United States Prevention, Pesticides EPA712-C-98-301
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3220
Porous Pot 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. I36etseq.).
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 835.3220 Porous pot 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 material used in developing this har-
monized OPPTS test guideline are the articles presented at paragraph (o)
of this guideline.
(b) Introduction. (1) This test is intended to simulate processes in
the aeration basin of the activated sludge sewage treatment process and
therefore give a measure of the extent of biodegradation or removal likely
to occur during sewage treatment.
(2) Information on the treatability, biodegradability, and/or removabil-
ity of a test compound can be assessed by either dissolved organic carbon
(DOC) measurements, specific chemical analysis, or a combination of the
two. DOC measurements, relative to the controls, can be used to calculate
the removal of the test compound or water-soluble degradation products
by the porous pot treatment. DOC measurements do not identify the water-
soluble components. Specific chemical analysis, on the other hand, is de-
signed to determine the concentration of the parent test compound in the
plant effluent and/or associated with the activated sludge solids and also,
if analytical standards for the degradation products are available, the con-
centrations of the products. From this information, the percent removal
of the test compound and the amount and identity of products resulting
from primary biodegradation can be determined. A specific chemical ana-
lytical method must have a limit of detection (LOD) of <0.1 mg/L for
water or <0.1 mg/kg for sludge solids.
(3) The feature that distinguishes this test from other activated sludge
simulation tests is the retention of the activated sludge in a porous liner,
which eliminates the need for a secondary clarifier and facilitates control
of a critical parameter, the sludge retention time (SRT).
(4) Porous pots can be completely sealed and tests using 14C- labeled
test compounds are possible. Carbon dioxide in the exhaust gas and bicar-
bonate in the effluent can be used together to assess the extent of min-
eralization, and levels of radiolabel in the sludge and in the aqueous phase
may also be determined.
(5) It is also possible to determine whether the test compound has
any adverse effect on normal sewage treatment processes by simulta-
neously measuring the efficiency of the pots in removing DOC.
(6) This guideline may involve hazardous materials, operations and
equipment. This standard does not purport to address all of the safety prob-
lems associated with its use. It is the responsibility of the user of this
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standard to establish appropriate safety and health practices and determine
the applicability of regulatory limitations prior to use. For specific hazard
statements see paragraph (g) of this guideline.
(c) Applicable ASTM standards. Refer to paragraph (o)(l) of this
guideline for the following standards:
(1) Dl 129-90 Standard Terminology Relating to Water.
(2) D1193-91 Standard Specifications for Reagent Water (Federal
Test Method and Standard No. 7916).
(3) D1293-84 Standard Test Methods for pH of Water.
(4) D2579-85 Standard Test Method for Total and Organic Carbon
in Water.
(5) D4375-90 Standard Terminology for Basic Statistics in Commit-
tee D-19 on Water.
(6) D4839-88 Standard Test Method for Total Organic Carbon in
Water by Ultraviolet, or Persulfate Oxidation or Both, and Infrared Detec-
tion.
(7) El78-80 Standard Practice for Dealing with Outlying Observa-
tions.
(d) Summary. (1) The test is designed to simulate processes in the
aeration basin of the activated sludge sewage treatment process and is per-
formed using a porous pot-type laboratory-scale activated sludge appara-
tus, based on an original design developed by the United Kingdom Water
Research Centre (WRC) (see paragraphs (o)(4) and (o)(5) of this guide-
line). The original design was modified (see paragraph (o)(7) of this guide-
line) and has been utilized in determining the effects of temperature, phos-
phate, and other growth media components on the growth of activated
sludge and the toxicity of treated effluents (see paragraphs (o)(8) and
(o)(10) of this guideline). It has also been used in the environmental safety
evaluation of a new product (see paragraph (o)(9) of this guideline). The
modified test system (Figure 1, under paragraph (h)(l)(i) of this guideline)
facilitates control of the SRT, and the effect of this fundamental parameter
on the efficiency of removal of surfactants in porous pots has been de-
scribed (see paragraph (o)(2) of this guideline).
(2) The test and control pots are filled with mixed liquor from an
activated sludge plant treating predominantly domestic sewage and then
operated as continuous-flow systems with primary effluent or settled do-
mestic sewage as feed.
(i) A solution or suspension of the test compound is dosed into the
test pot by means of a suitable micro-metering pump. The concentration
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of the test compound in the influent sewage is 10-20 mg C/L. A lower
concentration of the test compound may be used if a highly sensitive ana-
lytical method is available or if radiolabeled compound is used. The total
flow to the pot (sewage + test compound dosing solution) is controlled
to give the required hydraulic retention time.
(ii) A similar flow of sewage and a dosing solution of a suitable ref-
erence compound such as sodium benzoate are added to the control pots.
Benzoate biodegrades easily and completely in this test system, and is
added at such a concentration to ensure that the total organic carbon load
and the total sewage flow are the same in control and test pots. Reference
compounds may also have other uses (see paragraph (f)(5) of this guide-
line).
(3) Air is supplied to the pots through a diffuser stone to ensure ade-
quate oxygen transfer to the mixed liquor, and an additional flow through
a 5 mm open tube is provided to ensure complete mixing of the system.
The air flow should be sufficient to maintain and thoroughly mix the solids
in suspension and keep the concentration of dissolved oxygen (DO) above
2 mg/L at all times. In order to do this it is necessary to maintain an
air to influent flow ratio of 5 to 10/L on a volume basis.
(4) Sludge is wasted directly from the aeration chamber through the
base of the pot by means of a peristaltic pump. The pump is fitted with
a timer and operated intermittently to avoid problems caused by the low
flow rates required. Alternatively, sludge may be wasted manually by peri-
odically discarding the required volume.
(5) The levels of biodegradable materials remaining in the effluents
are dependent on the SRT and the growth kinetics of those organisms
that are involved in the metabolism of the compound under consideration.
The test is therefore, in effect, a kinetic study and consequently should
be conducted at a constant temperature. Further, by making measurements
at two or more temperatures, the biodegradability of the test compound
under summer and winter operating conditions may be established.
(6) The removal of test compound is determined by analysis of
effluents and comparison of the results obtained from pots containing test
compound to those from control pots treating only settled sewage and ben-
zoate. Primary biodegradation is assessed by specific analysis of the test
compound in effluents after correction for volatilization and adsorption of
the parent compound onto activated sludge. Further, analysis of DOC in
effluents provides a measure of ultimate biodegradation after corrections
have been applied for volatilization and adsorption of parent compound
and biodegradation products onto sludge.
(i) For materials that are insoluble or are sorbed or precipitated onto
the activated sludge additional information will be required to distinguish
between biodegradation and removal by these other processes. The addi-
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tional information may be obtained by analysis of the sewage sludge or
by using 14C-labeled test compound.
(ii) However, in the absence of this additional information, the DOC
and compound-specific analyses still provide data on the total amount of
parent or residual soluble materials present in the effluents without any
identification of these soluble components.
(7) The efficiency of the units may be assessed from the measure-
ments of DOC, sludge production, and ammoniacal nitrogen in the efflu-
ent. However, the latter parameter can only be used when the SRT is suffi-
ciently long for viable populations of nitrifying bacteria to become estab-
lished in the sludge. In each case a simple Student's t test is applied to
the data to determine if there is any significant difference between test
and control pots.
(e) Significance. (1) Secondary wastewater treatment using activated
sludge is one of the most important biological treatment processes in use
today. The porous pot test employs activated sludge from a domestic acti-
vated sludge plant to assess biodegradation and treatability of organic com-
pounds, and provides data that can be used to predict the fate of organic
compounds in full-scale plants.
(2) The porous pot system provides a laboratory-scale simulation of
activated sludge wastewater treatment because settled domestic sewage is
used as the feed and key control parameters are maintained in the ranges
typical of such treatment. These parameters include temperature, DO con-
centration, hydraulic retention time (HRT), and SRT. Mixed liquor volatile
suspended solids (MLVSS) are monitored but not controlled. The porous
pots are allowed to attain steady state at an MLVSS level commensurate
with the other key control parameters.
(f) Interpretation. (1) Because the porous pot test system is a simula-
tion of activated sludge wastewater treatment rather than a test to measure
"ready" or "inherent" biodegradability, there are no pass or fail criteria.
The levels of removal observed in the porous pot test should approximate
levels of removal expected in full-scale activated sludge treatment systems.
(2) Information on the physical/chemical properties of the test
compound will be useful for interpretation of results and in the selection
of appropriate test compound concentrations. These properties include
structure, composition, purity, molecular weight, water solubility, organic
carbon content, vapor pressure, octanol/water partition coefficient, adsorp-
tion isotherm, surface tension, and Henry's law constant.
(3) Information on the toxicity of the test compound or potential toxic
degradation products to activated sludge microorganisms may be useful
to the interpretation of low biodegradation results and in the selection of
appropriate test compound concentrations. The OECD Respiration Inhibi-
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tion Test under paragraph (o)(6) of this guideline can be used to indicate
such toxicity. Furthermore, chemical substances in solution or in the air
that may negatively affect the growth or metabolism of sludge microorga-
nisms, e.g., organic solvents, toxic metals, strong alkalis, and bactericides,
may result in low removals and should be avoided.
(4) Use of synthetic versus natural sewage is an important consider-
ation.
(i) It is sometimes assumed that use of synthetic sewage leads to more
reproducible results; however, the microbial population that develops dif-
fers from that which is present in full-scale activated sludge plants. Gen-
erally, the most rapidly growing microorganisms will dominate the more
slowly growing populations that are present in full-scale treatment plants.
Natural domestic sewage varies from source to source and in nutrient con-
tent. However, it provides both the nutrients needed to support the natural
microbial population and a continuous supply of fresh microorganisms to
the test system.
(ii) On some occasions, particularly during periods of heavy rainfall,
the strength of the primary effluent or settled domestic sewage from the
treatment plant may be too low to sustain a typical biomass (MLVSS)
concentration in the porous pot unit of 1.5 to 3.0 mg/L. This is not likely
to happen unless the DOC concentration in the feed is <20 mg DOC/
L. In this case, a blend made by supplementation of natural sewage with
synthetic sewage to achieve a DOC level of at least 200 mg/L and an
approximate 100:12:2 ratio of C:N:P may be desirable.
(5) Reference compounds may be useful in establishing the activity
of the activated sludge and in comparing results from different labora-
tories. While specific reference compounds cannot be recommended for
these purposes, data are available for several chemicals (see paragraphs
(o)(2) and (o)(7) of this guideline).
(g) Safety precautions. (1) This procedure involves the use of mixed
liquor and natural sewage from a domestic wastewater treatment plant.
Consequently, individuals performing this test may be exposed to micro-
bial agents that are dangerous to human health. It is recommended that
porous pots be operated in a separate room and that exhaust air be vented
outside the building.
(2) Personnel who work with sewage organisms may choose to keep
current with pertinent immunizations such as typhoid, polio, hepatitis B,
and tetanus.
(3) Effluent from the porous pots is treated with a chemical disinfect-
ant (chlorine bleach, 5 percent) or autoclaved prior to disposal. Safety
glasses and protective gloves should be worn when using sodium hypo-
chlorite to clean pot liners.
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(4) Unless shown to be nontoxic, all test compounds should be treated
as potentially harmful.
(h) Apparatus. The following apparatus are required to perform the
test:
(1) Porous pot aeration vessel.
(i) URPSL design (Figure 1).
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FIGURE i. URPSL POROUS POT
settled sewage
-10.5 mL/min.
test compound
-0.5 mL/min.
glass
or ABS
plastic
outer
vessel
porous
high density
polyethylene
liner
screw
thread
seal
solid
plastic
base
air supply
open ended tube
waste sludge
-0.45 mL/min.
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(ii) Engineering drawing of URPSL design (Figure 2).
FIGURE 2. ENGINEERING DRAWING OF URPSL POROUS POT
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(iii) The porous pot vessel liner is constructed from porous high den-
sity polyethylene sheets. The thickness ranges from 3.2 to 13.6 mm and
pore sizes are from 65 to 90 (im. The retention of the liner is about
20 (im and all particles above this size are retained in the system. A U.S.
manufacturer is Porex Technologies Corp., 500 Bohannon Rd., Fairburn,
GA 30213. A U.K. supplier is Porvair Technology Ltd., Clywedog Road
South, Wrexham Industrial Estate, Wrexham Cllyd, LL13 9XS, U.K. The
outer vessel can be constructed of glass or an impermeable plastic such
as acrylonitrile butadiene styrene (ABS) copolymer.
(2) Oil-free compressor for supplying compressed air to the aeration
vessel.
(3) Suitable pumps for dosing porous pots with test substance solu-
tions and sewage at the required rates (0-1.0 mL/min for test substance
solutions, 5-20 mL/min for sewage). An additional pump is required to
waste sludge from the pot, unless sludge is wasted manually. Low rates
of sludge wastage are attained using a pump set at a high flow rate but
operating intermittently. The actual flow is calculated as follows:
[pump throw (mL/min) x pumping time (sec)]
flow =
timer cycle (sec)
For example, when the pump is operating for 10 sec each minute, the
timer cycle is 1 min (60 sec), and the pump throw is 3 mL/min, the wast-
age rate would be 0.5 mL/min.
(4) 1-L sample bottles to hold test substance dosing solutions.
(5) Silicone rubber tubing: Bore = 0.5 mm ID.
(6) Polypropylene transmission tubing.
(7) Tube connectors.
(8) Diffuser stones.
(9) 25-mL measuring cylinders.
(10) 1-mL graduated pipets.
(11) Stopwatch.
(12) 40-mL sample bottles for collection of samples for waste sludge
and mixed liquor suspended solids determinations.
(13) Thermometer having range 0 to 50 °C.
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(14) 1- and 2-L measuring cylinders for each pot to collect waste
sludge.
(15) Timer for sludge wastage pump allowing intermittent operation,
unless sludge is wasted manually.
(16) Right-angled plastic tube to fit on one end of the air line to
ensure complete mixing of activated sludge.
(17) If pots are operated in the sealed mode (see paragraph (b)(4)
of this guideline), plastic tubing to fit on the pot effluent port as shown
in Figure 3 to balance the back pressure caused by the CCh traps.
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FIGURE 3. POROUS POT APPARATUS FOR SEALED MODE OF OPERATION
c
CD
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(i) Reagents and materials. The following reagents and materials
are required to perform the test:
(1) Activated sludge mixed liquor collected from aeration basin or
oxidation ditch of domestic wastewater treatment plant.
(2) Natural sewage feed: Primary effluent or settled domestic sewage
from a domestic wastewater treatment plant. Supplementation with the
synthetic sewage stock (see paragraph (i)(3) of this guideline) to achieve
at least 200 mg DOC/L may be desirable in some situations, as noted
in paragraph (f)(4)(ii) of this guideline.
(3) Synthetic sewage stock solution: Add 130 g glucose, 130 g nutri-
ent broth, 130 g beef extract, 130 g dipotassium hydrogen phosphate, and
25 g ammonium sulfate to 1 L of tap water. Dissolve by heating to just
below the boiling point and store in the refrigerator below 7 °C. Discard
if any visual evidence of biological growth (turbidity) is observed. Syn-
thetic sewage is created by adding 1 mL of this stock to 1 L of tap water.
(4) Compressed air (filtered for oil and water) for aeration of porous
pots.
(5) Test and reference compounds of known carbon content (for DOC
analyses) or composition (for specific analyses).
(6) Extraction apparatus and solvent for hydrophobic test compounds.
(7) Deionized or distilled water for preparation of test/reference
compound stock solutions.
(8) Glycerol for lubricating the rollers of the peristaltic pumps.
(9) Sodium hypochlorite solution.
(10) Stock solutions of test and reference compounds, (i) For com-
pounds that are sufficiently soluble and chemically stable, a stock solution
lOx times the strength of the dosing solution may be prepared and diluted
to the required strength each day.
(ii) If chemically unstable materials are being tested, it may be nec-
essary to prepare stock/dosing solutions immediately before use.
(iii) Note: Use of a suitable stable dispersion is required when testing
for insoluble compounds.
(11) Dosing solutions of test/reference compound, (i) To avoid bio-
degradation of the test/reference compound before it is introduced into the
test system, which might occur if the test/reference compound and sewage
are premixed, the test solution and the sewage are dosed into the porous
pot separately.
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(ii) The total flow into the pot (sewage flow + test/reference
compound flow, both in milliliters per minute) is calculated as follows:
[volume of porous pot (mL)]
total flow = F =
[required sewage retention time (h)] x 60 min/h
(iii) For a pot volume of 3 to 6 L, it is convenient to dose with
a solution of test/reference compound at about 0.5 mL/min.
(iv) If the total flow as calculated above is F (mL/min) and the re-
quired concentration in the influent sewage is C (mg/L), the concentration
of the solution (in mg/mL) to be dosed into the pot (at a rate of
0.5 mL/min) is given as follows:
Concentration of test/reference compound in dosing solution =
F (mL/min) x C (mg/L)/0.5 mL/min
(v) The dosing solution is usually prepared daily by diluting a suitable
stock solution.
(j) Procedure. (1) Mixed liquor should be maintained at the required
working temperature (±2 °C) throughout the test. When setting up the
test pots, test/reference compound and sludge wastage rates may initially
be set. The test should be started only after conditions are adjusted to
the values defined in the study plan and the pots have been operating
for some time under these conditions.
(2) Set up the number of pots required by the study plan. Each test
should have at least one control pot (pot fed settled sewage and benzoate
or other easily degradable reference compound) and it is recommended
(but not required) that each test compound be tested in duplicate.
(3) Fill the aeration vessel with mixed liquor to the level of the efflu-
ent overflow. The volume required is 3.8 L for a URPSL porous pot. The
initial MLVSS should be 1.5 to 3.0 g/L, and this level should be main-
tained throughout the test. This may necessitate supplementation of the
feed with synthetic sewage as described in paragraph (f)(4)(ii) of this
guideline.
(4) Start the aeration and set the air flow. The air flow should be
sufficient to maintain and thoroughly mix the solids in suspension and
keep the concentration of DO above 2 mg/L at all times. In order to do
this it is necessary to maintain an air to influent flow ratio of 5 to 10/
L on a volume basis.
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(5) Place 1 L of test/reference compound dosing solution in the dosing
vessel.
(6) Start the dosing pumps, lubricating the tubes with a small amount
of glycerol.
(7) Start the sludge wastage pump at the rate required to give the
desired SRT (the SRT (in days) is equal to the pot volume (L) divided
by the sludge wastage rate (L/day)). Alternatively, sludge may be wasted
manually by periodically discarding the required volume.
(8) Set the sewage dosing rate to give the required HRT and the test/
reference compound dosing rate at about 0.5 + 0.05 mL/min.
(9) Daily measurements of sewage flow rates should be made using
a 25-mL measuring buret and a stopwatch. The flow rates should be ad-
justed to within ±0.05 mL/min of the required flow.
(10) Dosing solution flow rates should be calculated from measuring
the volume left after 24 h of dosing.
(11) The dosing rates should be recorded and corrected to the nominal
value given in the study plan. The sewage flow should be adjusted if the
measured flow differs by more than 0.5 mL/min from the nominal value.
(12) Sludge that gathers around the rim of the porous liner should
be returned to the mixed liquor at least once per day by scraping with
a large spatula. This should always be done before taking a sample of
mixed liquor for MLVSS determination.
(13) The temperature, pH, and DOC of the mixed liquor should be
measured at least every other day.
(14) Periodically remove a 40-mL sample of mixed liquor from the
aeration vessel for MLVSS determination. Three times weekly is usually
sufficient.
(15) The volume of mixed liquor wasted from the porous pot should
be measured and recorded daily. Remove a representative 40-mL sample
from the sludge wastage bottle at least once per week and determine the
MLVSS level.
(16) The porous pot liner should be changed at the first sign of block-
ing of the pores, i.e., when the mixed liquor rises above the effluent over-
flow. To change the liner proceed as follows: Syphon the mixed liquor
into a suitable container and remove any solids from the inner surface
of the outer vessel. Place a fresh liner in the outer vessel. Return the mixed
liquor to the aeration vessel. Scrape off and transfer any sludge adhering
to the sides of the blocked liner. The blocked liner should be thoroughly
cleaned before reuse by immersion in a 20 percent solution of hypochlorite
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bleach for several hours. The liners must be thoroughly rinsed in clean
tap and deionized water before reuse.
(17) Sewage and effluent samples should be taken twice weekly dur-
ing the stabilization ("running-in") period for organic carbon analysis and
specific compound analysis if required. If necessary, ammoniacal nitrogen,
nitrate, nitrite, COD and BODs may also be determined.
(18) When the pots have attained steady state conditions, the effluents
are analyzed periodically to determine the extent of biodegradation/re-
moval of the test compound during treatment.
(19) If information on the effects of various operating conditions on
removal is required, e.g., temperature, SRT, HRT, etc., any changes should
be made gradually. Operate the pot for a period of at least three SRT
under the new conditions before collecting data to determine the effect
of the new condition(s).
(k) Sampling and analytical procedures—(1) Stabilization period.
Over the early period of the test, influent sewage and effluent samples
should be taken for organic carbon and ammoniacal nitrogen analysis to
monitor the overall performance of the pots. Specific analysis for test
compound or degradation products may also be performed on these sam-
ples if this is required. Normally these results are not used to assess either
the biodegradability or treatability of the test compound but simply to es-
tablish that the units have reached steady state, are operating properly,
and are acclimated to the test substance. However, in certain instances,
such as when information is desired on treatability of test compounds that
are released only intermittently to wastewater treatment systems, data gath-
ered during the stabilization period may be useful.
(2) Calculation period, (i) When the pots have achieved steady state,
the removal of the test compound is determined by specific compound
analysis, measurement of DOC, or both. At least 14 results should be ob-
tained over the calculation period at times when the process efficiency
of the control pots is high. The time period over which the measurements
are made is not critical but should cover at least 21 days with a maximum
sampling frequency of 1 sample per day.
(ii) The treatability of the test compound may be assessed by meas-
urement of DOC removal, removal of ammonia, sludge production, and
sludge activity. Of these parameters, DOC and NHs-nitrogen removal are
the most important. Note that when pots are being operated at short SRT
or reduced temperature, ammonia removal may be less than complete and
will be a less reliable indicator of efficiency. However, the critical assess-
ment of any adverse effect of the test compound on the process is always
based on the absence of any significant difference between the test
compound and control pots rather than the actual values of the observed
parameters.
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(3) DOC analysis, (i) DOC analysis for monitoring the porous pot
test is generally employed only for test compounds whose water solubility
exceeds the test concentration (e.g., a concentration equivalent to about
10 mg C/L).
(ii) Since precipitation as salts or sorption onto the sludge floe may
occur even with water-soluble test compounds, DOC removal doesn't nec-
essarily indicate biodegradation in all cases.
(iii) DOC analyses are carried out on supernatant samples removed
at the end of each cycle from the test compound and control pots. Samples
can either be filtered using 0.45 (im pore-size filters or centrifuged at
3,500 x g for 10 min. Precaution: An aliquot of the dosing solution should
be evaluated for adsorption of test compound to the filter or elution of
DOC from the filter itself.
(iv) The DOC concentration of aqueous samples is determined using
a suitable organic carbon analyzer (e.g., OI Corporation Model 700 TOC
Analyzer equipped with an autosampler) or equivalent.
(4) Specific compound analysis, (i) For the assessment of primary
biodegradability the porous pot method applies to water-soluble com-
pounds provided that a suitable method of specific analysis is available.
(ii) Insoluble compounds or compounds that sorb strongly onto the
activated sludge may also be examined by this procedure but it will be
necessary to determine the level of the test compound associated with the
activated sludge.
(iii) Nonpolar hydrophobic test compounds are usually isolated from
the sludge matrix by extraction with an immiscible solvent such as methyl-
ene chloride or hexane. The extract is dried, concentrated, and analyzed
by an appropriate instrumental method—GC, HPLC, GC-MS, or UV/visi-
ble spectroscopy.
(iv) Highly polar extractible or nonextractible test compounds that
are associated with the mixed liquor solids require specialized testing and
analytical procedures that cannot be fully documented in this guideline—
use of radiolabeled materials and special apparatus. However, the porous
pot operating system can be employed if appropriate mass balances can
be obtained.
(v) The porous pot test is not recommended for volatile compounds
(Henry's law constant >10 3 atm-m3/mol); however, it can be used for
compounds that are not completely volatilized. For compounds of mod-
erate volatility, volatilization losses during testing may be evaluated by
scrubbing aeration off-gases through a solvent train (usually three consecu-
tive traps containing acetone, methylene chloride, or hexane) or polymeric
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traps (e.g., Tenax or Sep-Pac). Specific compound analysis of each solvent
trap or polymeric trap is carried out.
(1) Treatment of results — (1) Calculation of results. The percentage
removal values obtained for the test compound during the calculation pe-
riod are calculated to 0. 1 percent as outlined below. Any seemingly atypi-
cal values obtained during the calculation period should be checked for
rejection using Dixon's test (refer to paragraph (o)(3) of this guideline),
and if rejection is statistically correct, the result should be omitted from
the calculation.
(2) Primary biodegradation/removal. Removal is defined by the fol-
lowing expression:
percent removal = [1 - (Cn/Co)] x 100
where
Co = mean concentration of test compound in the influent (mg/L)
CE = mean concentration of test compound in the effluent (mg/L).
(3) Ultimate biodegradation/removal. (i) The difference in DOC
level (in milligrams per liter) between the control and the test effluents
is assessed using a matched pairs hypothesis test. This is performed using
the following formula:
where
t is the Student's t value
Xi and X2 are the means of the two sets of data
S is the standard deviation of the paired differences
n is the number of paired sets of data.
(ii) The critical value for t is obtained from statistical tables for
(n - 1) degrees of freedom at the 0.05 significance level using a one-
tailed test.
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(iii) The percent removal is calculated from the mean difference:
percent removal = (Co - Xd)/C0 x 100
where
Co = concentration of test compound (expressed as milligrams of or-
ganic carbon per liter) in the test pot influent
= the mean difference between the test and control effluent DOC
levels over the calculation period expressed as milligrams per liter.
(iv) The 95% confidence limits for Xa are calculated as
±t x S/n1/2, where S is the standard deviation ((n - 1) degrees of freedom),
t is the t value at (n - 1) degrees of freedom (two tailed test, p = 0.05),
and n is the number of data pairs.
(v) 95% confidence limits for percent removal are calculated as
+ (95% confidence limit for Xd x 100)/C0.
(vi) Even if there is no significant difference between the test and
control effluent carbon levels, it is advisable to calculate the removals
since this will yield information on the variability of the results.
(vii) Efficiency may be estimated by determining organic carbon, am-
moniacal nitrogen, and specific compound removal. This calculation is
similar to that for primary biodegradation/removal (see paragraph (1)(2)
of this guideline).
(viii) A typical data set for two control pots and three test pots is
given in the following Table 1. and is used to illustrate the statistical analy-
sis of the data. It is first necessary to establish that the two control pots
are operating in parallel. As indicated in Table 1., the mean difference
in effluent DOC between the two control pots is 0.21 mg/L and the stand-
ard deviation is 0.53 (n = 17). The Student's t value is given by:
t = 0.21 [ (17)1/2/0.53] = 1.63
(ix) The critical t- value at the 0.05 significance level for a two-tailed
test and 16 degrees of freedom is 2.21 and since this is not exceeded
by the calculated value there is no significant difference between the con-
trols. (Note that a two-tailed test is used in this instance since there is
no preconception as to which pot will have the higher effluent concentra-
tion.)
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Table 1.—Sample test set
Effluent DOC (mg/L)
Control 1
9.6
9.5
9.2
7.8
7.5
7.3
6.7
11.8
12.6
18.0
11.1
8.8
8.5
10.8
12.0
11.1
14.2
Control 2
9.2
10.1
10.2
7.4
8.1
7.2
7.1
13.2
12.8
17.7
11.1
9.3
8.3
10.5
11.9
11.9
14.1
Control Mean
9.40
9.80
9.70
7.60
7.80
7.25
6.90
12.50
12.7
17.85
11.10
9.05
8.40
10.65
11.95
11.50
14.15
Test 1
10.9
9.8
11.9
8.4
9.1
9.2
7.9
13.2
11.6
13.8
9.2
7.5
7.6
9.8
9.7
10.0
13.7
Test 2
11.2
11.1
11.7
8.6
9.2
8.4
12.2
11.8
14.9
11.5
8.4
9.1
11.0
11.8
14.7
Tests
11.8
12.1
12.5
10.1
11.0
8.9
14.9
14.5
16.5
14.2
10.5
9.6
11.9
12.9
16.3
Mean of (control 2 - control 1) = 0.21
Standard deviation of (control 2 - control 1) = 0.53
(x) The test/reference compound data are treated in a similar manner
but the differences between each test pot effluent DOC level and the mean
control value are used. The statistical analysis for the three tests is summa-
rized in the following Table 2.:
Table 2.—Analysis of test data
Mean difference (test-mean control)
Standard deviation
Number of paired observations
Calculated student's t value
Critical student's t value
Percent removal
Test 1
-029
1 69
17
0.72
1.75
102.9
Test 2
041
1 25
15
1.27
1.76
95.9
Tests
1 88
1 10
15
6.62
1.76
81.2
95% confidence intervals for percent removal: Test 1 = 94.2-111.6; Test
2 = 84.0-107.8; Test 3 = 75.1-87.3.
(xi) For Test 1, the mean is calculated for all values of the difference
(test-control mean), and the calculated t-value is compared with critical
t-statistics using a one-tailed test for (n-1) degrees of freedom at the 0.05
probability level. A one-tailed test is used since it is only necessary to
establish if the test pot effluent has a significantly higher DOC than the
control pot. The converse is of no importance and is not normally ob-
served. Tests 2 and 3 are treated similarly.
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(xii) For Test 1, the critical t-value at the 0.05 probability level for
a one-tailed test and 17 paired observations (16 df) is 1.75, and since this
is not exceeded by the calculated value of 0.72, the difference between
Test 1 and the controls is not significantly different from zero.
(xiii) Only for Test 3 is there evidence for a non-zero difference;
i.e., that the effluent DOC for the test pot is significally greater than for
the controls. In this case, however, the result is highly significant since
the calculated t-value exceeds the critical value by a considerable margin.
If analysis for disappearance of parent compound had been conducted si-
multaneously with DOC analysis and showed complete removal, then these
results could be taken as evidence for formation of water-soluble degrada-
tion products in Test pot 3.
(m) Quality assurance. To assure the integrity of data developed
using this method and to comply with current regulatory requirements,
a quality assurance program meeting EPA, FDA, or OECD guidelines
should be followed. This may require replicates (three or more) to be run
for good laboratory practice (GLP) compliance and assessment of varia-
bility.
(n) Documentation to be provided. (1) A protocol giving a general
overview of the study goals and procedures must be prepared before the
study is initiated. If a substantive modification of this method is deemed
necessary for the test compound, deviation from the method should be
documented in the protocol.
(2) Final results of this study are to be documented in a final report.
The final report should include the following:
(i) Names of study, investigators, and laboratory.
(ii) A brief description of the test compound including its identifying
number in the log, chemical names, composition, and other appropriate
information.
(iii) Summary of test method including deviations from the written
method.
(iv) Summary of specific analytical methods, if employed.
(v) If applicable, tabular and graphical presentation of DOC removal
data as a function of time after test initiation. Data are expressed as percent
DOC removal (weekly mean for 24-h periods).
(vi) If applicable, tabular and graphical presentation of specific
compound analysis data as a function of time after test initiation. Data
are expressed as steady state concentrations of test compound in influents
and effluents, percent DOC removal, or primary biodegradation during
24-h periods.
20
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(vi) A listing of relevant references including all notebook pages con-
taining raw data from this study.
(vii) The following raw data should be recorded:
(A) Date.
(B) Pot number.
(C) Sewage flow.
(D) Temperature of mixed liquor.
(E) Volume of test compound dosing solution remaining.
(F) Time of replacement of dosing solution.
(G) pH of mixed liquor.
(H) Dissolved oxygen concentration (DOC) of mixed liquor.
(I) Weight of test compound used to prepare concentrated stock solu-
tions.
(J) Volume of activated sludge mixed liquor wasted.
(K) Date of start of calculation period.
(L) Change of porous pot liners.
(M) Faults with tubing, pumps, sewage or air supply.
(N) Signature of operator.
(O) Study number.
(P) Calibration of pH meter.
(Q) Calibration of DO meter.
(o) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) American Society for Testing and Materials (ASTM). Annual
Book of ASTM Standards, Volumes 11.01 and 11.02 on Water and Envi-
ronmental Technology, and Volume 14.02 on General Methods and Instru-
mentation (1993).
(2) Birch, R.R. Prediction of the fate of detergent chemicals during
sewage treatment. Journal of Chemical Technology and Biotechnology
50:411-422(1991).
(3) British Standard Institution. British Standard BS 5497. Precision
of test results, part 1, p. 12 (1987).
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(4) Department of the Environment. Standing Committee of Analysts.
National Water Council. U.K. 1983. Assessment of biodegradability 1981.
Methods for the examination of waters and associated materials. Continu-
ous Simulation (Activated Sludge) Test for the Assessment of
Biodegradability, Volume 1981. Her Majesty's Stationery Office, London.
(5) Hopwood, A.P. and A.L. Downing. Factors affecting the rate of
production and properties of activated sludge in plants treating domestic
sewage. Journal of the Proceedings of the Institute of Sewage Purification
64:435-452 (1965).
(6) Organization for Economic Cooperation and Development
(OECD). Activated Sludge Respiration Inhibition Test. OECD 209, OECD,
Paris (1981).
(7) Painter, H.A. and E.F. King. WRC porous pot method for assess-
ing biodegradability. WRC Technical Report TR70 (1978).
(8) Painter, H.A. and E.F. King. The effect of phosphate and tempera-
ture on the growth of activated sludge and on the biodegradation of
surfactants. Water Research 12: 909-915 (1978).
(9) Waters, J. et al. A new rinse conditioner active with improved
environmental properties. Tenside Surfactants Detergents 28:460-468
(1991).
(10) Watson, H.M. Important considerations in choosing a synthetic
feed for laboratory-scale wastewater treatment systems. Environmental
Toxicology and Risk Assessment: 2nd Volume, STP 1216. J.W. Gorsuch
et al., Eds., American Society for Testing and Materials, Philadelphia, PA,
pp. 228-239 (1993).
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