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50272-1QI
REPORT DOCUMENTATION 1,. REPORT NO.
PAGE EPA 560/6-82-003
4. Title and Subtitle
Chemical Fate Test Guidelines
7. Author(s)
9. Performing Organization Name and Address
Office of Pesticides and Toxic Substances
Office of Toxic Substances (TS-792)
United States Environmental Protection Agency
401 M Street, S.W.
Washington r D,C 20460
12. Sponsoring Organization Name and Address
3. Recipient's Accession No
PB82-233008
5. Report Date
[ August, 1982
6.
I .
8. Performing Organization Rept. No.
t - .- -.___. .. ..
j 10. Proiect/Task/Work Unit No.
I --.._.
11. Contract(C) or Grant(G) No.
(C)
(G)
; 13. Type of Report & Period Covered
i
I Annual Report
i
14.
15. Supplementary Notes
16. Abstract (Limit: 200 words)
These documents constitute a set of 21 chemical fate test guidelines (and, in
some cases, support documents) that may be cited as methodologies to be used
in chemical specific test rules promulgated under Section 4(a) of the Toxic
Substances Control Act (TSCA). These guidelines cover testing for physical
and chemical properties, transport processes and transformation processes.
The guidelines will be published in loose leaf form and updates will be made
available as changes are dictated by experience and/or advances in the state-
of-the-art .
17. Document Analysis a. Descriptors
b. Identififcrs/Open-Ended Terms
c. COSATI Field/Group
18. Availability Statement
Release unlimited
19. Security Class (This Report)
T In f.1 a ss i f ied
20. Security Class (This Page)
Unclassified
21. No of Pages
425
22. Price
(See ANSI-Z39.18)
See Instructions on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS-35)
Deoartment of Commerce
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PREAMBLE
The following guidelines describe methods for performing testing
of chemical substances under the Toxic Substances Control Act
(TSCA). These methods include the state-of-the-art for
evaluating certain properties, processes and effects of
chemical substances. They are intended to provide guidance
to test sponsors in developing test protocols for compliance
with test rules issued under Section 4 of the TSCA. They
may also provide guidance for testing which is unrelated
to regulatory requirements. Support documentation is
included for some of these guidelines. It is expected that
additional guidelines and support documentation will be
incorporated later as the state-of-the-art evolves or the
need for them warrants.
Since these guidelines are divided into three sections which
cover the diverse areas of health effects, environmental
effects and chemical fate testing, there are some differences
in the ways they are presented. These differences are
explained in an introduction prepared for each section.
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INTRODUCTION TO CHEMICAL FATR TRSTIMG
In these guidelines, methods have been categorized under the
general headings (1) physical and chemical properties,
(2) transport orocesses and, (3) transformation orocesses. These
categories are arbitrary and are only a convenient classification.
As complex microcosms are developed and validated for use in fate
studies, it may be appropriate to add a fourth category for them
since they often include the simultaneous evaluation of various
transport and transformation processes. The categorization and
numbering of the chemical fate guidelines allow for future
supplementations with methods for additional parameters (e.g. gas
phase photolysis) or other approaches to parameters already
addressed .
The environmental impact of a chemical substance depends on the
environment into which it is released, the concentration of the
chemical, the duration and nature of any exposure, and its
toxicity to organisms at risk or its effects on abiotic
structures or processes. Potential adverse effects on
populations or inanimate receptors at risk are highly dependent
upon the environmental fate of the chemical substance, where fate
is defined as the disposition of the substance resulting from
transport and transformation processes.
An assessment of the fate of a chemical released to the
environment will depend, in part, unon laboratory data used to
evaluate properties and processes which influence transport and
transformations. Test procedures identified in these guidelines
are the first in a series that will be needed to develop reliable
-------
and adequate data on the physical, chemical and environmental
persistence characteristics of chemical substances or mixtures.
Many of the fate guidelines contain complete descriptions of
appropriate laboratory procedures. For some of the guidelines,
it is believed that readily citable and widely available methods
described by the Organization for Economic Cooperation and
Development (OECD), the American Society for Testing and
Materials (ASTM) and others provide adequate examples, and in
those guidelines the reader is directed to appropriate sources.
Efforts are underway to develop and validate additional methods
of varying levels of complexity. This includes the development
of estimation techniques for many of the chemical and physical
characteristics to guide in the selection of test methods or to
preclude the necessity for certain laboratory testing. Also
under development are complex testing methods designed to provide
kinetic (rate) data and information on likely transformation
products in simulations of selected environments. Between these
two extremes, in method complexity, there is a need for
appropriate methods for additional properties and processes and
for improvements in existing guidelines. For example, it is
expected that methods for determining gas phase absorption
spectra, volatilization rates from soil and water, and gas phase
photolysis will be available soon. When their development and
validation have been completed, they will be added to these
guidelines. It is also anticipated that individual methods
described in these guidelines will be revised, discarded or
replaced, when appropriate.
11
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CHEMICAL FATE TESTING GUIDELINES
TABLE OF CONTENDS
Guideline Title
PHYSICAL AND CHEMICAL PROPERTIES
Absorption in Aqueous Solution,
ultraviolet/visible spectra
Roilinq Temperature
Density/Relative Density
Dissociation Constants in Water
Henry's Law Constant
Melting Temperature
Particle Size Distribution/Fiber Length and
Diameter Distributions
Partition Coefficient (n-Octanol/Water)
pH of Water Solution or Suspension
Water Solubility
Vapor Pressure
TRANSPORT PROCESSES
Soil 'Ti in-Layer Chromatoqranhv
Sediment and Soil Adsorption Isotherm
TRANSFORMATION! PROCESSES
Riodeqradation, Aerobic Aquatic
Riodeqradation, Ready
Biodeqradation, Anaerobic
Biodeqradation in Soil
Riodeqradation, Sewaqe ^reatment Simulations
Complex Formation Ability in Water
Hydrolysis as a ^mction of rH at 25 °C
Photolysis in Aqueous Solution in Sunlight
iii
Guideline
No.
Sunnort Document
No.
CG-1050
CG-iion
CG-1150
CG-1200
CG-1250
CG-1300
CG-1350
CG-1400
CG-1450
CG-ISOO
CG-1600
CS-1400
^S-1500
CS-1600
OG-i7nn
CG-1710
CG-2000
OG-2010
CG-2050
CG-2075
CG-2100
CG-4000
CG-5000
CG-6000
CS-1700
CS-1710
CS-2000
CS-2050
CS-SOOO
CS-6000
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PHYSICAL AND CHEMICAL PROPERTIES
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CG-1050
August, 1982
ABSORPTION IN AQUEOUS SOLUTION:
ULTRAVIOLET/VISIBLE SPECTRA
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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00-1050
ABSORPTION IN AQUEOUS SOLUTION;
ULTRAVIOLET/VISIBLE SPECTRA
I. PURPORT
This Test Guideline references methodology to develop ultraviolet
and visible absorption spectra of a chemical in aqueous
solution. ^he data may be used to evaluate the potential ^or
sunlight photochemical transformation in aqueous media.
IT. ^EST PROCEDURES
Examples of methods for determining the absorption spectra of
chemicals in solution are given in OECD Guideline No. 101,
(OECD), "UV-VIS Absorption Spectra" and in the U.S. EPA
Discussion of premanufacture testing (USEPA 1979). The U.S.
sales agent for the OECD guidelines is OECD Publications and
Information Center, Suite 1207, 2750 Pennsylvania Ave. NW,
Washington, DC 20006.
III. REFERENCE
OECD. 1981. Organization for Economic Cooperation and
Development. OECD Guidelines for Testing of Chemicals.
HSEPA. 1979. U.S. Environmental Protection Agency. Office
Toxic Substances. Toxic Substances Control: Discussion of
premanufacture testing policv and technical issues; request
comment. (44 FR 16267-8).
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CG-1100
August, 1982
BOILING TEMPERATURE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-1100
BOILING TEMPERATURE
I . PURPOSE
This Test Guideline references methodology to develop data on the
equilibrium boiling temperature of chemical substances and
mixtures at environmentally relevant pressures. The data may be
used to characterize the physical state of the material, to
evaluate the manner and extent that the chemical will be
transported in the environment, and as a guide in the selection
and design of other tests.
II. TEST PROCEDURES
Examples of methods for determining the boiling temperature or
boiling point range of chemical substances or mixtures are listed
in Table 1, 'lrnest Procedures for Equilibrium Boiling ^emperature"
and in OECD Guideline Mo. 103, (OECD), "Boiling Point/Boiling
Range." The codes to standardized bodies listed in Table 1 are:
ANSI American National Standards Institute
ASTM American Society for Besting and Materials
BSI British Standards Institution
CIPAC Collaborative International Desticides
Analytical Council
DIN Das 1st Norm (earlier Deutsche Tndustrienormen)
IP Institute of Petroleum
ISO International Organization for Standardization
USP United States Pharmacopeia XVIII
These Test Guidelines are available for purchase as follows:
(1) ANSI, BSI, DIN, and ISO standards are available from:
Sales Department, American National Standards Institute,
1430 Broadway, New York, NY. 10018.
(2) ASTM standards are available from: American Society for
Testing and Materials, 1916 Race St., Philadelphia, PA.
(3) CIPAC standards are available from: National Agricultural
Chemicals Association, 1155 Fifteenth Street, NW,
Washington, DC 20005.
-1-
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CG-1100
(4) IP standards are available from: Hayden and Son Ltd.,
Spectrum House, Alderton Cres., London NW4 3XX UK.
(5) OECD methods are available from: OECD Publications
and Information Center, Suite 1207, 2750 Pennsylvania
Ave. NW, Washington, DC 20006.
(6) USP standards are available from: U.S. Pharmacopeia,
Bethesda, MD 20014.
-2-
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CG-1100
Table 1. Test Procedures for Equilibrium Roiling temperature
Instrumental
Technique
Identification
Standard Method
or Other
Description
Applicability
Pure Pure Liquid
Liquid Solids Mixtures
& Impure
Liquids
Boiling temperature of liquid (Ebulliometric)
Cottrell distillation ASTM D 1088a
Ramsay/Young Ref. A pp. 56, 57
Immersed thermometer ASTM D 1120C
Osborn/Douslin
Herington/Martin/
Ambrose
Swietoslawski.
Ref. Bb pp. 222-225
Ref. Ab pp. 66-69
Ref. Ab pp. 66-69
Ref. Bb pp. 225-227
Ref. Db
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Condensation temperature of vapor (Ebulliometric)
Distillation flask
or Claissori flask
ASTM D 86/E 133d
ASTM D 1078/E 133
ASTM D 1160
Ref. Ab pp. 57-59
Ref. Bb pp. 218-221
Ref. Cb pp. 984,1000-
1003
X
X
X
X
X
X
X
X
X
X
X
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CG-1100
Hoover/John/Mellon Ref. Ab pp. 54-56 X X
Hickman/Weyarts Ref. Ab pp. 59, 71-74 X X
Fenske Ref. Ab p. 59 X X
Willingham et al./ Ref. Ab p. 60 X X
Stull
Swietoslawski Ref. Rb p. 228-230 X X
Fractionating column ASTM D 285 X X
ASTM D 2892 X X
USP I or II X X
ISO R 918e X X
Pressure equilibration (Static methods)
Isoteniscope ASTM n 2879 X X
Ref. Ab pp. 49-50 X X
Submerged bulb or
capillary
Sowoloboff/
Rosenblum Ref. Ab pp. 79-81 X
Smith/Menzies Ref. Ab pp. 79-81 X X
Garcia Ref. Ab pp. 79-83 X
Ref. Db X X
a Equivalent standard: ANSI 1088
b Reference A, Thomson, Douslin (1971); Reference R, Anderson (1971);
Reference C, Raw et al. 1970. Reference D, OECD 1979a,b
c Equivalent standard: ANSI 1120
d Equivalent standards: ANSI D86/E133, IP 123, BS 4349, DIN
51751, CIPAC MT70
e Equivalent standards: BS 4591. DIN 53171
-4-
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CG-1100
III. REFEREMCES
ANSI. (Latest Edition). American National Standards
Institute. Rook of Standards.
ASTM. (Latest Edition). American Society for Testing and
Materials. Annual Book of ASTM standards.
RSI. (Latest Edition). British Standards Institutute. Rook of
Standards.
CIPAC. 1970. Collaborative International Pesticides Analytical
Council. CIPAC Handbook, Volume 1. Analysis of Technical and
Formulated Pesticides.
DIN. (Latest Edition). Das 1st Norm. Rook of Standards.
IP. (Latest Edition). Institute of Petroleum. Rook of
Standards.
ISO. (Latest Edition). International Organization for
Standardization.
OECn. 1981. Organization for Economic Cooperation and
development. OECD Guidelines for Testing Chemicals.
USP. 1970. Pharmacopeia of the United States of America.
Eighteenth Revision.
-5-
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CG-1150
August, 1982
DENSITY/RELATIVE DENSITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-1150
DENSITY/RELATIVE DENSITY
I. INTRODUCTION
This Test Guideline references methodology to develop data
on density and relative density (specific gravity) of chemical
substances and mixtures. The data may be used to evaluate the
manner and extent that chemicals will be transported in the
environment and the places they will be deposited.
II. TEST PROCEDURES
Examples of methods for determining density and relative
density of gaseous, liquid, or solid chemical substances are
listed in Table 1, "Standard Density - Measurement Techniques
Referenced in this Test Guideline" and in OECD Guideline No. 109,
(OECD 1981), "Density of Liquids and Solids." The codes to
standardizing bodies listed in Table 1 are:
ANSI - American National Standards Institute.
ASTM - American Society for Testing and Materials.
BSI - British Standards Institution.
IP - Institute of Petroleum.
CIPAC - Collaborative International Pesticides Analytical
Counci1.
DIN - Das 1st Norm (earlier Deutsche Industrienormen).
API - American Petroleum Institute.
ISO - International Organization for Standardization.
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CG-1150
These Test Guidelines are available for purchase as follows:
(1) ANSI, BSI, ISO, and DIN standards are available from:
Sales Department, American National Standards Institute,
1430 Broadway, New York, NY 10018.
(2) ASTM standards are available from: American Society
for Testing and Materials, 1916 Race Street,
Philadelphia PA 19103.
(3) API methods are available from: American Petroleum
Institute 2101 L Street NW., Washington, DC 20037.
(4) IP methods are available from: Hayden and Son Ltd.,
Spectrum House, Alderton, Cres., London NW4 3XX U.K.
(5) CIPAC methods are available from: National Agricultural
Chemicals Association, 1155 Fifteenth Street, NW.,
Washington, DC 20005.
(6) OECD methods are available from: OECD Publications and
Information Center, Suite 1207, 2750 Pennsylvania Ave. NW,
Washington, DC 20006.
-2-
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CG-1150
Table 1
Standard Density-Measurement Techniques Referenced in this Standard
Cla
Technique Gas
ideal gas
calculation x
,gas density
balance X
'hydrometer
i i
i
hydrostatic
displacement
sink-float
comparator
pycnometer-
nar row-mouth
pycnometer-
wide-mouth
Sprengel-
Ostwald
Lipkin
bicapillaryi
Bingham
volumetric
flask :
thermometer
stoppered
t
capillary
stopper
i
Johnson and
Adams
i
1
gas comparison
pycnometer i
ss of Substance
1 Liq Solid
i |
1
I
i X
i i
i X chunks j
i
i chunks
> Standardizing Body and Identification Number
ANSI
Z77.12
Z11.84
Z11.147
D 891
K65.8
C 830
D 891
C 729
: X
i j
1 x
Z11.62
Z11.120
D 3505
i D 891
] X D 1217
: zii.119
powder ; D 153
I . icrystals1'
j X jchunks D 1076
(powder C 135
X Icrystals
ichunks K65.8
[powder
, crystals1
x
Ichunks
!
D 1076
D 153
D 1817
K65.8
D 891
| ipowder
i x crystals
J chunks ; ,
i
: powder
C 604
ASTM BSI CIPAC DTN ISO
;
D 1070(26)*
D 1298(23, 40)b 4714 MT3 ! 51757 R387
D 1657(23) 12791
D 891 (29) i -2,3 R649
D 792(35) 53*79 R1183
C 830(17) ;
D 891 (29) ! i
C 693(17)
C 729(17)
[
D 941 (23)C 4699 MT3 , 51757
D 1481 (23,40)
D 3505(29) ! 12798
D 891 (29) 12807
D 1217(23)
D 1480(23) :
D 153(28) ;
D 1076(37)
C 135(17) j
D 792(35)
D 1076(37) ! i
D 153(28) i
D 1817(37) ! 5093 MT3 12797
D 792(35) 12809
D 891 (29)
r * ,
C 604(17)
i | ]
1 ;
In parentheses by ASTM Test Standard number is the volume number in which the standard
appeared in the 1978 Annual Book of ASTM Standards.
b Adopted by American Petroleum Institute as API Standard No. 2547 and by the Institute of
Petroleum as IP Standard No. 160.
Adopted by the General Services Administration as Method 402, Federal Test Method Standard
791b.
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CG-1150
III. REFERENCES
ANSI. (Latest Edition). American National Standards
Institute. Book of Standards.
ASTM. (Latest Edition). American Society for Testing and
Materials. Annual Book of ASTM Standards.
API. (Latest Edition). American Petroleum Institute. Book of
Test Standards.
BSI. (Latest Edition). British Standards Institution. Book of
Standards.
CIPAC. 1970. Collaborative International Pesticides Council.
CIPAC Handbook, Volume 1. Analysis of Technical and Formulated
Pesticides.
DIN. (Latest Edition). Das 1st Norm. Book of Standards.
IP. (Latest Edition). Institute of Petroleum. Book of
Standards.
ISO. (Latest Edition). International Organization for
Standards. Book of Standards.
OECD. 1981. Organization of Economic Cooperation and
Development. OECD Guidelines for Testing of Chemicals.
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CG-1200
August, 1982
DISSOCIATION CONSTANTS IN WATER
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-1200
DISSOCIATION CONSTANTS IN WATER
I.
PURPOSE
This Test Guideline references methodology to develop data on
acid dissociation constants of chemical substances that are acids
or bases. The data may be used to evaluate the transport of a
substance in the environment, the kinds of reactions the
substance will undergo, the effects of pH on those reactions, the
probable sites and modes of action of the substance in humans and
the environment, and as a guide in the design of other tests for
physical and chemical properties and for effects on human health
and the environment.
II.
TEST PROCEDURES
Examples of methods for determining dissociation constants are
cited by Serjeant and Dempsey (1979), Perrin (1965, 1969), Kortum
Vogel and Andrussow (1961), and in OECD Guideline No. 112,
(OECD), "Dissociation Constants in Water." The U.S. sales
agent for this OECD Guideline is OECD Publications and
Information Center, Suite 1207, 1750 Pennsylvania Ave. NW,
Washington, DC 20006.
Ill
REFERENCES
Kortum G, Vogel W, Andrussow K. 1961.
for Organic Acids in Aqueous Solution.
Dissociation Constants
London: Rutterworths.
Kortum G, Vogel W, Andrussow K. 1960. Dissociation constants of
organic acids in agueous solution. Pure Appl Chem No. 2, 3.
186-536.
OECD. 1981.
Development.
Organization for Economic Cooperation and
OECD Guidelines for Testing of Chemicals.
Perrin DD. 1965.
Agueous Solution.
Dissociation Constants
London: Butterworths.
of Organic Bases in
Perrin DD. 1969. Dissociation Constants of Inorganic Acids in
Bases in Agueous Solution. London: Butterworths. Also, Pure
Appl Chem 20, No. 2.
Serjeant EP, Dempsey B. 1979. lonization Constants of Organic
Acids in Aqueous Solution. IUPAC Data Series No. 23. Oxford:
Pergamon Press.
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CG-1250
August, 1982
HENRY'S LAW CONSTANT
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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HENRY'S LAW
I. PURPOSE
This Test ^uideline references methodoloqv to develop data on
Henry's law constant of chemical substances. This data mav be
used to evaluate the potential for volatilization from water and
is essential in determininq rates of transfer from water.
IT. TEST PROCEDURES
An example of a method for determininq Henrv's law constant
described bv MacKay, Shiu, and Sutherland (1979).
III. REFERENCE
Mackav D, Shiu WY, and Sutherland RP. 1979. Determination of
air-water Henry's law constant for hvdronhobic pollutants.
Environ Sci Technol 11:333-337.
_ I
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CG-1300
August, 1982
MELTING TEMPERATURE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-1300
MELTING TEMPERATURE
I. INTRODUCTION
This Test Guideline references methodology to develop data
on the melting temperature or melting temperature range of
chemical substances and mixtures. The data may be used to assess
the potential for movement of materials in the environment, to
determine the physical state of the substance under environmental
conditions and to evaluate possible health and environmental
effects.
II. TEST PROCEDURES
Examples of methods for determining the melting temperature
or melting temperature range of chemical substances or mixtures
are listed in Table 1, "Standard Temperature Technique Referenced
in this Test Guideline" and in OECD Guideline No. 102, (OECD
1981), "Melting Point/Melting Range." The codes to standardized
bodies listed in Table 1 are:
ANSI - American National Standards Institute.
ASTM - American Society for Testing and Materials.
TAPPI - Technical Association for the ^ulp and Paper
Industry.
FTS - Federal Test Standards.
BSI - British Standards Institution.
IP - Institute of Petroleum.
CIPAC - Collaborative International Pesticides Analytical
Council.
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CG-1300
These Test Guidelines are available for purchase as follows:
(1) ANSI and BSI standards are available from: Sales
Department, American National Standards Institute,
1430 Broadway, New York, NY 10018.
(2) ASTM standards are available from: Testing and
Materials 1916 Race St., Philadelphia PA 19103.
(3) CIPAC standards are available from: National
Agricultural Chemicals Association, 1155 Fifteenth
Street, NW., Washington, DC 20005.
(4) FTS standards are identical to the corresponding ASTM
standards and should be obtained from the same source as
ASTM standards.
(5) IP standards are available from: Havden and Son Ltd.,
Spectrum House, Alderton Cres., London NW4 3XX U.K.
(6) OECD methods are available from: OECD Publications and
Information Center, Suite 1205, 2750 Pennsylvania Ave. NW,
Washington, DC 20006.
(7) TAPPI standards are available from: TAPPI Press,
Technical Association of the Pulp and Paper Industry.
One Dunwoody Park, Atlanta GA 30338.
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I
OJ
I
TABLE 1-STANDARD MELTING TEMPERATURE TECHNIQUES
REFERENCED IN THIS STANDARD
TECHNIQUE
Thiele Tube
Cooling Curve
Fisher-Johns
Microscope Hot Stage
Kofler Hot Bench
Drop Melting Point
Pour Point
Congealling Point
STANDARDIZING BODY1
ANSI/ASTM2'3
E 324-79 [30]
D 1519-685 (1974) [37]
D 87-77 [23] [20]
D 789-78a [36]
D 1457-78 [36]
D 21 16-79 [36]
D 2133-78 [36]
02117-64(1978) [35]
D 3451 -76 [27]
0127-63(1977) [23] [20]
097-66(1978) [23] [40]
D 938-71 (1976) [23]
TAPPI
T630-OS-71
T634-OS-70
FST4
7918*1402.5
791BH1401.4
791BK201.9
BSI
4695
5090
4452
5088
IP
55
133
15
76
CIPAC
MT2
MT1
1 Names of standardizing bodies are given in text.
2joint standard unless otherwise specified.
3|n brackets by ASTM Test Standard number is the volume number in which the standard appeared in the 1979 or 1980 Annual Book of ASTM
Standards. Numbers in parentheses indicate the year of last reapproval.
Identical to ANSI/ASTM standard.
5ASTM only.
n
o
i
M
U>
O
O
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CG-1300
III. REFERENCES
ANSI. (Latest Edition). American National Standards
Institute. Book of Standards.
ASTM. (Latest Edition). American Society for Testing and
Materials. Annual Book of ASTM Standard.
BSI. (Latest Edition). British Standards Institution. Book of
British Standards.
CIPAC. 1970. Collaborative International Pesticides Analytical
Council. CIPAC Handbook, Volume 1. Analysis of Technical and
Formulated Pesticides.
FTS. (Latest Edition). Federal Test Standards. Book of
Standards.
IP. (Latest Edition). Institute of Petroleum. Book of
Standards.
OECD. 1981. Organization for Economic Cooperation and
Development. OECD Guideline for Testing of Chemicals.
TAPPI. 1971. Technical Association of the Pulp and Paper
Industry. Book of Standards.
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CG-1350
August, 1982
PARTICLE SIZE DISTRIBUTION/FIBER LENGTH AND DIAMETER
DISTRIBUTIONS
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
CG-1350
PARTICLE SIZE DISTRIBUTION/FIBER LENGTH AND DIAMETER DISTRIBUTIONS
I. PURPOSE
This Test Guideline references methodology to (1) develop data on
the effective hydrodynamic radius or effective Stokes' radius
(Rs) and/or, (2) provide histograms of the length and diameter
distributions of fibers. The data may be used to evaluate the
transportation and sedimentation of insoluble particles in water
and air. In the special case of materials which can form fibers,
an additional set of measurements is also recommended to help
identify potential health hazards arising from inhalation or
ingest ion.
II. TEST PROCEDURES
Appropriate methods are described in OECD Guideline No. 110,
(OECD), "Particle Size Distribution/Fiber Length and Diameter
Distributions". The U.S. sales agent for the OECD guidelines is
OECD Publications and Information Center, Suite, 1207,
L750 Pennsylvania Ave. NW. Washington DC 20006.
III. REFERENCES
OECD. 1981. Organization for Economic Cooperation and
Development. OECD Guidelines for Testing of Chemicals.
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August, 1982
PARTITION COEFFICIENT (n-OCTANOL/WATER)
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Table of Contents
PAGE
I. INTRODUCTION 1
A. Background and Purpose 1
B. Definitions and Units 2
C. Principle of the Test Method 3
D. Applicability and Specificity 3
11. TEST PROCEDURES 4
A. Test Conditions 4
1. Special Laboratory Equipment 4
2. Temperature Control 4
3. Solvents 5
4. Concentration of Solute 5
5 . Equilibration Time 5
6. Octanol/Water Volume Ratio 5
7. Chemical Analysis of the Octanol and Water Phases 6
8. Emulsification and Ultracentrifugation 6
9. Equilibration Vessel 6
10. Speciation Effects 7
11. Prerinsing of all Transfer Vessels 8
B. Preparations,
1. Reagents and Solutions 8
a. Octanol and Water 8
b. Buffer Solutions 9
c . Presentation of the Solvents 9
d. Preparation of Test Solution 10
C. Performance of the Test 10
III. DATA AND REPORTING 14
A. Test Report 14
B. Specific Analytical and Recovery Procedures 14
C. Other Test Conditions 14
IV. REFERENCES 15
V. APPENDIX 1: DATA FORMAT SHEETS A-1
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PARTITION! COEFFICIENT (n-OCTANOL/WATER)
I. INTRODUCTION
A. Background and Purpose
Bioconcentration, the accumulation of a substance in living
tissues or other organic matter as a result of net chemical uptake
from the medium (e.g., water), is a factor in determining the
movement of a chemical in the environment and the potential
effects of the chemical on biota. Hydrophobic chemicals that are
present in the aqueous environment at subtoxic concentrations may
accumulate to toxic levels once inside organisms, presumably
through diffusion into nonpolar cell components, where they
accumulate because of their greater solubility. Further movement
of the substance in living tissues may occur as a result of
ingestion of lower trophic level organisms, i.e., food chain
effects.
The tendency of an organic chemical to bioconcentrate in
living cells can be inferred from the value of the octanol/water
partition coefficient, K (Neely et al. 1974). Chemicals with
K less than 10 will not significantly partition into, or tend to
accumulate in, living cells. Chemicals with KQW greater than 106
will tend to accumulate. Chemicals that exist in the environment
at subtoxic levels may bioconcentrate to toxic levels once inside
organisms.
This test guideline describes a detailed and commonly used
procedure for determining the octanol/water partition coefficient
of organic chemicals.
1
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B . Definitions and Units
The octanol/water partition coefficient (KQW) is defined as
the equilibrium ratio of the molar concentrations of a chemical in
n-octanol and water, in dilute solution. K-.. is a constant for a
\J W
given chemical at a given temperature. Since KQW is the ratio of
two molar concentrations, it is a dimensionless quantity. Some-
times K-... is reported as log 10 K^,,.
Cj W \J W
The mathematical statement of K-.. is-
(J W
K octanol
ow C .
water
where C is the molar concentration of the solute in n-octanol and
water at equilibrium at a given temperature.
According to Nernst (1891) the distribution law applies only
to individual molecular species in solution. If a molecule
dissociates or associates in octanol and water, then equation (1)
must be modified. In general, if a represents the fraction of the
total solute that is dissociated or associated, assuming that
either association or dissociation occurs in each solvent, then
oct. oct.
t\
ow (1 - a ^ ) C ^
water water
since (1-a) gives the fraction of unchanged molecules in each
phase. For the special case where no association takes place in
octanol, equation (2) reduces to
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K oct-
__
ow (1 - a water) C .
water
where a water represents the fraction of the total solute that has
dissociated in water.
C. Principle of the Test Method
The conventional method for determining the octanol/water
partition coefficient is carried out by distributing a chemical
between n-octanol and water in a vessel at constant temperature
and measuring the concentration in the two liquid phases after
equilibration. Numerous researchers use the conventional method
for determining K and have published papers using this method
O Wr
(e.g., Fujita et al. 1964; Hansch and Anderson 1967; Leo et al.
1971; Chiou et al. 1977).
D. Applicability and Specificity
The test guideline is designed to determine the octanol/water
partition coefficient of solid or liquid organic chemicals in the
range 10 to 10 . For chemicals whose values lie outside this
range, K should be characterized as less than 10 or greater than
O\n ~~"
10 with no further quantification.
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II. TEST PROCEDURES
A. Test Conditions
1. Special Laboratory Equipment
(1) A thermostatic bath, chamber, or room with a shaker and
temperature control as specified in Temperature Control
below;
(2) an ultracentrifuge with temperature control as specified
in Temperature Control below;
(3) stainless steel or glass centrifuge tubes with sealable
caps. Special glass centrifuge tubes can be used up to
approximately 12,0000 G and stainless steel tubes can be
used at high G values;
(4) a mechanical shaker; and
(5) a pH meter capable of resolving differences of 0.1 pH
unit or less.
2. Temperature Control
It is recommended that the temperature of the water bath, or
chamber, or room, and the ultracentrifuge be controlled to
(25 + 1)°C.
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3. Solvents
It is extremely important that n-octanol, purified as
described in Section II.B.I.a, and distilled or reagent grade
water, i.e., ASTM Type II water or an equivalent grade, be used
ASTM Type II water is described in ASTM D-1193-77, "Standard
Specification for Reagent Water."
4. Concentration of Solute
It is extremely important that all experiments be carried out
at solute concentration C < 0.01M (Molar) in octanol and water and
well below the solubility in either phase.
5. Equilibration Time
In general, 1 hour of gentle agitation is sufficient to reach
equilibrium. For surfactants, at least 16 hours is required to
reach equilibrium.
6. Octanol/Water Volume Ratio
It is recommended that the ratio of the volumes of the two
liquids be adjusted as appropriate for the relative solubility of
the chemical in octanol and water. By adjusting the volumes,
concentration errors (resulting from analytical errors) are
minimized and errors resulting from dividing large numbers by
small numbers are kept to a minimum.
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7. Chemical Analysis of the Octanol and Water Phases
In determining the KQW value for any given solute, it is
important that both the octanol and water phases be analyzed for
the chemical. An analytical method should be selected that is
most applicable to the analysis of the specific chemical.
Chromatographic methods are preferable because of their compound
specificity in analyzing the parent chemical without interference
from impurities. Whenever practicable, the chosen analytical
method should have a precision with +_ 5 percent.
8. Emulsification and Ultracentrifugation
It is important that gentle shaking be used to minimize the
formation of emulsions. Ultracentrifugation is necessary to
separate troublesome emulsions and to separate the octanol and
water phases. Therefore, it is very important that
Ultracentrifugation be carried out at 25°C for 20 minutes in a
temperature controlled ultracentrifuge. The acceleration (G)
value required to break the emulsion and to achieve complete
separation of the octanol and water phases can be determined by
trial-and-error experimentation.
9. Equilibration Vessel
If feasible, equilibration should be carried out in a
centrifuge tube (stainless steel or glass) with a scalable cap.
It is important that the centrifuge tubes be almost completely
full. In this way, partitioning with air will be minimized,
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especially for volatile chemicals, and the mixture will be
completely mixed.
Very hydrophobic chemicals, with KQW in the order of 10 to
10 , require relatively large volumes of the aqueous phase.
Hence, for these chemicals, it is recommended that equilibration
be carried out in a large ground-glass stoppered flask.
10 . Speciation Effects
The octanol/water partition coefficient, K_,_, has been
w W
defined in Section I.E. The mathematical statement of K is
O W
given by equation (1).
If the chemical does not associate or dissociate in octanol
and water, then use equation (1) and determine KQW at molar
concentrations C < 0.01M and C1 = 0.01C.
If the chemical associates in octanol or water or in both
liquids, then use equation (1) and determine KQW at molar
concentrations C < 0.01M, C^ = 0.1C, C2 = 0.01C, C3 =
0.001C When KQW is constant at two molar concentrations
differing by a factor of 10, then the effect of association has
been minimized or eliminated.
If a molecule dissociates or associates in octanol and water,
then it is extremely important that equation (1) be modified to
take into account such speciation changes as ionization,
aggregation, and hydration. For the special case, where no
association takes place in octanol and only dissociation takes
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place in water, equation (3) can be used. For chemicals that
reversibly ionize or pronate (e.g., carboxylic acids, phenols, or
anilines), use equation (3) with water buffered at pH 5.0, 7.0,
and 9.0. It is recommended that buffers described in Section
II.B.l.b be used.
11. Prerinsing of all Transfer Vessels
It is important that all transfer vessels be prerinsed with a
portion of the equilibrium phase prior to transfer for analysis.
This is especially important for very hydrophobic chemicals.
B. Preparations
1. Reagents and Solutions
a. Octanol and Water
Very pure jv-octanol can be obtained as follows: wash pure _n_-
octanol (minimum 98 percent pure) sequentially with 0.1N 112804,
with 0.1N NaOH, then with distilled water until neutral. Dry the
ji-octanol with magnesium sulfate and distill twice in a good
distillation column under reduced pressure [b.p. about 80°C at
0.27 kPa (2 torr)]. It is important that the octanol produced be
at least 99.9 percent pure. Alternatively, a grade equivalent to
Fisher Scientific Co. No. A-402 "Certified Octanol-1" can be
used. It is important that distilled or reagent grade (ASTM Type
II) water be used.
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b. Buffer Solutions
Prepare buffer solutions using reagent grade chemicals in
distilled or reagent grade water as follows:
pH 5.0 - To 250 mL of 0.1M potassium hydrogen
phthalate add 113 mL of 0.1M sodium
hydroxide; adjust final volume to 500 mL with
reagent grade water.
pH 7.0 - To 250 mL of 0.1 potassium dihydrogen
phosphate add 145 mL of 0.1M sodium hydroxide;
adjust final volume to 500 mL with reagent
grade water.
pH 9.0 - To 250 mL of 0.07M borax add 69 mL of 0.1M
HC1; adjust final volume to 500 mL with reagent
grade water.
Check the pH of each buffer solution at 25°C with a pH meter
and adjust to pH 5.0, 7.0, or 9.0, if necessary.
c. Presaturation of the Solvents
Before a partitioning experiment is carried out, prepare
octanol saturated with water and water saturated with octanol.
Add purified _n-octanol to a large stock bottle and sufficient
distilled water to saturate it. Shake the flask for 24 hours on a
mechanical shaker. Then allow sufficient time for the mixture to
stand so that the two phases separate. Repeat this procedure
using another large stock bottle containing distilled water and
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sufficient octanol to saturate it. The desired quantities of the
presaturated solvents can be taken from these stock bottles for
each partition experiment.
d. Preparation of Test Solution
9 "3
Prepare a 10 to 10 JM solution of the test material in
octanol.
C. Performance of the Test
(1) Add a small volume of the octanol test solution (1 to
5 mL) to a centrifuge tube with a sealable cap as
described in Section II.A.9.
(2) Add the required volume of water to the centrifuge tube
as described in Section II.A.6. The volume of water
required is variable, depending upon the amount of
chemical required for the analysis. Generally, 20-40 mL
of water should be sufficient. Make sure that the
centrifuge tube is almost completely full. In this way,
partitioning with air will be minimized. This is
important, especially when determining KQW for volatile
chemicals.
(3) Equilibrate the samples at 25 °C in a constant temperature
bath, chamber, or room by gently shaking the centrifuge
tube for 1 hour. Avoid vigorous shaking that may cause
troublesome emulsions to form. For surfactants, a
minimum of 16 hours of shaking is required as described
in Section II.A.5.
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(4) Centrifuge the samples at 25°C for 20 minutes to break
any emulsion and to separate the octanol and water
phases. Evidence for breaking the emulsion and
separation of the water and octanol phases can be
obtained using a turbidimeter. The acceleration (G)
value required to break the emulsion and to achieve
complete separation of the octanol and water phases can
be determined by trial-and-error experimentation.
(5) Sample the octanol and water phases as follows:
o Withdraw by pipet a known volume of the octanol phase
(approximately 1/2 or less of the total octanol phase)
and transfer to an analysis cell or diluting
solvent. Before transferring the aliquot of the
octanol phase, wipe the outside of the pipet with a
paper tissue.
o Remove by pipet the remainder of the octanol phase
including the interfacial layer and discard.
o Insert another clean pipet close to the bottom of the
centrifuge tube and carefully withdraw a known volume
of the aqueous phase. Wipe the bottom exterior part
of the pipet with a tissue and discharge the aqueous
sample directly into an analysis cell or extraction
solvent. Do not allow the extraction solvent to
contact the pipet stem.
(6) Select an analytical method that is most applicable to
the analysis of the specific chemical as described in
Section II.A.7. Determine the concentration in the
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octanol and water phases. Express the concentration of
the chemical in octanol and water in moles/liter (M).
(7) Determine the partition coefficient in triplicate (steps
1 through 7) at two concentrations o^ the test material
C < 0.01M and Ci = 0.1C as described in Section
II.A.10. Tf KOW is not constant at C and ^, then
association effects should be considered. therefore,
follow steps 1 through 7 at lower concentrations until
ow is constant at two concentrations differing by a
factor of 10 as described in Section TT.A.10.
(8) Very hydronhobic chemicals (with KOW on the order of 10
to 10") renuire relatively large volumes o^ the aqueous
phase as described in Section II. A.. 6 and section
T T . ?\ . 9 . Hence, ^or verv hvdronhobic materials,
equilibrate the octanol and water phases in a large
ground-qlass stoppered flask as described above in step
(3). For the final phase separation, transfer the two
phase mixture to centrifuge tubes that have been
prerinsed with some of the aqueous phase; centrifuge as
described in step (4); withdraw aliquots from each
centrifuge tube as described in step (5); and recombine
for analvsis. [Mote: Prerinse all transfer tubes with
the water phase.] Complete steps (6) and (7) to
determine K .
(9) For materials that reversiblv ionize or protonate,
determine KOW at pH 5.0, 7.0, and 9.0 as described in
Section TT.^.10. Follow steps (1) throuqh (7) using the
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buffered aqueous solutions described in Section
II.B.l.b. Using the acid dissociation constant and the
concentration of the chemical in the aqueous phase
[Cwater], the term a can be calculated. The concen-
tration of undissociated chemical can be determined from
a and Cwater'
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III . DATA AND REPORTING
A. Test Report
For each individual determination, report the octanol/water
partition coefficient at each concentration of the test substance,
including the molar concentration of chemical in each phase [C ,
and cwafer]' In addition, report the mean value of K , and the
standard deviation.
Summarize all the data on the data sheets listed in
Appendix 1.
B. Specific Analytical and Recovery Procedures
(1) Provide a detailed description or reference for the
analytical procedure used, including the calibration data
and precision; and
(2) if extraction methods were used to separate the solute
from the octanol and aqueous phases, provide a
description of the extraction data.
C. Other Test Conditions
Report the experimental (G) value required to break the
emulsion and to achieve separation of the octanol and water
phases.
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IV. REFERENCES
ASTM. 1978. Annual Book of ASTM Standards. American Society for
Testing and Materials. Philadelphia, Part 31, Method D 1193-77.
Chiou CT, Freed VH, Schmedding DW, Kohnert RL. 1977. Partition
coefficient and bioaccumulation of selected organic chemicals.
Environ Sci Tech 11:475.
Fujita T, Iwasa J, Hansch C. 1964. A new substituent constant
derived from partition coefficients. J Am Chem Soc 86:5175.
Hansch C, Anderson SM. 1977. The effect of intermolecular
hydrophobia bonding on partition coefficients. J Org Chem 23:2583.
Leo A, Hansch C, Elkins D. 1971. Partition coefficients and
their uses. Chem Rev 71:525.
Neely WB, Branson DR, Blau GE. 1974. Partition coefficient to
measure bioconcentration potential of organic chemicals in fish.
Environ Sci Tech 8:113.
Nernst W. 1891. Z Phys Chem 8:110.
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V. APPENDIX 1: DATA FORMAT SHEETS
Instructions
(1) If multiple pH values are required, complete multiple
copies of the Test Results Data page - one set of test
results should be reported for each pH tested. If only
unbuffered pure water is used, number 3 should be
checked.
(2) If Kow is not constant at C and Cj_, and the chemical
associates in water or octanol or both, additional
concentrations must be used (2, 3 . . .) until Kow is
constant at two molar concentrations differing by a
factor of ten. These additional concentrations should be
reported on duplicates of the Test Results page
substituting C.^ and 3 for C and C^.
(3) This test guideline is designed to determine the
octanol/water partition coefficient of the test chemical
in the range 10 to 10^. p0r chemicals outside this
range, the octanol/water partition coefficient should be
reported as Kow < 10 or Kow > 10" with no further
quantification.
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TEST RESULTS SUMMflRY
1. Chenicol lot nunber
2. pH of water
3. Unbuffered pure water II
Cone .
C
Cl
Det.' No.
1
2
3
1
2
3
4.
C0
ci
a n
o 1
(no 1 dm
Exp
Exp
Exp
Exp
Exp
Exp
-3
)
5.
cw
at
e r
In
oidn"3
Exp
Exp
Exp
Exp
Exp
Exp
^
J
Cone.
C
1
1
/*
Cl
Dei.
No.
1
2
3
1
Z
o
Partition
Coef-picient
6.
1 1 1 1 1 1 CXD 1 1 1
i i i i i i EXP rT i
i 1 1 Exp | | i
1 ! ! EXP I 1 !
1 ! 1 Exp 1 1 i
1 1 ! EXP 1 1 1
KOH
Mean
7.
1 1 1 1 1 1 Exp 1 I |
i L i i i i exP i i i
K
Standqrd Deviation
8.
1 1 1 1 I 1 Exp I 1 I
1 1 I 1 1 Exp 1 1 1
9. Partition coefficient nean (C and Ci
10. Partition coeff ictant
1C G n d C )
U. flece Ur».t von vo,l ue IG) 1 I I I
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August, 1982
PARTITION COEFFICIENT (n-OCTANOL/WATER)
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Table of Contents
Page
I . NEED FOR THE TEST 1
II. SCIENTIFIC ASPECTS 3
A. Rationale for the Use of the Octanol/Water Partition
Coefficient to Estimate Rioconcentration Potential... 3
B. Rationale for the Selection of the Test Method 4
1. The Conventional Method of Determining the
Octanol/Water Partition Coefficient KQW. 4
2. Other Experimental Methods of Determining K .... S
a. Reverse-Phase High-Pressure Liquid
Chromatography as a Method of Estimating KQw.. ' S
b. Thin-Layer Chromatography as a Method of
Estimating K 7
c. Estimation of K from Water Solubility Data.. 7
C. Rationale for the Selection of the Test Conditions... 9
1. Theory of the Distribution Law and the Octanol/
Water Partition Coefficient 9
2. Factors that Affect the Value of KQW 13
a. Effect of Temperature 13
b. Purity of the Solvents 13
c. Concentrations of Solute 14
d. Equilibration Time 14
e. Octanol/Water Volume Ratio 15
f. Chemical Analysis of the Octanol and Water
Phases 18
g. Emulsification and Ultracentrifugation 19
h. Equilibration Vessel 20
i. Speciation Effects 21
j. Presaturation of the Solvents 21
D. Reference Compounds 22
E. Test Data Required 22
F. Statistical Analysis of the Data 25
III . REFERENCES 26
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PARTITION COEFFICIENT (n-OCTANUL/WATER)
I. NEED FOR THE TEST
Bioconcentration, the accumulation of a substance in living
tissues or other organic matter as a result of net chemical uptake
from the medium (e.g., water), is a factor in determining the
movement of a chemical in the environment and the potential
effects of the chemical on biota. Hydrophobic chemicals that are
present in the aqueous environment at subtoxic concentrations may
accumulate to toxic levels once inside organisms, presumably
through diffusion into nonpolar cell components, where they
accumulate because of their greater solubility. Further movement
of the substance in living tissues may occur as a result of
ingestion of low trophic level organisms, i.e., food chain
effects.
The octanol/water partition coefficient KQW has been shown to
be a good predictor of the tendency of chemicals to bioconcentrate
in fish (Neely et al. 1974). Since 1974, KQW has been used as a
measure of bioconcentration potential in fatty tissues in aquatic
and other living organisms. The numerical value of the
octanol/water partition coefficient is one factor to be considered
in determining whether to conduct fish bioconcentration studies.
Other factors must also be taken into account. For example,
transformation rates (e.g., rates of biodegradation, hydrolysis,
photolysis, and oxidation) must also be considered. If a chemical
transforms readily by one of these processes, the potential for
bioconcentration will be reduced significantly and fish
bioconcentration studies may not be needed.
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The octanol/water partition coefficient has been introduced
by Hansch to correlate biological activity and chemical structure
(Hansch 1969; Hansch and Fujita 1964). Numerous papers have been
published by Hansch and his coworkers on this subject in the
ensuing years. A monograph has been published on the Hansch
approach (Gould 1972).
A recent publication has indicated that the sorption of
several hydrophobic pollutants on natural sediments can be related
to the octanol/water partition coefficient. Karickhoff et al.
(1979) showed that a reasonable estimate (within a factor of two)
of the sorption behavior of hydrophobic pollutants can be made
from knowledge of the particle size distribution and associated
organic content of the sediment and the octanol/water partition
coefficient.
Another recent publication has described a novel method for
estimating the distribution of a chemical in the environment
(Mackay 1979). KQW is used in this partitioning analysis. This
partitioning analysis will be used as a guide to ecological and
health effects testing.
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II. SCIENTIFIC ASPECTS
A. Rationale for the Use of the Octanol/Water Partition
Coefficient to Estimate Bioconcentration Potential
Intuitively, the absorption and fat storage of xenobiotic
chemicals in living organisms seem to be related to lipophilicity
or preferential solubility in fats as compared to water. By
definition, the octanol/water partition coefficient KQW expresses
the equilibrium concentration ratio of an organic chemical
partitioned between octanol and water in dilute solution. If one
assumes that octanol simulates fats in its solubilizing effect on
organic chemicals, then K should be a potential measure of the
OvV
ease of storage of organic chemicals in fats. For example, a
large value of K indicates that an organic chemical is not very
CjVr
soluble in water but soluble in octanol. Hence, this would
suggest the potential for a large storage of the organic chemical
in fats. Davies et al. (1975) reported human pesticide poisoning
by ci fat-soluble organophosphate, dichlofenthion. The octanol/
wateir partition coefficient KQW was found to be very high (1.37 X
10 ), which correlated with the high fat storage of this chemical.
Neely et al. (1974) found a pronounced correlation between
K and the bioconcentration in trout muscle. Specifically, these
o w
researchers obtained a linear correlation between the log of
bioconcentration and the log of calculated K for a series of
organic chemicals. Since that time, K has been used by
o w
researchers as an index of bioconcentration potential in living
-3-
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orqanisms. The Office of Pesticide Programs (OPP) [EPA 1975,
1978] has proposed, and the Organization for Economic Cooperation
and Development (OECD 1981) is using K as a measure of
bioconcentration potential in aquatic organisms.
B. Rationale for the Selection of the Test Method
1. The Conventional Method of Determining the Octanol/
Water Partition Coefficient KQW
The conventional method for determining a distribution
coefficient is carried out by distributing a chemical between two
immiscible liquids in a vessel and measuring the concentration of
the chemical in the two liquid phases after equilibration
(Glasstone 1946; Leo et al. 1971). This method can be applied to
the determination of the octanol/water partition coefficient
KQw. Mumerous researchers use the conventional method of
determining KQW and have published papers using this method (e.g.,
Fujita et al. 1964; Hansch and Anderson 1967; Leo et al. 1971;
Chiou et al. 1977). EPA (EPA 1975, 1978) has proposed, and OECD
(1981) is using the conventional method of determining K . Most
chemical companies that determine octanol/water partitioning use
the conventional method of determining KOW> Hence, the test-
guideline uses the conventional method of determining the
octanol/water partition coefficient KQW. It should be noted that
there is no validated standard test method for determining KQW
(e.g., an ASTM method). The method in this guideline was
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developed from a thorough review of the research literature on the
experimental determination of KQW and by talking to researchers
who have considerable experience in carrying out these
experiments.
2,. Other Experimental Methods of Determining K
pw
a. Reverse-Phase High-Pressure Liquid Chromatography
as a Method of Estimating K
ow
A rapid method based on reverse-phase high-pressure liquid
Chromatography has been developed by Veith (Veith and Morris 1978;
Veith et al. 1979) to estimate the octanol/water partition
coefficient of organic chemicals. Using the solvent mixture
water/methanol (15/85 v/v) as the elutant, the log of the
retention time [log (tR)] of organic chemicals on a permanently
bonded (C-18) reverse-phase high-pressure liquid chromatographic
system has been found to be linearly related to log KQW. This
rele.tionship has been expressed by the equation
log KQW = A log (tR) - B, (1)
where A and B are constants determined from the experimental data
for some organic chemicals. Using a mixture of the chemicals benzene,
bromobenzene, biphenyl, p,p'-DDE [2,2-bis(p-chlorophenyl)-l,1-
dichloroethylerie] and 2 , 4, 5 , 2 ' , 5 ' -pentachlorobiphenyl, A and B
were found to be 5.106 and 1.258, respectively, with a coefficient
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of determination of 0.975. It must be emphasized that this
correlation is limited with respect to being representative of the
organic chemicals encountered. This calibration mixture was
selected largely on the basis of the log KQW values reported in
the literature, and the correlation is linear over five orders of
magnitude of K . To determine the accuracy of this method of
estimating log K by comparison with data reported in the
literature, Veith and coworkers measured the retention time of 18
chemicals, and the standards and log KQW values were calculated
from the regression equation (1). The results indicated that log
K can be estimated to within (22.8 _+_ 20.0) percent when compared
with the values reported in the literature from measurements using
other methods. The percent error was calculated assuming the
literature value is the correct log KQW; these researchers had
some reservations about this assumption. It should be noted that
some of the greatest relative errors were observed with polar
chemicals that dissociate in water (e.g., m-chlorobenzoic acid,
2,4,5-trichlorophenol, and diphenylamine). This method has a
definite advantage, since the estimation of K can be made
rapidly and relatively easily in comparison to the determination
of KQW by the conventional method. Furthermore, KQW can be
estimated for individual chemicals in complex mixtures (e.g.,
solid wastes) without knowing the specific chemical structure of
each chemical.
-6-
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CS-1400
Other researchers have developed high-pressure liquid
chromatographic methods to determine K (Mirrless et al. 1976;
Yamena et al. 1977; Carlson et al. 1975; Hulshoff and Perrin 1976;
McCall 1975). However, these methods are based on a very limited
number of experiments and considerably more work is needed to
develop them.
b. Thin-Layer Chromatography as a Method of
Estimating KQW
It has been reported that thin-layer chromatography
can be used to estimate KQW (Mirrless et al. 1976; Hulshoff and
Perrin 1976). However, high-pressure liquid chromatography (HPLC)
is far superior to thin-layer chromatography (TLC) because of its
accuracy (i.e., definition of the peak, reproducibility, ease of
detection in many cases, and above all the range of applicability
(HPL2 is applicable over 5 orders of magnitude of K while TLC is
only applicable over 1.5 orders of magnitude of KQW) (Mirrless et
al. 1976).
c. Estimation of KQW from Water Solubility Data
The octanol/water partition coefficient is defined as the
ratio of the equilibrium molar concentration of the chemical in
octanol and water. Thus, low molecular mass (i.e., molecular
weight) organic chemicals with a low water solubility should have
-7-
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CS-1400
a high value of KQW (e.g., hydrophobia organic chemicals).
Therefore, there should be a correlation between KQW and water
solubility. Chiou et al. (1977) studied the relationship between
KQW and the water solubility, S, and found that, for 34 organic
chemicals, an excellent linear correlation was observed between
log K and log S that extended to more than eight orders of
magnitude in water solubility (10 to 10 ppm), and six orders of
magnitude in KQW (10 to 107). Chiou et al. (1977) found the
following regression equation
log KQW = 5.00 - 0.670 log S, (2)
where KQW is the octanol/water partition coefficient S is the
water solubility in mol/L, and the coefficient of determination
(r2) was 0.970 for these 34 chemicals. Thus, KQW can be estimated
from the experimental value of the water solubility of an organic
chemical. This method would have a definite advantage in that Kow
could be estimated directly from water solubility data without
having to experimentally measure KQW- Thus, the octanol/water
test guideline could eventually be eliminated, thereby reducing
the cost of testing. However, considerably more experimental work
is necessary to extend the correlation to a large number of
organic chemicals with different structures before it can be used
as a test guideline.
-8-
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CS-1400
C. Rationale for the Selection of Test Conditions
A detailed study of the theory of the distribution law, the
partition coefficient, and the published literature on the
conventional determination of K indicates that it is extremely
o\v
important that numerous factors (or test conditions) be
standardized. In order to establish these factors clearly, the
theory of the distribution law and its relation to these factors
are discussed in detail in the following sections.
1. Theory of the Distribution Law and the Octanol/Water
Partition Coefficient
The distribution coefficient or partition coefficient can be
derived using thermodynamic theory (Glasstone 1946). Consider a
mixture of two immiscible liquids that is shaken with a solute
(organic chemical). The solute distributes itself between the two
liquids in such a way that at equilibrium, in dilute solution, the
ratio of the concentrations of the solute in the two layers is a
constant at a given temperature. The tendency of a chemical to
distribute itself between two immiscible liquids with a constant
concentration ratio, in dilute solution, is a direct consequence
of the thermodynamic requirements for equilibrium. To illustrate
this, consider a pair of immiscible liquids A and B in contact
with each other containing the same solute in solution. The
chemical potential of a solute in solvent A is given by
VA = v °A + RT In aA, (3)
-9-
-------
CS-1400
o
where y A is the chemical potential of the solute in solvent A, y A
is the standard chemical potential of the solute in the same
solvent (i.e., the value of yA at aA = 1), while aA, the activity
of the solute in the solvent A is the effective concentration
taking into account intermolecular interactions of the solute in
the solvent. R is the gas constant and is equal to 8.314
joules/0K/mol, while T is the absolute temperature in °K.
Similarly, for solvent B
o
v B = y B + RT In aB, (4)
where all the quantities have the same significance as in equation
(3). At equilibrium between the layers Ay = 0; hence
Ay = y B - y A = 0,
and
UB = yA. (5)
Using equations (3) and (4) in (5) yields
y° + RT In an = y° + RT In a. (6;
Lj LJ f\ f\
O O
3B V A - V B
In =
RT
-10-
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CS-1400
However, at a given temperature, u B and y A are constants for a
given solute in a particular solvent; hence
ln
and
Equation (7) is the mathematical statement of the distribution law
that states that a substance will distribute itself between two
solvents until at equilibrium the ratio of the activities of a
chemical in the two layers is a constant at a fixed temperature,
irrespective of the absolute values of aA or aB. The activity aA
can be written as
aA = *A CA ' (8)
where uA is the activity coefficient and takes into account the
interaction between molecules A in solution, and CA is the molar
concentration. In dilute solution as
o,
(9)
*~i
hence,
limit
CA * 0 (a,) = C, .
A v A' A
-11-
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CS-1400
The same argument follows for the solute in solvents B and
C_, 0 (an) = C_ . Using these results in equation (7), the
r> * ts hi
distribution coefficient K, in dilute solution, becomes
limit
K = C * 0
For the specific case for the octanol/water partition
coefficient, B is the solvent n-octanol, A is the solvent water,
and K° = K . Thus, equation (11) becomes
\J Wr
Kow
water
According to Nernst (1891), the distribution law applies only
to individual molecular species in solution. If a molecule
dissociates or associates in octanol and water, then equation (12)
must be modified. In general, if a represents the fraction of the
total solute that is dissociated or associated, assuming that
either association or dissociation occurs in each solvent, then
K = aoct. Coct. , .
ow (1 - ex t ) C , ' UJ'
water water
since (1 - a) gives the fraction of unchanged molecules in each
phase. For the special case where no association takes place in
octanol, equation (13) reduces to
oct-
_
ow (1 - a _ ) C ,
water water
-12-
-------
CS-1400
2. Factors that Affect the Value of Knw
a. Effect of Temperature.
From the theory of the distribution law as outlined in
Section II.C.I, the distribution coefficient K is a function of
the temperature (equation (6)), and is a constant as a fixed
temperature (equation (7)). Since KQW is a distribution
coefficient, it should also vary with temperature and is a
constant at a fixed temperature. Hence, in carrying out
octanol/water partition coefficient experiments by the conven-
tional method, the temperature should be controlled. However,
variations due to temperature are small compared to those inherent
in the errors in the other measurements, e.g., the errors in
measuring the concentration of solute in octanol and water.
Therefore, for reasonably accurate determinations of KOW, it is
sufficient to control the temperature to +_ 1°C. Since most
physical properties of chemicals are reported at 25°C, this
guideline requires that K be determined at this temperature.
O W
b. Purity of the Solvents
Trace amounts of impurities present in n-octanol tend to
produce emulsions and must be removed (Fujita et al. 1964; Hansch
and Anderson 1967? Chiou et al. 1977). Emulsions give poor phase
separation find result in a wide scatter in the value of KQW. In
addition, impurities in octanol may affect the analysis for the
solute. Hence it is extremely important that the octanol be
-13-
-------
CS-1400
99.9 percent pure. Distilled or reagent qrade water (AS^M Type
II) should be used.
c. Concentration of Solute
From the theory of the distribution law, as outlined in
Section II.C.I, equations (12), (13), and (14) only apply in
dilute solution. Hence, it is extremely important that all
experiments be carried out at molar concentration C < 0.01M in
octanol and water.
d. Equilibration Time
For many chemicals, 5 minutes of gentle agitation of the two-
phase system established equilibrium and produced consistent
results (Leo et al. 1971). Studies by Craig and Craig (1950)
indicated that when the phases were of about equal volume,
equilibrium was rapidly attained. When high ratios of water to
octanol ( 100:1) were used, longer shaking was necessary to
establish equilibrium. High ratios of water to octanol are used
to determine KQw for very hydrophobic organic chemicals (Sections
Il.C.l.e and h as described below). Therefore, for most
chemicals, gentle agitation for 1 hour should be adequate to reach
equilibrium. For surfactants, at least 16 hours of agitaton is
necessary to reach equilibrium. This is an empirical observation
obtained by researchers who have carried out experiments with
surfactants. It is undoubtedly due to the nature of surfactant
chemicals.
-14-
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CS-1400
e. Octanol/Water Volume Ratio
Depending upon the solubility of the solute in octanol and
water, the ratio of the volume of octanol to water should be
adjusted. For hydrophobia solutes, which are very insoluble in
water, considerably more water than octanol should be used.
Adjustment of solvent volumes can decrease the effect of
analytical errors and consequently decrease the error in
determining KQW (Leo et al. 1971). The following example will
illustrate this point. Consider a chemical with a molecular mass
(i.e., molecular weight) MW (mg/mmol), KOW = 200. Twenty mg of
chenical are dissolved in 100 mL of octanol, and 100 mL of water
are added to this system. After equlibration, the mass of
chemical in each phase can be calculated as follows. The chemical
will partition with x mg in the water phase and (20-x) mg in the
octemol phase.
'oct.
(20-x)mg
MW (mg/mmol)
100 mL
<2°-x> Molar
100 MW
(20-x) mmol
100 MW
mL
x mg
MW(mg/mmol)
'water
100 mL
100 MW
Molar
100 MW
mmol
mL
-15-
-------
Since
CS-1400
ow
°ct'
,
water
200 =
(20-x)
100 MW
x
100 MW
(20-x)
Then x
20
201
= 0.0996 = 0.10 mg =
mass of chemical in the
water phase;
and
(20-x) = 19.9 ffl 20 mg = mass chemical in the octanol phase.
Consider an analytical error of ± 0.05 mg in the aqueous
phase (i.e., 0.10 - 0.05 = 0.05 and 0.10 + 0.05 = 0.15).
ow
.20
MW
100
0.05
MW
100
20
0.05
=
ow
.20
MW
100
0.15
MW
100
20
0.15
-16-
-------
CS-1400
Therefore, an analytical error of = 0.05 mg in the aqueous
phase means that KQW can range from 400 to 133, and a very large
error in KQW occurs.
Consider the same example as above, but now the solvents are
adjusted to 200 mL of water and 5 mL of octanol. After
equilibration, the mass of chemical in each phase is now:
(20-x)
MW
'oct.
(20-x)
5 MW
Molar
'H2°
MW
200
200 MW
Molar
ow
'oct.
'water
Then
200 =
20-x)
5 MW
200 MW
(20-x) (40)
x
x =
20
= 3.33 mg = mass of chemical in the water phase
and
20-x) = 16.7 mg = mass of chemical in the octanol phase.
-17-
-------
CS-1400
Now consider the same analytical error of ± 0.05 mg in the
aqueous phase (i.e., 3.33 - 0.05 = 3.28 and 3.33 + 0.05 = 3.38)
16.7_
"" T C. 1
= 203
j. jo
"200
16.7_
^ a -i
= 197
K
ow
MW
5
3.28
MW
16 *7 ( 4m
3.38 (40)
MW
5 16.7
3.28
MW
200
Now the analytical error of _+_ 0.05 mg in the aqueous phase means
that KQW can range from 197 to 203 and the error in KQW has been
reduced dramatically.
f. Chemical Analysis of the Octanol and Water Phases
Consider a partitioning experiment in which a chemical is
dissolved in octanol at a low concentration (less than 0.01
molar). The conventional partitioning experiment is carried out,
and only one phase is analyzed for the molar concentration of the
solute. Using a mass balance, the molar concentration of the
solute in the other phase is obtained by difference. However, if
there is a loss of chemical by adsorption to the surface of the
glass walls, a serious error will occur at this low concen-
tration. This is especially true for very hydrophobic chemicals
(Chiou et al. 1977) and for ionic solutes (Leo et al. 1971).
Therefore, it is important that both the octanol and water phases
be analyzed.
-i p_
-------
CS-1400
An analytical method should be selected that is the most
applicable to the analysis of the specific chemical. However,
large errors can occur as a result of traces of more-water-soluble
contaminants that are not analytically distinguishable from the
parent chemcal. This error is very significant when the analy-
tical method is ultraviolet absorption spectroscopy or radiometry,
since these methods can be nonspecific for many solutes.
Therefore, chromatographic methods are preferable because of their
compound specificity in analyzing the parent chemical without
interference from impurities (Karickhoff and Brown 1979).
Wherever practicable, the chosen analytical method should have a
precision with ± 5 percent.
g. Emulsification and Ultracentrifugation
Many chemicals can cause troublesome emulsions to form
between octanol and water and emulsification can result in large
errors in KQW (Leo et al. 1971; Chiou et al. 1977) This is
especially true for hydrophobic chemicals. Therefore, it is
important that gentle shaking be used to minimize the formation of
emulsions. In addition, incomplete separation of the two phases
is one of the most serious sources of error. To break any
emulsion formed and to separate completely the octanol and water
phases, it is extremely important that the two-phase system be
ultracentrifuged at 25°C for 20 minutes. The acceleration G value
required to break an emulsion and to separate completely the
octanol and water phases can be determined by trial-and-error
experimentation. Since the visual clarity of the two phases is
-19-
-------
CS-1400
not a dependable criterion of the absence of an emulsion and
complete separation of the two-phase system, it is recommended
that a turbidimeter be used to make sure that the emulsion is
broken and the octanol and water phases have been completely
separated.
h. Equilibration Vessel
To simplify the experimental procedure, it is recommended
that equilibration be carried out in a centrifuge tube (special
glass tubes can be used up to approximately 12,000 G and stainless
steel centrifuge tubes can be used at higher G values) with a
sealable cap. This will avoid a transfer step and volatile
chemicals can be handled easily. It is important that the
centrifuge tube be almost completely filled with the two-phase
mixture to minimize partitioning with air. This is expecially
important when determining KQW for volatile chemicals (Hansch and
Anderson 1967 ) .
Very hydrophobia chemicals, with KQW on the order of 10 to
10 , require relatively large volumes of the aqueous phase
(Section II.C.2.e). Hence, for these chemicals, it is recommended
that equilibration be carried out in a large ground-glass
stoppered flask.
-20-
-------
C S - 1 4 0 0
i. Speciation Effects
The details of speciation have been discussed in the theory
of the distribution law and the octanol/water partition
coefficient, Section II.C.I.
If the chemical does not associate or dissociate in octanol
and water, then the test guideline requires that equation (12) be
used and KQW be determined at concentrations C < 0.01M and
Cj :;: 0.1C. Under these experimental conditions, if KOW is
constant, then association or dissociation has been minimized or
elininated.
If the chemical associates in octanol or water or in both
liquids, then the test guideline requires that equation (13) be
user! and KQW be determined at concentations C < 0.01M, C^ = O.lC,
C2 =: 0.01C, C3 = 0.001C, When KQW is constant at two
concentrations differing by a factor of 10, then the effect o^
association has been minimized or eliminated.
For chemicals that reversibly ionize or protonate (e.g.,
cnrboxylic acids, phenols or anilines), the test guideline
requires that equation (14) be used with water buffered at pH 5.0,
7.0, and 9.0, the pHs of environmental concern.
j. Presaturation of the Solvents
Presaturation of octanol with water and water with octanol is
H *ir this test guideline. The preparation of these
,::>;rateii solutions is very simple to carry out. This requirement
-21-
-------
CS-1400
is extremely important when determining KQW for very hydrophobia
chemicals, since the ratio of water to octanol will be very
large. In this case, if the experiment is carried out without
presaturation of the water with octanol, then all the octanol will
dissolve in the aqueous phase and KQW cannot be determined.
D. Reference Compounds
It would be very desirable to have reference compounds that
cover a KQ range of 10 to 10 . These reference compounds would
provide the experimenter with comparative reference values to
determine how well the test has been conducted. Unfortunately,
these reference compounds are not currently available. When
appropriate reference compounds have been identified they will be
recommended for use in this test guideline. In the interim, it is
recommended that the book by Hansch and Leo (1979) be used for the
selection of potential reference compounds.
E. Test Data Required
The tendency of an organic chemical to partition out of water
into other environmental compartments containing hydrophobic
constituents (e.g., aquatic organisms) can be inferred from the
values of the octanol/water partition coefficient KOW- Chiou et
al. (1977) developed regression equations relating log KQW with
water solubility S (in mol/L) and bioconcentration in rainbow
trout (BF). Assuming log KQW is between 1 and 6, S and RF can be
-22-
-------
CS-1400
calculated; these results are summarized in Table 1 (note that S
has been converted to mol/L). Furthermore, assuming that the
average molecular mass (i.e., molecular weight) of an organic
chemical is 300 gm/mol, the water solubility can be converted to
ppm; these results are also summarized in Table 1. It is apparent
that for log KQW = 6 (i.e., KQW = 106), the water solubility will
be extremely low (9.7 x 10~^ ppm or 9.7 ppb) and the predicted RF
is 1.48 x ID1*. Hence, the data indicate that the chemical will
partition out of the water phase and into the fat of the fish
(i.e., the hydrophobic phase). For log Kow = 1 (i.e., Kow = 10),
the water solubility will be very high (2.80 x 10^ mg/L or
280 gm/L) and the predicted BF is 2.4. Hence, these data indicate
that the chemical will remain in the water phase and will not
partition significantly into the fat of the fish (i.e., the
hydrophobic phase). Therefore, the test guideline is designed to
determine the value of KQW in the range 10 to 106. Low molecular
mass organic chemicals with a K value less than 10 will not
part.ition significantly into or tend to accumulate in, any
hydrophobic environmental compartments. Low molecular mass
organic chemicals with KQW in excess of 106 will tend to
accumulate into all hydrophobic environmental compartments. For
low molecular mass organic chemicals outside the range 10 to 10 ,
the test guideline requires that K_,_ be characterized as 10 or
CJW
10° with no further quantification.
Specific analytical and recovery procedures should be
reported to determine whether acceptable data have been generated.
-23-
-------
CS-1400
Table 1. Summary of Calculated Values of Water Solubility and
Bioconcentration in Rainbow Trout as a Function of Log KQwa
Kow S(mol/L)b S(mg/L or ppm)c BFC
6 3.24 x 10~8 9.7 x 10~3 1.48 x 104
5 1 x 10~6 0.30 2.57 x 103
4 3.09 x 10~5 9.3 4.47 x 102
3 9.77 x 10~4 2.93 x 102 7.76 x 101
2 3.02 x 10"2 9.06 x 103 1.35 x 101
1 9.3 x 10'1 2.80 x 105 2.4
aRegression equations taken from Chiou et al. (1977).
^Water solubility
°Water solubility in ppm assuming a molecular mass of an organic
chemical is 300 gm/mol.
Bioconcentration in rainbow trout
-24-
-------
CS-1400
F. Statistical Analysis of the Data
Numerous researchers have published data on the determination
of the octanol/water partition coefficient by the conventional
method (e.g., Chiou et al. 1977; Davies et al. 1975; Fujita et al.
1964; Hansch and Anderson 1967; Leo et al. 1971). However, none
of these researchers has analyzed the data statistically and the
precision of KQW as determined by the conventional method has not
been clearly established. The precision is, in part, a function
of the nature of the specific chemical. As the hydrophobicity of
the chemical increases, KQW increases and the precision of KQW
decreases. Furthermore, the precision is also a function of the
analytical procedure used. In general, the lower the
concentration to be measured, the poorer is the precision of the
analytical procedure. Therefore, no reliable precision can be
stated at this time for determining KOW. Obviously, the precision
can be improved by making numerous replicate determinations.
However, in order to minimize cost, it has been decided to
determine KQW with three replicates. Therefore, it is important
that the submitter of the test results analyze the data
statistically. When a large number of chemicals have been
determined by the proposed method, the data will be analyzed
statistically and the level of precision can be defined for
various ranges of KQW.
-25-
-------
CS-1400
ITT. REFERENCES
Carlson RM, Carlson RE, Kopperman HL. 1975. Determination of
partition coefficients by liquid chromatography. J Chromatogr
107:219.
Chion Cm, Freed VH, Schmedding nw, Kohnert RL. 1977. Partition
coefficient and bioaccumulation of selected organic chemicals.
Environ Sci ^echnol 11:475.
Craig LC, Craig n. 1950. Tn: technique of organic chemistrv,
Vol. TIT, pt. I, Chapter 4. New York: Interscience Publishers,
Tnc.
Davies JE, Parquet A, Freed V, Hague R, Morgade C, Sonneborn RE,
Vaclavek C. 1975. Human poisonings by a fat-soluble
organophosphate insecticide. Arch Environ Health 30:608.
Fujita T, Iwasa J, Hansch C. 1964. A new substituent constant,
derived from partition coefficients. J Am Chem Soc 86:5175.
Glasstone S. 1946. Textbook of physical chemistry. New York:
Van Nostrand Co.
Gould RF, ed. 1972. biological correlations the Hansch
approach. Adv. Chem. Ser. No. 114. Washington, D.C.: American
Chemical Society.
Hansch C. 1969. A quantitative approach to biomedioal
structure-activity relationships. Ace Chem Res 2:232.
Hansch C, Anderson SM. 1967. ^he effect of intramolecular
hydrophobic bonding on partition coefficients. J Org Chem
23:2583.
Hansch C, Fujita T1. 1964. p - a - TT analvsis. A method for
the correlation of biological activity and chemical structure.
J Am Chem Soc 86:1616.
Hansch C, Leo A. 1979. Substituent constants for correlation
analysis in chemistry and biology. New York: J. Wiley & Sons.
Hulshoff A, Perrin JH. 1976. A comparison of the determination
of partition coefficients of 1,4-benzodiazepines by high-
performance liquid chromatography and thin-layer chromatogranhv.
J Chromatogr 129:263.
Karickhoff SW, Brown OS. 1979. Determination o^ octanol/water
distribution coefficients, water solubilities, and sediment/water
partition coefficients ^or hydrophobic organic pollutants. EPA-
600/4-79-032.
-26-
-------
CS-1400
Karickhoff SW, Brown DS, Scott TA. 1979. Sorption of
hydrophobia pollutants on natural sediments. Water Res 13:241.
Leo A, Hansch C, Elkins D. 1971. Partition coefficients and
their uses. Chem Rev 71:525.
Mackay D. 1979. Finding fugacity feasible. Environ Sci ^echnol
13:1218.
McCall JM. 1975. Liquid-liquid partition coefficients by high-
pressure liquid chromatography. J Med Chem 18:549.
Mirrless MS, Moulton SJ, Murphy CT, Taylor PJ. 1976. Direct
measurement of octanol-water partition coefficients by high-
pressure liquid chromatography. J Med Chem 19:615.
Mernst W. 1891. Z Phys Chem 8:110.
Meely WB, Branson DR, Blau GE. 1974. Partition coefficient
to measure bioconcentration potential of organic chemicals in
fish. Environ Sci Technol 8:113.
OECD. 1981. Organization for Economic Cooperation and
Development (OECD). Guidelines for Testing Chemicals: No. 107-
Part.ition Coefficient (n-Octanol/Water) . Director of
Information, OECD; 2 Rue Andre-Pascal, 75775 PARIS CEDEX 16,
France.
USEPA. 1975. U.S. Environmental Protection Agency. Office of
Pest.icide Programs. Proposed guidelines for registering
pest.icides in the United States. Fed Regist 40, 26802.
USEF'A. 1978. U.S. Environmental Protection Agency, Office of
Pest.icide Programs. Proposed guidelines for registering
pesticides in the United States. Fed Regist 43, 29696.
Veith GD, Morris RT. 1978. A rapid method for estimating log P
for organic chemicals. EPA-600/3-78-049.
Veith GD, Austin MM, Morris RT. 1979. A rapid method for
estimating log P for organic chemicals. Water Res 13:43.
Yamana T, Tsuja A, Miyamoto E, Kubo O. 1977. Movel method for
determination of partition coefficients of penicillins and
cephalosporins by high-pressure liquid chromatography. J Pharm
Sci 66:747.
-------
CG-1450
August, 1982
pH OF WATER SOLUTION OR SUSPENSION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
CG-1450
pH OF WATER SOLUTION OR SUSPENSION
I. PURPOSE
This Test Guideline references methodology to develop data on the
pH of aqueous solutions and suspensions of chemical substances
and mixtures. The data may be used to calculate the deqree of
acidity or basicity of an aqueous solutions or suspensions that
will be formed by a chemical substance and to evaluate the
resulting effects the substance will have on human health and the
environment. The pH may be used in the desiqn of other tests.
II. TEST PROCEDURES
The electrometric method is recommended for determininq the pH of
an ciqueous solution or suspension. The recommended apparatus
should consist of a pH meter and electrodes, meetinq performance
standards and specifications as described, for example, in
ANSI /ASTM E 70-7700 and in ASTM D 1293-78.
It i.s recommended that the water used in preparinq solutions or
suspensions meet the standards for ^ype I or Tyne IT reaqent
water specified by A.MSI/ASTM D 1193-77 and be free of CO2 For
chemical substances that are readily soluble in water, the ^est
Guideline recommends that a solution of 1% by mass be used. For
chemical substances that are soluble in water to an extent of
less than 1% by mass at 25°C, then an aqueous suspension of the
chemical substance should be tested.
The recommended test methods are available for purchase from the
American Society for Testinq and Materials, 1916 Race St.,
Philadelphia, PA 19103.
III. REFERENCE
ASTM. (Latest Edition). American Societv for Testinq and
Materials. Annual Rook of ASTM Standards.
-1-
-------
CG-1500
August, 1982
WATER SOLUBILITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
CG-1500
Contents
Page
I. INTRODUCTION 1
A. Background and Puroose 1
B. Definitions and Units 2
C. Principle of the Test Method 4
D. Applicability and Specificity 4
I T . TEST PROCEDURES S
A. Test Conditions 5
1. Special Laboratory Equipment 5
2. Purity of Water f>
3. Purity of Solvents 6
4. Seawater ~i
5. Agitation and Equilibration Time ~l
f>. Effects of Colliods and
Emulsions: Centrif ugat ion 7
7. Effect of'pH on Solubility 8
n. Analysis of Saturated Solutions n
9. Adsorption to Glass or Other Surfaces B
B. Preparation of Reagents and Solutions 9
1. Buffer Solutions 9
2. Artificial Seawater 10
C. Performance of the Test ID
1. Procedure for the Determination of
Solids and Liquids in Water at 25°C 12
2. Modification of Procedures
for Potential Problems 13
a. Interference of Soluble Impurities 13
b. Decomposition of the Test Compound 13
III. DATA AND REPORTING 14
A. Test Report 14
B. Specific Analytical Recovery Procedures IS
IV. REFERENCES 16
V. APPENDIX 1: DATA FORMAT SHEETS 17
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CG-1500
WATER SOLUBILITY
I. INTRODUCTION
A. Background and Purpose
The water solubility of a compound can he defined as the
equilibrium concentration of the compound in a saturated aqueous
solution at a qiven temperature and pressure. The water
solubility of a chemical is an important factor in determininq
the environmental movement and distribution of anv substance.
Chemicals that are relatively water soluble are more likely to be
widely distributed by the hydroloqic cycle than those which are
relatively insoluble.
Water provides the medium in which many orqanisms live, and
water is a major component of the internal environment of all
livinq orqanisms (except for dormant staqes of certain life
forms). Even orqanisms which are adapted to life in a qaseous
environment require water for normal functioninq. Water is thus
the medium throuqh which most other chemicals are transported to
anr into livinq cells. As a result, the extent to which
chemicals dissolve in water will be a major determinant for
movement throuqh the environment and entry into livinq systems.
The water solubility of a chemical has an effect on its
adsorption on and desorption from soils and sediments and on
volatilization from aqueous media. The more soluble a chemical
substance is, the more likely it is to desorb from soils and
serliments and the less likely it is to volatilize from water.
The extent of chemical transformations via hydrolysis,
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photolysis, oxidation, reduction, and biodeqradation in water
depends on the chemical being soluble in water (i.e., homogeneous
kinetics). Finally, the design of most chemical tests and many
ecological and health tests requires precise knowledge of the
water solubility of the chemical to be tested.
Procedures in this test guideline have been described to
enable sponsors to determine the water solubility -For solid and
liquid organic compounds.
B. Definitions and Units
(1) "Colloidal dispersion" is a mixture resembling a true
solution but containing one or more substances that are finely
divided but large enough to prevent passage through a
semipermeable membrane. It consists of particles which are
larger than molecules, which settle out verv slowly with time,
which scatter a beam of light, and which are too small for
resolution with an ordinary light microscope.
(2) A "concentration vs. time study" results in a graph
which plots the measured concentration of a given compound in a
solution as a function of elapsed time. Usually, it provides a
more reliable determination of eguilibrium water solubility of
hydrophobic compounds than can be obtained by single measurements
of separate samples.
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(3) "Concentration" of a solution is the amount of solute
in a given amount of solvent and can be expressed as a
weight/weight or weight/volume relationship. ^he conversion from
a weight relationship to one of volume incorporates density as a
factor. For dilute aqueous solutions, the density of the solvent
is approximately equal to the density of the solutions; thus,
concentrations in mg/dm are approximately equal to lO"-* g/10 g
or parts per million (ppm); ones in yg/dm are approximately
equal to 10~6 g/10^ q or parts per billion (ppb). In addition,
concentration can be expressed in terms of molarity, normality,
molality, and mole fraction. For example, to convert from
weight/volume to molarity one incorporates molecular mass as a
factor.
(4) "Density" is the mass of a unit volume o^ a material.
It is a function of temperature, hence the temperature at which
it is measured should be specified. For a solid, it is the
density of the impermeable portion rather than the bulk
density. For solids and liquids, suitable units of measurement
are g/cm . The density of a solution is the mass of a unit
volume of the solution and suitable units of measurement are
g/cin^.
(5) A.n "oversaturated (supersaturated) solution" is a
solution that contains a greater concentration of a solute than
is possible at equilibrium under fixed conditions o^ temperature
and pressure.
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(6) A "saturated solution" is a solution in which the
dissolved solute is in equilibrium with an excess of undissolved
solute; or a solution in equilibrium such that at a fixed
temperature and pressure, the concentration of the solute in the
solution is at its maximum value and will not change even in the
presence of an excess of solute.
(7) A "solution" is a homogeneous mixture of two or more
substances constituting a single phase.
C. Principle of the Test Method
The test method is based on the conventional method of
preparing saturated aqueous solutions. The method involves the
coating of the compound to the walls of a vessel, adding water
(i.e., very pure water, buffer solution, or artificial seawater),
and determining the concentration of the compound in the water as
a function of time at a fixed temperature. When the
concentration reaches a plateau, equilibrium has been achieved,
and the water is saturated v, ith the compound. Specific
procedures have bee .. incorporated in this test guideline to
measure the water solubility of very hydrophobia compounds and to
alleviate the problems of colloids and emulsions usually formed.
D. Applicability and Specificity
Procedures have been described in this test guideline to
determine the saturated water solubility for liquid or solid
compounds. The water solubility can be determined in very pure
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water, buffer solution for compounds that reversibly ionize or
protonate, or in artificial seawater as a function of temperature
(i.e., in the range of temperatures of environmental concern).
Water solubility is usually not useful for qases because their
solubility in water is measured when the qas above the water is
at a partial pressure of one atmosphere which is several orders
of magnitude greater than those existing under environmental
conditions. A more important parameter for gases is Henry's law
constant which is the ratio of the vapor pressure of the compound
to solution concentration at low partial pressures.
This test guideline is designed to determine the saturated
water solubility of a solid or liquid test chemical in the range
infinity to 10 parts per billion (ppb). ^or chemicals whose
solubilitv is below 10 ppb, the water solubility should be
characterized as "less than 10 ppb" with no further
que.nt if i cat ion.
II. DESCRIPTION OF THE TEST PROCEDURE
A. Test Conditions
1. Special Laboratory Equipment
(1) A thermostatic bath with temperature control (±1°C) in
the approximate range of 5-30°C;
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(2) an ultracentrifuge with temperature control (±1°C) in
the approximate range of 5-30°C and capable of
obtaining acceleration (G) values to 39,000 or higher;
(3) a pH meter capable of resolving differences of 0.1 pH
units or* less; and
(4) centrifuge tubes with scalable caps: special glass
tubes can be used up to approximately 12,000 G; tubes
to be used at G values > 12,000 should be made of
stainless steel.
2. Purity of Water
Reagent grade water, e.g., water meeting ASTM Type IIA
standards or an equivalent grade, is highly recommended to
minimize biodegradation and to minimize the effects of dissolved
salts on water solubility. ASTM Type IIA water is described in
ASTM D1193-77, "Standard Specification for Reagent Water".
3. Purity of Solvents
It is important that all solvents used for coating test
compounds on the walls of vessels and in separation and
analytical technique be reagent grade and contain no impurities
which will interfere with the determination of the test compound.
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4. Seawater
It is recommended that artificial seawater he used to
determine the saturated water solubility in seawater. The
preparation of artificial seawater is described in Section
II.B.2.
5. Agitation and Equilibration Time
It is important that contact time of test compounds with
water be sufficient to obtain a saturated solution. The lenqth
of time necessary will depend upon such variables as the size of
the vessel, the extent and degree of aqitation, the properties of
the compound and particle size. To increase the rate of solution
of hydrophobia compounds, mild agitation is recommended. For
hydrophobia compounds a minimum time of one day is required.
6. Effects of Colloids and Emulsions; Centrifugation
It is important that gentle shaking be used to minimize the
formation of colloids. The presence of colloids and emulsions
will lead to solubility values that are higher than those in a
true saturated solution. This is a common problem with
hyrlrophobic solids and liquids but can usually be overcome by
centrifugation. It is recommended that centrifugation be
conducted in tightly sealed tubes that are almost filled to
capacity to avoid partitioning with air.
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It is extremely important that centrifuqation be carried out
at two or three different G values (minimum of 12,000 G) for at
least 30 minutes at 25°C until concentration chanqes are small.
For hydrophobic compounds (solubility _<_ 10 ppm) , it is extremely
important that the acceleration G values differ by 10,000 G and
include a determination of 39,000 or hiqher.
7. Effect of pH on Solubility
It is recommended that all experiments be carried out at
pH's 5.0, 7.0, and 9.0 for any chemical which reversibly ionizes
or protonates (e.g., carboxylic acids, phenols, amines). Buffers
described in Section II.B.2. can be used.
8. Analysis of Saturated Solutions
Any suitable analytical method may be used; where
practicable, precision should be within ±5 percent. Preferred
analytical methods are those that are specific for the compound
to be tested, to the exclusion of other compounds.
Chromatoqraphic methods which incorporate separation, and
therefore,specification, are recommended.
9. Adsorption to Glass or Other Surfaces
Hydrophobic compounds have a tendency to adsorb to qlass or
other surfaces, e.q., stainless steel. Thus, when transferring
the solution to any qlass vessel or container, it is essential to
pre-rinse the surfaces of the vessel or container with the
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solution. Failure to do so will lead to solubility values that
are lower than those of true equilibrium water solubility because
the compound will adsorb to the unrinsed surface. However, when
hydrophobic compounds are extracted with organic solvent, the
extraction vessels should not be pre-rinsed since this would lead
to solubility values that are greater than those of true
equilibrium water solubility.
R. Preparation of Reagents and Solutions
1. Buffer Solutions
Prepare buffer solutions using reagent grade water as
fol lows:
pH 5.00 To 250 mL of 0.1M potassium hydrogen
phthalate add 113 mL of 0.1M sodium
hydroxide; adjust the final volume to
500 mL with reagent grade water.
pH 7.00 To 250 mL of O.lM potassium dihvdrogen
phosphate add 145 mL of O.lM sodium
hydroxide; adjust the final volume to
500 mL with reagent grade water.
pH 9.00 To 250 mL of 0.075M borax add 69 mL of
O.lM HCl; adjust the final volume to 500
mL with reagent grade water.
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Check the pH of each buffer solution with a pH meter at 25°C and
adjust to pH 5.0, 7.0, or 9.0, if necessary. If the pH of the
solution has changed by ±0.2 pH units or more after the addition
of the test compound, then a more concentrated buffer is required
for that pH determination. The sponsor should then choose a more
suitable buffer.
2. Artificial Seawater
Add the reagent-grade chemicals listed in Table 1 in the
specified amounts and order to 890 mL of reagent-grade water. It
is important that each chemical be dissolved before another one
is added.
C. Performance of the Test
Determine the saturated water solubility of the test
compound at 25°C in reagent grade water or buffer solution, if
appropriate. Under certain circumstances, it may be necessary to
determine the water solubility of a test compound at 25°C in
artificial seawater. The water solubility can also be determined
at other temperatures of environmental concern by adjusting the
temperature of the water bath to the appropriate temperature.
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Table l--Constituents of Artificial Seawater1
Chemical Amount
NaF 3 mg
SrCl2'6H20 20 mg
H3B03 30 mg
KBr l;00 mg
KC1 700 mg
CaCl2'2H20 1.47 g
Na2SO4 4.00 g
Mgd2'6H20 10.78 g
NaCl 23.50 g
Na2Si03'9H2O 20 mg
NaHC03 200 mg
If the resulting solution is diluted to 1 cubic decimeter (1
liter), the salinity should be 34 ± 0.5 g/kg and the pH 8.0 ±
0.2. The desired test salinity is attained by dilution at time
of use.
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1. Procedure for the Determination of
Solids and Liquids in Water at 25°C
Dissolve a sufficient amount of the solid compound in a
suitable volatile organic solvent and coat on the walls of a
«
vessel. Viscous liquids may be coated on vessels in a similar
fashion; non-viscous liquids do not require solvents. Remove the
solvent under reduced pressure or with a pure nitrogen gas
stream. When all the solvent is removed, add reagent grade water
or, for compounds which reversibly ionize or protonate, the
appropriate buffer solution and slowly stir or agitate the
mixture under temperature control. Mixing may be accomplished by
use of a teflon coated stirring bar and should be continued for a
minimum of 24 hours before aliquots are withdrawn. Prior to
taking aliquots, the mixture should be left to stand at constant-
temperature for at least one hour to permit separation of any
small particles. To determine the concentration of the compound
in the aqueous phase, aliquots should be centrifuged at two or
three different G values (minimum of 12,000 G) for at least 30
minutes at 25°C until concentration changes are small. The
concentration value so obtained is plotted against the time of
mixing. At a later time, aliquots are again taken and analyzed
in the same fashion to produce another data point on a
concentration vs. time plot. When the concentration reaches a
plateau, equilibrium is assumed. For hydrophobic compounds
(solubility _^ 10 ppm) it is extremely important that the
acceleration (G) values differ by 10,000 G and include a
determination at 39,000 G or higher.
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CG-1500
For more soluble compounds (solubility _>_ 500 ppm) coating
the walls of the vessel is not necessary and filtration may be
substituted for centrifugation. Use filters which are adequate
to remove suspended particles. If the concentration of the
solute exceeds 10 g/dm , then determine the density of the
solution. This can be done by weighing known volumes of the
solution at the same temperature as the constant temperature
bath. Sufficient solution should be used so that each
determination is made on a fresh aliquot. Carry out solubility
and density experiments in triplicate.
2. Modification of Procedures for Potential Problems
a. Interference of Soluble Impurities
Interference by soluble impurities in the test sample can be
avoided by the use of an analytical technique that is specific to
the compound being tested. If this is not practical,
interferences can sometimes be minimized by repeatedly preparing
saturated solutions from the same sample chemical until the
concentration of the impurity has been depleted.
b. Decomposition of the Test Compound
If the test compound decomposes in one or more of the
aqueous solvents required during the period of the test at a rate
such that an accurate value for water solubility cannot be
obtained, then it will be necessary to carry out detailed
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transformation studies e.q., hvdrolvsis. If decomposition is due
to aqueous photolysis, then it will be necessary to carry out
water solubility studies in the dark, under red or vellow liqhts,
or by any other suitable method to eliminate this transformation
process.
TTI. DATA AND REPORTING
A. Test Report
for each set of conditions, (e.q., temperature, nure water,
buffer solution, artificial seawater) required for the study,
provide the water solubility value for each of three
determinations, the mean value, and the standard deviation.
For compounds that decompose at a rate such that a precise
value for the water solubility cannot be obtained, provide a
statement to that effect.
For compounds with water solubility below 10 ppb, report the
value as "less than 10 ppb".
For compounds with water solubility qreater than 10 q/dm ,
report the density of the solution at each required temperature.
Summarize all the data in the data sheets listed in Anpendix 1
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B. Specific analytical and Recovery Procedures
(1) Provide a detailed description or references for the
analytical procedure used, including the calibration
data and precision; and
(2) if extraction methods were used to separate the solute
from the aqueous solution provide a description of the
extraction method as well as the recoverv data.
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IV.
ASTM. 1978. Annual Hook of ASTM Standards, American ^ocietv for
Testinq and Materials, Philadelphia, Pa., Part 31, Method n
1193-77.
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CG-1500
V. APPENDIX 1: DATA FORMAT SHEETS
Inst ruct ions
If multiple temperatures are required, complete multiple conies
of the Test Results pages, one set of test results reported for
each temperature at which the test was conducted.
If multiple pH values are required, complete multiple copies of
the Test Kesults paqes, one set of test results reported for each
pH tested.
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TEST RESULTS
J. Coating solven! (if used)
2. Solubility deternmed in I I I I
Reagent grodo water 101
Buffer solution 102
Rrtificiol seawoter 103
3. If *2 is buffer solution, pH of solution J_
4. flccelerotion (G) values for centrifugotion
5. If filtration was substituted for centrifugotion, describe the
tu,pe f i I tens used.
6. Concentration units
7. Tine units
8. Tenperature fT^C ±rT~l°C
flbbreo.
flbbreu.
Sonp 1 i ng
T i «e
9.
_
Run 1
Run 2
Run 3
Ploteou
13. Mean Saturated Equilibrium Concentration
14. Standard Deviation
12.
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TEST RESULTS
CONTINUED
('...port the density, of the solution for concen tro t i ons equo I to or
rj router than 10 gdn , and the tenperoture ot uhich it nc.s Measured,
15.
Run 1
16
|
m
\ \
Run
17.
2
Dens i tu,
1
Run
iqcn ,)
3
18.
1 1
1
Meon
19
1
,
1
Std. Dew.
20.
±1 1
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August, 1982
WATER SOLUBILITY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CS-1500
Contents
Page
I. NEED FOR THE TEST 1
II. SCIENTIFIC ASPECTS 3
A. Rationale for the Selection of the
Test Method 3
B. Other Methods of Determining
Water Solubility 7
1. Interf erometry 7
2. Nephelometry 8
3. Coupled Column Liquid
Chromatographv 10
C. Rationale for the Selection of
Test Conditions 11
1. Temperature 11
2. ourity of Water 1?
3. Purity of Solvents 13
4. Seawater 13
5. Agitation and Equilibrium Time 13
6. Effects of Colloids and Emulsions:
Centrif ugation 14
7. Effect of pH on Solubility 14
8. Analysis of Saturated Solutions 15
9. Adsorption to Glass or Other
Surfaces 15
D. Test Data Required 17
E. Statistical Analysis of the Data 17
III. REFERENCES 20
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WATER SOLUBILITY
I. NEED FOR THE TEST
The water solubility of a compound can be defined as the
equilibrium concentration of the compound in a saturated aqueous
solution at a given temperature. The water solubilitv is a
fundamental physical property of a compound. It is an essential
characteristic for determining that compound's movement and
distribution in the environment and, therefore, its potential
effects on living organisms. Highly soluble compounds are more
likely to be distributed by the hydrologic cycle than less
soluble compounds. The degree of water solubilitv of a compound
can affect Its adsorption and desorption on soils and sediments
and ease of volatility from aquatic systems. Substances which
are more soluble in water are more likely to desorb from soils
and sediments and less likely to volatilize from water.
Transformations such as hydrolysis, photolysis, oxidation,
reduction, and biodegradation in water proceed more rapidly when
the compound is dissolved in water (i.e., homogeneous
kiner,ics). The potential importance of these transformations is,
therefore, a function of the ability of a compound to dissolve in
water. The design of most chemical tests and many ecological and
health tests requires accurate knowledge of the water solubility
of the compound to be tested.
Virtually all modeling systems devised to determine the
distribution of a compound in the environment require water
solubility data. Although attempts have been made to correlate
water solubility with other physical parameters such as
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structure, enthalpy of fusion, melting point and molar volume, in
order to predict or calculate the water solubility of a compound,
it is generally agreed that an actual physical measurement of
water solubility is a far more accurate and desirable approach
(Mader and Grady 1970). In addition, values of water solubility
have been shown to correlate with the octanol/water partition
coefficient (KQW) for a number of compounds (Chiou et al. 1977,
Yalkowsky and Valvani 1979, 1980, 1981). These correlations
enable a quite accurate estimate of a compound's K to be made
from its water solubility.
Water provides the medium in which many organisms live, and
water is a major component of the internal portion of all living
organisms (except for dormant stages of certain life forms).
Even organisms which are adapted to life in a gaseous environment
require water for normal functioning. Water is thus the medium
through which most chemicals are transported to and into living
cells. As a result, the extent to which chemicals dissolve in
water will be a major determinant for movement through the
environment and entry into living systems.
Water solubility is an essential parameter for assessing the
environmental partitioning of all solid or liquid chemicals.
Water solubility is usually not useful for gases because their
solubility in water is measured when the gas above the water is
at a partial pressure of one atmosphere which is several orders
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of magnitude greater than those existing under environmental
conditions. Thus, water solubility of gases does not generally
apply to environmental assessment because the actual partial
pressure of a gas in the environment is extremely low.
II. SCIENTIFIC ASPECTS
A. Rationale for the Selection of the Test Method
Analytical methods for the determination of water solubilitv
consist of obtaining an equilibrium saturated aqueous solution of
the compound and analyzing the solution by some suitable physical
or chemical method. Equilibrium may be obtained by intimately
mixing the solute and solvent. Separation of the phases can then
be accomplished by filtration or decantation, usually followed by
cent rifugation.
The solubility of a compound in water at equilibrium is a
function of temperature, pressure, and purity of solute and water
(Mader and Grady 1970). A major practical difficulty is
determining that equilibrium has actally been attained. There
are two general methods to determine the attainment of
equilibrium. The concentration vs. time method (Mader and Gradv
1970) involves periodic sampling over meaningful periods o^ time;
when the concentration has reached a plateau, equilibrium has
been attain»d. The undersaturation/oversaturation method (Mader
and Grady 1970) refers to approaching equilibrium from both
possible non-equilibrium conditions. One of two identical
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solutions is heated to a temperature well above the required test
temperature so that the solubility is increased. Then both
solutions are placed in a thermostatic bath to equilibrate at the
required test temperature. The heated solution becomes
supersaturated (oversaturated) when its temperature decreases and
reaches equilibrium as the concentration of the dissolved solute
decreases to a plateau at the required test temperature. T'he
other solution is undersaturated and reaches equilibrium as the
concentration of dissolved solute increases to a plateau at the
required test temperature. If identical concentrations in
aliquots of these two samples are obtained after a period of
time, equilibrium has been reached. Research sponsored bv ^PA at
the Rattelle Institute demonstrated that the under/oversaturation
method will give poor analytical results if the compound tested
underqoes a phase transition in the temperature ranqe utilized
(EPA 1981). Thus, a low melting solid may recrystallize at a
very slow rate after being heated to elevated temperatures and
make separation of excess solute from the aqueous phase extremelv
difficult. Other potential problems include accelerated
decomposition of the test material at elevated temperatures and
dramatically different changes in solubility rates of impurities
vs. test compound with change in temperature.
The method described in the test guideline involves coating
the compound on the walls of a vessel, adding very pure water,
and determining the change in concentration of the compound in
the water over a period of time at a fixed temperature (Hague and
Schmedding 1975, Karickhoff and Rrown 1979). Typically, the
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concentration of a compound dissolved in the aqueous phase will
increase with time. As the solution proqresses toward
saturation, the rate of chanqe of concentration will decrease and
ultimately, the concentration will reach a plateau. ^he
formation of that plateau after a suitable period of time has
elapsed is generally considered an accurate indication that
equilibrium has been established. The coatinq of solids on the
walls of the vessel is considered to be more practical than
suspending the solid as small particles in water since the
coatinq technique minimizes the formation of microcrystals that
are difficult to remove.
The use of a sinqle procedure to cover the entire ranqe of
solubilities from infinity down to 10 ppb has the obvious
advantage of not havinq to chanqe procedures near the cut-off of
two ranqes. Sufficient flexibility has been incorporated into
the test guideline to accomodate a wide ranqe of solubilities.
The use of a "suitable solvent" for coating the vessel walls
allows the sponsor to choose a solvent that is more appropriate
for the test, compound in recognition of the possible variation in
the ohvsical properties of different compounds. Tvoically,
solvents such as acetone, methanol, ether, dichloromethane, and
hexane can be employed.
It has been shown that constant but erroneous solubility
values can be obtained when very small droplets or particles
remain dispersed in water (Biggar and Riggs 1974). To separate
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these collodial particles, centrifugation (or ultracentri-
fugation) is necessary (Biggar and Riggs 1974). If constant
concentration values are obtained after centrifugation at several
different acceleration (G) values, one can assume that removal of
collodial particles has been achieved. The centrifuge tubes
should be tightly sealed because the test compound can escape
from the solution through volatilization, esnecially for those
compounds with an appreciable vapor pressure. In addition, the
tubes should be filled almost to capacity to minimize
partitioning to air.
The less soluble the test compound, the ireater the error
introduced by the presence of suspended particles. For example,
the suspension of 100 yg/dm^ o? undissolved material is small i*
the true solubilitv is 100 mg/dm^ (error of 0.1 percent) but is
extremely large if the true solubility is 100 yg/dm (error of
100 percent). Thus, more extensive ultracentrifuge procedures
will be reguired for more hydrophobic compounds. It has been
demonstrated that reliable solubility determinations can be made
for very hydrophobic chemicals by centrifuging the suspension at
two or three different G values for one-half hour at constant
temperature until concentration changes are small (Biggar and
Riggs 1974). For several pesticides, centrifugation at 39,000 G
removed the colloidal particles corresponding to 0.1 urn particle
size. For DDT, it had been demonstrated that one hour of
centrifugation at 84,000 G removed all the collodial particles
(Bowman, Acree, and Corbett 1960). For chemicals that are
soluble in the range 500 to 10 ppm, aliquots of the suspension
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CS-1500
must he centrifuged at two or three different G values for at
least 30 minutes at constant temperature until concentration
changes are small. por hydrophobia chemicals (solubility < 10
ppm), it is extremely important that acceleration (G) values
differ by 10,000 r, and include a determination at 19,000 1 or
higier. When determining the solubility of relatively soluble
compounds (SSOO ppm), large solid particles can be conveniently
separated by filtration, and macropore filters may be employed as
par:: of the separation technique.
It is recommended that the analytical technique be selected
by the sponsor and/or testing laboratory in recognition of the
many different techniques available and the advantage of being
able to match one to the properties of the compound (e.g, the
degree of solubility, the snectroscooic nrooerties of the
compound, and its chromatographic behavior). Analytical
techniques that allow the quantitative determination of the test
compound to the exclusion of impurities are recommended to the
extent practicable. Therefore, chromatogranhic techniques are
recommended.
^ Other Methods of Determining Water Soluhili-.v
1. Tnterferometry
This method has been used to determine the solubilitv of
slightly soluble solids and liquids in water in the ppm range
(Adams 191S, Mader and Grady 1970). limited research has been
_ ~>
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CS-1500
done on this method, and the technique has only been applied to a
few compounds. It does not have general applicability.
2. *Tephelometry
The nephelometric (turbidity) method calls for preparation
of stable suspensions of the organic chemical at several
different concentrations exceeding the nominal solubility. ^he
turbidities of the resulting suspensions are then measured and
plotted against total concentration. ^ beer's Law relationship
is assumed; thus, a straight line drawn through the points yields
an intercept equal to the solubility, i.e., the concentration at
which the turbidity vanishes (Davis and Parke 1942).
In tests of this method a complete suspension r>f excess
solute could not be accomplished by mechanical means such as
sonication. In order to obtain a complete suspension, the
procedure requires that the compound be dissolved in a solvent
that is miscible with water. Initial amounts o^ dissolved solute
that are added tend to disperse in the water and form a true
solution. However, as the solubility limit is approached, turbid
zones are produced that persist briefly depending on the stirring
efficiency, and a permanent turbidity is formed at the solubility
limit.
In principle, this approach can be used to give a rough
initial estimate of the solubility. The resulting solubility is
subject to several conditions and limitations, including the
-8-
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inaccuracy and lack of; reproducibility of; the individual
t.urbiditv measurements, the instability of the oriqinal narticle
siz:s distribution (a function of time and dilution), and the
eff.?ct of the dispersinq agent (solvent) on the solubility.
These conditions and limitations are not independent of each
other. For example, the use of solvent pairs to form a liquid
having solvent oroperties intermediate between those of the
parent solvents is a well-recognized technique (e.g., as applied
with gradient elution HPLC). Thus, not only is it to be expected
that, the use of a dispersing agent might influence the true
solubilitv, but the solubility and hence the particle size
distribution might be expected to change as a function of
dilution of the stock solution. Also, in cases where the
solubility is strongly influenced by the presence of the
dispersing solvent, evaporative loss of the dispersing solvent
can load to continued precipitation of the solute and thus
.. ifluence the oarticle size distribution and apparent turbiditv.
The stability of the particle size distribution can also be
affected by the nature of the solute. With a solute that has a
relatively high mobility, it can be expected that the large
particles will grow at the expense of the smaller particles thus
altering th<> apparent turbidity. If the solute is volatile, an
appreciable fraction of the solute may be lost from the
: -> -">:-. ; m, and recrystal 1 izat ion may occur on the surface of the
;r. :r : 01 ]>,>.: surfaces external to the bulk liquid.
-9-
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CS-1500
Therefore, the nephelometric method has been rejected as a
general method for determining the water solubility of organic
compounds.
3. Coupled Column Liquid Chromatography
This is a recently published method used to determine the
aqueous solubility of hydrophobic polycyclic aromatic
hydrocarbons (May, Wasik, and Freeman 1978a,b). The method
consists of pumping water through a column containing glass beads
coated with the compound being studied. The beads are prepared
by adding them to a 0.1 percent methylene chloride solution of
the test compound and stripping the solvent with a rotary
evaporator. The coated beads are packed into stainless steel
tubes. Saturated solutions are generated by pumping water
through these thermostated columns at flow rates ranging between
0.1 and 5 mL/min. These saturated solutions are extracted by
flowing a measured volume of solution through a stainless steel
extactor column where the material is adsorbed to a special
column packing. Another solvent system is then used to elute the
test material from the extactor column onto an analytical column
where its concentration is measured by a standard liquid
chromatography detector system. It is necessary to calibrate the
detector signal by using solutions of known concentration.
Precision in the ppb range is excellent (±3 percent). The method
appears promising but needs more experimental work on a variety
of hydrophobic chemicals before it can be determined whether or
not to recommend the procedure.
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C. Rationale for the Selection of the
Test Conditions
1. Temperature
The equilibrium water solubility of a substance is a
constant at a fixed temperature and pressure. For most solid and
liquid compounds, an increase in temperature results in an
increase in water solubility. The rate of change in water
solubility with temperature is not linear for a given chemical
and varies dramatically from one substance to another (Mader and
Grady 1970). For many compounds the following relationship
describes concentration (water solubility) as a function of
temperature:
1 _ AH 1 1
X R~ (T ~T
f
where Xa is the mole fraction of compound A, AH is the enthalpy
of fusion, Tf is the thermodynamic temperature of fusion and R is
the universal gas constant (Bigger and Riggs 1974). The equation
can be put into the form log10C = - § + B, where C = molar
concentration, T is the thermodynamic temperature and A and B are
constants. By determining C as a function of T (e.g., at 5, 15,
25°C), the data can be used to determine A and B (i.e., plot
-11-
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CS-1500
vs. 1/T; the slope of the line is ^ and the intercept is
B). Thus, one can use this equation to estimate C at any
environmental temperature of concern. zvs a working estimate,
scientists often anticipate a doubling of solubility with an
increase of 10°C. The temperature chosen for this test guideline
is 25°C since this is the temperature traditionally used for
reporting physical properties. Solubility determinations mav be
requested at additional temperatures of environmental concern for
specific compounds or classes of compounds.
Since water solubility values will change with temperature
(Mader and Grady 1970), the temperature should be controlled to
±1°C. This is a condition easily achieved bv standard
temperature control devices for water baths and is a practical
range Cor temperature controlled centrifuges.
2. Purity of Water
Dissolved salts can affect the water solubilitv of a
compound, necessitating the use of very pure water (Mader and
Grady 1970). In addition, the water should be relatively free of
bacteria which may consume or alter the organic test material
during extended periods of testing. ASTM Type II reagent grade
water (ASTM 1978), or an equivalent grade containing less than I
mg/dm total organic carbon, has been recommended for use in this
test guideline to minimize biodegradation and the effects of
impurities.
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3. Purity of Solvents
Organic solvents that come into contact with the test
material arid aqueous solution must be as pure as practicable.
Trace organic impurities that are soluble in water can alter the
water solubility of the test material as well as interfere with
the analysis of the concentration of the test material.
4. Seawater
Organic compounds are often released to soawater. The
presence of dissolved salts will usually alter the true water
solubility of a compound. Typically, the water solubility of an
organic compound will be less in seawater than in pure water at a
fixed temperature (Long and McDevit 1952). Data on solubility in
seawater is needed to design ecological testing of marine species
and for modeling. Salinity varies in different marine
environments. In order to determine solubility under uniform
conditions, a formulation for artificial seawater is specified in
the test guideline (Kester et al. 1967, EPA 1975a).
5. Agitation and Equilibration Time
To increase the rate of solution of hydronhobic compounds,
mile agitation is recommended. The use of strong agitation,
while increasing the rate of solution, also increases the
formation of emulsions and colloids by producing very small
particles which remain suspended in solution. For hydrophobic
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compounds, experience has shown that a minimum of one day with
mild agitation is necessary to reach equilibrium, and several
weeks are often required (Haque and Schmedding 1975).
6. Effects of Colloids and Emulsions; Centrifugation
The use of a centrifuge allows the rapid separation of small
particles suspended in water. The size of the particles
separated depends on the acceleration factor (G value) and the
length of time the sample is centrifuged. Centrifugation at two
or three different G values will partition suspended particles to
two or three different distributions. When the water solubility
value obtained is relatively constant at two different G values,
this indicates that any suspended particles present are
relatively insignificant (Biggar and Higgs 1974). It is
important that Centrifugation be conducted in tightly sealed
tubes that are almost filled to capacity to avoid partitioning
with air and loss of chemical via volatilization.
7. Effect of pH on Solubility
It is known that the water solubility of some organic
compounds will alter with change in the pH of the solution
(Cheung and Biggar 1974). This change will not be dramatic
unless the compound contains readily ionizable or protonated
groups. For carboxylic acids, amines and compounds which
reversibly ionize or protonate in water, it is necessary to
-14-
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CS-1500
measure the water solubility at pH values of 5.0, 7.0, and 9.0,
since these pH values are representative of those found in
natural aquatic ecosystems, groundwater, and rainwater.
The specified buffer solutions contain an adequate
concentration, of buffer to neutralize approximately 100-500 ppm
of dissolved test compound (depending on its molecular weight)
without a significant change in pH and, therefore, will be
satisfactory for most test compounds. A change in pH of 0.2
units signifies that a more concentrated buffer solution is
needed. In such cases, the choice of buffer solution should be
made by the sponsor in order for it to be compatible with their
analytical scheme.
8. Analysis of Saturated Solutions
The diversity of compounds to be tested precludes the
specification of a limited number of analytical techniques; the
choice is best left to the sponsor. A great many analytical
procedures are potentially useful. Nonetheless, the use of a
compound-specific analytical procedure has been recommended for
use in this test guideline. Since chromatographic techniques
entail separation as well as quantification for many organic
compounds, these methods have been recommended. Where procedures
are available, chromatographic techniques are cost effective.
However, determining the proper chromatographic procedure can be
time consuming. Depending upon the physical properties exhibited
by the compound and its equilibrium concentration in water, other
-------
CS-lbOO
standard techniques can be more efficient with resnect to ease of
analysis, urecision, and cost. Whenever practicable, an
analytical procedure should be used havinq a precision within
±5 percent. Scientists are qenerally aware of the wide varietv
of standard techniques available todav and of the larqe number of
methods beinq developed, so that a discussion of even a small
percentaqe of analytical possibilities would serve little
purpose. This test quideline does require, however, that the
specific analvtical technique utilized be adequatelv described.
Some procedures involve the use of extractinq solvents and, when
so used, it is important that these extraction procedures be
adequatelv described and recovery information be submitted.
ct. .\dsorotion to Glass or Other Surfaces
Hydrophobia compounds will have a tendencv to adsorb to
qlass or other surfaces (e.q., stainless steel). Thus, when
transferrinq the test solution to any vessel or container, it is
essential to ore-rinse the surfaces with the solution. Failure
to do so will lead to solubility values that are lower than those
of true equilibrium water solubilitv. However, when hydrophobic
compounds are extracted with orqanic solvent, their containers
should not be pro-rinsed since this would lead to solubilitv
values that are qreater than those of true equilbrium water
-15-
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D. Test Data Required
Present analytical techniques allow the determination of the
concentration of organic compounds in water as low as the parts
per billion range and, in time, this capability may be
extended. However, using current techniques the reliability and
precision below 10 ppb will be ±50 percent at best. Because of
these inherent inaccuracies, the greatly increased costs
associated with quantification below in ppb would be difficult to
justify. Furthermore, organic substances that are so hydrophobic
that their water solubility is less than 10 ppb may disperse in
wat'?r forming micelles rather than being truly soluble. Thus,
for substances whose water solubility is below 10 ppb, the test
guideline requires that the water solubility be characterized as
"less than 10 ppb" with no further quantification.
When the solubility of a compound is equal to or greater
than 10 g/dnr', it is important that the density of the saturated
solution be determined experimentally and reported. For
solutions of such concentration, density is needed to convert
frori a concentration of g/dm to other units (e.g., ppm, mole
fraction, molality).
E. Statistical Analysis of the Data
Numerous researchers have published measurements of water
solubility using a variety of methods (Gunther et al. 1968). For
nany methods, good precision (i.e., repeatable values) can be
obtained but the comparision of one method to another may give
-17-
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larger differences than would be expected from the precision of
each one. Therefore, though precise, the water solubility data
may not be accurate (i.e., correct). For example, the water
solubility of DDT has been investigated by many researchers. It
was finally demonstrated that values obtained depended upon the
size distribution of suspended particles in the "saturated
aqueous solution". Suspended particle size distribution was a
function of time of centrifuging and acceleration (G) values
(Biggar and Riggs 1974). Therefore, significantly different-
values (each with good precision) could be obtained for the
different sets of conditions. This problem is more significant
for very hydrophobic compounds than for those which are more
water soluble. It is one of the reasons why the method does not
require quantification below 10 ppb.
The precision of the water solubility data generated by the
proposed general method has not been clearly established. As the
solubility decreases, the precision is expected to become
poorer. The precision is also a function of the nature of the
specific compound and the analytical procedure used. Therefore,
no reliable precision can be stated at this time for determining
the water solubility. Obviously, the precision can be improved
by making numerous replicate determinations. However, in order
to minimize costs, it has been decided to determine water
-18-
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CS-1500
solubilitv with three reolicates. therefore, it is extremelv
important that the submitter of the test results analvze the data
statistically. ^fter the water solubility of a larqo number of
compounds of various types has been determined by this method,
the level of precision can be defined for various ranqes.
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CS-1500
III. REFERENCES
Adams LH . 1915. The use of the interferometer for the analysis
of solutions. J Amer Chem Soc 37:1181-1194.
ASTM. 1978. American Society for Besting and Materials. Annual
book of standards. Part 31. Method D 1193. Philadelphia, PA.
Biggar JW, Rigqs RL. 1974. Apparent solubility of
organochlorine insecticides in water at various temperatures.
Hilgardia 42:383-391.
Bowman MC , Acree Jr. F, Corbett MK. 1960. Solubility of carbon-
14 DOT in water. J Agr Food Chem 8:406-408.
Cheung MW, Biggar JW. 1974. Solubility and molecular structure
of 4-amino-3 , 5 , 6-trichloropicolinic acid in relation to pH and
temperature. J Agr Food Chem 22:202-206
Chiou CT, Freed VH, Schmedding DW, Kohnert RL. 1977. Partition
coefficient and bioaccumulation of selected organic chemicals.
Environ Sci Technol 11:475.
Davis WW, Parke Jr TV. 1942. A nephelometric method
determination of solubilities of extremely low order. J Amer
Chem Soc 64:101-107.
EPA. 1975a. Environmental Protection Agency. Methods o^ acute
toxicity tests with fish, macroinvertebrates, and amphibians.
EPA-660/3-75-009. PB 242105.
Gunther FA, Westlake WE, Jaglan PS. 1968. Reported solubilities
of 738 pesticide chemicals in water. Residue Rev 20:1-148.
Haque R, Schmedding D. 1975. A method of measuring the water
solubility of hydrophobic chemicals. Bull Environ Contam Toxicol
14: 13-18.
Karickhoff SW, Brown DS . 1979. Determination of octanol/water
distribution coeficients, water solubilities, and sediment/water
partition coefficients for hydrophobic organic pollutants.
EPA-600/4-79-032.
Kester DA, Duedall IW, Connors DN, Pytkowicz RM. 1967.
Preparation of artificial sea water. Limnol and Oceanogr
12:17 6- 179.
Long FA, McDevit WF . 1952. Activity coefficients of
nonelectrolyte solutes in aqueous salt solutions. Chem Rev
51: 119-155.
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CS-1500
Mader WJ, Grady LT. 1970. The determination of solubility. In:
Techniques of Chemistry. Vol. 1, Part V, Chapter V.
A. Weissberger and B.W. Rossiter, Editor. Wiley Interscience.
Mew York, NY.
May WE, Wasik SP, Freeman DH. 1978. Determination of aqueous
solubility of polynuclear aromatic hydrocarbons by a coupled
column liquid chromatographic technique. Anal Chem 50:175-179.
May WE, Wasik SP, Freeman DH. 1978. Determination of solubility
behaviour of some polyaromatic hyrocarbons in water. Anal Chem
50:997-1000.
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CG-1600
August, 1982
VAPOR PRESSURE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-1600
Contents
Page
INTRODUCTION 1
A. Background and Purpose 1
B. Definitions and Units 2
C. Principle of the Test Methods 3
D. Applicability and Specificity 5
II., TEST PROCEDURES,
A. Test Conditions 6
B. Performance of the Tests 6
1. Isoteniscope Procedure 6
2,, Gas Saturation Procedure. . 7
II". DATA AND REPORTING 14
IV,, REFERENCES 15
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CG-1600
VAPOR PRESSURE
I. INTRODUCTION
A. Background and Purpose
Volatilization, the evaporative loss of a chemical, depends
upon the vapor pressure of chemical and on environmental
conditions which influence diffusion from a surface.
Volatilization is an important source of material for airborne
transport and may lead to the distribution of a chemical over
wide areas and into bodies of water far from the site of
release. Vapor pressure values provide indications of the
tendency of pure substances to vaporize in an unperturbed
situation, and thus provide a method for ranking the relative
volatilities of chemicals. Vapor pressure data combined with
water solubility data permit the calculation of Henry's law
constant, a parameter essential to the calculation of volatility
from water.
Chemicals with relatively low vapor pressures, high
adsorptivity onto solids, or high solubility in water are less
likely to vaporize and become airborne than chemcials with high
vapor pressures or with low water solubility or low adsorptivity
to solids and sediments. In addition, chemicals that are likely
to be gases at ambient temperatures and which have low water
solubility and low adsorptive tendencies are less likely to
transport and persist in soils and water. Such chemicals are
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CG-1600
less likely to biodegrade or hydrolvze and are prime candidates
for atmospheric oxidation and photolysis (e.g., smoq formation or
stratospheric alterations). On the other hand, nonvolatile
chemicals are less frequently involved in atmosphere transport,
so that concerns regarding them should focus on soils and water.
Vapor pressure data are an important consideration in the
design of other chemical fate and effects test; for example in
preventing or accounting for the loss of volatile chemicals
during the course of the test.
R. Definitions and Units
(1) "Desorntion efficiency" of a particular comnound
applied to a sorbent and subsequently extracted with a
solvent is the weight of the compound which can be
recovered from the sorbent divided by the weight of the
compound originally sorbed.
(2) "Pascal" (Pa) is the standard international unit of
vapor pressure and is defined as newtons per square
meter (N/m ). A Newton is the force necessary to qive
acceleration of one meter per second squared to one
"kilogram of mass.
(3) The "torr" is a unit of pressure which equals 133.3
pascals or 1 mm Hg at 0°C.
-2-
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C C, - \ 6 0 0
(4) "Vapor pressure" is the pressure at which n liquid or
solid is in equilibrium with its vapor at a given
temperature.
(5) "Volatilization" is the loss of a substance to the air
from a surface or from solution by evaporation.
G. Principle of the Test Methods
The isoteniscope procedure uses a standardised -echnigue
[ASTM 1978] that was developed to measure the vanor pressure of
certain liquid hydrocarbons. T'he sample is nurified within the
equipment by removing dissolved and entrained qases until the
measured vapor pressure is constant, a process called
"degassing.." Impurities more volatile than the samn"!n win tend
to increase the observed vapor nressure and thus must be
minimized or removed. Results are subject to only slight error
for samples containing nonvolatile impurities.
Gas Scituration (or transpiration) procedures use a current
of inert gas passed through or over the test material .slowly
enough to ensure saturation and subsequent analysis of either the
loss of material or the amount (and sometimes kind) of vapor
generated. Gas saturation procedures have been described by
Spencer and Cliath (1969). Results are easy to obtain and can be
qui-.e precise. The same procedures also can be used to study
volatilization from laboratory scale environmental simulations.
Vapor pressure is computed on the assumption that the total
pressure of a mixture of gases is equal to the sum r><~ the
-3-
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CG-1600
pressures of the separate or component gases and that the ideal
gas law is obeyed. The partial pressure of the vapor under study
can be calculated from the total qas volume and the weight of the
material vaporized. If v is the volume which contains w grams of
the vaporized material having a molecular weight M, and if p is
the pressure of the vapor in equilibrium at temperature T (K),
then the vapor pressure, p, of the sample is calculated by
p = (w/M)(RT/v)
-\ -I -1
where R is the gas constant (8.31 Pa m mol K ) when the
pressure is in pascals (Pa) and the volume is in cubic meters.
As noted by Spencer and Cliath (1970), direct vapor pressure
measurements by gas saturation techniques are more directlv
related to the volatilization of chemicals than are other
techniques.
In an effort to improve upon the procedure described bv
Spencer and Cliath (1969) and to determine the applicability o^
the gas saturation method to a wide variety of chemical tvpes and
structures, EPA has sponsored research and development work at
SRI International (EPA 1982). The procedures described in this
Test Guideline are those developed under that contract and have
been evaluated with a wide variety of chemicals of differing
structure and vapor pressures.
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CG-1600
D. Applicability and Specificity
A procedure for measuring the vapor pressure of materials
released to the environment ideally would cover a wide range of
vapor pressure values, at ambient temperatures. No single
procedure can cover this range, so two different procedures are
described in this Test Guideline, each suited for a different
part of the range. The isoteniscope procedure is for pure
liquids with vanor pressures from 0.1 to 100 kPa. por vapor
pressures of 10 to 10 Pa, a gas saturation procedure is to be
used.
With respect to the isoteniscope method, if compounds that
boil close to or form azeotropes with the test material are
present, it. is necessary to remove the interfering compounds and
use pure test material. Impurities more volatile than the sample
will tend to increase the observed vapor pressure above its true
value but the purification steps will tend to remove these
impurities. Soluble, nonvolatile impurities will decrease the
apparent vapor pressure. However, because the isoteniscope
procedure is a static, fixed-volume method in which an
insignificant fraction of the liquid sample is vaporized, it is
subject to only slight error for samples containing nonvolatile
impurities. That is, the nonvolatile impurities will not be
concentrated due to vaporization of the sample.
The gas saturation method is applicable to solid or liquid
chemicals. Since the vapor pressure measurements are made at
ambient temperatures, the need to extrapolate data from high
-5-
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CG-1600
temperatures is not necessary and high temperature extrapolation,
which can often cause serious errors, is avoided. The method is
most reliable for vapor pressures below 10 Pa. Above this
limit, the vapor pressures are generally overestimated, probably
due to aerosol formation. Finally, the gas saturation method is
applicable to the determination of the vapor pressure of impure
materials.
II. TEST PROCEDURES
A. Test Conditions
(1) The apparatus in the isoteniscope method is described
in Section II .B.1.
(2) The apparatus used in the gas saturation method is
described in Section II.B.2.
B. Performance of the Tests
1. Isoteniscope Procedure
The isoteniscope procedure described as ANSI/ASTM Method
D 2879-75 is applicable for the measurement of vapor pressures of
liquids with vapor pressures of 0.1 to 100 kilopascals (kPa)
(0.75 to 750 torr). The isoteniscope method involves placing
liquid sample in a thermostated bulb (the isoteniscope) connected
to a manometer and a vacuum pump. Dissolved and entrained gases
are removed from the sample in the isoteniscope by degassing the
-6-
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CG-1600
sample at reduced pressure. T'he vapor pressure of the sample at
selected temperatures is determined by balancing the pressure due
to the vapor of the sample against a known pressure of an inert
gas. The vapor pressure of the test compound is determined in
triplicate at 25 ± 0.5°C and at any other suitable temperatures
(± ).5°). It is important that additional vapor pressure
measurements tae made at other temperatures, as necessary, to
assure that there is no need for further degassing, as described
in -he ASTM method.
2. Gas Saturation Procedure
(1) The test procedures require the use of constant-
temperature box as depicted in Figure 1. The insulated
box, containing sample holders, may be of any suitable
size and shape. The sketch in Figure 1 shows a box
containing three solid sample holders and three liquid
sample holders, which allows for the triplicate
analysis of either a solid or liquid sample. The
temperature within the box is controlled to ± 0.5° or
better. Nitrogen gas, split into six streams and
controlled by fine needle valves (approximately 0.79 mm
orifice), flows into the box via 3.8 mm (0.125 in.)
i.. d. copper tubing. After temperature equilibration,
the gas flows through the sample and the sorbent trap
arid exits from the box. The flow rate of the effluent
-7-
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CC-1600
FIGURE 1 - SCHEMATIC DIAGRAM OF VAPOR SATURATION APPARATUS
Intuliltd taoi
N2ln
fift
<0i
09
XT*
09
(ft
<5?>
Iwlng
Light bulb
I
i ^F«O *nr
1 i t~t | ) > a^ f
000 \
Solid «»mpl« «nd
(oitwnl holdM
000 |JJ
000 ||||
000 (II
Liquid iwnpU *nd * III
y§
000
Coppw coll htn Mclunoei
] 000 f
*
/
Njoulto
Tlwac-way »ilv«
\
I OUl
-3-
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CG-1600
carrier gas is measurer) at room temperature with a
bubble flow meter or other suitable device. The flow
rate is checked frequently durinq the experiment to
assure that there is an accurate value for the total
volume of carrier qas. The flow rate is used to
calculate the total volume (at room temperature) of qas
that has passed through the sample and sorbent
[(vol/time) x time = volume]. The vapor pressure of
the test substance can be calculated from the total qas
volume and the mass of sample vaporized. If v is the
volume of qas that transported mass w of the vaporized
test material havinq a molecular weiqht M, and if o is
the equilibrium vapor pressure of the sample at
temperature T, then p is calculated by the equation
p = (w/M)(RT/v)
In this equation, R is the qas constant (8.31 Pa m^mol
K~l). The pressure is expressed in pascals (Da), the
volume in cubic meters (m^), mass in qrams and T in
kelvins (K). T = 273.15 + t, if t is measured in
degrees Celsius (°C).
(2) Solid samples are loaded into 5mm i.d. glass tubing
between qlass wool pluqs. Fiqure 2 depicts a drawinq
of a sample holder and absorber svstem.
-9-
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CG-1600
FIGURE 2 SOUD COMPOUND SAMPUNG SYSTEM
Sortawit'
.GlMS WOOi
Solid compound
-GlMt wool
N-, in
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CG-1600
(3) Liquid samples are contained in a holder as shown in
Figure 3. The most reproducible method for measuring
the vapor pressure of liquids is to coat the liquid on
glass beads and to pack the holder in the designated
place with these beads.
(4) At very low vapor pressures and sorbent loadings,
adsorption of the chemical on the glass wool separating
the sample and the sorbent and on the glass surafaces
may be a serious problem. Therefore, verv low loadings
should be avoided whenever possible. Incoming nitrogen
gas (containing no interfering impurities) passes
through a coarse frit and bubbles throuqh a 38 cm
column of liquid sample. The stream passes through a
glass wool column to trap aerosols and then through a
sorbent tube, as described above. The pressure dron
across the glass wool column and the sorbent tube are
negligible.
(5) With both solid and liquid samples, at the end of the
sampling time, the front and backup sorbent sections
are analyzed separately. The compound on each section
is desorbed by adding the sorbent from that section to
1.0 ml of desorption solvent in a small vial and
allowing the mixture to stand at a suitable temperature
until no more test compound desorbs. It is extremelv
important that the desorption solvent contain no
impurities which would interfere with the analytical
method of choice. The resulting solutions are analyzed
-11-
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CG-1600
FIGURE 3 - LIQUID COMPOUND SAMPLING SYSTEM
EFFLUENT OUT
SORBENT
TUBE
(5 mm ID)
CARRIER GAS IN
120mm BED OF GLASS BEADS
COATED WITH LIQUID
(28mm OD)
GLASS WOOL
LIQUID TRAP
-12-
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CG-1600
quantitatively by a suitable analytical method to
determine the weight of sample desorbed from each
section. The choice of the analytical method, sorbent,
and desorption solvent is dictated by the nature of the
test material. Commonly used sorbents include
charcoal, Tenax GC, and XAD-2. Describe in detail the
sorbent, desorption solvent, and analytical methods
employed.
(6) Measure the desorption efficiency for every combination
of sample, sorbent, and solvent used. The desorption
efficiency is determined by injected a known mass of
sample onto a sorbent and later desorbing it and
analyzing for the mass recovered. For each combination
of sample, sorbent, and solvent used, carry out the
determination in triplicate at each of three
concentrations. Desorption efficiency may vary with
the concentration of the actual sample and it is
important to measure the efficiency at or near the
concentration of sample under gas saturation test
procedure conditions.
(7) To assure that the gas is indeed saturated with test
compound vapor, sample each compound at three differing
gas flow rates. Appropriate flow rates will depend on
the test compound and test temperature. If the
calculated vapor pressure shows no dependence on flow
rate, then the gas is assumed to be saturated.
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CG-1600
III. DATA AND REPORTING
(1) Report the triplicate calculated vapor pressures for
the test material at each temperature, the average
calculated vapor pressure at each temperature, and the
standard deviation.
(2) Provide a description of analytical methods used to
analyze for the test material and all analytical
results.
(3) For the isoteniscope procedure, include the plot of o
vs the reciprocal of the temperature in K, developed
during the degasing step and showing linearity in the
region of 298.15 K (25°C) and any other required test
temperatures.
(4) For the gas saturation procedure, include the data on
the calculation of vapor pressure at three or more gas
flow rates at each test temperature, showing no
dependence on flow rate. Include a description of
sorbents and solvents employed and the desorption
efficiency calculations.
(5) Provide a description of any difficulties experienced
or any other pertinent information.
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IV. REFERENCES
ASTM. 1978. American Society for Testing and Materials. Annual
book of standards. Part 24. pp. 740-745.
EPA. 1982. U.S. Environmental Protection Agency. Office of
Pesticides and Toxic Substances. Evaluation of gas saturation
methods to measure vapor pressures. Final Report; EPA Contract
No. 63-01-5117 with SRI International, Menlo Park, California.
Spencer WF and Cliath MM. 1969. Vapor density of dieldrin.
J Agric Food C'hem 3:664-670.
Spencer WF and Cliath MM. 1970. Vapor density and apparent
vapor pressure of lindane. J Agric Food Chem 18:529-530.
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August, 1982
VAPOR PRESSURE
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Contents
Page
I . NEED FOR THE TEST 1
II. SCIENTIFIC ASPECTS 5
A. Test. Methods 5
B. Test Procedures 9
1 . Temperature of the Test 9
2. Sorbent for Gas Saturation Procedure 10
3. Gas Flow Rates in Gas Saturation
Procedure 10
4. Calculation of Vapor Pressure in Gas
Saturation Procedure 11
C. Test Data Required 12
III. REFERENCES 14
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VAPOR PRESSURE
I. NEED FOR THE TEST
The vapor pressure of a chemical is an important oarameter
in determining the environmental fate of the chemical.
The atmosphere is a major route for the widespread distribu-
tion of chemicals. There are several ways by which chemicals may
become airborne and subsequently be transported by wind currents.
Airborne solids and foamy emulsions are commonly observed, but
these are not believed to be major factors in atmospheric trans-
port because they involve particulate matter which may be of
sufficient size to settle out in a relatively short time (Seiber
et al. 1975). Aerosols, from spray applications, manufacturing
and formulation sites, and aerated waste treatment systems may
constitute more important sources of chemical for air transport
since very small droplets (5 micrometers or less in diameter) may
be formed and carried considerable distances (Edwards 1973).
However, it appears that volatilization from land and water
surfaces is the most important source of material for airborne
transport (Hartley 1969, Lichtenstein 1971, MacKav and Wolkoff
1973, Seiber et al. 1975).
Volatilization is the evaporative loss of a chemical com-
pound. Volatilization rates are dependent on the vapor pressure
of the chemical and the environmental factors which influence
diffusion .rom the evaporative surface. Harper et al. (1976,
p. 236) noted that "volatilization is probably the single largest
means by which pesticides are lost and transported over wide
areas and into bodies of water far from the application
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location." The airborne vapors of a hazardous chemical may
present a threat to plant and animal life exposed to those
vapors, not only in the area of chemical release hut also at
sites remote from the volatilization site. This occurs when
vapors are removed from the air, primarilv by precipitation with
rain or snow.
Volatilization rates are related to vapor pressure, which
varies with temperature. However, volatilization from soil or
water is also influenced bv other environmental conditions and
the effective vapor pressure may be considerably lower than the
potential vapor pressure. Nevertheless, vapor pressure is the
one common factor governing the tendency of a compound to
volatilize.
According to "kinetic theorv there is a continuous Alight of
molecules from the surface of a liquid or solid into the free
space above it. A.t the same time vapor molecules return to the
surface at a rate depending on the concentration of the vapor.
If there is no removal of vapor from the surface (for example, bv
air currents), equilibrium will be established where the rate of
vaporization is exactly equal to the rate of condensation. The
pressure exerted by the equilibrium vapor is known as the vapor
pressure (Daniel and liberty 1955) and is denendent upon
temperature.
Knowledge of the vapor pressure of a compound allows the
ranking of a chemical as relatively nonvolatile, highly volatile,
or of some intermediate volatility. When vapor pressure data are
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combined with solubility data to calculate Henry's Law constants,
as described by MacKay and Leinonen (1975) and Dilling (1977),
rates of the evaporation of dissolved chemicals from water can be
estimated.
Evaporation from an exposed surface will depend upon other
factors such as wind speed (which reduces the vapor density above
the surface) and adsorption (which may act to hold the substance
on the surface). Volatilization from aqueous systems also
depends on the solubility of the compound and its movement to the
water surface. In soils the rate of volatilization of a chemical
will depend upon such factors as adsorption on soil, solubility
in soil water, and on the amount of soil water and its rate of
evaporation. Volatilization from soils can become a diffusion
controlled process as mass transfer to the soil surface is
reduced by low water evaporation due to high humidity or to the
lack of soil water in a dry soil (Bailey et al. 1974).
Chemicals that have relatively low vapor pressures and that
sorb readily to solids or dissolve readily in water are not
likely to vaporize significantly at ambient temperatures. For
that reason, airborne transport is not a major transport mecha-
nism for these chemicals and assessment of them should be focused
on tieir chemical fate and environmental effects in soils,
sediments, and water. However, chemicals with high vapor pres-
sures or with relatively low water solubility and low adsorptiv-
ity to solids are less likely to reside only in soils, sediments
or water, since volatilization can be a potentially significant
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C S -1 6 0 0
factor in their environmental, transport. Chemicals that are
gases at ambient temperatures and that have low water solubilitv
and low adsorptive tendencies will be transported to a
significant degree in the atmosphere and are prime candidates for
photolysis and for involvement in adverse atmospheric effects
such as smog formation or stratospheric alterations. Further-
more, effects testing of those chemicals should also -FOCUS on
inhalation and surface contact as potential routes for direct
exposure.
An understanding of how a chemical is likely to partition
among the various environmental media (air, water, soil, and
sediment) is needed in judging whether or not a chemical will be
subject to various transformation possibilities, such as
oxidation by hvdroxyl radicals or ozone in the atmosphere. Vanor
pressure data can influence decisions on whether or not it is
appropriate to conduct photolysis, adsorption/desorption,
partition coefficient, and certain biodegradation tests. Vapor
pressure data are an important consideration in the design o^
other fate and effects tests, for example in preventing or
accounting for the loss of volatile materials during the course
of the test. Clearly, a knowledge of vapor pressure combined
with information on water solubility and adsorptive tendencies is
necessary in predicting environmental transport and in providing
guidance as to which persistence and effects tests need to be
considered and how those tests should be designed.
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II. SCI^MTIFIC ASPECTS
A. ^est Methods
A procedure for measuring the vapor pressure of materials
released to the natural environment ideally would cover a range
of vapor pressure values, at ambient temperatures, of about 10~^
Pa to 10 Pa (approximately 10 to 760 torr). Recause no sinqle
procedure can cover this range, two different procedures are
described, each suited for a different part of the range. The
isot.eniscope procedure (ASTM 1978) is for pure liquids with vanor
pressures from 0.1 to 100 kPa. For vapor pressures of 10~5 to
10 Pa, a gas saturation procedure may be used. The Knudsen
effusion procedure (Thomson and Douslin 1971) mav be used for low
vapor pressure values.
It is important that each of the tests be oerforrned under
conditions of normal laboratory room temperatures in order to
allow for careful control of the temperatures in thermostated
baths or chambers containing the test apparatus.
The isoteniscope procedure uses a standardized technique
that was developed to measure the vapor pressure of certain
liquid hvdrocarbons. It is applicable to pure liquids with vapor
pressures of 0.1 kPa (0.75 torr) or more at ambient temper-
atures. The sample is purified within the equipment by removing
dissolved and adsorbed gases until the measured vapor pressure is
constant. This process is called "degassing." The procedures do
not remove higher boiling impurities, decomposition products, or
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compounds that boil close to or form azeotropes with the material
under test. If compounds that boil close to or form azeotropes
with the test material are present, it is necessary to remove the
interfering compounds and use pure test material. Impurities
more volatile than the sample will tend to increase the observed
vapor pressure above its true value but the purification steps
will tend to remove these impurities. Soluble, nonvolatile
impurities will decrease the apparent vapor pressure. However,
because the isoteniscope procedure is a static, fixed-volume
method in which an insignificant fraction of the liquid sample is
vaporized, it is subject to only slight error for samples
containing nonvolatile impurities. That is, the nonvolatile
impurities will not be concentrated due to vaporization of the
sample.
Gas saturation (or transpiration) procedures use a current
of inert gas passed through or over the test material slowly
enough to ensure saturation and subsequent analysis of either the
loss of material or the amount (and sometimes kind) of vapor
generated (Bellar and Lichtenberg 1974, Thomson and Douslin
1971) .
The gas saturation procedures have been described by Spencer
and Cliath (1969). Results are easy to obtain and can be quite
precise. The same procedures can also be used to study
volatilization from laboratorv scale environmental simulations.
Vapor pressure is computed on the assumptions that the total
pressure of a mixture of gases is equal to the sum of the
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CS-1600
pressures of the separate or component gases and that the ideal
cgas law is obeyed. The partial pressure of the vapor under study
can be calculated from the total gas volume and the weight of the
material vaporized. If v is the volume which contains w grams of
the vaporized material having a molecular weight M, and if p is
the pressure of the vapor in eguilibrium at temperature T (K),
then the vapor pressure, p, of the sample is calculated by
p = (w/M)(RT/v)
where R is the gas constant (8.31 Pa M mol~^ K"-"- ) when the
pressure is in pascals (Pa) and the volume is in cubic meters.
As noted by Spencer and Cliath (1970), direct vapor pressure
measurements by gas saturation technigues are more directly
related to the volatilization of chemicals than are other
technigues.
In addition to the above methods, other procedures have been
described for the measurement of vapor pressure (Daniels et al.
1956, Glasstone 1946, Thomson and Douslin 1971). These include
boiling point procedures, effusion techniques, and many highly
specialized technigues that are restricted to the determination
of very precise vapor pressure values or to the measurement of
vapor pressures of specific kinds of materials. These highly
specialized methods do not have general applicability to either a
wide variety of chemicals or a relatively broad range of vapor
pressure values at ambient temperatures.
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CS-1600
Roiling point procedures, such as that using Ramsey and
Young apparatus, have very poor accuracy below 10^ Pa (Thomson
and Oouslin 1971) and provide inaccurate estimates of the vapor
pressures at ambient temperatures if there is a change of state
or a transition temperature between the boiling and ambient
temperatures.
Effusion techniques, particularly those employing the
Knudsen effusion apparatus, are used to measure vapor pressure
from about 10"^ to 1 Pa and have provided some good data (Hamaker
and Kerlinger 1969). Those procedures require working with
systems under vacuum and it is necessary to saturate the capsule
space with vapor during the measurement periods. "Hie lack of
equilibrium saturation has been postulated as a reason for
inaccurate published vapor pressure data (Soencer and Cliath
1970). However, it must be recognized that there are
laboratories which have employed Knudsen effusion techniques
successfully and which have considerable experience with the
method, especially for determining very low vapor pressure
values, such as 10~5 to 10~3 Pa. For such laboratories, the
Knudsen effusion methods are a satisfactory alternative to the
gas saturation method in the determination of low vapor pressure
values. However, it seems reasonable to require that the
laboratory using effusion methods supply documentation to
substantiate successful utilization of the effusion procedures
with other compounds.
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CS-1600
B . Test Procedures
1. Temperature of the Test
The test procedures generally require a thermostated hath or
test chamber temperature of 25 ± 0.5°C. Laboratories should be
able to carry out vapor pressure measurements without the need
for elaborate temperature control devices. Control of the bath
or chamber to ±0.5°C will permit substantial confidence in the
data without requiring unnecessarily costly apparatus.
The International TJnion of Pure and Applied Chemistry has
for many years (IIJPAC 1972) recommended the reporting of
physical-chemical properties measurements at the temperature of
2F)°C. A temperature of 25 °C is slightly above most laboratory
room temperatures and this allows for convenient adjustment and
maintenance of constant temperature baths and enclosures.
Because of the nature of the isoteniscope nrocedure, it is
necessary in that test to conduct some measurements at
temperatures above and/or below 25°C in order to determine
whether the sample needs further degassing. Also, for some
chemicals, it may be necessary to require vapor pressure data at
temperatures other than 25°C. Examples of when this requirement
may 'oe applicable include situations where there is evidence that
the vapor pressure may change significantly with relatively small
changes in ambient temperature or when the boiling temperature
for a chemcial is at an ambient temperature below 25°C.
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CS-1600
2. Sorbent for Gas Saturation Procedure
The choice of sorbent and desorption solvent is dictated by
the nature of the compound being evaluated. Charcoal sorbent is
inexpensive and may be desorbed with carbon disulfide, a conven-
ient solvent for use with a flame ionization detector. Many com-
pounds, however, do not desorb efficiently from charcoal and more
expensive sorbents, such as Tenax GC and XAD-2, are recommended.
The desorption efficiency of a particular compound from a sorbent
with a solvent is defined as the weight of the compound which can
be recovered from the sorbent divided by the weight of the com-
pound originally adsorbed. It is extremely important that the
desorption efficiency be measured for every combination of
sample, sorbent, and solvent used. Desorption efficiency may
vary with concentration, so it is important to measure it at or
near the concentration of the actual sample. It is sometimes
necessary to interpolate between two measured efficiencies.
3. Gas Flow Rates in Gas Saturation
Procedure
Accurate control of gas flow rates is essential to assure
that a known volume of carrier gas is passed through the system.
Very long sampling times are reguired for compounds with low
vapor pressures, and it is difficult to control very low flow
rates for very long times. It is necessary to use fine needle
valves to control the flow rates and to measure the flow rates
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frequently during the test period in order to make corrections
for variation which can occur, e.g. due to changes in atmospheric
pressure.
4. Calculations of Vapor Pressure in the nas
Saturation Procedure
The calculation of vapor pressure is straightforward. ^he
weiqht of the sample desorbed from a sorbent section is divided
ny the desorption efficiency to give the weight of the sample
collected by the sorbent trap. With the volume of carrier gas
calculated from the flow rate, the ideal qas law is used to
calculate the vapor pressure of the sample. To assure that the
carrier gas is indeed saturated with the compound vapor, each
compound is sampled at three different gas flow rates. If the
vapor pressure calculated shows no dependence on flow rate, then
: tie gas is assumed to be saturated. The method also assumes that
f-her-j are no interactions between vaporized sample and the
carrier gas and that the molecular weight of the vaporized sample
ii:; the same as for the sample liguid or solid. If there are anv
indications that these may not be valid assumptions, the vapor
should be analyzed both qualitatively and quantitatively usinq
such techniques as gas chromatography combined with mass
spec - rometry (Heller et al. 1975).
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C. Test Data Required
The Test Guideline requires that the average calculated
vapor pressure for the test material at each required test
temperature be reported, including the individual values from
triplicate determinations and the calculated standard deviation
for each average calculated vapor pressure. It might be
preferable for assessment purposes to require that each vapor
pressure determination be made in sufficient replication to
provide a given degree of reproducibility. However, the
precision attainable will vary not only with the number of
replications but also with the procedure employed and the test
chemical. For a given chemical, the only way to determine how
many replications of a given procedure are necessary to provide
vapor pressure data with some specified percision is to repeat
the procedure until the data provide that precision. This may
take a few or manv replications and a requirement for numerous
replications is not justified unless the specified precision is
needed for assessment purposes with an individual chemical. 'Hie
minimum requirement EPA would impose would be a statistical
analysis of vapor pressure data to provide standard deviation
calculations based on triplicate determinations.
For the isoteniscope method, the Test Guideline requires
that the vapor pressure data generated during the degassing
operation, including a plot of log p vs 1/T, be included to
provide evidence of successful degassing. For the gas saturation
method, the Test Guideline requires that the data showing that
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CS-1600
vapor pressure does not vary with flow rate be included to
provide evidence of saturation of the carrier gas with the sample
vapor. Furthermore, it is extremely important that the data also
include a complete description of all analytical techniques and
results, a description of the sorbents and desorption solvents
used and the desorption efficiency calculations.
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C S - 1 6 0 0
III. REFEREMCES
ASTM. 1978. American Society for Testing and Materials. Annual
book of standards, part 24. Philadelphia, PA. pp. 740-745.
Bailey GW, Swank RR Jr., and Nicholson HP. 1974. Predicting
pesticide runoff of agricultural land: A conceptual model.
J Environ Qual 3:95-102.
Bellar TA and Lichtenberg JJ. 1974. Determining volatile
organics at microgram-per-litre levels by gas chromatography.
J Am Water Works Assn 66:739-744.
Daniels F and Alberty RA. 1955. Physical chemistry, Mew York:
John Wiley and Sons, p. 157.
Daniels F, Mathews JH, Williams JW, Bender P, and Alberty RA.
1956. Experimental physical chemistry, New York: McGraw Hill
Book Co., op. 47-511, 370-373.
Dilling WL. 1977. Interphase transfer processes. I.
Evaporation rates of chloromethanes, ethanes, ethylenes, nro-
panes, and propylenes from dilute aqueous solutions. Comparisons
with theoretical prediction. Environ Sci Technol 11:405-409.
Edwards CA. 1973. Persistent pesticides in the environment.
Cleveland: CRC Press. p. 21.
Glasstone S. 1946. Textbook of physical chemistry, 2nd ed. "Tew
York: D. Van Mostrand Co., p. 446-449.
Hamaker JW and Kerlinger WO. 1969. Vapor pressure of
pesticides. Adv Chem Series 86:39-54.
Harper LA, White AW, Jr., Bruce RR, Thomas AW, and Leonard RA.
1976. Soil and micro climate effects of trifluralin
volatilization. J Environ Qual 5:236-242.
Hartley GS. 1969. Evaporation of pesticides. Adv Chem Series
86:115-134.
Heller SR, McGuire JM and Budde WL. 1975. Trace organics by
GC/MS. Environ Sci Technol 9:210-213.
International Union of Pure and Applied Chemistry. TUPAC,
1972. Commission on Thermodynamics and Thermochemistry. 1972.
A guide to procedures for the publication of thermodynamic
data. Pure and Appl Chem 29:397-407.
Lichtenstein EP. 1971. Environmental factors affecting fate of
pesticides. In: Degradation of Synthetic Organic Molecules on
the Biosphere. Washington DC: National Academy of Sciences, n.
192.
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MacKay D and Leinonen PJ. 1975. Rate of evaporation of low-
solufcility contaminants from water bodies to atmosphere.
Environ Sci Technol 9:1178-1180.
MacKay D and Wolkoff AW. 1973. Rate of evaporation of low-
solubility contaminants from water bodies to atmosphere.
Environ Sci Technol 7:611-614.
Seiber JM, Shafik TM and Enoa HF. 1975. Determination of
pesticides and their transformation products in water. In:
Haque R and Fried VH eds. Environmental dynamics of pesticides,
New York: Plenum Press, p. 18.
Spencer WF and Cliath MM. 1969. Vapor density of dieldrin.
J Agric Food Chem 3:664-670.
Spencer WF and Cliath MM. 1970. Vapor density and apparent
vapor pressures of lindane. J Agric Food Chem 18:529-530.
Thomson GW and Douslin DR. 1971. Vapor pressure. In: Physical
methods of chemistry, Vol. I. Part V., Weissberger A and Rossiter
BW, eds. TTew York: Wiley-Interscience, p. 46-89.
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TRANSPORT PROCESSES
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CG-1700
August, 1982
SOIL THIN-LAYER CHROMATOGRAPHY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-1700
Contents
Page
I . INTRODUCTION 1
A. Background and Purpose 1
B. Definitions and Units 2
C. Principle of the Test Method 4
D. Applicability and Specificity 6
11 . TEST PROCEDURES 7
A. Test Conditions 7
B. Test Procedures 7
III, DATA AND REPORTING 9
IV. REFERENCES 10
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CG-1700
SOIL THIN-LAYER CHROMATOGRAPHY
I. INTRODUCTION
A. Background and Purpose
Leaching of chemicals through soil is an important process
which affects a chemical's distribution in the environment. If a
chemical is tightly adsorbed to soil particles, it will not leach
through the soil profile but will remain on the soil surface. If
a chemical is weakly adsorbed, it will leach through the soil
profile and may reach ground waters and then surface waters.
Knowledge of the leaching potential is essential under certain
circumstances for the assessment of the fate of chemicals in the
environment.
Chemical leaching also affects the assessment of ecological
and human health effects of chemicals. If a chemical reaches
ground water, deleterious human health effects may arise due to
the consumption of drinking water. If a chemical remains at the
soil surface, deleterious environmental and human health effects
may arise due to an increased concentration of the chemical in
the zone of plant growth, possibly resulting in contamination of
human food supplies.
Soil thin layer chromatography (TLC) is a qualitative
screening tool suitable for obtaining an estimate of a chemical's
leaching ootential. This test is one of several tests which can
be jsed in obtaining a rough estimation of a chemical's leaching
potential.
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B. Definitions and Units
"Cation exchange capacity" (CEC) is the sum total of
exchangeable cations that a soil can adsorb. The CEC is
expressed in milliequivalents of negative charge per 100 grams
(meq/lOOg) or milliequivalents of negative charge per gram
(meq/g) of soil.
"Particle size analysis" is the determination of the various
amounts of the different particle sizes in a soil sample (i.e.,
sand, silt, clay) usually by sedimentation, sieving, micrometry
or combinations of these methods. The names and size limits of
these particles as widely used in the United States are:
very coarse sand 2.0 to 1.0 mm dia.
coarse sand 1.0 to 0.5 mm
medium sand 0.5 to 0.25 mm
fine sand 0.25 to 0.125 mm
very find sand 0.125 to 0.062 mm
silt 0.062 to 0.002 mm
clay <0.002 mm
11 Rf" is the furthest distance traveled by a test material on
a thin-layer chromatography plate divided by the distance
traveled by a solvent front (arbitrarily set at 10.0 cm in soil
TLC studies) .
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"Soil" is the unconsolidated mineral material on the
immediate surface of the earth that serves as a natural medium
for the growth of land plants; its formation and properties are
determined by various factors such as parent material, climate,
macro- and microorganisms, topography, and time.
"Soil aggregate" is the combination or arrangement of soil
separates (sand, silt, clay) into secondary units. These units
may be arranged in the profile in a distinctive characteristic
pattern that can be classified on the basis of size, shape, and
degree of distinctness into classes, type, and grades.
"Soil classification" is the systematic arrangement of soils
into groups or categories. Broad groupings are made on the basis
of general characteristics, subdivisions, on the basis of more
detailed differences in specific properties. The soil
classification system used today in the United States is the 7th
Approximation Comprehensive System. The ranking of subdivisions
under the system is: order, suborder, greatgroup, family and
series.
"Soil horizon" is a layer of soil approximately parallel to
the land surface. Adjacent layers differ in physical, chemical,
and biological oroperties or characteristics such as color,
structure, texture, consistency, kinds, and numbers of organisms
present, and degree of acidity or alkalinity.
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"Soil order" is the broadest category of soil classification
and is based on general similarities of physical/chemical
properties. The formation by similar genetic processes causes
these similarities. The soil orders found in the United States
are: Alfisol, Aridisol, Entisol, Histosol, Inceptisol, Mollisol,
Oxisol, Spodosol, Ultisol, and Vertisol.
"Soil organic matter" is the organic fraction of the soil;
it includes plant and animal residues at various stages of
decomposition, cells and tissues of soil organisms, and
substances synthesized by the microbial population.
"Soil pH" is the negative logarithm to the base 10 of the
hydrogen ion activity of a soil as determined by means of a
suitable sensing electrode couoled with a suitable reference
electrode at a 1:1 soil:water ratio.
"Soil series" is the basic unit of soil classification and
is a subdivision of a family. A series consists of soils that
were developed under comparable climatic and vegetational
conditions. The soils comprising a series are essentially alike
in all major profile characteristics except for the texture of
the "A" horizon (i.e. the surface layer of soil).
"Soil texture" refers to the classification of soils based
on the relative proportions of the various soil separates
present. The soil textural classes are: clay, sandy clay, silty
clay, clay loam, silty clay loam, sandy clay loam, loam, silt
loam, silt, sandy loam, loamy sand, and sand.
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C. Principle of the ^est Method
Before 1968, methods of investigating the mobility of
nonvolatile orqanic chemicals within soils were based on the use
of field analysis, soil adsorption isotherms, and soil columns.
In 1968, Hellinq and Turner introduced soil thin layer
chromatoqranhy (soil TT_,C) as an alternative procedure; it is
analogous to conventional TLC, with the use of soil instead of
silica qels, oxides, etc., as the adsorbent phase.
The papers by Helling (1968, 1971a, 1971b, 1971c) and
Helling and Turner (1968) were the basis of this test
guideline. The soil and colloid chemistry literature and the
analytical chemistry literature substantiates the experimental
conditions specified in the guideline.
The soil TLC offers many desirable features. First,
mobility results are reproducible. Mass transfer and diffusion
components are distinguishable. The method has relatively modest
requirements for chemicals, soils, laboratory space, and
equipment. It yields data that are amenable to statistical
analyses. A chemical extraction-mass balance procedure to elicit
information on degradation and chemical transformations occurring
at colloid interfaces can be incorporated into this test. The
ease with which the R^ and mass balance are performed will depend
upon the physical/chemical properties of the test chemical and
the availability of suitable analvtical techniques for measuring
the chemical.
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D. Applicability and Specificity
Soil TLC can be used to determine the soil mobility of
sparingly water soluble to infinitely soluble chemicals. In
general, a chemical having a water solubility of less than 0.5
ppm need not be tested since the literature indicates that these
chemicals are, in general, immobile (Goring and Hamaker, 1972).
However this does not preclude future soil adsorption/
transformation testing of these chemicals if more refined data
are needed for the assessment process.
Soil TLC may be used to test the mobility of volatile
chemicals by placing a clean plate over the spotted soil TLC
plate and then placing both plates in a closed chromatographic
chamber.
Soil TLC was originally designed for use with soils. The
literature shows no published use of this method with sediments
as the absorbent phase, probably due to the fact that sediment
surface properties change significantly during air drying. It is
extremely important that the TLC plate with the adsorbent be air
dried before leaching studies can be undertaken.
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II. TEST PROCEDURES
A. Test Conditions
(1) Equipment required: Distilled-deionized water adjusted
to pH 1 by boiling to remove CC^; clean glass plates
(TLC); glass rods or a variable thickness plate
spreader; masking tape; closed chromatographic
chambers; analytical instrumentation necessary and
appropriate for the detection and quantitative analysis
of the test chemical;
(2) the test procedure may be run at 23 +_ 5°C; and
(3) it is recommended that three replicate plates for each
soil be used.
B. Test Procedures
(1) To reduce aggregate size before or during seiving,
crush and grind the air dried soil very, very gently;
(2) sieve air dried soils with a 250 micrometer sieve;
(3) add water to the sieved soil until a smooth, moderately
fluid slurry is attained (approximately 3/4 ml F^O
added for each gram of soil);
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(4) spread the slurry evenly and quickly across the clean
glass plate using a variable thickness plate spreader,
a glass rod, or other available method. If a glass rod
is used, control the layer thickness by affixing
multiple layers of masking tape along the plate
edges. Soil layer thickness should be 0.50 - 0.75 mm;
(5) air dry the plates at 25°C for a minimum of 24 hours
after uniform slurry application is achieved;
(6) scribe a horizontal line 11.5 cm above the base through
the soil layer down to the glass so as to stop solvent
movement;
(7) spot the test chemical, in solution, 1.5 cm above the
base. For radiolabeled materials, 0.5 - 5 g
containing 0.01 - 0.03 Ci of C labeled compound may
be used;
(8) if the compound is volatile, it is extremely important
that a clean plate be placed over the soil TLC plate to
impede volatilization;
(9) immerse the plate with the base down at some angle from
the vertical in a closed chromatographic chamber
containing F^O at a height of 0.5 cm;
(10) allow the solvent front to migrate to the 11.5 cm line
before removing the plates from the chamber;
(11) determine the R£ values. Zonal extraction, plate
scanning, or any other method or combination of methods
suitable for detection of the parent test chemical may
be used; and
-------
C G -1 7 0 0
(12) determine the amount of the parent test chemical on the
entire soil TLC plate after test chemical migration.
Suiy method or combination of methods suitable for the
extraction and quantitative detection of the parent
test chemical may be used.
III. DATA AND REPORTING
Report the following information as shown in pigures 1 and 2,
(1) Temperature at which the test was conducted;
(2) amount of the test chemical applied and amount
recovered from the plates;
(3) detailed description of the analytical technique used
in the Rf determination, the chemical extraction, and
the quantitative recovery and analvsis of the parent
chemical;
(4) the mean frontal R^ value with the standard deviation
for each soil tested;
(5) a photograph or diagram of the ^Lf! plate which shows
the entire leaching pattern (from 1.5 to 11.5 cm);
(6) soil information: soil order, series, texture,
sampling location, horizon, general clay fraction
mineralogy; and
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(7) soil physical/chemical properties: percent sand,
percent silt and percent clay (particle size analysis);
percent organic matter; pH (soil to water ratio, 1:1);
and cation exchange capacity.
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FIGURE 1 - SOIL TLC DATA FORMAT
SOIL-
PLATE 1
PLATE 2
PLATE 3
QUANTITY
Rf
Amount Aooli«d
Amount Recovered
% Recovered
Mean Rf
Standard Deviation
PLATE 1
PLATE 2
PLATE 3
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Figure 2. Soil Physical, Chemical, and Classsi f ication
Data Format
Soil 1
Soil 2
SOIL ORDER:
SOIL SERIES:
SOIL TEXTURE:
HORIZON:
% SAfID :
% SILT:
% CLAV;
% ORGANIC MASTER:
pH (1:1 ^9°):
CEC (MEQ/100GMS):
FRACTION
MINERALOGY:
Alfisol
Crider
Silt Loam
Gallatin Couty, IL
A
1.2
86.6
12.2
1.74
7-20
13.5
75% Montmori llonite
5-20% Mica
5% Kaolinite
(36-120 cm depth)
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IV. REFERENCES
Goring CAT, Hamaker JW. 1972. Organic chemicals in the soil
environment. Vol. I & II. New York: Marcel Dekker, Inc.
Helling CS 1968. Pesticide mobility investigations using soil
thin-layer chromatography. Amer Soc Agron Abstracts p. 89.
Helling CS, Turner BC. 1968. Pesticide mobility: Determination
by soil thin layer chromatography. Science 162:562.
Helling CS, 1970. Movement of s-triazine herbicides in soils.
Residue Review 32:175-210.
Helling CS. 1971a. Pesticide mobility in soils I. Parameters
of soil thin layer chromatography. Soil Sci Soc Amer Proc
35:732-737.
Helling CS. 1971b. Pesticide mobility in soils II.
Applications of soil thin layer chromatography. Soil Sci Soc
Amer Proc 35:737-743.
Helling CS. 1971c. Pesticide mobility in soils III. Influence
of soil properties. Soil Sci Amer Proc 35:743-748.
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August, 1982
SOIL THIN-LAYER CHROMATOGRAPHY
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Contents
Page
I . NERD FOR THE TEST ..................................... 1
II . SCIENTIFIC ASPECTS OF SOIL LEACHING ................... 2
A. Introduction ...................................... 2
R. Basic Processes Affecting Soil Leaching ........... 2
C. Chemical Properties Affecting Leaching ............ 4
D. Soil Properties Affecting Leaching ................ 4
E . Types of Adsorptive Forces ........................ 7
F. Surface transformations ........................... 8
III. SCIENTIFIC ASPECTS OF THE TEST1 ........................ 10
A. Development of Soil ryrhin Layer
Chromatography (TLC) ............................ 10
R. Rationale for the Selection of Soil ^LC ........... 13
C. Rationale for Selection of Experimental
Conditions and Procedures ....................... 15
IV. REFERENCES
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SOIL THIN LAYER CHROMATOGRAPHY
I. NEED FOR THE TEST
Leaching of chemicals through soil is an important process
which affects a chemical's distribution in the environment. If a
chemical is tightly adsorbed to soil particles, it will not leach
through the soil profile but will remain on the soil surface. If
a chemical is weakly adsorbed, it will leach through the soil
profile and may reach ground waters and then surface waters.
Knowledge of the leaching potential is essential under certain
circumstances for the assessment of the. fate of chemicals in the
environment.
Chemical leaching also affects the assessment of ecological
and human health effects of chemicals. If a chemical reaches
ground water, deleterious human health effects may arise due to
the consumption of drinking water. If a chemical remains at the
soil surface, deleterious environmental and human health effects
may arise due to an increased concentration of the chemical in
the zone of plant growth, possibly resulting in contamination of
human food supplies.
Soil thin layer chromatography (TLC) is a qualitative
screening tool suitable for obtaining an estimate of a chemical°s
leaching potential. This test is one of several tests which can
be used in obtaining a rough estimation of a chemical's leaching
potential.
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II. SCIENTIFIC ASPECTS OF SOIL LEACHING
A. Introduction
Since chemical leaching in soils is affected by a large
number of interacting processes (Hamaker 1975) this section of
the support document will discuss these processes as they relate
to this phenomenon of soil leaching.
B. Basic Processes Affecting Soil Leaching
The general equation (Guenzi 1974) for chemical movement
through porous media under steady state soil-water flow
conditions is:
6C' - *C _ v _^cv
6t 6X2 6X
where B = soil bulk density (g/cm^)
o = volumetric water content (cm /cm )
S = amount of chemical adsorbed at the
soil/water interface (g/g soil).
t = time (sec.)
C1 = solution concentration of chemical
D1 = dispersion coefficient (cm /sec)
V = average pore-water velocity (cm/sec)
X = space coordinate measured normal to the
sect ion
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Most mass transport equations represent simplifications of
"real world" conditions. Equation 1 and similar mathematical
expressions try to describe the chromatographic distribution of
the chemical in the soil profile; however, they are gross
simplifications of a phenomenon affected by a number of complex
interacting processes including but not limited to precipitation,
evaporation, evapotranspiration and hydrodynamic dispersion.
In general, chemical leaching is dependent upon three major
processes: the mass transport of water (the direction and rate of
wat.er flow), diffusion, and the adsorption characteristics of the
chemical in soil (Guenzi 1974). Diffusion is the transport of
matter resulting from random molecular motion caused by molecular
thermal energy. This random motion will lead to the uniform
distribution of molecules in a closed system since there is net
movement from regions of higher to lower concentrations. In this
document, adsorption refers to the equilibrium distribution of a
molecule between a solid phase and a solution phase. As the
decree of adsorption increases, the concentration of the chemical
in the soil water and the soil air decreases. This equilibrium
prc'cess is governed by two opposing rate processes. The
adsorption rate is the rate to which molecules from the liquid
phase transfer into the adsorbed state in the solid phase. The
desorption rate is the opposite process, i.e., the rate at which
molecules transfer from the adsorbed state in the solid phase
into the liquid phase. In general, the mass transport,
diffusion, and adsorption processes produce the observed leaching
pattern of a chemical in soil.
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C. Chemical Properties Affecting Leaching
The nain process of the three processes discussed above
which determines a chemical's leachinq potential (as described
mathematically in equation 1) is adsorption. Adsorption is
qoverned by the properties of both the adsorbent and the
adsorbate. ^he important properties of the absorbate affectinq
adsorption by soil colloids (Bailey and White 1970) are: (1)
chemical structure and conformation (2) aciditv or basicity of
the molecule (pKg or pKb), (3) water solubility, (4) permanent
charqe, (5) polarity, (6) molecular size, and (7) nolariz-
ability. There are many ways in which each of these adsorbate
properties interact and are manifested in the overall adsorption
reaction (Railey and Vlhite 1970).
O. Soil Properties Affecting Leaching
Soil is the unconsolidated orqanic and mineral material on
the immediate surface of the earth which serves as a natural
medium for the qrowth o^ nlants. ^he combined actions of
climate, microorqanisms and macroorqanisms over lonq periods of
time on different narent qeologic and biotic materials form soils
that differ widely in their physical, chemical, and morpholoqical
characteristics. ^he wide variations in the amounts and types of
clay and orqanic matter, soil pH, primary and secondary minerals,
structure, texture, and exchanqe capacity create soils of
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substantial heterogeneity within the United States. There are
currently 10 Soil Orders, at least 43 Suborders, over 200 Great
Groups and over 7,000 soil series recognized in the United States
(Buckman and Brady 1969).
The soil properties affecting the adsorption and desorption
of organics include organic matter content, type and amount of
clay, exchange capacity, and surface acidity (Adams 1973; Bailey
and White 1970; and Helling 1970). Soil organic matter is a
primary soil parameter responsible for the adsorption of many
pesticides. Helling (1970) lists many examples where the organic
matter primarily influenced the adsorption of pesticides.
Although organic matter and clay are the soil components most
often implicated in pesticide adsorption, the individual effects
of either organic matter or clay are not easily ascertained.
Since the organic matter in most soil is intimately bound to the
clay as a clay-metal-organic complex (Stevenson 1973), two major
types of adsorbing surfaces are normally available to the
chemical, namely, clay-organic and clay alone. Clay and organic
matter function more as a unit than as separate entities and the
relative contribution of organic and inorganic surfaces to
adsorption will depend on the extent to which the clay is coated
with organic substances. Comparative studies between known clay
minerals and organic soils suggest that most, but not all,
pesticides have a greater affinity for organic surfaces than for
mineral surfaces (Stevenson 1973). Since typical soil studies
compare soils in which both clay and organic matter increase and
do not utilize multiple regression analyses to isolate the
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qoverninq narameter (Hellinq 1970), only qeneralizations
concerning the relative importance of clay and orqanic matter in
the adsorption process can be made.
The activity of protons in the bulk suspension (i.e., as
measured bv pH) and the activity of protons at or in close
proximity to the colloidal surface (i.e., the acidity in the
interfacial reqion) may differ siqnifioantly. T'he term "surface
acidity" as applied to soil systems is the acidity at or in close
proximity to the colloidal surface and reflects the ability o^
the system to act as a Lewis acid. Surface acidity is a
composite term which reflects both the total number r>f aridic
sites and their relative deqree of aciditv. Surface acidity is
nrobably the most important property of the soil or colloidal
system in determining the extent and nature of adsorption of
basic orqanic chemicals as well as determining if acid-catalyzed
chemical transformation occurs (Railey and White 1970). There is
overwhelming evidence, mainly ^rom infrared studies as well as
other studies, pointing to the fact that there is protonation O-F
basic chemicals by clays having hydrogen and aluminum as the
predominant exchangeable cation and by clays saturated with
alkali, alkaline earth, and transition metal cations. A summarv
of recent investigations indicates that the protonation of
chemicals in the interfacial reqion o^ clavs is a function r>f the
basicity of the molecule, the nature of the exchangeable cation
on ^.he clav, water content of the clay system, and the oriqin r>f
neqative charqe in the aluminosilicate clay (Bailey and White
1970) .
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In summary, the chemical properties discussed in (C) and the
soil properties discussed in (D) both govern the extent of
adsorption in soils.
E. Types of Adsorptive Forces
The specific type of interaction of organic molecules with
soil will depend on the specific chemical properties of the
organic molecule and the type of soil. These specific
interactions or adsorptive forces are usually classified as: van
der Waals forces, charge transfer, ion exchange, and hydrophobic
bonding (Adams 1975, Goring and Hamaker 1972).
The van der Waals forces arise from the fluctuations in a
molecule's electron distribution as the electrons circulate in
their orbitals. These fluctuations produce instantaneous dipoles
which cause that molecule's attraction to other atoms and
molecules. Charge transfer involves the formation of a donor-
acceptor complex between an electron donor molecule and an
electron acceptor molecule with partial overlap of their
respective molecular orbitals and a partial exchange of electron
density. Ion exchange refers to the exchange between counterions
balancing the surface charge on the soil colloid and the ions in
the soil solution. The driving force for this interaction is the
requirement for electroneutrality: the surface electric charge
must be balanced by an equal quantity of oppositely charged
counterions. In general, ion exchange is reversible, diffusion
controlled, stoichiometric and, in most cases, exhibits some
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selectivity or preferential adsorption for one ion over another
competing ion. Hydrophobic solvation, the process commonly
referred to as hydrophoric bonding, refers to the preference of
an organic molecule for a hydrocarbon solvent or hydrophobic
region of a colloid over a hydrophilic solvent. This preference
is due to the fact that hydrocarbon regions of a molecule have
greater solubility in liquid hydrocarbons (or most organic
solvents) than in water. In general, one or more of these
specific interactions or adsorptive forces may occur at the same
time depending on the presence and magnitude of the chemical and
soil properties discussed above.
F. Surface Transformations
A special type of interaction between organic molecules and
soils deals with the transformation of organic chemicals into new
compounds containing different chemical structures through the
catalytic activity of the soil colloid surfaces. Although
several theories exist to account for the mechanism of these
transformations, no scheme predicting the occurrence of such
surface reactions presently exists. Therefore, it is extremely
important that parent compound mass balances be performed and
reported in order to ascertain the extent of such transformations
during soil leaching experiments. Also, the leaching pattern (a
diagram or photograph of the TLC plate showing the position of
the chemical) can give a qualitative indication of the extent of
such transformations and should be reported. The scientific
literature shows that a number of chemicals and chemical classes
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undergo colloid surface induced chemical transformations. Poly-
(dirnethylsiloxane) fluids in intimate contact with many soils
undergo siloxane bond redistribution and hydrolysis, resulting in
the formation of low molecular weight cyclic and linear oligomers
(Buch and Ingebrightson 1979). S-triazines (White 1976) and
orga,nophosphorus pesticides (Yaron 1978, and Mingelgrin et al.
1977) undergo clay colloid induced hydrolysis. Benzene and
phenol polymerize into high molecular weight species by
adsorption and reaction at the surface of smectite saturated with
transition metal cations (Mortland and Halloran 1976). Gallic
acid, pyrogallol, protocatechuic acid, caffeic acid, orcinol,
ferulic acid, p-coumaric acid, syringic acid, vanillic acid and
p-hydroxybenzoic acid undergo oxidative polymerization in the
presence of various clay minerals (Wang and Li 1977, and Wang et
al. 1978). In general, testing methods that do not take into
account surface transformations should not be used in determining
the Leaching potential of chemicals.
In summary, the interfacial region is important in
determining the adsorption mechanism, the energy by which the
adsorbate is held, and in determining if the adsorbed chemical is
transformed. This information is important in determining the
persistence and ultimate toxicity of the molecule since the
transformation product(s) (1) may be more or less toxic than the
original compound, (2) may be more or less tightly bound than the
original compound, and (3) may have a water solubility either
greater than or less than the original compound, thereby
affecting its leaching and movement into the groundwater.
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III. SCIENTIFIC ASPECTS OF THE TEST
A. Development of Soil Thin Layer
Chromatography (TLC)
Before 1968, methods of investigating the mobility of
nonvolatile organic chemicals within soils were based on the use
of field analysis, soil adsorption isotherms, and soil columns.
In 1968, Helling and Turner introduced soil thin layer
chromatography (soil TLC) as an alternate procedure. It is
analogous to conventional TLC, with the use of soil instead of
silica gels, oxides, etc. as the adsorbent phase.
In their initial report, Helling and Turner used Lakeland
sandy loam, Chillum silt loam, and Hagerstown silty clay loam.
Medium sand ( 250 m dia.) was removed from Chillum and
Hagerstown soils and coarse sand ( 500 m) from Lakeland soil by
dry-sieving. Aqueous slurries were prepared and 0.50 mm (silt
loam, silty clay loam) or 0.75 mm (sandy loam) thick layers were
spread on TLC plates using conventional TLC apparatus. After
drying, six or seven radiolabelled pesticides were applied near
the base of a 20 x 20 cm plate and developed ten cm with water by
ascending chromatography. Pesticide movement was visualized by
autoradiography. Movement was expressed by the conventional Rf
designation, although this referred to the front of pesticide
movement rather than its maximum concentration. The soil TLC
data are most appropriately compared with other mobility data
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whi^h indicate the depth to which an organic chemical may be
leached. The ranking of pesticides in order of mobility is in
good agreement with general trends previously reported.
Absolute movement on soil TLC plates cannot be transposed
directly to field or soil column experiments. Since soil
stracture in the TLC system is considerably more homogeneous than
in most other systems, band spreading will be somewhat less than
in field or column regimes. Flow rates are also higher than
those occurring naturally. For example, infiltration into
Hagerstown silty clay loam was equivalent to rainfall of about
1.2 cm/hr (Helling 1970). High flow rates are usually associated
with increased mobility, as later correlations (Helling 1968)
bore out. In spite of these problems, monitoring data utilizing
certain reference chemicals has provided the necessary infor-
mation to relate soil TLC data to column and field data. In
general, Helling and Turner (1968) indicated that soil TLC
offered a rapid, simple, and inexpensive procedure for
establishing a general mobility classification of pesticides and
organic chemicals.
Simple chromatographic theory can be used to correlate
adsorption coefficients with soil TLC Rf values. If
chromatographic movement through a soil column is treated
according to the distillation theoretical plate theory (Block et
al. 1958, Martin and Synge 1941), a formula for Rf is obtained in
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terms of the relative cross-sectional areas of the liquid and
solid phases and partitioning of a chemical between solid and
liquid phases (Hamaker 1975):
Rf = ATj/(z\Tj + ^) = l/[ f 1 + a (ATj + Aq) 1 (2)
where Ae and AT are cross-sectional areas of solid and liquid
vS Lj
nhases and a is the ratio of volume concentration in the solid
nhase to that in the liquid phase. por saturated conditions which
will be assumed for a soil plate, AL + A<- = A (cross-sectional
area), this can be written:
Rf = I/Hi + a (A/[AL - 11)1 (3)
When reexpressed in terms of the pore fraction of the
soil 8, density of soil solids (d ) , and a soil adsorntion
coefficient K, this equation becomes:
Rf = (1 + K(ds) (l/e3-!)!- (4)
7 /3
This ratio, A/Ay / is set equal to 1/6" by analoqy to the
treatment of soil diffusion by Millinqton and Quirk (1961) where
it serves to correct ^or the tortuosity of flow throuqh the
porous medium. In this case, it serves to relate the pore volume
to the cross sectional area of the liquid phase in a saturated
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soil. In general, equation 4 has shown that an inverse
relationship exists between the soil adsorption coefficient K and
Rf (Hamaker 1975).
Riley (1976) presented a general relationship between the
soil/solution distribution coefficient K and the depth of
pesticide leaching. Relating the data of Riley (1976) with the
Rf values of Helling (1968, 1971a, 1971b, 1971c) and the average
K values of 'Soring and Hamaker (1972) for selected pesticides,
the general relationship shown in Table 1 was developed between
the soil/solution partition coefficient, Rf, and soil mobility.
B. Rationale for the Selection of Soil TLC
A number of laboratory tests - the soil thin layer
chromatography, soil adsorption isotherm, and soil columns - have
beer developed to obtain an estimate of a chemical's leaching
potential (Hamaker 1975). Soil TLC is the least expensive of the
available tests which measures leaching potential, and is widely
used; furthermore, it offers many desirable features. First,
mobility results are reproducible. Mass transfer and diffusion
components are distinguishable. The method has relatively modest
requirements for chemicals, soils, laboratory space, and
equipment. It yields data that are amenable to statistical
analyses. A chemical extraction-mass balance procedure to elicit
information on degradation and chemical transformations occurring
at colloid interfaces can be incorporated into this test. The
ease with which the Rf and mass balance are performed will depend
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Table 1. The General Relationship Between the Soil/Solution
Partition Coefficient K, Rg and Soil Mobility
K
0.1
1
10
io2
102'5
1Q3
IO4
Re Mobility class Distance surface applied chemical may leach
0.95 very Mobile
soil into subsoil.
0.25 Mobile Much of chemical leached into soil but peak
concentration in top 20 on soil.
0.10 Low mobility Only snail amount of leaching and pealc
concentration normally in top 5 en soil.
0 00
0.00
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upon the physical/chemical properties of the test chemical and
the availability of suitable analytical techniques for measuring
the chemica1.
C. Rationale for Selection of Experimental
Conditions and Procedures
The papers by Helling (1968, 1971a, 1971b, 1971c) and
Helling and Turner (1968) were the basis of this test
guideline. The soil and colloid chemistry literature and the
analytical chemistry literature substantiates the experimental
conditions specified in this Test Guideline as accepted, standard
procedures. A few of these conditions will be discussed in
greater detail below.
Soil Tr_,C can be used to determine the soil mobility of
sparingly water soluble to infinitely soluble chemicals. In
general a chemical having a water solubility of less than
0.5 ppm need not be tested since the literature indicates that
these chemicals are, in general, immobile (Goring and Hamaker
1972). However, this does not preclude advanced soil adsorption/
transformation testing of these chemicals if more refined data
are needed for the assessment process.
Soil TLC may be used to test the mobility of volatile
chemicals by placing a clean plate over the spotted soil TLC
plate and then placing both plates in a closed chromatographic
chamber.
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Soil TLC was originally designed for use with soils. The
literature shows no published use of this method with sediments
as the adsorbent phase, probably due to the fact that sediment
surface properties change significantly during air drying. It is
extremely important that the TLC plate with the adsorbent be air
dried before leaching studies can be undertaken.
Distilled-deionized H20 is required in order to minimize
competition effects for soil exchange sites by cationic and
anionic species normally present in tap and distilled H20.
It is extremely important that the test chemical be of the
purest grade available. Impurities may produce migration
patterns on the TLC plate independent of the parent chemical and
may be misinterpreted as transformation products. Transformation
product identification is an expensive analytical procedure that
may be unnecessarily required as a result of the presence of
impurities.
The sieving of soils will remove the coarse (500-2,000 \m)
and medium (250-500 vim) sand fractions. Published testing
results showed that removal of a portion of sand had no affect on
the mobility of a test compound but aided in achieving a more
cohesive uniform soil layer and more reproducible results
(Helling 1971a).
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Gentle crushing and grinding should be used to reduce soil
aggregate size. Fine particles (silt and clay) in excess of the
amount originally present may be created if excessive pressure is
exerted on the aggregates.
It is important that application of the soil slurry to clean
glass plates be done quickly to prevent particle size
segregration. A specific method of soil slurry application was
not identified since a number of methods which produce the
acceptable layer thickness are in use today.
Replication of the basic experimental unit was necessary in
order to estimate the standard deviation of the treatment mean.
Three replicates are considered to be the minimum number of
replicates for a statistically acceptable estimation. The soils
literature indicates that, in general, the standard deviation
should be less than 0.01 Rf units for soil TLC.
Since the available literature indicated that pesticide
mobility on soil TLC plates did not significatly change when
temperature varied from 2° to 25°C (Helling 1971a), only a room
temperature range was suggested.
The Soil Order, Series, and general clay fraction mineralogy
data may be found in Soil Survey Reports published after
approximately 1970. Pre-1970 reports may not contain mineralogy
data. Soil Survey Reports have been issued for most U.S.
counties and may be obtained from County Extension Offices; the
State Office of the U.S. Department of Agriculture Soil
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Conservation Service; or the USDA-Soil Conservation Service,
Publications and Information Division, P.O. Box 2890, Washington,
DC 20013. If mineralogy data are not printed in a report, the
State Office of the U.S. Department of Agriculture Soil
Conservation Service may be contacted for assistance in obtaining
general clay mineral data of a particular soil.
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Adams Jr. RS.. 1973. Factors influencing soil adsorption and
bioactivity of pesticides. Residue Rev 47:1-54.
Rai'ev GW, White TL. 1970. ^actors influencinq the adsorption,
desorption and movement of pesticides in soil. Residue Rev
32: ''9-92.
Rlock RJ, Ourrum EL, 7weiQ G. 1958. A manual of paper
chromatography and paper electrophoresis. Second Edition.
;\oarerriic Press, M.V'.
Ruch RR, Inqebriqtson ON. 1979. Rearrangement of poly-
('dirr:ethyl/s iloxane) fluids on soil. Environ Sci and "eehnol
1.3:676-679.
Ruck "nan nr>, Brady ?TC. 1969. 'T'he nature and properties of
soils. London: T'he Macmillan Company.
Gorinq CM, Hamaker ,TW eds. 1972. Orqanic chemicals in the soil
environment. Vol. I & II. New York: Marcel Oekker, Inc.
Guen7.i v/O ed. 1974. °esticides in soil and water. Madison, '?! :
f^oil Science Society of America, Inc.
Mama^er .TW 1975. ^he interpretation o^ soil leachinq
experiments. Tn Haque R and Freed VH eds. Environmental Science
Research vol. 6: Environmental dvnamics nf pesticides.
t:iell;.nq CS 1968. Pesticide mobilitv investiqations using soil
thin-laver chromatograohv. Amer Soc Agron Abstracts P. 89.
Helling CS, Turner RC. 1968. Pesticide mobilitv: Determination
bv soil thir laver chromatcgraphv. Science 162:562.
H=lL:nq CS. 1970. Movement of s-triazine herbicides in soils.
Residue Rev 32:175-210.
Helling CS. 1971a. Pesticide mobility in soils I. Parameters
of soil thin layer chromatography. Soil Sci Soc Amer Proc
3 5 : 7 I-, 2 - 7 3 7 .
'Telling CS. 19'?lb. Pesticide mobility in soils IT.
Applications of soil thin layer chromatoqraphv. Soil Sci Soc
-\rner Proc 35:737-743.
' '''.:'.'' CS. 1971 c. Pesticide mobilitv in soi. Is III. influence
nf- :-'"il properties. Soil Sci Soc Amer Proc 35:743-748.
A f!> ( Svnqe I?TJ^1. 1941. A new ^orm of chromatoqram
':.']' f.o 'iquid phases. Riochem J 35:1358.
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Milli'nqton RJ and Quirk JP. 1961. Dermeability of porous
solids. Trans Faraday Soc 57:1200.
Mingelgrin Tl, Saltzman S, Yaron B. 1977. A possible model
the surface induced hydrolysis of organophosphorus pesticides on
Xaolinite clays. Soil Sci Soc Amer Jour 41:519-523.
Mortland MM, Halloran LJ. 1976. Polymerization of aromatic
molecules on smectite. Soil Sci Soc Amer Jour 40:367-370.
Riley D. 1976. Physical loss and redistribution of pesticides
in the liquid phase. In: British Crop Protection Council
Symposium Proceedings, p. 109-115.
Shearer RC, Letey J, Farmer WJ, Klute A. 1973. Lindane
diffusion in soil. Soil Sci Soc Amer Proc 37:189-193.
Stevenson FJ. 1973. Organic matter reactions involving
pesticides in soil. In: Round and conjugated pesticide
residues. ACS Symposium Series Monograph 29/1976.
Wang TDC, Li,SW. 1977. Clay minerals as heterogeneous catalysts
in preparation of model humic substances. 7, P^lanzenernaehr
Bodenkd 140:669-676.
Wang TSC, Li SW, Ferna YIj. 1978. Catalytic polvmerir.ation O-P
phenolic compounds by clay minerals. Soil Sci 126:15-21.
White JL. 1976. Determination of susceptibility of s-tria^ine
herbicides to protonation and hydrolysis by mineral surfaces.
Arch Fnviron Contam ^oxicol 3:461-469.
Yaron B. 1978. Some aspects of surface interactions of clays
with organophosphorus pesticides. Soil Sci 125:210-216.
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CG-1710
August, 1982
SEDIMENT AND SOIL ADSORPTION ISOTHERM
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CONTENTS
Page
I . INTRODUCTION 1
A. Background and Purpose 1
B. Definitions and Units 1
C. Principle of the Test Method 5
D. Applicability and Specificity 7
11 . TEST PROCEDURES 8
A.. Test Conditions 8
1. Special Laboratory Equipment 8
2. Temperature 9
3. Replications 9
4. Soil Pretreatment 9
5. Sediment Pretreatment 10
6. Solid/Solution Ratio 10
7. Equilibration Time 11
8. Centrifuge Time 12
9. Storage of Solution 13
10. Solvents for Extraction 13
B. Test Procedure 13
1 . Equilibration 13
2. Centrif ugation 14
3. Chemical Extraction 14
4. Chemical Analysis 14
III . DATA AND REPORTING 15
IV. REFERENCES 21
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SEDIMENT AND SOIL ADSORPTION ISOTHERM
I. INTRODUCTION
A. Background and Purpose
The adsorption of chemicals to sediments and soils is an
important process that affects a chemical's distribution in the
environment. If a chemical is adsorbed to soil particles, it will
remain on the soil surface and will not reach ground water. If a
chemical is riot adsorbed, it will leach through the soil profile
and may reach ground waters and then surface waters. Similarly,
if ci chemical adsorbed to sediment, it will accumulate in the bed
and suspended load of aquatic systems. If a chemical is not
adsorbed to sediment, it will accumulate in the water column of
aquatic systems. Information on the adsorption potential is
needed under certain circumstances to assess the transport of
cherricals in the environment. This Test Guideline describes
procedures that will enable sponsors to determine the adsorption
isotherm of a chemical on sediments and soils.
B. Definitions and Units
The "cation exchange capacity" (CEC) is the sum total of
exchangeable cations that a sediment or soil can adsorb. The CEC
is expressed in milliequivalents of negative charge per 100 grams
(meq/lOOg) or milliequivalents of negative charge per gram (meq/g)
of soil or sediment.
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"Clay mineral analysis" is the estimation or determination of
the kinds of clay-size minerals and the amount present in a
sediment or soil.
"Organic matter" is the organic fraction of the sediment or
soil; it includes plant and animal residues at various stages of
decomposition, cells and tissues of soil organisms, and substances
synthesized by the microbial population.
"Particle size analysis" is the determination of the various
amounts of the different particle sizes in a sample (i.e., sand,
silt, clay), usually by sedimentation, sieving, micrometry, or
combinations of these methods. The names and diameter range
commonly used in the United States are:
Name diameter range
very coarse sand 2.0 to 1.0 mm dia.
coarse sand 1.0 to 0.5 mm
medium sand 0.5 to 0.25 mm
fine sand 0.25 to 0.125 mm
very fine sand 0.125 to 0.062 mm
silt 0.062 to 0.002 mm
clay <0.002 mm
The "pH" of a sediment or soil is the negative logarithm to
the base ten of the hydrogen ion activity of the sediment or soil
suspension. It is usually measured by a suitable sensing
electrode coupled with a suitable reference electrode at a 1/1
solid/solution ratio by weight.
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The adsorption ratio, "K^," is the amount of test chemical
adsorbed by a sediment or soil (i.e., the solid phase) divided by
the amount of test chemical in the solution phase, which is in
equilibrium with the solid phase, at a fixed solid/solution ratio.
"Sediment" is the unconsolidated inorqanic and orqanic
material that (a) is suspended in and beinq transported by surface
water, or (b) has settled out and has deposited into beds.
"Soil" is the unconsolidated mineral material on the
immediate surface of the earth that serves as a natural medium ^or
the qrowth of land plants. Its formation and properties are
determined by various factors such as parent material, climate,
macro- and microorganisms, topography, and time.
"Soil aggregate" is the combination or arranqement of soil
separates (sand, silt, clay) into secondarv units. These units
may be arranged in the soil profile in a distinctive characteris-
tic pattern that can be classified according to size, shape, and
degree of distinctness into classes, types, and arades.
"Soil classification" is the systematic arrangement of soils
into groups or categories. Rroad groupings are based on general
soil characteristics while subdivisions are based on more detailed
differences in specific properties. ^he soil classification
system used in this standard and the one used today in the United
States is the 7th Approximation-Comprehensive System. The ranking
of subdivisions under this system is: Order, Suborder, Great
group, family, and series.
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A "soil horizon" is a layer of soil approximately parallel to
the land surface. Adjacent layers differ in physical, chemical,
and biological properties such as color, structure, texture,
consistency, kinds and numbers of organisms present, and degree of
acidity or alkalinity.
"Soil Order" is the broadest category of soil classification
and is based on the general similarities of soil physical/
chemical properties. The formation of soil by similar general
genetic processes causes these similarities. The Soil Orders
found in the United States are: Alfisol, Aridisol, Entisol,
Histosol, Inceptisol, Mollisol, Oxisol, Spodosol, Ultisol, and
Vertisol .
"Soil series" is the basic unit of soil classification and is
a subdivision of a family. A series consists of soils that were
developed under comparable climatic and vegetational conditions.
The soils comprising a series are essentially alike in all major
profile characteristics except for the texture of the "A" horizon
(i.e., the surface layer of soil).
"Soil texture" is a classification of soils that is based on
the relative proportions of the various soil separates present.
The soil textural classes are: clay, sandy clay, silty clay, clay
loam, silty clay loam, sandy clay loam, loam, silt loam, silt,
sandy loam, loamy sand, and sand.
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C. Principle of the Test Method
The extent of adsorption of a chemical onto sediment or soil
is neasured, using this test guideline, by equilibrating aqueous
solutions containing different, but environmentally realistic,
concentrations of the test chemical with a known quantity of sedi-
ment or soil. After equilibrium is reached, the distribution of
the chemical between the water phase and the solid phase is
quantitatively measured by a suitable analytical method. Then,
sorption constants are calculated by using the Freundlich
equation:
x/m = C = KG
' s e
1/n
(1)
where
C = Equilibrium concentration of the chemical in the
solution phase
C = Equilibrium concentration of the chemical in the
S
solid phase
K = Freundlich adsorption coefficient
m = The mass of the solid in grams
1/n = Exponent where n is a constant
x = The mass in micrograms of the chemical adsorbed by m
grams of solid.
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Logarithmetic transformation of the Freundlich equation yields the
following linear relationship:
log Cs = log K + (1/n) log Ce (2)
In order to estimate the environmental movement of the test
chemical, the values K and 1/n are compared with the values of
other chemicals whose behavior in soil and sediment systems is
well-documented in scientific literature.
The adsorption isotherm (AI) test has many desirable
features. First, adsorption results are highly reproducible. The
test provides excellent quantitative data readily amenable to
statistical analyses. Also, it has relatively modest requirements
for chemicals, soils, laboratory space, and equipment. It allows
solution phase organic chemical determinations that are relatively
uncomplicated. A chemical extraction-mass balance procedure to
elicit information on chemical transformations occurring at
colloid interfaces can be incorporated into this test. The ease
of performing the isotherm test and mass balance will depend upon
the physical/chemical properties of the test chemical and the
availability of suitable analytical techniques to measure the
chemical.
The papers by Aharonson and Kafkafi (1975), Harvey (1974),
Murray (1975), Saltzman (1972), Weber (1971), and Wu (1975) served
as the basis for this Test Guideline. The soil and colloid
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chemistry literature and the analytical chemistry literature sub-
ste.ntiate the experimental conditions and procedures specified in
this guideline as accepted, standard procedures.
D. Applicability and Specificity
The AI Test Guideline can be used to determine the soil and
sediment adsorption potential of sparingly water soluble to
infinitely soluble chemicals. In general, a chemical having a
water solubility of less than 0.5 ppm need not be tested with soil
as the solid phase, since the literature indicates that these
chemicals are, in general, immobile in soils. (Goring and
Hamaker, 1972). However, this does not preclude future soil
adsorption/transformation testing of these chemicals if more
refined data are needed for the assessment process.
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II. TEST PROCEDURES
A. Test Conditions
1. Special laboratory equipment
a. Equilibrating solutions that contain, besides
the test chemical, 0.01M calcium nitrate
dissolved in sterilized, distilled-deionized H^
adjusted to neutral pH 7 by boiling to remove
C02.
b. Containers that are composed of material that
(1) adsorb negligible amounts of test chemical,
and (2) withstand high speed centrifugation.
The volume of the container is not a major
consideration; however, it is extremely
important that the amount of soil or sediment
and the solid/solution ratio used in the study
result in minimal container headspace. It is
also extremely important that the containers be
sterilized before use.
c. A 150 ym (100 mesh) stainless steel or brass
sieve.
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d. Drying oven, with circulating air, that can
attain 100°C.
e. Vortex mixer or a comparable device.
f. Rotary shaker or a comparable device.
g. High speed temperature-controlled centrifuge
capable of sedimenting particles greater than
0.5 ym from aqueous solution.
2. Temperature
It is recommended that the test procedure be performed at
23±5°C.
3. Replications
It is recommended that three replications of the experimental
treatments be used.
4. Soil Pretreatment
It is extremely important that these soil pretreatment steps
be performed under the following conditions:
a. Decrease the water content, air or oven dry
soils at or below 50°C.
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b. Reduce aggregate size before and during sieving,
crush and grind dried soil very gently.
c. Eliminate microbial growth during the test
period using a chemical or physical treatment
that does not alter or minimally alters the soil
surface properties.
d. Sieve soils with a 100 mesh stainless steel or
brass sieve.
e. Store all solutions and soils at temperatures
between 0 and 5°C.
5. Sediment Pretreatment
It is extremely important that these sediment pretreatment
steps be performed under the following conditions:
a. Decrease the F^O content by air or oven drying
sediments at or below 50°C. Sediments should
not be dried completely and should remain moist
at all times prior to testing and analysis.
b. Eliminate microbial growth during the test
period by using a chemical and/or physical
treatment that does not alter or minimally
alters the colloid surface's properties.
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c. Store at temperatures between 0 and 5°C.
6. Solid/Solution Ratio
It is recommended that the solid/solution ratio be equal to
or greater than 1/10. If possible, the ratios should be equal to
or greater than 1/5. The sediment or soil dry weight after drying
for a 24 hour minimum at 90°C is recommended for use as the weight
of the solid for ratio and data calculations.
7. Equilibration Time
The equilibration time will depend upon the length of time
needed for the parent chemical to attain an equilibrium distribu-
tion between the solid phase and the aqueous solution phase. It
is recommended that the equilibration time be determined by the
following procedure:
a. Equilibrate one solution containing a known
concentration of the test chemical with the
sediment or soil in a solid/solution ratio not
exceeding 1/10 and preferably equal to or
greater than 1/5. It is important that the
concentration of the test chemical in the
equilibrating solution (1) does not exceed one
half of its solubility and (2) should be 10 ppm
or less at the end of the equilibration period.
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b. Measure the concentration of the chemical in the
solution phase at frequent intervals during the
equilibration period.
c. Determine the equilibration time by plotting the
measured concentration versus time of sampling;
the equilibration time is the minimum period of
time needed to establish a rate of change of
solution concentration of 5 percent or less per
24 hours.
8. Centrifugation Time
Calculate the centrifugation time, tc, necessary to remove
particles from solution greater than approximately 0.5 pm
(5 x 10 m) equivalent diameter (which represents all particles
except the fine clay fraction) using the following equation:
tc(min) = 1.41 x 109 [log(R2/Ri)1/N2 (3
where
tc = centrifuge time in minutes
R2 = distance from centrifuge spindle to deposition
surface of centrifuge
Ri = distance from spindle to surface of the sample
N = number of revelations of the centrifuge per minute,
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9. Storage of Solutions
If the chemical analysis is delayed during the course of the
experiment, store all solutions between 0 and 5°C.
1C. Solvents for Extraction
It is extremely important that (1) the purity of the solvent
used to extract the chemical that is adsorbed on the sediment or
soil is analytical grade or better and (2) the minimum solubility
of the test chemical in the solvent is 10 g/1.
B. Test Procedure
1 . ^quilbration
Add six solutions containing different concentrations of the
test chemical to at least one gram of each solid. The initial
concentration of the test chemical in these solutions will depend
on the affinity the chemical has for the sediment or soil.
Therefore, after equilibrium is attained, it is extremely impor-
tant that the highest concentration of the test chemical in the
equilibrating solution (a) does not exceed 10 ppm, (b) is at least
one order of magnitude greater than the lowest concentration
reported, and (c) does not exceed one half of its solubility.
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CG-1710
a. Immediately after the solutions are added to the
solids, tightly cap the containers and
vigorously agitate them for several minutes with
a vortex mixture or similar device.
h. Shake the containers throughout the
equilibration period at a rate that suspends all
solids in the solution phase.
2. Centrifugation
When the equilibration time has expired, centrifuge the
containers for t minutes.
3. Chemical Extraction
a. After centrifugation, remove the supernatant
aqueous phase from the solid-solution mixture.
b. Extract the chemical adsorbed on the sediment or
soil colloid surfaces with solvent.
4. Chemical Analysis
Determine the amount of parent test chemical in the aqueous
equilibrating solution and organic solvent extractions. Use any
method or combination of methods suitable for the identification
and quantitative detection of the parent test chemical.
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C 0 - 1 7 1 0
III. REPORTING
Report, the following information using Cables 1 and 2 or a
similar format:
(1) Temperature at which the test was conducted.
(2) Detailed description of. the analytical technique(s)
used in the chemical extraction, recovery, and
quantitative analysis of the parent chemical.
(3) Amount of parent test chemical applied, the amount
recovered, and the percent recovered.
(4) Extent of adsorption by containers and the approach
used to correct the data for adsorption by
containers.
(5) The individual observations, the mean values, and
graphical plots of x/m as a function of Ce for each
sediment or soil for (a) the equilibration time
determination, and (b) the isotherm determination.
(6) ^he quantities K, n, and 1/n.
(7) Soil information: Soil Order, series, texture,
sampling location, horizon, general clay fraction
mineralogy.
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(8) Sediment information: sanroling location, general
clay fraction mineralogy.
(9) Pediment and soil physical-chemical nroperties:
percent sand, silt, and clay (particle size
analysis); percent organic matter; pH (1/1
); and cation exchange capacity.
(10) The procedures vised to determine the
physical/chemical properties listed above.
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TABLE 1
SEDIMENT AND SOIL ADSORPTION ISOTHERM DATA FORMAT
Sediment/Soil No. and Name
K
n
1/n
Temperature
Solid/Solution Ratio
Amount of Chemical Applied
Standard Deviation
Percent Recovered (Mean)
Standard Deviation
x/n
Replication 1
Replication 2
Replication 3
Mean
Standard Deviation
Adsorption Isotherm Determination
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1 (continued)
ce
Replication 1 :
Replication 2 :
Replication 3 :
Mean :
Standard Deviation :
Original Concentration :
Equilibration Tjme Determination
Sampling Time (hrs)* : 1 2 4 8 12 24 36 48 60 72
Ce :
x/m :
*Suggested Sampling Times.
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TABLE 2
SEDIMENT PHYSICAL, CHEMICAL, AND CLASSIFICATION DATA FORMAT
Sediment #1 Sediment #2
Lccation:
Percent SAND:
Percent SILT:
Percent CLAY:
Percent ORGANIC MATTER:
pH (1/1 Sediment H2O):
CEC (meq/lOOg):
CLAY MINERAL ANALYSIS:
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TABLE 3
SOIL PHYSICAL, CHEMICAL, AND CLASSIFICATION DATA FORMAT
Soil #1
Soil #2 Soil #3
SOIL ORDER:
SOIL SERIES:
SOIL TEXTURE:
LOCATION:
HORIZON:
Percent SAND:
Percent SILT:
Percent CLAY:
Percent ORGANIC MATTER:
pH (1:1 soil:H20):
CEC (meg/lOOg):
Alfisol
Crider
Silt Loam
Gallatin County, 111
A
1.2
6.6
12.2
1.74
7.20
13.5
CLAY MINERAL ANALYSIS: 75 percent Montmorillonite
5-20 percent Mica
5 percent Kaolinite
(25-120 cm depth)
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IV. REFERENCES
Aharonson V, Kafkafi U. 1975. Adsorption, mobility and
persistence of thiabendazole and methyl 2-benzimidasole carbamate
in soils. J Agr Food Chem 23:720-724.
Goring CAI, Hamaker JW. (eds). 1972. Organic chemicals in the
soil environment. Vol. I & II. New York: Marcel Oekker, Inc.
Harvey RG et al. 1974. Soil adsorption and volatility of
dinitroaniline herbicides. Weed Sci 22:120-124.
Murray DS et al. 1973. Comparative adsorption, desorption, and
mobility of dipropetryn and prometryn in soil. T \c\r Food Chem
23:578-581.
Saltzman SL et al. 1972. Adsorption, desorption o^: parathion as
affected by soil organic matter. J Agr Food Chem 20:1224-1226.
Weber JR. 1971. Model soil system, herbicide leaohinq, and
sorption. Weed Sci 19:145-160.
Wu CH et al. 1975. Napropamide adsorption, desorption, and
movement in soils. Weed Sci 23:454-457.
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CS-1710
August, 1982
SEDIMENT AND SOIL ADSORPTION ISOTHERM
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CS-1710
CONTENTS
Page
I . NEED FOR THE TEST 1
II. SCIENTIFIC ASPECTS OF SOIL LEACHING 2
A. Introduction 2
B. Basic Processes Affecting Soil Leaching 2
C. Chemical Properties Affecting Leaching 4
D. Soil Properties Affecting Adsorption 4
E. Types of Adsorptive Forces 7
F. Surface Transformations 8
III. SCIENTIFIC ASPECTS OF SEDIMENT-CHEMICAL TRANSPORT
AND ADSORPTION 10
A. Introduction 10
B. Basic Sedimentation Processes Affecting
Chemical Movement 10
C. Chemical Properties Affecting Adsorption 12
D. Sediment Properties Affecting Adsorption 12
IV. SCIENTIFIC ASPECTS OF THE TEST 14
A. Development of the Adsorption Isotherm 14
B. Rationale for the Selection of the Adsorption
Isotherm Test 15
C. Rationale for Selection of Experimental
Conditions and Procedures 17
V. REFERENCES 22
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SEDIMENT AND SOIL ADSORPTION ISOTHERM
I. NEED FOR THE TEST
The Sediment and Soil Adsorption Isotherm (AI) is a screening
test suitable for obtaining an estimate of the sediment adsorption
potential of a chemical and its soil leaching potential. The
adsorption affects the distribution of a chemical in the environ-
ment. Knowledge of the adsorption potential is essential under
certain circumstances for the assessment of the fate of chemicals
in the environment. If a chemical is tightly adsorbed to soil
particles, it will not leach through the soil profile but will
remain on the soil surface. If a chemical is weakly adsorbed, it
may leach through the soil profile and may reach ground waters and
then surface waters. Similarly, if a chemical is tightly adsorbed
to sediment, it will accumulate in the bed and suspended load of
aquatic systems. If a chemical is weakly adsorbed to sediment, it
ir:ay be found predominately in the water column of aquatic systems.
Since adsorption can affect the distribution of a chemical in
the environment, it may have a profound effect on a chemical's
effect on man, the ecosystem in question, and on species within
the ecosystem. If a chemical reaches ground and/or surface
waters, it may cause deleterious human health effects by
contaminating the drinking water. If a chemical remains at the
soil surface, it may cause deleterious environmental and human
health effects by contaminating the drinking water. If a chemical
remains at the soil surface, it may cause deleterious environmen-
tal iind human health effects due to it presence in the zone of
plant: growth that may result in contaminated feed and food.
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II . SCI^TTIFIC ASPECTS OF SOI
A . Introduction
The leaching of chemicals in soils is a-^ected bv several
interacting processes, including adsorption, that occur at the
soil-water interface. mhe inter-Facial region is important for two
reasons. First, it determines the adsorption mechanism and the
energv by which the chemical is held. Second, it may catalyze the
transformation of the original compound. transformation
product(s) are of particular concern, since they (1) mav be toxic
to a greater or lesser degree than the original compound, (2) may
be absorbed either greater than or less than the original
compound, and (3) may have a water solubility either greater than
or less than the original compound, thereby a^ecting i-f-.s leachina
and movement into the ground water. This section of the support
document will discuss these processes as they relate to leaching
and the AI Test Guideline.
R . Basic Processes Affecting Soil Leaching
The leaching of chemicals through soil is a complex
phenomenon consisting of several major processes (Hamaker 1975).
One general equation (Guenzi 1974) for chemical movement through
porous media under steady state soil-water flow conditions for
water in soil is:
H3S/63t + 3c'/3t = D' 32c* /3x2 - u3c'/3x (1)
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where R = soil bulk density (q/cm. }
a = volumetric water content (cm /cm )
S = mass fraction of test chemical adsorbed at the
soil/water interface (g test chemical/g soil)
t = time (s)
c1 = concentration of test chemical in solution (g/cm )
O
D' = dispersion coefficient (cm /s)
V = average pore-water velocity (cm/s)
X = space coordinate measured normal to the section
Most mass transport equations represent simplifications of
"real world" conditions that attempt to describe the chromato-
graphic distribution of the chemical in the soil profile. They
are gross simplifications of a phenomenon that is affected by
complex, interacting processes. In general, chemical leaching is
dependent upon three major processes: the mass transport of water
(the direction and rate of water flow), the rate of diffusion, and
the adsorption characteristics of the chemical in soil (Guenzi
1974) .
Diffusion is the transport of matter resulting from random
molecular motion, which is caused by molecular thermal energy.
This random motion leads to the uniform distribution of molecules
in a closed fluid system, since a net movement of molecules from
regions of higher to lower concentrations occurs.
Adsorption is the accumulation of molecules by the attractive
force's of the surface of a solid phase. when adsorption is a
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CS-1710
significant factor, there is a higher concentration of a chemical
in an extremely thin layer at the surface of a sediment or soil
than is present in the bulk aqueous solution associated with the
sediment and soil. The equilibrium adsorption-desorption process
is governed by two opposing rate processes. The adsorption rate
is the rate at which molecules from the liquid phase transfer into
the adsorbed state in the solid phase and the rate increases as
the concentration of dissolved species increases. The desorption
rate is the rate of the opposite process, i.e., the rate at which
molecules transfer from the adsorbed state in the solid phase into
the liquid phase. Equilibrium is established when the rates of
these two processes are equal.
C. Chemical Properties Affecting Adsorption
Adsorption is the main process that determines a chemical's
leaching potential as described mathematically in equation 1. It
is governed by the properties of both the solid phase and the
adsorbate. The important properties of the absorbate that affect
adsorption by soil (Bailey and White 1970) are: (1) chemical
structure and conformation (2) molecular size, (3) acidity or
basicity of the molecule (pK or pK. ), (4) water solubility, (5)
permanent charge, (6) polarity, and (7) polarizability.
D. Soil Properties Affecting Adsorption
The soil properties affecting the adsorption and desorption
of organics include the organic matter content, type and amount of
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region of clays is a function of the basicity o^ the molecule, the
nature of the exchangeable cation on the clay, water content of
the clay system, and the origin of negative charge in the alumino-
silicate clay (Bailey and White 1970).
E. ^vnes of Adsorptive Forces
The specific type of interaction that orqanic molecules have
with soil depends on the chemical properties of the organic
molecules and the tvpe o^ soil. ^hese interactions or adsorptive
forces are classified as: van der Waals forces, charge transfer,
ion exchange, and hydrophobic bonding (Adams 1975, Goring and
Hamaker 1972). In general, one or more of these specific interac-
tions or adsorptive forces may occur simultaneously.
The attractive van der Waals forces or polarizability forces
arise ^rom the random fluctuations in a molecule's electron dis-
tribution. These fluctuations theoretically produce instantaneous
dipoles due to the concentration o^ charges in one region o^ the
molecule and cause that molecule's attraction to other atoms and
molecules.
Charge transfer involves the -Formation o^ a donor-accentor
complex between an electron donor molecule and an electron
acceptor molecule with partial overlap of their respective
molecular orbitals and a partial exchange of electron density-
Ion exchange refers to the exchanqe between counterions
balancing the surface charge of the soil colloid and the ions in
-7-
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CS-1710
the soil solution. The driving force for this interaction is the
requirement for electroneutrality: the surface charge must be
balanced by an equal quantity of oppositely charged counterions.
In general, ion exchange is reversible, diffusion controlled, and
stoichiometric in most cases. It exhibits some selectivity or
preferential adsorption for one ion over another competing ion.
Hydrophobic bonding refers to the greater affinity of an
organic molecule for a hydrocarbon solvent or hydrophobic region
of a colloid than for a hydrophilic solvent. Hydrocarbon regions
of a molecule have greater solubility in liquid hydrocarbons (or
most organic solvents) than in water.
F. Surface Transformations
A special type of interaction between organic molecules and
soils deals with the transformation of organic chemicals into new
compounds containing different chemical structures through the
catalytic activity of the soil colloid surfaces. Although several
theories exist to account for the mechanism of these transforma-
tions, no scheme that predicts the occurrence of these surface
reactions presently exists. Therefore, it is extremely important
that mass balance calculations for the test chemical are performed
to ascertain the extent of these transformations during soil
leaching experiments.
The scientific literature shows that a number of chemicals
and chemical classes undergo colloid surface induced chemical
transformations. Poly-(dimethylsiloxane) fluids in intimate
-8-
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CS-1710
clay, exchange capacity, and surface acidity (Adams 1973, Bailey
and White 1970, and Helling 1970). The combined actions of
clirnate, micro-, and microorganisms over long periods of time on
different parent geologic and biotic materials form soils that
differ widely in their physical, chemical, morphological, and
adsorption characteristics. The amounts and types of clay and
organic matter, soil pH, primary and secondary minerals, struc-
ture, texture, and exchange capacity vary considerably for U..S.
soils. There are currently 10 Soil Orders, at least 43 Suborders,
over 200 Great groups and over 7,000 soil series recognized in the
United States (Buckman and Brady 1969).
Soil organic matter is a primary soil property responsible
for the adsorption of many chemicals. Helling (1970) lists many
examples where the organic matter primarily influenced the
adscrption of pesticides. Organic matter and clay are the soil
components most often implicated in pesticide adsorption.
However, the individual effects of either organic matter or clay
are not easily ascertained. Since the organic matter in most soil
is intimately bound to the clay as a clay-metal-organic complex
(Stevenson 1976), two major types of adsorbing surfaces are
normally available to the chemical, namely, clay-organic and clay
alone. Clay and organic matter function more as a unit than as
separate entities and the relative contribution of organic and
inorganic surfaces to adsorption will depend on the extent to
which the clay is coated with organic substances. Comparative
studies between known clay minerals and organic soils suggest that
most, but not all, pesticides have a greater affinity for organic
-5-
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CS-1710
surfaces than for mineral surfaces (Stevenson 1973). Since
typical studies compare soils in which both clay and organic
matter increase and do not use multiple regression analyses to
isolate the governing parameter (Helling 1970), only generaliza-
tions concerning the relative importance of clay and organic
matter can be made.
Surface acidity is another soil property affecting the
adsorption of many organic chemicals. Surface acidity is probably
the most important property of the soil or colloidal system in
determining the extent and nature of adsorption of basic organic
chemicals, as well as determining if acid-catalyzed chemical
transformation occurs (Bailey and White 1970). The activity of
protons in the bulk suspension, which is expressed by pH, and the
activity of protons at or in close proximity to the colloidal
surface (i.e., the acidity in the interfacial region) may differ
significantly. The term "surface acidity, " when applied to soil
systems, is the acidity at or in close proximity to the colloidal
surface that reflects the ability of the system to act as a Lewis
acid. Surface acidity is a composite term that reflects both the
total number of acidic sites and their relative degree of acidity.
Overwhelming evidence, mainly from infrared and other
studies, indicates that the clays that protonate basic chemicals
either have hydrogen and aluminum as the predominant exchangeable
cations, or are saturated with alkali metal, alkaline earth metal,
and transition metal cations. A summary of recent investigations
indicates that the protonation of chemicals in the interfacial
-6-
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CS-1710
contact wit.h many soil1? undergo siloxane bond redistribution and
hydrolysis that result in the formation of low molecular weiqht
cyci.ic and linear oliqomers (Ruch and Tnqebriqhtson 1979).
Substituted (White 1976) and organophosphorus pesticides (Yaron
1978, Minqelrin et al. 1977) underqo clay colloid induced
hydrolysis. Benzene and phenol polymerize into hiqh molecular
weicht chemicals by adsorption and reaction at the surface of
smectite saturated with transition metal cations (Mortland and
Halloran 1976). Gallic acid, nyroqallol, nrotocatechuic acid,
caffeic acid, orcinol, ferulic acid, p-coumaric acid, syrinqic
acid, vanillic acid, and p-hydroxybenzoio acid underqo oxidative
polymerization in the presence of various clav minerals (Wanq and
Li 1977, Wanq et al. 1978).
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CS-1710
III. SCIENTIFIC ASPECTS OF S^niMF/TT'-cn^MTCAL, ^RAMSPORT VTO
A. Introduction
The transport and adsorption of chemicals bv sediment are
affected by a larqe number of interacting processes. Furthermore,
chemical movement in aquatic systems, unlike soil systems, is
dependent not only on the extent of adsorption but on the movement
of sediment. ^his ^est Guideline will develop data on the extent
of a chemical°s adsorption onto sediments. Sediment movement can
be mathematicallv estimated in several wavs for a sneci^ic
situation provided hydrologic and meteorologio information is
available. ^his section o^ the support document wil 1. discuss the
transport and adsorption of chemicals by sediment as they relate
to the Test Guideline.
B ^asic Sedimentation Processes Affecting Chemical
Movement
Sediment is the unconsolidated inorganic and organic material
that is being transported or has been transported by and deposited
in beds from water. Synthesis, erosion, transportation, and
deposition of sediment are natural processes that have occurred
throughout geologic time. T'he extent of biologic activity and fh<=>
extent of erosion will govern the amount of sediment that enters a
watershed (Chow 1964). In general, everv sediment particle that
passes a particular cross section of a water body must satisfy two
-10-
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CS-171U
conditions: (a) it must have been eroded or synthesized somewhere
in the watershed above the cross section and (b) it must have been
transported by the flow of water from the place of erosion or
synthesis to the cross section. (Chow 1964). The wash load is
the finer sediment fraction that the flow can easily carry in
large quantities. The bed-material load is the coarser sediment
fraction that is difficult to move by the flow and is limited in
its movement by the transporting ability of the flow between
source and section. The bed load is the sediment in the bed layer
that cannot be suspended in the water column for fluid-dynamic
reasons. The basic difference between wash load and bed-material
load can best be visualized in a concrete-lined channel. If the
flow is large and fast, the flow condition is not in any way
affected by adding small amounts of a fine and easily transported
material. This added material, the wash load, moves in suspension
with the flow at the same average velocity and does not settle.
If the flow velocity and discharge are now reduced and/or if the
material is increased in size and rate, sediment will begin to
deposit on the channel bottom and a granular sediment bed will
develop. These sedimenting particles are designated as the bed-
material load.
Sediment particle size is the single most important physical
parameter affecting sediment transport and deposition. The
exemplified differences between wash load, bed-material load, and
bed load show that different sediment sizes behave differently in
the same hydrologic system. Similar quantitative differences in
behavior exist between different sizes of the bed-material load.
-11-
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CS-1710
The grain size distributions in the bed and in transport are quite
often different, even within the size range of the bed-material
load. Also, many streams have heterogeneous beds with individual
bars having very different composition and appearance. Finally,
many stream and river beds exhibit another type of segregation in
which all coarse particles are concentrated in lenses, or layers,
at a greater or lesser depth below the bed surface.
C. Chemical Properties Affecting Adsorption
A chemical's adsorption potential is governed by the
properties of both the sediment phase and the adsorbate. The
important properties of the adsorbate affecting adsorption onto
sediments are basically the same properties affecting adsorption
onto soil colloids as discussed in Section II.C. They are: (I)
chemical structure and conformation, (2) acidity or basicity of
the molecule (pK or pK,) , (3) water solubility, (4) permanent
charge, (5) polarity, (6) molecule size, and (7) polarizability.
D. Sediment Properties Affecting Adsorption
The inorganic chemical composition of sediments includes most
primary and secondary minerals. In general, it is composed of
mineral fragments having particle sizes ranging from clay and silt
to sand, gravel, and boulders. The mineralogy of original source
rocks, together with chemical weathering processes, mechanical
weathering processes, and precipitation processes, determine the
ultimate size, weight, shape, and, therefore, the adsorptive
-12-
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CS-1710
capacity of inorganic sediment particles. The mineral content
contributes to the sorting phenomena. Heavy minerals will deposit
at higher flow velocities compared to lighter materials of equal
size. Hard minerals, such as quartz, will resist abrasion to a
greater degree than soft, chemically unstable minerals, such as
gypsum or limestone. Some minerals and rocks disintegrate along
crystal faces or cleavage planes to form platelike particles while
others form equidimensional particles.
The organic sediment particles are composed of plant and
animal tissue in various stages of chemical and microbial
decomposition. The highly varied composition and the continuous
fluctuation of the hydrologic conditions found in many aquatic
systems create heterogeneous sediments with a widely range of
characteristics.
A review of the literature on the adsorption of chemicals
reveals that pesticide-soil studies dominate the literature. Most
of "he principles applicable to the adsorption and transformation
of chemicals in soils (Section II, Parts C, D, R, and F) are
applicable to sediments. In general, sediments have a finer
texture than soils. They contain more amorphous organic matter
due to the biotic activity in water and contain more clay due to
the erodability of the finer soil components. Therefore, sedi-
ments generally show higher sorption tendencies than soils (Pionke
and Chesters 1973, Lotse et al. 1968). However, Pionke and
Chesters (1973) state that little information exists on sediment-
chemical interactions to indicate differences from typical soil-
chemical interactions.
-13-
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CS-1710
IV. SCIENTIFIC ASPECTS OF
A. Development of the Adsorption Isotherm
The fact that solids can remove salts and color from solution
by adsorption has been known from the earliest times. For
example, Aristotle knew that seawater lost some of its taste by
filtration through sand (Forrester and Giles 1971b) . Since then,
the phenomenon of adsorption has been used to solve various water
purification problems. In the 17th and 18th centuries, seawater
purification by soil was used on the Rarbary Coast to produce
fresh water (Forrester and Giles 1971b) . During the 19th century,
a number of soil chemists published studies on the adsorntion of
both basic and acidic compounds onto soils. In 1881, van Remmelen
published the first solute-solid adsorption diaqram based on his
studies of sulphuric acid adsorption by metastannic acid
(Forrester and Giles 1972). The only previously nublished
adsorption diagrams were those for gas-solid adsorption plotted by
Chappius and by Kayser in 1880. The term "adsorption" appeared
for the first time in the English language in an 1882 abstract of
Kayser' s work in Nature (Forrester and Giles 197la). T>ie term
"isotherm" was first used by Ostwald in 1885 to describe the plot
of pressure (abscissa) versus the amount of gas adsorbed
(ordinate) (Forrester and Giles 1971a) . Soon afterwards, the term
"adsorption isotherm" v/as adopted to describe solute-solid adsorp-
tion diagrams. In the first decade of this century, adsorption
isotherms began appearing frequently in the published literature,
often as adjuncts to other investigations. Since then, the
-11-
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CS-1710
isotherm has been used for innumerable adsorption studies usinq an
extremely wide variety of solutes, solvents, and solids over the
entire ranqe of experimentally obtainable nressures and
temperatures.
Since adsorption is the major retention mechanism ^or most
organic and inorganic compounds in soils (Section TT), the
magnitude of any mathematical or empirical estimation of a chemi-
cal's soil leaching potential (e.g., equation (1)) is, in general,
proportional to the magnitude of the adsorption coepficient.
Therefore, one need only estimate the adsorption coefficient to
obtain an estimate of a chemical's leaching potential in soil.
Similarly, a chemical's distribution in aquatic system will, in
general, depend upon the magnitude of its adsorption to
sediment. Therefore, an estimate of the adsorption potential is
needed to estimate a chemical's distribution in the water column.
R. Raticinale for the Selection of the Adsorption Isotherm
Test
Soil thin layer chromatography, soil AT, and soil columns
have been developed to obtain an estimate of a chemical's leaching
potential (Hamaker 1975). ^he M test is applicable to obtaininq
both sediment adsorption potential data and soil adsorption and
mobility data.
This ^est Guideline is developed upon the basic principles
comprising most adsorption isotherm tests found in scientific
literature. ^he extent of adsorption o^ a chemical onto sediments
-------
CS-1710
or soils is measured by equilibrating aqueous solutions containing
different but environmentally realistic concentrations of the test
chemical with a known quantity of sediment or soil. *\fter
equilibrium is reached, the distribution of the chemical between
the water phase and the solid phase is quantitatively measured bv
a suitable analytical method. ^hen, sorption constants are
calculated by usinq the Freundlich equation:
x/m = Cs = KCe
1/n
where
Cp =equilibrium concentration o^ the chemical in the
solution phase
Cg =equilibrium concentration of the chemical in the solid
phase
K =Freundlich adsorption coefficient
m =the mass of the solid in qrams
1/n =exponent where n is a constant
x =the mass in micrograms of the chemical adsorbed bv m
qrams of solid.
A logarithmic transformation of the Freundlich equation vields the
following linear relationship:
log C = loa K + (1/n) log r (2)
-16-
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CS-1710
In order to estimate the environment.a 1 movement of the chemical in
surface water svstems and soils, the values K and 1/n are compared
with the values of other chemicals whose behavior in soil and
sediment systems is well-documented in scientific literature.
1T1he adsorption isotherm (Al) test was selected because it
contains many desirable features. pirst, adsorption results are
hiqHlv renrodncible. ^he test provides excellent quantitative
dat i readily amenable to statistical analyses. Also, it has
relatively modest requirements ^or chemicals, soils, laboratorv
space, and equipment. It allows solution phase orqanic chemical
de4:erminations that are relativelv uncomplicated. A chemical
extraction-mass balance procedure to elicit information on
chemical transformations occurrinq at colloid interfaces can be
Incorporated into this test. The ease of performinq the isotherm
test and mass balance will depend upon the phvsical/chemica1
properties of the test chemical and the availabilitv o^ suitable
analytical techniques to measure the chemical.
C. Rationale for the Selection of Experimental Conditions
and Procedures
The papers by Aharonson and Kafkafi (1975), Harvev (1974),
Murray (1975),, Saltzman (1972), Weber (1971), and Wu (1975) served
as the basis 'cor this ^est Guideline. mhe soil and colloid
ohem:stry literature and the analytical chemistry literature
-.instantiate the experimental conditions and procedures specified
in t': e suqqested Test Guideline as accepted, standard procedures.
itionale for the selection o^ these conditions and procedures
v.?; 1 ! >->e disc issod in greater detail below.
-17-
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CS-1710
The AI Test Guideline can be used to determine the soil
adsorption potential of sparingly water soluble to infinitely
water soluble chemicals. In general, a chemical having a water
solubility of less than 0.5 ppm need not be tested, since the
literature indicates that these chemicals are, in general,
immobile (Goring and Hamaker 1972). However, this does not
preclude future soil adsorption/transformation testing of these
chemicals if more refined data are needed for the assessment
process.
The 0.01M calcium nitrate is required to insure colloid
flocculation during the experiment.
Distilled-deionized F^O and glassware are required to
minimize competition effects for exchange sites by unidentified
cationic and anionic species normally present in tap and distilled
H2O.
Sterile water and glassware are required to minimize the
potential for microbial growth in the test containers.
The absorption isotherm standard can be used to determine the
adsorption potential of volatile chemicals, since the standard
requires the use of containers that are capped and the use of a
solid/solution ratio that minimizes container headspace.
A room temperature range of 23±5°C was adopted since the
available literature indicated that pesticide adsorption in
general does not significantly change in the temperature range of
1° to 30°C.
-18-
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CS-1710
Replication of the basic experimental unit was necessary in
order to estimate the standard deviation of the treatment mean.
Three replicates are considered to be the minimum number of
replicates for a statistically acceptable estimation.
It is important that gentle crushing and grinding be used to
reduce soil aggregate size. Fine particles (silt and clay) in
excess of the amount originally present may be created if exces-
sive pressure is exerted on the aggregates.
Soil sieving with a 100 mesh stainless steel or brass sieve
is required to remove debris and coarse fragments. Removal of
these components should improve the reproducibility of the adsorp-
tion test and will aid in obtaining a more uniform sample.
In order to obtain reproducible results, it is necessary
to: (1) sterilize the soil or sediment to prevent microbial
growth and degradation of the test chemical; (2) choose the
appropriate sterilization technique to make sure that the soil or
sediment surface properties are not altered; and (3) store the
pretreated soil or sediment at 0 to 5°C to minimize microbial
effects.
It is extremely important that the solvent chosen for
extraction be: (1) analytically pure or better; and (2) the
minimum solubility of the test chemical in the solvent be at least
10 g/1. The first condition is necessary to minimize the effects
of solvent impurities on the analytical determination of the
concentration of chemical on the soil or sediment and in aqueous
-19-
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CS-1710
solution. mhe second condition is necessary to make sure that the
test chemical is essentially completely extracted so that an
accurate value of the concentration of chemical adsorbed on the
soil or sediment and in aqueous solution is obtained.
In carrying out the adsorption experiments, it is recommended
that the solid/solution ratio be approximately equal to 1/10 or
greater. This ratio will give the best reproducible results.
However, for certain chemicals which do not adsorb readily, it is
preferable to use the ratio of approximatelv 1/5 or slightly
greater to give the best reproducible results. However, it is not
practical to use ratios much greater than 1/5, since the mixture
would be too viscous and true equilibrium would not be achieved.
Hence, under these conditions, the experimental results would be
erroneous.
Tn general, a majoritv of chemicals should attain equilibrium
between the solid and solution phases within 24 to 48 hours. For
unknown reasons, however, many chemicals require longer equilibra-
tion periods. Since it is not presently possible to predict the
proper equilibration period for a chemical, the "est Guideline
recommends that the equilibration period be determined as a part
of: the test procedure for all chemicals.
In general, the test chemical should be of the purest grade
readily available. Impurities may produce mass balance data
indicating the presence of transformation products. Transforma-
tion product identification is an expensive analytical procedure
that may be unnecessarily required due to the presence of
impurities.
-20-
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CS-1710
The Soil Order, series, and general clay fraction mineralogy
data may be found in Soil Survey Reports published after approxi-
mately 1970. Pre-1970 reports may not contain mineralogy data.
Soil survey reports have been issued for most U.S. counties and
may be obtained from County Extension Offices; the State Office of
the U.S. Department of Agriculture Soil Conservation Service; or
the USDA-Soil Conservation Service, Publications and Information
Division, P.O. Box 2890, Washington, DC 20013. If mineralogy
data are not printed in a report, the State Office of the U.S.
Department of Agriculture Soil Conservation Service may be con-
tacted for assistance in obtaining general clay mineral data of a
particular soil. The Test Guideline does not require soil mineral
analysis since general clay mineralogy data may already exist for
the test soil.
-21-
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CS-1710
V. RFFKRF^TCFS
Adams RS Jr. 1973. Factors influencinq soil adsorntion and
hioact.ivity of pesticides. Residues Rev 47:1-54.
Aharonson "T, Kafkafi U. 1975. Adsorption, mobility, and
persistence of thiabendazole and methy 2-ben^.imidasole carbamate
in soils. J Aqr Food Chem 23:720-724.
Bailey GW, VThite JL. 1970. Factors influencing the adsorption,
desorption, and movement of pesticides in soil. Residue
32:29-92.
Block RJ Durrum FL, 7,weiq G. 1958. A manual of naper
chromatography and paper electrophoresis. Second Edition.
York: Academic Press.
Buch RR, Inqebriqhtson ON. 1979. Rearranqement of only-(dimethyl/
siloxane fluids on soil. Fnviron Sci and Technol 13:676-679.
Ruckman HO, Brady TTC. 1969. The nature and properties OF sgils.
London: The Macmillan Company.
Chow U^1. (ed) . 1964. Handbook o^ applied hydrology. *Tew vork:
McGraw-Hill Book Co.
Forrester SO Giles CM. 1971a. The qas-solid adsorption isotherm:
A historical survey up to 1918. Chemistry and Industry, pp. 831-
839.
Forrester SO, Giles CM. 1971b. Prom manure heaps to monolavers:
^he earliest development of solute-solid adsorption studies.
Chemistry and Industry, pp. 1314-1321.
Forrester SO, Giles CM 1972. From manure heaps to monolayers: One
hundred years of solute-solvent adsorption isotherm studies.
Chemistry and Industry, pp. 318-325.
Gorinq CAI, Hamaker ..71V. (eds). 1972. Organic chemicals in the soil
environment. Vol. I & II. Mew York: Marcel Oekker, Inc.
Guenzi WD. (ed). 1974. Pesticides in soil and water. Madison,
WI: Soil Science Society of America, Inc.
Hamaker .TW. 1975. The interpretation of soil leaching
experiments. Fnviron Sci Res 6:115-133.
Harvey RG et al. 1974. Soil adsorption and volatility of
dinitroaniline herbicides. Weed Sci 22:120-124.
Hellinq CS. 1970. Movement of s-triazine herbicides in soils.
Residue Rev 31:175-210.
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CS-1710
Lots 5 EG, Graetz DA, Chesters G, Lee QR, ^ewland LW. 1068. Lindane
adsorption by lake sediments. Environ Sci T'echnol 5:353-357.
Martin AJP, Synge RLM . 194]. A new form of ehromatonram emplovinq
two Liquid phases. Riochem J 35:1358.
MillLnqton R J , Quirk JP. 1961. Permeability of norous solids.
Trans Faraday Soc 57:1200.
Mingelgrin U, Saltzman S, Varon R. 1977. A possible model for the
surface induced hydrolysis or organophosphorous pesticides on
kaolinite clavs. Soil Sci Soc Amer Jour 41:519-523.
Mortland MM, Halloran L J . 1976. Polymerization of aromatic
molecules on smectite. Soil Sci Soc Amer Jour 40:367-370.
Murray DS et al. 1973. Comparative adsorption, desorption, and
mobility of dipropetryn and prometrvn in soil. J Aqr Food Chem
23:578-581.
Pionke HR, Chesters G. 1973. Pesticide-sediment-water
interactions. J Environ Oual 2(1): 29-45.
Riley O. 1976. Physical loss and redistribution of pesticides in
the liquid phase. In: Rritish crop protection council symposium
proceedings , PP. 109-115.
Salt.2,man SL et al. 1972. Adsorption, Desorption of narathion as
affected by soil organic matter. J Aqr Food Chem 20:1224-1226.
Stevenson F J . 1976. Organic matter reactions involving pesticides
in soil. In; Bound and conjugated pesticide residues. ACS
Symposium Series Monograph 29.
Wang ^OC, Li SW. 1977. Clay minerals as heterogeneous catalysts in
preparation of model humic substances. 7,. Pf lanzenernaehr Rodenkd
140:669-676.
Wang TSC, Li SW, Ferng YL. 1978. Catalytic polymerization of
phenclic compounds by clay minerals. Soil Sci 126:15-21.
Weber JB . 1971. Model soil system, herbicide leaching, and
sorption. Weed Sci 19:145-160.
White JL. 1976. Determination of susceptibility of s-triazine
herbicides to protonation and hydrolysis bv mineral surface. Arch
Environ Contam Toxicol 3:461-469.
Wu CH et al. 1975. Napropamide adsorption, desorption, and
movement in soils. Weed Sci 23:454-457.
Varon B. 1978. Some aspects of surface interactions of clavs with
organophosphorus pesticides. Soil Sci 125:210-216.
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TRANSFORMATION PROCESSES
-------
CS-2000
August, 1982
AEROBIC AQUATIC BIODEGRADATION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
CS-2000
Contents
Page
I. NEED FOR THE TEST 1
II. SCIENTIFIC ASPECTS 4
A. Test Methods 4
B. Test Conditions 10
1. Incubation Temperature 10
C. Test Procedures 10
1. Reference Compounds 10
2. Inhibited Systems 11
3. Replication 12
4. Sampling Frequency and Duration 12
5. Filtration 13
D. Test Data 13
III. REFERENCES 14
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CS-2000
AEROBIC AQUATIC RIODEGRAOATIOM
I . *JEKD FOR
"^he transformation of orqanic substances by livinq orqanisms
is an important factor in determininq their environmental fate.
Orqanic substances may be transformed by nonbioloqical as we! 1. as
bioloqical mechanisms, such as nhotolysis, hvdrolvsis and
oxidation. ^here is little doubt, however, that biodeqradat ion
is the predominant mechanism for the transformation of manv
orqanic compounds in soil and water. Evidence indicates that
microorqanisms are responsible for convertinq many complex
orqanic substances to inorqanic products { Alexander 1^73,
Howard et al. 1975 p. 37).
Riodeqradat ion is often the most desirable mechanism
decomposinq orqanic substances. ^his is especially true if
biodeqradat ion is rapid and if deqradation products are inorqanic
molecules and metabolites that may be used for enerqy and
microbial qrowth . Photochemical deqradation and other chemical
processes usually do not completely mineralize orqanic
substances , and resultinq products of unknown toxicitv and /or
persistence may be generated (Alexander 1967).
Laboratory evaluations ^or determininq biodeqradabilitv are
an important part of testinq to indicate whether a substance is
likely to persist in the presence of microorqanisms in the
natural environment or in bioloqical treatment processes. T f the
substance does not persist, it mav be necessary to determine
whether the substance deqrades to innocuous molecules or whether
some relativelv persistent and toxic intermediate is ^ormed . It
-1-
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CS-2000
also may be important to obtain better estimates for
biodegradation rates under various environmental conditions in
order to more precisely assess the persistence of a substance.
The assessment of risk from environmental exposure to organic
substances depends upon estimates of environmental concentrations
of the parent substances and potentially toxic transformation
products. Because biodegradation plays a vital role in the
transformation of most organic compounds in the environment,
knowledge of biodegradation rates and products is an important
element in the assessment process.
Chemical substances can enter natural waters in a variety of
ways. These include runoff from land, discharges of industrial
wastes, home and commercial use with disposal into sewers, spills
and leaks, leaching from landfills and transfer from the
atmosphere through rainfall or particulate deposition. Surface
waters normally contain bacterial populations which are
continually replenished from sewer outfalls and land runoff and
which are capable of the uptake and metabolism of many of these
chemical substances. Microbes account for a rapid turnover and
substantial breakdown of such substances, particularly those
organic substances of relatively low molecular weight
(R. T. Wright 1979).
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It is therefore important to obtain an understanding of
whether or not chemical substances which are released to or which
may be transported to aerobic surface waters will be degraded by
the microbial populations in those waters. The most cost-
effective way to obtain that knowledge is through laboratory
studies using aerated water containing the test substance and
representative microorganisms.
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II. SCIENTIFIC ASPECTS
A. Test Methods
Laboratory techniques which are used to study microbial
degradation processes in aerobic aquatic environments include
(1) those which test for the biodegradability potential of a
substance without any attempt to carefully simulate any
particular portion of the aquatic environment and (2) those which
do attempt to simulate natural water bodies (e.g. a given lake or
stream) in an effort to determine the rate and extent of
biodegradation at a specific site.
The purpose of the procedure in this guideline is to screen
for the biodegradation potential of substances in aerobic aqueous
environments in general. The method is applicable to a wide
variety of substances and is not intended to simulate any
particular aquatic environment. Those procedures which do
attempt to simulate a specific site (for example by carefully
controlling most of the important variables such as pH, salinity
and nutrient concentration) are more appropriately employed at a
higher tier or step in a testing program.
The screening methods for biodegradability potential in
aerobic/ aquatic environments may be subdivided into tests for
ready biodegradability and for inherent biodegradability, as in
the OECD Level I and Level II methods (OECD 1979). Methods to
test for ready biodegradability are designed so that positive
results are unequivocal and lead to the reasonable assumption
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that the substance will undergo rapid and ultimate biodegradation
in the environment (biodegradation to inorganic compounds and
products associated with the normal metabolic processes of
microorganisms). This assumption is supported by the features of
test methods used for this purpose such as exclusion of organic
substrates other than the test substance and the absence of any
adaptation steps. Methods to test for inherent biodegradability,
on tine other hand, using more favorable conditions, are designed
to assess if a substance has any potential for biodegradation.
The method in this guideline is fundamentally a method to
test for ready biodegradability with an option to employ some
features of methods for inherent biodegradability such as an
adaptation of the microorganisms to the chemical substance. Such
flexibility is highly desirable for the purposes of Test Rules
for specific chemical substances. A review of the available
information on a specific substance may reveal patterns of
disposal that make the inclusion of a method with an adaptation
step advisable. If a substance is being released to the aquatic
environment on a rather continuous basis and at some steady
concentration, then the natural microbial population will have
ample opportunity to adapt to the transformation of the substance
and laboratory studies should include an adaptation step.
Laboratory methods which have been employed to screen for
biodegradation potential in aerobic aquatic environments include
(1) those which follow the uptake of dissolved oxygen by the
-5-
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microbial population, (2) river water die-away tests and,
(3) aerobic culture tests. These have been discussed in some
detail by Howard et al. (1975, p. 49-117).
Dissolved oxygen methods included dilution methods such as
the standard BOD test (APHA 1975) and respirometric techniques.
The dilution methods employ closed bottles containing appropriate
dilutions of the test substance in inoculated water which
generally contains a buffered essential salts mixture. The
uptake of dissolved oxygen by the microoganisms is followed by
chemical analyses for dissolved oxygen in a series of replicate
bottles over a period of time, or by the use of an oxygen-
sensitive electrode. Respirometric methods follow the uptake of
dissolved oxygen by manometric techniques, commonly in a system
where the carbon dioxide evolved by the microorganisms is trapped
in an alkaline solution contained in a well or side arm. The
respirometric methods are more difficult to set up and interpret
than the dilution methods and they require the use of relatively
costly equipment which must be recalibrated frequently. For
these reasons, the dilution methods are preferable for screening
purposes.
The dilution methods of the OECD Guidelines (1981) are
validated methods with proven reproducibility. The OECD Closed
Bottle Test (OECD 1981) is a modification of the standard BOD
test (APHA 1975). For those investigators who are more familiar
with the procedures of the standard BOD test, that test should be
an acceptable substitute for the OECD method provided that the
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CS-200U
BOD test is continued for a nominal 28-day incubation period. In
either method, the use of an oxygen-sensitive electrode is
preferred to chemical analysis for dissolved oxygen because
repetitive dissolved oxygen readings may be made on a single
bottle and, in addition, the electrode is more accurate and less
subject to interferences than the chemical titration methods
(Reynolds 1969, Hwang and Forsberg 1973). The oxygen uptake
methods are highly desirable in the screening level group of
methods because they are the only simple methods available which
can handle volatile substances.
River water die-away methods, although used by some for'
bioc'egradability screening, are, in fact, static simulation tests
which employ raw water collected from a river or lake and follow
the disappearance of an added amount of the test substance.
Variations include the use of added nutrients and/or
microorganisms to fortify the natural water. Generally,
biodegradation is followed by using an analytical method which is
specific for the test substance. Although a number of
investigators have used die-away methods to study
biodegradability, there is no standard version of the procedure
and the use of specific natural waters tends to make the methods
less amenable to standardization than other screening methods.
The difficulties of standardization combined with the requirement
for chemical-specific analytical methods preclude the inclusion
of river water die-away methods in a screening-level or base-set
of test methods.
V
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Aerated culture methods generally employ flasks or bottles
containing an aqueous medium plus the test substance and a
suitable inoculum. The vessels may be placed on a shaker to
promote aeration of the aqueous medium and contact of the
microorganisms with both substrate and dissolved oxygen, or they
may be aerated by an air stream bubbled into the liquid medium.
Analysis for biodegradation may be by analytical methods which
are specific for the test substance (e.g. colorimetric or
chromatographic techniques), by the use of appropriately
radiolabeled compounds, or by the use of such non-specific
procedures as following the loss of dissolved organic carbon
(DOC) or the evolution of respiratory carbon dioxide
The use of an analytical method specific for the test
substance or of an appropriately labeled test substance would be
too costly for requirement in an initial evaluation of
biodegradation in aerobic waters. Thus, this screening-level
guideline method and other screening-level or first tier methods
are limited to aerated culture methods which employ non-specific
analytical methods to follow the biodegradation of the test
substance. This guideline method, which is based on work by
Gledhill (1975), is a shake flask method which uses the test
substance as the sole carbon source in a mineral nutrient medium,
and follows biodegradation by both DOC analyses and C02
evolution. This method includes an adaptation step as a routine
procedure. The guideline method has been evaluated in several
laboratories and has proven reproducibility.
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type of aerated culture method is that, emplovinq
continuous culture, with the bacterial population density
requlated bv automatic additions of fresh medinrn and test
substance to the reaction vessel. ^dvantaqes of such procedures
are that toxic products and metabolic wastes will not accumulate
and -.heir effects on biodeqradation processes will not increase
with time. ^hese systems are more like those natural aquatic
environments where a continual input of chemical and removal or
dilution of toxic products is likely. mhese methods invariably
require the use of special equipment and specific analytical
procedures or radiolabeled test substances, and thev tend to
simulate rather specific kinds of aquatic environments. For
these reasons, such methods were rejected for inclusion in the
screeninq level of test methods for aerobic aquatic
biodeqradation.
The method cited in the quideline is applicable to orqanic
substances. Substances which are hiqhly volatile cannot be
studied readily in the aerated systems and are restricted to the
Closed Rottle or ROD test. Substances with very low water
solubility (less than a ^ew mq/L) cannot be used for those
methods that rely on noc measurements. ^he information already
known about a specific substance will aid in the specification o^
the most appropriate biodeqradation method(s) to use for the
substance.
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B. Test Conditions
1. Incubation Temperatures
The incubation temperatures specified in the guideline
method are at or slightly above most laboratory room temperatures
and this allows for convenient adjustment and maintenance of
constant temperature baths and enclosures. For some substances
it may be necessary to require biodegradation data at
temperatures other than those specified. Examples of when this
requirement may be applicable include situations where there is
evidence that the substance is being released in significant
amounts to environmental sites where the ambient temperatures are
commonly well below or above the guideline temperatures.
C. Test Procedures
1. Reference Compounds
Reference compounds are suggested to evaluate the
biodegradation potential of the microbial inoculum. For that
purpose it is necessary to use a reference compound that will be
biodegradable under the test conditions but not so readily
biodegradable that the material is completely degraded within a
small fraction of the normal test period. For that reason, some
traditional reference materials such as glucose and mixtures of
glucose and glutamic acid are not appropriate since they would
biodegrade too rapidly. Aniline appears to be a good general
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choi::e for the aqueous aerobic methods. Sodium citrate, phthalic
acid and trimellitic acid are also suitable reference substances
and, like aniline, will exhibit ultimate biodegradation in this
test method. However, for some purposes, the use of a specific
reference compound that is analogous to the test substance may be
requ:. red.
2. Inhibited Systems
Tests which rely on DOC removal to evaluate biodegradation
require the use of inhibited systems which allow determinations
to be made with regard to whether or not such losses were due to
non-hiological processes such as adsorption and volatilization.
Estimates of test substance removed by sorption and
volatilization can be achieved in two ways: (1) use of
uninoculated flasks or (2) use of inoculated flasks that contain
the test substance and a metabolic inhibitor such as HgCl2 to
prevent microbial activity (inhibited systems). The first option
is more difficult because it requires the maintenance of sterile
conditions in units which are aerated. The use of a chemically
inhibited system provides a practical method for controlling
microoial activity and estimating loss of the test substance by
sorptlon or volatilization.
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3. Replication
Three inoculated cultures containing test substance are
desirable at each incubation temperature, which follows the
recommendation for the use of 2 to 4 replicates in biodegradation
testing (Gledhill 1975). The precision of the test data is
related to the number of replicates, and three replicates are
considered to be the minimum for statistically acceptable mean
and standard deviation calculations.
4. Sampling Frequency and Duration
Samples should be taken according to a schedule appropriate
to the rates of degradation of the test substance and the
reference compound. They should be sufficiently frequent to
establish plots of degradation vs time, in order to properly
judge the nature of the biodegradation and whether or not it is
possible to identify such aspects as an adaptation phase, a
degradation phase and a plateau. A nominal test time of 28 days
was selected to allow for a reasonable period for observations
with more slowly degraded substances, to permit some adaptation
to occur, and to be consistent with the requirements of the OECD
(1981). Tests may be terminated prior to 28 days if an end-point
plateau is observed and if that plateau is consistent (± 10%)
over 3 consecutive days.
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5. Filhration
The use of membrane -filters with 0.45 micrometer (urn) nore
dianeter to prepare samples for DOC analysis is recommended to
standardize the process of sample preparation and to insure that
all particulate matter ^ 0.45 pm is excluded from the sample.
D. Test Data
The written, tabular and qraphical d=ita represent the
minimum acceptable information necessary to evaluate the
biodeqradation of the test substance under the conditions o^ the
quideline.
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TIT . REFERENCES
Alexander M. 1967. Pollutants that resist the microbe. New
Scientist. 35:439.
Alexander M. 1973. Nonbiodeqradable and other recalcitrant
molecules. Riotech Rioenqr 15:61-647.
APHA. 1979. TVmerican Public Health Association. Standard
Methods for the Examination of Water and Wastewater, 14th
edition. Washington, n.C. p. 543-550.
Gledhill WE. 1975. Screeninq test for assessment of ultimate
biodeqradabilitv: linear alkvlbenzene sulfonates. Appl
Microbiol 30:922-929.
Howard PH, Saxena .T, Durkin PR, Ou TV71. 1975. Review and
evaluation of available techniques for determininq persistence
and routines of deqradation o^ chemical substances in the
environment. Office of Toxic Substances, U.S. EOA-560/5-75-
006. H.S. Nat. ^echn. Inform. Service PR Rpt. NO. 243825.
Hwanq CP, ^orsberq CR. 1973. Polaroqraphic method for nitrate
and dissolved oxyqen analvses. Water and Sewaqe Works
April 71-74.
OECH. 1979. Orqanization for Economic Cooperation and
Development. Chemicals testing proqramme final report, expert
qroup on deqradation/accumulation.
OECD. 1980. Orqanization for Economic Cooperation and
Development. Chemicals 'T'estinq Proqramme Ring-mest TT.
OECD. 1981. Organization for Economic Cooperation and
Development. OECD Guidelines for ^estinq of Chemicals.
Paris.
Revnolds .TF. 1969. Comparison studies of Winkler vs oxvgen
sensor. J Water Poll Control Fed 41:2002-2009.
Wright R.T . 1979. Natural, heterotrophic activitv in estuarine
and coastal waters. In Microbial Degradation of Pollutants in
Marine Environments Rourguin AW and Pritchard PH, eds, EPA-
600/9-79-012.
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August, 1982
AEROBIC AQUATIC BIODEGRADATION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Contents
I . INTRODUCTION 1
A.. Purpose 1
R. Definitions 1
C. Principle of the ^est Method 2
D. Prerequisites 3
E. Guidance Information 3
F. Reference Substances 3
G. Reproducibilitv 4
H. Sensitivity 4
I. Possibility of Standardization 4
J. Possibility of Automation 5
II. ^EST PROCEDURES 5
A. Preparations 5
1. Apparatus 5
2. Reagents and Stock Solutions 7
3. Soil Inoculum 7
4. Acclimation Medium 9
R. Procedures 9
C. Analytical Measurements 12
III. DA^A AND REPORTING 12
A. Treatment of Results 12
R. ^est Report 15
IV. REFERENCES 16
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AEROBIC AQUATIC BIODEGRADATION
I. INTRODUCTION
A. Purpose
This Guideline is designed to develop data on the rate and
extent of aerobic biodegradation that might occur when chemical
subtances are released to aquatic environments. A high
biodeqradability result in this test provides evidence that the
test substance will be biodegradable in natural aerobic
freshwater environments.
On the contrary, a low biodegradation result mav have other
causes than poor biodegradability of the test substance.
Inhibition of the microbial inoculum by the test substance at the
test concentration may be observed. In such cases further work
is needed to assess the aerobic aquatic biodegradabilitv and to
detsrmine the concentrations at which toxic effects are
evident. An estimate of the expected environmental concentration
will help to put toxic effects into perspective.
R. Definitions
1. Adaptation is the process by which a substance induces
the synthesis of any degradative enzymes necessary to catalvze
the transformation of that substance.
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CG-2000
2. Ultimate Biodegradability is the breakdown of an organic
compound to CC^/ water, the oxides or mineral salts of other
elements and/or to products associated with normal metabolic
processes of microorganisms.
3. Ready Biodegradability is an expression used to describe
those substances which, in certain biodegradation test
procedures, produce positive results that are unequivocal and
which lead to the reasonable assumption that the substance wil
undergo rapid and ultimate biodegradation in aerobic aquatic
environments.
C. Principle of the Test Method
This Guideline Method is based on the method described by
William Gledhill (1975). The method consists of a two-week
inoculum buildup period during which soil and sewage
microorganisms are provided the opportunity to adapt to the test
compound. This inoculum is added to a specially equipped
Erlenmeyer flask containing a defined medium with test
substance. A reservoir holding barium hydroxide solution is
suspended in the test flask. After inoculation, the test flasks
are sparged with CC^-free air, sealed and incubated with shaking
in the dark. Periodically, samples of the test mixture
containing water soluble test substances are analyzed for
dissolved organic carbon (DOC) and the Ba(OH)2 from the
reservoirs is titrated to measure the amount of CC>2 evolved.
Differences in the extent of DOC disappearance and C02 evolution
2
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CG-2000
between control flasks, containing no test substance, and flasks
containing test substance are used to estimate the degree of
ultimate biodegradation.
D * Prerequisites
The total organic carbon (TOC) content of the test substance
should be calculated or, if this is not possible, analyzed, to
enable the percent of theoretical yield of carbon dioxide and
percent of DOC loss to be calculated.
E. Guideline Information
Information on the relative proportions of the major
components of the test substance will be useful in interpreting
the results obtained, particularly in those cases where the
res lit lies close to a "pass level".
Information on the toxicity of the chemical may be useful in
the interpretation of low results and in the selection of
appropriate test concentrations.
F. Reference Substances
Where investigating a chemical substance, reference
compounds may be useful and an inventory of suitable reference
compounds needs to be identified. In order to check the activity
of the inoculum the use of a reference compound is desirable.
Aniline, sodium citrate, dextrose, phthalic acid and trimellitic
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acid will exhibit ultimate biodegradation under the conditions of
this Test Guideline method. These reference substances must
yield 60 percent of theoretical maximum Cr>2 and show a removal
of 70 percent DOC within 28 days. Otherwise the test is
regarded as invalid and should be repeated using an inoculum from
a different source.
G. Reproducibility
The reproducibility of the method has not yet been
determined; however it is believed to be appropriate for a
screening test which has solely an acceptance but no rejective
function.
H. Sens itivity
The sensitivity of the method is determined by the ability
to measure the endogenous CO2 production of the inoculum in the
blank flask and by the sensitivity limit of the dissolved organic
carbon analysis. If the test is adapted to handle C-labelled
test substances, test substance concentrations can be much lower.
I. Possibility of Standardization
This possiblity exists. The major difficulty is to
standardize the inoculum in such a way that interlaboratory
reproducibility is ensured.
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J. Possibility of Automation
None at present, although parts of the analyses may be
automated.
II. TEST PROCEDURES
A. Preparations
1. Apparatus
The shake flask apparatus (Figure 1) contains 10 mL of 0.2N
Ba(OH)2 in an open container suspended over 1-liter of culture
medium in a 2-liter Erlenmeyer flask. The Ba(OH)2 container is
made by placing a constriction just above the 10 mL mark of a 50
mL heavy duty centrifuge tube and attaching the centrifuge tube
to a 2 mm I.D. x 9 mm O.D. glass tube by means of 3 glass support
rods. The centrifuge tube opening is large enough to permit Cr>2
to diffuse into the Ba(OH)2> while the constriction permits
transferal of the flask to and from the shaker without Ba(OH)2
spillage into the medium. For periodic removal and addition of
base from the center well, a polypropylene capillary tube,
attached at one end to a 10 ml disposable syringe, is inserted
through the 9 mm O.D. glass tube into the Ba(OH)2 reservoir. The
reservoir access port is easily sealed during incubation with a
serum bottle stopper. Two glass tubes are added for sparging,
venting, and medium sampling. The tops of these tubes are
connected with a short section of flexible tubing during
incubation.
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#10 RUBBER STOPPER
VENT TUBE
9 MM 0,D, X 3 MM I,D. TUBE
RESERVOIR FOR BA(OH)2
2 MM O.I), POLYPROPYLENE TUBE
0,2 N 1U(OII)2J TO ML
AERATION AND SAMPLING TUBE
1000 ML MEDIUM
o
n
i
M
o
o
o
FIGURE 1, SHAKE FLASK SYSTEM FOR
CARBON DIOXIDE EVOLUTION
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2. Reagents and Stock Solutions
Stock solutions, I, II, and III (Table 1)
Yeast Extract
Vitamin-free Casamino Acids
70% 02 in nitrogen or CC^-free air
0.2N Ba(OH)2
0.1 N HC1
20% H2SO4
Phenolphthalein
Dilution water - distilled, deionized water (DIM)
3. Soil Inoculum
A fresh sample of an organically rich soil is used as the
inoculum in the ultimate biodegradation test. Soil is collected,
prepared, and stored according to the recommendations of Pramer
and Bartha (1972). The soil surface is cleared of Litter and a
soil sample is obtained 10-20 cm below the surface. The sample
is screened through a sieve with 2-5 mm openings and stored in a
polyethylene bag at 2-4°C for not more than 30 days prior to
use. The soil is never allowed to air dry, and should not be
frozen during storage.
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TABLE 1
MEDIUM EMPLOYED FOR ASSAY OF CO0 EVOLUTION
SOLUTION'
II'
III
COMPOUND
NH4C1
KNO-,
K2HPO4.3H2O
NaH2PO4.H2O
KC1
MgS04
FeS04.7H2O
CaCl2
ZnCl0
MnCl2.4H2O
CuClo
CoCl-
H3BO3
MoO-
STOCK SOLUTION
CONG. (g/L)
35
15
75
25
10
20
1
5
0.05
0.5
0.05
0.001
0.001
0.0004
a _
= Each liter of test medium contains 1 mL of each solution.
b _
= Final pH is adjusted to 3.0 with 0.10 N HC1,
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4. Acclimation Medium
Acclimation medium is nrenared bv addina, ^or each liter r>f
dis-.il.led, deionized water (niw): 1 mL each of solutions T, IT,
and Til (Table 1), 1.0 qm of soil inoculum (prepared accordinq to
3, above), 2.0 mL of aerated mixed linuor (obtained ^rom an
activated sludqe treatment nlant not more than 2 days nrior to
commencinq the acclimation phase, and stored in the interim at
4°C) and 50 mL, raw domestic influent sewaqe. ""his medium is
mixed for 15 minutes and filtered throuqh a qlass wool pluq in a
glass funnel. The ^iltrate is permitted to stand for 1 hour,
ref iltered throuqh qlass wool, and supplemented with 25 mq/Tj each
of ">ifco vitamin-^ree casamino acids and veast extract.
Appropriate volumes are added to 2-liter ^rlenmeyer flasks. Test
comnounds are added incrementally durinq the acclimation neriod
at concentrations equivalent to 4, 8, and 8 mq/L carbon on davs
0, 7, and 11, resnectively. On day 14, the medium is refiltered
through qlass wool prior to use in the test. For evaluating the
biodeqradability of a series o^ functionallv or sfructurallv
related chemicals, media from all inoculum ^lasks mav be combined
before final filtration.
T^ . Procedures
Inoculum (100 mL of acclimation medium) is added to qOO mL
OIW containing 1 mL each of solutions T, II, and III (Table 1) in
a 2-liter ^rlenmeyer flask. Test compound equivalent to 10
mq/liter carbon is added to each of the replicate flasks
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CG-2000
containing the test medium. Ten mL of 0.2 N Ba(OH)2 are added to
the suspended reservoir in each flask and duplicate 10 mL samples
of Ra(OH)2 are also saved as titration blanks for analysis with
test samples. Flasks are sparged with CO2-free air (for volatile
test materials, sparging is done prior to addition of the
chemical), sealed, and placed on a gyrotary shaker (approximately
125 rpm) at 20 to 25 °C in the dark. For each set of experiments,
each test, reference, inhibited and control system should be
analyzed at time zero and at a minimum of four other times from
time zero through day 28. Sampling must be made with sufficient
frequency to allow for a smooth plot of biodegradation with
time. Sampling times should be varied by the investigator as
deemed appropriate to match the rate of degradation of the test
substance. Tests may be terminated when biodegradation reaches a
plateau and is consistent (± 10%) over 3 consecutive days or on
day 28, whichever occurs first. For chemicals which are water
soluble at the test concentration, an adequate volume (5-10 mL)
of medium is removed for DOC analysis. Each sample for DOC
analysis should be filtered through a membrane filter of 0.45
micrometer pore diameter before DOC analysis. For all test and
reference compounds, Ba(OH)2 from the center well is removed for
analysis. The center well is rinsed with 10 mL CO2~free DIW and
is refilled with fresh base. Rinse water is combined with the
Ba(OH)2 sample to be analyzed. Flask are resealed and placed on
the shaker. On the day prior to terminating the test, 3 mL of
20% H2S04 are added to the medium to release carbonate bound CO2.
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For each set of experiments, each test substance should be
tested in triplicate.
For each set of experiments, one or two reference compounds
are included to assess the microbial activity of the test
medium. Duplicate reference flasks are prepared by adding
reference compound equivalent to 10 mg/liter carbon to each of
two flasks containing the test medium. Reference compounds which
are; positive for ultimate biodegradability include: sodium
citrate, dextrose, phthalic acid, trimellitic acid and aniline.
For each test set, triplicate controls receiving inoculated
medium and no test compound, plus all test and reference flasks,
are: analyzed for CC>2 evolution and DOC removal. Results from
analysis of the control flasks (DOC, CO2 evolution, etc.) are
subtracted from corresponding experimental flasks containing test
compound in order to arrive at the net effect due to the test
compound.
A test system containing a growth inhibitor should be
established as a control for each substance tested for
bicdegradation by this method. ^hat inhibited system must
contain the same amount of water, mineral nutrients, inoculum and
test substance used in the uninhibited test systems, nlus 50 mg/L
mercuric chloride (HgCl2) to inhibit microbial activity.
Flasks should be incubated in the dark to minimize both
photochemical reactions and algal growth. Appropriate sterile
controls or controls containing a metabolic inhibitor, such as 50
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CG-2000
mg/L HgCl2, are needed to correct for interferences due to non-
biological degradation. With volatile organic materials,
sparging with CO2~free air is performed only once, just prior to
addition of the test chemical. Analyses for CO2 evolution and
DOC removal are conducted within 2-3 hours of sampling to
minimize interferences which may occur in storage. All glassware
should be free of organic carbon contaminants.
C. Analytical Measurements
The guantity of CO2 evolved is measured by titration of the
entire Ba(OH)2 sample (10 mL Ra(OH)2 + 10 mL rinse water) with
0.1 N HC1 to the phenolphthalein end point. Ba(OH)2 blanks are
also supplemented with 10 mL CO2~free HIW and titrated in a
similar manner. Samples (5 mL) for DOC are centrifuged and/or
filtered and supernatant or filtrate analyzed by a suitable total
organic carbon method.
III. DATA AND REPORTING
A. Treatment of Results
Test compound (10 mg carbon) is theoretically converted to
0.833 mmol CO2. Absorbed CO2 precipitates as BaCO-^ from Ba(OH)2,
causing a reduction in alkalinity by the eguivalent of Ifi.fiV mL
of 0.1 N HC1 for complete conversion of the test compound carbon
to CO2. Therefore, the percent theoretical CO2 evolved from the
test compound is calculated at any sampling time from the formula:
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CG-2000
chosen, the investigator may use lower test substance
concentrations if those concentrations are more representative of
env:.ronmental levels.
B. Test Report
For each test and reference compound, the following data
should be reported.
Information on the inoculum, including source, collection
date, handling, storage and adaptation possibilities (i.e., that
the inoculum might have been exposed to the test substance either
before or after collection and prior to use in the test).
Results from each test, reference, inhibited (with
and control system at each sampling time, including an average
result for the triplicate test substance systems and the standard
deviation for that average.
Average cumulative percent theoretical CO2 evolution over
the test duration.
Dissolved organic carbon due to test compound at each
sampling time (DTF-DCF).
Average percent DOC removal at each sampling time.
Twenty-eight day standard deviation for percent CO2
evolution and DOC removal.
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IV. REFERENCES
Gledhill WE. 1975. Screening test for assessment of ultimate
biodegradability: Linear alkyl benzene sulfonate. Appl
Microbiol 30:922-929.
Pramer D, Bartha R. 1972. Preparation and processing of soil
samples for biodegradation testing. Environ Letters 2:217-224.
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CG-2000
CO2 evolution = [ (TF-CF)/16.67] 100 (for 10 mg/L test compound
carbon)
where:
TF = mL 0.1 N HC1 required to titrate Ba(OH)2 samples from the
test flask
CF = mL 0.1 N HC1 required to titrate Ra(OH)2 samples from the
control flask.
The cumulative % CO2 evolution at any sample time is
calculated as the summation of the % CO2 evolved at all sample
points of the test.
The percent DOC disappearance from the test compound is
calculated from the following equation:
DOC Removal = [1-(DTFV - DCFV)/(DTF^ - DCF_)] 100
X. J\ O O
whe re:
DTP = Dissolved organic carbon from test flask
DCF = Dissolved organic carbon from control flask
o = Day zero measurements
x = Day of measurements during test.
The difference between the amount of 0.1 N HC1 used for the
Ba(OH)2 titration blank samples and the Ba(OH)2 samples from the
control units (no test compound) is an indication of the activity
of nhe microorganisms in the test system. In general, this
-13-
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CG-2000
difference is approximately 1-3 mL of 0.1 N HC1 at each sampling
time. A finding of no difference in the titration volumes
between these two samples indicates a poor inoculum. In this
case, the validity of the test results is guestionable and the
test set should be rerun beginning with the acclimation phase.
CC>2 evolution in the reference flasks is also indicative of
the activity of the microbial test system. The suggested
reference compounds should all yield final CC>2 evolution values
in the range 80-100% of theoretical CO2- If, for any test set,
the percent theoretical CCU evolution value for the reference
flasks is outside this range, the test results are considered
invalid and the test is rerun.
Inhibition by the test compound is indicated by lower Cru
evolution in the test flasks than in the control flasks. If
inhibition is noted, the study for this compound is rerun
beginning with the acclimation phase. During the test phase for
inhibitory compounds, the test chemical is added incrementally
according to the schedule: Day 0 - 0.5 mg/liter as organic
carbon, Day 2-1 mg/liter C, Day 4-1.5 mg/liter C, Day 7-2
mg/liter C, Day 10-5 mg/liter C. For this case, the Ba(OH)2 is
sampled on Day 10, and weekly thereafter. The total test
duration remains 28 days.
The use of C-labelled chemicals is not required. If
appropriately labelled test substance is readily available and if
the investigator chooses to use this procedure with labelled test
substance, this is an acceptable alternative. If this option is
-14-
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CS-2050
August, 1982
ANAEROBIC BIODEGRADATION
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-4000
August, 1982
COMPLEX FORMATION ABILITY IN WATER
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
CG-4000
COMPLEX FORMATION ABILITY IN WATER
I. PURPOSE
This Test Guideline references methodology to develop data on the
ability of a chemical substance to form soluble metal complexes.
It is applicable only to pure chemical substances and is not
applicable to the determination of mercury complexes. The data
may be used to evaluate the potential increase in availability to
food chains or drinking water of metals that might otherwise
become inaccesible.
TI. TEST PROCEDURES
Appropriate methods are found in OECn Guideline NO. 108, (OECn),
"Complex Formation Ability in Water". The U.S. sales agent for
the OECD guidelines is OECD Publications and Information Center,
Sui-:e 1207, 1750 Pennsylvania Ave. NW, Washington, DC 20006.
III., REFERENCE
OECO. 1981. Organization for Economic Cooperation and
Development. OECO Guidelines for Testing of Chemicals.
-1-
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CG-5000
August, 19B2
HYDROLYSIS AS A FUNCTION OF pH AT 25°C
OFFICE OF TOXIC SUBSTANCES
OFFICE: OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CG-5000
CONTENTS
I. INTRODUCTION 1
A. Background and Purpose 1
B. Definitions and Units 2
C. Principle of the Test Method 5
D. Applicability and Specificity 6
11 . TEST PROCEDURES 7
A. Test Conditions 7
1. Special Laboratory Equipment 7
2. Purity of Water 7
3. Sterilization 7
4. Precautions for Volatility 8
5 . Temperature Controls , ., 8
6. pH Conditions 8
7. Concentration of Solutions of '"-.emical
Substances 8
8. Effect of Acidic and Basic Groups 8
9. Buffer Catalysis 9
10. Photosensitive Chemicals 9
11 . Chemical Analysis of Solutions 10
B. Preparations 10
1 . Reagents and Solutions 10
a. Buffer Solutions 10
b. Additional Buffer Solutions 11
c. Adjustment of Buffer Concentrations 12
d. Preparation of Test Solution 13
C. Performance of the Test 14
1. Procedure 1 14
2. Procedure 2 14
3. Procedure 3 15
4. Analytical Methodology 15
III. DATA AND REPORTING 16
A. Treatment of Results 16
B. Specific Analytical and Recovery Procedures 16
C . Test Data Report 17
IV. REFERENCES 18
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CG-5000
HYDROLYSIS AS A FUNCTION OF pH AT 25°C
I. INTRODUCTION
A. Background and Purpose
Water is one of the most widely distributed substances in the
environment. It covers a large portion of the earth's surface as
oceans, rivers, and lakes. The soil also contains water as does
the atmosphere in the form of water vapor. As a result of this
ubiquitousness, chemicals introduced into the environment almost
always come into contact with aqueous media. Certain classes of
these chemicals, upon such contact, can undergo hydrolysis, which
is one of the most common reactions controlling chemical stability
and is, therefore, one of the main chemical degradation paths of
these substances in the environment.
Since hydrolysis can be such an important degradation path
for certain classes of chemicals, it is necessary, in assessing
the fate of these chemicals in the environment, to know whether,
at what rate, and under what conditions a substance will
hydrolyze. Some of these reactions can occur so rapidly that
there may be greater concern about the products of the
transformation than about the parent compounds. In other cases, a
substance will be resistant to hydrolysis under typical
environmental conditions, while, in still other instances, the
substance may have an intermediate stability that can result in
the necessity for an assessment of both the original compound and
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CG-5000
its transformation products. The importance of transformation of
chemicals via hydrolysis in aqueous media in the environment can
be determined quantitatively from data on hydrolysis rate
constants. This hydrolysis Test Guideline represents a test to
allow one to determine rates of hydrolysis at any pH of
environmental concern at 25°C.
B. Definitions and Units
1. "Hydrolysis" is defined as the reaction of an organic
chemical with water, such that one or more bonds are broken and
the reaction products of the transformation incorporate the
elements of water (H^O).
2. "Elimination" is defined in this Test Guideline to be a
reaction of an organic chemical (RX) in water in which the X group
is lost. These reactions generally follow the same type of rate
laws that hydrolysis reactions follow and, thus, are also covered
in this Test Guideline.
3. A "first-order reaction" is defined as a reaction in
which the rate of disappearance of the chemical substance being
tested is directly proportional to the concentration of the
chemical substance and is not a function of the concentrations of
any other substances present in the reaction mixture.
4. The "half-life" of a chemical is defined as the time
required for the concentration of the chemical substance being
tested to be reduced to one-half its initial value.
-2-
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CG-500U
Hydrolysis refers to a reaction of an organic chemical with
water such that one or more bonds are broken and the reaction
products incorporate the elements of water (t^O)- This type of
transformation often results in the net exchange of a group X, on
an organic chemical RX, for the OH group from water. This can be
wr i tten
RX + HOH -» ROH + HX.
Another result of hydrolysis can be the incorporation of both H
and OH in a single product. An example of this is the hydrolysis
of epoxides, which can be represented by
v/
\ --OH
0 + HOH *
A i°H
The hydrolysis reaction can be catalyzed by acidic or basic
species, including OH~ and H-^O"1" (H ). The promotion of the
reaction by HgO+ or OH~ is called specific acid or specific base
catalysis, respectively, as contrasted with general acid or base
catalysis encountered with other cationic or anionic species.
Usually, the rate law for chemical RX can be written as:
kB[OH~] [RX] + k'N [H20] [RX] ,
where k», kg and k'« are the second-order rate constants for acid
and base catalyzed and neutral water processes, respectively. In
dilute solutions, such as are encountered in following this Test
Guideline, water is present in great excess and its concentration
-3-
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CG-5000
is, thus, essentially constant during the course of the hydrolysis
reaction. At fixed pH, the reaction, therefore, becomes pseudo
first-order, and the rate constant (k) can be written as:
kB[OH~] + kN, (2)
where kN is the first-order neutral water rate constant. Since
this is a pseudo first-order process, the half-life is independent
of the concentration and can be written as:
= 0.693/kh. (3;
At constant pH, Equation 1 can be integrated to yield the first
order rate expression
C = - (kt/) + log1(J CQ, (4)
where C is the concentration of the test chemical at time t and CQ
is the initial chemical concentration (t = 0).
At a given pH, Equation 2 contains three unknowns, kA, kg,
and kN. Therefore, three equations (i.e., measurements at three
different pH's at a fixed temperature) are required if one wishes
to solve for these quantities. Making suitable approximations for
-4-
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CG-5000
quantities that are negligible, the expressions for kA/ kg, and kN
using values of k, measured at pH 3, 7, and 11 are:
kA = 103 [kh (3) - kh (7) + 10~4 kh (11)]
kB = 103 [kh (11) - ^ (7) + 10~4 kh (3)] (5)
*N = \ (7) - 10 4 [T^ (3) + 1^ (11)]
The above calculated rate constants can be employed in equation 2
to calculate the hydrolysis rate of a chemical at any pH of
environmental concern.
The above equations apply whether the test chemical has one
hydrolyzable group or several. In the latter case, the rate may
be written:
-- = k. [RX] + k0 [RX] + ---- + k [RX]
dt 12 n
+ ..... kn) [RX] = k^ [RX]. (6)
Equation 6 applies to the hydrolysis rate of a molecule having n
hydrolyzable groups, each of which follows first-order reaction
kinetics. The measured k-. is now the sum of the individual
reaction rates and is the only rate constant required in this Test
Guideline .
C. Principle of the Test Method
Procedures described in this Test Guideline enable sponsors
to obtain quantitative information on hydrolysis rates through a
-5-
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CG-5000
determination of hydrolysis rate constants and half-lives of
chemicals at pH 3.00, 7.00, and 11.00 at 25°C. The three measured
rate constants are used to determine the acidic, basic, and
neutral rate constants associated with a hydrolytic reaction.
These latter constants can then be employed in determining the
hydrolysis rates of chemicals at any pH of environmental concern
at 25°C.
D. Applicability and Specificity
There are several different common classes of organic
chemicals that are subject to hydrolysis transformation, including
esters, amides, lactones, carbamates, organophosphates, and alkyl
halides. Processes other than nucleophilic displacement by water
can also take place. Among these are elimination reactions that
exhibit behavior similar to hydrolysis and, therefore, are also
covered in this Test Guideline.
-6-
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CG-5000
II. TEST PROCEDURES
A. Test Conditions
1. Special Laboratory Equipment
(1) A thermostatic bath that can be maintained at a
temperature of 25 ± 1°C.
(2) A pH meter that can resolve differences of 0.05
pH units or less.
(3) Stoppered volumetric flasks (no grease) or
glass ampoules that can be sealed.
2. Purity of Water
Reagent-grade water (e.g., water meeting ASTM Type IIA
standards or an equivalent grade) is highly recommended to
minimize biodegradation. ASTM Type IIA water is described in ASTM
D-:,.193-77, "Standard Specification for Reagent Water."
3,. Sterilization
It is extremely important to sterilize all glassware and to
use aseptic conditions in the preparation of all solutions and in
carrying out all hydrolysis experiments to eliminate or minimize
biodegradation. Glassware can be sterilized in an autoclave or by
any other suitable method.
-7-
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CG-5000
4. Precautions for Volatility
If the chemical is volatile, it is extremely important that
the reaction vessels be almost completely filled and sealed.
5. Temperature Controls
It is important that all hydrolysis reactions be carried out
at 25°C and the temperature is controlled to ±1°C.
6. pH Conditions
It is recommended that all hydrolysis experiments be
performed at pH 3.00, 7.00, and 11.00 ± 0.05 using the appropriate
buffers described in Section II.B.I.a.
7. Concentration of Solutions of Chemical Substances
It is extremely important that the concentration of the test
chemical be less than one-half the chemical's solubility in water
and not greater than 10 M.
8. Effect of Acidic and Basic Groups
Complications can arise upon measuring the rate of hydrolysis
of chemicals that reversibly ionize or are protonated in the pH
range 3.00 to 11.00. Therefore, for these chemicals, it is
recommended that these hydrolysis tests be performed at pH 5.00,
7.00, and 9.00 ± 0.05 using the appropriate buffers described in
Sections II.B.I.a and II.B.l.b. If a test chemical reversibly
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CG-5000
ionizes or protonates in the pH range 5.00 to 9.00, then it is
recommended that additional hydrolysis tests should be carried out
at pH 6.00 and 8.00 ± 0.05 using the buffers described in Section
II.B.l.b.
9. Buffer Catalysis
For certain chemicals, buffers may catalyze the hydrolysis
reaction. If this is suspected, it is extremely important that
hydrolysis rate determinations be carried out with the appropriate
buffers and that the same experiments be repeated at buffer con-
centrations lowered by at least a factor of five. If the
hydrolysis reaction produces a change of greater than 0.05 pH
units in the lower concentration buffers at the end of the mea-
surement time, then it is extremely important that the test
chemical concentrations also be lowered by at least a factor of
five. Alternatively, test chemical concentrations and buffer con-
centrations may both be lowered simultaneously by a factor of
five. A sufficient criterion for minimization of buffer catalysis
is an observed equality in the hydrolysis rate constant of two
different solutions differing in buffer or test chemical
concentration by a factor of five.
10. Photosensitive Chemicals
The solution absorption spectrum can be employed to determine
whether a particular chemical is potentially subject to photolytic
transformation upon exposure to light. For chemicals that absorb
-9-
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CG-5000
light of wavelengths greater than 290 nm, it is important that the
hydrolysis experiment be carried out in the dark, under amber or
red safelights, in amber or red glassware, or employing other
suitable methods for preventing photolysis. The absorption spec-
trum of the chemical in aqueous solution can be measured by OECD
Test Guideline No. 101, "UV-VIS Absorption Spectra."
11. Chemical Analysis of Solutions
In determining the concentrations of the test chemicals in
solution, any suitable analytical method may be employed, although
methods which are specific for the compound to be tested are pre-
ferred. Chromatographic methods are recommended because of their
compound specificity in analyzing the parent chemical without
interferences from impurities. Whenever practicable, the chosen
analytical method should have a precision within ±5 percent.
B. Preparation
1. Reagents and Solutions
a. Buffer Solutions
Prepare buffer solutions using reagent-grade chemicals and
reagent-grade water as follows:
(1) pH 3.00 use 250 mL of 0.100M potassium
hydrogen phthalate;
111 mL of 0.100M hydrochloric acid;
-10-
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CG-5000
and adjust volume to 500 mL with
reagent-grade water.
(2) pH 7.00 use 250 mL of 0..100M potassium
dihydrogen phosphate;
145 mL of 0.100M sodium hydroxide; and
adjust volume to 500 mL with reagent-
grade water.
(3) pH 11.00 use 250 mL of 0.0500M sodium
bicarbonate;
113 mL of 0.100 M sodium hydroxide;
and adjust volume to 500 mL with
reagent-grade water.
b. Additional Buffer Solutions
For chemicals that ionize or are protonated as discussed in
Section II.A.8, prepare buffers using reagent-grade water and
reagent-grade chemicals as follows:
(1) pH 5.00 use 250 mL of 0.100 M potassium
hydrogen phthalate;
113 mL of 0.100M sodium hydroxide; and
adjust volume to 500 mL with reagent-
grade water.
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CG-5000
(2) pH 6.00 use 250 mL of 0.100M potassium
dihydrogen phosphate;
28 mL of 0.100M sodium hydroxide; and
adjust volume to 500 mL with reagent-
grade water.
(3) pH 8.00 use 250 mL of 0.100M potassium
dihydrogen phosphate;
234 mL of 0.100M sodium hydroxide; and
adjust volume to 500 mL with reagent-
grade water.
(4) pH 9.00 use 250 mL of 0.0250M borax (Na2B4O7);
23 mL of 0.100M hydrochloric acid; and
adjust volume to 500 mL with reagent-
grade water.
c. Adjustment of Buffer Concentrations
(1) The concentrations of all the above buffer
solutions are the maximum concentrations to be
employed in carrying out hydrolysis
measurements. If the initial concentration of
the test chemical is less than 10 M, it is
extremely important that the buffer
concentrations be lowered by a corresponding
amount; e.g., if the initial test chemical
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CG-500U
concentration is 10 M, then reduce the
concentration of the above buffers by a factor
of 10. In addition, for those reactions in
which an acid or base is not a reaction
product, then employ the minimum buffer
concentration necessary for maintaining the pH
within ±0.05 units.
(2) Check the pH of all buffer solutions with a pH
meter at 25°C and adjust the pH to the proper
value, if necessary.
<3. Preparation of Test Solution
If the test substance is readily soluble in water, prepare an
aqueous solution of the chemical in the appropriate buffer and
determine the concentration of the chemical. Alternatively, a
solution of the chemical in water may be prepared and added to an
appropriate buffer solution and the concentration of the chemical
then determined. In the latter case, it is important that the
aliquot be small enough so that the concentration of the buffer in
the final solution and the pH of the solution remain essentially
unchanged. Do not employ heat in dissolving the chemical. It is
extremely important that the final concentration not be greater
than one-half the substance's solubility in water and not greater
than 10~3M.
If the test chemical is too insoluble in pure water to permit
reasonable handling and analytical procedures, it is recommended
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CG-50UO
that the chemical be dissolved in reagent-grade acetonitrile and
buffer solution then added to an aliquot of the acetonitrile
solution. Do not employ heat to dissolve the chemical in
acetonitrile. It is extremely important that the final
concentration of the test substance not be greater than one-half
_3
the chemical's solubility in water and not greater than 10 M. In
addition, it is extremely important that the final concentration
of the acetonitrile be one volume percent or less.
C. Performance of the Test
Carry out all hydrolysis experiments by employing one of the
procedures described below. Prepare the test solutions as
described in Section II.B.I at pH 3.00, 7.00, and 11.00 ±0.05, and
determine the initial test chemical concentration (C ) in
triplicate. Analyze each reaction mixture in triplicate at
regular intervals, employing one of the following procedures:
1. Procedure 1
Analyze each test solution at regular intervals to provide a
minimum of six measurements with the extent of hydrolysis between
20-70 percent. Rates should be rapid enough so that 60-70 percent
of the chemical is hydrolyzed in 672 hours.
2. Procedure 2
If the reaction is too slow to conveniently follow hydrolysis
to high conversion in 672 hours but still rapid enough to attain
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CG-5000
at least 20 percent conversion, take 15 to 20 time points at
regular intervals after 10 percent conversion is attained.
3. Procedure 3
If chemical hydrolysis is less than 20 percent after 672
hours, determine the concentration (C) after this time period.
If the pH at the end of concentration measurements employing
any of the above three procedures has changed by more than 0.05
units from the initial pH, repeat the experiment using a solution
having a test chemical concentration lowered sufficiently to keep
the pH variation within 0.05 pH units.
4. Analytical Methodology
Select an analytical method that is most applicable to the
analysis of the specific chemical being tested (Section II.A.11).
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CG-5000
III. DATA AND REPORTING
A. Treatment of Results
(1) If Procedures 1 or 2 were employed in making
concentration measurements, use a linear regression analysis with
equation 4 to calculate k-^ at 25 °C for each pH employed in the
hydrolysis experiments. Calculate the coefficient of
n
determination (R ) for each rate constant. Use equation 3 to
calculate the hydrolysis half-life using k, .
(2) If Procedure 3 was employed in making rate
measurements, use the mean initial concentration (C ) and the mean
concentration of chemical (C) in equation 4 to calculate k-u for
each pH used in the experiments. Calculate the hydrolysis half-
life using k^ in equation 3.
(3) For each set of three concentration replicates,
calculate the mean value of C and the standard deviation.
(4) For test chemicals that are not ionized or
protonated between pH 3 and 11, calculate k,, kg, and kN using
equation 5.
B. Specific Analytical and Recovery Procedures
(1) Provide a detailed description or reference for the
analytical procedure used, including the calibration data and
precision.
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CG-5000
(2) If extraction methods were used to separate the
solute from the aqueous solution, provide a description of the
extraction method as well as the recovery data.
C. Test Data Report
(1) For procedures 1 and 2, report k^, the hydrolysis
half-life (tL/J, and the coefficient of determination (R ) for each
pH employed in the rate measurements. In addition, report the
individual values, the mean value, and the standard deviation for
each set of replicate concentration measurements. Finally, report
*A' *B' and kN*
(2) For Procedure 3, report k^ and the half-life for
each pH employed in the rate measurements. In addition, report
the individual values, the mean value, and the standard deviation
for each set of replicate concentration measurements. Finally,
report kA, kg, and kN.
(3) If, after 672 hours, the concentration (C) is the
same as the initial concentration (C ) within experimental error,
then kn cannot be calculated and the chemical can be reported as
being persistent with respect to hydrolysis.
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CG-5000
IV. REFERENCES
ASTM. 1978. Annual Book of ASTM Standards. American Society for
Testing and Materials. Philadelphia, PA., Part 31, Method D1193-77.
OECD. 1981. Organization of Economic Cooperation and Development.
OECD Guidelines for Testing Chemicals: No. 101 - UV-VIS Absorption
Spectra.
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CS-5000
August, 1982
HYDROLYSIS AS A FUNCTION OF pH AT 25°C
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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CS-50UO
Contents
Page
I. NEED FOR THE TEST 1
II. SCIENTIFIC ASPECTS 2
A. General Background 2
B. Rationale for Selection of Test Method 8
C. Rationale for the Selection of
Experimental Conditions 10
1. Parity of Water 10
2. Sterilization 11
3. Precautions for Volatility 11
4. Temperature 11
5. pH 12
6. Initial Concentration of Chemicals 14
7. Ionized or Protonated Groups 15
8. Buffers 15
9. Light Sensitive Compounds 16
10. Chemical Analysis of Solutions 16
D. Test Data Required 17
E. Statistical Analysis of the Data 17
III. REFERENCES 20
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CS-5000
HYDROLYSIS AS A FUNCTION OF pH AT 25°C
I. NEED FOR THE TEST
Water is one of the most widely distributed substances in
the environment. It covers a large portion of the earth's
surface as oceans, rivers and lakes. The soil also contains
water as does the atmosphere in the form of water vapor. As a
result of this ubiquitousness, chemicals introduced into the
environment almost always come into contact with aqueous media.
Certain classes of these chemicals, upon such contact, can
undergo hydrolysis which is one of the most common reactions
controlling chemical stability and is, therefore, one of the main
chemical degradation paths of these substances in the
environment. There are several different, common classes of
chenicals which are subject to this type of degradation including
esters, amides, lactones, carbamates, organophosphates, alkyl
halr.des, epoxides, etc. Processes other than nucleophilic
displacement by water can also take place. Among these are
elimination reactions which exhibit rate behavior similar to
hydrolysis and, thus, are also covered in this Test Guideline.
Since hydrolysis can be such an important degradation path
for certain classes of chemicals, it is necessary, in assessing
the fate of these chemicals in the environment, to know whether,
at what rate, and under what conditions a substance will
hydrolyze. Some of these reactions can occur so rapidly that
there may be greater concern about the products of the
_ i _
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CS-5000
transformation than about the parent compounds. In other cases a
substance will be resistant to hydrolysis under typical
environmental conditions, while in still other instances, the
substance may have an intermediate stability which can result in
the necessity for an assessment of both the original compound and
its transformation products. The importance of transformation of
chemicals via hydrolysis in aqueous media in the environment can
be determined quantitatively from data on hydrolysis rate
constants. This hydrolysis Test Guideline represents a procedure
to allow one to determine the rate constants for acid catalyzed,
base catalyzed, and neutral hydrolysis at 25°C. The results can
be used to calculate the rate constant for hydrolysis at any pH
of environmental concern at this temperature. Future Test
Guidelines will extend the temperature ranqe of testing
conditions and will cover other environmental factors which might
affect hydrolysis such as general acid-base catalysis involving
transition metal ions or nucleophilic species, salt effects in
sea water, etc. In addition, more advanced Test Guidelines may
be concerned with determining the identity and fate of the
transformation products.
II. SCIENTIFIC ASPECTS
A. General Background
Hydrolysis refers to a reaction of an organic chemical with
water such that one or more bonds are broken and the reaction
products incorporate the elements of water (H^O). This type of
transformation often results in the net exchange of a group X, on
-2-
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an organic chemical RX, for the OH group from water. This can be
written as:
RX + HOH > ROH + HX.
Another result of hydrolysis can be the incorporation of both H
and OH in a single product. An example of this is the hydrolysis
of epoxides which can be represented by:
N
O + HOH
OH
-OH
The hydrolysis reaction can be catalyzed by acidic or basic
species, including OH~ and HoO (H ). The promotion of the
reaction by H.,0 or OH~ is called specific acid or specific base
catalysis, as contrasted to general acid or base catalysis
encountered with other cationic or anionic species,
respectively. The rate law for hydrolysis of chemical RX usually
can be written
-d[RX] = kh[RX] = kB[OH ][RX] (1)
dt
+kA[H+][RX] + k'N[H20][RX],
where k,, kg, and k'N are the second-order rate constants for
acid and base catalyzed and neutral water processes,
respectively. In the environment, hydrolysis of organic
chemicals occurs in dilute solution. Under these conditions,
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CS-5UOO
water is present in large excess, and the concentration of water
is essentially constant during the hydrolysis reaction. Thus, at
fixed pH, this process follows pseudo first-order kinetics and
the rate of disappearance of the chemical is dependent only on
its concentration. The hydrolysis rate constant can thus be
written as:
k = k[OH~] + k[H+] + k' (2)
where k, and kg are the second-order rate constants and k,, is a
first order-rate constant. At a given pH, expression (2)
contains three unknowns, k,, kg, and k,,. Therefore, three
equations (i.e., measurements at three different pH ' s at a fixed
temperature) are required to solve for these quantities. Making
suitable approximations for quantities which are negligible, the
expressions for kA, kg, and kN using values of k^ measured at
three environmental pH ' s (e.g., pH = x, x+y, and x+y+z) are (Mill
et al. 1981a)
kA - 10X [kh(x) - kh(x+y)] + 10X~Z kh ( x+y+z)
k = k(x+y)-10-yk(x)-10~zk(x+y+z) (3)
At a fixed temperature, the determination of rate constants at
three different pH ' s , therefore, can be used to determine k^ , kR ,
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CS-5000
and k., . For example, using the three measurements at pH 3.0,
7.0, and 11.0 required by the protocol (x=3, y=4, z=4), one
obtains :
kA = 103 [kh(3)-kh(7) + 10 kh(ll)]
kB = 103 [kh(ll) - kh (7) + 10~4 kh(3)]
kN = kh(7) ~ 10~4[*h(3) + kh(11)]' (4)
Once the above three rate constants have been calculated,
equation (2) can be used to calculate rate constants at any pH of
environmental significance at a fixed temperature. After k, has
been determined, the half-life of a chemical is easily obtained
since at fixed pH the hydrolysis reaction is pseudo-first order,
and the half -life of the substrate is independent of its
concentration. Thus:
= 0.693/kh. (5)
The dependence of rate on pH can be conveniently expressed
graphically. Figure (1) shows a typical log k, vs. pH plot for
substances which undergo acid, water, and base-promoted
hydrolysis (Mill et al . 1981a). Most pH-rate profiles are found
to have one or two areas of curvature corresponding to pH values
where two kinds of rate processes contribute to the overall
hydrolysis rate;. For molecules for which acid catalyzed
processes do not play an important role, the low pH region will
have near zero slope, while the same will be true in the high pH
region if base-catalyzed hydrolysis is unimportant. The lower
curve in Figure (1) results when kj, «k,[H+] and kg[OH~].
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pH
Figure .1 pH Dependence of k for Hydrolysis by (a) Acid-, (b) Water-, and ^c) Base-Promoted Processes
O
tn
I
01
o
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The equations discussed in this section are equally
applicable to molecules having one or many hydrolyzable groups.
In the latter case, the rate of reaction may be written as:
+ k2[RX] + + kn[RX]
dt
= (k: + k2 + kn) [RX] = kh[RX]
The above equation applies to the hydrolysis rate of a
molecule having n hydrolyzable groups, each of which reacts
according to first-order kinetics. The measured k^, which is
still the rate constant for the disappearance of RX, is now the
sum of the individual hydrolysis rate constants. Measurements of
concentration of any particular reaction product vs. time,
permits the determination of the rate constants associated with
this product (Frost and Pearson 1961). This determination is not
required in this Test Guideline.
Details of the hydrolysis reactions of various types of
compounds can be found in many kinetics texts (e.g., Laidler
1965, Frost and Pearson 1961). Discussions of hydrolysis from an
environmental point of view have also been published (Mabey and
Mill 1978, Tinsley 1979). Finally, examples of experiments in
which hydrolysis rate constants were measured in an
environmentally relevant fashion can be found in the papers by
Wolfe et al. (1976, 1977), Smith et al. (1977, 1978).
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B. Rationale for Selection of Test Method
An extensive amount of information has been published on the
hydrolysis of a wide variety of organic chemicals. However, most
of the literature relating to hydrolysis of chemicals in the
environment concerns pesticides. Many of these data are
incomplete for the range of pH and temperature of environmental
concern. Effects of buffer salts are often unrecognized. A
detailed literature search indicates that numerous factors must
be considered in determining the hydrolysis rate constants of
certain organic chemicals. At present, no validated procedure
exists for determining the hydrolysis rate constant at
environmental pH's and temperatures. The proposed method in this
Test Guideline was developed from a detailed review of the
literature on hydrolysis, from consultations with researchers
having considerable experience in carrying out these types of
measurements, and from the results of a contract with SRI
International on optimization of hydrolysis protocols (Mill
et al. 1981). The method was selected on the basis of the
following criteria: (1) The test method should be based on the
fundamentals of the kinetics of hydrolysis. (2) The test method
should yield quantitative data in the pH and temperature range of
environmental concern. (3) The effects of buffer salts on the
rates of hydrolysis should be minimized. (4) The test should be
designed to insure that only hydrolysis takes place. For
example, the experiment should be designed to make sure that
other processes such as biodegradation, loss by volatilization,
or photolysis are eliminated.
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The test method requires that rate constants be measured in
buffered, distilled water solutions rather than in natural waters
and, thus, makes the assumption that hydrolysis rates are the
same in the two media. This assumption has been confirmed by the
published data to date [e.g., Smith et al. (1977, 1978), Wolfe
et al. (1976, 1977), Zepp et al. (1975)], in which comparable
rates of hydrolysis were found for various chemicals in sterile
natural waters and in buffered distilled water at the same
temperature and pH.
Assuming a sufficient number of time points, n, are taken,
the; percent standard deviation in the measurement of the
hydrolysis rate constant, k-. , is proportional to l/(n)2and 1/t,
wheire t represents the number of half-lives over which
measurements were taken (Mill et al. 1981a). The number of half-
lives, or in. other words the extent of conversion, is thus a more
important factor than the number of time points in reducing the
error in k, . In order to obtain a reasonably good determination
of k-L, and, also to clearly demonstrate that the hydrolysis
reaction is following first-order kinetics, the reaction should
be followed for at least one half-life (50% conversion). For a
reaction which can attain at least 60% conversion in four weeks,
Procedure 1 which requires a minimum of six data points is
adequate. If the reaction is too slow to follow to high
conversion, but at least 20% hydrolysis is attained in four
weeks, the number of data points must be increased in order to
decrease the uncertainty of the rate constant determination.
Thus, for example, a reaction which is only 30% complete at the
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end of concentration sampling has a percent standard deviation
twice that of a reaction which is followed until it is 60%
complete. To offset this increase in error, the number of data
*\
points must be increased (2) or fourfold. The Procedure 2
requirement of 15 to 20 data points is an approximately threefold
increase in the number of measurements and represents a
compromise between the need to reduce uncertainty in a slow
hydrolysis and the need to avoid an undue number of experimental
determinations. If less than 20% of the chemical is hydrolyzed
at the end of four weeks, a significant reduction in the
uncertainty in k, becomes more difficult to attain than is deemed
necessary for the procedure described in this Test Guideline. In
this case, a determination of only one data point is required.
C. Rationale for the Selection of Experimental Conditions
1. Purity of Water
Dissolved impurities can catalyze or affect the rate of
hydrolysis. In addition, the water should be free of bacteria
which may consume or alter the organic test material during the
prolonged periods of testing which may occur in the course of a
rate determination. Thus, very pure water [e.g., water
comparable to that meeting ASTM Type II specification (ASTM
1979), or an equivalent grade], is required in this Test
Guideline.
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2,, Sterilization
It is extremely important to sterilize all glassware, to use
aseptic conditions in the preparation of all solutions, and in
carrying out all hydrolysis experiments to eliminate or minimize
biodegradation. Glassware can be sterilized in an autoclave or
by any other suitable method.
3. Precautions for Volatility
Loss of a test chemical through volatilization will result
in the determination of excessively large rate constants.
Therefore, for volatile chemicals, it is extremely important that
the reaction flasks be filled almost completely and sealed in
order to avoid this type of loss.
4. Temperature
Since hydrolysis rates are a function of temperature, it is
extremely important that the temperature of a hydrolysis reaction
be kept constant during the course of measurement. The
relationship between temperature and rate constant can be found
in any chemical kinetics text (e.g., Laidler 1965, Benson 1960).
In aquatic systems, temperatures commonly encountered range
from close to freezing in some lakes during winter to 30°C in
some ponds during summer. Since this test method is only a
screening test, the rate constant for hydrolysis is, ordinarily,
required only at 25°C. This temperature was chosen since it is
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in the temperature range of environmental concern, is the
temperature at which most physical and chemical properties are
reported, and, being at the higher end of the environmental
temperature range, allows one to measure rates which are close to
an upper limit for environmental hydrolysis of a particular
substance and, thus, are more practical to determine within 672
hours.
In general, hydrolysis rates can be expected to vary by
factors of 2 to 5 with each 10°C change in temperature (Mill
et al. 1981a). Therefore, variations of temperature of ±1°C can
typically lead to changes of hydrolysis rate on the order of ±7
to ±18%. This is an acceptable fluctuation for the proposed
method and, thus, temperature control is set at ±1°C in the Test
Guideline. This is a condition easily achieved by standard
control devices for water baths.
5. j>3.
It is recommended that hydrolysis experiments be carried out
at pH's of 3.00, 7.00, and 11.00. The first and last pH's are
not ordinarily found under environmental conditions, and the rate
constants measured at these pH's cannot therefore be directly
used to yield environmentally relevant rates. The three measured
rate constants are instead used in expression (4) to calculate
kA, kg, and KN. These latter constants are then used in
expression (2) to determine the hydrolysis rate at 25°C of a test
chemical at pH's commonly found in the environment.
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Although the above procedure is less direct than performing
rate measurements at environmental pH's such as 5, 7, and 9 as
required previously (EPA 1979), an examination of equation (1)
indicates that the rates of hydrolysis at pH 3 and 11 are as much
as two orders of magnitude greater than at pH 5 and 9. This
reduces the possibility of the intrusion of unwanted processes
such as biodegradation, failure of control equipment,
intermittent power failure, etc., which could tend to be a large
factor in the errors associated with the slower rate processes at
pH 5 and 9. The potential increase in accuracy of the pH 3 and
11 measurements is great enough so that the indirect measurement
of environmentally relevant hydrolysis rates described in this
Tesit Guideline can actually lead to more reliable results than a
direct measurement (Mill et al. 1981). In addition, the
potential decrease in measurement time can lead to a large
reduction in the cost and effort of obtaining a rate constant.
For these reasons, measurements at pH 3 and 11 are specified in
the Test Guideline rather than pH 5 and 9.
The pH of a test solution cannot vary by more than 0.05
units. Examination of equation (1) shows that such a variation
can lead to changes in the rate of as-much as 12 percent. This
is considered a maximum acceptable variation for this hydrolysis
test method.
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6. Initial Concentration of Chemicals
Typical concentrations of trace organics in the environment,
except perhaps near release sites, do not ordinarily exceed
10~^M (Mill 1981a). Changes in concentration at these low levels
are difficult to monitor, however, so that the required 10 M
concentration represents a compromise between the desireability
of easily following the reaction rate and the desire to reproduce
environmental conditions. Simple first-order processes at higher
concentrations are expected to remain the same at lower
concentrations so that the rate constants found at 10 M will be
valid at environmental concentrations. In addition, since
hydrolysis is almost always first-order (Laidler 1965, Frost and
Pearson 1961), the actual half-life of the chemical is a function
only of the rate constant and not the initial concentration.
If the test chemical is not sufficiently soluble to permit
reasonable handling and analytical procedures, then test
solutions can be prepared from a chemical dissolved in reagent
grade acetonitrile. The solution can then be diluted with buffer
to an appropriate concentration. It is extremely important that
the final acetonitrile concentration be no more than one volume
percent in order to avoid acetonitrile solvent effects (Smith
et al. 1977, 1978; Mabey and Mill 1978).
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7. Ionized or Protonated Groups
Certain chemicals reversibly ionize or are protonated in the
pH range 3 to 11. As a result, complications can arise when
measuring hydrolysis rates. The pH rate profiles of these
substances will he more complicated than typical profiles and
will often have a maximum or minimum. For chemicals which
exhibit ionization or are protonated in the pH range 3 to 11, it
is not possible to use kA, kR and kN to obtain rate constants at
environmental pH's. In this case, it is therefore necessary to
measure k^ at pH 5,7, and 9 directly. If the test chemical
ionizes or is protonated between pH 5 and 9, the resulting
complexity of the pH rate profile necessitates 2 additional
measurements at pH 6 and 8.
8. Buffers
Nucleophi1ic salts can increase hydrolysis rates through a
general acid or base catalyzed process (Jencks 1969), the
magnitude of which is dependent on the nature and concentration
of the salt. Thus, the results from experiments which utilize
high concentrations of buffer are suspect unless carefully
examined for buffer catalysis effects. The buffers listed for pH
3, 7, and 11 in this method were tested and used at low
concentrations in order to minimize this type of catalysis (Mabey
and Mill 1978). The hydrolysis of some chemicals, however, may
still be affected by nucleophilic buffers at the concentrations
specified in this Test Guideline. If there is any reason to
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believe that this is occurring, it is recommended that hydrolysis
experiments be carried out at buffer concentrations reduced by a
factor of five. If the pH changes by more than 0.05 pH units at
the end of these latter experiments, then the buffer capacity has
been exceeded. Therefore, it is extremely important that the
test chemical concentrations then also be reduced by a factor of
five. Alternatively, one may reduce the buffer and test chemical
concentration by the above factors simultaneously.
9. Light Sensitive Compounds
Chemicals which absorb light at wavelengths greater than 290
nm may be subject to photolysis, especially over the relatively
long periods of time in which the hydrolysis experiments may be
run. Therefore, it is recommended that precautions be taken, to
insure that these substances are protected from light, including
sunlight. Appropriate measures could include use of an amber or
red safelight, use of amber or red colored glassware, or any
other suitable technique which will eliminate the possibility of
photolytic transformation.
10. Chemical Analysis of Solutions
The analytical techniques employed in the determination of
test chemical concentrations are left to the sponsor to select.
This is in recognition of the many different techniques available
and the practical advantage of being able to make particular use
of one of the properties of the compounds; e.g., the NMR or UV
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spectrum of the substance, or the chroraatographic behavior.
Analytical techniques that permit the determination of the test
compound to the exclusion of impurities or reaction products are
recommended to the extent practicable. Therefore,
chromatographic techniques are particularly desirable. Whenever
practicable, an analytical procedure having a precision of ±5%
should be used. The Test Guideline requires that the specific
technique utilized be adequately described.
D. Test Data Required
The rate constant data required will be used to help assess
the environmental fate and persistence of the test materials. It
is essential data which is needed to make a risk assessment. If
hydrolysis is a relatively important transformation process and
the initial risk assessment indicates that the material poses a
threat to the health of humans and/or to the environment, then
advanced tests may be necessary to obtain more extensive data.
E. Statistical Analysis of the Data
In the case of Procedure 1, assuming 7 points have been
measured over the course of one half-life and the error in
individual concentration measurements is approximately 5%, one
can anticipate an uncertainty in the rate constant of about twice
that of the individual measurements (Mill et al. 1981a). This is
an ideal case, however, in which temperature is assumed to have
remained strictly constant and that no adventitious processes
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have occurred. In cases in which errors due to environmental
fluctuations (e.g., temperature) are larger than the
uncertainties due to individual measurements, a case of
cumulative errors arises which can lead to greater uncertainties
than the aforementioned factor of two (Mandel 1964, Daniels and
Johnston 1921). As was pointed out in section B, greater
uncertainties are also expected when employing Procedure 2 in
which the total measurement time covers less than one half-
life. Finally, Procedure 3 (in which only one point is measured
over less than 20% conversion) yields the greatest degree of
uncertainty in the rate constant.
The exact magnitudes of all of the aforementioned errors are
a function of the details of the hydrolysis kinetics of the
particular chemical being tested. These uncertainties have not
been clearly established and, thus, the Test Guideline makes no
specific requirements for degree of precision in rate constants
other than that of a ±5 percent uncertainty in individual
concentration measurements, if at all possible. The final
precision can be improved by making numerous replicate
determinations. The minimum requirement EPA would impose would
be a statistical analysis of the data to provide standard
deviations based on triplicate determinations. When a large
number of chemicals have been determined by the proposed methods
and the practical effect of factors such as fluctuations of
temperature over a one degree range, measurement of very slow
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reactions over a long time period, measurement of rates of
relatively insoluble, highly volatile chemicals, etc. have been
better defined, the level of precision in the rate constants can
be given for chemicals having varying ranges of hydrolysis rates
and differing physical properties.
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ITT. REFERENCES
. 1979. American Society ^or Besting and Materials. Annual
book of standards. Part 31. Standard specification for water.
Philadelphia, PA: pp 20-22.
Renson sw. 1960. mhe foundations of chemical kinetics. rTew
York: McGraw-Hill Rook Co.
Oaniels E, Johnston EH. 1921. The thermal decomposition of
gaseous nitrogen pentoxide. J Am Chem Soc 43:53-71.
Frost AA, Pearson RG . 1961. Kinetics and mechanism. New Vork :
John Wiley and Sons, Inc.
Jencks wr1 . 1969. Catalysis in chemistry and enzymology.
New York: McGraw-Hill Rook Co.
Laidler K J . 1965. Chemical kinetics. Mew ^or\ : McGraw-Hill
Rook Co.
Mahev W, Mill rp. 1978. Critical review o^ hvdrolysis of organic
compounds in water under environmental conditions. J Phvs Chem
nata 7:383-415.
Mandel J. 1964. ^he statistical anaylsis of exnerimental
data. New York: John Wiley and Sons, Inc.
Martell Ap . 1963. Metal chelate compounds as acid catalvsts in
solvolysis reactions. Adv Chem Ser 37:161-173.
Mill ^, ^1ahey WR , Romherger nc, Chou ^-W, Hendry OG, Smith J" .
1981a. Laboratory protocols for evaluating the fate of organic
chemicals in air and water. Athens, GA. n.s. Environmental
Protection Agency. EPA
Mill T, Rawol R, Cartridge I, Mabey WR. 1981. Evaluation and
optimization of hydrolysis screening protocols. Ora-Pt final
report. Washington, H.C., TJ.S. Environmental Protection
Agency. EPA 560/5-81-004.
Smith JH, Mabev WR, Rohonos N, et al. 1977. Environmental
pathways of selected chemicals in freshwater systems. Part I.
Rackground and experimental procedures. Athens, GA: n.s.
Environmental Protection Agency. EPA-600/7-77-113 .
JH, Mabev WR , Rohonos N et al. 1978. Environmental
pathways of selected chemicals in freshwater systems. Part IT.
Laboratory studies. Athens, GA. H.S. Environmental Protection
Agency. EPA-600/7-78-074.
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Tiislev TJ. 1979. Chemical concepts in pollutant behavior.
York: John Wiley and Sons, Inc.
n.S. Environmental Protection ^qency, Office of ^oxic Substances
1979a. Toxic substances control act premanuf acturinq testing o^
new chemical substances. ^ederal Reqister, March 16, 1979,
44 FR 16268.
WoL-Ffi ML, 7,enp RG, R-auqhman GL, ^incher RC, Gordon J^. 1976.
Chemical and photochemical transformation of selected pesticides
in aquatic svstems. Athens, G^ TT.S. environmental Protection
EPA-600/3-76-067.
Wo'L^e ML, ^enn RG, Gordon .T^, Rauqhman GL, Cline HM . 1977.
Kinetics of chemical deqradation of malathion in water. Environ
Sci mechnol 11:88-93.
7epp RG, wol^e ML, Gordon, J^, Rauqhman GL. 197^. Hvnamics
2,4-n esters in surface waters. Hydrolysis, photolysis, and
vaporization. Environ Sci ^echnol 9:1144-11^0.
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CG-6000
August, 1982
PHOTOLYSIS IN AQUEOUS SOLUTION IN SUNLIGHT
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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Contents
Page
I. INTRODUCTION 1
A. Background and Purpose 1
B. Definition and Units 2
C. Principle of the 'T'est Method 5
D. Applicability and Specificity 6
11 . TES^ PROCEDURES 7
A. Test Conditions 7
1 . Special Laboratory Equipment 7
2. Purity of Water 8
3. Sterilization 8
4. pH Effects 9
5. Chemical Analysis of Solutions 9
6. Volatile Chemical Substances 9
7. Control Solution 10
8. Absorption Spectrum as a Criterion for
Performing the Photolysis in Aqueous Solution
in Sunlight Test 10
B. Preparations 11
1. Reagents and Solutions 11
a. Preparation of Test Chemical Solution 11
b. Preparation of Buffer Solution 12
C. Performance of the Test 12
1. Procedures 13
a. Procedure 1 14
b. Procedure 2 14
c. Procedure 3 14
d. Analytical Methodology 15
III. DATA AND REPORTING 15
A. Treatment of Results 15
B. Specific Analytical and Recovery Procedures 17
C. Other Test Conditions 17
D. Test Data Report 18
IV. REFERENCES 19
V. APPENDIX 1: DATA FORMAT SHEETS 20
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PHOTOLYSIS IM AQUEOUS SOL'ITTON TNT s> r-'M GH
I. INTRODUCTION
.A. background and Purpose
Numerous chemicals have entered natural aquatic svstons from
a variety of sources. For example, chemical wastes have been
discharged directly into natural water bodies. Chemicals have
leached into natural water bodies from landfills. Pestjcides
have been applied directly into water bodies. nestioi.des have
been applied to soils and veqetation and have subsequently
leached into water bodies. Pollutants present in aqueous media
can undergo photochemical transformation in the environment
(i.Q., in sunlight by direct photolysis or bv sensitised
photolysis). \s a result, there has been considerable interest
in Photolysis in solution, especiallv the photolvsis of
pesticides. However, most of these studies have been qualitative
in nature and involved the identification of photolvsis
products. Quantitative data in the form of rate constants* and
half-lives are needed to determine the importance of nhotochemica1
transformation of pollutants in aqueous media. ^his
* The ^IPA is developing test methods to determine the transport
and transformation of chemicals in the environment. ^hese
methods will yield rate constants and equilibrium constants which
can be extrapolated to a variety of environmental scenarios.
Thus, this data can be used in models along with production,
volume, distribution, etc. to predict an expected environmental
concentration
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Guideline describes a method for determining direct photolysis
rate constants and half-lives of chemicals in water in the
presence of sunlight.
B. Definitions and Units
Direct photolysis is defined as the direct absorption of
light by a chemical followed by a reaction which transforms the
parent chemical substance into one or more products.
Numerous papers have been published on the photolysis of
chemicals in solution. However, only recently has work been
published on the determination of rate constants and half-lives
for direct photolysis of chemicals in water under environmental
conditions, i.e., in sunlight.
Zepp and Cline (1977) published a paper on the rates of
direct photolysis in aquatic environments. The rates of all
photochemical processes in a water body are affected by solar
spectral irradiance at the water surface, radiative transfer from
air to water, and the transmission of sunlight in the water
body. It has been shown that in dilute solution (i.e., with the
absorbance of a chemical less than 0.02 in the reaction cell at
all wavelengths greater than 290 nm) at shallow depths, the
kinetic expression for direct photolysis of a chemical at a molar
concentration C is
d< k c = k c , (i)
j : y is. \_- /\. v
dt a p
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where k = 4>k , (2)
P a
o is the reaction quantum yield of the chemical in dilute
solution, and k = )k , , the sum of k , values of all wavelengths
<;) a A a A
of sunlight that are absorbed by the chemical. The term k
represents the photolysis rate constant in sunlight in the units
of reciprocal time. Integrating equation 1 yields
C k t
° P
= -
i
Iog10
where C is the molar concentration of chemical at time t during
photolysis and CQ is the initial molar concentration. Ry
measuring the concentration of chemical as a function of the time
t during photolysis in sunlight, k can be determined using
equation 3. Since equation 1 is a first-order rate equation, the
half-life for direct photolysis in sunlight is given by
0.693. f
k
P
Zepp and Cline (1977) derived equations that describe the
direct photolysis rates of pollutants in aquatic environments.
These equations translate readily obtained laboratory data, such
as the quantum yield ^ as measured by Zepp (1978) and extinction
coefficients of the chemical in aqueous solution, into rate
constants and half-lives for photolysis in sunlight. Rate
constants and half-lives can be calculated (by computer) as a
function of season, latitude, time-of-day, depth in water bodies,
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and the ozone layer. Several published papers concerning the
photolysis of chemicals in sunlight verified this method (Zepp et
al. 1977, Wolfe et al. 1976, Zepp et al. 1976, Zepp et al. 1975,
Smith et al. 1977, 1978).
The absorbance of a chemical substance is the logarithm
(base 10) of the ratio of the intensity of light entering a cell
containing a solution of chemical to that leaving the cell.
A photolysis "day" is defined as the period of time from
sunrise to sunset when sunlight photolysis of a chemical may take
place. A fraction of a day is defined as a fraction of the
daylight period. Based on this definition, the term k , used in
equation 3, is then the sunlight photolysis rate constant
expressed in the units day . The half-life of the chemical is
defined as the time in days corresponding to the disappearance of
one-half of the initial concentration of the chemical in
sunlight. The half-life can be calculated using equation 4. The
rate constant and half-life determined by this method are
relevant to the day mid-way between the beginning and termination
of the photolysis experiment and, therefore, represent an
"average" rate constant and half-life for the chemical during a
certain period of time. Obviously, the longer the experiment,
the greater the difference in the average value of the rate
constant and half-life during the experimental time period.
Therefore, all photolysis data reported should carry appropriate
information on the duration of the experiment.
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C. Principle of the Test Method
This ^est Guideline is based on the principles developed by
Zenp and Cline (1977) and the n.S. EPA Premanufacture Test
Guideline (1979). A simple aqueous photolysis screeninq test has
been developed to determine rate constants and half-lives in the
presence of sunliqht using equations (3) and (4). Sunlight was
chosen as the irradiation source because of its relevancy as well
as its low cost in comparison to artificial liqht sources. A.
small error (approximately 3%) is introduced in this methodology
assuning an absorbance of 0.1 instead of 0.02 and first order
kinetics are still applicable. Therefore, this screening test
method is applicable to homogeneous chemical solutions having an
absorbance of less than 0.10 in the reaction cell at all
wave'enqths greater than 290 ran and at shallow depths. ^he
experiments have been designed to make sure that only photolysis
occurs. For example, the experiments have been designed to
eliminate biodegradation and volatilization. The experiment must
be carried out during a warm time of the year (i.e., May, June,
July, or August in the northern hemisphere, weather permitting)
and the measured rate constant and half-life is characteristic of
that time of year and the latitude of the site where the
experiment was carried out.
T'his preliminary screening test has a limitation in that it
fails to measure sunlight intensities incident on the sample
during photolysis. Sunlight actinometers are being developed to
evaluate sunlight intensities. The screening test will then be
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modified to use simultaneous photolysis o^ a chemical and
actinometer to evaluate sunlight intensities on the sample. The
modified procedure will quantify sunlight photolysis of a
chemical at a specific time of year and latitude and will give a
useful measure of seasonal variation of photolysis.
D. Applicability and Specificity
This ^est Guideline is applicable to all chemicals which
have uv-visible absorption maxima at 290nm or greater. Some
chemicals absorb light significantly below 290nm and conseguentlv
will not undergo direct photolysis in sunlight (e.g., alkanes
alkenes, alkvnes). Some chemicals have absorption maxima
significantly below 290nm but have measureable absorption tails
above the baseline in their absorption spectrum at wavelennths
greater than 290nm. Photolysis experiments should also be
carried out *or these chemicals. ^his screening test method is
applicable to the photolysis of chemicals in dilute solution in
which the absorbance is less than 0.10 in the reaction cell at
all wavelengths greater than 290nm and at shallow depths.
Furthermore, these experiments are limited to the direct
photolysis of chemicals in air-saturated pure water. The water
must be air-saturated to simulate environmental conditions.
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II. TEST PROCEDURES
A. Test Conditions
1. Special Laboratory Equipment
(1) A variable wavelength uv-visihle spectrophotometer
capable of measuring accurate absorbances at 0.10
(absorbance) units or less. Refer to ORCn Guideline
No. 101, "UV-VIS Absorption Spectra", for details on
the use of the spectrophotometer;
(2) a pH meter capable of resolving differences of 0.1 pH
unit or less; and
(3) the absorption spectrum of the chemical substance in
aqueous solution (as determined by OECD Guideline
No. 101) can be used to determine the type of reaction
vessel to be employed for these photolysis
experiments. It is strongly recommended that quartz
vessels be used for the photolysis of chemical
substances which absorb at wavelengths below 340nm.
Chemical substances that absorb at wavelengths greater
than 340nm may be tested in borosilicate glass vessels.
Thin walled borosilicate or quartz tubes are recommended.
Disposable culture tubes (13 x 100 mm) with teflon lined screw
caps or quartz tubes with ground glass stoppers (no grease) may
be used as reaction vessels. Tubes of 11 mm i.d. are
recommended. For some chemical substances it may difficult to
-7-
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CG-6000
analyze the concentration of the chemical substance in reaction
vessels of such small volume. For these chemical substances
larger reaction vessels are recommended providing that the cell
walls are thin and the pathlength of radiation through the vessel
is less than 0.5 meter. Reaction vessels should be filled as
completely as possible and sealed to minimize volatilization.
2. Purity of Water
Reagent grade water, e.g., water meeting ASTM Type II A
Standards, or an equivalent grade, is highly recommended to
minimize biodegradation. ASTM Type II A water is described in
ASTM D 1193-77, "Standard Specification for Reagent Water". It
is important to saturate water with bacteria-free air just prior
to the preparation of the test and control solutions to simulate
environmental conditions. The air can be filtered through a
0.2 urn (pore size) filter to remove bacteria.
3. Sterilization
It is extremely important to sterilize all glassware and to
use aseptic conditions in the preparation of all solutions and in
carrying out all photolysis experiments to eliminate or minimize
biodegradation. Glassware can be sterilized in an autoclave or
by any other suitable method.
-8-
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CG-6000
4. pH Effects
It is recommended that all photolysis experiments be carried
out a.t pHs 5.0, 7.0, and 9.0 for any chemical which reversibly
ioni2,es or protonates (e.g., carboxylic acids, phenols, and
amines). Buffers described in Section II.B.l.b. may be used.
5. Chemical Analysis of Solutions
In determining the concentration of the chemical in
solution, an analytical method should be selected which is most
applicable to the analysis of the specific chemical substance.
Chromatographic methods are generally recommended because of
their chemical specificity in analyzing the parent chemical
substance without interference from impurities. Whenever
practicable the chosen analytical method should have a precision
of ± 5 percent.
6. Volatile Chemical Substances
Special care should be taken when testing a volatile
chemical so that the chemical substance is not lost due to
volatilization during the course of the photolysis experiment.
Thus, it is important to effectively seal the reaction vessels.
Tubes with ground-glass stoppers (no grease) or with plastic
screw tops with teflon inserts are recommended. In addition, the
reaction vessels should be as completely filled as is possible to
prevent volatilization to any air space.
-9-
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CG-6000
7. Control Solution
It is extremely important to take certain precautions to
prevent loss of chemical from the reaction vessels by processes
other than photolysis. For example, biodeqradation and
volatilization can be eliminated or minimized by use of sterile
conditions and minimal air space in sealed vessels. Hydrolysis
is a process which cannot be minimized by such techniques. Thus,
control vessels containing test substance which are not exposed
to sunlight are required. By suitable analysis of the
concentration of test substance in the control vessels,
corrections, if any, can be made to the measured photolysis
rates. If hydrolysis is found to be significant, hydrolysis
studies should be carried out first. (Test Guideline CG-5000).
8. Absorption Spectrum as a Criterion for
Performing the Photolysis in Aqueous Solution
in Sunlight Test
The Photolysis in Aqueous Solution in Sunlight ^est is
applicable to all chemicals which have uv-visible absorption
maxima at 290mm, or greater. Some chemicals have absorption
maxima significantly below 290nm but have measureable absorption
tails above the baseline in their absorption spectrum at
wavelengths greater than 290nm. Photolysis experiments must also
be carried out for these chemicals. ^he absorption spectrum of
the chemical in aqueous solution can be measured by OECD Test
Guideline No. 101, "UV-VIS Absorption Spectra".
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CG-6000
B. Preparations
1. Reagents and Solutions
a. Preparation of Test Chemical Solution
Prepare homogeneous solutions with the chemical at less than
one-half its solubility in water and at a concentration such that
the absorbance is less than 0.10 in the photolysis reaction
vessel at wavelengths greater than 290nm. For verv hvdrophobic
chemicals, it is difficult and time consuming to prepare aqueous
solutions. To facilitate the preparation of aqueous solutions
containing very hydrophobic chemicals and to allow for easier
analytical measurement procedures, the following procedure may be
used to aid in the dissolution of the chemical. Dissolve the
pure chemical in reagent grade acetonitrile. Add pure water as
decri.bed under Test Conditions, Section IT. A. 2., or buffer
solution as described under Preparations, Section IT.B.l.b., for
chemical substances which reversible ionize or protonate, to an
aliquot of the acetonitrile solution. no not exceed one volume-
percent of acetonitrile in the final solution. Place the
reaction solution in the photolysis reaction vessels.
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CG-6000
b. Preparation of Buffer Solutions
Prepare buffer solutions using reagent grade chemicals and
pure water as described under Test Conditions, Section TI.A.2.,
as follows:
pH 5.0 0.1 molar sodium acetate (NaC 2^13(^2) adjusted
to pH 5.0 with 0.1 molar acetic acid
(CH3C02H).
pH 7.0--0.01 molar potassium dihydrogen phosphate
(KH2PO4) adjusted to pH 7.0 with 0.1 molar
sodium hydroxide (NaOH)
pH 9.0--0.025 molar sodium tetraborate (^21340-7)
adjusted to pH 9.0 with 0.1 molar hydro-
chloric acid (HC1)
The pH of all buffer solutions must be checked with a pH meter at
25°C and adjusted to the proper value if necessary.
C. Performance of the Test
For all experiments, prepare an aqueous solution of the
chemical substance and a sufficient number of samples in quartz
or borosilicate glass vessels to perform all the required
tests. Fill the vessels as completely as possible and seal
them. Prepare three control samples in the absence of
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CG-6000
ultraviolet-, liqht and totally exclude liqht hv wrappina the
vessels with aluminum foil or by other suitable methods. 'Hiese
samples are analvzed for the chemical substance immediatelv after
completion of the experiment to measure the loss of chemical in
the absence of liqht. Place the samples, includinq the controls,
outdoors in an area free of shade and reflections of sunliqht
from windows and buildinqs. ^lace the samples on a black, non-
reflective backqround and inclined at approximately 30° from the
horizontal with the upper end pointinq due north. Conduct the
photolysis exneriments during a warm time of year (i.e., May,
June, July, and August in the northern hemisphereweather
periritting) and start the experiments initially before sunrise.
Record the date and time the experiment was begun, the date and
time completed, the time of sunrise and sunset on all days when
photolysis experiments were performed, the times exposure was
stopped and restarted for intermittent exposure, the weather
conditions during this period, and the latitude of the site.
chemical substances that reversibly ionize or protonate, carry
out photolysis experiments at pH 5.0, 7.0, and 0.0 as described
under Test Conditions, Section II.A.4.
1. Procedures
Use one of the following procedures, depending on how fast
the chemical substance photolyzes.
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CG-6000
a. Procedure 1
If the chemical substance degrades 50-80% within 28 days,
measure the concentration of the chemical substance, in
triplicate, at time t = O and periodically (at least three data
points at approximately equal time intervals) at 12 o'clock noon
until at least 50% of the substance has been consumed. Determine
the concentration of test chemical from three, freshly opened,
reaction vessels for each time point. Determine the
concentration in each of the three control solutions as soon as
the photolysis experiments are completed.
b. Procedure 2
If the chemical substance degrades in the range of 20-50% in
28 days, determine the concentration of the chemical substance,
in triplicate, at time t = 0. Determine the concentration of the
three separate reaction vessels and the three control vessels
after 28 days of photolysis.
c. Procedure 3
For chemical substances Lhat degrade in sunlight 50-80%
within two days, place the samples outside before sunrise and
analyze triplicate samples of the concentration of the chemical
substance at t = 0, and in three, freshly opened reaction vessels
after sunset the first day, and again, in three, freshly opened,
reaction vessels after sunset the second day. Determine the
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CG-6000
concentration in each of the three control solutions as soon as
the: photolysis experiments are completed. Carry out the above
experiment on clear sunnv days a total of three times.
d. Analytical Methodoloqv
Select an analvtioal method which is most annlicahle to the
analysis of: the specific chemical heinq tester) [Section Tl.A.S.l.
ITT. OAT\ A?n RCPORTTM^,
A. T>-f>atment of Results
If loss of test substance in the control vessels has
occurred, use this data to make corrections to the measured
ohotolvsis rate. "Jote the site of ohotolvsis and its latitude
an>: the weather con'! it ions. For Procedures 1 and 2 note the
dates and t Lines of actual exposure includinq times of sunrise and
surset and, in case the cells are moved to prevent freezinq or
for other reasons, make sure that these times are recorded and
that the cells are kent in a dark olace when exnosure is not in
oroqress.
(1) For chemical substances which deqrade 50-^0% within ?R
davs, use a concentration C, which corresponds to no
more than SO^ of the initial concentration of chemical
substance remaininq, and the corresnondinq time t, in
days, alonq with the initial molar concentration C , in
-IS-
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CG-6000
equation 3 to calculate k in days . From the
analysis of the three samples at time t, calculate a
mean value of C and a value of k . Calculate the half-
life, t , using the value of k in equation 4;
(2) for chemical substances which degrade 20-50% in 28
days, use the mean concentration C remaining at t 28
days along with C to calculate k . Use the same
procedure as described above to calculate the value of
k and t . If less than 20% of the chemical substance
degrades in 28 days, report C and CQ and the mean
concentration of C and C . In this case the apparent
half-life is greater than 3 months; and
(3) for chemical substances which degrade 50% or more in
the first day, as described in Procedure 3, calculate a
full day k value using the concentration C of chemical
substance remaining after sunset the first day along
with CQ using equation 3. For chemical substances
which degrade less than 50% by the end of the first day
but 50% or more by the end of the second day, calculate
k using the mean concentration of chemical substances
remaining after sunset the second day. Repeat these
calculations for the three separate full-day photolysis
experiments. Calculate a mean value of k from the
results of the three separate experiments. Calculate
the half-life, t , using the mean value of k in
Rquation 4. If loss of test substance in the control
-16-
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CG-6000
vessels has occurred, use this data to make corrections
to the measured photolysis rate. Note the dates of
photolysis, the latitude, and the site.
3. Specific Analytical and Recovery Procedures
(1) Provide a detailed description or reference for the
analytical procedure used, including the calibration
data and precision; and
(2) if extraction methods were used to separate the solute
from the aqueous solution, provide a description of the
extraction method as well as the recovery data.
C. Other Test Conditions
(1) Report the size, shape, approximate cell wall
thickness, and type of glass used for the reaction
vessels;
(2) report the initial pH of all test solutions;
(3) for all Procedures, report the dates of photolysis, the
time of sunrise and sunset on each photolysis day, the
site of photolysis and its latitude, and the weather
conditions. For Procedures 1 and 2 submit the dates
and times of actual exposure, and the duration of
exposure, and, for intermittent exposure, the fraction
of each day photolyzed; and
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CG-6000
(4) if acetonitrile was used to solubilize the test
substance, report the percent, by volume, of
acetonitrile which was used.
D. Test Data Report
(1) For each photolysis experiment, report the initial
concentration (CQ), and the mean value for test and
control solutions.
(2) \ fter the completion of the photolysis experiments,
report the concentration of chemical in each test and
control vessel, the time(s) for experiment termination
and the mean concentration value
(3) For Procedures 1 and 2, report the value of k and the
half-life, t , calculated using the value of k_.
(4) For Procedure 3, from the analysis of triplicate
samples, report a value of k for each series of
experiments. Report these data for the three separate
full-day photolysis experiments. Report the mean value
of k from the three separate full-day experiments.
Report the half-life, t , calculated from the mean
value of k .
Summarize all the data in the data sheets listed in Appendix 1
-------
CG-6000
IV. REFERENCES
ASTM. 1978. Annual book of ASTM standards. American Society
for Testing and Materials. Part 31, Method D 1193-77.
Philadelphia, PA.
OEC"). 1981. OECD guidelines for testing chemicals: No. 101-
UV-VIS absorption spectra. Director of Information, OECD; 2, rue
Andre-Pascal, 75775 Paris CEDEX 16, France.
Smith JH, Mabey WR, Bohonoe N, Holt BR, Lee SS, Chou T-W,
Bomberger DC, and Mill T. 1977. Environmental pathways of
selected chemicals in freshwater systems. Part I. Background
and experimental procedures. EPA-600/7-77-113 .
h JH, Mabey WR, Bohonos N, Holt BR, Lee SS, Chou T-W,
Bomberger DC, and Mill T. 1978. Environmental pathways of
selected chemicals in freshwater systems. Part II. Laboratory
studies. EPA-600/7-78-074.
USEPA. 1979. U.S. Environmental Protection Agency. Office of
Toxic Substances. Toxic Substances Control: Discussion of
premanufacture testing policy and technical issues. Request for
comment. Federal Register 44, 16240.
Wolfe NL, ?iepp RG, Baughman GL, Fincher CR, and Gordon JA.
1976. Chemical and photochemical transformation of selected
pesticides in aquatic systems. EPA-600/3-76-067 .
Zepp RG and Cline DM. 1977. Rates of direct photolysis in
aquatic environment. Environ Sci and Tech 11:359.
Zepp RG. 1978. Quantum yields for reactions of pollutants in
dilute "aqueous solution. Environ Sci and Tech 12:327.
Zepp RG, Wolfe NL, Azarraga LV, Cox RH, and Pape CW. 1977.
Photochemical transformation of the DDT and methoxychlor
degradation products, DDE and DMDE, by sunlight. Arch Environ
Contam Toxicol 6:305.
Zepp RG, Wolfe NL, Gordon JA, and Fincher RC . 1976. Light-
induced transformations of methoxychlor in aquatic systems. J
Agr:_ Food Chem 24:727.
Zepp RG, Wolfe NL, Gordon JA, and Baughman GL. 1975. Dynamics
of 2,4-D esters in surface water. Hydrolysis, photolysis, and
vaporization. Environ Sci Tech 9:1145.
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CG-60UO
V. APPRMDTX 1: DzyTA FORMAT SHEEm
Instructions
1. On tho first Test 'Results page, time in columns ri, f>, fi, and
9 are to be based on a 24-hour clock. Times of sunrise and
sunset are to be recorded for all photolvsis davs. Stop and
start times for intermittent exposure are to be recorded only
when exposure is not continuous from sunrise to sunset for
any day of the experiment (columns 8 and 9). More than one
line may be required to record this information if several
intermittent exposure periods occur on the same day.
Exposure duration (column in) is the total number of hours
and fractions of hours of sunlight exnosure of the samples
from sunrise to sunset for everv day of photolvsis. Tf
photolysis is carried out uninterrupted for a complete dav,
the number of hours in column 10 equals the number of hours
in column 7. The fraction of day exposed (column 11) is
obtained by dividing the value in column in bv the value in
column 7 for each photolysis day.
2. To report data for procedures 1 or 1, complete the first and
second Test Results pages. To report data for procedure 3,
complete the first, third, an^ fourth mest Results pages.
For procedure 3, multiple copies of the first Test Results
cage should be used, one COPV for each determination.
-20-
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CG-6UOO
3. If Tiultlnle pii values are required, comolete ono sot of the
appropriate Test Results paqes Cor each oH tested.
4. Tf multlole test substance initial concentrations are used,
complete one set of: the appropriate Test Results naqes, Tor
each concentration tested.
-21-
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TEST RESULTS
1 . Photo I LJS i a si+e
2. Lot i tuda CO Degrees f~~1 N |"~]
3. Oeterri i not i on nunber (if procedure 3 was used) I |
Exposure '/. Doulight
Durotion Mre. Exposed
IHra)
10. 11.
Oofe
(DUMHTTI
4.
-_
-
-
-
-
--
Tine
of
Sunr i ee
IHrMin)
5.
-
-
E
T i fie
of
Sunset
IHrMin)
6.
X
P 0 S U fl
Totol
OouJ ight
Hours
7.
E
PERIODS
I nt ern i t tent
Exposure IHrMin)
Slop Start
8. 9.
o
in
i
-------
TEST RESULTS
CONTINUED
PROCEDURES 1 or 2
i
fo
U3
13. pH | | | | 14. X flcsionitnle used in solvent sqaten | | ]\ \ '/,
T i no
(Hr.Mm)
15.
IT
> 1 t 1 O 1
_..
-._
Run
16.
1
Exp
C
Run
17.
0
2
N C
Exp
E
N T R fl T
Run
18.
__
I
3
0 N
Exp
-
Ir
lofdrt'3)
Mean
19.
Exp
Std. Dew.
20.
-
--
Fxn
_._
Concentration of controls ot the end of experiment:
Runl Run 2 Run3 Moon Std.Dev
21. Exp 22. Ex£ 23. Exp 2'1. Exp 25. Fxp
n~i~r~n m n~mn nj nzxn m nznnu en nznzn nfi
n
a
en
O
O
O
2G.
21.
T i n o fron cojjj nn 15 used
Kp ldo.|s"') CJIUCO E
to_cojculatu
^p LU 20-
Hie following
Ha If-I ife (t
1/2'
ft
1'tiyc
-------
TEST RESULTS
CONTINUED
PROCEDURE 3
29. pH | | I I 30. X flce-foni+r i le ueed in eoluent elusion I I I I I
Dei er n i no t i on 1
Elapsed
Tine
CONCENTRRT ION Inoldn"3)
Run 1 Run 2 Run 3 Mean
31. Exp 32. Exp 33. _ _ Exp 3U. Exp
Std. Dew.
35. Exp
Concentration of controls at Ihe end of experinani:
. _ _ __ _ . _ _ __
ri i i i i m i i i i i i nu i i i i M nn n i i i i
_ _
i i i i i \ m
, _ __. _ .
H2. Molf-l .fa lt,/2) I I I I I I doMB
_ .
Exp [~T~1 on DoM
CONCENTRRT ION
Ex H7.
Concanlroiion of controle a-f the end of exparmen-fi
_ _ _ _
en n~rm en i i i i u an cnnzn en i i i i i i en
5'I. Uulf-Sifs (
n
o
i
OT
O
C)
O
53. Kp n J I ! I
Page 3
-------
TEST RESULTS
CONTINUED
PROCEDURE 3
004 ern i no~f i on 3
CONCENTRflTION Ifioldn"3)
i i i i i i m i i i i i i m
Concent ro-f i on of controls o+ -fhe Qnd of experinonij
60. 61. . 62, ^
r en rn~m m m i i i
_________ from date
65. Kp I I I I I I Exp FT! on Do4 D 66. Holf-life lt,/2) I I I I I I do4a
Procedure 3
67. Kp Meon I I I I I "I Exp I I I doqs
"1
G8. Ho IF- 1 i fa I I I I l"71
o
o
I
CTi
o
o
o
i'MJC 4
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CS-6000
August, 1982
PHOTOLYSIS IN AQUEOUS SOLUTION IN SUNLIGHT
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
CS-600U
Contents
Daqe
I. NRED FOR ^HR TEST 1
IT. SCIEMTI t-'TC AS^C^S ?
A. Rationale for the Selection of the Test Method.... ?
1. Historical Discussion 2
2. Selection of the Test Method S
3. theoretical Aspects of the selected
Test Method 7
'3. Rationale for the Selection of the
Test Conditions 0
1. Soecial I.aboratorv Equipment 0
2. Parity of Water 11
1. .Sterilization 12
4. Concentration in Solution 12
5. Absorotion Spectrum 11
£>. oH Effects 14
7. Outdoor T^xnerimental Conditions IS
f]. Chemical Analysis of Solutions 16
Q. nrecautions Tor Losses Due to
processes Other Than Photolysis 17
C. ^est rv-ata Required 17
D. Statistical Analysis of Data IB
III. REFHRENCn.S 20
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CS-6000
PHOTOLYSIS IN AQUEOUS SOLUTION IN SUNLIGHT
I. NEED FOR THE TEST
The majority of the earth's surface is covered by water in
the form of oceans, seas, rivers, lakes, streams, ponds, etc. As
a result, chemicals are likely to enter aqueous media and can
then undergo transformation via direct aqueous Photolysis.
Direct aqueous photolysis represents the transformation of a
chemical substance by direct absorption of radiant enerqy
(sunlight) into new chemicals different from their precursors.
Chemical substances which are present in aqueous media photolyze
at different rates depending upon the solar irradiance an-1 the
chemical substance's molar extinction coefficient at each
wavelength of solar radiation and its photolysis quantum vield at
the wavelengths of concern. Chemical substances which photolyze
rapidly under environmental conditions have relatively short
Lifetimes in the environment. Consequently, the Agency's
assessment may focus on the degradation products to a qreater
extent than on the parent compound. On the other hand, if the
chemical substance is resistant to photolysis as well as to all
the other possible transformation processes, the assessment
should focus on the parent chemical.
A cost-effective aqueous photolysis test is needed to assess
quantitatively the transformation of chemical substances in
sunlight. The importance of direct photolysis in sunlight as a
transformation process of chemical substances in aqueous media in
the environment can be determined quantitatively from data on
photolysis rate constants and half-lives.
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CS-6UOO
The photolysis in aqueous solution test represents a
screening test to allow one to determine how rapidly photolysis
will take place in aqueous media under certain environmental
conditions. If the photolysis test data indicate that photolysis
is a relatively important transformation process and the initial
assessment indicates that there is a threat to the health of
humans and/or to the environment, then detailed tests mav he
required to obtain more precise aqueous nhotolvsis data over a
wide ranqe of environmental conditions. ^hese more detailed
tests will also he concerned with determining the identity and
fate of the transformation oroducts.
IT. SCIENTIFIC ASPECT0)
A. Rationale for the Selection of the Test Method
1. Historical Discussion
The scientific literature contains a number of nublications
dealing with the photolysis in solution of various chemical
substances. nnfortunatelv, for one or more reasons, most of the
data contained in the literature is of little or no use to E^A in
determining aqueous photolysis rate constants and half-lives.
Reasons for this include: (1) Many of the publications deal
primarily with the products which form from direct photolysis
reactions and the detailed mechanisms involved rather than
photolysis rates. (?) The publications qive no auantitative data
on the rates of photolysis under environmental conditions, i.e.,
-2-
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CS-6000
many researchers used sources of liqht which do not simulate
sunlight or performed experiments in solvents other than air-
satr. rated water. (3) ^ome studies are not based upon the
fundamental laws of photochemistry. (4) Manv publications renort
the effects of certain sensitizers which are environmentally
unimportant, or the effect of other chemicals present in
solution, on the chemical substance being studied. (5) Many of
the studies suffer from poor experimental design, overlooking the
controls necessary to make sure that photolysis and not
biodegradation, volatility, or other competing processes are
taking place to remove or transform the chemical substance. The
following paragraphs cite a few representative examples which
illustrate the points mentioned above, as well as the publication
which forms the basis for this Test Guideline.
A paper by Grunwell and "Rrickson (1973) deals with the
photochemistry of parathion. This report is of little relevance
in the evaluation of photolysis rates of chemical substances for
several reasons. The main emphasis of this work is in the
identification of major products formed by the photolysis of
parathion rather than in the measurement of rate constants and
half-lives. Photolysis was performed at three wavelengths (254,
300, and 350 nm), of which only the last two are environmentally
relevant. The chemical was dissolved in solutions of 20 percent
water and either 80 percent ethanol or tetrahydrofuran (by
weight), neither of which is environmentally relevant.
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CS-6000
A paper by Langford et al. (1973) claims environmental
relevance by working under environmental conditions. Radiation
of 350 nm as well as sunlight was used. Pure water and river
water were used as solvents. The authors presented no
quantitative data on the rate of photolysis o^ nitrilotriacetic
acid, the chemical they studied. Detailed quantitative
measurement of the photolysis rate constant and half-life, along
with adequate controls, would be necessary to make this research
useful for the purposes of this Test Guideline.
Benson et al. (1971) photolyzed chlordane with both mercurv
arc radiation and sunlight. Acetone was used as both a solvent
and photosensitizer. Sunlight photolysis was done only on the
pure compound applied as a thin layer under nuartz qlass. ^he
main emphasis of this work was to look at the chemical structure
of the reaction products. Considering that rate constants were
not measured, acetone was used as both a solvent and sensitizer,
and a mercury arc lamp was used as radiation source, this
publication has minimal applicability as a test method to screen
for photolysis rates.
Su and 7abik (1972) studied the photochemistrv of
arylamidine derivatives in distilled natural water. ^ high
pressure mercury arc, filtered to remove radiation below 286 nm
was used. Products of the reaction were determined but no
kinetic studies were performed and no rate constants or ha].-F-
lives were reported.
-A-
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CS-6000
'I1ancini (1978) presented a theoretical -Framework ^or the
first order photodecomposition of picloram in aqueous solution
and tested the framework using some experimental data on the
photolysis of picloram in sunlight. In reality, the Mancini
framework is only an empirical approach describing the rate of
photolysis of picloram in sunlight.
7,epp and Cline (1977) published a paper on direct nhotolvsis
in aqueous environments with equations for the determinations of
direct photolysis rates in sunlight. ^his paper avoids the
problems illustrated above and serves as a basis for the nronosed
photolysis in aqueous solution in sunlight test. ^hese equations
translate readily obtained laboratory data into rate constants
and half-lives for sunlight photolysis. ^hotolysis half-lives
can be calculated as a function of season, latitude, tine o^ dav,
depth in water bodies, and thickness of the atmospheric ozone
layer. Several published papers concerning the photolysis r>f
chemicals in sunlight have verified this method. (T^olfe et al .
1976, Smith et al. 1977, 1978, 7,epp et al. 1975, 1976).
2. Selection of the ^est Method
The method in this Test Guideline was developed From a
thorough review of the research literature on the exnerimental
determination of aqueous photolysis rate constants and bv talkinq
to researchers who have considerable experience in carrvinq out
these experiments. In the development of this ^est Guideline on
aqueous photolysis, the principles outlined by 7,epp and Cline
(1977) have been taken into account.
-5-
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CS-6000
The proposed test method for the measurement of direct solar
photolysis of a chemical substance in aqueous solution is based
upon four fundamental criteria. These criteria are: (1) "Hne
test method should be based upon the fundamentals of
photochemistry. (2) The test method should yield quantitative
data on direct photolysis rates of chemical substances in aqueous
media. (3) Sunlight should be used as the irradiation source
because of its obvious relevance as well as its low cost in
comparison to artificial light sources (7,epp 1980). (4) ^he test
method should be designed to account for degradation or chemical
losses by mechanisms other than photolysis. For example, the
experiments should be designed to account for or minimize
hydrolysis, biodegradation, and volatilization as factors in the
estimation of test substance losses. ^he proposed method has a
limitation since it does not measure sunlight intensity on the
sample during photolysis. A careful study has been made on the
use of insolation methods for measuring solar irradianoe [e.g.,
radiometry, photometry, and actinometry (Mill et al. 1981)1. The
most suitable method for measuring sunlight intensity was -Found
to be actinometry. ^his method has the advantage that it
conforms to the geometry of the reaction cell and measures the
actinic flux directly, under known sensitivity conditions. When
reference compounds (i.e., sunlight actinometers) are developed,
the proposed test will be modified to use simultaneous photolysis
of a chemical substance and an actinometer to evaluate sunlight
intensities on the sample. The modified procedure will quantify
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sunliqht photolysis of a chemical substance ah a specific time o^
year and latitude and will give a useful measure of seasonal
variation of photolvsis.
This photolvsis in aqueous solution mRst Guideline allows
one to determine how rapidly a chemical will photolvze in
sunliqht. ^uture ^est Guidelines need to he developed to obtain
detailed data on the direct photolysis of a chemical under a wide
varietv of environmental conditions. ^hese detailed tests will
provide improved translation of laboratory data into rate
constants and half-lives for photolysis in sunliqht as a ^unction
of season, latitude, time of day, depth in water bodies, ef^e-ct
of ether dissolved materials (e.q., clays or humic acids) or
suspended solids (e.q., sediments) in water, and the thickness o^
the ozone layer.
3. rpheoretical 7\spects of the Selected
Test Method
'T'he theorv of the method of 7,epp and Cline (1977) is briefly
discussed to show that the proposed test method is based upon the
fundamental criteria qiven in Section 11.^.2. T'hese discussions
lay the foundation for the proposed method, show how the method
can be used to obtain direct sunliqht photolysis rate constants
and half-lives, and indicate what test conditions must be
standardized in order to obtain meaningful aqueous Photolvsis
rate data.
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For the direct photolysis of a chemical substance, the rate
of decrease of the concentration of the chemical with time is
q i v e n by
= 4 I (1)
dt a
where C is the molar concentration of the chemical substance, t
is time, 4> is the photolysis quantum yield of the chemical
substance, and I is the absorbed radiation intensity. For the
direct sunlight photolysis of a chemical substance in dilute
aqueous solution (an absorbance of less than 0.10 units)9 in pure
water at shallow depth (less than 0.5 meter), the kinetic
expression for direct photolysis is
= 4 k C = k C , (2)
dt a P
where k equals Zk ,, the sum of the k , values for all
^ a A a A
wavelengths of sunlight that are absorbed by the chemical
substance, and k represents the photolysis rate constant in
sunlight (summed over all wavelengths of sunlight) in units of
reciprocal time. The expression in equation (2) is a first order
rate equation. Integration of equation (2) yields
a Zepp and Cline showed that the kinetics are first order under
the conditions of absorbance less than 0.02. Only a small
error (approximately 3%) is introduced in the proposed test
method assuming an absorbance of 0.1 and first order kinetics
is still applicable.
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C k t
, o p . _.
Iog10 = x- (3
1 C 2.30
where C is the molar concentration of chemical at time t during
photolysis and C is the initial molar concentration. It then
follows that the half-life for a first order equation is
t =T (4)
P
Thus, by measuring the initial molar concentration of a
chemical substance and measuring C as a function of the time, t,
it is possible to calculate both the sunlight photolysis rate,
k_, and the half-life, t , for that chemical substance.
B. Rationale for the Selection of the
Test Conditions
1. Special Laboratory Equipment
(1) A variable wavelength uv-visible absorption
spectrophotometer. This instrument, which must be
capable of measuring accurate absorbances to 0.10 or
less,is necessary for use in the aqueous photolysis
screening test to measure an accurate uv-visible
absorption spectrum of each chemical substance. There
are two reasons why accurate absorption spectra are
necessary. These are: one; the theory upon which this
Test Guideline is based, as discussed in detail in
Section II.A.3., is only applicable at low
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absorbances. therefore, a complete uv-visible
absorption spectrum of the aqueous solution of each
chemical substance, at the concentration at which it is
being tested, is required to make sure that these
conditions are met so that the photolysis experiments
are valid and two; the determination of which chemical
substances must be tested in the aqueous photolysis
screening test is based upon information obtained from
the chemical substance's uv-visible absorption spectrum
in aqueous solution. Test chemical substances which
have absorption maxima at wavelengths r>f 2^0 nm or
greater, as determined from the chemical substance's
uv-visible absorption spectrum, are applicable. ^he
iustification for the use of a chemical substance's
absorption spectrum as a means nf determining whether a
chemical substance should be tested for aqueous
photolysis is discussed in Section TT.R.5.
'2) In this test method, special reaction vessels are
necessary to contain the reaction solutions during
photolysis. Reaction vessels of 11 mm inside diameter
are recommended as they are inexpensive and easily
obtained in the form of culture tubes. For some
chemical substances it may be difficult to analyze the
concentration of the chemical substance in the small
volume present in 11 mm i.d. reaction vessels. ^or
such chemical substances the use of larger reaction
vessels is permissible as long as the pathlength is
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less than 0.5 meter (see Section II.A.3.). Reaction
vessels of either quartz or thin walled borosilicate
glass may be used. The absorption spectrum of the
chemical in aqueous solution as determined by OECD
Guideline No. 101 (OECD 1981), can be used to determine
the type of reaction vessel to be employed for these
photolysis experiments.
Ml reaction vessels must be capable of beinq sealed
(without the use of qrease) and must be filled as completely as
possible to prevent volatilization or other losses of the test
substance or water (see Section IT..R.9.). Grease must be avoided
because it might absorb or react with the substance being
tested. Caps lined with teflon inserts must be used to avoid
adsorption of hydrophobic chemicals.
2. Purity of Water
Pure water is used because dissolved impurities could
sensitize or otherwise affect the rate of photolysis. In
addition, the water needs to be sterile because bacteria may
consume or alter the chemical substance during the prolonged
periods of testing which may occur in the course of a rate
determination. Thus, pure water [e.g., ASTM Type IT A ( AS^M
1979)], is recommended in this Test Guideline. furthermore, it
is important that the water be saturated with air prior to
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preparation of the test and control solutions to simulate
environmental conditions. It is important that this air be
filtered through a 0.2 ym (pore size) filter to remove bacteria.
3. Sterilization
Sterilization is necessary to kill all bacteria and
therefore limit or reduce biodegradation which could interfere
with the photolysis rate determination. The presence of bacteria
in either the test solutions or controls may cause biodegradation
of the test substance. ^his may make concentration
determinations difficult and less accurate, calculations
difficult, and in general increase sources of error in the
experiment. Thus, it is extremely important to use aseptic
conditions in carrying out all photolysis experiments to minimize
biodegradation.
4. Concentration of Solution
Solutions of chemical substances used in this ^est Guideline
must be prepared at low concentrations in order to both
approximate environmental conditions and to allow first-order
kinetics assumptions to apply (see Section II.A.3.).
If the chemical substance is too difficult to dissolve in
pure form to permit reasonable handling and analytical
procedures, then test solutions can be prepared more easily from
a chemical dissolved in reagent grade acetonitrile. The final
acetonitrile concentration in the test solution should be no more
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than one volume percent in order to avoid acetonitrile solvent
effects (Smith et al. 1977, 1978). Acetonitrile was chosen as a
solvent as it is soluble in water, is non-nolar and thus
effective in dissolving many substances which are insoluble in
water, it does not absorb radiation over the wavelenath ranqe of
290 to SOOnm, and it causes minimal solvent effects or shifts in
atasorbance wavelength for test substances.
5. Absorption Spectrum
The absorption spectrum of the chemical substance is used as
a criterion for determining the necessity of performing this
aqueous photolysis test. Solar radiation reaching the earth's
surface has a sharp cutoff at a wavelength of approximatelv 290
nm (Leighton 1961, 7epn and Iline 1977). Photolysis does not
occur unless there is absorption of radiant energy. ^hus, if an
aqueous solution of a chemical substance does not absorb liqht at
a wavelength of 290 nm or greater, it will not undergo direct
photolysis under natural conditions. ^he uv-visible absorption
spectrum of a chemical substance in aqueous solution will give a
good indication of whether it would be useful to carrv out this
aqueous photolysis test.
If the absorption spectrum o^ a chemical substance, as
determined in OKCD Test Guideline No. 101 (OKCn, 1981), exhibits
an absorption maximum at a wavelenqth of 290 nm or greater, the
chemical substance may undergo direct photolysis in sunlight.
Thus, this Test Guideline is applicable to all chemicals which
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have an absorption maximum at a wavelength of 290 nm or
greater. If the chemical substance absorbs radiation only at
wavelengths appreciably below 290 nm, then it cannot undergo
direct photolysis in sunlight and therefore need not be tested.
\ few examples of classes of chemicals that do not need to be
tested in this ^est Guideline are alkanes, alkenes, and alkynes
because they only absorb uv radiation substantially below
290 nm. It is possible that some chemicals will absorb radiation
mainly at wavelengths below 290 nm but may have an absorption
tail that extends above 290 nm. Photolysis experiments should be
carried out for these chemicals.
6. pH Effects
The molecular structure of a chemical substance which
ionizes or nrotonates is a function of the pH. A.S a result, the
absorption spectrum and consequently the rate of photolysis may
change with pH. In general, the pH range of environmental
concern is from 5 to 9; hence, for chemical substances that
reversibly ionize or protonate (e.g., carboxylic acids, phenols,
and amines), the aqueous photolysis test should be carried out at
pHs of 5.0, 7.0, and 9.0. Since buffers could influence the rate
of photolysis, the recommended buffers for use in this T'Rst
Guideline were carefully chosen to be transparent to radiation
between 290 and 800 nm and are kept at very low concentrations to
avoid buffer effects which may cause transformation of the
substance by, for example, catalysis.
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7. Outdoor Experimental Conditions
It is important that the photolysis reaction vessels
containing the chemical substance be placed in an area free from
shade and reflections and on a black, non-reflectinq, background
to insure that they receive direct and sky radiation from the
sun. The reaction vessels should be tilted at 30° from
horizontal with the upper end pointing due north so that they
present a large surface area and minimum pathlength to the sun
and create minimal internal reflections.
It is recommended that the photolysis experiments be carried
out during the warm time of the year (i.e., May, June, July, and
August in the northern hemispheretemperature permitting). This
period of time was chosen because the solar intensity is a
maximum and consequently the rate of photolysis will be a
maximum. Thus, the kinetics of photolysis will be easier to
follow, especially for chemicals that photolyze slowly.
Furthermore, in many parts of the Hnited States, the temperature
falls below 0° C during the winter months. therefore, if the
photolysis experiments are carried out in the winter, the dilute
aqueous reaction solution would freeze, the tubes would break,
and the samples would be destroyed. It may be possible to avoid
this problem by placing the reaction tubes in a housing
transparent to the appropriate wavelenghs of radiation and
thermostated to control the temperature at 25 ± 5° C. Thus, with
this device it may be possible to carry out photolysis experi-
ments at any time of the year.
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8. Chemical Analysis of Solutions
The analytical techniques employed in the determination of
the concentration of the test substances are left to selection by
the sponsor. This is in recognition of the many different
techniques available and the practical advantage of being able to
make particular use of one of the properties of the substances;
e.g., the NMR or UV spectrum of the substance, or its
chromatographic behavior. Analytical techniques that permit the
determination of the test compound to the exclusion of impurities
or photolysis reaction products are recommended to the extent
practicable. Therefore, chromatographic techniques are
particularly desirable. Whenever practicable, an analytical
procedure should be used which has a precision of ±5%. The
specific technique which is utilized should be adequately
described.
9. Precautions for Losses r>ue to
Processes Other "Hian Photolysis
Undetected loss of a test substance through volatilization,
hydrolysis, or other processes during the course of the
photolysis experiment will result in the determination o^
excessively large rate constants for aqueous photolysis.
Therefore, for volatile chemical substances, it is important that
the reaction vessels and control vessels be -filled as completely
as possible and sealed in order to avoid evaporative lossre-s, ^o
correct for possible losses, control solutions of test substance,
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in darkened vessels, are placed side by side with the photolysis
vessels and the contents of the control vessels are analyzed at
the end of the experiment. Tn this way the loss rate for
processes other than photolysis may be determined and substracted
from the overall rate of disappearance of the chemical substance
to qive the corrected direct photolysis rate.
C. ^est Data Required
This Test Guideline is designed to obtain direct photolysis
rate constants and half-lives (of less than one day to 3 months)
for chemical substances in aqueous solution. ^hese data will be
used to assist in the determination of the environmental fate of
the chemical substance. Tt is important for each photolysis
experiment to keep a complete record of the time the vessels are
exposed to solar radiation including the times o^ sunrise and
sunset.. For each experiment, the initial concentrations (CQ) of
three test solutions and the mean value of the initial
concentrations are required. During the course of the
experiment, the concentration of chemical substance in each test
vessel must be reported at each time that it is measured. After
completion of each photolysis experiment the concentrations of
chemical substance in each test vessel and control and the mean
values of the concentrations of both test and control solutions
must be reported. These data are needed to calculate appropriate
photol.ysis rate data and to make sure that the test substance is
riot lost by other processes (e.g., biodegradation or volatility),
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or, if lost, is accounted for and that only the rate of
photolysis, kn, is determined. Using the value of k_, the half-
life (t^) can be calculated and reported.
The rate constant and half-life determined in this Test
Guideline are relevant to the day midway between the beginning
and the end of the experiment and thus represent an "average"
value for the test chemical substance during a certain period of
time. Therefore, all photolysis experiments should include the
duration of exposure. Since the rate of photolysis can vary with
a number of conditions, the latitude, dates of exposure, weather
conditions, and nH for all test and standard solutions, it is
important that these data be reported.
n. Statistical Analysis of Data
Several groups o^ researchers have published experimental
data on the determination of direct aqueous photolysis rate
constants and half-lives of chemical substances using solar
radiation (Smith et al. 1978, Wolfe et al. 1976, 7,epp et al.
1975, 1976, 1977). However, the precision in measuring the
sunlight rate constant has not been clearly established.
In general, when measuring direct photolysis rate constants
or half-lives by this Test Guideline, there are many factors
which will influence the values obtained. For the purposes of
this Test Guideline it is impossible to accurately evaluate the
effects of these factors on the rate constant data obtained.
Solar intensity may vary due to ozone layer thickness,
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meteorological conditions, tine of day and year, latit'ide, etc.
Therefore, no reliable precision can be stated at this time for
the determining the sunlight photolysis rate constant. ^or the
purpose of this Test Guideline and to minimize costs, the test
procedure is limited to the determination of triplicate samples
arid a statistical analysis of the data.
As stated above, the variability in the rate data is a
function of the variability in the solar intensity. As sunlight
actinorneters are developed to quantify solar intensities, the
test method will be modified to use simultaneous photolysis of a
chemical substance and an actinometer to evaluate sunlight
intensity on the sample.
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III. REFERENCES
ASTM 1970. American Society for Testinq Materials. Pronosod
standard practice for conducting aqueous photolysis test. Draft
document. Philadelphia, PA: ASTM.
Benson WR, Lombardo P, Eqrv U, Ross, RD, Rarron RP, Masthrook
DW, Hansen EA. 1971. Chlordane photoalteration products: Their
preparation and identification. J Aqr Food Chem 19:857-862.
Grunwell .TR, Erickson RH. 1973. Photolysis of parathion. New
products. J Aqr Food Chem 21:929-931.
Lanqford CH, Winqham M, Sastrin VS. 1973. Liqand photooxidation
in copper (IT) complexes of nitrilotriacetic acid. Environ Sci
and Technol 7:870-822.
Leiqhton PA. 1961. Photochemistry of air pollution. New York:
Academic Press, Inc.
Mancini JL. 1973. Analysis framework for ohotodecomposit ion in
water. Environ Sci and Technol 12:1274-1276.
Mill T, Davenport TE, nulin DE, Mahey WR and Bawol R. 1981.
Evaluation and optimisation of photolysis screeninq protocols.
U.S. Environmental Protection Aqency. EPA 560/5-81-003.
OECD. 1981. Orqanization for Economic Cooperation and
Development. OECD quidelines for testinq chemicals:
No. 101-UV-VI3 absorption spectra. Director of Information,
OECD; 2, rue Andre-Passal, 75775 Paris CEDEX 16, Prance.
Smith JH, Mabey WR, Bohonos N, Holt BR, Lee SS, Chou TW,
Bomberqer DC, Mill T. 1977. Environmental pathways of selected
chemicals in freshwater systems. Part I. Backqround and
experimental procedures. Athens, GA: n.S. Environmental
Protection Aqency. EPA 600/7-77-113.
Smith .TH, Mabey WR, Bohonos M, Holts BR, Lee SS, Chou ^T,
Bomberqer DC, Mill T. 1978. Environmental pathway of selected
chemicals in freshwater systems. Part II. Laboratory studies.
Athens, GA: U.S. Environmental Protection Aqency. EPA 600/
7-78-074.
Su CC, Zabik MJ. 1972. Photochemistry of bioactive compounds.
Photolysis of arvlamidine derivatives in water. J Aqr Food
Chem 20:320-323.
Wolfe ML, Zeop RG, Bauqhman ^L, Pincher RC, Gordon JA. 1976.
Chemical and photochemical transformation of selected pesticides
in aquatic systems. Athens, GA: U.S. Environmental Protection
Aqency. EPA 600/3-767-067.
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Zepp RG, Wolfe ML, Gordon JA, Baughman GL. 1975. Dynamics of
2,4-D esters in surface water. Hydrolysis, photolysis, and
vaporization. Environ Sci and Technol 9:1144-1150.
Zepp RG, Wolfe ML, Gordon JA, Fincher RC. 1976. Light-induced
transformations of raethoxchlor in aquatic systems. J Agr Food
Chem 24:727-733.
Zepp RG, Wolfe ML, Azarraga LV, Cox RH, Pape LW. 1977.
Photochemical transformation of the DDT and methoxychlor
degradation products, DDE and DMDE, by sunlight. Arch Environ
Contam Toxicol 6:305-314.
Zepp RG and Cline DM. 1977. Rates of direct photolysis in
aquatic environment. Environ Sci and Technol 11:359-366.
Zepp RG. 1980. Experimental approaches to environmental
photochemistry. The handbook of environmental chemistry.
O. Hutzinger, Editor. Springer-Verlag.
1HJ . S . GOVERNMENT PRINTING OFFICE: 1982-360-997/2219
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