United States Prevention, Pesticides EPA712-C-08-004
Environmental Protection And Toxic Substances October 2008
Agency (7101)
4>EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3240
Simulation Test—Aerobic
Sewage Treatment: A.
Activated Sludge Units
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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 835.3240: Simulation test—aerobic sewage treatment: A. activated
sludge units
(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
the activated sludge process. 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, it is assumed that results can be extrapolated to predict elimination in
full-scale treatment plants, provided that critical parameters such as temperature, sludge
retention time (SRT) and initial test substance concentration are in the same range.
Normally tests are performed at only one nominal concentration of sludge solids or one
nominal SRT. However, variants of the principle test method are also described in which
test substance is present at low concentrations (e.g. ng/1) considered more typical
of wastewaters; and in which the SRT is controlled within much narrower limits as
happens in full-scale treatment plants.
(c) Overview—(1) General Concepts. In the 1950s it was realized that the newly
introduced surfactants caused excessive foaming in wastewater treatment plants and in
rivers. They were not fully removed in aerobic treatment and in some cases limited the
removal of other organic matter. This instigated many investigations into how surfactants
could be removed from wastewaters and whether new substances produced by industry
were amenable to wastewater treatment. Because it would have been impractical and very
costly to distribute each new material and to monitor large-scale treatment plants, model
units have been developed that represent the two main types of aerobic biological
wastewater treatment (activated sludge and percolating, or trickling, filtration).
(2) Activated sludge units, (i) Model activated sludge units have been described
ranging in size from 300 ml up to about 2000 ml. Some closely mimic full-scale plants,
having sludge settlement tanks with settled sludge being pumped back to the aeration
tank, while others provide no settlement facilities; e.g. Swisher (see paragraph (p)(l) of
this guideline). The size of the apparatus is a compromise. On the one hand, it must be
large enough for successful mechanical operation and for the provision of sufficient
volume of samples without affecting the operation, while on the other hand it should not
be so large that it demands excessive space and materials.
(ii) Two forms of apparatus that have been extensively and satisfactorily used are
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the Husmann units (see paragraph (p)(2) of this guideline) and porous pot units (see
paragraphs (p)(3) and (p)(4) of this guideline), first used in the study of surfactants; these
are described in this guideline. Others have also been used satisfactorily, e.g. Eckenfelder
(see paragraph (p)(5) of this guideline). Because of the relatively high cost and effort of
applying this test, simpler and cheaper screening tests, now embodied in 301 A-F, were
investigated in parallel. Experience with many surfactants and other chemical substances
have shown that substances that passed the screening tests (i.e. were readily
biodegradable) also degraded in the activated sludge simulation test. Some of those
failing the screening tests passed the inherent biodegradability tests (302 A, B) but only
some of this latter group were degraded in the activated sludge test, while those substances
that failed tests for inherent biodegradability did not degrade in the activated sludge test
(see paragraphs(p)(6), (p)(7) and (p)(8) of this guideline).
(iii) For some purposes simulation tests carried out under a single set of operating
conditions are sufficient. The results are expressed as a percentage removal of the test
substance or of dissolved organic carbon (DOC). A description of such a test is given in
this guideline. However, unlike the previous 303A (see paragraph (a)(2) of this guideline),
which described only one type of apparatus treating synthetic sewage in the coupled mode
using a relatively crude method of sludge wastage, this text offers several variations.
Alternatives to the type of apparatus, mode of operation, sewage and sludge wastage
removal are described. This text closely follows that of the ISO Standard 11733 (see
paragraph (p)(9) of this guideline), which was carefully scrutinized during its preparation,
though the method has not been subject to a ring test.
(iv) For other purposes the concentration of the test substance in the effluent needs
to be known more accurately and for this a more extensive method is needed. For
example, the sludge wastage rate must be more precisely controlled throughout each day
and throughout the period of the test, and units have to be run at several wastage rates. For
a fully comprehensive method, tests should also be run at two or three different
temperatures: such a method is described by Birch (see paragraphs (p)(10) and (p)(ll) of
this guideline) and summarized in paragraph (n) of this guideline. However, present
knowledge is insufficient to decide which of the kinetic models is (are) most appropriate for
describing the biodegradation of substances in wastewater treatment and in the aquatic
environment generally. The application of Monod kinetics, given in paragraph (n) as an
example, is limited to substances present at 1 mg/1 and above, but in the opinion of some,
even this remains to be substantiated. Tests at concentrations more truly reflecting those
found in wastewaters are described in paragraph (m) of this guideline.
(3) Percolating (trickling) filters, (i) Much less attention has been given to
model percolating filters, perhaps because they are more cumbersome and less compact
than activated sludge plant models. Gerike et al (see paragraph (p)(12) of this guideline)
developed trickling filter units and operated them in the coupled mode (see paragraphs
(a)(2) and (p)(12) of this guideline). These filters were relatively large (height 2 m;
volume 60 1) and each required as much as 2 1/h of sewage. Baumann et al (see paragraph
(p)(13) of this guideline) simulated trickling filters by inserting polyester fleece strips into
1 m tubes (14 mm int. diameter) after the strips had been immersed in concentrated
activated sludge for 30 min. The test substance as sole C source in a mineral salts solution
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was fed down the vertical tube and biodegradation was assessed from measurements of
DOC in the effluent and CC>2 in the issuing gas.
(ii) Biofilters have been simulated in another way (see paragraph (p)(14) of this
guideline): the inner surfaces of rotating tubes, inclined at a small angle to the horizontal,
were fed with sewage (250 ml/h) with and without the test substance, and the collected
effluents were analyzed for DOC and/or the specific substance. This method is described
in part B of OECD 303 (cited in paragraph (a)(2) of this guideline) and the corresponding
U.S. EPA Fate, Transport and Transformation Test Guidelines no. 835.3260, on biofilms
(cited in paragraph (p)(15) of this guideline).
(d) Principle of the test. (1) This method is designed to determine the
elimination and the primary and/or ultimate biodegradation of water-soluble organic
substances by aerobic microorganisms in a continuously operated test system simulating
the activated sludge process. An easily biodegradable organic medium and the organic
test substance are the sources of carbon and energy for the microorganisms.
(2) Two continuously operated test units are run in parallel under identical
conditions that are chosen to suit the purpose of the test. Normally the mean hydraulic
retention time (HRT) is 6 h and the mean sludge age (= sludge retention time or SRT) is 6
to 10 d. Sludge is wasted by one of two methods. The test substance is normally added at a
concentration of between 10 mg/1 and 20 mg/1 as DOC to the influent (organic medium) of
one of the units. The second unit is used as a control to determine the biodegradation of the
organic medium.
(3) In frequently taken samples of the effluents, the DOC (preferably) or chemical
oxygen demand (COD) is determined, together with the concentration of the test substance
(if required) by specific analysis, in the effluent from the unit receiving the test substance.
The difference between the effluent concentrations of DOC or COD in the test and control
units is assumed to be due to the test substance or its organic metabolites. This difference is
compared with the influent concentration of DOC or COD due to the added test substance
in order to determine the extent of its elimination.
(4) Biodegradation normally can be distinguished from bioadsorption by careful
examination of the elimination vs. time curve, and usually can be confirmed by applying a
test for ready biodegradation using an acclimated inoculum taken from the activated sludge
unit receiving test substance.
(e) Applicable ASTM standards. Refer to the documents referenced in paragraph
(p)(16) of this guideline for the standards 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).
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(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 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) General. The purity, water solubility,
volatility and sorption characteristics of the test substance should be known to enable
correct interpretation of results. Normally, volatile and insoluble substances cannot be
tested unless special precautions are taken. 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. theoretical oxygen demand (ThOD), DOC and COD.
(2) Poorly water-soluble substances, (i) Few reports seem to have been published
on subjecting poorly water-soluble and insoluble substances to tests simulating wastewater
treatment (see paragraphs (p)(17), (p)(18) and (p)(19) of this guideline).
(ii) There is no single method of dispersal of the test material that is applicable to all
insoluble substances. Two of the four methods described in ISO 10634 (see paragraph
(p)(20) of this guideline) appear suitable for attempting to disperse test substances for
activated sludge simulation testing; they are the methods using emulsifying agents and
ultrasonic energy. The stability over at least a 24-h period of the resulting dispersion should
be established. Suitably stabilized dispersions, contained in a constantly stirred reservoir,
would then be dosed to the aeration tank separately from the domestic (or synthetic)
sewage.
(iii) If the dispersions are stable, investigate how the test substance concentration
can be determined in the dispersed form. It is unlikely that DOC will be suitable, so that a
specific analytical method for the test substance would have to be established that could be
applied to effluents, effluent solids and activated sludge. The fate of the test substance in
the simulation of the activated sludge process would then be determined for both liquid and
solid phases. Thus, a mass balance would be established to decide whether the test
substance had been biodegraded. However, this would indicate only primary
biodegradation. Demonstration of ultimate biodegradation should be attempted by applying
a respirometric test for ready biodegradability (301 B, C or F) using as the inoculum sludge
exposed to the test substance in the simulation test.
(3) Volatile substances—(i) Applicability. The application of wastewater
treatment simulations to volatile substances is both debatable and problematic. As with
poorly water-soluble test substances, very few reports seem to have been published
describing simulation tests using volatile substances. A conventional type of complete-
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mixing apparatus is adapted by sealing the aeration and settling vessels, measuring and
controlling the air flow using flowmeters, and passing the exit gas through traps to collect
volatile organic matter. In some cases, a vacuum pump has been used to draw the exit gas
through a cold trap or a purge-trap containing Tenax and silica gel for gas
chromatographic analyses. The test substance present in the trap can be determined
analytically.
(ii) Stripping test. The test is carried out in two parts. The units are first operated
without sludge but with the synthetic wastewater plus test substance being pumped into the
aeration tank. Influent, effluent and exit gas samples are collected and analyzed for the test
substance for a few days. From the data collected, the percentage (Rvs) of the test material
stripped from the system may be calculated.
(iii) Biological test. Then the biological (i.e. with sludge) test is performed under
operating conditions identical to those in the stripping study. DOC or COD measurements
are also made to check that the units are performing efficiently. Occasional analyses are
made to determine the test substance in the influent, effluent and exit gas in the first part of
the test; and after acclimation, more frequent analyses are made. Using the data obtained
after steady-state removal is achieved, the percentage removal of the test substance from
the liquid phase by all processes (Rx) (physical and biological) may be calculated, as well
as the proportion (Rv) stripped from the system.
(iv) Calculations.
(A) In the non-biological (stripping) test, the percentage (Ryp) of the test material
stripped from the system is
%RVP = SVP/SIP xioo
where RVP = removal of test substance by volatilization (%),
SVP = test substance collected in trap expressed as equivalent concentration in
liquid phase (mg/1),
SIP = test substance concentration in influent (mg/1).
(B) In the biological test, the percentage (Rv) of the test material stripped from the
system is
%Rv = Sv/Si x 100
where RV = removal of test substance by volatilization in biological test (%),
Sv = test substance collected in trap in biological test, expressed as equivalent
concentration in liquid influent (mg/1),
Si = test substance concentration in influent (mg/1).
(C) In the biological test, the percentage (Rx) of the test substance removed by all
processes is given by:
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%RT = (l-SE/Si) xlOO
where SE = concentration of test substance in the (liquid) effluent (mg/1).
(D) Thus, the percentage (RBA) removed by biodegradation plus adsorption can be
calculated from:
%RBA = (RT-RV)
Separate tests should be carried out to determine whether the test substance is
adsorbed; if it is, then a further correction may be made.
(E) A comparison between the proportion of test substance stripped from the
biological (Ry) and non-biological test (Ryp) systems indicates the overall effect that
biological treatment has had on the emission of the test substance into the atmosphere.
Example: Benzene
Sludge retention time = 4 days
A synthetic sewage; retention time = 8h
SIP = Si = 150 mg/1
S Vp = 150 mg/1 ( SEP = 0)
Sv = 22.5 mg/1
SE = 50
Thus, RVp = 1 00%, Rv = 15%
RT = 100% and RBA = 85%.
If benzene is assumed not to be adsorbed onto sludge, the results suggest that it is removed
by a combination of stripping and (mostly) biodegradation.
(4) Inhibitory substances — (i) 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.
(ii) A substance (or wastewater) may not be degraded or removed in the simulation
test and may even have an inhibitory effect on the sludge microorganisms. Other substances
may be biodegraded at low concentrations but inhibitory at higher concentration. Inhibitory
effects may have been revealed at an earlier stage or may be determined by applying a
toxicity test, using an inoculum similar or identical to that used in the simulation test (see
paragraph (p)(21) of this guideline). Useful microbial toxicity tests include inhibition of
oxygen uptake (OECD Guideline 209; ISO Standard 8192)(see paragraph (p)(22) of this
guideline) and inhibition of growth of sludge organisms (ISO 15522)(see paragraph (n)(24)
of this guideline).
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(iii) In the activated sludge simulation test, if the test substance inhibits activity of
the activated sludge, the percentage removal of DOC (and biochemical oxygen demand or
BOD; COD; or NH4+ ) of the organic medium on treatment will be decreased by the
presence of the test substance. If this occurs, the test should be repeated reducing the
concentration of the test substance until a level is reached at which no inhibition occurs,
and perhaps further reducing the concentration until the test substance is biodegraded.
However, if the test substance (or wastewater) has adverse effects on the process at all
concentrations tested, the indications are that the substance is difficult, if not impossible, to
treat biologically. In this case it may be worth repeating the test with activated sludge from
a different source and/or subjecting the sludge to a more gradual acclimation.
(iv) Conversely, if the test substance is removed by biodegradation at the first
attempt in this test, its concentration could be increased if it is considered useful to know
whether the substance could be inhibitory at some higher level. It also should be
remembered that the activated sludge population can change, so that with time the
microorganisms may become more tolerant of an inhibitory substance.
(v) The overall percentage removals RO, for BOD, DOC and COD for the test and
control units are calculated as shown in this paragraph:
where I = influent concentration of BOD, DOC or COD, for test or control vessels (mg/1)
E = respective effluent concentrations (mg/1).
I and E must be corrected for the DOC due to the test substance in the test units; otherwise
the calculations of percentage inhibition will be incorrect.
The degree of inhibition caused by the presence of the test material can be calculated from:
% inhibition = 100 (Rc - Rt)/ Rc
where RC = percentage removal in the control vessels
Rt = percentage removal in the test vessels
(g) Pass levels. (1) Because this test is intended to simulate activated sludge
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 activated sludge treatment systems.
(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.
(3) Relatively few substances (e.g. surfactants) are present in sewage at the
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concentrations (about 10 mg C/l) used in this test. Some substances may be inhibitory at
these concentrations, while the kinetics of removal of others may be different at low
concentrations. A more accurate assessment of their biodegradation could be made by using
modified methods, using realistically low concentrations of the test substance, and the data
collected could be used to calculate kinetic constants. However, the necessary experimental
techniques have not yet been fully validated and neither have kinetic models, which
describe the biodegradation reactions, been established.
(h) Reference substances. To ensure that the experimental procedure is being
carried out correctly, it is useful to include one or more reference substances, i.e. a
substance whose behavior is known, when test substances are investigated. Examples of
such substances are adipic acid, 2-phenyl-phenol, 1-naphthol, diphenic acid and 1-
naphthoic acid (see paragraphs (p)(6), (p)(7) and (p)(8) of this guideline).
(i) Reproducibility of test results. (1) There have been far fewer reports of
simulation tests than of tests for ready biodegradability. Reproducibility between
(simultaneous) replicates is good (within 10-15%) for test substances degraded by 80% or
more, but for less well-degraded substances, variability is greater. Also, with some
borderline substances widely disparate results (e.g. 10% vs. 90%) have been recorded on
different occasions within the 9 weeks allowed in the test.
(2) Little difference has been found in results obtained with the two types of
apparatus (see paragraph (c)(2)(ii) of this guideline), but some substances have been more
extensively and consistently degraded in the presence of domestic sewage than with OECD
synthetic sewage.
(j) Description of the test method—(1) General. The test system for one test
substance consists of a test unit and a control unit; but when only specific analyses are
performed (i.e. primary biodegradation is determined), only a test unit is required. One
control unit can be used for several test units receiving either the same or different test
substances. In the case of coupling (see paragraph (1)(5) of this guideline) each test unit
should have its own control unit. The test system may be either a Husmann unit (see Figure
1), or a porous pot (see Figures 2 and 3). In both cases, storage vessels of sufficient size for
the influents and effluents are needed, as well as pumps to dose the influent, either mixed
with solution of the test substance or separately.
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Figure 1. Activated sludge treatment model test system: Husmann unit
Air
B
\,
'Air
I
t
H
V
C
G
D
I
F
A. Storage vessel
B. Dosing pump
C. Aeration chamber (3 1 capacity)
D. Settling vessel
E. Air lift pump
F. Collection vessel
G. Aerator
H. Air flow meter
Figure 2. Activated sludge treatment model test system: porous pot
A. Storage vessel
B. Dosing pump
C. Porous aeration vessel
D. Outer impermeable vessel
c
F
G
Air
E. Collection vessel
F. Diffuser
G. Air flow meter
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Figure 3. Detailed diagram of typical porous pot aeration vessel
Internal diameter of porous pot
14cm
Internal diameter of
outer pot
10.6cm
Side of cone outer
9.9cm
Side of cone inner
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(2) Activated sludge model system: Husmann unit. Each unit consists of an
aeration vessel with a known capacity of about 3 1 of activated sludge and a separator
(secondary clarifier) that holds about 1.5 1 (see Figure 1). To some extent the volumes can
be changed by adjusting the height of the separator. Vessels of different sizes are
permissible if they are operated with comparable hydraulic loads. If it is not possible to
keep the temperature in the test room in the desired range, the use of water-jacketed vessels
with temperature controlled water is recommended. An airlift pump or a dosing pump is
used to recycle the activated sludge from the separator to the aeration vessel, either
continuously or intermittently at regular intervals.
(3) Activated sludge model system: porous pot. (i) The porous pot system (see
Figures 2 and 3) consists of an inner, porous cylinder with a conical bottom held in a
slightly larger vessel of the same shape, but made of an impervious plastic material. A
suitable material for the porous vessel is porous polyethylene of maximum pore size 90 jim
and 2 mm thickness. Separation of the sludge from the treated organic medium is effected
by differential passage through the porous wall. Effluents collect in the annular space from
where it overflows into the collecting vessel. No settlement occurs and hence there is no
sludge return. The whole system may be mounted in a thermostatically controlled water
bath.
(ii) The feature that distinguishes this test from other activated sludge simulation
tests is the retention of the activated sludge by the porous liner, which eliminates the need
for a secondary clarifier and facilitates control of a critical parameter, the SRT.
(iii) 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, dissolved oxygen (DO) concentration, 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.
(iv) Porous pots can be completely sealed and tests using 14C-labeled test substances
are possible. Carbon dioxide in the exhaust gas and bicarbonate in the effluent can be used
together to assess the extent of mineralization, and levels of radiolabel in the sludge and in
the aqueous phase may also be determined.
(4) Aeration, (i) For aeration of the sludge in the aeration vessels of both systems,
suitable techniques are required, for example sintered cubes (diffuser stones) and
compressed air. The air should be cleaned, if necessary, by passing it through a suitable
filter and washing. Sufficient air should pass through the system to maintain aerobic
conditions and to keep sludge floes in suspension at all times during the test.
(ii) An oil-free compressor is needed for supplying compressed air to the aeration
vessel.
(5) Pumps. Suitable pumps are needed for dosing activated sludge units with test
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substance solutions and sewage at the correct rates (0-1.0 mL/min for test substance
solutions, 5-20 mL/min for sewage). An additional pump is needed 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 shown in this paragraph:
flow = [pump throw (mL/min) x pumping time (sec)1
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 wastage rate is 0.5 mL/min.
(6) Filtration apparatus or centrifuge. A device is needed for filtering samples
that uses membrane filters of suitable porosity (nominal aperture diameter 0.45 |im) and
that adsorbs soluble organic substances and releases organic carbon to a minimum degree.
If filters are used that release organic carbon, first wash the filters carefully with hot water
to remove teachable organic carbon. Alternatively, a centrifuge capable of producing
40,000 m/s2 may be used.
(7) Other equipment, (i) Analytical apparatus as needed, to determine
(A) DOC and total organic carbon (TOC), or COD;
(B) Specific substance;
(C) Suspended solids, pH, oxygen concentration in water;
(D) Temperature, acidity and alkalinity;
(E) Ammonium, nitrite and nitrate, if the test is performed under nitrifying
conditions.
(ii) Additional apparatus
(A) 1-L sample bottles to hold test substance dosing solutions.
(B) Silicone rubber tubing: Bore = 0.5 mm ID.
(C) Polypropylene transmission tubing.
(D) Tube connectors.
(E) Diffuser stones.
(F) 25-mL measuring cylinders.
(G) 1-mL graduated pipets.
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(H) Stopwatch.
(I) 40-mL sample bottles for collection of samples for waste sludge and mixed
liquor suspended solids determinations.
(J) Thermometer having range 0 to 50°C.
(K) 1- and 2-1 measuring cylinders for each pot to collect waste sludge.
(L) Timer for sludge wastage pump allowing intermittent operation, unless sludge is
wasted manually.
(M) Right-angled plastic tube to fit on one end of the air line to ensure complete
mixing of activated sludge.
(N) If porous pots are operated in the sealed mode, plastic tubing to fit on the pot
effluent port as shown in Figure 3 to balance the back pressure caused by the CO2 traps.
(8) 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 (d)
of this guideline, on applicable ASTM standards.
(9) Organic medium—(i) General. Synthetic sewage, domestic sewage or a
mixture of both can be used as the organic medium. It has been shown (see paragraphs
(p)(8) and (p)(ll) of this guideline) that the use of domestic sewage alone often gives
increased percentage DOC removal and even allows the removal and biodegradation of
some substances that are not biodegraded when synthetic sewage is used. Also, the constant
or intermittent addition of domestic sewage often stabilizes the activated sludge, including
the crucial ability to settle well. Thus, 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 organic medium may require the
addition of a suitable buffer (sodium hydrogen carbonate or potassium dihydrogen
phosphate) if it is of low acidity or alkalinity, to maintain a pH of about 7.5 ± 0.5 in the
aeration vessel during the test. The amount of buffer to be added, and when to add it, has to
be decided in each individual case. When mixtures are used either continuously or
intermittently, the DOC (or COD) of the mixture must be kept at an approximately constant
value, e.g. by dilution with water.
(ii) Synthetic sewage. Dissolve in each liter of tap water peptone, 160 mg; meat
extract, 110 mg; urea, 30 mg; anhydrous dipotassium hydrogen phosphate (K2HPO4), 28
mg; sodium chloride (NaCl), 7 mg; calcium chloride dihydrate (CaQ2-2H2O), 4 mg;
magnesium sulfate heptahydrate (Mg2SO4'7H20), 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 another composition, with about the same DOC concentration that is
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closer to real sewage. If a less-concentrated influent is required, dilute the synthetic sewage
with tap water (for example 1:1) to obtain a concentration of about 50 mg/1. Such a weaker
influent will allow better growth of nitrifying organisms and this modification should be
used if the simulation of nitrifying wastewater plants is to be investigated. Synthetic sewage
may be prepared with 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 not entirely satisfactory,
e.g. the nitrogen concentration is very high and 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, prior to
primary sedimentation, from the overflow channel of the primary sedimentation tank, or
from the feed to the activated sludge plant, and be largely free of coarse particles. The
sewage can be used after storage for several days (but storage time generally should not
exceed 7 d) at about 4°C, if it is first established 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.
(iv) Activated sludge. Collect activated sludge for inoculation from the aeration
tank of a well operated wastewater treatment plant or from a laboratory-scale activated
sludge unit, treating predominantly domestic sewage.
(10) Stock 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).
For insoluble and volatile substances, see paragraphs (f)(2) and (f)(3) of this guideline.
Determine the DOC and total organic carbon (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.
(11) Other reagents and materials, (i) Compressed air (filtered for oil and water)
for aeration of porous pots.
(ii) Extraction apparatus and solvent for hydrophobic test substances.
14
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(iii) Glycerol for lubricating the rollers of the peristaltic pumps.
(iv) Sodium hypochlorite solution.
(k) 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 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 activated sludge 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.
(1) Procedure—(1) Preparation of the inoculum, (i) Inoculate the test system at
the beginning of the test with either activated sludge or an inoculum containing a low
concentration of microorganisms. Keep the inoculum aerated at room temperature until it is
used, and use it within 24 h. In the first case, take a sample of activated sludge from the
aeration tank of an efficiently operated biological wastewater treatment plant, or a
laboratory treatment plant, that receives predominantly domestic sewage. If nitrifying
conditions are to be simulated, take sludge from a nitrifying wastewater treatment plant.
Determine the concentration of suspended solids and, if necessary, concentrate the sludge
by settling so that the volume added to the test system is minimal. Ensure that the starting
concentration of dry matter is about 2.5 g/1.
(ii) In the second case, use 2 ml/1 to 10 ml/1 of an effluent from a domestic
biological wastewater treatment plant as inoculum. To maximize microbial diversity, it may
be helpful to add inocula from various other sources, for example surface water. In this
case, the activated sludge will develop and grow in the test system.
(2) Husmann unit procedure—(i) Dosage of organic medium. (A) Ensure that
influent and effluent containers and tubing between influent and effluent vessels are
thoroughly cleaned, to remove microbial growth initially and for the duration of the test.
Assemble the test systems in a room where the temperature is controlled (normally in the
range 20-25°C) or use water-jacketed test units. Prepare a sufficient volume of the required
organic medium (see paragraph (j)(9) of this guideline). Initially fill the aeration vessel and
the separator with the organic medium, then add the inoculum. Start the aeration such that
the sludge is kept in suspension and in an aerobic state, and begin dosing the influent and
recycling the settled sludge. Dose organic medium from storage vessels into the aeration
15
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vessels of the test and control units, and collect the respective effluents in similar storage
vessels. To get the normal HRT of 6 h, the organic medium is pumped at 0.5 1/h. To
confirm this rate, measure the daily amount of organic medium dosed by noting the
reduction in volumes of the medium in the storage vessels. The dosing mode may need to
be modified if, for example, the study objective is to determine the effects of intermittent
release or shock loading of substances.
(B) If the organic medium is prepared for use for a period >1 d, it should be cooled
to about 4°C, or another appropriate method should be used to prevent microbial growth
and biodegradation outside the test units. If synthetic sewage is used, it is possible to
prepare, and store at about 4°C, a concentrated stock solution (e.g. lOx the normal
concentration. This stock solution can be mixed with the appropriate volume of tap water
immediately before use. Alternatively, it can be pumped directly while the appropriate
amount of tap water is pumped separately.
(ii) Dosage of test substance. (A) Add an appropriate volume of the stock solution
of test substance to the storage vessel of the influent, or dose it directly with a separate
pump into the aeration vessel. The normal mean test substance concentration in the influent
should be between 10 and 20 mg/1 as DOC, with an upper concentration of no more than 50
mg/1. If the water solubility of the test substance is low (see paragraph (e)(2) of this
guideline) or if toxic effects are considered likely (see paragraph (e)(4) of this guideline),
reduce the concentration to 5 mg/1 as DOC or even less, but only if a suitable specific
analytical method for test substance is available. Dispersed test substances that are poorly
soluble in water may be added using special dosing techniques: see paragraph (e)(2) of this
guideline.
(B) Start adding the test substance after a period in which the system has stabilized
and is removing DOC of the organic medium efficiently (i.e. by about 80%). It is important
to check that all units are working equally efficiently before the addition of test substance;
if they are not, it usually helps to mix the individual sludges and to re-dispense equal
volumes to individual units. When an inoculum of (about) 2.5 g/1 (dry weight) activated
sludge is used, the test substance may be added from the start of the test since directly
adding increasing amounts from the beginning has the advantage that the activated sludge
may be better able to adapt to the test substance. However test substance is added, it is
recommended that the relevant flow rate and/or the volumes in the storage vessel(s) be
measured at regular intervals.
(iii) Handling of activated sludge. (A) The concentration of activated sludge solids
normally stabilizes between limits during the test, independent of the inoculum used, in the
range of 1 to 3 g/1 (dry weight) depending on the quality and concentration of the organic
medium, operating conditions, the nature of the microorganisms present and the influence
of the test substance.
(B) Either determine the suspended solids in the aeration vessels at least weekly and
discard surplus sludge to maintain the concentration at 1 g/1 to 3 g/1 (dry weight), or control
the mean SRT at a constant value usually in the range 6 days to 10 days. If, for example, a
sludge retention time of 8 days is selected, remove daily 1/8 of the volume of the activated
16
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sludge in the aeration vessel and discard it. Perform this action on a daily basis or,
preferably, by means of an automatic intermittently operating pump. Maintaining the
concentration of suspended solids at a constant level, or within narrow limits, does not
ensure a constant SRT, which is the operating variable that determines the value of the
concentration of test substance in the effluent.
(C) Throughout the test, remove, at least daily, any sludge adhering to the walls of
the aeration vessel and the separator so that it is resuspended. Check and clean regularly all
tubes and tubing to prevent growth of biofilm. Recycle the settled sludge from the separator
to the aeration vessel, preferably by intermittent pumping.
(D) Poor settling and loss of sludge may occur in the Husmann units. These may be
rectified by employing one or more of the actions listed in paragraphs (l)(2)(iii)(D)(7)
through (l)(2)(iii)(D)(<5) of this guideline for both test and control units:
(1) Fresh sludge or a flocculant (for example 2 ml/vessel of 50 g/1 Feds) can be
added at regular intervals, e.g. weekly, but ascertain that no reaction or precipitation of the
test substance occurs with
(2) The air-lift pump can be replaced by a peristaltic pump, thus enabling a sludge
recirculation flow that equals the influent flow to be used and allows development of an
anaerobic zone in the settled sludge. The geometry of the air-lift pump limits the minimum
flow rate of returned sludge to be about 12x that of the influent;
(3) Sludge can be pumped intermittently from the separator to the aeration vessel
(e.g. 5 min every 2.5 h to recycle 1 1/h to 1.5 1/h);
(4) A nontoxic anti-foaming agent (e.g. silicone oil) at minimal concentration can be
used to prevent loss by foaming;
(5) Air can be passed through the sludge in the separator in short, shock bursts (e.g.
10 sec every hour);
(6) The organic medium may be dosed at intervals into the aeration vessel (e.g. for 3
min to 10 min every hour).
(3) Porous pot procedure, (i) 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 substance) and it is recommended (but not required) that each
test substance be tested in duplicate.
(ii) Fill the aeration vessel with activated sludge to the level of the effluent
overflow. The volume required is 3.8 1 for a typical porous pot (see Figure 3). The initial
MLVSS should be 1.5 to 3.0 g/L, and this level should be maintained throughout the test.
This may necessitate supplementation of the feed with synthetic sewage.
(iii) Start the aeration and set the air flow. The air flow should be sufficient to
17
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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 10/1 on a volume basis.
(iv) Place 1 1 of test/reference substance dosing solution in the dosing vessel.
(v) Start the dosing pumps, lubricating the tubes with a small amount of glycerol.
(vi) Start the sludge wastage pump at the rate required to give the desired SRT. The
SRT (in days) is equal to the pot volume (1) divided by the sludge wastage rate (1/d).
Alternatively, sludge may be wasted manually by periodically discarding the required
volume.
(vii) Set the sewage dosing rate to give the required HRT and the test/reference
substance dosing rate at about 0.5 ± 0.05 ml/min.
(viii) Daily measurements of sewage flow rates should be made using a 25-ml
measuring buret and a stopwatch. The flow rates should be adjusted to within ± 0.05
mL/min of the required flow.
(ix) Dosing solution flow rates should be calculated from measuring the volume left
after 24 h of dosing.
(x) 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.
(xi) 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.
(xii) The temperature, pH and DOC of the mixed liquor should be measured at least
every other day.
(xiii) Periodically remove a 40-ml sample of mixed liquor from the aeration vessel
for MLVSS determination. Three times weekly is usually sufficient.
(xiv) 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.
(xv) The porous pot liner should be changed at the first sign of blocking of the
pores; i.e., when the mixed liquor rises above the effluent overflow. To change the liner,
proceed as explained in this paragraph: siphon 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
18
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cleaned before reuse by immersion in a 20 percent solution of hypochlorite bleach for
several hours. The liners should be thoroughly rinsed in clean tap and the deionized water
before reuse.
(xvi) Sewage and effluent samples should be taken twice weekly during the
stabilization ("running-in") period for organic carbon analysis and specific compound
analysis if required. If necessary, ammoniacal nitrogen, nitrate, nitrite, COD and 5-d BOD
may also be determined.
(xvii) When the pots have attained steady-state conditions, the effluents are
analyzed periodically to determine the extent of biodegradation/removal of the test
substance during treatment.
(xviii) 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).
(4) Sampling and analysis, (i) At regular intervals measure the DO concentration,
temperature and pH of the activated sludge in the aeration vessels. Ensure that sufficient
oxygen is always available (>2 mg/1) and that the temperature is kept in the required range
(normally 20°C to 25°C). Keep the pH at 7.5 ± 0.5 by dosing small amounts of inorganic
base or acid into the aeration vessel or into the influent, or by increasing the buffering
capacity of the organic medium (see paragraph (j)(9)(i) of this guideline). When
nitrification occurs acid is produced, the oxidation of 1 mg N producing the equivalent of
about 7 mg COs"2. The frequency of measurement depends on the parameter to be measured
and the stability of the system, and may vary between daily and weekly.
(ii) Measure the DOC or COD in the influents to the control and test vessels.
Measure the test substance concentration in the influent by specific analysis, or estimate it
from the concentration in the stock solution (see paragraph (j)(10)(i) of this guideline), the
volume used and the amount of sewage dosed into the test unit. It is recommended that the
concentration of the test substance be calculated in order to reduce the variability of the
concentration data.
(iii) Take suitable samples from the collected effluent (e.g. 24-h composites) and
filter through a membrane of pore size 0.45 jim, or centrifuge them at 40,000 m/s2 for 15
min. Centrifuging should be used if filtering is difficult. Determine DOC or COD at least in
duplicate to measure ultimate biodegradation and, if required, primary biodegradation by an
analysis method specific for the test substance.
(iv) The use of COD may give rise to analytical problems at low concentrations and
is therefore recommended only if a sufficiently high test concentration (about 30 mg/1) is
used. Also, for strongly adsorbing substances, it is recommended that the amount of
adsorbed substance in the sludge be measured using an analytical technique specific for the
test substance.
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(v) The frequency of sampling depends on the expected duration of the test. A
recommended frequency is three times per week. Once the units are operating efficiently,
allow from 1 week to a maximum of 6 weeks after the test substance has been introduced,
for adaptation to reach steady state. Preferably obtain at least 15 valid measurements during
the plateau phase (see paragraph (m)(2)(iv) of this guideline), which normally lasts 3
weeks, for the proper evaluation of the test substance. The test may be terminated if a
sufficient degree of elimination is reached (e.g. >90%) and these 15 values, which represent
analyses carried out each weekday over 3 weeks, are available. Normally, the test duration
should not exceed 12 weeks after addition of the test substance.
(vi) If the sludge nitrifies and the effects of the test substance on nitrification are to
be studied, analyze samples from the effluent of the test and control units at least once per
week for ammonium and/or nitrite plus nitrate.
(vii) All analyses, but especially the nitrogen determinations, should be performed
as soon as possible. If analyses have to be postponed, store the samples at about 4°C in the
dark in full, tightly closed bottles. If samples have to be stored for more than 48 h, preserve
them by freezing, acidification (e.g. 10 ml/1 of a 400 g/1 solution of sulfuric acid), or
addition of a suitable preservative (e.g. 20 ml/1 of a 10 g/1 solution of mercury(II) chloride).
Ensure that the preservation technique does not influence results of analysis.
(5) Coupling of test units, (i) If coupling is to be used, exchange daily the same
amount of activated sludge (150 ml to 1500 ml for aeration vessels containing 3 1 of liquor)
between the aeration vessels of the test unit and its control unit. If the test substance
adsorbs strongly onto the sludge, change only the supernatant of the separators. In both
cases use a correction factor to calculate the test results.
(ii) In order to try to equalize the microbial populations in sludges in a test unit
receiving organic medium plus a test substance, and in a control unit, receiving only
organic medium, a daily interchange of sludge was introduced (see paragraph (p)(24) of
this guideline). The procedure was called coupling and the method is referred to as the
Coupled Units Test. Coupling was initially performed using Husmann activated sludge
units but it has also been done with porous pot units (see paragraphs (p)(3) and (p)(8) of
this guideline). No significant differences in results were found between noncoupled and
coupled units, whether Husmann or porous pot, suggesting that there is no advantage in
expending the time and energy needed in coupling the test units.
(iii) Sludge exchanges can give the appearance of considerable removal, since
some of the test substance in transferred and the concentrations of test substance in the test
and control effluents become more nearly equal. Thus, correction factors have to be used
which depend on the fraction exchanged and the mean HRT. More details of the
calculation have been published (see paragraph (p)(24) of this guideline).
(iv) Calculate the corrected DOC or COD elimination percentage using the formula
in this paragraph:
Dtc = (Dt- 100-a-r/12)/(l-a-r/12)
20
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where Dtc = corrected % DOC or COD elimination
Dt = determined % DOC or COD elimination
a = interchange fraction of the volume of the activated sludge units
r = mean HRT (h)
If, for example, half of the volume of the aeration tank is exchanged (a = 0.5) and the mean
HRT is 6h, the correction formula is:
Dtc = 4Dt- 100
3
(m) Data and reporting—(1) Treatment of results, (i) Calculate the percentage of
DOC or COD elimination of the test substance for each time using the equation
Dt = G. - (E -En) x 100
Cs
where Dt = % elimination of DOC or COD at time t
Ct = DOC or COD in the influent due to the test substance, preferably estimated
from the stock solution (mg/1)
E = measured DOC or COD value in the test effluent at time t (mg/1)
E0 = measured DOC or COD value in the control effluent at time t (mg/1)
(ii) The degree of DOC or COD elimination of the organic medium in the control
unit is helpful information in assessing the activity of the activated sludge during the test.
Calculate the percentage elimination from the equation
DB = CM-En x 100
CM
where DB = % elimination of DOC or COD of the organic medium in the control unit at
time t
CM = DOC or COD of the organic medium in the control influent (mg/1)
Optionally, calculate the percentage elimination DOC or COD due to the organic medium
plus test substance in the test unit from the equation
DT = CT-E x 100
CT
where DT = % elimination of total test influent DOC or COD
CT = DOC or COD of total test influent or calculated from stock solutions (mg/1)
(iii) Calculate the removal of the test substance if measured with a specific
analytical method at each time assessment from the equation
21
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DST = Si-Se x 100
Si
where DST = % primary elimination of test substance at time t
S; = measured or estimated test substance concentration in the test influent (mg/1)
Se = measured test substance concentration in test effluent at time t (mg/1)
(iv) If the coupling mode has been used, compensate for the dilution of the test
substance in the aeration vessel by the sludge exchange using a correction factor (see
paragraph (l)(5)(iv) of this guideline).
(2) Expression of test results—(i) Elimination curve. Plot the percentage
elimination Dt (or Dtc) and Dst, if available, vs. time (see Figure 4). From the shape of the
elimination curve of the test substance (as parent or as DOC), conclusions may be drawn
about the removal process.
Figure 4. Example of an elimination curve
Polyethylene glycol 400
Test Concentration 20 mg/1 as DOC
120
0
0
10 15 20
Time (Day)
25
30
(ii) Adsorption. If high DOC elimination for the test substance is observed from
the beginning of the test, the test substance is probably eliminated by adsorption onto the
activated sludge solids. It is possible to prove this by determining the adsorbed test
substance by specific analysis. It is not usual for the elimination of DOC of adsorbable
substances to remain high throughout the test; normally, removal is high initially but
22
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gradually falls to a lower, equilibrium value. However, acclimation can occur over time,
possibly encouraged by the sorption to sludge, 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.
(iii) 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.
(iv) 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.
(v) Mean degree of elimination of test substance. Calculate the mean value from
the elimination values (Dt) of the test substance at the plateau phase. Rounded to the
nearest whole number, it is the degree of elimination of the test substance. It is also
recommended to calculate the 95% confidence interval of the mean value.
(vi) Elimination of organic medium. Plot the elimination percentage of the DOC
or COD of the organic medium in the control unit (DB) vs. time. Indicate the mean degree
of elimination in the same way as for the test substance.
(vii) Indication of biodegradation. If the test substance does not sorb
significantly to activated sludge and the elimination vs. time curve has the typical shape
of a biodegradation curve with lag period, then increasing percentage degradation and
finally the plateau phase, the measured elimination can safely be attributed to
biodegradation. If a 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
substances or perform additional static biodegradation tests based on parameters clearly
indicative of biological processes. Such tests are the oxygen uptake methods (OECD
301C, 301D and 301F) or a test with measurement of carbon dioxide production (301B or
310), using a pre-exposed inoculum from the simulation test. 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.
(3) 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)(l)(ii) of this guideline). Consider the test to be valid if the
degree of DOC or COD elimination in the control unit is >80% after two weeks and no
23
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unusual observations have been made.
(ii) If a readily biodegradable (reference) substance has been used, the degree of
biodegradation (Dt) should be >90%.
(iii) If the test is performed under nitrifying conditions, the mean concentration in
the effluents should be <1 mg/1 for ammonia-N and <2 mg/1 nitrite-N.
(iv) If these criteria are not met, repeat the test using an inoculum from a different
source, include a reference substance, and review all experimental procedures.
(4) 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.
(5) Test report. The test report should include the information in paragraphs
(m)(5)(i) through (m)(5)(iii) of this guideline:
(i) Test substance
(A) Identification data;
(B) Physical state and, where relevant, physical/chemical properties.
(ii) Test conditions
(A) Type of test system; and any modifications for testing insoluble or volatile
substances;
(B) Type of organic medium;
(C) Proportion and nature of industrial wastewaters in sewage, if known;
(D) Inoculum, nature and sampling site(s), concentration and any pre-treatment;
(E) Test substance stock solution: DOC and TOC content; how prepared, if a
suspension; test concentration used; reasons if outside range of 10-20 mg DOC/1; method of
addition; date first added; any changes;
(F) Mean SRT and mean HRT; method of sludge wastage; methods of overcoming
bulking, loss of sludge, etc.;
(G) Analytical techniques employed;
(H) Test temperature;
24
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(I) Sludge qualities: MLVSS, sludge volume index (SVI), bulking (if observed);
(J) Any deviations from standard procedures and any circumstances that may have
affected results.
(iii) Test results
(A) All measured data, including DOC, COD, specific analyses, pH, temperature,
oxygen concentration, suspended solids, N compounds, as appropriate;
(B) All calculated values of Dt (or Dtc), DB, DSt, in tabular form and as elimination
curves;
(C) 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;
(D) Discussion of results.
(n) Tests At low (ug/1) concentration range—(1) Background. Many substances
are normally present in the aquatic environment, even in wastewaters, at very low
concentrations (ng/1). At such concentrations, they probably do not serve as primary
substrates resulting in growth, but are more likely to degrade as nongrowth, secondary
substrates, concurrent with a variety of naturally occurring carbon compounds.
Consequently the degradation of such substances will not fit kinetics models for substrates
that support growth (see paragraph (o) of this guideline). There are many models that could
be applied and, under the conditions prevailing in wastewater treatment systems, more than
one may be operative at a given time.
(2) Procedure. Until research sheds more light on kinetics of biodegradation at low
substrate concentrations in activated sludge treatment, the procedure given in the main text
(see paragraph (1) of this guideline) can be followed, but only for primary biodegradability,
using suitably low test substance concentrations (<100 |ig/l) and a validated analytical
procedure. The percentage biodegradation may be calculated provided that abiotic
processes (adsorption, volatility, etc.) are taken into account. Examples can be found in the
studies cited in paragraphs (p)(25) and (p)(26) of this guideline, which used a fill-and-draw
system with 4-h cycle. These studies reported pseudo first-order biodegradation rate
constants for 5 substances in synthetic sewage initially at 5-100 |ig/l. For ultimate
biodegradability, 14C-labeled test substance may be used. A description of this is beyond
the scope of this guideline since there are as yet no agreed procedures, although a proposed
method for ISO standard 14592 (see paragraph (p)(27) of this guideline) contains guidance
on the use of 14C-labelled substances.
(3) Two-stage test—(i) General. More recently a simpler two-stage test was
proposed (see paragraphs (p)(28), (p)(29) and (p)(30) of this guideline), and is described
below in paragraphs (n)(3)(ii) through (n)(3)(iv). In it, a semi-continuous activated sludge
25
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(SCAS) test is followed by short-term kinetic tests on samples withdrawn from the SCAS
units.
(ii) SCAS test. (A) The SCAS system is operated with known sludge wastage rates
and is fed a modified OECD synthetic sewage or domestic sewage. The synthetic sewage
was modified (because of changing pH value and poor sludge settleability) by addition of
phosphate as buffer, yeast extract, iron (III) chloride and trace element salts, and its COD
was increased to about 750 mg/1 by increasing the concentration of peptone and meat
extract. The units are operated on a 24-h cycle: aeration for 23 h, wastage of sludge,
settlement, and withdrawal of supernatant (effluent), followed by addition of synthetic
sewage plus test substance at up to 100 jig/1. Once per week 10% of the total sludge is
replaced by fresh sludge in order to maintain a balanced microbial population.
(B) The concentrations of test substance initially and at the end of aeration are
measured and the test is continued until a constant removal of test substance is attained; this
takes from one week to several months.
(iii) Short-term test. A short test (e.g. 8 hours) is applied to determine the (pseudo)
first-order kinetic rate constant for the decay of the test substance in activated sludges of
known but different origins and histories. In particular, sludge samples are taken from the
SCAS reactors - at the end of an aeration period when the concentration of organic
substrate is low - during the course of an acclimatization experiment (see paragraph
(n)(3)(ii) of this guideline). Sludge may also be taken from a parallel SCAS unit not
exposed to the test substance, for comparison. Mixtures of sludge and the test substance
added at two or more test substance concentrations in the range 1-50 jig/1 are aerated,
without the addition of synthetic sewage or other organic substrate. Test substance
remaining in solution is determined at regular intervals, e.g. hourly, depending on the
degradability of the substance, for a period not longer than 24 h. Samples are centrifuged
before analysis.
(iv) Calculations. (A) Data from the SCAS units are used to calculate the
percentage removal of test substance (see paragraph (m)(l)(iii) of this guideline). Also, an
average rate constant KI, normalized for concentration of suspended solids, can be
calculated as shown in this paragraph:
KI = 1/t • In (Ce / CO • 1/SS (1/g h)
where t = aeration time (23 h)
Ce = concentration at end of aeration period (|ig/l)
C; = concentration at beginning of aeration (|ig/l)
SS = concentration of activated sludge solids (jig/1)
(B) In the short term test the log (% concentration remaining) is plotted against time
and the slope of the initial part of the plot (i.e. at 10-50% degradation) is equivalent to KI,
the (pseudo) first-order rate constant. The constant is normalized with respect to the
concentration of sludge solids by dividing by the concentration of sludge solids. As
reported, test results should also include details of initial concentrations of the test
26
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substance and suspended solids, sludge retention time, sludge loading and source, and
details of pre-exposure (if any) to the test substance.
(4) Variability of results. Variability and other facets of biodegradation kinetics
were discussed at a 1996 workshop (see paragraph (p)(31) of this guideline). From such
studies, reported and projected, a clearer view of kinetics operating in wastewater treatment
plants should be forthcoming, to enable a better interpretation of existing data as well as
suggest more relevant designs for future test guidelines.
(o) Effects of SRT on treatability of substances—(1) Background. The method
described in the main text (paragraphs (a) through (1) of this guideline) was 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. The conditions of operation of the activated sludge units and choice of
influent allow rather wide variations in concentration of the test substance in the effluent.
Normally tests are performed at only one nominal concentration of sludge solids or one
nominal SRT, and the sludge wastage regimes described can cause the value of SRT to vary
considerably during the test, both from day to day and during a day.
(2) General. In this variant (see paragraphs (p)(10) and (p)(ll) of this guideline)
the SRT is controlled within much narrower limits throughout each 24-h period, as happens
in full-scale treatment plants, which results in a more constant test substance concentration
in effluents. Domestic sewage is recommended since it gives more consistent and higher
percentage removals. Also, the effects of varying the SRT are investigated, and in a more
detailed study, the effects of a range of temperature on effluent concentration may be
determined.
(3) Kinetic models. There is no general agreement yet on which kinetic models
operate when substances biodegrade in wastewater treatment. The Monod model of
bacterial growth and substrate utilization was selected for application here, since the
method was intended for application only to substances produced in high tonnages,
resulting in concentrations in sewage of above 1 mg/1. The validity of the simplified model
and the assumptions made was established using a series of alcohol ethoxylates having
varying degrees of primary biodegradability (see paragraphs (p)(ll) and (p)(32) of this
guideline).
(4) Principle of the test, (i) This variant method follows closely much of the text in
paragraphs (a) through (1) of this guideline and only those details that differ are given
hereafter.
(ii) Activated sludge porous pot units, designed to facilitate the nearly continuous
wastage of mixed liquor allowing very precise control of the SRT, are operated in the non-
coupled mode over a range of SRT and, optionally, over a range of temperature. The
retention time is usually 2 to 10 d and the temperature between 5 and 20°C. Sewage,
preferably domestic, and a solution of the test substance, are dosed separately to the units at
rates to give the required sewage retention time (3 to 6 h) and the required concentration of
27
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test substance in the influent. Control units receiving no test substance are operated in
parallel for comparative purposes.
(iii) Other types of apparatus can be used but great care should be exercised to
ensure that good control of SRT is achieved. For example, when using a model test system
that incorporates a settler, allowance for loss of solids via the effluent may be necessary.
Further, special precautions to avoid errors due to variation in the quantity of sludge in the
settler should also be taken.
(iv) The units are operated at each selected set of conditions and, after steady state
has been reached, the average steady-state concentrations in the effluents of test substance
and, optionally, DOC, are obtained over a period of about three weeks. Besides assessing
the percentage removal of test substance and, optionally, DOC, the relationship between
operating conditions and the test substance concentration in the effluent is expressed in
graphical form. From this, tentative kinetic constants may be calculated and the conditions
under which the test substance can be treated may be predicted.
(5) Apparatus, (i) A suitable unit is the modified porous pot system (see Figure 5).
It consists of an inner vessel (or liner) constructed from porous polypropylene of 3.2 mm
thickness and pore size of approximately 90 jim, the joint being butt-welded. This makes a
more robust unit than the one shown in Figure 3 (see also paragraph (j)(3)(i) of this
guideline). The liner is fitted into an impervious polyethylene outer vessel, which consists
of two parts: a circular base in which holes are bored to accommodate two air lines and a
sludge wastage line, and an upper cylinder that screws onto the base and that has an outlet
placed so as to give a known volume (3 1) in the porous pot vessel. One of the air lines is
fitted with a diffuser stone and the other is open-ended and set at right angle to the stone in
the pot. This system produces the necessary turbulence to ensure that the contents of the pot
are completely mixed, as well as providing concentrations of DO >2 mg/1.
(ii) The appropriate number of units are maintained at controlled temperatures in the
range of 5 to 20°C (+/- 1°C), either in a water bath or in a constant-temperature room.
Pumps are required to dose to the aeration vessels the solution of test substance and settled
sewage at the required rates (0-1.0 ml/min and 0-25 ml/min, respectively), as well as a third
pump to remove waste sludge from the aeration vessels. The necessary very low flow rate
of waste sludge is achieved by using a pump set at a higher rate and operated intermittently
by the use of a timer switch, e.g. operating for 10 seconds per min, with a pump delivery
rate of 3ml/min yielding a wastage rate of 0.5 ml/min.
(6) Inoculum. Paragraph (l)(l)(i) of this guideline applies, but use only activated
sludge at about 2.5 g/1.
(7) Number of test units. For a simple test, i.e. to measure percentage removal,
only a single SRT is required, but in order to acquire data necessary to calculate tentative
kinetic constants, data for 4 or 5 SRT values are required. Values between 2 and 10 days
are usually chosen. Practically, it is convenient to perform a test at 4 or 5 SRT values
simultaneously at one temperature. In extended studies the same SRT values, or perhaps a
different range of values, are used at other temperatures within the range 5 to 20°C. For
28
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primary biodegradation (the main use), only one test unit per set of conditions is normally
required. However, for ultimate biodegradability a control unit is required, for each set of
conditions, which receives sewage but not test substance. If the test substance is thought to
be present in the sewage used in the test, it is necessary to use control units when assessing
primary biodegradation, and to make the necessary correction in the calculations.
(8) Dosage of organic medium and test substance. Paragraphs (l)(2)(i) and
(l)(2)(ii) of this guideline apply generally, but note that the test substance solution is dosed
separately and that various sludge wastage rates are used. Also, frequently (e.g. twice per
day) monitor and adjust to within +/- 10%, if necessary, the flow rates of influent, effluent
and sludge wastage. If difficulties are encountered in the analytical methods when domestic
sewage is used, perform the test with synthetic sewage, but it must be assured that different
media give comparable kinetic data.
(9) Handling of activated sludge units. Paragraph (l)(2)(iii) of this guideline
applies generally, but control SRT only by constant wastage of sludge.
(10) Sampling and analysis. Paragraph (1)(4) of this guideline applies generally,
except that the concentration of the test substance should be determined and DOC
determined optionally; also, COD should not be used.
(11) Calculation of kinetic constants—(i) It is more realistic to quote the mean
steady-state concentration of the test substance in the effluent and to describe how this
varies with operating conditions than to quote percentage removal due to primary
biodegradation. This can be done by consideration of Equation [6] of this guideline, which
can yield values for KS, |im and ©sc, the critical sludge retention time.
(ii) Alternatively, approximate values of Ks and m may be obtained using a simple
computer program to fit the theoretical curve calculated from Equation [2] of this guideline
to the experimental values obtained. Although any given solution will not be unique, a
reasonable approximation of Ks and |imcan be obtained.
(iii) By assuming Monod kinetics apply and considering a mass balance of active
solids and substrate across the activated sludge system (see paragraph (p)(10) of this
guideline), the steady state expressions shown in this paragraph can be obtained:
J_ = [% • S^ - Kd Equation [1]
©s Ks + Si
Si = Ks-Q +KH-Q.) Equation [2]
0~ • (|im - Kd) -~1
where Si = concentration of substrate in effluent (mg/1)
Ks = half-saturation constant, the concentration at which |i = |im/2 (mg/1)
|i = specific growth rate (d"1)
|im = maximum value of m (d"1)
Kd = specific decay rate of active solids (d"1)
29
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0s = mean SRT (d)
Examination of this equation leads to the conclusions in paragraphs (o)(ll)(iii)(A) through
(A) The effluent concentration is independent of that in the influent (S0); hence, the
percentage biodegradation varies with the influent concentration, S0.
(B) The only plant-control parameter affecting Si is the sludge retention time, S.
(C) For a given concentration in the influent, S0, there will be a critical sludge
retention time, such that:
J_ = j±s_l_So_ - Kd Equation [3]
0SC KS + So
where 0SC = critical sludge retention time, below which the competent microorganisms will
be washed out of the plant.
(D) Since the other parameters in equation [2] are associated with growth kinetics,
temperature is likely to affect the effluent substrate level and the critical SRT, i.e. the sludge
retention time needed to obtain a certain degree of treatment, will increase with decreasing
temperature.
(iv) From a mass balance of solids in the porous pot system, and assuming that the
solids concentration in the plant effluent, X2, is low compared with that in the aeration
vessel, Xi,
0S = V-Xi _ Equation [4]
(Qo - Qi) • X2 + Qi • Xi
and
0S = v-x, = V
Qi • Xi Qi
where 0s = mean SRT (d)
V = volume of the aeration vessel (1)
Xi = concentration of solids in aeration vessel (mg/1)
X2 = concentration of solids in effluent (mg/1)
Qo = fl ow rate of influent (1/d)
Qi = flow rate of waste sludge (1/d)
Thus, it is possible to control the sludge retention time at any preselected value by
the control of the waste sludge flow rate, Qi.
(v) The main purpose of the test is thus to allow the effluent concentration, and
hence the levels of test substance in the receiving waters, to be estimated. By plotting Si,
30
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vs. 0s, the critical sludge retention time, 0sc, can sometimes be readily evaluated; e.g.
curve 3 in Figure 6. When this is not possible, 0sc may be calculated, together with
approximate values of |im and Ks, by plotting Si, vs. Si -0s. Rearrangement of equation
(1) gives
0s = K£ + Si
0S • Kd Hm Hm
Equation [5]
If Kd is small, then 1 + 0S
1 and [5] becomes
Si • 0S = Kj, + Si
Urn Urn
Equation [6]
Thus, the plot should be a straight line (see Figure 7) of slope l/|im and intercept Ks/|im;
also 0S ~l/Hm-
Figure 5. Modified porous pot for SRT control
Test substance
Influent
Plastic outer vessel
Porous
polypropylene liner
Seal
Screw thread
Solid plastic base
Diffuse? stone
rzu
Effluent
Jr supply
Water sludge
31
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Figure 6. Effluent concentration vs. SRT for three temperatures and five SRT
values
CURVE3:5°C
CURVE 2: 10°C
CURVE 1: 15°C
.^ 0
4 6
SRT - Days
32
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Figure 7. Regression line for SRT • Si vs Si at T= 5° C
30 —
25^
U3 2°-
H
*
Wi *z
ij
?
d>
S io-
cs
O
O
1 5-
S
§* 0
x
X'"
MMax: 0.3
Ks; 1
/
xx"
x"k
m l ' 1 1 1
02468 1
Effluent concentration (81) (mg/l)
33
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(12) Variability of results, (i) It is common experience that variable values of
kinetic parameters for individual substances are obtained. It is thought that the conditions
under which the sludge has been grown, as well as the conditions prevailing in the type of
test system used (e.g. see paragraph (o)(4)(iii) of this guideline), have a large effect on the
resulting values. One aspect of this variability has been discussed by Grady et al. (see
paragraph (p)(33) of this guideline), who have suggested that the terms extant and intrinsic
should be applied to two extreme conditions representing the limits of physiological state
that a culture may attain during a kinetics experiment. If this physiological state is not
allowed to change during the test, the kinetic parameter values reflect the conditions in the
environment from which the microorganisms were taken; these values are called extant or
currently existing. At the other extreme, if conditions in the test are such as to permit the
full development of the protein synthesizing system allowing the maximum possible
growth rate, the kinetic parameters obtained are said to be intrinsic, and are dependent only
on the nature of the substrate and the types of bacteria in the culture. As a guide, extant
values will be obtained by keeping the ratio of concentration of substrate to competent
microorganisms (S0/X0) low, e.g. 0.025, and intrinsic values arise when the ratio is high,
e.g. at least 20. In both cases, S0 should equal or exceed the relevant value of Ks, the half-
saturation constant.
(ii) Variability and other facets of biodegradation kinetics were discussed at a 1996
workshop (see paragraph (p)(31) of this guideline). From such studies, reported and
projected, a clearer view of kinetics that apply in wastewater treatment plants should
eventually be forthcoming. This will enable a better interpretation of existing data, as well
as suggest more relevant designs for future test guidelines.
(p) References.
(1) Swisher, R. D. (1987). Surfactant biodegradation, 2nd ed. Marcel Dekker, New
York, NY. 1085pp.
(2) German Government (1962). Ordinance of the degradability of detergents in
washing and cleaning agents. Bundesgesetzblatt, Pt.l, No. 49:698-706.
(3) Painter, H. A. and E.F. King (1978). WRC porous pot method for assessing
biodegradability. Technical Report No. 70, Water Research Centre, Medmenham, UK.
(4) Painter, H. A. and E.F. King (1978). The effect of phosphate and temperature on
growth of activated sludge and on biodegradation of surfactants. Wat. Res. 12, 909-915.
(5) Eckenfelder, Jr., W.W. and R.R. Cardenas, Jr. (1966). Scale-up from laboratory
activated sludge and trickling filter units to prototype design. Biotechnol. Bioengr. 8, 389-
404.
(6) Gerike, P. and W.K. Fischer (1979). A correlation study of biodegradability
determinations with various chemicals in various tests. Ecotox. Environ. Saf 3, 157-173.
(7) Gerike, P. and W.K. Fischer (1981). A correlation study of biodegradability
34
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determinations with various chemicals in various tests. II. Additional results and
conclusions. Ecotox. Environ. Saf. 5, 45-55.
(8) Painter, H. A. and D. Bealing (1989). Experience and data from the OECD
activated sludge simulation test. In (B.N. Jacobsen, H. Muntau, G. Angeletti, H.A. Painter
and DJ. Bealing, eds.) Laboratory tests for simulation of water treatment processes, pp.
113-138. CEC Water Pollution Report 18.
(9) ISO 11733 (1995). Evaluation of the elimination and biodegradability of organic
substances in an aqueous medium - activated sludge simulation test. International
Organization for Standardization, Geneva.
(10) Birch, R. R. (1982). The biodegradability of alcohol ethoxylates. XIII Tornado
Com. Espafiol. Deterg., 33-48.
(11) Birch, R. R. (1984). Biodegradation of nonionic surfactants. J. Am. Oil. Chem.
Soc. 61, 340-343.
(12) Gerike, P., W.K. Fischer and W. Holtmann (1980). Biodegradability
determinations in trickling filter units compared with the OECD confirmatory test. Wat.
Res. 14, 753-758.
(13) Baumann, U., G. Kuhn and M. Benz (1998). Einfache Versuchsanordnung zur
Gewinnung gewasserokologisch relevanter Daten. UWSF - Z. Umweltchem. Okotox. 10,
214-220.
(14) HMSO (1982). Methods for the examination of waters and associated
materials. Assessment of biodegradability, 1981, pp. 91-98. Her Majesty's Stationery
Office, London.
(15) US EPA (2007). Simulation test - aerobic sewage treatment: B. Biofilms.
Fate, Transport and Transformation Test Guidelines 835.3260, U.S. Environmental
Protection Agency, Washington, DC.
(16) 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.
(17) Horn, J.A., I.E. Moyer and J.H. Hale (1970). Biological degradation of tertiary
butyl alcohol. Proc. 25th Ind. Wastes Conf. Purdue Univ., 939-854.
(18) Fitter P. and J. Chudoba (1990). Biodegradability of organic substances in the
aquatic environment. CRC Press, Boston, MA.
(19) Stover, E.L. and D.F. Kincannon (1983). Biological treatability of specific
organic compounds found in chemical industry wastewaters. J. Wat. Pollut. Contr. Fed. 55,
97-109.
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(20) ISO 10634 (1995). Water quality-guidance for the preparation and treatment of
poorly water-soluble organic compounds for the subsequent evaluation of their
biodegradability in an aqueous medium. International Organization for Standardization,
Geneva.
(21) Reynolds, L., J. Blok, A. de Morsier, P. Gerike, H. Wellens and WJ. Bontinck
(1987). Evaluation of the toxicity of substances to be assessed for biodegradability.
Chemosphere 16, 2259-2277.
(22) OECD (1984). Activated sludge, respiration inhibition test, no. 209, adopted 4
April 1984. Organization for Economic Cooperation and Development, Paris.
(23) ISO 15522 (1999). Water quality-determination of the inhibitory effect of
water constituents on activated sludge microorganisms. International Organization for
Standardization, Geneva.
(24) Fischer, W., P. Gerike and W. Holtmann (1975). Biodegradability
determinations via unspecific analyses (chemical oxygen demand, DOC) in coupled units
of the OECD confirmatory test. I. The test. Wat. Res. 9, 1131-1135.
(25) Nyholm, N., B.N. Jacobsen, B.M. Pedersen, O. Poulsen, A. Dambourg and B.
Schultz (1992). Removal of micropollutants in laboratory activated sludge reactors.
Biodegradability. Wat. Res. 26, 339-353.
(26) Jacobsen, B.N., N. Nyholm, B.M. Pedersen, O. Poulsen and P. Ostfeldt (1993).
Removal of organic micropollutants in laboratory activated sludge reactors under various
operating conditions: Sorption. Wat. Res. 27, 1505-1510.
(27) ISO 14592 (ISO/TC 1477 SC57 WG4, N264) (1998). Water Quality -
Evaluation of the aerobic biodegradability of organic compounds at low concentrations in
water. International Organization for Standardization, Geneva.
(28) Nyholm, N., F. Ingerslev, U.T. Berg, J.P. Pedersen and H. Frimer-Larsen
(1996). Estimation of kinetic rate constants for biodegradation of chemicals in activated
sludge wastewater treatment plants using short-term batch experiments and |ig/l range
spiked concentrations. Chemosphere 33, 851-864.
(29) Berg, U.T. and N. Nyholm (1996). Biodegradability simulation studies in semi-
continuous activated sludge reactors with low (|ig/l range) and standard (ppm range)
chemical concentrations. Chemosphere 33, 711-735.
(30) Nyholm, N., U.T. Berg and F. Ingerslev (1996). Activated sludge
biodegradability simulation test, Environmental Project No. 337. Danish Environmental
Protection Agency, Ministry of Environment and Energy, Copenhagen.
(31) Hales, S.G, T.C.J. Feijtel, H. King, K. Fox and W. Verstraete, eds. (1997).
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Biodegradation kinetics: generation and use of data for regulatory decision making.
Proceedings of the SETAC Europe Workshop held at Port Sunlight, UK, 4-6 September
1996. SETAC Europe, Brussels.
(32) Birch, R. R. (1991). Prediction of the fate of detergent chemicals during
sewage treatment. J. Chem. Tech. Biotechnol. 50, 411-422.
(33) Grady, C.P.L., B.F. Smets and D.S. Barbeau (1996). Variability in kinetic
parameter estimates: a review of possible causes and a proposed terminology. Wat. Res. 30,
742-748.
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