United States Prevention, Pesticides EPA712-C-98-085
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3210
Modified SCAS Test
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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 guideline 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), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.3210 Modified SCAS test.
(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
796.3340 Ready Biodegradability: Modified OECD Screening Test and
OECD guideline 302 A Inherent Biodegradability: Modified SCAS Test.
(b) Introductory information—(1) Prerequisites, (i) Water solu-
bility.
(ii) The organic carbon content of the test material must be estab-
lished.
(2) Guidance information, (i) Information on the relative proportions
of the major components of the test material will be useful in interpreting
the results obtained, particularly in those cases where the result lies close
to the "pass level."
(ii) Information on the toxicity of the chemical may be useful to the
interpretation of low results and in the selection of appropriate test con-
centrations.
(3) Qualifying statements, (i) The method is only applicable to those
organic test materials which, at the concentration used in the test:
(A) Are soluble in water (at least 20 mg DOC/L (DOC = dissolved
organic carbon)).
(B) Have negligible vapor pressure.
(C) Are not inhibitory to bacteria.
(D) Do not significantly adsorb on glass surfaces.
(E) Are not lost by foaming from the test solution.
(ii) This test has been found suitable by the OECD Expert Group
Degradation/Accumulation for determining the inherent biodegradability of
organic chemicals under aerobic conditions.
(4) Recommendations. Test chemicals giving a result of greater than
20 percent loss of DOC in this test may be regarded as inherently bio-
degradable, whereas a result of greater than 70 percent loss of DOC is
evidence of ultimate biodegradability. The use of a compound specific ana-
lytical technique on 14 C-labeled test substance may allow greater sensitiv-
ity. In these last cases a lower level may be regarded as evidence of inher-
ent biodegradability.
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(5) Standard documents. This test guideline has been based on the
paper cited under paragraph (e)(l) of this guideline.
(c) Method—(1) Introduction, purpose, scope relevance, applica-
tion and limits of test, (i) The method is an adaptation of the Soap and
Detergent Association semicontinuous activated sludge (SCAS) procedure
for assessing the primary biodegradation of alkyl benzene sulfonate. The
method involves exposure of the chemical to relatively high concentrations
of microorganisms over a long time period (possibly several months). The
viability of the microorganisms is maintained over this period by daily
addition of a settled sewage feed.
(ii) Because of the long detention period (36 h) and the intermittent
addition of nutrients the test does not simulate those conditions experi-
enced in a sewage treatment plant. The results obtained with the test sub-
stance indicate that it has a high biodegradation potential, and for this
reason it is most useful as a test of inherent biodegradability.
(iii) Since the conditions provided by the test are highly favorable
to the selection and/or adaptation of microorganisms capable of degrading
the test compound, the procedure may also be used to produce accli-
matized inocula for use in other tests. The test is applicable to water solu-
ble, nonvolatile, organic chemicals that are not inhibitory to bacteria at
the test concentration.
(2) Reference substances. In some cases when investigating a new
substance reference substances may be useful; however, specific reference
substances cannot yet be recommended. Data on several compounds used
in ring tests are provided (see Table 1 under paragraph (d)(l)(ii) of this
guideline) primarily so that calibration of the method may be performed
from time to time and to permit comparison of results when another meth-
od is employed.
(3) Principle of the test method, (i) Activated sludge from a sewage
treatment plant is placed in an aeration (SCAS) unit. The test compound
and settled domestic sewage are added, and the mixture is aerated for
23 h. The aeration is then stopped, the sludge allowed to settle and the
supernatant liquor is removed. The sludge remaining in the aeration cham-
ber is then mixed with a further aliquot of test compound and sewage
and the cycle is repeated.
(ii) Biodegradation is established by determination of the DOC con-
tent of the supernatant liquor. This value is compared with that found for
the liquor obtained from a control tube dosed with settled sewage only.
(4) Quality criteria—(i) Reproducibility. The reproducibility of this
modification of the method based on removal of DOC has not yet been
established. When primary biodegradation is considered, very precise data
is obtained for materials that are extensively degraded. The results reported
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in paragraph (e)(l) of this guideline suggest 95 percent confidence limits
of less than ±3 percent, and this includes interlaboratory tests. As would
be expected, wider confidence limits are obtained for less biodegradable
materials.
(ii) Sensitivity. The sensitivity of the method largely depends on the
precision of the determination of DOC and the level of test compound
in the liquor at the start of each cycle. At the end of the aeration period
about 10 mg/L of DOC remain in the supernatant liquor of the control
experiment. Assuming that the DOC determination is within ±5 percent
and a level of 20 mg/L of carbon as test material is added at the start
of the aearation period, then the assessment of the extent of biodegradation
should be within ±6 percent for the range 80-100 percent biodegradation.
(iii) Specificity. The method is applicable to any nonvolatile, water-
soluble, organic compound.
(iv) Possibility of standardization. Since the method uses a feed of
real settled sewage, absolute standardization is not possible unless this feed
were replaced by an artificial one. However, since the method is designed
to give an indication of the biodegradability potential of a chemical and
is not a simulation test, such standardization is unnecessary.
(v) Possibility of automation. Automation of this method would be
possible but would be expensive. As the method is not labor intensive,
the exercise would offer few advantages.
(5) Description of the test procedure—(i) Preparations. (A) The
aeration units are cleaned and fixed in a suitable support. The air inlet
tubes are connected to the supply manifold. A small laboratory scale air
compressor is used to aerate the units, and the air is presaturated with
water to reduce evaporation losses from the units.
(B) A sample of mixed liquor from an activated sludge plant treating
predominantly domestic sewage is obtained. Approximately 150 mL of
the mixed liquor are required for each aeration unit.
(C) The organic carbon analyzer is calibrated using potassium hydro-
gen phthalate.
(D) Stock solutions of the test compounds are prepared: the con-
centration normally required is 400 mg/L as organic carbon which gives
a test compound concentration of 20 mg/L carbon at the start of each
aeration cycle if no biogradegradation is occurring.
(E) The organic carbon content of the stock solutions is measured.
(ii) Test conditions. A high concentration of aerobic microorganisms
is used, and the effective detention period is 36 h. The carbonaceous mate-
rial in the sewage feed is oxidized extensively within 8 h of the start of
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each aeration cycle. Thereafter, the sludge respires endogeneously for the
remainder of the aeration period, during which time the only available
substrate is the test compound unless this is also readily metabolized.
These features, combined with daily reinoculation of the test when domes-
tic sewage is used as the medium, provide highly favorable conditions
for both acclimatization and biodegradation.
(iii) Performance of the test. (A) A sample of mixed liquor from
a suitable activated sludge plant is obtained and aerated during transpor-
tation to the laboratory. Each aeration unit is filled with 150 mL of mixed
liquor and the aeration is started. After 23 h, aeration is stopped, and the
sludge is allowed to settle for 45 min. The tap is opened and 100 mL
of the supernatant liquor withdrawn. A sample of settled domestic sewage
is obtained immediately before use, and 100 mL are added to the sludge
remaining in each aeration unit. Aeration is started anew. At this stage
no test materials are added, and the units are fed daily with domestic sew-
age only until a clear supernatant liquor is obtained on settling. This usu-
ally takes up to 2 weeks, by which time the DOC in the supernatant liquor
at the end of each aeration cycle should be less than 12 mg/L.
(B) At the end of this period the individual settled sludges are mixed,
and 50 mL of the resulting composite sludge are added to each unit.
(C) Settled sewage (100 mL) is added to the control units and
95 mL plus 5 mL of the appropriate test compound stock solution
(400 mg/L) to the test units. Aeration is started again and continued for
23 h. The sludge is then allowed to settle for 45 min and the supernatant
drawn off and analyzed for DOC.
(D) The fill and draw procedure under paragraph (c)(5)(iii)(A) of this
guideline is repeated daily throughout the test.
(E) Before settling it may be necessary to clean the walls of the units
to prevent the accumulation of solids above the level of the liquid. A sepa-
rate scraper or brush is used for each unit to prevent cross contamination.
(F) The DOC in the supernatant liquors is determined daily, although
less frequent analysis is permissible. Before analysis the liquors are filtered
through washed 0.45 (im membrane filters and certifuged. Temperature
of the sample must not exceed 40 °C while it is in the centrifuge.
(G) The length of the test for compounds, showing little or no biodeg-
radation is indeterminate, but experience suggests that this should be at
least 12 weeks.
(d) Data and reporting—(1) Treatment of the results, (i) The re-
sults of analysis for DOC in the supernatant liquors of the test units and
the control units are plotted against time. As biodegradation is achieved
the level found in the test will approach that found in the control. Once
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the difference between the two levels is found to be constant over three
consecutive measurements, three further measurements are made and the
percentage biodegradation of the test compound is calculated by the fol-
lowing equation:
Percent biodegradation = 100 [OT - (Ot - OC)]/OT
where
OT = concentration of test compound as organic carbon added to the settled
sewage at the start of the aeration period.
Ot = concentration of DOC found in the supernatant liquor of the test at the
end of the aeration period.
Oc = concentration of DOC found in the supernatant liquor of the control.
(ii) The level of biodegradation is therefore the percentage elimination
of organic carbon, under the following Table 1:
Table 1—Examples of Results of SCAS Test on Various Compounds Used in the OECD/EEC
Ring Test
Test compound
4-Acetylaminobenzene sulfonate
Tetrapropylenebenzene sulfonate
4-Nitrophenol
Diethylene glycol
Aniline
0T (mg/L)
17.2
17.3
16.9
16.5
16.9
Ot - Oc(mg/L)
2.0
8.4
0.8
0.2
1.7
Percent-
age bio-
degrada-
tion/bio-
elimina-
tion
85.0
51.4
95.3
98.8
95.9
Duration of test 40 days.
Results Found for Cyclopentane Tetracarboxylate
O (mn/LI CO - 01 (mn/LI Percentage biodegradation/
UT (,mg/i_;
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(e) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) A Procedure and Standards for the Determination of the
Biodegradability of Alkyl Benzene Sulfonate and Linear Alkylate
Sulfonate. Journal of the American Oil Chemists Society 42:986-993
(1965).
(2) [Reserved]
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