United States Prevention, Pesticides EPA712-C-96-031
Environmental Protection and Toxic Substances August 1996
Agency (7101)
&EPA Product Properties
Test Guidelines
OPPTS 830.7050
UV/Visible Absorption
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This document is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks or paper copies.
This guideline is available in ASCII and PDF (portable document format)
from the EPA Public Access Gopher (gopher.epa.gov) under the heading
"Environmental Test Methods and Guidelines."
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OPPTS 830.7050 UV/Visible absorption.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are the OPPT guideline under 40 CFR
796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra and
OECD guideline 101 UV-VIS Absorption Spectra.
(b) Introductory information—(1) Guidance information, (i) Mo-
lecular formula.
(ii) Structural formula.
(2) Standard documents. The spectrophotometric method is based
on national standards and consensus methods which are applied to measure
the absorption spectra.
(c) Method—(l)(i) Introduction, purpose, scope, relevance, appli-
cation and limits of test. (A) The primary environmental purpose in deter-
mining the ultraviolet/visible (UV/VIS) absorption spectrum of a chemical
compound is to have some indication of the wavelengths at which the
compounds may be susceptible to photochemical degradation. Since photo-
chemical degradation is likely to occur in both the atmosphere and the
aquatic environment, spectra appropriate to these media will be inform-
ative concerning the need for further persistence testing.
(B) Degradation will depend upon the total energy absorbed in spe-
cific wavelength regions. Such energy absorption is characterized by both
molar absorption coefficient (molar extinction coefficient) and band width.
However, the absence of measurable absorption does not preclude the pos-
sibility of photodegradation.
(ii) Definitions and units. The UV-VIS absorption spectrum of a so-
lution is a function of the concentration, Ci, expressed in moles per liter
(mol/L), of all absorbing species present; the path length, d, of the
spectrophotometer cell, expressed in centimeters; and the molar extinction
coefficient, 8i, of each species. The absorbance, A, of the solution is then
given by:
For a resolvable absorbance peak, the band width X is the wavelength
range, expressed in nanometers (nm = 10-9m), of the peak at half the
absorbance maximum.
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(iii) Reference substances. (A) The reference substances need not
be employed in all cases when investigating a new substance. They are
provided primarily so that calibration of the method may be performed
from time to time and to offer the chance to compare the results when
another method is applied.
(B) Reference compounds appropriate for the calibration of the sys-
tem are:
(7) Potassium dichromate (in 0.005 mol/L, tbSCU solution) from
J.A.A. Ketelaar, under paragraph (e)(2) of this guideline:
loge 3.56 3.63 3.16 3.50
Mn nm 235 257 313 350
(2) Fluoranthene (in methanol) from C.R.C. Atlas of Spectral Data,
under paragraph (e)(3) of this guideline:
loge 4.75 4.18 4.73 3.91 3.92
Mn nm 237 236 288 339 357
(3) 4-nitrophenol (in methanol) from C.R.C. Atlas of Spectral Data,
under paragraph (e)(3) of this guideline:
log e 3.88 4.04
X in nm 288 311
Refer to paragraph (e)(l) of this guideline for more information.
(iv) Principle of the test method. This method utilizes a double-
beam spectrophotometer which records only the absorption differences be-
tween the blank and test solutions to give the spectrum of the chemical
being tested.
(v) Quality criteria—reproducibility and sensitivity. (A) Reproduc-
ibility and sensitivity, need not be measured directly. Instead, the accuracy
of the system in measuring the spectra of reference compounds will be
defined so as to assure appropriate reproducibility and sensitivity. It is
preferable to use a recording double-beam spectrophotometer to obtain the
UV/vis spectrum of the test compound. Such an instrument should have
a photometric accuracy of ±0.02 units over the absorbance range of 0 to
2 units. It should be capable of recording absorbances at wavelengths of
200 to 750 nm with a wavelength accuracy of ±0.5 nm. The cells em-
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ployed with the instrument must necessarily be transparent over this wave-
length range and must have a path length determined to within 1 percent.
To ensure that the instrument is performing satisfactorily, spectra for test
solutions of K2Cr2Oy (for absorbance accuracy) and holmium glass (for
wavelength accuracy) should be run periodically.
(B) In the event that a recording double-beam instrument is not avail-
able, it will be necessary to determine the absorbance of the test solution
in a single-beam instrument at 5-nm intervals over the entire wavelength
range and at 1-nm intervals where there are indicated absorbance maxima.
Wavelength and absorbance tests should be done as with the double-beam
instrument.
(2) Description of the test procedure—(i) Preparation of test solu-
tions. (A) Solutions should be prepared by accurately weighing an appro-
priate amount of the purest form of the test substance available. This
should be made up in a concentration which will result in at least one
absorbance maximum in the range 0.5 to 1.5 units.
(B) The absorption of a compound is due to its particular chemical
form. It is often the case that different forms are present, depending on
whether the medium is acidic, basic, or neutral. Consequently, spectra
under all three conditions are required where solubility and concentration
allow. Where it is not possible to obtain sufficient concentrations in any
of the aqueous media, a suitable organic solvent should be used (methanol
preferred).
(C) The acid medium should have a pH of less than 2, and the basic
medium should be at least pH 10. The solvent for the neutral solution,
and for preparing the acidic and basic ones, should be distilled water,
transparent to UV radiation down to 200 nm. If methanol must be used,
acidic and basic solutions can be prepared by adding 10 percent by volume
of HC1 or NaOH in aqueous solution ([HC1], [NaOH] = 1 mol/L).
(D) In theory, all chemical species other than that being tested are
present in both beams and would therefore not appear in the recorded spec-
trum of a double-beam instrument. In practice, because the solvent is usu-
ally present in great excess, there is a threshold value of wavelength below
which it is not possible to record the spectrum of the test chemical. Such
a wavelength will be a property of the solvent or of the test medium.
In general, distilled water is useful from 200 nm (dissolved ions will often
increase this), methanol from 210 nm, hexane from 210 nm, acetonitrile
from 215 nm, and dichloromethane from 235 nm.
(ii) Blank solutions. A blank must be prepared containing the solvent
and all chemical species other than the test chemical. The absorption spec-
trum of this solution should be recorded in a manner identical to that of
the test solution and preferably on the same chart. This "baseline" spec-
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trum should never record an absorbance reading varying more than ±0.05
from the nominal zero value.
(iii) Cells. Cell pathlengths are usually between 0.1 cm and 10 cm.
Cell lengths should be selected to permit recording of at least one maxi-
mum in the absorbance range of 0.5 to 1.5 units. Which set of cells should
be used will be governed by the concentration and the absorbance of the
test solution as indicated by the Beer-Lambert law. The cells should be
transparent over the range of the spectrum being recorded, and the path-
lengths should be known to an accuracy of at least 1 per cent. Cells should
be thoroughly cleaned in an appropriate manner (chromic acid is useful
for quartz cells) and rinsed several times with the test or blank solutions.
(iv) Performance of the test. Both cells to be employed should be
rinsed with the blank solution before filling. The instrument should be
set to scan at a rate appropriate for the required wavelength resolution
and the spectrum of the blank recorded. The sample cell should then be
rinsed and filled with the test solution and the scanning repeated, pref-
erably on the same chart, to display the baseline. The test should be carried
out at 25 ° C.
(d) Data and reporting — (1) Treatment of results, (i) The molar
absorption coefficient, e, should be calculated for all absorbance maxima
of the test substance. The formula for this calculation is:
8 = A/Ci x d
where the quantities are as defined at paragraph (d)(l)(ii) of this guideline.
(ii) For each peak which is capable of being resolved, either as re-
corded or by extrapolated symmetrical peaks, the bandwidth should be re-
corded.
(2) Test report, (i) The report should contain a copy of each of the
three spectra (three pH conditions). If neither water nor methanol solutions
are feasible, there will be only one spectrum. Spectra should include a
readable wavelength scale. Each spectrum should be clearly marked with
the test conditions.
(ii) For each maximum in each spectrum, the 8 value and bandwidth
(when applicable) should be calculated and reported, along with the wave-
length of the maximum. This should be presented in tabular form.
(iii) The various test conditions should be included, such as scan
speed, the name and model of the spectrophotometer, the slit width (where
available), cell type and path length, the concentrations of the test sub-
stance, and the nature and acidity of the solvent medium. A recent test
spectrum on appropriate reference materials for photometric and wave-
length accuracy should also be submitted (see paragraph (d)(2)(i) of this
guideline).
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(e) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Milazzo, G. et al., Analytical Chemistry, 149:711 (1977).
(2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin 8
(Cambridge, 1955).
(3) Chemical Rubber Company, Atlas of Spectral Data (Cliffland,
Ohio).
(4) Organization for Economic Cooperation and Development, Guide-
lines for The Testing of Chemicals, OECD 101, UV-VIS Absorption Spec-
tra, OECD, Paris, France.
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