United States Prevention, Pesticides EPA712-C-98-298
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.1110
Activated Sludge
Sorption Isotherm
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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 835.1110 Activated sludge sorption isotherm.
(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 material used in developing this har-
monized OPPTS test guideline are the articles referenced under paragraphs
(f)(l) through (f)(10) of this guideline.
(b) Introduction. The sorption of chemical compounds to activated
sludge biomass in biological wastewater treatment systems is an important
process that affects the distribution of the compounds between solid, aque-
ous, and vapor phases. If a chemical compound is sorbed to sludge bio-
mass, it may be removed from the system along with other solids by clari-
fication. If a compound is not sorbed, it will remain in the aqueous phase
where it is subject to removal via biodegradation, chemical interactions,
and/or volatilization. A nonsorbing, nonbiodegradable, noninteracting,
nonvolatile compound will pass through a biological treatment system un-
affected. Information on sorption potential is needed to assess the possibil-
ity for the removal of chemical compounds in biological wastewater treat-
ment systems. This test guideline describes a procedure for the determina-
tion of the sorption potential of activated sludge solids for removal of
specific chemical compounds.
(c) Discussion. (1) This guideline describes a procedure for measur-
ing the extent to which a chemical compound distributes itself between
activated sludge as the sorbent and water as the solvent. The equation
describing that relationship is called a sorption isotherm. The chemical
compound solubilized at a constant concentration in the solvent is con-
tacted with a measured quantity of the sorbent for a time period sufficient
to attain sorption equilibrium. Data are obtained for various sorbent dos-
ages. The amount of compound remaining in solution is determined analyt-
ically and the amount of compound sorbed is calculated for each sorbent
dosage. A range of sorbent dosages is selected to achieve optimal spread
of the data and to reflect biomass concentrations in an actual wastewater
treatment system.
(2) There are several common models for describing a sorption iso-
therm. Historically, each model has been based on either an empirical or
a theoretical equation. The three most common models are the Freundlich,
the Langmuir, and the Brunauer, Emmett, and Teller (BET). The
Freundlich model is most widely used to describe sorption in dilute
wastewater systems and is employed in this test guideline.
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(i) The basic form of the Freundlich equation is as follows:
X/M = K • Ce1/n
where
Co = initial concentration of the chemical compound in solution ex-
pressed in grams per liter
Ce = final concentration of the chemical compound in solution ex-
pressed in grams per liter
X = amount of test compound sorbed =
[(Co x solution volume (L)) - (Ce x solution volume (L))]
M = mass of sorbent expressed in grams
K = Freundlich sorption coefficient
1/n = exponent where n is a constant
(ii) K and 1/n are the sorption constants and are unique for each iso-
therm. In order to determine K and 1/n graphically, the Freundlich equa-
tion is plotted on a log-log scale to yield a linear relationship. X/M is
plotted along the Y-axis and Ce along the X-axis. The data will fit the
logarithmic form of the Freundlich equation, as follows:
log X/M = log K + 1/n log Ce
(iii) K is the X/M intercept when Ce = 1, and 1/n is the slope of
the line when plotted. The logarithmic plot of the equation is termed the
isotherm plot.
(3) In order to assess the potential for relative sorptive removal of
the chemical compound, the values K and 1/n are compared with the val-
ues for other chemicals whose behavior in activated sludge treatment sys-
tems is documented.
(4) A sorption isotherm is dependent on the conditions under which
it is determined. Sorption can be greatly affected by changes in tempera-
ture, pH, and test chemical concentration. Therefore, each of these param-
eters must be held constant during a sorption isotherm test.
(d) Sorption isotherm test—(1) General, (i) The papers by
Aharonson and Kafkafi (1975), Goring and Hamaker (1972), Harvey
(1974), Lieberman (1986), Murray (1975), Saltzman (1972), Weber
(1971), and Wu (1975), under paragraphs (f)(l) through (f)(8) of this
guideline, served as the basis for this test guideline. For additional infor-
mation on conducting sorption isotherm experiments, the reader is referred
to OPPTS 835.1220, Sediment and Soil Adsorption/Desorption Isotherm.
The soil and colloid chemistry literature and the analytical chemistry lit-
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erature substantiate the experimental conditions and procedures specified
in this guideline as accepted, standard procedures. These procedures have
been modified slightly to allow the use of activated sludge solids.
(ii) Any procedure designed to determine the sorption potential of
activated sludge for the test compound should use lyophilized, desiccated
sludge solids as the sorbent. Lyophilization freeze-dries the microorga-
nisms in sludge and allows the sorption potential to be measured without
interference by biodegradation of the test compound. After lyophilization
many of the microorganisms are still viable, but desiccation (drying in
an oven) should inactivate the remaining microorganisms. The flocculation
and settling properties of lyophilized, desiccated sludge resemble those of
active sludge biomass upon rehydration. Photomicrographs of this material
reveal the presence of structurally intact microorganisms (see the report
referenced in paragraph (f)(9) of this guideline). Furthermore, sludge sub-
jected to this treatment has been shown to have a sorption capacity similar
to that of viable sludge solids (see paragraph (f)(10) of this guideline).
(iii) Lyophilization is an expensive process with a significant capital
investment. For the purpose of this test guideline, it is assumed that the
lyophilization process will be contracted to a qualified vendor, for which
cost should be less significant (less than $500 for several grams of sludge).
(2) Activated sludge collection and preparation, (i) The activated
sludge to be used as a sorbent should be collected from the most con-
centrated source available within an activated sludge treatment process;
i.e., the return activated sludge (RAS) line. If the solids concentration of
the sludge to be collected and the amount of sorbent required for the iso-
therm test are known, the volume of sludge that needs to be collected
can be determined. About 2 to 3x the volume calculated should be col-
lected as a safety measure. The shelf life of lyophilized sludge has not
been determined systematically, but based on empirical observation it
should be approximately 6 mo. As a result, excess sludge may be col-
lected, prepared, and held for future testing as outlined under paragraphs
(d)(2)(ii) through (d)(2)(v) of this guideline.
(ii) The sludge solids to be lyophilized need to be separated from
the wastewater present in the sludge. In the first step collected sludge is
allowed to settle for 15 to 30 min before the supernatant is decanted. Care
must be taken to remove only the supernatant and leave the solids.
(iii) Further mechanical separation (e.g. centrifugation) of the solids
from the water is required in order to achieve a sludge suitable for
lyophilization. To accomplish this, settled solids are placed in a centrifuge
vessel and centrifuged for 5 min. After centrifugation the supernatant is
decanted and the sludge solids remain in the centrifuge vessel.
(iv) Washing of the solids is recommended to remove any color or
matrix materials. Washing can be accomplished by filling the centrifuge
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vessel with laboratory-grade water and suspending the sludge solids in the
water, followed by centrifugation and decantation of the supernatant.
Sludge solids should be washed and centrifuged 3 times. They can be
stored at 4 °C until lyophilization can be undertaken by a vendor, but
sludge should not be held for longer than 24 hr prior to starting
lyophilization because changes in the sludge are likely to occur after this
even when the sludge is held at 4 °C.
(v) After the lyophilization process is complete, the solids will be
in the form of a dry cake. This cake should be broken into a dry powder
by gently passing it through a mesh screen. The powder should be des-
iccated at 103 °C for 3 h or more prior to use as a sorbent.
(3) Procedure—(i) Test conditions. (A) The isotherm test should
be run at constant temperature and pH. Temperature control can be accom-
plished using a bath or constant temperature room. pH should be controlled
using buffers. Preliminary tests should be run to assure that there are no
interactions between test compound and buffer (such as complexation with
the buffer) that could affect sorption of the test compound. This test is
accomplished by dosing the test compound into a mixture of buffer and
clean water at the isotherm test's target concentration. The dosed mixture
should be mixed for a time period equal to the anticipated duration of
the isotherm test. Analytical tests and visual observation of the test
compound in the buffered mixture should be performed to verify that no
chemical interactions or buffer-catalyzed degradation of the test compound
has occurred.
(B) A sorption isotherm plot can be generated with two sorbent dos-
ages. However, the accuracy of the isotherm plot is increased if more data
points are generated. Generally, six to eight points are needed to produce
an accurate plot.
(C) Normal quality assurance/quality control practices (QA/QC)
should be employed when conducting the isotherm test in order to assure
that valid data are generated. A test solution control and a sorbent control
should be included as part of the isotherm test. The test solution control
consists of a sample of the test compound dosed at the target concentration
into buffered water with no sorbent present. This control is used to verify
the initial concentration of test compound for each reaction vessel and
to indicate if any degradation or interaction of the test compound with
buffer occurs during the test period. The sorbent control consists of a sam-
ple of the sorbent in buffered water without test compound. Analysis of
this sample will indicate if any color or matrix interferences were caused
by the sorbent.
(ii) Equilibration. (A) Weigh sludge solids into six to eight individ-
ual isotherm reaction vessels to yield final concentrations of sludge solids
that will achieve varying degrees of sorption of the test compound. Test
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vessel volume is not specified in this test guideline because it is not critical
as long as the vessel is large enough to allow for good mixing. The vessels
should be of a constant and known volume.
(B) Place a stirring bar or other mixing device in each reaction vessel.
Contents of test vessels must not be mixed by bubbling gas through them
because this could remove test compound by stripping.
(C) If necessary, bring the buffered test substance/solids mixtures in
the reaction vessels up to the correct volume with measured quantities
of laboratory-grade water and buffer.
(D) Stir mixtures in the reaction vessels for 0.5 h. Stirring should
be vigorous enough to keep the sorbent in suspension without being so
vigorous as to break it up.
(E) Measure the pH and temperature of the contents of each reaction
vessel while stirring to verify the target conditions for the test.
(F) Dose each reaction vessel and the test solution control while stir-
ring by pipeting in a calculated volume of test compound. This will bring
the solution in the vessel up to a final specified volume and test compound
concentration. The dosing process should be completed quickly and with
precision, in order to achieve a uniform starting time for all reaction ves-
sels.
(G) Stir the test solutions until sludge and test compound have
reached equilibrium. How long this takes may have to be determined by
trial and error.
(iii) Centrifugation. (A) At the end of the stirring period, transfer
a representative sample of the mixture from each reaction vessel into a
clean centrifuge tube. The volume of mixture that needs to be collected
is determined by the analytical method employed to detect and quantify
the test compound. Contents of the reaction vessels should be stirred con-
tinuously while samples are being collected.
(B) Centrifuge the contents of each tube at 2,000 x g for 5 min in
order to provide adequate separation of solid and liquid phases for subse-
quent analysis.
(4) Analysis for test compound, (i) After centrifugation, remove a
volume of the supernatant sufficient for analysis from each tube, pass it
through a 0.45 (im pore-size filter previously shown not to sorb test
compound, and place the filtrate in a clean sample bottle.
(ii) Analyze the aqueous phase for the presence and amount of test
compound using an appropriate analytical method. QA/QC procedures
should be employed for verification of analytical precision.
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(iii) If the analysis for test compound must be delayed for any reason
after the samples have been filtered, store samples at 0 to 4 °C. Samples
should not be stored for more than 24 h before analysis.
(5) Analysis of test data, (i) After the isotherm test has been per-
formed and analysis for test compound is complete, tabulate the data gen-
erated using the following format, representative of a Freundlich model:
Data Collection Format
M
Activated sludge
Ce
Test chemical
n/l
y/L
X
Test chemical sorbed
(Ce x sol'n volume in L)]
mass in grams
X/M
Grams test chemical
sorbed per gram sludge
(ii) Plot X/M vs. Ce on log-log paper, and calculate the line of best
fit to yield the factors K and 1/n for the isotherm.
(e) Reporting. Report the following information:
(1) Test conditions: Temperature and pH at which the test was con-
ducted.
(2) Detailed description of the analytical techniques used in the recov-
ery and quantitative analysis for test compound.
(3) Amount of test compound dosed (Co x solution volume), and the
amount recovered in each reaction vessel (Ce x solution volume).
(4) Volume of each reaction vessel and total volume of the sorbent/
sorbate mixture.
(5) QA/QC data such as duplicate analyses, background interferences,
spikes, matrix spikes, etc.
(6) Graphical plots of log X/M as a function of log Ce, and the values
of K and 1/n determined from the plots.
(7) Sludge solids information: Sampling location, observations, cal-
culations for volume sampled, lyophilization vendor, sample custody, de-
scription of the lyophilized sludge, desiccation time.
(8) Any unusual observations made during the experiments.
6
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(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Aharonson, N. and U. Kafkafi. Adsorption, mobility and persist-
ence of thiabendazole and methyl-2-benzimidazole carbamate in soils.
Journal of Agricultural and Food Chemistry 23:720-724 (1975).
(2) Goring, C.A. and J.W. Hamaker, Eds. Organic chemicals in the
soil environment, vol. I and II. Marcel Dekker, NY (1972).
(3) Harvey, R.G. et al. Soil adsorption and volatility of dinitroaniline
herbicides. Weed Science 22:120-124 (1974).
(4) Lieberman, R.J. Technical status report for adsorption of azo dyes
onto activated sludge solids. Report prepared under EPA contract no. 68-
03-3183. USEPA Risk Reduction Engineering Laboratory, Cincinnati, OH
(1986).
(5) Murray, D.S. et al. Comparative adsorption, desorption, and mo-
bility of dipropetryn and prometryn in soil. Journal of Agricultural and
Food Chemistry 23:578-581 (1973).
(6) Saltzman, S.L. et al. Adsorption, desorption of parathion as af-
fected by soil organic matter. Journal of Agricultural and Food Chemistry
20:1224-1226(1972).
(7) Weber, J.B. Model soil system, herbicide leaching, and sorption.
Weed Science 19:145-160 (1971).
(8) Wu, C.H. et al. Napropamide adsorption, desorption, and move-
ment in soils. Weed Science 23:454-457 (1975).
(9) USEPA. Prediction of the Fate of Organic Chemicals in Activated
Sludge Wastewater Treatment Processes. EPA/600/2-85-102 (1985).
(10) Hopkins, B.T. et al. Activated sludge adsorption methodology:
effect of biomass inactivation and incubation conditions. Presented at the
Society of Environmental Chemistry and Toxicology (SETAC) 13th An-
nual Meeting, November 8-12, Cincinnati, OH. Abstract no. WA4A18
(1992).
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