calcuUited
assuming a QC check sample concentration of 20 p ^ . Note: These criteria are based
directiy upon the method performance data in Table 3. Where necessary, the limits
for recovay have been lxx)adened to assure applicability of the limits to
concentrations below those used to develop Table 3.

TABLE 3: METHOD ACCURACY AND PREQSION AS FUNCTIONS OF

CONCENTRATION
Accuracy ,as Single analyst OveraU
Parameter recovery, X' precision, s' precision, S'
(ftg/L) (ftg/L)
Benzene (1920+0.57 (r09X+0.59 « lX+0.56
Chlorobenzene 0.95C-K).02 0.09X+0.23 0.17X+0.10
1,2-Dichlorobenzene 0.93C4O.52 0.17X+0.04 0.22X+0.53
1,3-Dichlorobenzene 0.96C-0.04 0.15X+0.10 0.19X+0.09
1,4-Dichlorobenzene 0.93C+0.09 0.15X+0.28 0.20X+0.41
Ethylbenzene 0.940+0.31 0.17X+0.46 0.26X+0.23
0.94C+0.65 0.09X+0.48 0.18X+0.71
[Toluene
X'= Expected recovery from one or more measurements of a sample containing a concentrauon of
C, in ^g/L.
s'= Expected single analyst standard deviation of measurements at an average concentration tound
in X, in ng/L.
S*= Expected interlaboratory standard deviation of measurements at an average concentration touna
of X, in jig/L.
C= True value for the Concentration, in ^lg/L.
X= Average recovery found for measurements of samples containing a concentration of C, m
^g/L.

286
References

United States EnvUonmental Protection Agency. (1992). Methods for Chemical

Analysis of Water and Wastes. Methcxi #602. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency,
Cincinnati, Ohio.

American Pubtic Healtii Asscxnation. (1992). >>tf^"<1f^^d MethcxLs for tiie

Examination of Water and Wastewater. 18th ed. Methcxi 6210. American
Public Health Asscx:iation, American Water Works Association, and Water
Environment Federation, Washington DC, 6-17.

287
METHOD #: 120.1 Approved for NPDES (Editorial Revision 1982)
TITLE: Satinity
ANALYTE:
Salirtity

INSTRUMENTATION: Conductivity Meter

INTRODUCTION:
Salinity is an important unitiess property of indusuial and namral waters. It was origuiaUy
conceived as a measure of tiie mass of dissolved salts in a given mass of solution. The
experimental determination of the content by drying and weighing presents some
difficulties due to the loss of some components. The only retiable way to determine tiie
true or absolute salirtity of a natural water is to make-complete chemical analysis.
However, this method is time-consuming and cannot yield the precision necessary for
accurate work. Thus, to determine satinity, one normally uses indirect metiicxis involving
the measurement of a physical property such as conductivity, density, sound speed, or
refractive index. From an empirical relationship of salinity and the physical property
detemtined for a stanciard solution it is possible to calcitiate salinity. The resultant salinity
is no more accurate than the empirical relationship. The precision of the measurement of a
physical property wiU determine the precision in salinity. Following are the precisions of
various physical measurements and the resultant satinity presentiy attainable with
commercial instmments:

Precision of Precision of
Property Measurement Salinity
Conductivity ± 0.0002 ± 0.0002
Density ± 3.0 x 10-6 g/cm^ ± 0.004
Sound speed ± 0.02 m/s ±0.01

Although conductivity has the greatest precision, it responds only to ionic solutes.
Density, although less precise, responds to all ctissolved solutes. Because of its high
sensitivity and ease of measurement, the conductivity methcxi is most commonly used to
detemtine salinity. In conductivity, an electrical current is measured. This current is
carried by both anions and cations in solution inducting those due to salts, but to different
degrees. Thus the salinity of a sample is related to the conductivity of a sample depending
on the cations and anions of the given salt or salts.

1.0 Scope and AppUcation

1.1 This methcxi is applicable to cirinking, surface, and saline water, domestic and
industrial wastes and acid rain (atmospheric deposition).

2.0 Summary of Metiiod

2.1 The salinity of a sample is measured by use of a self-contained conductivity meter,
Wheatstone bridge-type, or equivalent
2.2 Samples are preferable analyzed at 20°C. If not,tiiemeter wiU display an error.

288
3.0 Comments

3.1 Insdnment must be standardized with KCl solution before datiy use.
3.2 Conductivity ceU must be kept clean.
3.3 Temperanire variations and corrections represent the largest source of potential
error.

4.0 Sample Handling and Preservation

4.1 Analyses can be performed either in the field or laboratory.

4.2 If analysis is not completed wititin 24 hours of sample coUection, sample should
be ftitered through a 0.45 micron filter and stored at 4°C. Ftiter and apparams
must be washed with high quaUty distiUed water and pre-rinsed with sample
before use.
5.0 Apparams
5.1 Conductivity Meter preferably witiitemperaturecompensation
5.1.1 Orion Model 160
6.0 Reagents
6.1 Conductivity water: Prepared reagent-grade water. The conductivity should be
small compared to the value being measured
6.2 Standard potassium chloride solution, KCl, 0.01(X)M. Dissolve 745.6 mg
anhydrous KCl in conductivity water and dtiute to 1(XX) mL in a class A
volumetric flask at 25°C. This is the standard reference solution which at 25°C
has a conductivity of 1412 |imhos/cm It is satisfactory for most samples when
the cell has a constant between 1 and 2 cm-i. Store in a glass-stoppered
borositicate glass bottie.

7.0 CeU CaUbration

7.1 The analyst should use the standard potassium chloride solution (6.2) and Table I
to check the accuracy of the ceU constant and conductivity bridge.
7.2 A seawater with a conductivity at 15°C equal to that of a KCl solution containing
a mass of 32.4356 g in a mass of 1 kg of solution is defined as having a practical
saUnity of 35. This value was determined as an average oftiu-eeindependent
laboratory stucties.

8.0 Prcx:edure
8.2 Conductivity meters often uidicate salinity directiy. Commercial probes
commonly contain atemperaturesensor.
8.2.1 Model 160 (Orion)
8.2.1.1 Select 20°C as a reference temperamre.
8.2.1.2Select salinity by pressuig tiie S/cm,Sal key.
8.2.1.3After immersing tiie conductivity cell into tiie sample,tiiemeter
displays tiie sample's salinity and temperature.

289
9.0 Calculation

9.1 Express saluiity as parts pertiiousand(ppt) read duectiyftx)mtiiemeter display.

10.0 Accuracy

10.1 Precision and accuracy are not avatiable.

Table I: Temperature of O.OIM KCl

versus conductivity values.
Conductivity 0.01 M KCl
°C }imhos/cm
ii 1305
22 1332
23 1359
24 1386
25 1413
26 1441
27 1468
28 1496
References
American PubUc Health Association. (1992). Standard Methcxis fortiieExamination of
Water and Wastewater. 18th ed Method 2520. American PubUc Health Asscx:iation,
American Water Works Association, and Water Environment Federation, Washington
DC, 2-46.
Orion Research Incorporated. (1990). Model 160 Conductivity Meter Instmction Manual.
Mcxiel 160. Orion Research Inc. Laboratory Prcxiucts Group, The Scrafft Center,
Boston, Ma.
Urtited States Environmental Protection Agency. (1992). Methods for Chemical Analysis
of Water and Wastes. Methcxi #120.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

290
METHOD #: 370.1 Approved for NPDES (Editorial Revision 1978)
TITLE: SiUca, Dissolved (Colorimetric)
ANALYTE:
Stiica, Si02

INSTRUMENTATION: HACH DR/2000

INTRODUCTION:
SiUcon ranks next to oxygen in abundance in the earth's cmst. It appears as the oxide
(sitica) in quartz and sand and is combined with metals in the form of many complex
stiicate minerals, particularly igneous rcx:ks. Degradation of sitica containing rocks results
in the presence of stiica in natural waters as suspended particles, in a coUoidaJ or polymeric
state, and as siUcic acids or siUcate ions. Volcanic and geothermally heated waters often
contain an abundance of sitica. A more complete ctiscussion of the cxxurrence and
chemistry of stiica in natural waters is avatiable The sitica (Si02) content of natural water
most commortiy is in the 1 to 30 mg/L range, although concentrations as high as 100 mg/L
are not unusual and concentrations exceecUng 1000 mg/L are found in some brackish
waters and brines. SiUca in water is undesirable for a number of industrial uses because it
forms ctifficitit-to-remove sitica and siUcate scales in eqitipment, particularly on
high-pressure steam-turbine blades. Stiica is removed most often by the use of strongly
basic anion-exchange resins in the deionization prcx:ess, by ctisttilation, or by reverse
osmosis. Some plants use precipitation with magnesium oxide in either the hot or cold time
softening prcx:ess such as diatoms.

1.0 Scope and Application

1.1 This method is appUcable to drinking, surface and saline waters, domestic and
industrial wastes.
1.2 The working range of the metiicxi is approximately 0 to 1(X) mg siUca/L.
2.0 Summary of Metiiod
2.1 SiUca and phosphate in the sample react with molybdate ion under conditions to
form yeUow siUcomolybdic acid complexes and phosphomolybcUc acid
complexes. Adctition of ciuic acid desux)ystiiephosphate complexes. Stiica is
then determined by measuring the remaining yellow color.

3.0 Interferences
3.1 Excessive color andl/or mrbidity interfere. Correct by running blanks prepared
without adcUtion of the ammonium molybdate solution.
3.2 Large amounts of iron and sulfide interfere.
3.3 Contact witii glass should be minimized, siticafreereagents should be used as
much as possible. A blank should be mn.
3 4 At levels of 50 mg/L phosphate, interference is not a problem. At 60 mg/L
phosphate, interference of minus 2% is observed. At 75% mg/L, tiie interference
is minus 11%.
4.0 Sampting and Storage

291
 4.1 CoUect samples in clean plastic or glass botties. Analyze as soon as possible after
coUecnon. Store samples up to seven days at 4°C (39°F) or below. Warm
samples to roomtemperaturebefore analyzing.
5.0 Apparams

5.1 Hach DR / 2000 Specux)photometer

5.2 Bottie, square mixing, 25 nti mark
6.0 Reagents

6.1 Acid Reagent Powder Ptilows for high range Stiica

6.2 Citric acid piUows
6.3 Holmium Trichloride Powder PiUows
6.4 Molybdate Reagent Powder PiUows for high range Stiica
6.5 DI water
7.0 Prcx^edure

7.1 Entertiiestored program for high range silica (SIO2) 656 READ / ENTER. The
display wiU show DIAL nm TO 452.
7.1.1 Rotate the wavelength dial until tiie smaU cUsplay shows 452 nm. Press
READ / ENTER. The display wiU show mg/1 Si02
7.2 FiU a sample ceU with 25 ml of sample.
7.3 Add the contents of one molybdate Regent Powder PiUow for high range Stiica.
7.4 Add the contents of one acid Reagent Powder Ptilow fcjr high range Silica. Swirl
to mix.
7.5 Press SHlh'l TIMER. A ten minute reaction pericxi wiU begin.
7.6 Press SHIFT ABS. The display wiU show Abs. Press ZERO. The display wiU
show: WATTtiien0.000 ABS.
7.7 Prepare the Holmium Trichloride solution.
7.7.1 Add the contents of one Holntium Trichloride Powder Ptilow to 25 ml of
DI water and cap. Solution may be kept indefinitely if capped.
7.8 Place the capped square mixing bottie with the Holntium Trichloride solution into
the ceU holder and closetiieUght shield
7.9 Starting at 460 nm, slowly tum the wavelength conux)l ctial to decrease tiie
wavelength. Watch the ctisplay for the peak absorbance reading. This should
cxjcur between 450-454 nm. Without moving the wavelength ctial, remove the
holmium trichloride solution from the cell holder.
7.10 When thetinierbeeps, add the contents of one Ciuic Acid Powder PiUow to the
sample ceU (the prepared sample). Swirl to mix.
7.11 Press SHIFT CONC and SHIFT TIMER. A 2 ntin reaction period wtil begin.
7.12 When thetimerbeeps, the display wiU show: 0.0 mg/L SIO2 H. FiU a second
sample ceU witii 25 nti of DI water.
7.13 Place tiie blank intiieceU holder. Close tiie Ught shield
7.14 Press ZERO. The display wtil show: WATTtiien0.0 mg/L SIO2 H.
7.15 Wititintiu^eminutes after the secondtimerbeeps, placetiieprepared sample in
the ceU holder. Close tiie Ught shield The results wtil be displayed in mg/L
stiica.

292
8.0 Calculations

8.1 The results are obtained duectiy from tiie specuxjphotometer.

9.0 Accuracy

9.1 In a single laboratory, using a standard solution of 50.0 mg/L Si02 and two
representative lots of reagent witiitiieDR / 2(XX), a suigle operator obtained a
standard deviation of ± 0.45 mg/1 stiica.
References

American PubUc Healtii Association. (1992). Standard Methods fortiieExamination of

Water and Wastewater. 18tiied. Metiiod 4500. American PubUc Healtii Asscxriation,
American Water Works Asscxnation, and Water Environment Federation, Washington
DC, 4-117.
Hach Company. (1992). DR/2000 Spectrophotometer Handbook. Metiiod 8185. Hach
Company, Loveland, Co.
United States Environmental Protection Agency. (1992). Methods for Chemical Analvsis
of Water and Wastes. Methcxi #370.1. Environmental Monitoring and Support
Laboratory, Urtited States Environmental Protection Agency, Cincinnati, Ohio.

293
TITLE: SoUds

INTRODUCnON
SoUds refCT to matter suspended or dissolved ui water or wastewater. SoUds may affect
water or effluent quaUty adversely in a number of ways. Waters witii high dissolved soUds
generaUy are of mferior palatabtiity and may induce an unfavorable physiological reaction
m theti-ansientconsumer. Fortiiesereasons, a Umit of 500 mg dissolved soUds/L is
desuable for drinking waters. Highly mineraUzed waters also are unsuitable for many
mdustrial appUcations. Waters high in suspended solids may be aestiietically
unsatisfactory for such purposes as batiiing. SoUds analyses are important intiieconti-ol
of biological and physical wastewatertteattnentprocesses and for assessing compUance
witii regulatory agency wastewater effluent limitations.

"Total sotids" istiietermappUed totiiematerial residue left intiievessel after evaporation

of a sample and its subsequent drying in an oven at a defuied temperamre. Total solids
includes "total suspended solids,"tiieportion of total solids retained by a ftiter, and "total
dissolved solids,"tiieportiontiiatpassestiiroughtiiefilter. The type of filter holder, the
pore size, porosity, area, andtiiicknessof tiie ftiter and the physical nature, particle size,
and amount of material deposited on thefilterare the principal factors affecting separation
of suspended from cUssolved soUds.

"Dissolved solids" is the portion of solidstiiatpassestiu-ougha ftiter of 2.0 p.m (or

smaUer) nominal pore size under specified conctitions. "Suspended soUds" is the portion
retained on the ftiter.

"Fixed sotids" is thetermappUed to the residue of total, suspended, or dissolved solids

after heating to dryness for a specified time at a specifiedtemperature.The weight loss on
igttition is caUed "Volattie sotids." Determinations of fixed and volattie soUcis do not
cUstinguish precisely between inorganic and orgartic matter because the loss on ignition is
not confined to organic matter. It includes losses due to decomposition or volattiization of
some mineral salts. Better characterization of organic matter can be made by such tests as
total orgaitic cartx)n , BOD, and COD.
Provided in this manual are the insuiictions for Total SoUds and Total Suspended Solids.
To determine Total Dissolved Solids (TDS), one should conduct both of these tests, and
the difference is TDS. Also, it is possible to determine TDS by collecting thefilteredwater
used in preparing TSS.
Strictiy speaking, the definition of total soUds includes materialfloatingon water (e.g.
wcxxi, plastics, grease) as weU as larger particulate matter maintained in suspension by
tiu-bulence (e.g. gravel, deuims). However, these fractions (floatables, settieable solids)
are commonly not measured as total solid because they are low-tech devices to treat them
apartfromconventional solids (skimmers, baffles, settUng chambers).
Provided in this manual istiiemethcxis for determining Total Volatile SoUds (TVS) and
Total Fixed SoUds (TFS). It should be knowntiiatto detemtine TVS and TFS, Total
Solids must be done in a porcelain dish but witii tiie same procedure. TS ashed at 550°C
wiU bum aUtiieorganic matter (TVS) sotiiatwhat is remaining is inorganic matter (TFS).
Also, it is possible to detemtine Total Fixed Dissolved SoUds (TFDS) and Total Fbced
Suspended SoUds (TFSS)fromtiieTSS ftiter used previously. The procedure is tiie same
as TSS buttiiefilteris placed ui a porcelain dish attiiebeginning of any determinations.

294
After dryuig at 105°C,tiiefilteris ashed at 550° C. What is remaining is TFSS, and tiie
difference is Total Volatile Suspended SoUds (TVSS). To determine TVDS, subu-act
TVSS from TVS
References

American PubUc Healtii Association. (1992). Standard Metiiods fortiieExamination of

Water and Wastewater. 18tiied. Metiiod 2540. American PubUc Healtii Association,
American Water Works Asscxnation, and Water Environment Federation, Washington
DC, 2-53.

295
METHOD*: 160.3 Approved for NPDES (Issued 1971)
TITLE: Total SoUds (Gravimeuic, Dried at 103-105°C)
ANALYTE:
Residue, Total

INSTRUMENTATION: Drying Oven

1.0 Scope and AppUcation

1.1 This method is appUcable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The practical range oftiiedetemtination isfrom10 mg/L to 20,000 mg/L.
2.0 Summary of Methcxi

2.1 A weU mixed aUquot of the sample is quantitatively transferred to a pre-weighed

evaporating cUsh and evaporated to dryness at 103-105°C.
3.0 Definitions
3.1 Total Residue is defined as the sum of the homogenous suspended and cUssolved
materials in a sample.
4.0 Sample Handling and Preservation
4.1 Preservation of the sample is not practical; analysis should begin as scxjn as
possible. Refrigeration or icing to 4°C, to minimize microbiological
decomposition of solids, is recommended.

5.0 Interferences
5.1 Nonrepresentative particulate such as leaves, sticks, fish and lumps of fecal matter
should be excluded from the sample if it is determined that their inclusion is not
desired in thefinalresult.
5.2 Floating oil and grease, if present, should be included in the sample and cUspersed
by a blender device before aliquoting, if the inclusion is desu^ in the final result

6.0 Apparatus
6.1 Evaporating dishes, aluminum, 57 X 16 mm. CommerciaUy available.

7.0 Procedure
7.1 Heat tiie clean evaporating dish to 103-105°C for one hour. Cool, weigh and
store in desiccator untti ready for use. If volattie solids are to be detemtined later,
a porcelain dish should be used See Fixed and Volatile SoUds.
— 7 . i Weightiireealuminum pans to 4 decimal places. Transfer a thoroughly mixed
measured aliquot of sample to each oftiiepre-weighed dishes and evaporate to
dryness in a drying oven.

296
 7.2.1 Choose an aliquot ot sample sutticient to contam a residue of at least 2:> mg
usuaUy 20 ml. To obtain a weighable residue, successive aUquots of
sample may be added to tiie sarne dish.
7.2.2 If evaporation is performed in a drying oven, the temperature should be
lowered to approxunately 98°C to prevent boiUng and splattering of tiic
sample
sample.
7.3 Dry the evaporated sample for at least 1 hour at 103-105°C. Ccx)l in a desiccator |
and weigh. The ESL waits at least 12 hours before reweighine.j Repeat
the cycle of drying at 103-105°C, ccx)Ung, desiccating and weighuig unul a
constant weight is obtained or until loss of weight is less than 4% of the previous
weight, or 0.5 mg, whichever is less.
8.0 Calculation

8.1 Calcitiate total residue as follows:

(A-B)X1,000
Total residue, mg/L =
where:
A = weight of sample + cUsh in mg
B = weight of cUsh in mg
C = volume of sample in mL

9.0 Accuracy
9.1 Precision and accuracy data are not avatiable attitistime.

References
United States Envux)nmental Protection Agency. (1992). Metiipq? fpr Chgmical Ar>^Y§i$
of Water and Wastes. Metiiod #160.3. Environmental Monitoring and Support
Laboratory, United States Envux)nmental Protection Agency, Cuicuuiati, Ohio.
American PubUc Healtii Association. (1992). Standard Methods for tiie Examination of
Water and Wastewater. 18tiied. Metiiod 2540. American Public Healtii Association,
American Water Works Association, and Water Environment Federation, Washmgton
DC, 2-53-58.

297
METHOD #: 160.2 Approved for NPDES Gssued 1971)
TITLE: Total Suspended SoUds (Gravunetric, Dried at 103-105°Q
ANALYTE:
Residue, Non-Ftiterable
INSTRUMENTATION: Drying Oven
1.0 Scope and AppUcation
1.1 This methcxi is appUcable to drinking, surface, and saUne waters, domestic and
industrial wastes.
1.2 The practical range of the determination is 4 mg/L to 20,0(X) mg/L.
2.0 Summary of Method
2.1 A weU-mixed sample is ftitered through a glassfiberfilter,and the residue
retained on the ftiter is dried to constant weight at 103-105°C.
3.0 Definitions
3.1 Residue, non-filterable, is defmed as those sotids which are retained by a glass
fiberfilterand dried to constant weight at 103-105°C.
4.0 Sample HandUng and Preservation
4.1 Non-representative particulates such as leaves, sticks, fish, and lumps of fecal
matter should be excludedfromtiiesample if it is detemtinedtiiattheir inclusion is
not desired in thefinalresult.
4.2 Preservation of the sample is not practical; analysis should begin as soon as
possible. Refrigeration or icing to 4°C, to minunize microbiological
decomposition of sotids, is recommended

5.0 Interferences
5.1 FUtration apparatus,filtermaterial, pre-washing, post-washing, and dryuig
temperamre are specified becausetiiesevariables have been shown to affect tiie
results.
5.2 Samples high in Ftiterable Residue (dissolved solids), such as saline waters,
brines and some wastes, may be subject to a positive uiterference. Care must be
taken in selecting tiie fUtering apparams sotiiatwashing oftiiefilterand any
dissolved solids uitiiefilter(7.5) minimizestiiispotential uiterference.

6.0 Apparams
6 1 Glassfiberftiter discs, witiiout organic binder, such as Mtitipore AP-40, Reeves
Angel 934-AH, Gelman type A/E, or equivalent. NOTE: Because of tiie physical
nanue of glassfiberfilters,tiieabsolute pore size cannot be conUDtied or
measured Terms such as "pore size", collection effiaencies and effective
retention are used to definetitisproperty in glass fiber filters. Values for tiiese

298
 parameters vary fortiieftiters Usted above.
^•^ S!l^^^?.PP°^* ^il^^S apparams witii reservou- and a coarse (40-60 microns)
fritted disc as a ftiter support NOTE: Manyfiinneldesigns are avatiable in glass
or porcelain Some of tiie most common are Hu-sch or Buchner funnels
membranefilterholders and Gooch cmcibles. AU are avatiable witii coake fritted
6.3 Suction flask.
6.4 Drying oven, 103-105°C.
6.5 Desiccator.
6.6 Analytical balance, capable of weighing to 0.1 mg.
7.0 Prcx^ediue

7.1 Preparation of glass fiber ftiter disc: Place the glass fiber filter on the
membrane filter apparatus or insert into bottom of a suitable Gooch cmcible
witii wrinkled surface up. While vacuum is appUed, washtiiedisc witii tiiree
successive 20 mL volumes of distiUed water. Remove allfracesof water by
continuuig to apply vacuum after water has passedtiuxjugh.Remove ftiter fix)m
membrane ftiter apparams or botii cmcible and ftiter if Gooch cmcible is used,
and dry in an oven at 103-105°C for one hour. Remove to desiccator and store
untti needed Repeattiiedrying cycle until a constant weight is obtained (weight
loss is lesstiian0.5 mg). Weigh immediately before use. After weighing, handle
the ftiter or cmcible/filter with forceps or tongs only.
7.2 Selection of Sample Volume for a 4.7 cm diameterfilter,filter100 mL of sample.
If weight of capmred residue is lesstiian1.0 mg, tiie sample volume must be
increased to provide at least 1.0 mg of residue. If otiier ftiter diameters are used,
start witii a sample volume equal to 7 niL/cm2 offilterarea and coUect at least a
weight of residue proportional to the 1.0 mg stated above. NOTE: If during
function oftillsinitial volumetiiefiltt^tionrate dropsrapidly,or if ftio^on time
exceeds 5 to 10 minutes, the following scheme is recommended: Use an
unweighed glassfiberftiter of choice affbced intiieftiter assembly. Add a known
volume of sample to the ftiter funnel and reccjrd the time elapsed after selected
volumes have passedtiux>ughthe ftiter. Twenty five mL uicrements for timing
are suggested Continue to record thetimeand volume increments imtti ftiuation
rate dropsrapicUy.Add aciditional sample if the ftiter funnel volume is inadequate
to reach a reduced rate. Plot the observed time versus volume filtered Select the
proper fUtration volume as that just short oftiietime a significant change in
filtrationrateoccurred.
7.3 Weigh three pans with ftiters in them and record their weight to 4 decimal places.
7.4 Assemble the fUtering apparams and begin suction. Wet thefilterwith a smaU
volume of cUsttiled water to seat it against the fritted support.
7.4 Shake the sample vigorously and quantitativelyttansferthe predetermined sample
volume selected in 7.2 (20 ml) to thefilterusing a graduated cyUnder. Remove
aU traces of water by continuing to apply vacuum after sample has passed
through.
7.5 With suction on, washtiiegraduated cyUnder,filter,nonftiterable residue and
filter fiinnel waU with three portions of distiUed water allowing complete drainage
between washing. Remove aU traces of water by continuing to apply vacuum
after water has passed through. NOTE: Total volume of wash water used should
equal approximately 2 mL per cm2. For a 4.7 cm ftiter the total volume is 30
mL.

299
 7.6 CarefuUy remove the filter from the ftiter support and place in aluminum pan.
Altematively, remove cmcible and ftiter from cmcible adapter. Do this two
more times. Dry all for at least one hour at 103-105°C. The ESL waits at
least 12 hours before reweighing.|(Jcx)l in a desiccator and weigh. Repeat
the drying cycle untti a constant weight is obtained (weight loss is less than 0.5
mg).
8.0 Calculations

8.1 Calculate non-filterable residue (TSS) as foUows:

(A - B) X 1,000
Non-ftiterable residue, mg/L =

where:
A = final weight of filter and pan+ residue in mg
B = initial weight of ftiter and pan in mg
C = mL of sample filtered
8.2 Total Dissolved Solids (TDS) = Total Solids (TS) - Total Suspended SoUds
(TSS)

9.0 Accuracy
9.1 Precision data are not avatiable at this time.
9.2 Accuracy data on actual samples cannot be obtained
References
American PubUc Healtii Association. (1992). Standard Methods for tiie Examination of
Water and Wastewater. 18tiied. Metiiod 2540. American PubUc Healtii Association,
American Water Works Association, and Water Environment Federation, Washuigton
DC, 2-57.
United States Envux)nmental Protection Agency. (1992). Metiipds for Chgmic^ Analysis
of Water and Wastes. Metiiod #160.2. Envuonmental Monitonng and Support
Laboratory, United States Envux)nmental Protection Agency, Cincinnati, Ohio.

300
METHOD #: 160.4 Approved for NPDES (Issued 1971)

TITLE: Total Volatile SoUds (TVS) and Total Fixed SoUds (TFS)
ANALYTE:
Residue and Volattie
INSTRUMENTATION: Muffle Fumace
1.0 Scope and Application
1.1 This method determines the weight of soUd material combustible at 550°C.
1.2 The test is useful in obtaining aroughapproximation of the amount of organic and
inorgartic matter present in the solidfractionsof sewage, activated sludge,
industrial wastes, or bottom sectiments.
2.0 Summary of Methcxi
2.1 The residue obtained from the determination of total solids is ignited at 550°C in a
muffle fumace. The loss of weight on ignition is reported as mg/L total volattie
solids (TVS) and that which is remaining is total fixed solids (TFS).
3.0 Comments
3.1 The test is subject to many errors due to loss of water of cnystaUization, loss of
volatile organic matter prior to combustion, incomplete oxidation of certain
complex organics, and decomposition of mineral salts during combustion.
3.2 The results should not be considered an accurate measure of organic carbon in the
sample, but may be useful in the control of plant operations.
3.3 The principal source of error in the determination is fatiure to obtain a
representative sample.
4.0 Sample HandUng and Preservation
4.1 Preservation of the sample is not practical; analysis should begin as soon as
possible. Refrigeration or icing to 4°C, to minimize microbiological
decomposition of solicis is recommended.

5.0 Apparams
5.1 Evaporating Dishes, porcelain
5.2 Fumace at 550°C
5.3 Tongs
5.4 Scale capable of measuring 0.1 mg
6.0 Procedure
6.1 Heat the clean evaporating dish to 550°C for one hour to remove organic matter
that may be adhering totiiedish. Cool, dessicate and store until use.
6.2 The procedure for Total solids should be foUowed substimting porcelain dishes
for the aluminum pans.

301
 6.3 After Total SoUds has been calculated,tiieporcelaui dishes should be placed ui tiie
muffle fumace at 550°C for one hour.
6.4 Reweigh porcelain ctishes
7.0 Calculation

7.1 Total Fixed Solids is calculated by

TFS Ul mg/L =weight remaining after SSQ *>C ashin^rmpV weight of dishrmp^ X IQQQ
sample volume, ml

7.2 Total Volattie SoUds is calculated by

TVS Ul mg/L =
(Weight of dish + residue before ignition, mg^ - (weight of residue + dish after ignition, mg) X IQQQ
sample volume, ml

Precision and Accuracy

5.1 A coUaborative smdy involving three laboratories examirting four samples by
means of ten replicates showed a standard deviation of ± 11 mg/L at 170 mg/L
volattie residue concenttation.
References
American Pubtic Health Asscx:iation. (1992). Standard Methcxis for the Exantination of
Water and Wastewater. 18th ed. Methcxi 2540. American PubUc Healtii Asscx:iation,
American Water Works Asscxnation, and Water Environment Federation, Washington
DC, 2-53.
United States Environmental Protection Agency. (1992). Metiiods for Chentical Analvsis
of Water and Wastes. Method #160.4 and #160.3. Environmental Monitoring and
Support Laboratory, United States Environmental Protection Agency, Cincinnati,
Ohio.

302
METHOD #: 160.4 Approved for NPDES (Issued 1971)

TITLE: Total Volatile Suspended Solids (TVSS) and Total Fixed Suspended Solids
(TFSS)

ANALYTE:
Residue and Volattie
INSTRUMENTATION: Muffle Fumace
1.0 Scope and AppUcation

1.1 This metiiod detemtines the weight of solid material combustible at 550°C.
1.2 The test is useful in obtaining aroughapproximation of the amount of organic and
inorgartic matter present in the suspended solidfi^ctionof sewage, activated
sludge, industrial wastes, or bottom sectiments.
2.0 Summary of Method
2.1 The residue obtained from the determination of total suspended soUcis is ignited at
550°C in a muffle fumace. The loss of weight on igitition is reported as mg/L
volatile suspended residue, that which is remaining isfixedvolattie suspended
solids.
3.0 Comments
3.1 The test is subject to many errors due to loss of water of crystaUization, loss of
volatile orgaitic matter prior to combustion, incomplete oxidation of certain
complex organics, and decomposition of mineral salts during combustion.
3.2 The resitits should not be considered an accurate measure of organic carbon in the
sample, but may be usefiti in the control of plant operations.
3.3 The principal source of error in the determination is fatiure to obtain a
representative sample.
4.0 Sample HandUng and Preservation
4.1 Preservation of the sample is not practical; analysis should begin as soon as
possible. Refrigeration or icing to 4°C, to minimize microbiological
decomposition of solids is recommended.

5.0 Apparams
5.1 Evaporating Dishes, porcelain
5.2 Fumace at 550°C
5.3 Tongs
5.4 Scale capable of measuring 0.1 mg
6.0 Procedure
6.1 Heat tiie clean evaporating dish to 550°C for one hour, to removetiieorganic
matter that may be adhering totiiedish. Cool, desiccate and store untti use.

303
 6.2 The prcxjedure for Total Suspended SoUds should be foUowed substimting
porcelain dishes for the aluminum pans.
6.3 After Total Suspended SoUcis has been calculated, the porcelain dishes should be
placed in the muffle fumace at 550°C for one hour.
6.4 Reweigh porcelain ctishes

7.0 Calculation
7.1 Total Fixed SoUds is calculated by
TFSS Ul mg/L =
weight remaining on filter after 550 '^C ashing(mgV weight of dish(mg^ X 1000
sample volume, ml

7.2 Total Volatile SoUds is calculated by

TVSS in mg/L =
(Weight of dish + filter before ignition, m?) - (weight of filter + dish after ignition, me) X IQOQ
sample volume, ml

8.0 Accuracy
8.1 A coUaborative smdy involving three laboratories examirting four samples by
means oftenreplicates showed a standard deviation of ± 11 mg/L at 170 mg/L
volattie residue concenttation.

References
American PubUc Healtii Association. (1992). Standard Methods fortiieExamination of
Water and Wastewater. 18tiied. Metiiod 2540. American PubUc Healtii Association,
American Water Works Association, and Water Environment Federation, Washington
DC, 2-57.
United States Environmental Protection Agency. (1992). Method? fpr Chemical Analysis
nf Water and Wastes. Metiiod #160.4 and #160.3. Envuonmental Monitonng and
Support Laboratory, United States Envuonmental Protection Agency, Cuicinnati,
Ohio.

304
TITLE: Temperature

INSTRUMENTATION: Thermometer

INTRODUCTION:
Temperattu^e reacUngs are used ui tiie calculation of various forms of aUcaUnity, in stucUes of
samration and stabtiity witii respect to calcium carbonate, ui tiie calculation of saUnity, and
in general laboratory operations. In Umnological smdies, watertemperaturesas a function
of deptii often are required Elevatedtemperaturesresulting from discharges of heated
water may have significarit ecological impact Identification of source of water supply,
such as deep weUs, often is possible by temperature measurements alone. Indusuid plants
often require data on water temperature for process use cjr heat-transmission calculations.

1.0 Scope and AppUcation

1.1 This methcxi is appUcable to aU waters and otiier fluids.

2.0 Summary of Methcxi

2.1 Laboratory and Other Non-Depth Temperature Measurements. NormaUy,

temperature measurements may be made with any gcxxi mercury-fiUed Celsius
thermometer. As a minimum, the thermometer should have a scale marked for
every 0.1 °C, with markings etehed on the capiUary glass. The thermometer
should have a minimal thermal capacity to pemtitrapidequtiibration. PeriocUcaUy
check the thermometer against a precision thermometer certified by the National
Instimte of Standards and Technology (NIST, formerly National Bureau of
Standards)* that is used with its certificate and correction chart For field
operations use a thermometer having a metal case to prevent breakage.
2.2 Depth temperature required for Umnological smdies may be measured with a
reversing tiiermometer, thermophone, or thermistor. The thermistor is most
convertient and accurate; however, higher cost may preclude its use. CaUbrate any
temperature measurement devices with a NIST-certified thermometer before field
use. Thetiiermometercommonly used for depth measurements is of the reversing
type. It often is mounted on the sample collection apparatus so that a water
sample may be obtained sunultaneously. Correct readings of reversing
themK)meters for changes due to differences betweentemperatureat reversal and
temperature at time of reading.

3.0 Apparams
3.1 A mercury fiUed Celsius thermometer, reversing thermometer,tfiermophone,or
thermistor if deptiitemperattueare going to be done.

4.0 Prcx:edure
4.1 Make readings witiitiietiiermometeror device unmersed in water long enough to
permit complete equiUbration. Report results to tiie nearest 0.1 or 1.0°C,
depending on neeci

5.0 Calculation

305
 5.1 Results of non-depthtempCTaturesare direct.
5.2 If a reversing thennometer is useci, the correct values are obtained by:
AT = [ (T* -1) (T' + Vo)/K] X [ 1 -h (T' -t )(T' + Vo)/K] +L
where:
AT = correction to be added algebraicaUy to uncorrected reading,
T' = uncorrected reading at reversal,
t = temperature at which thermometer is read,
Vo = volume of smaU bulb end of capiUary up to 0°C graduation,
K = constant depending onrelativethermal expansion of mercury and glass (usual
value of K = 6100), and
L = caUbration cxnrection of thermometer depencUng on T.
6.0 Accuracy
6.1 Accuracy ciata are not avatiable at this time.
References
American PubUc Healtii Association. (1992). Standard Methods fortiieExamination of
Water and Wastewater. 18tii ed. Metiiod 2550. American Public Healtii Association,
American Water Works Asscx:iation, and Water Environment Federation, Washington
DC, 2-59.

306
TITLE: Turbidity

ANALYTE:
TurbicUty

INSTRUMENTATION: Turbidimeter

INTRODUCTION
The term turbid is appUed to waters containing suspended matter that interferes witii the
passage of Ught through the water or in which visual deptii is restticted. The turbidity may
be caused by a wide variety of suspended materials, which range in size fiom coUoicial to
coarse dispersions, depencUng upon the degree of turbulence. In lake or otiier waters
existing under relatively quiescent conditions, most of tiie turbidity wtil be due to coUoidal
and extremely fine dispersions. In rivers underflcxxlconctitions, most of the turbicUty wiU
be due to relatively coarse cUspersions.

TurbicUty may be caused by a wide variety of materials. In glacier-fed rivers and lakes
most of the turbicUty is due to coUoicial rcx:k particles prcxiuced by the grinding action of the
glacier. The beautiful blues and greens of the lakes andriversin Glacier National Park are
typical examples. As rivers descend from mountain areas onto the plains, they receive
contributions of turbicUty from farming and other operations that cUsturb the soti. Under
flcxxi conctitions, great amounts of topsoti are washed to receiving stteams. Much of this
material is inorganic in nature but considerable amounts of organic matter are included. As
the rivers progress toward the cx:ean, they pass through urban areas where domestic and
industrial wastewaters, treated or untreated, may be added. The domestic waste may add
great quantities of organic and some inorganic materials that contribute turbicUty. Certain
industrial wastes may add large amounts of organic substances and others inorgartic
substances that prcxiuce turbidity. Stteet washings contribute much inorganic and some
orgartic turbicUty. Organic materialsreachingriversserve as fcxxi for bacteria, and the
resulting bacterial growth and other nticroorganisms that feed upon the bacteria produce
adcUtional turbicUty. Inorganic nutrients such as nitrogen and phosphoms present in
wastewater cUscharges and agricultural mnoff stimulate the growth of algae, which also
contribute turbicUty. Highly eutrophic natural waters are green to brown from algae in
suspension.

From tiie above considerations, it is safe to say tiiat tiie materials causuig ttutidity may
range from purely inorgartic substances to tiiose that are largely cwganic in nature. This
cUsparity in the nature of the materials causing turbicUty makes it impossible to estabtish
hard and fast mles for its removal.

Turbidity is an important consideration in public water suppUes for tiuee major reasons.
AestiieticaUy, consumers of pubtic water suppUes expect and have arightto demand
turbidity-fiee water. Laymen are awaretiiatdomestic wastewater is highly ttui)id. Any
ttutidity in tiie drinking water is automatically associated witii possible wastewater
pollution and tiie healtii hazards occasioned by it. This fear has a sound basis historicaUy,
as anyone knows who is farrtiUar witii tiie waterbome epidemics tiiat formeriy plagued tiie
water works uidustty. Second, filtt-ation of water isrenderedmore difficult and costiy
when ttubidity uicreases. The use of slow sand filters has become unpractical ui most
areas because high nu-bidity shortens filter mns and increases cleanuig costs. Satisfactory
operation of rapid sand filters depends upon effective removal of mrbidity by chemical
coagulation before tiie water is admitted to tiie filters. Fatiure to do so results in short filter

307
mns and production of an inferior-quality water, urtiessfiltersof special constmction are
used. Finally, disinfection of public water suppUes is usuaUy accompUshed by means of
chlorine or ozone. To be effecrtive, there must be contaa between the agent and the
orgartisms that the cUsinfectant is to kiU. In waters with high turbicUtyresultingfrom
(jrganic matter, chentical disinfection is more expensive because some disuifectants (e.g.
chlorine)are consumed in the oxiciation of the organic matter. This demand must be
overcome in order for the cUsinfectant to achieve an effective concenttation. Further more,
nticroorgartisms adhering to irregularly shaped or porous matter can be protected from fuU
contact with a disinfectant For this and aestheticreasonsthe U.S. Environmental
Protection Agency has placed a lintit of 1 urtit of turbidity as the maximum amount
aUowable in public water suppUes.

TurbicUty measurements are of particular importance in thefieldof water supply. They

have Untited use in the field of domestic and industrial waste treatment. Knowledge of the
turbicUty variation inraw-watersuppUes is of prime importance to the environmental
engineer. The values are used in conjunction with other information to determine whether a
supply requires special treatment by chentical coagulation and ftiuation before it may be
used for a pubtic water supply. Many large cities, such as New York, Boston, and Seattie,
have upland or mountain suppUes whose turbicUties are so low that treatment other than
chlorination is not required
Water suppUes obtained fromriversusually require chenticalflocculationbecause of high
turbidity. TurbicUty measurements are used to determine the effectiveness of the treatment
prcxiuced with different chemicals and the dosages needed. Thus they aid in selection of
the most effective and economical chentical to use. Such information is necessary to design
faciUties for feeding the chemicals and fortiieu-storage. Turbidity measurements help to
gauge the amount of chenticals needed from day to day in the operation of treatment works.
This is particularly important on "flashy"riverswhere no unpoundment is provided.
Measurement of turbicUty in settied water prior to ftittation is useful ui conuolUng chemical
dosages so as to prevent excessive loading of rapid sand filters. FuiaUy, turbidity
measurements oftiiefilteredwater are needed to check on faultyfilteroperation .
The suspended-solids determination is usually employed ui wastetteattnentplants to
determine tiie effectiveness of suspended-soUds removal. The determination is slow and
time-consuming, and in plants employing chenticaltteattnent,changes in chemical dosages
have to be maderatiierfrequentiy.Turbidity measurements can be used to advantage,
because of tiie speed witii whichtiieycan be made, to gaintiienecessary information. By
tiieir use, chemical dosages can be adjusted to usetiieminimum amount of chemical whtie
prcxiucing a high-quality effluent
Turbidity is also a criterion used to expresstiieuophic state of namral waterbodies.
Nuuient rich waters produce algae whose mass often exceeds suspended morganic matter,
whereas nuttient poor waters often have outstanding clarity. The deptii of light penett^tion
can have a profound effect on species diversity and productivity of waterbodies.
Turijidity is determined as uiterference totiiepassage of Ught by matter in suspension.
Most laboratory metiiods eitiier measure residual Ughttiiatpassestiiorougha sample water
column directiy opposite tiie Ught source or measuretiieUghtreflectedfromthe sample at
rieht anglesfromtiielight source. Eitiier technique is adequate if standardizwi. Methods
usinc direct visual comparison with standards lack accuracy. There is also a low tech field
method, witii a very long history,tiiatestimates mrbidity. It requues lowenng a

308
standardized disk (Secchi disk) into water on a measured and marked tine, and noting tiie
depth at which the cUsk cUsappears and reappears.

1.0 Scope and AppUcation

1.1 This rnethod is applicable to drinking, surface, and saline waters in tiie range of
turbidity from 0 to 450 formazine turbidity units (FTU). Higher values may be
obtained with dtiution of the sample.
NOTE 1: Nephelomettic Turbidity Units (NTU) are considered comparable to tiic
reported Formazuie TurbicUty Units (FTU) and Jackson Turbidity Units (JTU).
1.2 There are several methcxis in detemtining turbicUty: one instrumental and two
visual. This manual covers a variation of tiie instmmental metiicxL The two
visual methcxis are by the Jackson cancUe turbidimeter and bottie standards.
1.2.1 The Jackson cancUe methcxi utiUzes a candle. A series of adcUtions is added
to a container in front of or on top of a cancUe. Readings are taken when the
Ught from the cancUe can no longer be seen.
1.2.2 Bottie standards uses comparisons between a bottie containing a standard
amount versus a bottie containing the sample. Comparisons are made by
transntittable Ught or visual perception.

2.0 Summary of Methcxi

2.1 The methcxi is based upon a comparison of the intensity of Ught scattered by the
sample under defined conctitions with the intensity of Ught scattered by a stanciard
reference suspension. The higher the intensity of scattered Ught, the higher the
turbicUty. A standard suspension of Formazine, prepared under closely defined
conctitions, is used to caUbrate the instrument
2.1.1 Formazine polymer is used as the turbicUty reference suspension for water
because it is morereproduciblethan other types of standards previously
used for turbicUty standards.
2.1.2 A commercially available polymer standard is also approved for use for
the National Interim Primary Drinking Water Regulations. This standard is
identified as AMCO-AEPA-1 available from Amco Standard International,
Inc.

3.0 Sample Handling and Preservation

3.1 Preservation of the sample is not practical; analysis should begin as scx)n as
possible. Refrigeration or icing to 4°C, to minimize microbiological
decomposition of sotids, is recommended

4.0 Interferences
4.1 The presence of floating debris and coarse sediments which settle out rapidly wiU
give low reacUngs. Finely divided air bubbles wiU affect the results in a positive
manner.
4 2 The presence of true color, tiiat is tiie color of water which is due to dissolved
substances which absorb tight, wiU cause ttutidities to be low, altiiough tiiis
effect is generally not significant witii finished waters.

5.0 Apparams

309
 5.1 The sample mbes to be used witii tiie avatiable uisttiiment must be of clear
colorless glass. They should be kept scmpulously clean, botii uiside and out and
discarded whentiieybecome scratehed or etehed. They must not be handled at aU
wheretiietightsttikestiiem,but should be provided witii sufficient extta lengtii
or witii a protective case, sotiiattiieymay be handled. Disposable mbes are
commerciaUy avatiable.
5.2 The Hach 2000/DR is in wide use and has been found to be reUable. However,
titis test can not be used for EPAreportingprocesses but may be used ui day to
day ui-plant operations. EPA usestiieNephelomettic metiiod #180.1.
6.0 Reagents

6.1 Turbidity-free water: Pass distilled watertiuougha 0.45|im pore size membrane
filter if such ftitered water shows a lower mrbiditytiiantiiedistiUed water.
6.2 Stock formazinettu-biditysuspension: Solution 1: Dissolve 1.00 g hydrazine
sulfate, (NH2)2H2S04, in distilled water and dilute to 100 mL ui a volumeuic
flask. Solution 2: Dissolve 10.00 g hexametiiylene-tett^mine ui distilled water and
dtiute to 100 mL in a volumetticflask.In a 100 mL volumeuicflask,mix 5.0 mL
Solution I with 5.0 mL
Solution 2. AUow to stand 24 hours at 25 ± 3*C,tiiendilute totiiemark and ntix.
6.3 Stanciard formazine turbidity suspension: The turbicUty of tiiis suspension is
defined as 4(X) FTU units. Dilute portions of the stanciard turbicUty suspension
with turbicUty-free water as required. CommerciaUy prepared stcx:k stanciards are
avatiable. The Hach DR/2000 has intemal caUbrations so preparation of stanciards
is not needed.
6.3.1 A new stock turbicUty suspension should be prepared each month. The
stanciard turbicUty suspension and dtiute turbicUty standarcis should be
prepared weekly by dilution of the stcx^k turbicUty suspension.
7.0 Prcx^edure

7.1 Turbidimeter caUbration: The Hach 2000/DR is available with the caUbration
automaticaUy done. The use of the above standarcis can be used to vaUdate the
intemal caUbration.
7.2 The Hach Turbidimeter has an acceptable range between 0 and 450 FTU. If
samples are higher than this, cUlution will have to be undertaken.
7.3 OntiieHACH 2000/DR select Metiiod 750.
7.4 Rotate the wavelength to 450 nm. Press Read/Enter.
7.5 Pour 25 nti of deionized water into the sample ceU. Place the blank into the seU
holder and press Zero. The screen wtil report 0. FTU TURBIDITY.
7.6 Pour 25 ml of sample into another ceU. ImmecUately place in the ceU holder.
Press Read/Enter.
7.7 The screen wiU showtiieresults in FTU units. NOTE: Picture insttiictions are
provided.
8.0 Calculation
8.1 Multiply samplereadingsby appropriate dilution to obtainfinalreading.
«,2 Results are obtauied du-ectly from HACH meter.

310
9.0 Accuracy

9.1 In a single laboratory, usuig a standard solution of 140 FTU and one
representative lot reagent witiitiieDR/2000, a single operator obtained a standard
deviation of± 2 FTU.
References
Hach Company. (1992). DR/2000 Specuophotometer Handbook. Metiiod 8237. Hach
Company, Loveland, Co.
Sawyer, C. N., and McCarty, P. L. (1994). Chemistt^^ for Envu-onmental Engineering.
4tii ed. McGraw-HiU, Inc., New York, N.Y., 439.
Urtited States Environmental Protection Agency. (1992). Methcxis for Chentical Analvsis
of Water and Wastes. Methcxi #180.1. Environmental Monitoring and Support
Laboratory, United States Environmental Protection Agency, Cincinnati, Ohio.

311
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