United States Prevention, Pesticides EPA712-C-08-005
Environmental Protection And Toxic Substances October 2008
Agency (7101)
4>EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3260
Simulation Test—Aerobic
Sewage Treatment: B.
Biofilms
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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
(OPPTS), United States Environmental Protection Agency for use in the testing
of pesticides and toxic substances, and the development of test data to meet the
data requirements of the Agency under the Toxic Substances Control Act (TSCA)
(15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and
Cosmetic (FFDCA) (21 U.S.C. 346a).
OPPTS developed this guideline through a process of harmonization of
the testing guidance and requirements that existed for the Office of Pollution
Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code
of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in
publications of the National Technical Information Service (NTIS) and in the
guidelines published by the Organization for Economic Cooperation and
Development (OECD).
For additional information about OPPTS harmonized guidelines and to
access this and other guidelines, please go to http://www.epa.gov/oppts and
select "Test Methods & Guidelines" on the left side menu.
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OPPTS 835.3260: Simulation test— aerobic sewage treatment: B. biofilms
(a) Scope—(1) Applicability. This guideline is intended for use in testing pursuant
to the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this harmonized OPPTS
guideline is Organization for Economic Cooperation and Development (OECD) test
guideline 303 (adopted 22 January 2001), available from Source OECD at
http ://masetto. sourceoecd.org/.
(b) Purpose. The test method described in this guideline is designed to determine
the elimination and primary and/or ultimate biodegradation of water-soluble organic
substances by aerobic microorganisms in a continuously operated test system simulating
percolating (trickling) filters. The method is designed to ascertain whether the substances
tested (usually those known to be inherently but not readily biodegradable) can be
biodegraded within the limits imposed in typical wastewater treatment plants. The results
are expressed in terms of percentage removal and percentage biodegradation. Because this
is a simulation test, the levels of removal observed should approximate levels of removal
expected in full-scale treatment systems based on analogous principles (e.g. trickling
filters). Percentage removal obtained in this test can therefore be used to calculate probable
environmental concentration, for application in hazard assessment.
(c) Overview. (1) Simulation tests are normally applied to chemical substances that
have failed a screening test for ready biodegradability (301 A to F) (see paragraph (n)(l) of
this guideline), but have passed a test for inherent biodegradability. Simulation tests can
also be applied to any substance about which more information is required, especially high-
tonnage substances, and normally the activated sludge test is run (OECD 303A). In some
circumstances, however, specific information is required on the behavior of a substance in
wastewater treatment involving biofilms; i.e. in percolating or trickling filters, rotating
biological contactors, or fluidized beds. To meet this need various simulation test systems
have been developed.
(2) Gerike et al. (see paragraph (n)(2) of this guideline) used large, pilot-scale
trickling filters that they used in the coupled mode. These filters took up much space and
required relatively large volumes of sewage or synthetic sewage. Truesdale et al. (see
paragraph (n)(3) of this guideline) described smaller filters (6 ft x 6 in. diameter) that were
fed surfactant-free natural sewage but still required rather large volumes. As many as 14
weeks were required for the development of a "mature" biofilm and an additional 4-8
weeks were needed after first introduction of the test surfactant before acclimation took
place.
(3) Baumann et al. (see paragraph (n)(4) of this guideline) developed a much
smaller filter that used polyester "fleece" previously steeped in activated sludge as the inert
medium supporting the biofilm. The test substance was used as the sole source of carbon
and biodegradability was assessed from measurements of dissolved organic carbon (DOC)
in the influent and effluent, and from the amount of CO2 in the exit gas.
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(4) A quite different approach was employed by Gloyna et al. (see paragraph (n)(5)
of this guideline) who invented the rotating tubular reactor. On the internal surface of the
rotating tube a biofilm was grown on the known surface area by passage of influent
introduced at the top end of the tube, inclined at a small angle to the horizontal. This reactor
has been used to study the biodegradability of surfactants (see paragraph (n)(6) of this
guideline), as well as to investigate the optimal thickness of biofilm and diffusion through
the film (see paragraph (n)(7) of this guideline). The latter authors further developed the
reactor, including modifying it to be able to determine CC>2 in the exit gas.
(5) The rotating tubular reactor has been adopted by the Standing Committee of
Analysts (UK) as a standard method for assessing both the biodegradability of substances
(see paragraph (n)(8) of this guideline) and the treatability and toxicity of wastewaters (see
paragraph (n)(9) of this guideline). The method described here has the advantages of
simplicity, compactness, reproducibility and the need for relatively small volumes of
organic medium.
(d) Principle of the test. (1) Synthetic or domestic sewage, and the test substance,
in admixture or alone, are applied to the internal surface of a slowly rotating inclined tube.
A layer of microorganisms, similar to those present on biofilter media, is built up on the
internal surface. The operating conditions of the reactor are selected to give adequate
elimination of organic matter and, if required, oxidation of ammonium.
(2) Effluent from the tube is collected and either settled and/or filtered before
analysis for DOC and/or the test substance by a specific method. Control units receiving no
test substance are operated in parallel under the same conditions for comparative purposes.
The difference between the concentrations of DOC in the effluent from the test and control
units is assumed to be due to the test substance and its organic metabolites. This difference
is compared with the concentration of the added test substance (as DOC) to calculate the
elimination of test substance.
(3) Normally biodegradation can be distinguished from biosorption by careful
examination of the elimination-time curve. Confirmation can usually be obtained by
applying a test for ready biodegradation (based on oxygen uptake or CO2 production) using
an acclimated inoculum taken at the end of the test from the reactors receiving test
substance.
(e) Applicable American Society for Testing and Materials (ASTM) standards.
Refer to the documents referenced in paragraph (n)(10) of this guideline for the standards
referenced in paragraphs (e)(l) through (e)(7) of this guideline.
(1) Dl 129-90 Standard Terminology Relating to Water.
(2) Dl 193-91 Standard Specifications for Reagent Water (Federal Test Method and
Standard No. 7916).
(3) D1293-84 Standard Test Methods for pH of Water.
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(4) D2579-85 Standard Test Method for Total and Organic Carbon in Water.
(5) D4375-90 Standard Terminology for Basic Statistics in Committee 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 Detection.
(7) E178-80 Standard Practice for Dealing with Outlying Observations.
(f) Information on the test substance. (1) The purity, water solubility, volatility
and sorption characteristics of the test substance should be known to enable correct
interpretation of results. Normally, volatile and poorly soluble substances cannot be tested
unless special precautions are taken. See paragraphs (f)(2) and (f)(3) of U.S. EPA Fate,
Transport and Transformation Test Guidelines no. 835.3240, on activated sludge units
(cited in paragraph (n)(l 1) of this guideline).
(2) The chemical structure, or at least the empirical formula, should also be known
in order to calculate theoretical values and/or check measured values of parameters, e.g.
DOC and theoretical oxygen demand.
(3) Information on the toxicity of the test substance to microorganisms may be
useful for selecting appropriate test concentrations and may be essential for the correct
interpretation of low biodegradation values. See paragraph (f)(4) of U.S. EPA Fate,
Transport and Transformation Test Guidelines no. 835.3240 (cited in paragraph (n)(l 1) of
this guideline).
(g) Pass levels. (1) Because this test is intended to simulate wastewater treatment
rather than measure "ready" or "inherent" biodegradability, there are no pass or fail
criteria. The levels of removal observed should approximate levels of removal expected
in full-scale treatment systems based on analogous principles (e.g. trickling filters).
(2) Percentage removal obtained in this test can therefore be used to calculate
probable environmental concentration, for application in hazard assessment. Results tend to
follow an all-or-nothing pattern. For example, in several studies of pure substances the
percentage removal of DOC was found to be >90% in more than three quarters and >80%
in over 90% of substances that showed any significant degree of biodegradability.
(h) Reference substances. To ensure that the experimental procedure is being
performed correctly, it is useful to occasionally test reference substances whose behavior is
known. Such substances include adipic acid, 2-phenylphenol, 1-naphthol, diphenic acid and
1-naphthoic acid.
(i) Reproducibility of test results. The relative standard deviation within tests was
found by a laboratory in the UK to be 3.5%, and between tests it was 5% (see paragraph
(n)(8) of this guideline).
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(j) Safety precautions. (1) This procedure involves the use of natural sewage from
a domestic wastewater treatment plant. Consequently, individuals performing this test may
be exposed to microbial agents that are dangerous to human health. It is recommended that
test units 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 test units should be treated with a chemical disinfectant (chlorine
bleach, 5%) or autoclaved prior to disposal. Safety glasses and protective gloves should be
worn when using sodium hypochlorite to clean porous pot liners.
(4) Unless shown to be nontoxic, all test substances should be treated as potentially
harmful.
(k) Description of the method - (1) Rotating tubular reactors, (i) The test
apparatus (see Figure 1) consists of a bank of acrylic tubes each 30.5 cm long and with 5
cm internal diameter, supported on rubber-rimmed wheels contained within a metal
supporting frame. Each tube has an outside lip, approximately 0.5 cm deep, to keep it on
the wheels, the internal surface is roughened with coarse wire wool, and there is a 0.5 cm-
deep internal lip at the upper (feed) end to retain the liquid. The tubes are inclined at an
angle of approximately one degree to the horizontal to achieve the required contact time
when the test medium is applied to a clean tube. The rubber wheels are rotated using a
slow, variable-speed motor. The temperature of the tubes is controlled by operating the test
units in a constant-temperature room.
(ii) By enclosing each tube reactor inside a slightly larger, capped tube and ensuring
that connections are gas-tight, CC>2 in the exit gas can be collected in an alkaline solution
for subsequent measurement (see paragraph (n)(7) of this guideline.
(iii) A 24-h supply of organic medium with added test substance (if applicable), for
each tube, is contained in a 20-1 storage vessel (see A in Figure 2). If required, the test
substance solution may be dosed separately. Near the bottom of each storage vessel there is
an outlet that is connected by suitable tubing, e.g. silicone rubber, via a peristaltic pump (B)
to a glass or acrylic delivery tube that enters 2-4 cm into the higher (feed) end of the
inclined tube (C). Effluent is allowed to drip from the lower end of the inclined tube to be
collected in another storage vessel (D). The effluent is settled or filtered before analysis.
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Figure 1. Rotating Tubes
PLAN VIEW
5 IDLING WHEELS
Drive
Motor
Reduction
Gears
VIEW A
DRIVEN WHEELS 2 AND 4
IDLING WHEELS 1,3, AND 5
VIEW
30,5cm
ft-h-1-1-
Internal
Range
X—_ Tilting
Mechanism
Bevel Gear Drive
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Figure 2. Flow diagram of rotating tubular reactor test system
A
B
A: Feed tank
B: Peristaltic pump
C: Rotating tube
D: Effluent collection vessel
D
(2) Filtration apparatus or centrifuge. A device is required for filtering samples
that uses membrane filters of suitable porosity (nominal aperture diameter 0.45 jim) and
that adsorbs soluble organic compounds and releases organic carbon to a minimum degree.
If filters are used that release organic carbon, wash the filters carefully with hot water to
remove teachable organic carbon, before using them. Alternatively, a centrifuge capable of
producing 40,000 m/s2 may be used.
(3) Other equipment. Analytical apparatus is required as needed, to determine
- DOC and total organic carbon (TOC), or chemical oxygen demand (COD);
Specific substance, if required;
Suspended solids, pH, oxygen concentration in water;
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- Temperature, acidity and alkalinity;
- Ammonium, nitrite and nitrate, if the test is performed under nitrifying
conditions.
(4) Water, (i) Tap water should contain less than 3 mg DOC/1. Determine the
alkalinity if it is not already known.
(ii) Deionized water should contain less than 2 mg DOC/1. See also paragraph (e) of
this guideline, on applicable ASTM standards.
(5) Organic medium, (i) General. Synthetic sewage, domestic sewage or a mixture
of both may be used as the organic medium. It has been shown that the use of domestic
sewage alone often gives increased percentage removal of DOC (in activated sludge units)
and even allows the biodegradation of some substances that are not biodegraded when
OECD synthetic sewage is used. Thus, the use of domestic sewage is recommended.
Measure the DOC (or COD) concentration in each new batch of organic medium. The
acidity or alkalinity of the organic medium should be known. The medium may require the
addition of a suitable buffer (sodium hydrogen carbonate or potassium hydrogen
phosphate), if it is of low acidity or alkalinity, to maintain a pH of about 7.5 +0.5 in the
reactors during the test. The amount of buffer, and when to add it, has to be decided in each
individual case.
(ii) Synthetic sewage. Dissolve the following in each liter of tap water: peptone,
160 mg; meat extract, 110 mg; urea, 30 mg; anhydrous dipotassium hydrogen phosphate
(KH2PO4), 28 mg; sodium chloride (NaCl), 7 mg; calcium chloride dihydrate (CaCl2
2H2O), 4 mg; magnesium sulphate heptahydrate (MgSO4.7H2O), 2 mg. This OECD
synthetic sewage is an example and gives a mean DOC concentration in the influent of
about 100 mg/1. Alternatively, use other compositions, with about the same DOC
concentrations, that are closer to real sewage. This synthetic sewage may be prepared in
distilled water in a concentrated form and stored at about 1°C for up to one week. When
needed, dilute with tap water. This medium is unsatisfactory, e.g. the nitrogen
concentration is very high and the carbon content relatively low, but nothing better has
been suggested. Some investigators add more phosphate as buffer and extra peptone.
(iii) Domestic sewage. Use fresh settled sewage collected daily from a wastewater
treatment plant receiving predominantly domestic sewage. It should be collected from the
overflow channel of the primary sedimentation tank, or from the feed to the activated
sludge plant, and be largely free from coarse particles. The sewage can be used after
storage for several days at about 4°C, if it is proved that the DOC (or COD) has not
significantly decreased (i.e. <20%) during storage. In order to limit disturbances to the
system, the DOC (or COD) of each new batch should be adjusted before use to an
appropriate constant value, e.g. by dilution with tap water.
(6) Lubricant. Glycerol or olive oil may be used for lubricating the peristaltic pump
rollers: both are suitable for use on silicone rubber tubing.
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(7) Stocks solutions of test substance, (i) For substances of adequate solubility,
prepare stock solutions at appropriate concentrations (e.g. 1 to 5 g/1) in deionized water, or
use synthetic sewage without the organic nutrient portion (e.g. peptone or meat extract).
This method is not suitable for volatile substances without modifications to the tubular
reactors (see paragraph (k)(l)(ii) of this guideline). Determine the DOC and TOC of the
stock solution and repeat the measurements for each new batch. If the difference between
the DOC and TOC is greater than 20%, check the water solubility of the test substance.
Compare the DOC or the concentration of the test substance measured by specific analysis
of the stock solution with the nominal value, to ascertain whether recovery is good enough
(normally >90% can be expected). Ascertain, especially for dispersions, whether DOC can
be used as an analytical parameter or an analytical technique specific for the test substance
is required. Centrifugation of the samples is required for dispersions. For each new batch,
measure the DOC, COD or the test substance by specific analysis.
(ii) Determine the pH of the stock solution. Extreme values indicate that the
addition of the substance may have an influence on the pH of the activated sludge in the
test system. In this case neutralize the stock solution to obtain a pH of 7 ± 0.5 using small
amounts of inorganic acid or base, but avoid precipitation of the test substance.
(1) Procedure - (1) Preparation of organic medium for dosing, (i) Ensure that all
influent and effluent containers and tubing from influent vessels and to effluent vessels are
thoroughly cleaned to remove microbial growth, initially and throughout the test.
(ii) If synthetic sewage is used (see paragraph (k)(5)(ii) of this guideline), prepare it
freshly each day either from the solids or from the concentrated stock solution by
appropriate dilution with tap water. Measure the required amount in a cylinder and add to a
clean influent vessel. Also, where necessary, add the required amount of the stock solution
of the test substance or reference substance to the synthetic sewage before dilution. If it is
more convenient or necessary to avoid loss of the test substance, prepare a separate diluted
solution of the test substance in a separate reservoir and deliver this to the inclined tubes via
a different dosing pump.
(iii) Alternatively (and preferably), use settled domestic sewage (see paragraph
(k)(5)(iii) of this guideline) collected freshly each day if possible.
(2) Operation of rotating tubular reactors, (i) Two identical tubular reactors are
required for the assessment of one test substance, and they are assembled in a constant
temperature room normally at 22 ± 2°C.
(ii) Adjust the peristaltic pumps to deliver 250 ± 25 ml/h of the organic medium
(without test substance) into the inclined tubes, which are rotated at 18 ± 2 rpm. Apply the
lubricant (see paragraph (k)(6)) to the pump tubes initially and periodically through the test
to ensure proper functioning and to prolong the life of the tubing.
(iii) Adjust the angle of inclination of the tubes to the horizontal to produce a
residence time of 125 ± 12.5 sec. for the feed in a clean tube. Estimate the retention time by
adding a non-biological marker (e.g. NaCl or an inert dye) to the feed. The time taken to
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reach peak concentration in the effluent is taken to be the mean retention time; when the
biofilm is fully developed, the retention time can increase by up to about 30 min.
(iv) These rates, speeds and times have been found to give adequate removals
(>80%) of DOC (or COD) and to produce nitrified effluents. The flow rate should be
changed if removal is insufficient or if the performance of a particular treatment plant is to
be simulated. In the latter case, adjust the rate of dosing of the organic medium until the
performance of the reactor matches that of the treatment plant.
(3) Inoculation. Airborne inoculation may be sufficient to start the growth of
micro-organisms when synthetic sewage is used; otherwise add 1 ml/1 of settled sewage to
the feed for 3 days.
(4) Measurements. At regular intervals check that the dose-rates and rotating
speeds are within the required limits. Also, measure the pH of the effluent, especially if
nitrification is expected.
(5) Sampling and analysis, (i) The method, pattern and frequency of sampling are
selected to suit the purpose of the test. For example, take snap (or grab) samples of influent
and effluent, or collect samples over a longer period, e.g. 3-6 h. In the first period, without
test substance, take samples twice per week. Filter the samples through membranes or
centrifuge at about 40,000 m/sec2 for about 15 min (see paragraph (k)(2) of this guideline).
It may be necessary to settle and/or coarse-filter the samples before membrane filtration.
Determine DOC (or COD) at least in duplicate and if required biological oxygen demand
(BOD), ammonium and nitrite/nitrate.
(ii) All analyses should be performed as soon as possible after collection and
preparation of the samples. If analyses have to be postponed, store the samples at about 4°C
in the dark in full, tightly stoppered bottles. If samples have to be stored for more than 48 h,
preserve them by deep-freezing, acidification or by addition of a suitable toxic substance
(e.g. 20 ml/1 of a 10 g/1 solution of mercury (II) chloride). Ensure that the preservation
technique does not influence the results of analysis.
(6) Running-in period. During this period, the surface biofilm grows to reach an
optimal thickness. This usually takes about 2 weeks and should not exceed 6 weeks. The
removal of DOC (or COD) increases and reaches a plateau value (see paragraph (m)(2) of
this guideline). When the plateau has been reached at a similar value in both tubes, one is
selected to be a control for the remainder of the test, during which their performance should
remain consistent.
(7) Introduction of test substance. At this stage add the test substance to the other
reactor at the required concentration, usually 10-20 mg C/l. The control continues to
receive the organic medium alone.
(8) Acclimation period. Continue the twice weekly analyses for DOC (or COD)
and, if primary biodegradability is to be assessed, also measure the concentration of the test
substance by specific analysis. Allow from one to six weeks (or longer under special
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conditions) after the test substance has first been introduced for acclimation to occur.
When the percentage removal reaches a maximum value, obtain 12-15 valid values in the
plateau phase over about 3 weeks, for evaluation of the mean percentage removal. The test
is considered finished if a sufficiently high degree of elimination is reached. Normally, do
not exceed a test duration of more than 12 weeks after the first addition of test substance.
(9) Sloughing of the film. The sudden removal of large quantities of excess film
from the tubes, or sloughing, takes place at relatively regular intervals. To ensure that the
comparability of results is unaffected, allow tests to cover at least two full cycles of
growing and sloughing.
(m) Data and reporting - (1) Treatment of results. Calculate the percentage DOC
(or COD) elimination of the test substance at sampling time (Dst) using equation [1].
Dst=100[Cs-(Et-E0t)]/Cs% [1]
where Cs = concentration of DOC (or COD) in the influent due to the test substance,
preferably estimated from the concentration in, and volume added, of the stock
solution (mg/1);
Et = measured DOC (or COD) in the test effluent at time t (mg/1);
Eot = measured DOC (or COD) in the control effluent at time t (mg/1).
Repeat the calculation for the reference substance, if applicable.
(2) Performance of the control reactor, (i) The degree of DOC (or COD)
elimination (Dmt) for the organic medium in the control reactors at each sampling time is
helpful information in assessing the biodegradative activity of the biofilm during the test.
Calculate the percentage elimination from equation [2].
Dmt=100(l-Eot/Cm)% [2]
where Cm = DOC (or COD) of the organic medium in the control influent (mg/1).
Eot = measured DOC (or COD) in the control effluent at time t (mg/1).
(ii) Calculate the percentage removal (Dpt) of the parent (test) substance at each time
point, if measured by a specific analytical method, from equation [3].
Dpt = 100(1-Set/Si) % [3]
where S; = measured or, preferably, estimated concentration of test substance in the
test influent (mg/1)
Set = measured test substance concentration in the test effluent at time t (mg/1)
If the analytical method gives a positive value in unamended sewage equivalent to
Sm mg/1, calculate the percentage removal of test substance at each time point using
equation [4].
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Dpt = 100 (Si - Set + Sm)/( Si + Sm) % [4]
where S; = measured or, preferably, estimated concentration of test substance in the
test influent (mg/1)
Set = measured test substance concentration in the test effluent at time t (mg/1)
Sm = measured concentration of test substance in unamended sewage (mg/1)
(3) Expression of test results. Plot the percentage elimination Dst, Dmt, and Dpt
(from equation [3] or equation [4]), if available, versus time. See paragraph (m)(2) of U.S.
EPA Fate, Transport and Transformation Test Guidelines no. 835.3240 (cited in
paragraph (n)(ll) of this guideline). Take the mean (expressed to the nearest whole
number) and standard deviation of the 12-15 values for Dst (and for Dpt, if available),
obtained in the plateau phase, as the percentage removal of the test substance. From the
shape of the elimination curve, some conclusions may be drawn about the removal
processes.
(4) Adsorption. If high DOC elimination is observed for the test substance at the
beginning of the test, it is probably eliminated by adsorption onto the biofilm. It may be
possible to prove this by determining the adsorbed test substance on solids sloughed from
the film. It is not usual for the elimination of DOC of adsorbable substances to remain
high throughout the test; normally, removal is high initially but gradually falls to a lower,
equilibrium value. However, acclimation can occur over time, such that removal could
remain or increase to a high level by the end of the measurement period, and reflect some
combination of sorption and biodegradation.
(5) Lag phase. As in static screening tests, many test substances exhibit a lag
phase before biodegradation becomes evident. In the lag phase, acclimation or adaptation
of the degrading bacteria takes place with little or no evident removal of the test
substance. As growth of bacteria at the expense of the test substance occurs, its
biodegradation becomes measurable. Normally, the lag phase is considered to end and the
degradation phase begins when 10% of the initial amount of test substance is removed
(after allowing for adsorption, if it occurs). This value is somewhat arbitrary but has
become common practice. The lag phase is often highly variable and poorly reproducible.
(6) Plateau phase. The plateau phase of an elimination curve in a continuous test
is defined as that phase in which the maximum degradation takes place. The plateau phase
should last at least 3 weeks and have at least 15 measured removal values.
(7) Mean degree of elimination of the test substance. Calculate the mean value
from the elimination values Dst (and Dpt, if available) of the test substance at the plateau
phase. Rounded to the nearest whole number (1%), it is the degree of elimination of the test
substance. It is also recommended to calculate the 95% confidence interval of the mean
value. In a similar way calculate the mean degree of elimination of the organic medium in
the control vessel (Dmt).
(8) Indication of biodegradation. (i) If the test substance does not adsorb
significantly onto the biofilm and the elimination curve has the typical shape of a
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biodegradation curve with lag, degradation and plateau phases, the measured elimination
can safely be attributed to biodegradation. If on the other hand high initial removal is
observed, this test cannot differentiate between biological and abiotic elimination
processes. In such cases, and in other cases where there is any doubt about biodegradation
(e.g. if stripping takes place), analyze adsorbed test substance on samples of the film or
perform additional static tests for biodegradability based on parameters clearly indicative of
biological processes. Such tests include ready biodegradability tests based on oxygen
uptake (OECD 301C, 301D and 301F) or CO2 production (OECD 301B or 310) (see
paragraph (n)(l) of this guideline); use as inoculum pre-exposed biofilm from the
appropriate test system.
(ii) If both DOC removal and specific substance removal have been measured,
significant differences between the percentages removed (the former being lower than the
latter) indicate the presence in the effluents of intermediate organic products, which may
be more difficult to degrade than the parent substance.
(9) Validity of test results, (i) Information on the normal biodegradation behavior
of the inoculum is obtained from the degree of elimination of the organic medium in the
control unit (see paragraph (m)(2)(i) of this guideline). Consider the test to be valid if the
degree of DOC (or COD) elimination (Dmt) in the control units is >80% after 2 weeks
operation and no unusual observations have been made.
(ii) If a readily biodegradable (reference) substance has been tested, the degree of
biodegradation should be >90% and the difference between duplicate values should not be
greater than 5%. If these two criteria are not met, review the experimental procedures
and/or obtain domestic sewage from another source.
(iii) Similarly, differences between biodegradation values from duplicate units (if
used) treating a test substance should not differ by more than 5%. If this criterion is not met
but the removals are high, continue analysis for a further three weeks. If removal is low,
investigate the inhibitory effects of the test substance if not known, and repeat the test at a
lower concentration of test substance, if that is feasible.
(10) 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
variability.
(11) Test report. The test report should include the following:
(i) Test substance
Identification data;
- Physical state and, where relevant, physical/chemical properties.
(ii) Test conditions
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- Type of test system; and any modifications for testing insoluble or volatile
substances;
- Type of organic medium;
- Proportion and nature of industrial wastes in sewage, if used and if known;
Method of inoculation;
- Test substance stock solution - DOC and TOC content; how prepared, if a
suspension; test concentration(s) used, reasons if outside range 10-20 mg DOC/1;
method of addition; date first added; any changes in concentration;
- Mean hydraulic residence time (with no growth); rotational speed of tube;
approximate angle of inclination, if possible;
- Details of sloughing; time and intensity;
Test temperature and range;
Analytical techniques employed.
(iii) Test results
All measured data for DOC, COD, specific analyses, pH, temperature, N
compounds, if relevant;
All calculated data for Dt (or Dtc), DB, Ds, in tabular form and as elimination
curves;
- Information on lag and plateau phases, test duration, degree of elimination
of the test substance and reference substance (if included) and that of the organic
medium in the control unit, together with statistical data and statements on
biodegradability and validity of the test;
- Discussion of results.
(n) Literature.
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(3) Truesdale, G.A., K. Jones and K.G. Vandyke (1959). Removal of synthetic
detergents in sewage treatment processes: trials of a new biologically attackable material.
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(4) Baumann, U., G. Kuhn and M. Benz (1998). Einfache Versuchsanordnung zur
Gewinnung gewasserokologisch relevanter Daten. UWSF - Z. Umweltchem. Okotox. 10,
214-220.
(5) Gloyna, E.F., R.F. Comstock and C.E. Renn (1952). Rotary tubes as
experimental trickling filters. Sewage Ind. Waste 24, 1355-1357.
(6) Kumke, G.W. and C.E. Renn (1966). LAS removal across an institutional
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trickling filter. J. Am. Oil Chem. Soc. 43, 92-94.
(7) Tomlinson, T.G. and D.H.M. Snaddon (1966). Biological oxidation of sewage
by films of microorganisms. Int. J. Air Wat. Pollut. 10, 865-881.
(8) HMSO (1982). Methods for the examination of waters and associated materials.
Assessment of biodegradability, 1981. Her Majesty's Stationery Office, London.
(9) HMSO (1984). Methods for the examination of waters and associated materials.
Methods for assessing the treatability of chemicals and industrial wastewaters and their
toxicity to sewage treatment processes, 1982. Her Majesty's Stationery Office, London.
(10) ASTM (1993). Annual Book of ASTM Standards, Volumes 11.01 and 11.02 on
Water and Environmental Technology, and Volume 14.02 on General Methods and
Instrumentation. American Society for Testing and Materials, Philadelphia, PA.
(11) US EPA (2007). Simulation test—aerobic sewage treatment: A. Activated
sludge units. Fate, Transport and Transformation Test Guidelines 835.3240, U.S.
Environmental Protection Agency, Washington, DC.
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