United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–298

April 1996

Fate, Transport and

Transformation Test

Guidelines

OPPTS 835.1110

Activated Sludge

Sorption Isotherm

‘‘Public Draft’’
i

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Public Draft Access Information: This draft guideline is part of a

series of related harmonized guidelines that need to be considered as a

unit. For copies: These guidelines are available electronically from the

EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the

OPP Public Docket at (703) 305–5805 or by e-mail:

guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office

of Pesticide Programs, Field Operations Division (7506C), Environmental

Protection Agency, 401 M St. SW., Washington, DC 20460. In person:

bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending

electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp:

fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks

or paper copies. This guideline is also available electronically 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 835.1110 Activated sludge sorption isotherm.

(a) Scope—(1) Applicability. This guideline is intended to meet testing 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 harmonized OPPTS test guideline are the articles referenced under paragraphs

(f)(1) through (f)(8) 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, aqueous, and vapor phases. If a chemical compound is sorbed to sludge biomass, it may be removed from the system along with other solids by clarification. 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 unaffected. Information on sorption potential is needed to assess the possibility for the removal of chemical compounds in biological wastewater treatment systems. This test guideline describes a procedure for the determination of the sorption potential of activated sludge solids for removal of

specific chemical compounds.

(c) Discussion. (1) This guideline describes a procedure for measuring 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 contacted with a measured quantity of the sorbent for a time period sufficient

to attain sorption equilibrium. Data are obtained for various sorbent dosages. The amount of compound remaining in solution is determined analytically 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 isotherm. 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 . Ce

1/n

where

C0 = initial concentration of the chemical compound in solution expressed in grams per liter

Ce = final concentration of the chemical compound in solution expressed in grams per liter

X = amount of test compound sorbed =

[(C0 × solution volume (L)) – (Ce × 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 isotherm. In order to determine K and 1/n graphically, the Freundlich equation 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 values for other chemicals whose behavior in activated sludge treatment systems is documented.

(4) A sorption isotherm is dependent on the conditions under which

it is determined. Sorption can be greatly affected by changes in temperature, pH, and test chemical concentration. Therefore, each of these parameters 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)(1) through (f)(8) of this

guideline, served as the basis for this test guideline. For additional information on conducting sorption isotherm experiments, the reader is referred

to OPPTS 835.1220, Sediment and Soil Sorption Isotherm. The soil and

colloid chemistry literature and the analytical chemistry literature substan-
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tiate 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 microorganisms 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.

(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 concentrated 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 isotherm test are known, the volume of sludge that needs to be collected

can be determined. About 2 to 3× the volume calculated should be collected 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 collected, 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

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
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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 desiccated 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 accomplished 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 dosages. 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 sample 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 individual isotherm reaction vessels to yield final concentrations of sludge solids

that will achieve varying degrees of sorption of the test compound. Test

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.
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(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 stirring 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 vessels.

(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 continuously while samples are being collected.

(B) Centrifuge the contents of each tube at 2,000 × g for 5 min in

order to provide adequate separation of solid and liquid phases for subsequent 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 μm 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.

(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.
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(5) Analysis of test data. (i) After the isotherm test has been performed and analysis for test compound is complete, tabulate the data generated using the following format, representative of a Freundlich model:

Data Collection Format

M Ce X X/M

Activated sludge Test chemical Test chemical sorbed

[(C0 × sol’n volume in L) –

(Ce × sol’n volume in L)] Grams test chemical

sorbed per gram sludge mass in grams g/L

mass in grams

................................ ....................................... ........................................... .......................................

................................ ....................................... ........................................... .......................................

................................ ....................................... ........................................... .......................................

................................ ....................................... ........................................... .......................................

(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 conducted.

(2) Detailed description of the analytical techniques used in the recovery and quantitative analysis for test compound.

(3) Amount of test compound dosed (C0 × solution volume), and the

amount recovered in each reaction vessel (Ce × 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, calculations for volume sampled, lyophilization vendor, sample custody, description of the lyophilized sludge, desiccation time.

(8) Any unusual observations made during the experiments.

(f) References. The following references should be consulted for additional background material on this test guideline.
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(1) Aharonson, N. and U. Kafkafi. Adsorption, mobility and persistence 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 mobility 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 affected 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 movement in soils. Weed Science 23:454–457 (1975).